rologie i - European Congress of Virology

Contents5 th European Congress of VirologyRédacteurs associés et comitéde lecture permanentFrancis Barin (Tours)Yves Gaudin (Gif-sur-Yvette)Denis Gerlier (Lyon)Étienne Thiry (Liège)Noël Tordo (Paris)Astrid Vabret (Caen)Comité de lectureOuvertMembres fondateursHenri Agut (Paris)Marc Éloit (Maisons-Alfort)Anne Flamand (Gif-sur-Yvette)Marc Girard (Lyon)Jean-Marie Huraux (Paris)Didier Ingrand (Clamart)Jean-Jacques Lefrère (Paris)Jean-Michel Pawlotsky (Créteil)Anciens membresHubert Laude (Jouy-en-Josas)Comité scientifiqueAbdenour Benmansour (Jouy-en-Josas)Michèle Bouloy (Paris)Françoise Brun Vezinet (Paris)Bruno Canard (Marseille)Dominique Challine (Créteil)Caroline Denesvre (Nouzilly-Tours)François Denis (Limoges)Philippe Despres (Paris)Jean Dubuisson (Lille)Antoine Gessain (Paris)Jacques Izopet (Toulouse)André Jestin (Ploufragan)Isabelle Jupin (Paris)Mario Keller (Strasbourg)Marie-Edith Lafon (Bordeaux)Bruno Lina (Lyon)Patrice Morand (Grenoble)Nadia Naffakh (Paris)Nicole Pavio (Maisons-Alfort)Hélène Peigue-Lafeuille (Clermont-Ferrand)Bruno Pozzetto (Saint-Étienne)Philippe Roingeard (Tours)Christine Rouzioux (Paris)Rob Ruigrok (Grenoble)Ali Saib (Paris)Camille Sureau (Paris)Sylvie van der Werf (Paris)Correspondants étrangersVincent Deubel (Phnom-Penh)Esteban Domingo (Madrid)Claude Fauquet (Saint-Louis)Otto Haller (Fribourg)Souleymane Mboup (Dakar)Laurent Roux (Genève)Robert Snoeck (Louvain)Mark A. Wainberg (Montréal)S1S4S6S8S9S10S11S12S13S14S15S19S20S36S117S285CommitteesList of Keynote SpeakersList of ChairpersonsWelcomeAwardsYoung scientists travel awardsParticipating Organizations and SupportExhibitorsExhibition mapFacilitiesGeneral InformationProgramme at a glanceScientific ProgrammeWednesday, 11 th of SeptemberThursday, 12 th of SeptemberFriday, 13 th of SeptemberSaturday, 14 th of SeptemberOral Presentations’ AbstractsPoster Presentations’ AbstractsAuthor indexS2Virologie, Vol. 17, supplément 2, septembre 2013

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i r o l o g i eList of Keynote SpeakersALTMEYER Ralf (China)BALDANTI Fausto (Italy)BAMFORD Dennis (Finland)BARCLAY Wendy (United Kingdom)BARTENSCHLAGER Ralf (Germany)BARZON Luiza (Italy)BECKER Stephan (Germany)BENKIRANE Monsef (France)BOUCHER Charles (The Netherlands)BUCHY Philippe (Cambodia)BUTCHER Sarah J. (Finland)CAVAZZANA-CALVO Marina (France)CONIBEAR Tim (United Kingdom)COSSET François-Loïc (France)DOMINGO Esteban (Spain)DROSTEN Christian (Germany)DUPREX Paul (United Kingdom)DURANTEL David (France)ECHAVARRIA Marcela (Argentina)ELLIOTT Richard (United Kingdom)FAILLOUX Anna-Bella (France)FASEL Nicolas (Switzerland)FAURE Mathias (France)FAZAKERLEY John (United Kingdom)FOUCHIER Ron (The Netherlands)GACK Michaela (USA)GASTAMINZA Pablo (Spain)GAUDIN Yves (France)GERNA Giuseppe (Italy)GOODFELLOW Ian (United Kingdom)GUY Bruno (France)HAAGMANS Bart (The Netherlands)HELENIUS Ari (Switzerland)HOFFMANN Jonathan (Madagascar)JACKSON Nicholas (France)JIMENEZ Miguel-Angel (Spain)KIRCHHOFF Frank (Germany)LERUEZ Marianne (France)MARTINA Byron (The Netherlands)MAVINGUI Patrick (France)MORFIN Florence (France)NEYTS Johan (Belgium)NIESTERS Bert (The Netherlands)NJOUOM Richard (Cameroon)OPENSHAW Peter (United Kingdom)OSTERHAUS Albert (The Netherlands)PALUDAN Soren (Denmark)PARANHOS-BACCALÀ Gláucia (France)PFEIFFER Julie (USA)REVELLO Maria Grazziella (Italy)RIMMELZWAAN Guus(The Netherlands)ROOSSINCK Marilyn (USA)SAKUNTABHAI Anavaj (France)SALEH Maria-Carla (France)SCHNURIGER Aurélie (France)SCHULZ Thomas (Germany)SCHUTTEN Martin (The Netherlands)SINKINS Steven (United Kingdom)SMITH Geoffrey (United Kingdom)SNIJDER Eric (The Netherlands)TRKOLA Alexandra (Switzerland)ULBERT Sebastian (Germany)URBAN Stephan (Germany)VAN DER POEL Wim (The Netherlands)VERHOEYEN Els (France)VIGNUZZI Marco (France)VINJE Jan (USA)WEISSENHORN Winfried (France)WOLF Dana (Israël)S4Virologie, Vol. 17, supplément 2, septembre 2013

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i r o l o g i eList of ChairpersonsAGUT Henri (France)ARNBERG Niklas (Sweden)AVSIC ZUPANC Tatjana (Slovenia)BALDANTI Fausto (Italy)BARTENSCHLAGER Ralf (Germany)BARTOSCH Birke (France)BARZON Luiza (Italy)BERGSTROM Tomas (Sweden)BERKHOUT Ben (The Netherlands)BOUTOLLEAU David (France)CAMPADELLI-FIUME Gabriella (Italy)CATTANEO Roberto (USA)COYLE Peter (United Kingdom)DE LAMBALLERIE Xavier (France)DOMINGO Esteban (Spain)DUPREX Paul (United Kingdom)ELLIOTT Richard (United Kingdom)ENJUANES Luis (Spain)FLECKENSTEIN Bernhard (Germany)GERLIER Denis (France)GINOCCHIO Christine C. (USA)GREBER Urs (Switzerland)HALLER Otto (Germany)HELENIUS Ari (Switzerland)HORVAT Branka (France)KIRCHHOFF Frank (Germany)KOOPMANS Marion (The Netherlands)KUIKEN Thijs (The Netherlands)LAFON Monique (France)LANDOLFO Santo (Italy)LANG Jean (France)LUNDKVIST Ake (Sweden)MAVINGUI Patrick (France)MAVROMARA Penelope (Greece)MCKNIGHT Aine (United Kingdom)MERTENS Thomas (Germany)MURIAUX Delphine (France)NETTELBECK Dirk (Germany)NEYTS Johan (Belgium)NIESTERS Bert (The Netherlands)ORTIN Juan (Spain)OSTERHAUS Albert (The Netherlands)PAIXAO Paulo (Portugal)PALU Giorgio (Italy)PALUDAN Soren (Denmark)PAPA Anna (Greece)PARDIGON Nathalie (France)PENIN François (France)PFEIFFER Julie (USA)PINSCHEWER Daniel (Switzerland)PLYUSNIN Alexander (Finland)POZZETTO Bruno (France)PRADEL Florence (France)PUCHHAMMER Elisabeth (Austria)REY Felix (France)ROQUE AFONSO Anne-Marie (France)ROSA CALATRAVA Manuel France)ROTTIER Peter (The Netherlands)SALEH Maria-Carla (France)SCHULZ Thomas (Germany)SCHUTTEN Martin (The Netherlands)SMITH Geoffrey (United Kingdom)SVENSSON Lennart (Sweden)TOMMASINO Massimo (France)VABRET Astrid (France)VAN DEN BERG Thierry (Belgium)VAN DER POEL Wim (The Netherlands)VAN DER WERF Sylvie (France)VICTOIR Kathleen (France)VOLCHKOV Viktor (France)VON MESSLING Veronika (Germany)ZOULIM Fabien (France)S6Virologie, Vol. 17, supplément 2, septembre 2013

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i r o l o g i eWelcomeWelcome to the European Congress of Virology 2013Lyon Convention Centre, France11-14 September 2013Dear Participants to the 5th ECV, Dear Colleagues and FriendsFor more than a decade, Virology in Europe has increased in strength and coordination. This was initiated through theorganization of a regular “European Congress of Virology” (ECV) and was reinforced by the foundation of the “EuropeanSociety for Virology” (www.eusv.eu) in Rome, April 24, 2009.Following meetings in Glasgow (2000), Madrid (2004), Nürnberg (2007) and Como (2010), the French community of virologistsis pleased to welcome you in Lyon for the 5th European Congress of Virology (ECV), September 11-14,2013. The 5th ECV is combined with the annual meeting of the European Society for Clinical Virology. It brings togetherboth junior and senior investigators from throughout Europe and from all spheres of Virology, including basic, medical,clinical, veterinary and plant Virology.The city of Lyon and the region “Rhône-Alpes” are remarkable in France in many aspects. Research in the basic, clinicaland veterinary domains is very active with some remarkable pieces such as the oldest Veterinary School in Europe orthe only High-Security Laboratory (BSL4) in France built by Charles Mérieux. In addition, a unique network of start-ups,biotechs and major world leading companies investing in vaccine, diagnosis and drugs, offers a unique potential totranslate basic research into applications for the benefit of Public and Animal Health. Last but not least, Lyon is a WorldHeritage city, at the crossroads of many European influences and a key place to enjoy the essentials of the French wayof life, gastronomy, wine, art and culture.We are delighted to receive you for the 5th ECV in Lyon and have done our best to make it a scientifically stimulatingand socially enjoyable event.The Convenors:Dimitri LavilletteCNRS, LyonBruno LinaHCL-UCBL, LyonNoël TordoInstitut Pasteur, ParisS8Virologie, Vol. 17, supplément 2, septembre 2013

i r o l o g i eAwardsAward of the European Society for VirologyAri HELENIUSInstitute of Biochemistry, ETH Zurich, SWITZERLANDFriday 13 th September 201312h30 – 13h15 (Amphitheater)Pathways of animal virus entryESV Junior AwardMichaela GACKDepartment of Microbiology and Immunology, Harvard Medical School, Boston, USAFriday 13 th September 201317h30 – 18h00 (Amphitheater)Essential Role of the Phosphatase PP1 in RIG-I and MDA5 Mediated Antiviral Innate ImmunityESCV Gardner LectureHeine-Medin awardPaul DUPREXDepartment of Microbiology, Boston University School of Medicine,Boston University, Massachusetts, USAWednesday 11 th September 201313h30 – 14h15 (Amphitheater)Illuminating the “isolate, attenuate and vaccinate” paradigmJan Felix DREXLERInstitute of Virology / University of Veterinary Medicine Hannover, Hannover, GERMANYThursday 12 th September 201317h30 – 18h00 (Amphitheater)Abbott AwardIrene GÖRZERDepartment of Virology, Medical University of Vienna, Vienna, AustriaSaturday 14 th September 201309h45 – 10h00 (Amphitheater)Level and kinetics of Plasma Torque Teno Virus DNA after Lung Transplantation as a Marker to GuideImmunosuppressive TherapyAward “Excellence in Virology” of the Italian Society for VirologyGiuseppe GERNALaboratori Sperimentali di Ricerca, Area Trapiantologica, Fondazione IRCCS Policlinico, San Matteo, ITALYSaturday 14 th September 20138h30 – 9h00 (Amphitheater)Virologic and immunologic monitoring of human cytomegalovirus infections in the immunocompetent and immunocompromisedhost: perspectives for the development of a subunit glycoprotein pentameric vaccineVirologie, Vol. 17, supplément 2, septembre 2013S9

5 th European Congress of VirologyYoung Scientists Travel AwardsESV (European Society for Virology)BAUDIN Maria (SWEDEN)CABANILLAS Laura (SPAIN)CONTRANT Maud (FRANCE)DUPONT Jean Baptiste (FRANCE)GUPTA Shawon (SWEDEN)HAGBOM Marie (SWEDEN)KARAMICHALI Eirini (GREECE)LI EN Hsieh (TAIWAN)LOPES Ana M (PORTUGAL)LUTEIJN Rutger (THE NETHERLANDS)NGUYEN Duy Tien (THENETHERLANDS)NIKOLICValentina (SERBIA)NYGÅRDAS Michaela (FINLAND)OUDE MUNNINK Bas(THE NETHERLANDS)POLAT Ceylan (TURKEY)ROZEN GAGNON Kathryn (FRANCE)SCORDEL Chloé (FRANCE)SUBISSI Lorenzo (FRANCE)VU TRA MY Phan (VIETNAM)WANG I Hsuan (SWITZERLAND)STROTTMANN Daisy (BRAZIL)ESCV (European Society for Clinical Virology)GARD Lilli (THE NETHERLANDS)LI Xumeng (FINLAND)MADSEN Tina Vasehus (DENMARK)SADEGHI Mohammadreza (FINLAND)SCHWAIGER Julia (AUSTRIA)SIMON Benedikt (AUSTRIA)VLAHAVA Virginia Maria (GREECE)GfV (Gesellshaft für Virologie) - GermanyBOGDANOW BorisEIFLER MartinKIRCHHOFF JanaKOEHLER ChristianSEDIRI HannaSGM (Society for General Microbiology) - United KingdomBAYOUMY AmrNISHIYAMA ShokoO RIORDAN BernadetteSHAMI AshjanWEISHEIT SabineSIV (Società Italia di Virologia) - ItalyARTUSI SaraCORONA AngelaMANTELLI BarbaraMARTINELLI MariannaMAZAHERI ElhamPELLEGRINELLI LauraSALDAN AldaSPANEVELLO FrancescaS10Virologie, Vol. 17, supplément 2, septembre 2013

i r o l o g i eParticipation Organisations and PatronagesThe Following Organisations Take Part and Support the Congress• European Society for Virology (ESV)EUROPEAN SOCIETYforVIROLOGY• European Society for Clinical Virology (ESCV)• American Society for Microbiology• Association Journées Francophones de Virologie• Danish Society for Virology• European Society for Veterinary Virology• Gesellschaft für Virologie• Hellenic Society of Virology• Hungarian Society for Microbiology• International Society for Neurovirology• Israel Society of Microbiology• Italian Society for Virology (SIV)• Netherlands Society for Microbiology• Norwegian Society for Virology• Polish Society of Virology• Spanish Society of Virology• Society for General Microbiology• Society of Microbiology of the Czech Republic• Swedish Molecular Virology Network• Swedish Society for Microbiology• Swiss Society for Microbiologya n tigoneCOMMUNAUTÉSDE RECHERCHEACADÉMIQUESANTÉEUROPEANSOCIETY FORVETERINARYVIROLOGYVirologie, Vol. 17, supplément 2, septembre 2013S11

i r o l o g i eExhibitorsThe industrial support has been essential for the organization of the 5th European Congress of Virology. We wish toacknowledge the following Companies for their support and participation through different means of sponsorships.GOLD SPONSORSSILVER SPONSORSThe Women’s Health CompanyOTHERS SPONSORSABBOTT MOLECULARALTONA DIAGNOSTICSAPPLIED MATHSBECKMAN COULTERCEPHEIDCOPANDIAGENODEDIASORINEUROBIOEUROGENTECFAST-TRACT DIAGNOSTICSFOCUS DIAGNOTICSINGENINTERNATIONAL AIDS SOCIETYLABQUALITY AND R-BIOPHARMFRANCELUMINEXMERIALMIKROGENMP BIOMEDICALSMWE MEDICAL WIRE & EQUIPMENTNOVATEC IMMUNODIAGNOTISCAQCMDROCHE DIAGNOSTIC FRANCESANOFI R&DSIGMA-ALDRICH CHIMIETRANSGENES12Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyExhibitorsEXHIBITION & POSTER AREAForum 5 & 6POSTER AREA2524232221201918171615 bisPOSTERAREA910111213POSTERAREA15148 765432 1Booth numbersALTONA DIAGNOSTICS 5 FAST-TRACT DIAGNOSTICS 22APPLIED MATHS 25 FOCUS DIAGNOTICS 23MIKROGEN 24 HOLOGIC 3BD 12 LABQUALITY and R-BIOPHARM FRANCE 6BIO-RAD 11 LUMINEX 4BIOMÉRIEUX 10 ESCV 16CEPHEID 2 MP BIOMEDICALS 15COPAN 19 MWE MEDICAL WIRE & EQUIPMENT 20DIAGENODE 14 NOVATEC IMMUNODIAGNOSTICA 21DIASORIN 13 QCMD 8EUROBIO 18 QIAGEN 9EUROPEAN SOCIETY FOR VIROLOGY (ESV) 15bis ROCHE DIAGNOSTIC FRANCE 7EUROGENTEC 1 SIGMA-ALDRICH CHIMIE 17Virologie, Vol. 17, supplément 2, septembre 2013S13

i r o l o g i eFacilities for the 5th EuropeanCongress of VirologyCentre des congrès de Lyon by levelLevel 2ROOMPRESTIGEGRATTE-CIELROOMGRATTE-CIEL 1, 2, 3Level 1ROOM TÊTE-D’ORLevel 0MAIN ENTRANCELevel -1AMPHITHEATERWELCOMEDESKPREVIEW ROOM(for speakers)Level -2EXHIBITION AREAPOSTER AREABREAKSS14Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyOral PresentationsThe congress language is English. All the speakers are invited to check at the Preview Room located in Salle BELLE-COUR 3 Level -1 to download their PowerPoint presentations. Personal laptops cannot be accepted.PostersLocation: Posters will be displayed in the Exhibition Area, Level -2, Forum 5 - 6.Set up: of posters on Wednesday, September 11 th , from 11h30 to 15h00Removal: Saturday, September 14 th from 16h45 onwards.Materials for the mounting of posters will be provided at the POSTER Welcome Desk.Check-in: Upon arrival, you are requested to check-in at the POSTER Welcome Desk.Poster size: A0 maximum size (i.e. 1.19m high & 0.84m wide) or (46.9” high & 33.1” wide), portrait orientation.Your attendance (or of one co-author) is mandatory at the following Poster Sessions:SESSION 1: Thursday, September 12 th from 17h15 to 19h00 (posters with even reference)SESSION 2: Friday, September 13 th from 17h15 to 19h30 (posters with odd reference)Opening Hours of the Industrial ExhibitionWednesday 11 th of SeptemberThursday 12 th of SeptemberFriday 13 th of SeptemberSaturday 14 th of September14h00 to 20h008h30 to 19h008h30 to 19h308h30 to 17h00WIFI AccessFree wifi is accessible from the exhibition area (Forum 5&6 - Level -2) under the name: ECV2013S16Virologie, Vol. 17, supplément 2, septembre 2013

i r o l o g i eGeneral Information (Access)How to reach the Lyon Conference and Convention CentreBy carThe Lyon Convention Centre is situated near the Lyon Périphérique Nord (Northern ring road) -Exit: “Porte de SaintClair”- and 5 minutes from a junction connecting with a major European motorway network.Lyon Motorways:A7, Marseille / A6, Paris / A42, Genève / A43, Chambéry - GrenobleAddress of the Lyon Convention Centre:50, quai Charles de Gaulle - 69463 Lyon Cedex 06 - FranceGPS coordinates of the Lyon Convention Centre:45°47,0829’/ 4°51,1488’By trainLyon - Part-Dieu and Lyon - Perrache are the two train stations in the city, while the third is at Lyon - Saint Exupéryairport.The Lyon Convention Centre is located approximately 20 min away from the Lyon - Part-Dieu station and 35 min awayfrom the Lyon - Perrache station by public transportTGV trains run between Lyon and Paris every hour or every half hour.By public transportThe Lyon Conference and Convention Centre is served by the public transports of the city.Three bus and trolley lines connect the Lyon Convention Centre (“Cité internationale – Centre de Congrès” stop)directly to the Part-Dieu TGV station, the Métro (Saxe-Gambetta/Foch), and the City Centre (Bellecour):• C1 (Train station «Part-Dieu» - Cuire) - a fast trolley bus with a dedicated lane connects the Cité internationale withthe Part-Dieu Station• C4 (Jean Macé – Cité internationale) connects with metro lines A (“Foch” stop) and D (“Saxe-Gambetta” stop)• C5 (Bellecour Antonin Poncet - Rillieux Semailles/Vancia Château Bérard) direct connection with the presqu’île areaet the place Bellecour (downtown)From the Lyon - Part-Dieu TGV stationBus C1, towards Cuire - Stop: Cité internationaleTicket: €1.60From the Lyon - Perrache TGV stationMétro A, towards Vaulx-en-Velin La Soie - Stop FochConnexion with Bus C4, towards Cité internationale - Stop: Cité internationaleTicket: €1.60By bikeVélo’v provides 4000 bikes for rental at its 340 pick-up and drop-off, self-service bike stations, found all across Lyonand Villeurbanne at intervals of 300 m on average.Service available 24/7.No less than 3 Vélo’v stations available on the Cité Internationale site.Virologie, Vol. 17, supplément 2, septembre 2013S17

5 th European Congress of VirologyAirport ConnectionsAt 25 km from the city centre, the Lyon - Saint Exupéry airport offers flights to over a 100 different destinations servingFrance and abroad and is served by numerous airline companies.The Lyon - Part-Dieu station is located less 30 min away from the Lyon - Saint Exupéry airport using the new tramwayline RhônexpressAirport shuttle service and transportation30 minutes from the City Center using the Tram Express Rhonexpress.1 Single Ticket: 13.5 €TaxiGroupement des Taxis de l’Aéroport Lyon - Saint Exupéry: +33 (0)4 72 22 70 90Taxi Radio: +33 (0)4 72 10 86 86Maison des Taxis du Rhône: +33 (0)4 72 72 03 03Congress Parking facilityOn-site CAR PARKS (not free – 3,350 spaces):- P0: located about 100 m away from the Convention Centre- P1: located about 50 m away from the Convention Centre-P2: located about 50 m away from the Convention CentreS18Virologie, Vol. 17, supplément 2, septembre 2013

PROGRAMME AT A GLANCEWednesday 11th of September Thursday 12th of September Friday 13th of September Saturday 14th of SeptemberWS 25 WS 17 WS 5 WS 12 WS 15 WS 10 WS 6 WS 28 WS 26 WS 19 WS 11 WS 28h30 8h30 8h30Respiratoryviral infectionsVector borneviral infectionsViral (immuno)pathogenesisViral geneexpressionHighlypathogenicvirusesVirus entry:envelopeglycoproteinsreceptors,endocytosisRestrictionfactors of viralinfection,interfering RNA& immuneresponseGastrointestinalviralinfections10h30 10h30 10h30Viral infectionsin transplantand immunocompromisedpatientsViral evolutionandquasispeciesVirus exit:assembly,maturation andreleaseOncolyticviruses andgene therapyp. S54 p. S51 p. S47 p. S49 p. S75 p. S72 p. S70 p. S77 p. S101 p. S99 p. S97 p. S9510h30 10h30 10h30Coffee break Coffee break11h 11h 11hCoffee break10h3013hRegistrationPlenary 2 Plenary 3 Plenary 4INTERACTION OF VIRUSES WITH PATHOGENS HOST RESTRICTION AND BARRIERS THE UNIVERSE OF VIRUSES11h OR ENDOSYMBRIONTS 11h TO VIRAL INFECTION11hJulie PFEIFFER (USA)Marilyn ROOSSINCK (USA)Nicolas FASEL (Switzerland)13h 13h13h15Frank KIRCHHOFF (Germany)Ron FOUCHIER (Netherland)Steven SINKINS (UK)p. S56 Ari HELENIUS (Switzerland) - ESV AWARDp. S10413h p. S80Opening - Welcome session13h30 Symposium SymposiumPlenary 1Symposium Symposium Symposium 13h15Inst. Pasteur FinoviFROM BASIC VIROLOGY TO THERAPY13h30 13h30 Lunch LunchFond.Mérieux Rhône-Alpes13h30 Lunch BioMérieux LunchQiagen Sanofi 14h30Networks Viral hepatitisPaul DUPREX (USA) - ESCV Gardner LectureJohan NEYTS (Belgium)14h45 14h45 Pasteurp. S58 p. S81 p. S82Dennis BAMFORD (Finland)Soren PALUDAN (Denmark) Christian DROSTEN (Germany)Maria-Carla SALEH (France) Richard ELLIOTT (UK)15h45 Esteban DOMINGO (Spain)WS 22 WS 20 WS 21 WS 1Marina CAVAZZANA-CALVO (France)WS 23 WS 18 WS 4 WS 14 WS 9 WS 13 WS 7 WS 16 14h45P. S37 15h15h45 16h45Coffee break16h15WS 8 WS 3 WS 24 WS 27 17h1517h15 p. S115 p. S111 p. S113 p. S10916h15 16h45-17hFinal words - Next meeting presentationp. S67 p. S64 p. S59 p. S62 p. S86 p. S89 p. S83 p. S92Antiviraltherapy andresistance toacute infectionInnateimmunityagainst virusesTypingofvirusesNeurotropicvirusesViral diagnosisin chronicinfections andduring pregnacyEmergingdiseases inveterinaryvirologyAdaptiveimmunity andvaccinesJan Felix DREXLER (Germany)Virus structure,dynamicimaging andtrafficking18h15 17h30 17h30 Locations: Amphitheater18h Heine Medin AWARD18hESV Junior AWARD15hViral replicationstrategiesMichaela GACK (USA)p. S41 p. S39 p. S43 p. S45 Room Gratte-ciel 1,2, 3Antiviraltherapy andresistance tochronicinfectionCellular factorscontroling viralinfection androle of hostgeneticsZoonoticvirusesInnovativemethods in viraldiagnosisVirusepidemiologyVirus discoveryandmetagenomicsOncogenicmechanisms ofviruses18h 18hGeneralGeneral18h15 Poster - Cheese and Wine partyPoster and Wine partyRoom Prestige Gratte-CielAssemblyAssemblyGet together - Cheese and Wine party20h ESCV20hESVRoom Tête d'Or20hPoster20h Lyon City Hall reception 20h Gala dinner - Lyon Convention CentreForum 5&6 - Poster Area21h 00h

Scientific Programme -Wednesday 11 th September 201313h00 – 13h30 OPENING CEREMONY – WELCOME SESSION Amphitheater, Level 0Peter COYLE (President ESCV), Otto HALLER (President ESV) Bruno LINA (Lyon, FRANCE),Dimitri LAVILLETTE (Lyon, FRANCE), Noël TORDO (Paris, FRANCE)13h30 – 15h45 PLENARY SESSION 1: “FROM BASIC VIROLOGY TO THERAPY” Amphitheater, Level 0 (p. S37)Chairpersons: Giorgio PALÙ (Padova, ITALY), Felix REY (Paris, FRANCE)13h30 – 14h15 Illuminating the “isolate, attenuate and vaccinate” paradigm,Paul DUPREX (Boston, USA) - ESCV GARDNER LECTURE14h15 – 14h45 Treatment of viral infections, still many viruses to go, Johan NEYTS (Leuven, BELGIUM)14h45 – 15h15 Theoretical and experimental antiviral approach by combining mutagenic agents and inhibitorsEsteban DOMINGO (Madrid, SPAIN)15h15 – 15h45 Using retroviruses for human genetic therapy: progresses and difficultiesMarina CAVAZZANA-CALVO (Paris, FRANCE)15h45 – 16h15 COFFEE BREAK IN THE EXHIBITION AREA Forum 5 & 6 - Level -216h15 – 18h15WORKSHOP SESSIONSWORKSHOP 3: “INNATE IMMUNITY AGAINST VIRUSES” Room Gratte-Ciel 1, 2, 3 - Level 2 (p. S39)Chairpersons: Denis GERLIER (Lyon, FRANCE), Maria-Carla SALEH (Paris, FRANCE)16h15 – 16h4516h45 – 17h0017h00 – 17h1517h15 – 17h3017h30 – 17h4517h45 – 18h0018h00 – 18h15Keynote: Manipulation of autophagy by Measles virus, Mathias FAURE (Lyon, FRANCE)Activation of RIG I by incoming RNA virus nucleocapsids containing a 5’ triphosphorylatedgenome, Michaela WEBER, (Marburg, GERMANY)Sensing of infected cells in absence of virus particles by plasmacytoid dendritic cells isinhibited by the ribonuclease Erns of classical swine fever virus, Nicolas RUGGLI(Mittelhäusern, SWITZERLAND)Integrins synergize with TLRs to initiate a specific branch of the innate response to herpessimplex virus, Gabriella CAMPADELLI FIUME (Bologna, ITALY)Severe acute respiratory syndrome virus with E protein deleted was attenuated because Eprotein was responsible for the induction of an inflammatory response mediated by NF-K-Bactivation, Luis ENJUANES (Madrid, SPAIN)A reverse genetics approach to study the determinants for dsRNA binding and PKR inhibitionin the NS1 protein of influenza A virus, Kristina L. SCHIERHORN (Berlin, GERMANY)The matrix protein of rabies virus binds to RelAp43 to suppress NF-K-B dependent geneexpression related to innate immunity, Youcef BEN KHALIFA (Paris, FRANCE)S20Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyWorkshop 8: “ANTIVIRAL THERAPY AND RESISTANCE TO ACUTE VIRAL INFECTION”Amphitheater, Level 0 (p. S41)Chairpersons: Johan NEYTS (Leuven, BELGIUM), Manuel ROSA-CALATRAVA (Lyon, FRANCE)16h15 – 16h45 Keynote: Antiviral Therapy and Resistance during acute viral infectionCharles BOUCHER (Rotterdam, THE NETHERLANDS)16h45 – 17h00 Potent inhibition of diverse Coronaviruses by targeting membrane bound viral RNA synthesisEveline KINDLER (St. Gallen, SWITZERLAND)17h00 – 17h15 Discovery of low molecular weight compounds inhibiting Chikungunya virus RNA replicationTero AHOLA (Helsinki, FINLAND)17h15 – 17h30 Humanized antibodies able to protect mice from tick borne encephalitisNina TIKUNOVA (Novosibirsk, RUSSIA)17h30 – 17h45 Characterization of human cytomegalovirus microRNA temporal expression profile and targetprediction by dynamic expression analysis, Marta TREVISAN (Padova, ITALY)17h45 – 18h00 Non liposomal delivery of anti-rabies virus siRNAs counteracts viral growth and spread in vitroTobias HALBACH (Munich, GERMANY)18h00 – 18h15 Inhibition of Pyrimidine Biosynthesis Pathway suppresses viral growth through innate immunityPierre Olivier VIDALAIN (Paris, France)WORKSHOP 24: “TYPING OF VIRUSES (MOLECULAR EPIDEMIOLOGY, TAXONOMY)”Room Prestige Gratte-Ciel, Level 2 (p. S43)Chairpersons: Anna PAPA (Thessaloniti, GREECE), Marie ROQUE-AFONSO (Paris, FRANCE)16h15 – 16h45Keynote: Genome diversity and evolution of vector-borne viruses, Luisa BARZON (Padova, ITALY)16h45 – 17h00 Mumps outbreak in the Netherlands, 2010-2012, Sigrid GOUMA (Bilthoven, THE NETHERLANDS)17h00 – 17h15 Molecular epidemiology of Hepatitis A virus in France, 2004-2012, Muriel MACÉ (Villejuif, FRANCE)17h15 – 17h30 Prevalence of human enterovirus and parechovirus genotypes in Amsterdam between 20072011 and comparison to surveillance data, Katja WOLTHERS (Amsterdam, THE NETHERLANDS)17h30 – 17h45 Characterization of complete rotavirus genomes using a single reverse transcriptionpolymerase chain reaction (RT PCR), Christian KOEHLER (Leipzig, GERMANY)17h45 – 18h00 First report on rotavirus genotypes from the Democratic Republic of São Tomé and Príncipe:high prevalence of G8P[6] genotype, Claudia ISTRATE (Lisbon, PORTUGAL)18h00 – 18h15 Taxonomy of Circoviridae: restructuring and expansion, Philippe BIAGINI (Marseille, FRANCE)WORKSHOP 27: “NEUROTROPIC VIRUSES” Room Tête d’Or, Level 1 (p. S45)Chairpersons: Tomas BERGSTROM (Gothenburg, SWEDEN), Monique LAFON (Paris, FRANCE)16h15 – 16h45 Keynote: Neurotropic Viruses, John FAZAKERLEY (Surrey, UNITED KINGDOM)16h45 – 17h00 Viral DNA containing PML NBs (DCP NBs) are a hallmark of the nuclear associated antiviralresponse controlling the latency of a herpesvirus, Patrick LOMONTE (Villeurbanne, FRANCE)17h00 – 17h15 Molecular interactions of tick borne encephalitis virus with human neural cellsDaniel RUZEK (Brno, CZECH REPUBLIC)17h15 – 17h30 Understanding the role of NS1 protein in the neuropathogenicity of Japanese encephalitis virus(Flaviviridae), Melissanne DE WISPELAERE (Paris, FRANCE)17h30 – 17h45 Activation of the potentially neuropathogenic HERV W/MSRV type endogenous retrovirusduring severe infectious mononucleosis and past Epstein Barr virus infection: the missing linkwith multiple sclerosis? Antonina DOLEI (Sassari, ITALY)17h45 – 18h00 Perturbation of neuronal signaling pathways by the rabies virus, Nicolas WOLFF (Paris, FRANCE)18h00 – 18h15 Infection and crossing of an in vitro cellular model of human blood brain barrier by a largearray of neurotropic enteroviruses, Romain VOLLE (Clermont Ferrand, FRANCE)18h15 – 20h00 GET TOGETHER AND POSTER SESSION Forum 5 & 6, Level -2Virologie, Vol. 17, supplément 2, septembre 2013S21

Scientific Programme -Thursday 12 th September 201308h30 – 10h30 WORKSHOP SESSIONSWORKSHOP 5: “VIRAL (IMMUNO)PATHOGENESIS” Room Prestige Gratte-Ciel, Level 2 (p. S47)Chairpersons: Branka HORVAT (Lyon, FRANCE) & Thomas MERTENS (Ulm, Germany)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: Vaccine induced immunity to influenza: Some considerationsGuus F. RIMMELZWAAN (Rotterdam, THE NETHERLANDS)Consumptive coagulopathy and tissue fibrin deposition in seasonal, pandemic and highlypathogenic avian influenza infection, Marco GOEIJENBIER (Rotterdam, THE NETHERLANDS)Bioluminescence Imaging of Cytomegalovirus (CMV) Dissemination in the Guinea Pig ModelDemonstrates a Novel Hot Spot for Infection & Impaired Tropism Associated with a Viral Gprotein Coupled Receptor Mutant, Alistair MCGREGOR (College Station, USA)Herpes Simplex Virus Type 1 infection of enteric nervous system causes neuronal damage throughtoll like receptor 2 mediated macrophages recruitment, Ignazio CASTAGLIUOLO (Padova, ITALY)Ross River Virus is able to persist in tissues of infected macaque similarly to Chikungunyavirus despite lower viremia during acute phaseLaurence DUPUIS MAGUIRAGA (Fontenay aux Roses, FRANCE)MicroRNA expression signatures in chronic viral hepatitis progressionAlessandro SINIGAGLIA (Padova, ITALY)Characterization and functional analysis of the murine gammaherpesvirus 68 microRNAsAmr BAYOUMY (Edinburgh, UNITED KINGDOM)WORKSHOP 12: “VIRAL GENE EXPRESSION – TRANSCRIPTION, TRANSLATION” Room Tête-d’Or, Level 1 (p. S49)Chairpersons: Richard ELLIOTT (Glasgow, UNITED KINGDOM) & Peter ROTTIER (Utrecht, THE NETHERLANDS)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: Norovirus translation: a novel paradigm for viral protein synthesisIan GOODFELLOW (Cambridge, UNITED KINGDOM)Identification of an RNA translational regulator element at the 3’ end of the TGEV genomeFernando ALMAZAN (Madrid, SPAIN)TIA 1 is a cellular restriction factor for tick borne encephalitis virus that binds viral RNAand is recruited to perinuclear sites of viral replication, Alessandro MARCELLO (Trieste, ITALY)Structure of the full length hepatitis C virus IRES in solution, Marc JAMIN (Grenoble, FRANCE)A dynamic G quadruplex region regulates the HIV 1 LTR promoter, Rosalba PERRONE (Padua, ITALY)A transcriptional pre initiation complex encoded by beta and gamma herpesviruses requiredfor late viral gene expression, Henri GRUFFAT (Lyon, FRANCE)HCMV pp150 constitutes a Cyclin A2 CDK dependent switch controlling the cell cycle anddifferentiation dependent onset of lytic gene expression, Boris BOGDANOW (Berlin, GERMANY)S22Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyWORKSHOP 17: “VECTOR BORNE VIRAL INFECTIONS (PARTNERSHIP WITH EU PROGRAMMES EUROWN,VECTORIE, WINGS)” Room Gratte-Ciel 1, 2, 3 – Level 2 (p. S51)Chairpersons: Luisa BARZON (Padova, ITALY), Nathalie PARDIGON (Paris, FRANCE)08h – 08h1008h10 – 08h2008h20 – 08h3009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote 1: Epidemiology of WN in the Mediterranean region: towards better diagnostics andsurveillance Miguel-Angel JIMENEZ (Madrid, SPAIN)Keynote 2: West Nile virus strains in Europe are as virulent as the North American counterpartsMartina BYRON (Rotterdam, THE NETHERLANDS)Keynote 3: Development of vaccines against WNV, Sebastian ULBERT (Leipzig, GERMANY)Impact of vector ecology on virus amplification and spillover into humans and livestock: thecase of West Nile virus in Europe, Jordi FIGUEROLA (Sevilla, SPAIN)Characterization of Human West Nile Virus strains isolated in Northern Italy,Luisa BARZON (Padova, ITALY)Recombinant vaccinia MVA expressing E and prM/M proteins of West Nile Virus for vaccinegeneration Gerd SUTTER (Munich, GERMANY)Sylvatic origin and geographic spread of St. Louis encephalitis virus, Sandra JUNGLEN(Bonn, GERMANY)Clonal expansion of tick borne encephalitis virus in a disease hot spot at lake Vänern, SwedenTomas BERGSTRÖM (Gothenburg, SWEDEN)Novel insights into the defence response in ticks against tick borne encephalitis virusSabine WEISHEIT (Pirbright, UNITED KINGDOM)WORKSHOP 25: “RESPIRATORY VIRUS INFECTIONS” Amphitheater, Level 0 (p. S54)Chairpersons: Fausto BALDANTI (Pavia, ITALY) & Astrid VABRET (Caen, FRANCE)08h30 – 09h00 Keynote: Respiratory virus infections: from pathogenesis to pandemicsPeter Openshaw (London, UNITED KINGDOM)09h00 – 09h15 Growth and characterization of different HRV C types in 3D human airway epitheliareconstituted in vitro, Komla SOBO (Geneva, SWITZERLAND)09h15 – 09h30 Replication of Human Rhinovirus C in a Standard Cell Line, Anna GÖRANSSON (Kalmar, SWEDEN)09h30 – 09h45 Novel system for analysis of virus host interactions in primary human airway epitheliumHulda R. JONSDOTTIR (St.Gallen, SWITZERLAND)09h45 – 10h00 In vitro and in vivo characterization of A(H1N1)pdm09 influenza virus withHA 222 polymorphism Jean Sebastien CASALEGNO (Lyon, FRANCE)10h00 – 10h15 Genetic variation in influenza A virus: mutations associated with immune reactionsand severity of disease, Laura KAKKOLA (Helsinki, FINLAND)10h15 – 10h30 Analyses of Influenza Vaccine Break through Infections and Estimation of Influenza VaccineEffectiveness based on a Sentinel Platform, Monika REDLBERGER FRITZ (Vienna, AUSTRIA)10h30 – 11h00 COFFEE BREAK IN THE EXHIBITION AREA Forum 5 & 6, Level -211h00 – 13h00 PLENARY SESSION 2: “INTERACTION OF VIRUSES WITHPATHOGENS OR ENDOSYMBRIONTS” Amphitheater, Level 0 (p. S56)Chairpersons: Roberto CATTANEO (Rochester, USA), Bernhard FLECKENSTEIN (Erlangen, GERMANY)11h00 – 11h30 How gut microbes enhance enteric virus infectivity, Julie PFEIFFER (Dallas, USA)11h30 – 12h00 Ecology of viruses of endophytic fungi, Marilyn ROOSSINCK (Pennsylvania, USA)12h00 – 12h30 Leishmania RNA virus-a backseat driver to metastatic leishmaniasisNicolas FASEL (Epalingues, SWITZERLAND)12h30 – 13h00 Wolbachia and arbovirus inhibition in mosquitoes, Steven SINKINS (Oxford, UNITEDKINGDOM)Virologie, Vol. 17, supplément 2, septembre 2013S23

5 th European Congress of Virology13h00 – 15h00 LUNCH BREAK (BUFFET) IN THE EXHIBITION AREA Forum 5 & 6, Level -213h30 – 14h45 SYMPOSIUM BIOMERIEUX Molecular Scientific Room Prestige Gratte-Ciel, Level 2 (p. S58)Chair: Christine C. GINOCCHIO (Lake Success, USA)13h30 – 13h45 Erythrovirus B19 infections in Immunocompromised patients, Marianne LERUEZ (Paris, FRANCE)13h45 – 14h00 Evaluation of Parvovirus B19 R-gene qPCR assay for Erythrovirus B19 detection andquantification in Blood and Bone Marrow samples, Aurélie SCHNURIGER (Paris, FRANCE)14h00 – 14h15 How molecular methods have improved Adenoviruses diagnosis. Monitoring in bone marrow orstem cell transplant recipients, Marcela ECHAVARRIA (Buenos Aires, ARGENTINA)14h15 – 14h30 Goals and Potential impact of a complete Molecular Laboratory Automation for virus detectionFlorence MORFIN (Lyon, FRANCE)15h00 – 17h15 WORKSHOP SESSIONSWORKSHOP 4: “ADAPTIVE IMMUNITY AND VACCINES” Room Prestige Gratte-Ciel, Level 2 (p. S59)Chairpersons: Ben BERKHOUT (Amsterdam, THE NETHERLANDS) & Daniel PINSCHEWER (Geneva, SWITZERLAND)15h00 – 15h30Keynote: Increasing vaccine immunogenicity via manipulation of vaccinia virus Bcl-2-likeimmunomodulatory proteins, Geoffrey L. SMITH (Cambridge, United Kingdom)15h30 – 15h4515h45 – 16h0016h00 – 16h1516h15 – 16h3016h30 – 16h4516h45 – 17h0017h00 – 17h15Cowpox virus protein CPXV012 avoids MHC I antigen presentation by blocking ATP bindingto TAP Rutger LUTEIJN (Utrecht, THE NETHERLANDS)The role of antigen avidity in memory inflation upon MCMV infectionLisa BORKNER (Braunschweig, GERMANY)Specificity and predictability of CD4+ T cell responses after tick borne encephalitis virusinfection and vaccination are strongly influenced by structural features of viral proteinsJulia SCHWAIGER (Vienna, AUSTRIA)A new lentiviral vector tool to evaluate measles virus escape from infection- or vaccine-inducedantibodies, Camille LEVY (Lyon, FRANCE)Design of Single Round Infection Alphaviruses: New Approaches of Vaccine DevelopmentIlya FROLOV (Birmingham, USA)Respiratory vaccination with live attenuated measles virus: studies towards identificationof the optimal site of vaccine delivery, Rik DE SWART (Rotterdam, THE NETHERLANDS)Comparison of neutralizing and cross neutralizing antibodies induced by Cervarix andGardasil® Human Papillomavirus Prophylactic Vaccines: an independent study,Laura SQUARZON (Padova, ITALY)WORKSHOP 14: “VIRUS STRUCTURE, DYNAMIC IMAGING AND TRAFFICKING” Room Tête d’Or, Level 1 (p. S62)Chairpersons: Paul DUPREX (Boston, USA), François PENIN (Lyon, FRANCE)15h00 – 15h30 Keynote: Paramyxoviral ultrastructures revealed by electron cryotomographySarah J. BUTCHER (Helsinki, FINLAND)15h30 – 15h45 Real time cell biological analysis of vaccinia replication, N. Bishara MARZOOK (Sydney, AUSTRALIA)15h45 – 16h00 Intracytoplasmic transport of hepatitis B virus capsids and their dissociation from microtubuleQuentin OSSEMAN (Bordeaux, FRANCE)16h00 – 16h15 Tracking genomes of DNA viruses in the cytosol and nucleus by click chemistry and superresolution Microscopy, I Hsuan WANG (Zurich, SWITZERLAND)16h15 – 16h30 Dengue Virus Requires KDEL Receptor to Exit from the ER, Pei Gang WANG (Hong Kong, CHINA)16h30 – 16h45 Recruitment of the host plant heat shock protein 70 by Tomato yellow leaf curl virus coatprotein is required for virus infection, Rena GOROVITS (Rehovot, ISRAEL)16h45 – 17h00 Murine leukemia virus targets innate like B 1 B cells to establish infection in miceXaver SEWALD (New Haven, USA)17h00 – 17h15 Biophysical analyses of the central region of HSV1 pUL36, Stephane ROCHE (Gif sur Yvette, FRANCE)S24Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyWORKSHOP 18: “EMERGING VIRAL DISEASES IN VETERINARY VIROLOGY (PARTNERSHIP WITH ESVV)”Room Gratte-Ciel 1,2,3 - Level 2 (p. S64)Chairpersons: Wim VAN DER POEL (Lelystad, THE NETHERLANDS) & Thierry VAN DEN BERG (Uccle, BELGIUM)15h00 – 15h3015h30 – 15h4515h45 – 16h0016h00 – 16h1516h15 – 16h3016h30 – 16h4516h45 – 17h0017h00 – 17h15Keynote: Emerging diseases in veterinary virologyWim H. M. VAN DER POEL (Lelystad, THE NETHERLANDS)Avian Influenza Virus Hemagglutinins H2, H4, H8, and H14 support a highly pathogenicphenotype Juergen STECH (Greifswald Insel Riems, GERMANY)Vaccination of chickens with neuraminidase recombinant virus replicon particles confersprotection against avian influenza virus, Gert ZIMMER (Mittelhäusern, SWITZERLAND)Influenza A virus infection dynamics in permanently infected pig farms: evidence of recurrentinfections, co circulations of different virus subtypes and reassortment eventsGaëlle SIMON (Ploufragan, FRANCE)Different strategies of host infection by bovine respiratory virusesJana KIRCHHOFF (Hannover, GERMANY)Schmallenberg virus – two years after the incursion into the UKFalko STEINBACH (Addlestone, UNITED KINGDOM)Modulation of the interferon signalling pathway by bluetongue virusVirginie DOCEUL (Maisons Alfort, FRANCE)Study of the virulence of Serotypes 4 and 9 of the Orbivirus African horse sickness virus in twomouse Models, Maria Ana DE LA GRANDIÈRE (Liège, BELGIUM)Workshop 23: “VIRAL DIAGNOSIS IN CHRONIC INFECTIONS AND DURING PREGNANCY”Amphitheater, Level 0 (p. S67)Chairpersons: Peter COYLE (Belfast, Ireland) & Hubert NIESTERS (Groningen, THE NETHERLANDS)15h00 – 15h30 Keynote: Diagnosis and prognosis of human cytomegalovirus (HCMV) infection in pregnancyMaria GRAZIA REVELLO (Torino, ITALY)15h30 – 15h45 Human Cytomegalovirus specific hyperimmune globulin for prevention of congenital infectionfollowing primary infection in pregnancy, Milena FURIONE (Torino, ITALY)15h45 – 16h00 Phylogenetic analysis of human parvovirus B19 in pregnant women in Lyon (FRANCE)Yahia MEKKI (Lyon, FRANCE)16h00 – 16h15 Determination of Rubella Virus Specific Humoral and Cell Mediated Immunityin pregnant women with negative of equivocal rubella specific IgG,Christelle VAULOUP FELLOUS (Villejuif, FRANCE)16h15 – 16h30 A delay in the maternal antibody response to human cytomegalovirus (HCMV) gH/gL/pUL128130 131 complex is associated with virus transmission to the fetus,Giuseppe GERNA (Pavia, ITALY)16h30 – 16h45 Development of a multiplex PCR approach for detection of dual infections and recombinantsinvolving HIV variants, Pierre CAPPY (Rouen, FRANCE)16h45 – 17h00 Clinical Evaluation of BioPlex 2200 HIV Ag Ab: An Automated Screening MethodProviding Discreet Detection of HIV 1 p24, HIV 1 Antibody, and HIV 2 Antibody,François SIMON (Paris, FRANCE)17h00 – 17h15 Impact of the coexistence of anti HBs antibodies on HBsAg quantification in chronic hepatitisB carriers Vincent THIBAULT (Paris, FRANCE)17h30 – 18h00 ESCV Heine-Medin AWARD, Jan Felix DREXLER (Bonn, GERMANY) Amphitheater, Level 018h00 – 20h00 GENERAL ASSEMBLY – ESCV Room Tête d’Or, Level 118h00 – 20h00 POSTER – CHEESE AND WINE PARTY Forum 5 & 6, Level -220h00 – 21h00 LYON CITY HALL RECEPTION Hôtel de Ville de LyonVirologie, Vol. 17, supplément 2, septembre 2013S25

Scientific Programme -Friday 13 th September 201308h30 – 10h30 WORKSHOP SESSIONSWORKSHOP 6: “RESTRICTION FACTORS OF VIRAL INFECTION, INTERFERING RNA & IMMUNE RESPONSE”Room Prestige Gratte-Ciel, Level 2 (p. S70)Chairpersons: Santo LANDOLFO (Turin, ITALY) & Soren PALUDAN (Aarhus, DENMARK)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: Restriction Factors are key determinants of viral susceptibility,Monsef BENKIRANE (CNRS-UPR1142, France)Defining the relationship between miRNAs and cytoplasmic viruses,Simone BACKES (New York, USA)Regulation of virus endocytosis by miRNAs, Pierre Yves LOZACH (Laval, CANADA)Host microRNA molecular signatures associated with human influenza A virus infections revealan unanticipated antiviral activity for miR 146a, Olivier TERRIER (Lyon, FRANCE)Requirement of the autophagy initiating protein kinase Ulk1 for late stages of human cytomegalovirusreplication, Sabine FEICHTINGER (Erlangen, GERMANY)Hantavirus infection confers resistance to Natural Killer cell mediated killing and Hantavirusnucleocapsid protein inhibits granzyme B and caspase 3, Shawon GUPTA (Stockholm, SWEDEN)Inflammatory cytokines promote viral infection of polarized cellsNicola FLETCHER (Birmingham, UNITED KINGDOM)WORKSHOP 10: “VIRUS ENTRY: ENVELOPE GLYCOPROTEINS, RECEPTORS, ENDOCYTOSIS”Room Gratte-Ciel 1, 2, 3 - Level 2 (p. S72)Chairpersons: Niklas ARNBERG (Umea, SWEDEN), Urs GREBER (Zurich, SWITZLERLAND)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: Intermediate structures in the fusion associated transition of vesiculovirusglycoprotein Yves GAUDIN (Gif sur Yvette Cedex, FRANCE)Dipeptidyl peptidase 4: Entry portal for the emerging human coronavirus EMCBerend Jan BOSCH (Utrecht, THE NETHERLANDS)Human hepatitis B and D Viruses exploit sodium taurocholate co transporting polypeptide(NTCP) to bind and enter hepatocytes in a species specific manner, Yi NI (Heidelberg, GERMANY)LDL Receptor and its family members are the cellular receptors of VSV and VSV G pseudotypedviral vectors, Menachem RUBINSTEIN (Rehovot, ISRAEL)Changes in SV40 binding to its receptor GM1 affects vacuolization and plaque formation in CV1monkey cells, Nasim MOTAMEDI (New Haven,USA)Identification of functional networks in E1E2 glycoproteins unveils Hepatitis C virus fusionmechanisms and new therapeutic opportunities, Florian DOUAM (Lyon, FRANCE)Targeted antiviral siRNA screen identifies the poxvirus genome uncoating factorSamuel KILCHER (Zurich, SWITZERLAND)S26Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyWORKSHOP 15: “HIGHLY PATHOGENIC VIRUSES” Amphitheater, Level 0 (p. S75)Chairpersons: Ake LUNDKVIST (Stockholm, Sweden), Viktor VOLCHKOV (Lyon, FRANCE)08h30 – 09h00 Keynote: Transport and assembly of filoviruses, Stephen BECKER (Marburg, Germany)09h00 – 09h15 Mopeia virus and recombinant Lassa virus containing mutations into the exonuclease of thenucleoprotein, but not wild type Lassa virus, induce a strong release of CXC and CC chemokinesby human antigen presenting cells Sylvain BAIZE (Lyon, FRANCE)09h15 – 09h30 Soluble proteins of Ebola virus bind and activate dendritic cells and macrophages causingrelease of pro and anti-inflammatory cytokines, Beatriz ESCUDERO PÉREZ (Lyon, FRANCE)09h30 – 09h45 Crimean Congo Hemorrhagic Fever Virus infected human monocyte deriveddendritic cells basolaterally transmits infection to human epithelial cellsCecilia ANDERSSON (Stockholm, SWEDEN)09h45 – 10h00 Pathogenesis of emergent Henipavirus infection, Branka HORVAT (Lyon, FRANCE)10h00 – 10h15 A virus like particle system for Crimean Congo Hemorrhagic Fever virusStéphanie DEVIGNOT (Marburg, GERMANY)10h15 – 10h30 Marburg and Ebola Virus Vaccines: Antibodies are necessary for ProtectionAndrea MARZI (Hamilton, USA)WORKSHOP 28: “GASTROINTESTINAL VIRAL INFECTIONS” Room Tête d’Or, Level 1 (p. S77)Chairpersons: Julie PFEIFFER (Dallas, USA) & Lennart SVENSSON (Linköping, SWEDEN)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: Genotypic and Epidemiologic Trends of Viral Gastroenteritis InfectionsJan VINJE (Atlanta, USA)Lewis negative phenotype is a strong restriction factor for genotype P[8] rotavirus infectionsJohan NORDGREN (Linköping, SWEDEN)The cholinergic anti-inflammatory pathway attenuates the innate inflammatory responseto rotavirus Infection, Marie HAGBOM (Linköping, SWEDEN)Emerging and common genotypes of Rotavirus among children with severe diarrhea in Italy,2007 2012 Franco Maria RUGGERI (Rome, ITALY)Unexplained diarrhea in HIV 1 infected individuals,Bas OUDE MUNNINK (Amsterdam, THE NETHERLANDS)Rotavirus infections in young children in Ho Chi Minh City, VietnamPhan VU TRA MY (Ho Chi Minh, VIETNAM)Molecular dating in the evolution of primate bocaviruses, Igor BABKIN (Novosibirsk, RUSSIA)10h30 – 11h00 COFFEE BREAK IN THE EXHIBITION AREA Forum 5 & 6, Level -211h00 – 13h15PLENARY SESSION 3: “HOST RESTRICTION AND BARRIERS TO VIRAL INFECTION”Amphitheater, Level 0 (p. S80)Chairpersons: Gabriella CAMPADELLI FIUME (Bologna, ITALY), Otto HALLER (Freiburg, GERMANY)11h00 – 11h30 Innate sensing and IFN induction during infection with DNA viruses,Soren PALUDAN (Aarhus, DENMARK)11h30 – 12h00 RNA virus persistence in Drosophila is controlled by siRNAs produced from de novosynthesized virus cDNA, Maria-Carla SALEH (Paris, FRANCE)12h00 – 12h30 Viral adaptations preceding the AIDS pandemic, Frank KIRCHHOFF (Ulm,GERMANY)12h30 – 13h15 Pathways of animal virus entry, Ari HELENIUS (Zurich, SWITZERLAND) ESV AwardVirologie, Vol. 17, supplément 2, septembre 2013S27

5 th European Congress of Virology13h15 – 15h00 LUNCH BREAK (BUFFET) IN THE EXHIBITION AREA Forum 5 & 6, Level -213h30 – 14h45 SYMPOSIUM QIAGEN Room Prestige Gratte-Ciel, Level 2 (p. S81)ACHIEVING CLINICAL AND OPERATIONAL EXCELLENCE THROUGH WORKFLOW OPTIMISATIONChairpersons: Martin SCHUTTEN (Rotterdam, the Netherlands), David BOUTOLLEAU (Paris, FRANCE)13h30 – 14h00 Keynote: Modernizing in house developed molecular assays, the need for automationMartin SCHUTTEN (Rotterdam, the Netherlands)14h00 – 14h20 Detection and quantification of CMV DNA in peripheral blood and amniotic fluid in pregnancycomplicated by primary CMV infection, Fausto BALDANTI (Pavia, ITALY)14h20 – 14h40 Improving laboratory efficiency and output: Comparison of artus QS-RGQ and establishedin-house workflows Tim CONIBEAR (London, United Kingdom)13h30 – 14h45 SYMPOSIUM SANOFI PASTEUR Room Tête D’Or, Level 1 (p. S82)NEW PARADIGM FOR DENGUE PREVENTIONChairpersons: Nicholas JACKSON (Lyon, FRANCE) - Patrick MAVINGUI (Lyon, FRANCE)13h30 – 13h4513h45 – 14h0014h00 – 14h1514h15-14h3014h30-14h45Characterization of the Sanofi Pasteur dengue vaccine candidate: hypotheses andinvestigations to explain results of the Phase IIb proof-of-concept efficacy trial in RatchaburiBruno GUY (Sanofi Pasteur, Lyon, FRANCE)Role of the vector in dengue epidemic dynamicPatrick MAVINGUI (Lyon, France)Arbovirus emergence caused by small genetic changes in the viral genomeAnna-Bella FAILLOUX (Paris, FRANCE)Functional genomic study of asymptomatic versus symptomatic dengue viral infectionAnavaj SAKUNTABHAI (Paris, FRANCE)Conclusion, Nicholas JACKSON (Lyon, FRANCE)15h00 – 17h15 WORKSHOP SESSIONSWORKSHOP 7: “CELLULAR FACTORS CONTROLLING VIRAL INFECTION AND ROLE OF HOST GENETICS”Chairpersons: Frank KIRCHHOFF (Ulm, GERMANY) & Aine McKNIGHT (London, UNITED KINGDOM)Room Prestige Gratte-Ciel, Level 2 (p. S83)15h00 – 15h30Keynote: Host cell factors and pathways promoting and restricting hepatitis C virus replicationRalf BARTENSCHLAGER (Heidelberg, GERMANY)15h30 – 15h45 Hepatitis C virus infection modulates the central carbon metabolism of the cell,Olivier DIAZ (Lyon, France)15h45 – 16h00 Coxsackievirus Hijacks Host miRNA 126 to Mediate Cross talk of ERK and Wnt/beta cateninSignal Pathways for Its Replication, Decheng YANG (Vancouver, CANADA)16h00 – 16h15 Human respiratory syncytial virus infection is modulated by Streptococcus pneumoniaDuy Tien NGUYEN (Rotterdam, THE NETHERLANDS)16h15 – 16h30 Gene loss and adaptation to hominoids underlie the ancient origin of HIV 1,Lucie ETIENNE (Seattle, USA)16h30 – 16h45 A novel mechanism of human cytomegalovirus evasion from restriction factore IFI16,Valentina DELL’OSTE (Turin, ITALY)16h45 – 17h00 Use of exome sequencing and RNAi to understand the influence of host factors on theoutcome of influenza Infection, Rachael WASH (Cambridge, UNITED KINGDOM)17h00 – 17h15 Characterization of one novel interferon stimulated gene participating in the type I interferonblock to HIV1 infection, Caroline GOUJON (London, UNITED KINGDOM)S28Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyWORKSHOP 9: “ANTIVIRAL THERAPY AND RESISTANCE TO CHRONIC VIRAL INFECTION”Amphitheater (p. S86)Chairpersons: Henri AGUT (Paris, FRANCE) & Christine GINOCCHIO (Lake Success, USA)15h00 – 15h30 Keynote: Cell-cell transmission governs spread and immune escape of HIV-1Alexandra TRKOLA (Zurich, SWITZERLAND)15h30 – 15h45 Simultaneous Hepatitis C virus (HCV) NS3 protease and NS5B polymerase resistance testing by analternative error correction 454 deep sequencing pipeline, F. Xavier LÓPEZ LABRADOR (Valencia, SPAIN)15h45 – 16h00 Experience of HCV resistance after 2 years antiproteases clinical practice; detection ofresistance mutations after long period of undetectability, Christophe RAMIERE (Lyon, FRANCE)16h00 – 16h15 Mutagenesis of HIV 1 reverse transcriptase ribonuclease H domain defines residuescontributing to ribonuclease H activity inhibition by diketo acids, Angela CORONA (Cagliari, ITALY)16h15 – 16h30 New anti CMV drugs alone or in combination: efficacy in cell culture and placental explantsDeborah ANDOUARD (Limoges, FRANCE)16h30 – 16h45 Generation and Characterization of defined HCMV UL97 and UL54 mutations associatedwith drug resistance, Lena FISCHER (Tübingen, GERMANY)16h45 – 17h00 Herpes simplex virus infection control with novel small interfering RNA poolsHenrik PAAVILAINEN (Turku, FINLAND)17h00 – 17h15 Biophysical characterization and antiviral effects of non canonical DNA structuresin the herpes simplex virus type 1 Genome, Sara ARTUSI (Padova, ITALY)WORKSHOP 13: “VIRAL REPLICATION STRATEGIES” Room Gratte-Ciel 1, 2, 3 - Level 2 (p. S89)Chairpersons: Luis ENJUANES (Madrid, SPAIN), Juan ORTIN (Madrid, SPAIN)15h00 – 15h3015h30 – 15h4515h45 – 16h0016h00 – 16h1516h15 – 16h3016h30 – 16h4516h45 – 17h0017h00 – 17h15Keynote: Influenza polymerase host adaptation, Wendy Barclay (London, UNITED KINGDOM)Processive RNA synthesis, repair and capping activities embedded in a SARS Coronavirusmulti protein complex, Lorenzo SUBISSI (Marseille, France)Crystal structure of the N0 P complex of Nipah virus and of VSV provide new insights intothe encapsidation mechanism of the Paramyxoviridae and Rhabdoviridae, Filip YABUKARSKI(Grenoble, FRANCE)Role of host factors in enterovirus RNA replication, Frank VAN KUPPEVELD(Utrecht, THE NETHERLANDS)Phosphatidylinositol 4 kinase III beta enhances human rhinovirus replicationby recruiting the oxysterol binding protein to ER Golgi derived membranes,Pascal S ROULIN (Zurich, SWITZERLAND)A Structural Basis for the Oligomer Assembly and Host Chromatin Interactionof Two Gamma2 Herpesviral LANA Proteins and Their Role in Viral Persistence,Magdalena WEIDNER GLUNDE (Hannover, GERMANY)Unscheduled mitotic entry of HCMV infected cells leads to chromosomal damage and mitoticcell death, Martin EIFLER (Berlin, GERMANY)BET proteins as cofactors for gammaretroviral integration,Saumyashree GUPTA (Hannover, GERMANY)Virologie, Vol. 17, supplément 2, septembre 2013S29

5 th European Congress of VirologyWORKSHOP 16: “ZOONOTIC VIRUSES (PARTNERSHIP WITH EU PROGRAMMES ANTIGONE, PREDEMICS)”Room Tête d’Or, Level 1 (p. S92)Chairpersons: Thijs KUIKEN (Rotterdam, THE NETHERLANDS) & Sylvie VAN DER WERF (Paris, FRANCE)15h00 – 15h3015h30 – 15h4515h45 – 16h0016h00 – 16h1516h15 – 16h3016h30 – 16h4516h45 – 17h0017h00 – 17h15Keynote: Coronaviruses: designed to emerge?, Eric J. SNIJDER (Leiden University Medical Center,THE NETHERLANDS)Phylodynamics and dispersal of domestic dog rabies in an African city,Herve BOURHY (Paris, FRANCE)Arboviruses in Europe, an increasing threat, Natalie CLETON (Bilthoven, THE NETHERLANDS)A charge dependent interaction is necessary for Rift Valley fever virus like particle entryinto its host cell Maria BAUDIN (Umeå, SWEDEN)Functional comparison of the SARS CoV and bat borne SARS like Coronavirusspike glycoproteins: entry process and interplay with chiropteran cells,Markus HOFFMANN (Hannover, GERMANY)Efficient replication of the novel human betacoronavirus EMC on primary human epitheliumhighlights its zoonotic potential, Ronald DIJKMAN (St. Gallen, SWITZERLAND)Identification of cellular factors involved in HEV replication using an in vivo model of HEVinfection in swine and a proteomic approach, Sophie ROGEE (Maisons Alfort, France)Binary virus host interactions using an infectious protein complementation assaySandie MUNIER (Paris, FRANCE)17h30 - 18h00Essential Role of the Phosphatase PP1 in RIG-I and MDA5 Mediated Antiviral Innate ImmunityMichaela GACK, Boston, USA, ESV Junior Award Amphitheater, Level 0 (p. S94)18h00 – 20h00 GENERAL ASSEMBLY – ESV Amphitheater, Level 017h15 – 20h00 POSTER – CHEESE AND WINE PARTY Forum 5 & 6, Level -219h30 – 00h00 GALA DINNER Forum 4, Level -2S30Virologie, Vol. 17, supplément 2, septembre 2013

Scientific Programme -Saturday 14 th September 201308h30 – 10h30WORKSHOP SESSIONSWORKSHOP 2: “ONCOLYTIC VIRUSES AND GENE THERAPY” Room Tête d’Or, Level 1 (p. S95)Chairpersons: Penelope MAVROMARA (Athens, GREECE) & Dirk NETTEBECK (Heidelberg, GERMANY)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: New lentiviral vector pseudotypes offer exciting perspectives for T, B, DC and HSCmediated immuno and gene therapy, Els VERHOEYEN (Lyon, FRANCE)Mantle cell lymphoma radiovirotherapy: high resolution in vivo imaging documentscritical role of measles virus entry through the signaling lymphocyte activation moleculeRoberto CATTANEO (Rochester MN, USA)Cell surface receptor targeting expanded: from oncolytic measles viruses to lentiviral and AAVvectors Anke RASBACH (Langen, GERMANY)Preclinical therapy of disseminated carcinomas and of Gliomas with retargeted oncolyticherpesviruses. Gabriella CAMPADELLI FIUME (Bologna, ITALY)Gene therapy with a replicative HSV vector expressing LIF limits loss of oligodendrocytesand modulates autoimmunity during experimental demyelinating diseaseMichaela NYGÅRDAS (Turku, FINLAND)Efficient generation gf Human Natural Killer Cells by viral transformationBernhard FLECKENSTEIN (Erlangen, Germany)Lentiviral delivery of multiple short hairpin RNAS: A gene therapy approach for HIV 1 infectionFrancesca SPANEVELLO (Padova, ITALY)WORKSHOP 11: “VIRUS EXIT: ASSEMBLY, MATURATION AND RELEASE”Room Prestige Gratte-Ciel, Level 2 (p. S97)Chairpersons: Ari HELENIUS (Zurich, Switzerland), Delphine MURIAUX (Montpellier, FRANCE)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: ESCRTing enveloped viruses out of cells, W. WEISSENHORN (Grenoble, FRANCE)Intermolecular RNA interactions and the selective packaging of influenza A genomic segmentsRoland ROLAND MARQUET (Strasbourg, FRANCE)Characterization of a 3D in vitro model for assembly of very low density HCVUrsula ANDREO (NEW YORK, USA)Infectivity of an alphacoronavirus depends on the tyrosine based sorting motif in its S proteincytoplasmic Domain, Christel SCHWEGMANN WESSELS (Hannover, GERMANY)Association of apolipoprotein B with Hepatitis C virus is involved in viral infectivityand is dependent on the liver enriched protein Cideb, Christophe RAMIÈRE (Lyon, FRANCE)Essential role of the Fusion protein during Sendai virus particle formationManel ESSAIDI LAZIOSI (Geneva, SWITZERLAND)A comparative functional RNA interference screen identifies factors differentially required forlipid droplet homeostasis and life cycle of Flaviviridae members, Gualtiero ALVISI (Padua, ITALY)Virologie, Vol. 17, supplément 2, septembre 2013S31

5 th European Congress of VirologyWORKSHOP 19: “VIRAL EVOLUTION AND QUASISPECIES” Room Gratte-Ciel 1, 2, 3 - level 2 (p. S99)Chairpersons: Esteban DOMINGO (Madrid, SPAIN) & Alexander PLYUSNIN (Helsinki, FINLAND)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: Monitoring short-term virus evolution in vivo by deep sequencing transmitted viralsubpopulations Marco VIGNUZZI (Paris, FRANCE)Low fidelity strains of alphaviruses with fitness defects in vitro and in vivoKathryn ROZEN GAGNON (Institut Pasteur, Paris, FRANCE)Factors Influencing the Fixation of Beneficial Mutations in Bacteriophage Qbeta Evolvedat Increased Error Rate, Laura CABANILLAS (Torrejón de Ardoz, SPAIN)Response of hepatitis C virus to long term passage in the presence of interferon alpha.Multiple mutations and a common phenotype, Celia PERALES (Madrid, SPAIN)Codon specific positive selection and atypical nucleotide substitution patterns in prolongedpoliovirus infection, Hovi TAPANI (Helsinki, FINLAND)Evolutionary implications of distinct recombination patterns observed within Human enterovirusA species, Alexander LUKASHEV (Moscow, RUSSIA)Evolution of the hemagglutinin of pandemic H1N1 (2009): maintaining optimal receptor bindingby compensatory substitutions, Erik DE VRIES (Utrecht, THE NETHERLANDS)WORKSHOP 26: “VIRAL INFECTIONS IN TRANSPLANT AND IMMUNOCOMPROMISED PATIENTS”Amphitheater, Level 0 (p. S101)Chairpersons: Thomas SCHULZ (Hannover, GERMANY) & Paulo PAIXAO (Lisbon, PORTUGAL)08h30 – 09h0009h00 – 09h1509h15 – 09h3009h30 – 09h4509h45 – 10h0010h00 – 10h1510h15 – 10h30Keynote: Virologic and immunologic monitoring of human cytomegalovirus infections in theimmunocompetent and immunocompromised host: perspectives for the development of asubunit glycoprotein pentameric vaccine Giuseppe GERNA (Pavia, Italy) - Excellence in Virology,SIV AWARDEEHepatitis E virus: an underestimated opportunistic pathogen in recipients of allogeneichematopoietic stem cell transplantation, Suzan PAS (Rotterdam, THE NETHERLANDS)Prolonged rhinovirus infection in hematological immunocompromised patients with impaired Tcell Immunity, Antonio PIRALLA (Pavia, ITALY)Factors for Adenovirus Infection and Disease in Pediatric Hematopoietic Stem Cell Transplantpatients Linda FEGHOUL (Paris, FRANCE)Level and kinetics of Plasma Torque Teno Virus DNA after Lung Transplantation as a Markerto Guide Immunosuppressive Therapy, Irene GOERZER (Vienna, AUSTRIA)The A5.1 mutation in the MICA gene of the kidney donor is associated to aprotective effect towards BK polyomavirus infections after renal transplantationCeline BRESSOLLETTE BODIN (Nantes, FRANCE)Improved detection Reveals Active Beta Papillomavirus Infection in Skin Cancer from OrganTransplant Recipients, Cinzia BORGOGNA (Novara, ITALY)10h30 – 11h00 COFFEE BREAK IN THE EXHIBITION AREA Forum 5 & 6, Level -211h00- 13h00 PLENARY SESSION 4: “THE UNIVERSE OF VIRUSES” Amphitheater, Level 0 (p. S104)Chairpersons: Marion KOOPMANS (Bilthoven, THE NETHERLANDS) & Geoffrey SMITH (London, UNITED KINGDOM)11h00 – 11h30 Order to the Viral Universe, Dennis BAMFORD (Helsinki, FINLAND)11h30 – 12h00 Viral reservoirs and the promise of pandemic preparedness, Christian Drosten (Bonn, GERMANY)12h00 – 12h30 Emerging bunyaviruses, Richard ELLIOTT (Saint Andrews, UNITED KINGDOM)12h30 – 13h00 Emerging respiratory viruses, Ron FOUCHIER (Rotterdam, THE NETHERLANDS)13h00 – 15h00 LUNCH BREAK (BUFFET) IN THE EXHIBITION AREA Forum 5 & 6 - Level -213h15 – 14h30 SYMPOSIUM FINOVI - VIRAL HEPATITIS Room Prestige Gratte-Ciel, Level 2 (p. S105)Chairpersons: Birke BARTOSCH (Lyon, France) & Ralf BARTENSCHLAGER (Heidelberg, GERMANY)S32Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of Virology13h15 – 13h3013h30 – 13h4513h45 – 14h0014h00 – 14h15Entry and entry inhibition of Hepatitis B Virus, Stephan URBAN (Heidelberg, GERMANY)A role for SIGMA-1 receptor in early steps of viral RNA replication at the onset of Hepatitis Cvirus infection Pablo GASTAMINZA (Madrid, SPAIN)Cell entry process of in vitro and in vivo produced hepatitis C virus (HCV)François-Loïc COSSET (Lyon, FRANCE)Beyond the “stealthy” character: HBV is a weak inducer of innate immunity, but also a potentinhibitor David DURANTEL (Lyon, FRANCE)13h15 – 14h30 SYMPOSIUM INSTITUT PASTEUR – FONDATION MERIEUXRoom Gratte-Ciel 1, 2, 3 – Level 2 (p. S107)“FROM PUBLIC HEALTH AND SURVEILLANCE TO DRUG DISCOVERY”Chairpersons: Florence PRADEL (Lyon, FRANCE), Kathleen VICTOIR (Paris, FRANCE)13h15 – 13h30 Pilot pneumonia multicentric study in the GABRIEL Network of Fondation MÉRIEUX:The relevance of the respiratory viruses, Gláucia PARANHOS-BACCALÀ (Lyon, FRANCE)13h30 – 13h45 Factors associated with severity of influenza virus infections in Africa and Asia: the InstitutPasteur International Network Research Project, Richard NJOUOM (Yaounde, CAMEROON)13h45 – 14h00 Identification of viral pathogens and host-biomarkers in overlapping febrile childhoodillnesses in a malaria-endemic region of Madagascar, Jonathan HOFFMANN (Antananarivo,MADAGASCAR)14h00 – 14h15 Enterovirus 71 Epidemic in Cambodia, Philippe BUCHY Phnom Penh, CAMBODIA)14h15 – 14h30 Suramin inhibits Enterovirus 71 replication in vitro and in vivo by blocking virus entry intotarget cells through binding of the naphthalene trisulfonic acid group to the viral capsid,Ralf ALTMEYER (Shanghai, CHINA)14h45 – 16h45WORKSHOP SESSIONSWORKSHOP 1: “ONCOGENIC MECHANISMS OF VIRUSES” Room Tête d’Or, Level 1 (p. S109)Chairpersons: Massimo TOMMASINO (Lyon, FRANCE) & Fabien ZOULIM (Lyon, FRANCE)14h45 - 15h1515h15 – 15h3015h30 – 15h4515h45 – 16h0016h00 – 16h1516h15 – 16h3016h30 – 16h45Keynote: How does Kaposi Sarcoma-associated Herpes virus cause a vascular tumour?Thomas F. SCHULZ (Hannover, Germany)Identification of novel target genes of Epstein Barr Virus miRNAsMarjolein HOOYKAAS (Utrecht, THE NETHERLANDS)Alternative splicing signatures discriminate ATL cells from untransformed CD4+ counterpartsderiving from HTLV 1 infected individuals, Morgan THENOZ (Lyon, FRANCE)High risk human papillomavirus E6 induces expression of the histone demethylase KDM2Bby repressing miR 146a in human keratinocytes, Elektra PETA (Padova, ITALY)Induction of the tumor marker Fascin by latent membrane protein 1 (LMP1) dependson NF-kappaB and could contribute to invasion, Andrea K. KRESS (Erlangen, GERMANY)Cell cycle modulation by Marek’s Disease Virus: the tegument protein VP22 triggers S Phasearrest and DNA damage in proliferating cells, Laëtitia TRAPP FRAGNET (Nouzilly, FRANCE)The human bocavirus is associated with lung and colorectal cancers and persists in solidVirologie, Vol. 17, supplément 2, septembre 2013S33

5 th European Congress of Virologytumours, Oliver SCHILDGEN (Cologne, GERMANY)WORKSHOP 20: “VIRUS EPIDEMIOLOGY” Room Gratte-Ciel 1, 2, 3 - Level 2 (p. S111)Chairpersons: Tatjana AVSIC-ZUPANC (Ljubljana, SLOVENIA) & Bruno POZZETTO (Saint Etienne, FRANCE)14h45 – 15h15 Keynote: HCMV maternal-fetal transmission: epidemiology and experimental modelingDana WOLF (Jerusalem, ISRAEL)15h15 – 15h30 HCMV reactivation of breastfeeding mothers of preterm infants: from transmissionto prevention, Klaus HAMPRECHT (Tuebingen, GERMANY)15h30 – 15h45 Prevalence and clinical association of Human Rhinovirus in the first years of life in anunselected birth cohort, Katja WOLTHERS (Amsterdam, THE NETHERLANDS)15h45 – 16h00 Molecular Characterization of HCV Genotype 4d Infections in Kayseri RegionCeylan POLAT (Izmir, TURKEY)16h00 – 16h15 Factors Challenging Epidemiology of Poliomyelitis in the Polio Free CountriesNatalya ROMANENKOVA (Saint Petersburg, RUSSIA)16h15 – 16h30 Seroepidemiological and phylogenetic characterization of measles outbreaks in Ireland, 2004to 2012, Bernadette O RIORDAN (Dublin, IRELAND)16h30 – 16h45 30 years of MMR vaccinations in Finland: Successful elimination but what about the future?Mia KONTIO (Helsinki, FINLAND)WORKSHOP 21: “VIRUS DISCOVERY AND METAGENOMICS (PARTNERSHIP WITH EU PROGRAMME EMPERIE)”Room Prestige Gratte-Ciel, Level 2 (p. S113)Chairpersons: Albert OSTERHAUS (Rotterdam, THE NETHERLANDS) & Veronika VON MESSLING (Langen, GERMANY)14h45 – 15h1515h15 – 15h4515h45 – 16h0016h00 – 16h1516h15 – 16h3016h30 – 16h45Keynote 1: EMPERIE: where do we go? Albert D.M.E. OSTERHAUS(Rotterdam, THE NETHERLANDS)Keynote 2: Identification and characterization of MERS-CoVBart L. HAAGMANS (Rotterdam, THE NETHERLANDS)Novel pathogenic viruses and their zoonotic potential, Marta CANUTI(Amsterdam, THE NETHERLANDS)Discovery of rodent coronaviruses related to major human and animal pathogensJan Felix DREXLER (Bonn, GERMANY)Virome analysis of French bat species in contact with humans: identification of a large majorityof novel mammalian related viruses, Laurent DACHEUX (Paris, FRANCE)Identification of novel cetacean poxviruses in animals stranded in Southern EnglandFalko STEINBACH (Addlestone, UNITED KINGDOM)S34Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyWORKSHOP 22: INNOVATIVE METHODS IN VIRAL DIAGNOSISAmphitheater, Level 0 (p. S115)Chairpersons: Xavier de LAMBALLERIE (Marseille, FRANCE) & Elisabeth PUCHHAMMER (Vienna, AUSTRIA)14h45 – 15h1515h15 – 15h3015h30 – 15h4515h45 – 16h0016h00 – 16h1516h15 – 16h30Keynote: Molecular diagnostics in the future: how will it look like and what are the challenges?Hubert G.M. NIESTERS (Groningen, THE NETHERLANDS)Validation of a flavivirus protein microarray for simultaneous detection of and differentiationbetween west Nile, Japanese encephalitis, Usutu and dengue virus immunoglobin G and Mantibodies, Natalie CLETON (Bilthoven, THE NETHERLANDS)Current mumps outbreak in Belgium: is laboratory confirmation by IgM useful?Liesbeth SEAUX (Ghent, BELGIUM)Human Papillomavirus Genotyping: Comparison of the seegene Anyplex II HPV28 with thePGMY CHUV Assay, Roland SAHLI (Lausanne, SWITZERLAND)MxA: a diagnostic marker of viral infection in children, Ilka ENGELMANN (Lille, FRANCE)Application of surface plasmon resonance imaging (SPRi) for the detection of single virusesand virus like particles (VLPs), Victoria SHPACOVITCH (Dortmund, GERMANY)16h45 – 17h00 FINAL WORDS – NEXT MEETING PRESENTATION Amphitheater, Level 0Virologie, Vol. 17, supplément 2, septembre 2013S35

Virologie 2013, 17 (supplément 2) : 36-116Oral Presentations SummaryWednesday 11 th September 2013p. S37 PLENARY SESSION 1: “FROM BASIC VIROLOGY TOTHERAPY”p. S39 WORKSHOP 3: “INNATE IMMUNITY AGAINST VIRUSES”p. S41 WORKSHOP 8: “ANTIVIRAL THERAPY AND RESISTANCETO ACUTE VIRAL INFECTION”p. S43 WORKSHOP 24: “TYPING OF VIRUSES (MOLECULAREPIDEMIOLOGY, TAXONOMY)”p. S45 WORKSHOP 27: “NEUROTROPIC VIRUSES”Thursday 12 th September 2013p. S47 WORKSHOP 5: “VIRAL (IMMUNO)PATHOGENESIS”p. S49 WORKSHOP 12: “VIRAL GENE EXPRESSION – TRANSCRIP-TION, TRANSLATION”p. S51 WORKSHOP 17: “VECTOR BORNE VIRAL INFECTIONS(EUROWN, VECTORIE & WINGS)”p. S54 WORKSHOP 25: “RESPIRATORY VIRUS INFECTIONS”p. S56 PLENARY SESSION 2: “INTERACTION OF VIRUSES WITHPATHOGENS OR ENDOSYMBRIONTS“p. S58 SYMPOSIUM BIOMERIEUX MOLECULAR SCIENTIFICp. S59 WORKSHOP 4: “ADAPTIVE IMMUNITY AND VACCINES”p. S62 WORKSHOP 14: “VIRUS STRUCTURE, DYNAMIC IMAGINGAND TRAFFICKING”p. S64 WORKSHOP 18: “EMERGING DISEASES IN VETERINARYVIROLOGY (ESVV)”p. S67 WORKSHOP 23: “VIRAL DIAGNOSIS IN CHRONIC INFEC-TION AND DURING PREGNANCY”Friday 13 th September 2013p. S70 WORKSHOP 6: “RESTRICTION FACTORS OF VIRAL INFEC-TION, INTERFERING RNA & IMMUNE RESPONSE”p. S72 WORKSHOP 10: “VIRUS ENTRY: ENVELOPE GLYCOPRO-TEINS, RECEPTORS, ENDOCYTOSIS”p. S75 WORKSHOP 15: “HIGHLY PATHOGENIC VIRUSES”p. S77 WORKSHOP 28: “GASTROINTESTINAL VIRAL INFEC-TIONS”p. S80 PLENARY SESSION 3: “HOST RESTRICTION AND BAR-RIERS TO VIRAL INFECTION”p. S81 SYMPOSIUM QIAGENp. S82 SYMPOSIUM SANOFI PASTEURp. S83 WORKSHOP 7: “CELLULAR FACTORS CONTROLLINGVIRAL INFECTION AND ROLE OF HOST GENETICS”p. S86 WORKSHOP 9: “ANTIVIRAL THERAPY AND RESISTANCETO CHRONIC VIRAL INFECTION”p. S89 WORKSHOP 13: “VIRAL REPLICATION STRATEGIES”p. S92 WORKSHOP 16: “ZOONOTIC VIRUSES (ANTIGONE, PRE-DEMICS)”Saturday 14 th September 2013p. S95 WORKSHOP 2: “ONCOLYTIC VIRUSES AND GENE THE-RAPY”p. S97 WORKSHOP 11: “VIRUS EXIT: ASSEMBLY, MATURATIONAND RELEASE”p. S99 WORKSHOP 19: “VIRAL EVOLUTION AND QUASISPECIES”p. S101 WORKSHOP 26: “VIRAL INFECTIONS IN TRANSPLANTAND IMMUNOCOMPROMISED PATIENTS”p. S104 PLENARY SESSION 4: “THE UNIVERSE OF VIRUSES”p. S105 SYMPOSIUM FINOVIp. S107 SYMPOSIUM INSTITUT PASTEUR – FONDATIONMERIEUXp. S109 WORKSHOP 1: “ONCOGENIC MECHANISMS OF VIRUSES”p. S111 WORKSHOP 20: “VIRUS EPIDEMIOLOGY”p. S113 WORKSHOP 21: “VIRUS DISCOVERY AND METAGENO-MICS (EMPIRIE)”p. S115 WORKSHOP 22: “INNOVATIVE METOHDS IN VIRAL DAI-GNOSIS”doi:10.1684/vir.2013.0526S36 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyWednesday 11 th September 2013,13h30 – 15h45PLENARY SESSION 1: “FROM BASIC VIROLOGY TOTHERAPY”Chairpersons: Giorgio PALÙ (Padova, ITALY)& Felix REY (Paris, FRANCE)Amphitheaterviruses, (many of which cause life-threatening infections) there are nodrugs at hand. [including viruses such as the dengue fever virus (and otherflaviviruses), Chikungunya virus, enterovirus 71 and other enteroviruses(including rhinoviruses that cause exacerbations of asthma and COPD)rabies virus, HEV, coronaviruses and arenaviruses, bunyaviruses and filoviruses].Ideally, potent and broad-spectrum (i.e., pan-genus or pan-familyvirus activity) antiviral drugs should be developed whereby one drug couldbe used for the treatment of a number of such viral infections. We willreview recent evolutions in the search for much needed inhibitors of viralinfections for which today no treatment or prophylaxis is available.13h30 – 14h15GARDNER AWARD of the European Society of Clinical VirologyIlluminating the “isolate, attenuate and vaccinate” paradigmPaul DUPREXDepartment of Microbiology, Boston University School of Medicine, BostonUniversity, 72 East Concord Street, Boston, Massachusetts, 02118,USAUnderstanding the myriad of interactions which occur when a pathogeninfects a primary target cell, spreads within an organism, establishes a systemicinfection and positions itself for transmission to the next susceptiblehost is critical for the development of novel therapeutics. Such studiesdemand access to unpassaged “real world” clinical isolates which aregrown in disease-relevant cells. This ensures that no untoward laboratoryadaptedmutations find their way into the genome as such changes can leadto erroneous conclusions when in vivo pathogenesis experiments are performed,particularly if the model of disease uses an outbred species whereanimal-to-animal variations are greater. Recognizing the importance ofthese issues we have developed a range of reverse genetics systems basedon unpassaged clinical isolates which can be used to produce recombinantviruses that illuminate pathogenesis both macroscopically and microscopically.These viruses express fluorescent proteins from an additionaltranscription unit which allows infected cells to be detected with unprecedentedlevels of sensitivity. Such studies can fundamentally change thetextbook views on how viruses initiate an infection and spread from cellto-cellin the host. This in turn allows vaccines to be developed rationallyand the mechanism of action of antiviral agents to be studied.14h15 – 14h45Treatment of viral infections, still many viruses to goJohan NEYTSRega Institute for Medical Research, KU Leuven, B-3000 Leuven, BEL-GIUMToday, small molecule antiviral drugs are available for the treatment ofinfections with herpesviruses, HIV (25 compounds approved most usedin fixed-dose drug combinations). For HCV two protease inhibitors wererecently approved, that are now being used in combination with pegylatedinterferon + ribavirin. A number of other highly potent HCV inhibitorsare in development. It is expected that within the next 4 to 5 years alloral, pan-genotype and safe combination HCV therapies will be availablethat may results in a >90 cure rate. Ribavirin, has besides the use inHCV management, been approved for the treatment of infections with therespiratory syncytial virus (with questionable efficacy) and Lassa virus.The large number of drugs that are available against HIV (and the manydrugs that are in clinical development for the treatment of chronic HCVinfections) demonstrates that even for viruses with a short genome, manyexcellent molecular targets exist for inhibition of viral replication. Antiviraldrugs would urgently be needed for the treatment of infections withadeno- and polyomaviruses in immunodeficient patients. For most RNA14h45 – 15h15Theoretical and experimental antiviral approach by combining mutagenicagents and inhibitorsEsteban DOMINGOCentro de Biología Molecular “Severo Ochoa” (CSIC-UAM) UniversidadAutónoma de Madrid, SPAINLethal mutagenesis is an antiviral strategy that aims at extinguishingviruses by increasing the mutation rate during viral replication. Excessmutagenesis is achieved with base or nucleoside analogues that are convertedinto nucleoside-triphosphates and incorporated specifically by the viralpolymerases. Despite an established advantage of combination therapiesto control variable RNA viruses, model studies with several viruses indicatethat when a mutagenic agent and an inhibitor participate in therapy asequential inhibitor-mutagen administration can be more effective than thecorresponding combination. The advantage is manifested in a more pronounceddecrease of viral load and a higher probability of virus extinction.In contrast, with either two mutagens or two inhibitors, a combined administrationis more effective than sequential designs (Iranzo et al. PNAS108: 16008, 2011; Perales et al. PLoS Pathogens 5: e1000658, 2009; TIM20: 595, 2012). The availability of a full length, infectious hepatitis Cvirus (HCV) that can replicate extensively in cell culture has allowed theisolation and characterization of several interferon alpha-resistant HCVmutants, and to study their behaviour regarding response to the currentstandard-of-care therapy (Perales et al. J. Virol. 87: 7593, 2013). This cellculture system is currently being used to explore antiviral designs for HCVbased on lethal mutagenesis, using ribavirin as mutagenic agent (Ortega-Prieto et al. PLoS One, in press), and a number of inhibitors, includingdirectly-acting antiviral agents. Recent experimental results and explorationof parameters (derived from a theoretical model), that may affect theefficacy of sequential versus combination treatments for HCV will be presented.Prospects for a clinical application of lethal mutagenesis will beevaluated. In particular, emphasis will be made on the adaptive capacity ofviral quasispecies (dynamic viral mutant clouds), that often leads to selectionof inhibitor-escape mutants, and how lethal mutagenesis may partlyovercome such adaptive potential.15h15 – 15h45Using retroviruses for human genetic therapy: progresses and difficultiesMarina CAVAZZANA-CALVO 1,2,4,5 , Fabien Touzot 1,2,4,5 , ChantalLagresle 1,2,4,5 , Laure Caccavelli 2,5 , Emmanuelle Six 1,5 , JohannaBlondeau 2,5 , Alain Fischer 1,3,4 , Salima Hacein-Bey-Abina 2,4,51 U768 INSERM, Paris, FRANCE; 2 Département de Biothérapie, AP-HP,Hôpital Universitaire Necker-Enfants Malades, Paris, FRANCE; 3 Unitéd’Immuno-Hématologie Pédiatrique, Hôpital Universitaire Necker-Enfants Malades, Paris, FRANCE; 4 Université Paris Descartes, SorbonneParis Cité, IMAGINE Institue, Paris, FRANCE; 5 CIC Biothérapie GHUOuest, INSERM-APHP, Paris, FRANCEVirologie, Vol 17, supplément 2, septembre 2013S37

5 th European Congress of VirologyGene therapy has become an attractive alternative therapeutic strategy toallogenic hematopoietic stem cell transplantation for primary immunodeficiencies.Clinical trials using gammaretroviral vectors have demonstrated the proofof principle for SCID-X1, Adenosine deaminase, Wiskott-Aldrich syndromeand chronic granulomatous disease, but have also highlightedlimitations of the technology. New strategies based on self-inactivated vectorsthat can achieve more robust corrections with less risk of insertionalmutagenesis are being developed.Methods for integration site recovery that allow for efficient monitoringof integration site abundance while minimizing methodologicalbiases have been set up. Clustering of integration sites was thus comparedbetween these different vectors showing a significant lowerintegration frequency near “dangerous genes” when SIN vectors areused.Despite this improvement hematopoietic, gene therapy approach presentsspecific issues as the use of patients’ bone marrow stem and precursorcells which may have an abnormal composition with unpredictableconsequences. Thus the long-term clinical results of on-going trials usingself-inactivated vectors are eagerly awaited with the expectation that theywill better define the role of gene therapy with respect to conventionalHSCT.S38 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyWednesday 11 th September 2013,16h15 – 18h15WORKSHOP 3: “INNATE IMMUNITY AGAINST VIRUSES”KEYNOTE:Chairpersons: Denis GERLIER (Paris, FRANCE) &Maria-Carla SALEH (Paris, FRANCE)Room Gratte-Ciel 1, 2, 3Manipulation of autophagy by measles virusMathias FAURECentre de Recherche International en Infectiologie (CIRI), INSERMU1111, UCBL-1, CNRS UMR5308, ENS-Lyon, FRANCEMacroautophagy, referred to as autophagy, is a lysosomal catabolicpathway, which allows the sequestration of portions of the cytoplasmwithin vesicles called autophagosomes, leading to their ultimate degradationwithin autolysosomes. In mammalian cells, regulation of autophagyinvolves dozens of proteins including those of the ATG (AuTophaGyrelated)family. Essential for the maintenance of cellular homeostasis,autophagy is also a cell-autonomous defense mechanism that allows thetargeting of pathogens, including viruses, towards lysosomes. In addition,autophagy may potentiate innate and adaptive antiviral immune responses.However, several viruses have evolved strategies in order to escape orhijack autophagy to facilitate their replication.Infection with measles virus leads to a paradox: despite the developmentof an effective immune response, which eliminates the virus and leads tothe development of a lifelong immunity, immunosuppression occurs. Wehave shown that CD46, the receptor of the attenuated strains of measlesvirus, is directly connected to autophagy via a specific molecular pathway.Furthermore, we found that, during infection, two other pathways lead tothe induction of autophagy, which are both independent of CD46 and fromeach other. These pathways contribute to the ability of measles virus toexploit the autophagic process in order to replicate, through the delay ofvirus-induced cell death. Thus, subtle relationships between measles virusand autophagy could contribute to either control the infection or to increaseinfectivity.The antiviral pathogen recognition receptor RIG I is known to bind nakeddsRNA containing a triphosphate (ppp) 5 ′ terminus. It remained unclear,however, whether RIG I can also recognize RNAs packaged into nucleocapsids,the first viral structure to enter the host cell. We show that incomingnucleocapsids of viruses containing a 5 ′ ppp dsRNA “panhandle” structuretrigger an antiviral signaling cascade that commences with RIG I and terminateswith activation of the transcription factor IRF 3. Independent ofmammalian cofactors or of viral polymerase activity, RIG I interacted withthe viral nucleocapsids, underwent a conformational switch, and homooligomerized. Enzymatic probing, as well as super resolution GSDIMmicroscopy (Ground State Depletion Microscopy followed by IndividualMolecule return) suggest that RIG I associates with the 5 ′ ppp panhandlestructure on the viral nucleocapsids.These results define the entry of nucleocapsids into the cytoplasm as thefirst RIG I sensitive step in infection, and establish viral nucleocapsids witha5 ′ ppp dsRNA panhandle as a pathogen associated molecular pattern forRIG I (Weber et al., Cell Host and Microbe, 2013).REF 002Sensing of infected cells in absence of virus particles by plasmacytoiddendritic cells is inhibited by the ribonuclease Erns of classical swinefever virusNicolas RUGGLI, Sylvie PYTHON, Markus GERBER, Rolf SUTER,Artur SUMMERFIELDInstitute of Virology and Immunology, Mittelhäusern, SWITZERLANDPlasmacytoid dendritic cells (pDC) have been shown to sense efficientlyhepatitis C virus infected cells using a virion free pathway. We found thatpDC can also sense infection with classical swine fever virus (CSFV),another member of the Flaviviridae, by direct contact with infected cells,a process that is independent of virus particles. This process is much moreefficient in terms of interferon alpha induction when compared to directstimulation by virus particles. By employment of virus replicon particlesor infectious RNA that can replicate but not form de novo virions, a transferof virus from the donor cell to the pDC was excluded. Activation ofpDC by infected cells is mediated by a contact dependent RNA transfer topDC, which is sensitive to a TLR7 inhibitor. Interestingly, the Erns proteinof CSFV does efficiently prevent this process, which requires intactribonuclease function of Erns in intracellular compartments. The presentstudy underlines the importance of pDC activation by infected cells andidentifies a novel pathway of viral escape from the interferon system.ORAL COMMUNICATIONSREF 001Activation of RIG I by incoming RNA virus nucleocapsids containinga5 ′ triphosphorylated genomeMichaela WEBER 1 , Ali GAWANBACHT 2 , Matthias HABJAN 2 ,Andreas RANG 3 , Christoph BORNER 4,5 , Anna Mareike SCHMIDT 2,5 ,Sophie VEITINGER 6 , Ralf JACOB 6 , Stéphanie DEVIGNOT 1 , GeorgKOCHS 2 , Adolfo GARCÍA SASTRE 7,8,9 , Friedemann WEBER 1,2,51 Philipps University Marburg, Institute for Virology, Marburg,GERMANY; 2 University Freiburg, Department of Virology, Freiburg,GERMANY; 3 University Hospital Charité, Institute of Virology, Berlin,GERMANY; 4 University Freiburg, Institute of Molecular Medicine,Freiburg, GERMANY; 5 Albert Ludwigs University Freiburg, Centre forBiological Signalling Studies (BIOSS), Freiburg, GERMANY; 6 PhilippsUniversity Marburg, Department of Cell Biology and Cell Pathology,Marburg, GERMANY; 7 Mount Sinai School of Medicine, Department ofMicrobiology, New York, USA; 8 Mount Sinai School of Medicine, Departmentof Medicine, Division of Infectious Diseases, New York, USA; 9 MountSinai School of Medicine, Global Health and Emerging Pathogens Institute,New York, USAREF 003Integrins synergize with TLRs to initiate a specific branch of the innateresponse to herpes simplex virusGabriella CAMPADELLI FIUME, Tatiana GIANNIDepartment of Experimental Diagnostic and Specialty Medicine, AlmaMater Studiorum – University of Bologna, Bologna, ITALYIntegrins are exquisite signaling molecules that regulate a number of cellularprocesses. They are also very popular among viruses as cellularreceptors. We have discovered that the av3 integrin serves the additionalfunction of sensor of herpes simplex virus (HSV) and initiator of abranch of the innate response which culminates in the secretion of a specificset of cytokines, including IFNa and . This function is exerted throughinteraction and synergy with TLR2.Here we report that av3 integrin is not the only one integrin that servesthe function of innate response sensor. We found that av6 and av8 canserve as HSV receptors, each independently of the other, via interactionwith gH/gL. av6 but not av8 integrins can activate specific branches ofthe innate response. Further yet, integrins synergize also with TLR4. Thesignalling activity which culminates in the innate response involves theVirologie, Vol 17, supplément 2, septembre 2013S39

5 th European Congress of Virologyphosphorylation of specific thyrosine residues in the cytoplamic tail of integrins. Remarkably, the synergistic integrin TLR innate activity is seenin response not only to HSV, but also bacterial components.Our studies support the following conclusions. (i) Integrins constitute anovel class of pattern recognition receptors. (ii) Inasmuch as integrinsserve as receptors for HSV entry, they couple virus entry with initiation ofa branch of the innate response, and thus ensure activation of host defencemechanisms. (i) The role of integrins as pathogens’ sensors is not restrictedto HSV, but is broader.REF 004Severe acute respiratory syndrome virus with E protein deleted wasattenuated because E protein was responsible for the induction of aninflammatory response mediated by NF KB activationLuis ENJUANES, Jose L. NIETO TORRES, Jose M. JIMÉNEZGUARDEÑO, Jose A. REGLA NAVAS, Carlos CASTAÑO, RaulFERNANDEZ, Marta L. DEDIEGODepartment of Molecular and Cell Biology, National Center of Biotechnology(CNB CSIC), Darwin 3, Madrid, SPAINDeletion of E gene from SARS CoV led to an attenuated virus (SARS CoVE). Stress response and unfolded protein response genes were upregulatedin cells infected by SARS CoV E in relation with the infection bySARS CoV with E protein. The expression of proinflammatory cytokineswas reduced in lungs of mice infected with a mouse adapted SARS CoVMA15 E compared to lungs infected with wt virus. The induction ofvirus induced inflammatory responses may be mediated by the activationof five signaling pathways (IRF3/7, ATF 2/Jun, AP 1, NF KB, and NFAT), but in infections by SARS CoV with and without E protein the onlypathway differentially activated was NF KB. Interestingly, the addition ofan inhibitor of NF KB led to a reduced inflammatory response after SARSCoV infection, significantly inhibiting the proinflammatory cytokine response.A reduction in neutrophil migration to sites of lung inflammationwas observed in mice infected with SARS CoV MA15 E, what probablycontributed to the lower degree of inflammation detected and to SARSCoV E attenuation. The mutant SARS CoV missing E protein providedprotection against challenge with homologous and heterologous pathogenicSARS CoVs in hamsters and transgenic mice. Furthermore, SARSCoV MA15 E provided complete long term protection against a virulentmouse adapted SARS CoV in both young and old Balb/c mice. These dataindicate that SARS CoV E is a very promising vaccine candidate andthat inhibitors of NF KB may protect against SARS CoV infection.REF 005A reverse genetics approach to study the determinants for dsRNAbinding and PKR inhibition in the NS1 protein of influenza A virusKristina L. SCHIERHORN 1 , Katrin HOEGNER 2 , JuliaDZIECIOLOWSKI 3 , Susanne HEROLD 2 , Stephan PLESCHKA 3 ,Thorsten WOLFF 11 Robert Koch Institute, Influenza and other respiratory viruses, Berlin,GERMANY; 2 Justus Liebig University Gieβen, Department for InternalMedicine II, Section of Infectious Diseases, Gieβen, GERMANY; 3 JustusLiebig University, Institute for Medical Virology, Gieβen, GERMANYThe non structural protein 1 (NS1) of influenza viruses functions in inhibitingthe type I IFN mediated antiviral state in infected cells. WhileNS1 proteins of influenza A and B viruses (A/NS1 and B/NS1) haveonly 20% sequence identity, they have a conserved N terminal dsRNAbinding domain (RBD) and share functions as inhibition of the antiviralkinase PKR. We have previously shown that distinct basic amino acidsin the RBD of B/NS1 are required for dsRNA binding and inhibition ofPKR. In contrast, A/NS1 has been suggested to silence PKR by a physicalinteraction involving a region outside the RBD. To evaluate whetherthe two influenza virus types in fact inhibit PKR by different means, weconducted a systematic analysis of the RBD of A/NS1 using reverse genetics.Among a panel of constructed mutants we identified 3 NS1 proteinswith single basic amino acid exchanges eliminating dsRNA binding. Twoof the corresponding mutant viruses showed a 10 fold reduction in viralreplication, possibly due to strong IFN induction. The third mutant viruswas severely attenuated for replication by 4 logs, which was paralleledby strong activation of PKR, but very low IFN secretion. Significantly,replication of this mutant virus was largely rescued on PKR knock downcells illustrating the strong impact of PKR on viral propagation. Thus, thisstudy highlights the crucial role of the RBD of A/NS1 for PKR inhibitionand viral replication. Moreover, we suggest that PKR has a major role inrestricting influenza virus propagation among the more than 400 knowntype I IFN stimulated factors.REF 006The matrix protein of rabies virus binds to RelAp43 to suppress NFKB dependent gene expression related to innate immunityYoucef BEN KHALIFA 1 , Sophie LUCO 1,2 , Mehdi ARCHAMBAUD 2 ,Olivier DELMAS 1 , Jonathan GRIMES 3 , Hervé BOURHY 11 Institut Pasteur, Unité Dynamique des Lyssavirus et Adaptation à l’hôte,Paris, France; 2 Université Paris Diderot, Sorbonne Paris Cité, CellulePasteur, Paris, France; 3 Division of Structural Biology, Welcome TrustCenter for human Genetics, University of Oxford, Oxford, ENGLANDThe activation of expression of genes involved in innate immune responseis controlled by the activity of transcription factors such as the proteins ofthe NF KB family. Recently, we identified a sixth member of this family,RelAp43, which is involved in the activation of INF transcription duringviral infection (Luco et al., PLoS Pathog 2012). Since, we showed that thematrix (M) protein of wild isolates of rabies virus such as Tha (M Tha) isable to interact with RelAp43 and to efficiently suppress NF kB dependentreporter gene expression, in response to activation by tumor necrosis factor(TNF), in contrast with the vaccine strain SAD. Co immunoprecipitationassays revealed that the central part of M Tha is required for this interaction.Comparison of the corresponding amino acid sequence of the M Tha andM SAD, identified 4 differences, two of which induce the loss of bindingof M Tha to RelAp43 but are not sufficient to restore the NF KB dependentreporter gene expression. To fully restore this effect, additional mutationsare required. Moreover, we selected a subset of genes involved in antiviralresponse and we identified four genes as particularly down regulated by MTha compared to M SAD: CYCLD, NFKB1, STAT1 and TRIM25. Finally,we used an approach that relies on small interfering RNA of RelAp43 in thepresence of M Tha or M SAD to study the modulation of genes involved ininnate immunity and in NF KB signaling. Thus, RABV M protein appearsas a potent viral immune modulatory factor that prevents NF KB dependentgene expression by fixation of RelAp43.S40 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyWednesday 11 th September 2013,16h15 – 18h15WORKSHOP 8: “ANTIVIRAL THERAPY ANDRESISTANCE TO ACUTE VIRAL INFECTION”Chairpersons: Johan NEYTS (Leuven, BELGIUM)& Manuel ROSA-CALATRAVA (Lyon, FRANCE)Amphitheatermembrane spanning protein of the viral replication complex corroboratethat K22 interferes with membrane bound viral RNA synthesis. This novelapproach is also applicable to diverse animal and human CoVs, includingthe recently identified HCoV EMC, and efficient inhibition can be achievedat the entry port of human CoV infection, the human airway epithelium.Collectively, we identified the recruitment of cellular membranes for viralreplication as a promising target for antiviral intervention. As replication atcellular membranes is a key step in the life cycle of many positive strandedRNA viruses, this approach may serve as a paradigm for the developmentof antiviral drugs to combat many other important virus infections.KEYNOTE:Antiviral Therapy and Resistance during acute viral infectionCharles BOUCHERDepartment of Virology Erasmus Medical Centre, Erasmus UniversityRotterdam, THE NETHERLANDSThe viral inoculum required for a successful viral infection is generallyquite small. Very early in HIV infection a nearly homogeneous viral wildtype population is found. The absence of viral variation enhances the efficacyof therapy because selection of preexisting resistance viruses is verylow. For example treatment with a low genetic drug (oseltamivir) veryrarely results in selection of single mutant resistant influenza. Under certainconditions selection of drug resistant may occur, for Influenza, HCV orHIV viruses the initial selected resistant viruses have a reduced replicativepotential. Continuous selection can lead to multiple additional compensatorymutations resulting in resistant viruses with a compensated replicationpotential. As a result of large scale spread use of antiviral drugs such compensatedresistant viruses can be transmitted and start sub epidemics (asreported for HIV) or can be the start of a global epidemic of resistance asobserved for Influenza in 2008. We study the rate of transmission of HIVand Influenza and subsequent evolution and its clinical significance.ORAL COMMUNICATIONSREF O07Potent inhibition of diverse Coronaviruses by targeting membranebound viral RNA synthesisEveline KINDLER 1 , A. LUNDIN 2 , R. DIJKMAN 1 , T. BERGSTROM 2 ,N. KANN 3 , B. ADMIAK 2 , C. HANNOUN 1 , H.R. JÓNSDÓTTIR 1 ,D. MUTH 4 , M.A. MÜLLER 4 , C. DROSTEN 4 , V. THIEL 1 ,E. TRYBALA 21 Institute of Immunobiology,Kantonal Hospital, St.Gallen, SWITZER-LAND; 2 Department of Clinical Virology, University of Gothenborg,Gothenborg, SWEDEN; 3 Chalmers University of Technology, Gothenborg,SWEDEN; 4 Institute of Virology, University of Bonn, Bonn,GERMANYThe concept of targeting key functions of the viral replication cycle ledto the development of potent antiviral drugs against HIV, influenza, andrecently HCV. Although targeting of multiple viral functions or enzymaticactivities resulted in highly active antiviral therapies, virus resistanceremains a concern. Therefore, there is an need to identify novel and druggabletargets to improve antiviral therapy. By screening more than 16 ′ 000compounds for anti human coronavirus 229E activity, we identified apotent inhibitor designated K22 that specifically targets membrane boundcoronavirus (CoV) RNA synthesis. Formation of virus induced doublemembrane vesicles, a hallmark of CoV replication, was impaired uponK22 treatment, which manifested a near complete inhibition of viral RNAsynthesis. Resistance mutants containing amino acid substitutions in aREF O08Discovery of low molecular weight compounds inhibiting Chikungunyavirus RNA replicationTero AHOLA 1 , Pasi KAUKINEN 1 , Finny VARGHESE 1 , MaximBESPALOV 2 , Krister WENNERBERG 2 , Andres MERITS 3 , BeateKUMMERER 41 University of Helsinki, Helsinki, FINLAND; 2 Institute for MolecularMedicine Finland, Helsinki, FINLAND; 3 University of Tartu, Tartu, ESTO-NIA; 4 University of Bonn Medical Centre, Bonn, GERMANYChikungunya virus (CHIKV) (genus Alphavirus) causes in humans highfever, body rash, headache and severe joint pain that can persist for monthsor years. The re emergence of CHIKV caused a large epidemic on LaRéunion Island in 2005 2006 and the virus subsequently spread to Indiaand South East Asia causing millions of infections. Endemic CHIKV infectionshave also been reported in Italy and France. CHIKV is transmitted inhumans by the bite of infected yellow fever mosquito (Aedes aegypti) orAsian tiger mosquito (Aedes albopictus). Currently, there is no vaccine orspecific drugs available to treat CHIKV infection. In this study, a CHIKVreplicon cell line expressing the virus replicase proteins and replicatingviral RNA was used in an antiviral screening assay with approximately3000 compounds. We identified several low molecular weight compoundsthat target the intracellular step of CHIKV replication. The most activecompounds were validated in CHIKV infection assay in human Huh7cells in combination with toxicity counter screening. We identified six hitcompounds, which showed dose dependent inhibition of CHIKV and therelated Semliki Forest virus with half maximal inhibitory concentration(IC50) below 3 micro Molar. As some of the compounds strongly modifiedthe localization of viral membrane bound replication complexes inCHIKV infected cells, they may affect general membrane properties. Therefore,some of the inhibitors could have broad spectrum antiviral activity.Isolation and analysis of drug resistant mutant viruses is currently beingpursued.REF O09Humanized antibodies able to protect mice from tick borne encephalitisNina TIKUNOVA 1 , Andrey MATVEEV 1 ,IvanBAIKOV 1 , LeonidMATVEEV 1 , Oleg STRONIN 21 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk,RUSSIA; 2 Federal State Unitary Company Microgen Scientific IndustrialCompany for Immunobiological Medicines, Tomsk, RUSSIAAntibodies are used for therapy for a long time due to their exceptionalproperties – high specificities, availability of effector functions, andinvolvement of natural mechanisms in pharmacokinetics of administratedantibodies. There is currently no specific therapeutic strategy approved foruse in human tick borne encephalitis (TBE) in Europe. Specific immunoglobulinproduced from donor blood is used for TBE treatment in Russiabut this preparation has some disadvantages. Recombinant therapeuticantibodies offer an obvious alternative to donor’s immunoglobulin.Virologie, Vol 17, supplément 2, septembre 2013S41

5 th European Congress of VirologySeveral neutralizing mouse monoclonal antibodies (MAbs) specific to glycoproteinE of TBE virus were tested in the model animal protectionexperiments using lethal doses of this virus. Mouse MAb demonstratedprotective activity in the absence of antibody dependent enhancementwas selected. Variable domains of this mouse Mab and constant domainsof human immunoglobulin were used to construct humanized antibody.This humanized antibody produced by CHO cell line shown nanomolaraffinity and ability to neutralize TBE infectivity in vitro. Moreover, thisantibody demonstrated protective activity when administrated 123daysbefore infection and therapeutic activity when administrated 1 2 days postinfection in TBE virus infected mice, and this activity was higher thantherapeutic activity of commercially available specific immunoglobulinfrom donor blood.REF O10Characterization of human cytomegalovirus microRNA temporalexpression profile and target prediction by dynamic expression analysisMarta TREVISAN 1 , Carlotta ALBONETTI 1 , Enrico LAVEZZO 1 ,Tiziana SANAVIA 2 , Alessandro SINIGAGLIA 1,3 , Barbara DICAMILLO 2 , Giorgio PALÙ 1 , Luisa BARZON 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Department of Information Engineering, University of Padova,Padova, ITALY; 3 IOV Istituto Oncologico Veneto, Padova, ITALYTargets for human cytomegalovirus (HCMV) miRNAs in host transcriptomeare still largely unknown. Aim of this study was to characterize theeffects of HCMV miRNAs on host human miRNA and mRNA temporalexpression profiles after infection, identifying candidate gene targets.To this aim, tandem microarray analysis of miRNA and mRNA temporalexpression was done in time series of MRC 5 cells infected with HCMVTowne strain. The majority of HCMV miRNAs was found to be expressedsince the earliest stages or within 24 hours post infection and continuedto accumulate over time. Differentially expressed mRNAs were selectedaccording to the area of the region bounded by the expression profilesrelated to control and post infection cases. Identification of HCMV miR-NAs targets was done by integrating the prediction of sequence basedalgorithms with Pearson correlation between viral miRNAs and cellularmRNAs temporal profile to detect significant negatively correlated targetgenes. This method, for instance, permitted to identify 55 significantlycorrelated target for HCMV miR US25 2 5p among the 1034 sequencebased selected candidate genes. Experimental validation of some selectedpredicted targets was done by standard lucyferase activity assays and westernblot analysis. In conclusion, an integrated analysis of viral miRNAsand host mRNA using a meta consensus approach based both on sequenceprediction methods and on correlation analysis of dynamic expressiondata allowed the prediction and identification of host targets for HCMVmiRNAs.REF O11Non liposomal delivery of anti rabies virus siRNAs counteracts viralgrowth and spread in vitroTobias HALBACH, Johannes HARDER, Konstantin SPARRER,Thomas CARELL, Karl Klaus CONZELMANNLMU, Munich, GERMANYRabies virus (RABV) infection continues to be a threat throughout theworld, with more than 55,000 human deaths each year. The majority ofrabies cases occur in rural regions of Africa and South Asia, where RABVinfection is endemic and access to rapid medical treatment is hindered.After the first symptoms of rabies emerge, post exposure treatment andvaccinations are not potent anymore and the outcome of the disease isalmost exclusively fatal. Therefore, the need for an efficient antiviral therapyagainst rabies is still a major issue. RNA interference (RNAi), whichsilences expression of specific target genes could represent a promisingtool for treating RABV infections in mammalian hosts. However, difficultiesincluding delivery of siRNA/miRNA, short term efficiency, theemergence of resistant subpopulations and the resistance of viral RNPsagainst RNAi limit the potential of this method. Here, we developed anovel non liposomal siRNA delivery system that can counteract RABVgrowth and spread in vitro. Using siRNAs that were covalently linked toan arachidonoyl ethanol amide (anandamide) ligand via their 3‘end wecould specifically target cells expressing the receptor for the anandamideligand (the cannabinoid receptor), which is predominantly present on neuronaland immune cells. Indeed, RABV infected human immune cells andmouse neuronal cells that were treated with the anandamide modified siR-NAs showed a strong inhibitory effect against RABV infection. Adaptingthis system to an in vivo model, it might represent a promising tool to limitRABV infections in vivo.REF O12Inhibition of Pyridimine Biosynthesis Pathway Suppresses ViralGrowth Through Innate ImmunityPierre Olivier VIDALAIN 1,2 , Marianne LUCAS HOURANI 1,2 , DanielDAUZONNE 3,4 , Pierre JORDA 3,4 , Gaëlle COUSIN 3,4 , AlexandruLUPAN 5,6 , Olivier HELYNCK 5,6 , Grégory CAIGNARD 1,2 , GenevièveJANVIER 1,2 , Gwénaëlle ANDRÉ 7,8 , Nicolas ESCRIOU 1,2 , PhilippeDESPRÈS 9 ,YvesJACOB 10,11 , Hélène MUNIER LEHMANN 5,6 ,Frédéric TANGY 1,21 Unité Génomique Virale et Vaccination, Institut Pasteur, Paris, FRANCE;2 CNRS, UMR 3569, Paris, FRANCE; 3 Institut Curie, Paris, FRANCE;4 CNRS, UMR 176, Paris, FRANCE; 5 Unité de Chimie et Biocatalyse,Institut Pasteur, Paris, FRANCE; 6 CNRS, UMR 3523, Paris, FRANCE;7 Unité de Biochimie Structurale, Institut Pasteur, Paris, FRANCE; 8 CNRS,URA 2185, Paris, FRANCE; 9 Unité Interactions moléculaires FlavivirusHôtes, Institut Pasteur, Paris, FRANCE; 10 Unité de Génétique Moléculairedes Virus à ARN, Institut Pasteur, Paris, FRANCE; 11 Dana Farber CancerInstitute, Center for Cancer Systems Biology (CCSB) and Department ofCancer Biology, Boston, USASearching for stimulators of the innate antiviral response is an appealingapproach to develop novel therapeutics against viral infections. Here weestablished a cell based reporter assay to identify compounds stimulatingexpression of interferon inducible antiviral genes. We screened a total of41,353 small molecules and selected DD264 for its immuno stimulatoryand antiviral properties. While searching for its mode of action, we identifiedDD264 as an inhibitor of pyrimidine biosynthesis, establishing ayet unsuspected link between this pathway and the expression of antiviralgenes. Furthermore, we found that antiviral activity of DD264 orbrequinar, a well known inhibitor of pyrimidine biosynthesis pathway, isstrictly dependent on cellular gene transcription and required InterferonRegulatory Factor 1 (IRF1). Altogether, our results better explain the antiviralproperty of pyrimidine biosynthesis inhibitors and unravel a novelpathway that induces cell resistance to RNA virus infections.S42 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyWednesday 11 th September 2013,16h15 – 18h15WORKSHOP 24: “TYPING OF VIRUSES (MOLECULAREPIDEMIOLOGY, TAXONOMY)”KEYNOTE:Chairpersons: Anna PAPA (Thessaloniti, GREECE)& Marie ROQUE-AFONSO (Paris, FRANCE)Room Prestige Gratte-CielGenome diversity and evolution of vector-borne virusesLuisa BARZON, Giorgio PALÙDepartment of Molecular Medicine, University of Padova, Padova, ITALYVector-borne viruses, e.g., WNV, TBEV, DENV, CHIKV, and CCHFV,are emerging pathogens in Europe and worldwide. Although genomesequences of vector-borne viruses are highly conserved and typically showslow rate of evolutionary changes over space and time because of the adaptiveconstrains resulting from alternate virus cycling between invertebratevectors and vertebrate hosts, the emergence of these viruses in new ecologicalniches is characterized by the occurrence of mutations to adapt tonew vectors and hosts that may also affect fitness, transmissibility, andvirulence. Like other RNA viruses, vector-borne viruses exist in nature asquasispecies and this is an important factor that contributes to the abilityof these viruses to adapt to new environments.An overview on genome diversity and evolution of some relevant vectorborneviruses causing disease in Europe will be presented by reportingdata from epidemiological surveys and experimental in vitro and in vivostudies. Genotypic and phenotypic correlates will be also discussed.ORAL COMMUNICATIONSREF O13Mumps outbreak in the Netherlands, 2010 2012Sigrid GOUMA 1,2 , Daphne GIJSELAAR 1 , Jeroen CREMER 1 , HeinBOOT 1 , Jussi SANE 1,3 , Susan HAHNÉ 1 , Marion KOOPMANS 1,2 , RobVAN BINNENDIJK 11 National Institute for Public Health and the Environment (RIVM), Bilthoven,THE NETHERLANDS; 2 Erasmus Medical Center, Rotterdam, THENETHERLANDS; 3 European Center for Disease Prevention and Control(ECDC), Stockholm, SWEDENIntroduction: During the three mumps outbreak seasons in the Netherlands(September 2009 – August 2012), 1557 mumps cases have beenreported. Most patients were vaccinated young adults. Here we describe thegenetic diversity among circulating strains during the mumps outbreak andlook for possible correlations between sequence types and epidemiologicaland virological data.Methods: 808 mumps PCR positive specimens were used for phylogeneticanalyses of the SH gene (316 bp) and the HN gene (1749 bp). Salivaryviral load was determined by real time PCR (n=289). ANOVA was usedfor statistical analysis (preliminary).Results: The first G5 mumps strain detected was G5 NLD1, with an SHsequence identical to outbreak strains from New York. In March 2010,a first co circulating variant of this G5 strain was detected (G5 NLD2),that differs two nucleotides in the SH gene from G5 NLD1. Throughoutthe mumps outbreak, G5 NLD2 became the most dominant strain (n=520),compared to G5 NLD1 (n=176). G5 NLD2 is shed much longer after onsetof clinical symptoms as compared to G5 NLD1 (p0.2). Phylogenetic clusters formed by HNsequences differ from SH gene clustering.Conclusion: Viral shedding is higher in patients infected with G5 NLD2as compared to G5 NLD1, which suggests a virological advantage of G5NLD2 in viral transmission. Whether these differences are caused by particularmutations in the viral genome is currently investigated, but analysisof the HN gene indicates that G5 NLD1 and G5 NLD2 might be twoseparate lineages.REF O14Molecular epidemiology of Hepatitis A virus in France, 2004 2012Muriel MACÉ 1 , Anne Marie ROQUE AFONSO 1,2,31 AP HP, Hôpital Paul Brousse, Virologie, Villejuif, FRANCE; 2 Univ ParisSud, UMR S 785, Villejuif, FRANCE; 3 INSERM U785, Villejuif, FRANCEFrom 2002, the French Reference Centre for HAV receives samples fromthe whole country on a voluntary basis for strains surveillance and tospecifically investigate outbreaks.Methods: HAV genotype was determined by the phylogenetic analysis ofa 400 bp nucleotide segment of the VP1/2A junction amplified from serumor stool samples of case patients. Epidemiological data (age, sex, geographicarea of living) and HAV risks factors were collected (i.e internationaltravel, contact with another hepatitis A case, at risk communities). Epidemiologicaland sequence data were analyzed using Bionumerics software,version 6.6 (Applied Maths, Sint Martens Latem, Belgium).Results: From 2004 to 2012, 1944 HAV RNA positive samples were includedfor analysis. Sub genotype IA was evidenced in 59.9% of circulatingisolates, IB in 23.6%, IIIA in 15.6% and IIA in 0.9%. The highest diversitywas observed within IB sequences, mainly associated with internationaltravels.Strains belonging to 3 clusters persisted several years due to transmissionwithin specific communities. These “autochthonous” strains representedup to 70% of the strains identified each year: two were mainly transmittedwithin the travelling community and one strain was mainly associated withthe homosexual community.Conclusion: Surveillance of circulating HAV strains in France through thelast decade shows a great diversity linked to international travels. However,some of these strains tend to become autochthonous by spreading withinspecific communities.REF O15Prevalence of human enterovirus and parechovirus genotypes inAmsterdam between 2007 2011 and comparison to surveillance dataKatja WOLTHERS 1 , Victoria JANES 2 , Rene MINNAAR 1 , GerritKOEN 1 , Hetty VAN EIJK 1 , Karen DE HAAN 1 , KimberleyBENSCHOP 31 Laboratory of Clinical Virology, Department of Medical Microbiology,Academic Medical Center, Amsterdam, THE NETHERLANDS; 2 EmmaChildren’s Hospital, Amsterdam, THE NETHERLANDS; 3 Laboratory forInfectious Diseases and Screening, Center for Infectious Diseases, NationalInstitute of Public Health and the E, Bilthoven, THE NETHERLANDSInfections with enteroviruses (EVs) and human parechovirusus (HPeVs)are endemic worldwide. However, comprehension of the worldwide circulationof the different genotypes is unclear. EV/HPeV epidemiologywas monitored in the Amsterdam region between 2007 and 2011 by PCRand direct genotyping. A literature study of existing surveillance data wasconducted for comparison.In our region, 241 EVs and 157 HPeVs could be typed from stool orCSF samples of 398 patients. EV B strains (74% of the EV strains) werefrequently found, followed by EV A (24%), and EV D strains (2%). AsVirologie, Vol 17, supplément 2, septembre 2013S43

5 th European Congress of Virologya single genotype, HPeV1 accounted for 22% of all combined EV/HPeVstrains. The distribution of EV types detected in CSF reflected the distributionin stool with E30 as the dominant strain. However, HPeV distributionin CSF did not reflect the distribution in stool. While HPeV1 dominatedin stool, HPeV3 was the only type found in CSF.The literature study showed that the 10 most frequently isolated viruses,based on virus culture, were E30, CBV5, E6, E9, E4, E11, CAV9, CBV1,E18 en CBV3, six of which were frequently detected in our region usingPCR over the 5 years studied. With our methods, more HPeV and EV Astrains were found.Comparison of regional epidemiological data with worldwide EV/HPeVdistribution is difficult, since extensive surveillance studies are sparse anddetection methods vary. Therefore, uniform data collection is essential toaid prediction of outbreaks and disease management.REF O16Characterisation of complete rotavirus genomes using a single reversetranscription polymerase chain reaction (RT PCR)Christian KOEHLER, Antje RUECKNER, Thomas W. VAHLENKAMPInstitute of Virology, Faculty of Veterinary Medicine, University of Leipzig,Leipzig, GERMANYRotaviruses cause gastroenteritis with watery diarrhoea in young childrenas well as in different animal species. One of the currently used humanrotavirus vaccines is based on a pseudotyped bovine rotavirus. Belongingto the family Reoviridae rotaviruses feature a double stranded, segmentedRNA genome and are therefore capable of rearrangement and reassortment.To enhance our knowledge about the epidemiological characteristicscomplete sequences of all eleven rotavirus genome segments are needed.In the current study published bovine rotavirus genomes were selectedfor alignments in order to generate consensus nucleotide sequences at the5 ′ and 3 ′ ends from each genome segment. Based on these consensussequences an assay was developed which enabled the amplification of alleleven segments of the rotavirus genome in a single reverse transcriptionpolymerase chain reaction (RT PCR). The developed RT PCR proved to beapplicable for a broad range of animal rotaviruses as complete genotypeconstellations of equine, porcine and bovine rotaviruses could be determinedwith this assay and classic Sanger sequencing methods. The assaywas also used to compare the nucleotide sequence of the common cellculture adapted bovine rotavirus strain B223 with its original isolate andcurrently circulating strains in the bovine population in Germany. By itsdesign this assay could also be a helpful support for a many molecularapproaches (e.g. protein expression, cloning or reverse genetics) and willfurther increase our knowledge about the coherences of animal and humanrotavirus infections.REF O17First report on rotavirus genotypes from the Democratic Republicof São Tomé and Príncipe: high prevalence of G8P[6] genotypeClaudia ISTRATE 1 , Sumit SHARMA 2 , Johan NORDGREN 2 , SandraVIDEIRA E CASTRO 1 , Ângela LOPES 1 , João PIEDADE 1 , AhmedZAKY 3 , António LIMA 3,4 , Edgar NEVES 3 , José VEIGA 3,4,5 , AidaESTEVES 11 Unidade de Microbiologia Médica, Grupo de Virologia, UPMM, Institutode Higiene e Medicina Tropical, Lisbon, PORTUGAL; 2 Division ofMolecular Virology, Department of Clinical and Experimental Medicine,Medical Faculty, University of Linköping, Linköping, SWEDEN; 3 InstitutoMarquês de Valle de Flôr, Lisbon, Portugal; 4 Ministério de Saúde daRépublica Democrática de São Tomé e Príncipe, São Tomé, DEMOCRA-TIC REPUBLIC OF SÃO TOMÉ AND PRÍNCIPE; 5 Centro de Saúde deMé Zóchi, Trindade, DEMOCRATIC REPUBLIC OF SÃO TOMÉ ANDPRÍNCIPERotavirus (RV) infection is responsible for about 500,000 deaths annually,the death toll affecting specifically the children in developing countriesof Africa and Asia. Our study was focused on RV epidemiology in theDemocratic Republic of São Tomé and Príncipe (DRSTP), a small, lowincome country off the western equatorial coast of Central Africa.Faecal specimens (n=237) were collected between August and December2011 from children under 5 years of age with acute gastroenteritis. Samplespositive for RV by rapid test (CerTest, Spain) were G and P typed byhemi nested type specific multiplex PCR (1), subgrouped (SG) for theVP6 gene by real time PCR and, finally, some strains were sequenced forphylogenetic analysis.A high prevalence (36.7%) of RV infection was shown. G8P[6] genotypewas identified in 71% of the positive samples, while G1P[8] representedonly 8.4%. A strong association of G8P[6] and G1P[8] strains with VP6SGI and SGII, respectively, was demonstrated. Phylogenetic analysis showedthat the VP7 and VP4 genes of G8P[6] RV from DRSTP cluster withcorresponding reference genotype sequences with bootstrap values =99%.Moreover, geographic grouping within each genotype was also evident.The present study, the first, to our knowledge, from DRSTP, showed ahigh prevalence of RV infection as well as the circulation of an unusualRV strain, G8P[6]. Our results underline the importance of RV surveillancein DRSTP, especially in view of the introduction of RV vaccines, highlyrecommended by WHO.Iturriza Gomara, et al. J Clin Virol 2004; 31: 259.REF O18Taxonomy of Circoviridae: restructuring and expansionPhilippe BIAGINI 1 , Mya BREITBART 2 , Eric DELWART 3 , JoaquimSEGALÉS 4 , Daniel TODD 5 , Arvind VARSANI 61 UMR 7268, French Blood Agency, Aix Marseille University and CNRS,Marseille, FRANCE; 2 University of South Florida, Saint Petersburg, USA;3 Blood Systems Research Institute, San Francisco, USA; 4 CReSA andUniversity of Barcelona, Barcelona, SPAIN; 5 Agri Food and BiosciencesInstitute, Belfast, UNITED KINGDOM; 6 University of Canterbury, Christchurch,NEW ZEALANDViruses with circular single stranded DNA genomes infect human and nonhuman hosts (animals, insects, bacteria, plants). On the basis of molecularand structural considerations, they are grouped into distinct taxonomicfamilies.The family Circoviridae was named after porcine circovirus which wasbiochemically characterised in 1982. Official description of the family waspublished in 1995, with several viral species grouped into a single genus,Circovirus. The progressive characterization of new viruses identified inbirds and pigs led to the subsequent creation of two genera, Circovirus andGyrovirus, incorporating distinct species.According to latest results obtained by metagenomics and next generationsequencing, it is however evident that the genetic diversity characterisingviruses with circular single stranded DNA genomes is extremely wide.This is exemplified by the recent discovery of viruses, tentatively namedcycloviruses, sharing significant similarities to circovirus isolates (genomeorganisation, presence of Rep and Cap genes..) despite a high geneticdiversity. Such findings are in favour with a restructuring of the familyCircoviridae.Taxonomic aspects, updates, and future directions are exposed.S44 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyWednesday 11 th September 2013,16h15 – 18h15WORKSHOP 27: “NEUROTROPIC VIRUSES”Chairpersons: Tomas BERGSTROM (Gothenburg, SWEDEN)& Monique LAFON (Paris, FRANCE)Room Tête d’OrKEYNOTE:Neurotropic VirusesJohn FAZAKERLEYThe Pirbright Institute, Surrey, UNITED KINGDOMMany viruses are capable of causing developmental CNS abnormalities,encephalitis, meningitis or neuritis in humans and other animals. Rubellainduced brain abnormalities; Herpes simplex virus temporal lobe encephalitis;shingles; progressive multifocal leukoencephalopathy; mumps andechovirus meningitis; Japanese, Venezuelan and West Nile encephalitides;rabies and canine distemper all provide prominent examples. Viruses havealso been suggested to be causative or contributing agents in multiple sclerosis,Parkinson’s disease, schizophrenia, motor neuron disease, dementiaand other neurological diseases. Viruses access the nervous system directlyfrom the blood or along nerves. In the absence of an ability to studydisease progress in the human CNS, understanding of the pathogenesis ofmany CNS virus diseases has been led by studies in experimental animalmodel systems. Importantly, the CNS has highly specialized long-livedcells and highly specialized immune responses. Many viruses replicatewell in immature neurons but some replicate less well, or unproductively,in mature neurons where, in the absence of the propensity of highlyspecialized CNS cells to undergo apoptosis, infection can become persistent.The resting CNS is an immunosuppressive environment but strongimmune responses can develop and there are well-documented examplesof the detailed mechanisms of both protective and destructive responses.This talk will provide an overview, illustrate key paradigms, present somerecent findings and outline outstanding questions.ORAL COMMUNICATIONSREF O19Viral DNA containing PML NBs (DCP NBs) are a hallmark of thenuclear associated antiviral response controlling the latency of a herpesvirusPatrick LOMONTE 1,2 , Catez FRÉDÉRIC 1 , Antoine ROUSSEAU 3 ,Nancy SAWTELL 4 , Marc LABETOULLE 31 Centre de Génétique et de Physiologie Moléculaire et CellulaireUMR5534 CNRS/UCBL1, Villeurbanne, France; 2 Laboratoired’excellence, LabEX DEVweCAN, Lyon, France; 3 Institut de VirologieMoléculaire et Structurale, CNRS UPR 3296, Gif sur Yvette, FRANCE;4 Division of Infectious Diseases, Cincinnati Children’s Hospital MedicalCenter, Cincinnati, USAHerpes simplex type 1 (HSV 1) is a neurotropic virus establishing latencyin trigeminal ganglia (TG) sensory neurons. During infection, the switchbetween the lytic cycle and latency is characterized by a change inviral gene transcription program. Nuclear architecture, including nucleardomains, is determinant for the control of gene expression and is likely toinfluence the fate of viral infection. We recently performed a fluorescentin situ hybridization (FISH) based study to analyse features of the HSV1 genome within TG neurons of latently infected mice. High resolutionimaging showed that viral genomes were present during latency withinnuclear domains containing the promyelocytic leukemia nuclear bodies(PML NBs or ND10) associated proteins, PML, Daxx, ATRX and SUMO2/3, that we named DNA containing PML NBs (DCP NBs). Infections ofPML KO mice demonstrated that PML protein was required for DCP NBsformation. We then analyzed the expression of a viral long non codingRNA, namely LAT, produced during latency, and showed that DCP NBsrestrict its expression, supporting the idea that DCP NBs are repressorsof viral transcription. Using the same approach, we investigated the DCPNBs formation at the initial stages of latency establishment, and confirmedthe affinity of PML NBs associated proteins towards viral genomes.In conclusion, our data support a role of DCP NBs in the physical andfunctional control of HSV 1 latency. This is the first study that describes atthe viral genome level, the role of PML NBs in the control of the biologyof a persistent virus.REF O20Molecular interactions of tick borne encephalitis virus with humanneural cellsDaniel RUZEK 1,2 , Martin PALUS 2,3 , Jana ELSTEROVA 2,3 , MarieVANCOVA 2,3 , Tomas BILY 21 Veterinary Research Institute, Brno, CZECH REPUBLIC; 2 Institute ofParasitology, Academy of Sciences of the Czech Republic, Ceske Budejovice,CZECH REPUBLIC; 3 Faculty of Science, University of SouthBohemia, Ceske Budejovice, CZECH REPUBLICTick borne encephalitis (TBE) virus causes severe encephalitis withserious sequelae in humans. However, the pathogenesis of TBE remainspoorly understood. TBE is a generalized infection. The virus invades thecentral nervous system after initial extraneuronal replication – virus replicationis first detected within draining lymph nodes, this is followed bydevelopment of viremia and during the viremic phase virus enter into thebrain. Our study was focused on the immunopathological features of thehost immune system during TBE, on the interaction of the virus with neuralcells, and on the role of the host blood brain barrier in the neuropathogenesisof TBE. We infected primary human neurons, astrocytes, perycites,and brain microvascular endothelial cells and examined their susceptibilityto the infection, changes in global gene expression and virus mediatedcytopathic effect including ultrastructural (3D cryoelecton microscopy)and apoptotic changes of the cells. We observed a number of ultrastructuralchanges in the infected cells that included e.g. enlargement of Golgiapparatus, presence of induced vesicles (smooth membrane structures)and convoluted membranes or disorganization of the rough endoplasmaticreticulum, as well as early features of apoptosis. Expression of keyinflammatory cytokines/chemokines was analyzed by real time RT PCRand ELISA. Moreover, changes in the expression of miRNA in infectedhuman neurons were investigated. Taken together, we contributed to thecharacterization of the complex molecular interactions of TBE virus withhuman neural cells.REF O21Understanding the role of NS1 protein in the neuropathogenicityof Japanese encephalitis virus (Flaviviridae)Melissanne DE WISPELAERE, Marie Pascale FRENKIEL, PhilippeDESPRESInstitut Pasteur, Paris, FRANCEJapanese Encephalitis Virus (JEV) is a member of the Flaviviridae familyand is transmitted by Culex species mosquitoes. This zoonotic arboviruscauses human encephalitis and remains an endemic disease in the AsiaPacific region. JEV has a positive sense RNA genome encoding threestructural proteins (C, prM, E), and seven non structural proteins (NS1,NS2A, NS2B, NS3, NS4A, NS4B, NS5). The Flavivirus NS1 is a multifunctionalglycosylated protein, that is secreted to the extracellular mediumVirologie, Vol 17, supplément 2, septembre 2013S45

5 th European Congress of Virologyduring viral infection. NS1 has been implicated in viral RNA replication(Khromykh et al. Journal of Virology 1999), and regulation of the hostimmune response (Chung et al. PNAS 2006). A larger NS1 related protein,NS1’, is produced via ribosomal frameshifting and plays a criticalrole in JEV neuroinvasiveness (Melian et al. Journal of Virology 2010).In the present study, we identified a single amino acid substitution in NS1that attenuates the virulence of JEV strain RP9 in a mouse model. Usinga molecular clone of JEV RP9, we demonstrated that this mutation abolishesthe neuroinvasiveness but not the neurovirulence of JEV RP9 inthree week old Balb/c mice. Interestingly, this mutation had no impact onviral RNA replication, and did not have any effect on JEV RP9 infectiouscycle in cell culture. Studies are ongoing to assess whether this mutationaffects JEV NS1 capacity to subvert the antiviral immune responses.REF O22Activation of the potentially neuropathogenic HERV W/MSRV typeendogenous retrovirus during severe infectious mononucleosis andpast Epstein Barr virus infection: the missing link with multiple sclerosis?Antonina DOLEI 1 , Giuseppe MAMELI 1 , Giordano MADEDDU 2 ,Alessandra MEI 1 , Elena ULERI 1 , Luciana PODDIGHE 1 , IvanaMAIDA 2 , Sergio BABUDIERI 2 , Caterina SERRA 1 , MariastellaMURA 21 Department of Biomedical Sciences, University of Sassari, Sassari,ITALY; 2 Department of Clinical and Experimental Medicine, Universityof Sassari, Sassari, ITALYThe etiology of multiple sclerosis (MS) is still unclear. Proposed viral cofactors are the Epstein Barr virus (EBV), and the potentially neuropathogenicHERV W/MSRV/Syncytin 1 endogenous retroviruses. The ascertainedEBV/MS links are history of late primary infection, possibly leading toinfectious mononucleosis (IM), and high titers of pre onset IgG againstEBV nuclear antigens (anti EBNA IgG). There is no evidence of MS specificEBV expression during MS, while a continuous HERV W expressionoccurs. We found repeatedly extracellular HERV W/MSRV and MSRVspecific mRNA sequences in MS patients, and MRSV presence/load strikinglyparalleled MS stages and phases. To verify whether HERV W mightbe activated in vivo, young adults hospitalized for IM were analyzed forexpression of HERV W/MSRV transcripts and proteins. Healthy controlswere either EBV() or latently EBV infected with/without high titers ofanti EBNA IgG. The results show that HERV W/MSRV activation occursin blood mononuclear cells of patients hospitalized for IM. When healthycontrols are stratified for high, low, or no anti EBNA IgG titers, this tightlycorrelates to the extent of HERV W/MSRV activation. Thus, the data indicatethat the two main links between EBV and MS (IM and high antiEBNA IgG titers) are paralleled by activation of the potentially neuropathogenicHERV W/MSRV. These novel findings suggest that the activationof HERV W/MSRV is the missing link between EBV and MS, and mayopen new avenues of intervention.REF O23Perturbation of neuronal signaling pathways by the rabies virusNicolas WOLFF 1,3,4 , Pierre MAISONNEUVE 1,3,6 , ElouanTERRIEN 1,3,6 , Célia CAILLET 1,3,4 , Mireille LAFAGE 1,2,5 , FlorenceCORDIER 1,3,4 , Christophe PRÉHAUD 1,2,5 , Henri BUC 1 , MurielDELEPIERRE 1,3,4 , Monique LAFON 1,2,51 Institut Pasteur, Paris, FRANCE; 2 Unité de NeuroImmunologie ViraleDépartement de Virologie, Paris, FRANCE; 3 Unité de RMN des BiomoléculesDépartement de Biologie Structurale et Chimie, Paris, FRANCE;4 CNRS UMR3528, Paris, FRANCE; 5 CNRS UMR3015, Paris, FRANCE;6 Université Pierre et Maris Curie, Paris, FRANCEThe human tyrosine phosphatase PTPN4 and the Microtubule associatedSer/Thr kinase 2 (MAST2) are two enzymes expressed in neurons. WhilePTPN4 is an anti apoptotic protein, MAST2 inhibits neurogenesis andneuroprotection. The PDZ domain of these two enzymes is specifically targetedby the cytoplasmic domain of the envelope glycoprotein (G protein)of the rabies virus (RABV) during neuron infection (Préhaud et al, 2010).As a result, the complexes formed by the PDZ of the two enzymes and theirrespective ligands are disrupted, triggering drastic effect on cell signalingand cell commitment either towards death or survival. By targeting MAST2PDZ, the G protein of virulent RABV alters the intracellular trafficking ofPTEN (Terrien et al., 2012) and promotes survival, whereas the G proteinof attenuated RABV induces neuronal cell death by targeting PTPN4 PDZ.We recently demonstrated that the catalytic activity of PTPN4 is regulatedby its PDZ domain and that the viral sequence interfered with this allostericregulation.We provided structural and biological evidences that the RABV Gproteins act as competitors endowed with specificity and sufficient affinityin a vital cellular process. The disruption of critical endogenousprotein protein interactions by viral protein altered drastically intracellularprotein trafficking and catalytic activity controlling the cellularhomeostasis.Terrien E, et al. Science Signal 2012; 5 (237): ra58.Babault, et al. Structure 2011; 19: 1518 1524.Préhaud, C et al. Science Signal 2010; 3 (105): ra5.REF O24Infection and crossing of an in vitro cellular model of human bloodbrain barrier by a large array of neurotropic enterovirusesRomain VOLLE 1,2 , Christine ARCHIMBAUD 1,2 , CécileHENQUELL 1,2 , Audrey MIRAND 1,2 , Martine CHAMBON 1,2 , HélènePEIGUE LAFEUILLE 1,2 , Jean Luc BAILLY 1,21 Université d’Auvergne, Faculté de Médecine, EPIE EA4843, ClermontFerrand, France; 2 CHU de Clermont Ferrand, Laboratoire de virologie,Centre National de Référence Entérovirus/Parechovirus – Laboratoireassocié, Clermont Ferrand, FRANCEHuman enteroviruses (EVs) are associated with neurological conditions(meningitis, encephalitis, and acute flaccid paralysis) but theirentry into the central nervous system (CNS) remains poorly understood.Infection of the blood brain barrier may represent an access pathwayto the CNS common to most neurotropic EVs. We used a humanbrain microvascular endothelial cell line (hCMEC/D3) to investigate thishypothesis.Cytotoxic effects in non polarized hCMEC/D3 cells were assessed byRT qPCR, virus infectivity, and infection kinetics for 88 EV strains(43 serotypes). Two virus groups were characterized in the screeninganalysis. Cytotoxic viruses, in contrast to replicating but poorly cytotoxicEVs, caused statistically significant cell death through necrosisand apoptosis. Confocal and electron microscopy observations showedpatterns of intra cellular injuries common to both virus groups: actindepolymerization, mitochondria relocation in the perinuclear space, andinduction of vesicular structures suggestive of viral factories. Endothelialbarriers generated in vitro by culture of hCMEC/D3 cells on a microporousmembrane were productively infected by cytotoxic EVs, whichaltered the barrier permeability within 24 hours. Poorly cytotoxic EVshad no effect on barrier permeability over at least 96 hours of productiveinfection.This study provides consistent data indicating that human cerebral microvascularendothelial cells are susceptible to a large array of EV types andsuggests two pathways for the infection of brain parenchyma from virusespresent in the blood.S46 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyThursday 12 th September 2013, 8h30 – 10h30WORKSHOP 5: “VIRAL (IMMUNO)PATHOGENESIS”Chairpersons: Branka HORVAT (Lyon, FRANCE)& Thomas MERTENS (Ulm, GERMANY)Room Prestige Gratte-CielEpidemiological studies relate influenza infection with vascular diseases.The hypothesis that influenza infection has procoagulant effects on humanshas been investigated in experimental animal models. However, thesestudies used animal models only susceptible to mouse adapted influenzaviruses and therefore results are hard to translate to human infection.Since ferrets are good models for human influenza we used ferrets tostudy and compare effects on blood coagulation in a controlled setting.Ferrets were inoculated with either a mock, seasonal, pandemicor avian influenza strain. All influenza infected animals showed significantalterations in clotting times compared to mock inoculated ferrets.Specifically on day 4 post infection, we observed a four second rise inboth prothombin time and activated partial thromboplastin time. D Dimerconcentrations were increased in all 3 influenza groups with the highestconcentrations in the pandemic influenza group. Von Willebrand factoractivity increased early in the infection suggesting endothelial cellactivation. Mean thrombin antithrombin complex levels were raised inboth pandemic and avian influenza infected ferrets in the first days afterinfection. At the tissue level, fibrin staining showed intracapillary fibrindeposition especially in H5N1 infected ferrets. To our knowledge thisis the first study that visualized hemostatic alterations in influenza virusinfection in an animal model resembling human disease both at the circulatoryand tissue level. These alterations may be the result of consumptivecoagulopathy.KEYNOTE:Vaccine induced immunity to influenza: Some considerationsGuus F. RIMMELZWAANDepartment of Viroscience, Erasmus Medical Center, Rotterdam, THENETHERLANDSFor the induction of protective immunity against seasonal influenza, commonlyinactivated vaccine preparations are used. These vaccines are highlyefficacious provided that the virus strains used in the vaccine antigenicallymatch the epidemic strains. The use of these inactivated seasonalinfluenza vaccines afford little or no protection against antigenicallydistinct influenza viruses, including potentially pandemic influenza Aviruses of novel subtypes and even may hamper the induction the protectiveimmunity against pandemic influenza, so-called heterosubtypicimmunity, otherwise induced by natural infection with seasonal influenzaviruses. Ideally, influenza vaccines induce more broadly protective immunityagainst viruses of various subtypes. Recent research aims at definingconserved viral proteins or conserved regions of viral proteins as targets forcross-protective humoral and cellular immune responses to aid the developmentof more broadly protective vaccines. Especially immunologicallynaïve individuals such as children

5 th European Congress of VirologySince HSV 1 can persistently infect the enteric nervous system (ENS)causing gut dysmotility 1 , we investigated the role of Toll like receptor(TLR)2 in HSV 1 induced ENS alterations.C57/Bl6 (WT) and TLR2KO mice were inoculated with HSV 1 orreplication deficient HSVdeltaICP27 intranasally and 4 weeks (W) laterintragastrically (IG). Macrophages or CD8 lymphocytes were depletedby clodronate liposomes or anti CD8. One 2W after viral IG injectionwe determined in the ileum a) monocyte chemotactic protein 1 (MCP 1)level by ELISA, b) infiltrating immune cells by fluorescent activated flowcytometry, c) ENS integrity by immunohistochemistry and by changes inisometric muscle tension following electric field stimulation.HSV 1 or HSVdeltaICP27 IG injection in WT mice caused loss of neuronalHuC/D and peripherin, altered cholinergic transmission, increasedMCP 1 and recruited activated macrophages (CD11b+F4/80+CD80+).Clodronate mediated macrophages depletion entirely prevented HSV 1induced ENS anomalies. TLR2KO mice failed to up regulate MCP 1 andto recruit macrophages following HSV 1 exposure but HSV 1 reactiveCD3+CD8+INFgamma+ lymphocytes infiltrated the myenteric plexuses.CD3+CD8+ cells but not macrophages depletion prevented HSV 1 inducedENS alterations in TLR2KO mice.Our study provides evidences that TLR2 signaling is essential for theoptimal development of anti viral immune responses following HSV 1infection of ENS. However, TLR2 driven recruitment of inflammatorycells causes neuronal damage and gut dysfunction.1. Brun, et al. Gastroenterology 201 ; 138: 1790.REF O28Ross River Virus is able to persist in tissues of infected macaquesimilarly to Chikungunya virus despite lower viremia during acutephaseLaurence DUPUIS MAGUIRAGA 1,2 , Thibault LARCHER 3 , CandiceBARBARIT 3 , Andres MERITS 4 , Andreas SUHRBIER 5 , JohnAASKOV 6 , Roger LE GRAND 1,2 , Pierre ROQUES 1,21 CEA, division of Immuno virologie/Institute of Emerging Diseasesand Innovative Therapies (iMETI)/, Fontenay aux Roses, FRANCE;2 UMR E1, Université Paris Sud 11, ORSAY, FRANCE; 3 INRA, UMR 703,Oniris, Nantes, FRANCE; 4 Institute of Technology, University of Tartu,Tartu, ESTONIA; 5 Queensland Institute of Medical Research, Queensland,AUSTRALIA; 6 Queensland University of Technology, Brisbane,AUSTRALIAThe chikungunya virus (CHIKV) and the Ross River virus (RRV), twoAlphaviruses provoke persistent myalgia/arthritis in humans. We showedthat chronic CHIKV infection in macaque (Macaca fascicularis) involvedmacrophages. To better characterize the alphavirus arthritis developmentmechanism in human, we compared RRV and CHIKV infections withinprimate model. We infected macaques with an infectious molecular cloneof the strain LR2006 OPY 1, we previously used, and with two strains ofRRV: a) T48, a molecular clone derived from a mosquito isolate obtainedin 1948 and b) QML 1, an 1999’ Australian clinical strain. We thenfollowed the clinical, viral and immunological parameters in peripheralblood at different time point during the infection and on the tissues at theeuthanasia. In situ Hybridization and Immuno histochemistry allowed to1) identify sites of viral replication and viral reservoirs tissue at the earlyand late infection associated with CHIKV and RRV and viral titrationand Q PCR together with Cytokine follow up by ELISA 2) assess themechanisms associated with arthralgia and/or persistence of these alphaviruses.Our study is the first comparison of in vivo CHIKV and RRVinfections in primates, showing that despite different replication kinetic,both pathogens are able to induce in vivo local inflammation and tissuedamage during arthralgia. Different pro inflammatory cytokines balancesshowed that processes however might be distinct. Our alphaviruses chronicinfection model can therefore be used to assess treatment strategy forthese specific tissue damages.REF O29MicroRNA expression signatures in chronic viral hepatitis progressionAlessandro SINIGAGLIA 1,2 , Enrico LAVEZZO 1 , Tiziana SANAVIA 3 ,Barbara DI CAMILLO 3 , Melania SCARPA 1 , IgnazioCASTAGLIUOLO 1 , Fabio FARINATI 4 , Giorgio PALÙ 1 , LuisaBARZON 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 IOV Istituto Oncologico Veneto, Padova, ITALY; 3 Departmentof Information Engineering, University of Padova, Padova, ITALY;4 Department of Surgical and Oncological Sciences, University of Padova,Padova, ITALYSeveral studies investigated miRNA expression in hepatocellular carcinoma,but little is known on the changes in liver miRNA expression duringthe different stages of chronic viral hepatitis. This study, which investigatedmiRNA expression profile by microarray analysis in liver biopsies from 40patients with chronic B and C viral hepatitis, identified a coherent pictureof miRNAs that were significantly associated with viral aetiology and withhistological features of chronic liver disease. In fact, data on differentiallyexpressed miRNAs that were identified in this study were consistent withvalidated targets and activity of each miRNA. Among modulated miRNAs,the miR 200a/200b/429 cluster appeared relevant in liver disease and itsup regulation was associated with different histological markers of diseaseprogression (i.e., fibrosis, inflammation and necrosis, steatosis); few miR-NAs were differentially expressed in HBV and HCV infected livers andwere conducible to specific viral activity; advanced fibrosis/cirrhosis wasassociated with the overexpression of fibrosis related miRNAs, the underexpression of miRNAs with tumor suppressor activity, and with a miRNAsignature consistent with retention of the epithelial phenotype and inhibitionof epithelial to mesenchymal transition. The most significant miRNAsassociated with severe fibrosis were investigated and confirmed in a mousemodel of acute liver fibrosis. In conclusion, this study described miRNAssignatures associated with chronic viral hepatitis progression that couldbe potentially used as markers of disease.REF O30Characterisation and functional analysis of the murine gammaherpesvirus68 microRNAsAmr BAYOUMY, Finn GREY, Bernadette DUTIARoslin Institute, University of Edinburgh, Edinburgh, UNITED KING-DOMMicroRNAs (miRNAs) regulate gene expression post transcriptionally byaltering the stability of their target mRNA. Murine gammaherpesvirus68 (MHV 68) encodes at least 15 miRNAs derived from a cluster of8 tRNA like molecules. The miRNAs are located in a genomic regionknown to encode latency associated transcripts, but their functions areunknown. MHV 68 naturally infects murid rodents and is closely relatedto Kaposi’s sarcoma associated herpesvirus and Epstein–Barr viruswhich are associated with lymphoproliferative tumours in humans. Thestrict species specificity of gammaherpesviruses has limited research onthe human viruses mainly to in vitro studies. Infection of mice with MHV68 provides a unique small animal model to investigate in vivo pathogenicfeatures that are difficult to assess in humans. During latency, MHV 68expresses a protein encoded by ORF73 named murine latency associatednuclear antigen (mLANA). In this study, a recombinant MHV 68 viruscontaining beta lactamase gene fused to ORF73 was used to detect andisolate latently infected mouse splenocytes by flow cytometry. This systemfacilitates the characterisation of viral miRNAs expression in latentlyinfected cells. At day 14 post infection which coincides with a peak splenicviral load, ten viral miRNAs were detected in mLANA positive splenocyteswith variable expression levels. In order to investigate the functionsof these miRNAs, a mutant virus that lacks the miRNAs was constructed.The phenotypic analysis of this mutant virus will allow us to investigatethe role of viral miRNAs in pathogenesis.S48 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyThursday 12 th September 2013, 8h30 – 10h30WORKSHOP 12: “VIRAL GENE EXPRESSION –TRANSCRIPTION, TRANSLATION”Chairpersons:Richard ELLIOTT (Glasgow, UNITED KINGDOM)& Peter ROTTIER (Utrecht, THE NETHERLANDS)Room Tête d’OrThis RNA motif presented high homology in sequence and secondarystructure with the translation inhibitor GAIT element that is present at the3 ′ end of several mRNAs coding proinflammatory proteins. In response toIFN-, the GAIT complex (EPRS, hnRNP Q, L13a, and GAPDH) interactswith the GAIT element and suppresses the translation. Similarly tothe cellular GAIT element, the presence of the viral motif at the 3 ′ UTR ofan mRNA encoding the luciferase gene inhibited its translation in the presenceof the GAIT complex, suggesting that the viral GAIT motif could beinvolved on translation silencing. It could be postulated that the viral GAITcould act either promoting the viral genome translation silencing duringthe translation replication switch or interfering with the host inflammatoryresponse.KEYNOTE:Norovirus translation: a novel paradigm for viral protein synthesisIan GOODFELLOWUniversity of Cambridge, UNITED KINGDOMNoroviruses are now recognized as the major cause of viral gastroenteritisin the developed world. Despite this impact, our understanding of the biologyof these viruses lags significantly behind that of other RNA viruses.Human noroviruses remain unable to replicate in the currently availablecell culture systems. Murine norovirus is currently used as model for elucidatingmolecular details of noroviruses replication due to the fact thatit can be propagated in cell culture, has reverse genetics systems and anavailable animal model.Noroviruses and other members of the Caliciviridae use a novel mechanismof viral genome translation not yet observed in any other animalRNA virus. This mechanism relies on the interaction of cellular translationinitiation factors with a virus-encoded protein covalently linked tothe viral RNA genome, VPg. In collaboration with Stephen Curry andStephen Mathews (Imperial College) we have determined the structure ofthe norovirus VPg protein. Mutational analysis has then been used to identifyfunctional sites within VPg and to identify those residues importantfor the interaction with initiation factors. Further insights into the role ofVPg in viral translation have been obtained from proteomic analysis ofhost factors associated with VPg. To further understand the effect of virusinfection on the host cell translation initiation apparatus we have begunto use quantitative proteomic approaches and a number of other methodsto understand the effect of norovirus infection on the host cell translationapparatus.ORAL COMMUNICATIONSREF O31Identification of an RNA translational regulator element at the 3 ′ endof the TGEV genomeFernando ALMAZAN, Silvia MARQUEZ JURADO, Nogales AITOR,Enjuanes LUISDepartment of Molecular and Cell Biology, National Center of Biotechnology(CNB CSIC), Darwin 3, Madrid, SPAINCoronavirus (CoV) replication and transcription are complex processesthat require the specific recognition of RNA cis acting signals located atthe ends of the viral genome. To identify cell proteins involved in CoVreplication, transmissible gastroenteritis CoV (TGEV) genome ends wereused as baits for RNA affinity chromatography purification. Nine proteinspreferentially bound to the 3 ′ end were identified, from which, a functionalrole on CoV RNA synthesis was demonstrated for hnRNP Q, glutamyl prolyltRNA synthetase (EPRS), arginyl tRNA synthetase (RRS), and poly(A)binding protein (PABP). The RNA motifs interacting with PABP, EPRSand RRS were identified. The PABP bound to the poly(A), while the EPRSand RRS bound to a 32 nt motif located at 411 nt from the 3 ′ genome end.REF O32TIA 1 is a cellular restriction factor for tick borne encephalitis virusthat binds viral RNA and is recruited to perinuclear sites of viralreplicationAlessandro MARCELLO, Amelina ALBORNOZ, Lisa MIORINICGEB, Trieste, ITALYTick borne encephalitis virus (TBEV) replication compartments are formedof 80 nm vesicles where the viral genomes are replicated and aconfined extravesicular space where replicated viral RNA is retained fortranslation and assembly (Miorin et al. J Virol 2013). These structurescontribute to the early delay in interferon signalling observed in infectedcells (Miorin et al. Virus Res 2012). In this work we investigated thehost cell factor T intracellular antigen 1 (TIA 1), a stress induced RNAbinding protein involved in the repression of initiation of translation ofcellular mRNAs through the formation of stress granules (SG). TIA 1interacts with viral RNA as shown by RNA immuneprecipitation of TIA1 in TBEV infected cells. During TBEV infection, cytoplasmic TIA 1 isrecruited at sites of viral replication with concomitant depletion from SGs.This effect is TIA 1 specific since G3BP1, another SG protein, remainsin SGs and doesn’t re localize to sites of viral replication. Moreover, heatshock induction of TIA 1 SGs, but not G3BP1 SGs, is inhibited in TBEVinfected cells. TIA 1 depletion leads to an increase of TBEV RNA levelsas well as extracellular infectivity. Taking advantage of a TBE luciferasereplicon system we could observe increased signal also at early timepoints consistent with TIA 1 mediated inhibition at the level of translation.These results indicate that TIA 1 is a cellular restriction factor duringTBEV replication that binds viral RNA and is recruited to extravesicularsites of replicated viral RNA.REF O33Structure of the full length hepatitis C virus IRES in solutionMarc JAMIN 1 , Julien PÉRARD 1 , Cédric LEYRAT 1 , FlorenceBAUDIN 1,2 , Emmanuel DROUET 11 UMI3265 UJF EMBL CNRS UVHCI, Grenoble, FRANCE; 2 EuropeanMolecular Biology Laboratory (EMBL), Heidelberg, ALLEMAGNEThe 5 ′ untranslated region of the hepatitis C virus genome contains aninternal ribosome entry site that initiates cap independent translation ofthe viral RNA. Atomic structures of different fragments of this large RNAregion have been determined but, until now, the structural characterizationof the entire internal ribosome entry site remained limited to cryo electronmicroscopy reconstructions of the internal ribosome entry site bound to differentcellular partners. Here we report an atomic model of free full lengthhepatitis C virus internal ribosome entry site refined by selection againstsmall angle X ray scattering data that incorporates the known structures ofdifferent fragments. We found that an ensemble of conformers reproducessmall angle X ray scattering data better than a single structure suggestingin combination with molecular dynamics simulations that hepatitis Cvirus internal ribosome entry site is an articulated molecule made of rigidVirologie, Vol 17, supplément 2, septembre 2013S49

5 th European Congress of Virologyparts that move relative to each other. Principal component analysis on anensemble of physically accessible conformers of hepatitis C virus internalribosome entry site revealed dominant collective motions in the molecule,which may underlie the conformational changes occurring in the internalribosome entry site molecule upon formation of the initiation complex.Structure of the full length hepatitis C virus IRES in solution. Julien Pérard,Cédric Leyrat, Florence Baudin, Emmanuel Drouet and Marc Jamin (2013)Nature Communications 4, 1612 doi:10.1038/ncomms2611.REF O34A dynamic G quadruplex region regulates the HIV 1 LTR promoterRosalba PERRONE 1 , Matteo NADAI 1 , Ilaria FRASSON 1 , ElenaBUTOVSKAYA 1 , Manlio PALUMBO 2 , Giorgio PALÙ 1 , Sara N.RICHTER 11 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Department of Pharmaceutical and Pharmacological Sciences, Universityof Padua, Padua, ITALYG quadruplexes are non canonical nucleic acid structures that may form inG rich sequences. G quadruplexes act as silencer elements in the promoterregions of human oncogenes and genes; putative G quadruplexforming sequences are also present in promoter regions of other mammals,yeasts and prokaryotic cells. Here we show that the HIV 1 LTR promoterexploits G quadruplex mediated transcription regulation like eukaryoticand prokaryotic promoters. Computational analysis on 953 HIV 1 strainssubstantiated a much conserved G rich sequence corresponding to Sp1 andNF B binding sites. Biophysical and biomolecular experiments provedthat two mutually exclusive parallel intramolecular G quadruplexes, eventuallystabilized by ligands, like TMPyP4 and BRACO 19, primarily foldin this region. Mutations abrogating G quadruplex formation incrementedpromoter activity in cells, whereas treatment with G quadruplex ligandsimpaired promoter activity. Striking parallelism between HIV 1 LTR andeukaryotic regulatory oncogene promoter G quadruplexes encompassesparallel like topology, one nucleotide loop, Sp1 binding sites and promoteractivity shaping. These findings open up the possibility of inhibitingthe HIV 1 LTR promoter by G quadruplex interacting small molecules,therefore envisaging development of anti HIV 1 drugs with unprecedentedmechanism of action.REF O35A transcriptional pre initiation complex encoded by beta and gammaherpesviruses required for late viral gene expressionHenri GRUFFAT, Valentin AUBRY, Thibaut DESCHAMPS, EvelyneMANETCIRI/INSERM, Lyon, FRANCEDuring their productive cycle, herpesviruses exhibit a strictly regulatedtemporal cascade of gene expression that can be divided into three generalstages: immediate early (IE), early (E), and late (L). Promoter complexitydiffers strikingly between IE/E genes and L genes. IE and E promoterscontain cis regulating sequences upstream of a TATA box whereas L promotersare mostly constituted of a unique cis element. In the case of the and herpesviruses, this element is a TATT motif, which is found in theposition where the consensus TATA box of eukaryotic promoters usuallylocalizes.We have previously shown that Epstein Barr virus encodes a protein calledBcRF1 that is absolutely required for the expression of the viral L genes.BcRF1 has structural homology with TBP (TATA Binding Protein) andinteracts specifically with the TATT box. However, although necessary forthe expression of the viral L genes, BcRF1 is not sufficient, suggestingthat other viral proteins are also required.Here, we present the identification and characterization of a viral proteincomplex necessary and sufficient for the expression of the late viralgenes. This minimal viral complex is composed of five different proteinsin addition to BcRF1 which appears to be responsible for its stabilization.Our results clearly show that some herpesviruses encode their own transcriptioncomplex responsible for the expression of the late viral genes.REF O36HCMV pp150 constitutes a Cyclin A2 CDK dependent switch controllingthe cell cycle and differentiation dependent onset of lytic geneexpressionBoris BOGDANOW 1 , Henry WEISBACH 1 , Jens VON EINEM 2 ,Sebastian VOIGT 3 , Michael WINKLER 4 , Christian HAGEMEIER 1 ,Lüder WIEBUSCH 11 Charité Universitätsmedizin, Berlin, GERMANY; 2 Institut für Virologie,Ulm, GERMANY; 3 Robert Koch Institut, Berlin, GERMANY; 4 DeutschesPrimatenzentrum, Göttingen, GERMANYHerpesviruses are able to deliver a multitude of pre made tegument proteinsto their host cells upon entry. The interplay between incoming tegumentproteins and cell specific regulatory factors dictates the outcome of herpesvirusinfections at the cellular level. Here we report a novel type oftegument host cell interaction that is essential for the cell cycle and differentiationstate dependent onset of human cytomegalovirus (HCMV) lyticgene expression. The major tegument 150 kD phosphoprotein (pp150) ofHCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in itscyclin A2 dependent phosphorylation. Alanine substitution of the RXL/Cymotif prevents this interaction and allows the virus to fully escape thecyclin dependent kinase (CDK) mediated block of immediate early geneexpression in S/G2 phase and cyclin A2 overexpressing cells. The cyclinA2 CDK pp150 axis is further required for establishing of HCMV quiescencein undifferentiated NTera2 cells, thereby acting synergistically withhiston deacetylase activity. Consistent with the known nucleocapsid bindingfunction of pp150, its RXL/Cy dependent phosphorylation affectsonly gene expression of the parental virion, suggesting a cis acting, virusparticle associated mechanism of control. The pp150 homologues of otherprimate and mammalian CMVs lack an RXL/Cy motif and accordinglyeven the nearest relative of HCMV, chimpanzee CMV, starts its lytic cyclein a cell cycle independent manner. Thus, HCMV has evolved a sensor ofcyclin A2 CDK activity as a new level of self limitation and adaptation toits human host.S50 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyThursday 12 th September 2013, 8h30 – 10h30WORKSHOP 17: “VECTOR BORNE VIRAL INFECTIONS”KEYNOTES:Partnership with the FP7-EU programsEuroWN, Vectorie & WingsChairpersons: Luiza BARZON (Padova, ITALY)& Nathalie PARDIGON (Paris, FRANCE)Room Gratte Ciel 1, 2, 3Epidemiology of WN in the Mediterranean region: towards betterdiagnostics and surveillanceMiguel-Angel JIMENEZINIA, Cisa, Madrid, SPAINWest Nile virus (WNV) is one of the most relevant emerging pathogensof the World. Public health authorities worldwide have incorporated thispathogen as one of their priorities in their agendas and road maps. Thismeans surveillance, preparedness and contingency plans at National andInternational levels, to tackle with the challenge of monitoring West Nilevirus circulation in vast geographic areas, enabling capabilities to detectand diagnose efficiently every case of WNV infection.WNV cycle involves different birds as vertebrate hosts and different typesof mosquitoes as vectors. Spillover from this cycle may affect other vertebratehosts, such as horses and humans. Understanding the risk of spilloverand epidemics requires a basic knowledge on how biotic (mosquito andvertebrate species composition and diversity, phenology, diet, habitats,etc.) and abiotic (geography, climate, etc.) components affect WNV transmissiondynamics.Although having common characteristics, Europe/Mediterranean regionis not a homogeneous region in terms of eco-epidemiology for WNV, butrather exhibit a “patchy” pattern in terms of virus transmission and persistencein the field. To define these patterns precisely is the objective of theepidemiological studies carried out in each area. A good model of study isgiven by the project EuroWestNile, with teams located in different areasof WNV transmission looking at the main factors governing the epidemiologyin each location. From their comparison, it is expected to produce a listof “common” and “differential” factors governing WNV epidemiology ineach transmission setting, using parameters accurately determined locally.Epidemiological models, diagnostic tools and surveillance programs mustbe adapted to the specific situation at which they target.West Nile virus strains in Europe are as virulent as the North AmericancounterpartsByron E.E. MARTINA, Stephanie M. Lim, Penelope KORAKA, AlbertDME OSTERHAUSDepartment of Virology, Erasmus Medical Center, Rotterdam, THENETHERLANDSThe severity of West Nile virus (WNV) infection in immunocompetentanimals is highly strain dependent, ranging from avirulent to highly neuropathogenic.Reasons for susceptibility to WNV-associated neuroinvasivedisease in a minority of human cases remain unexplained. Furthermore,until recently, WNV outbreaks in Europe were limited compared to the outbreaksrecorded in the US. It is also remarkable that many of the outbreaksin humans caused by lineage 1 and 2 in Europe, were not preceded by massivebird mortality. Virulence is a multi-factorial process and many aspectsneed to be studied in order to elucidate the pathogenic force of viruses.Several parameters can be used to describe virulence such as replicationrate, immune-interfering properties, lethal dose 50, median survival time,tropism for particular cells or tissues, as well as the viral burden present ininfected tissues. In addition, virulence factors in mice differ from those thatdetermine pathogenicity in birds. It is believed that a weak immune responseallows WNV replication in the periphery and development of highviremia with an increased chance of infection of the CNS. In addition, toWNV replication in the brain, the cells involved in supporting infection inthe CNS and the resultant response to WNV may tip the balance betweenrecovery and severe neuroinvasive disease. Evidence exists that both gliaand neurons are involved in WNV pathogenesis as well as virus-relatedvirulence factors.Development of vaccines against WNVSebastian ULBERTFraunhofer Institute for Cell Therapy and Immunology, Leipzig, GER-MANYWest Nile Virus (WNV) is an emerging pathogenic and mosquito-borneflavivirus with increasing distribution worldwide. During the last coupleof years the virus became endemic in several European countries. Birdsare the natural hosts of WNV, but also mammals, including humans, can beinfected. In most cases, WNV infections remain asymptomatic or lead toflu-like symptoms. However, especially older and immunocompromisedindividuals are at risk to develop severe neurological complications such asencephalitis or meningitis, which happens in approx. 1% of the cases. Currentlyavailable WNV-vaccines include formaldehyde-inactivated virusand viral vector systems, but are only for veterinary use. Despite majorresearch efforts, there is no human vaccine on the market yet. Due tothe need to protect older people, any vaccine technology against WNVhas to especially focus on effectiveness and safety. The lecture will givean overview on WNV-vaccine development and will present EU-fundedresearch that is being carried out on this topic by the scientific consortiaVECTORIE, WINGS and EUROWESTNILE.ORAL COMMUNICATIONSREF O37Impact of vector ecology on virus amplification and spillover intohumans and livestock: the case of West Nile virus in EuropeJordi FIGUEROLA 1 , Josue MARTINEZ DE LA PUENTE 1 , AnnapaolaRIZZOLI 2 , Alexander PLATONOV 3 , Hanna BIN 4 , Joaquin MUÑOZ 1 ,Gioia CAPELLI 5 , Santiago RUIZ 6 , Roberto ROSA 2 , David ROIZ 1 , AnaVAZQUEZ 7 , Antonio TENORIO 7 , Maripaz SANCHEZ SECO 7 , RamonSORIGUER 11 EBD CSIC, Sevilla, SPAIN; 2 Fondazione Edmund March, S. Micheleall’Adige, ITALY; 3 Central Institute of Epidemiology, Moscow, RUSSIA;4 Central Virology Laboratory Ministry of Health, Ramat Gan, ISRAEL;5 Instituto Zooprofilattico Sperimentale delle Venezie, Legnaro, ITALY;6 Servicio de Control de Mosquitos, Huelva, SPAIN; 7 Instituto de SaludCarlos III, Madrid, SPAINOutbreaks of zoonotic diseases are recurrently occurring across Europe.Usually interpreted as the result of virus introductions into new areas, inmany cases outbreaks are the result of environmental conditions favouringvirus amplification or spillover into humans or livestock. Understandingthe risk of emergence of vector borne zoonotic virus implies improvingour knowledge of the interactions between vectors and vertebrates. Thisis specially relevant for West Nile virus, a flavivirus causing encephalitisamong humans that has demonstrated its capacity to overwinter in Europeand produce until recent years, reduced outbreaks in humans and horses.The virus is able to replicate in most avian species, and mosquitoes getinfected after feeding on viraemic birds. However, most mammal speciesare dead end hosts. To understand the potential of virus amplificationwe analysed the blood meal origin of mosquitoes trapped in Israel, Italy,Virologie, Vol 17, supplément 2, septembre 2013S51

5 th European Congress of VirologyRussia and Spain. Results show that some mosquito species feed mainlyon birds (with a large potential for virus amplification) while others feed onmammals only. Important differences in diet composition also occurredbetween localities, probably due to vertebrate community composition.In Spain, WNV transmission risk for birds human and horses, was estimatedbased on mosquito abundance, vectorial competence and bloodmeal origin. Estimated risk fitted reasonably well interspecific differencesin antibody prevalence and virus prevalence in mosquitoes. Vector ecologymay contribute to understand risk of zoonotic virus outbreaks acrossEurope.REF O38Characterization of Human West Nile Virus strains isolated in NorthernItalyLuisa BARZON 1,2 , Monia PACENTI 2 , Elisa FRANCHIN 1,2 , LauraSQUARZON 1 , Alessandro SINIGAGLIA 1,3 , Samuele ASNICAL 1 ,Enrico LAVEZZO 1 , Stefano TOPPO 1 , Silke CORBACH SOEHLE 4 ,Ignazio CASTAGLIUOLO 1 , Giulia MASI 1 , Sebastian ULBERT 5 ,Margherita CATTAI 2 , Riccardo CUSINATO 2 , Giorgio PALÙ 1,21 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Clinical Microbiology and Virology, Padova University Hospital,Padova, ITALY; 3 IOV Istituto Oncologico Veneto, Padova, ITALY;4 Institut für Labortierkunde, Universität Zürich, Zurich, SWITZERLAND;5 Vaccine Technologies Unit, Fraunhofer Institute for Cell Therapy andImmunology, Leipzig, GERMANYWest Nile Virus (WNV), a flavivirus, is intrinsically maintained in anenzootic cycle between mosquitos as vectors and wild birds serving asreservoir hosts. WNV can also infect and cause disease in humans andhorses. The virus is widely distributed in Africa, Europe, the Middle East,Asia and the Americas and is able to cause neuroinvasive disease withthe potential for severe courses especially in the elderly and immunocompromisedhumans. WNV infections increasingly occur in mediterraneancountries with tendency to spread to central and northern Europe. Thus,safe and efficacious vaccines are urgently sought for WNV prophylaxis inhumans and animals. Replication deficient Modified Vaccinia virus Ankara(MVA) can be exploited as versatile viral vector in medical and veterinaryvaccine development.Here, we have generated and evaluated recombinant MVA delivering theWNV antigens E (envelope) and prM/M (precursor membrane/membrane)and fulfilling the requirements to undergo clinical testing in humans. Thestructural proteins of the WNV envelope are highly relevant vaccine antigensfor the induction of WNV specific antibody and T cell responses.Infections of human and equine cell cultures with recombinant MVAdemonstrated efficient synthesis and secretion of WNV envelope proteinsin mammalian cells non permissive for MVA replication. Primeboost immunizations in BALB/c mice induced high levels of WNV specificantibodies. Moreover, vaccinations with recombinant MVA in HLAA2.1/HLA DR1 transgenic H 2 class I/class II knockout mice resulted inthe induction and efficient expansion of WNV specific CD8+ T cells. Thus,results from vaccinations in two different mouse models demonstratedsolid immunogenicity of MVA WNV vector vaccines. Further evaluationin different animal models is warranted to evaluate protective efficacyagainst WNV and to demonstrate the potential of the recombinant MVAas candidate vaccines in humans.West Nile virus (WNV) is an emerging pathogen that is circulating in Northeastern Italy and causing disease in humans since 2008. Aim of this studywas the investigation of the spread of WNV strains in north eastern Italytaking advantage of the special surveillance programme for WNV infectionthat was activated in Veneto Region in 2008 and enhanced in 2010.The pathogenic potential of isolated WNV strains was also investigated.During 2008 2009, several human cases of WNV disease caused by anendemic lineage 1a strain, named Ita09, were identified in areas surroundingthe Po river. Since 2010, cases have been recorded in nearby northernareas, where, in 2011, both lineage 1a and 2 were detected. Two novelhuman lineage 1a strains were identified in 2011 by full genome sequencingand phylogenetic analysis. One of these strains, called Livenza, wasresponsible of a relatively large outbreak with over 50 human infections in2012. Both the Ita09 and Livenza strains had a prolin at codon 249 of theNS3 protease/helicase, which has been associated with increased WNVtransmissibility and virulence. In vitro experiments demonstrated higherstability and activity of the NS3 protease/helicase with prolin at codon 249than other NS3 mutants. Pathogenicity studies on the WNV Ita09 straindemonstrated high neuroivasive potential and lethality in mouse models.In the mouse model, different organs were involved by WNV infectionwith the highest viral load detected in brain, stomach, and gut. Activationof markers of innate immune response was demonstrated in infectedorgans.REF O39Recombinant vaccinia MVA expressing E and prM/M proteins of WestNile Virus for vaccine generationGerd SUTTER 1 , Martina KASERER 1 , Asisa VOLZ 1 , Sylvia JANY 1 ,Stephanie LIM 2 , Byron MARTINA 21 Institute for Infectious Diseases and Zoonoses,Chair for Virology, LMU,Munich, GERMANY; 2 Virosience Lab, Erasmus Medical Center, Rotterdam,THE NETHERLANDSREF O40Sylvatic origin and geographic spread of St. Louis encephalitis virusSandra JUNGLEN 1 , Anne KOPP 1 , Thomas GILLESPIE 2 , DanielHOBELSBERGER 3 , Alejandro ESTRADA 4 , James HARPER 5 ,Richard MILLER 5 , Isabella ECKERLE 1 , Marcel MÜLLER 1 , LarsPODSIADLOWSKI 6 , Fabian LEENDERTZ 3 , Christian DROSTEN 11 Institute of Virology, Bonn, GERMANY; 2 Emory University, Atlanta,USA; 3 Robert Koch Institute, Berlin, GERMANY; 4 University of Mexico,Mexico City, MEXICO; 5 University of Michigan, Ann Arbour, USA;6 Institute of Evolutionary Biology and Ecology, Bonn, GERMANYSt. Louis encephalitis virus (SLEV) is the prototypic mosquito borne flavivirusin the Americas. The virus is transmitted between Culex mosquitoesand birds in North and South America. The geographic and ecologicalorigins of SLEV remain obscure.In an ecological investigation in a tropical rainforest in Palenque NationalPark, Mexico, we discovered an ancestral variant of SLEV in Culexnigripalpus mosquitoes.The tentatively named SLEV Palenque strains were distinct from all presentlyknown SLEV strains showing only 94.2 – 95.7% amino acid identityto epidemic SLEV strains and forming a highly distinct phylogenetic cladewithin the SLEV species. Cell culture studies of SLEV Palenque versusepidemic SLEV (MSI 7) revealed no growth differences in insect cells, buta clear inability of SLEV Palenque to replicate in cells from several SLEVhost species permissive for MSI 7 replication (birds, cotton rats, free tailedbats). Only cells from nonhuman primates and neotropical fruit batswere moderately permissive. Phylogeographic reconstruction suggestedthat the common ancestor of all epidemic SLEV strains has existed in anarea between southern Mexico and Panama circa 330 years before present.Expansion of the epidemic lineage occurred in two waves, the first representingemergence near the area of origin and the second involving almostparallel appearances of virus in the lower Mississippi and Amazonas deltaregions. We were able to link the emergence of a major epidemic arboviruswith anthropogenic ecosystem invasion during colonial times.S52 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF O41Clonal expansion of tick borne encephalitis virus in a disease hot spotat lake Vänern, SwedenTomas BERGSTRÖM 1 , Peter NORBERG 1 , Anette ROTH 1 ,Peter NOLSKOG 1 , John PETTERSSON 2 , Thomas JAENSSON 2 ,Mats HAGLUND 3 , Gert OLSSON 3 , Sirkka VENE 3 ,ÅkeLUNDQVIST 31 Department of Infectious Diseases, University of Gothenburg, Gothenburg,SWEDEN; 2 Department of Systematic Biology, Uppsala University,Uppsala, SWEDEN; 3 Center for Disease Control, Karolinska Institute,Stockholm, SWEDENAfter a presumed entry over the Baltic Sea to the southeast of Sweden, TickBorne Encephalitis Virus (TBEV) is a recently introduced and emergingzoonotic infection in the region of Western Gotaland, Sweden. Almostall of the 200 human cases recorded at present are located in the closeproximity of lakes or rivers. The aim of the current study was to understandthe mechanisms of spread responsible for this expansion, which areunknown. After the first TBE patient was reported in 1997, close to 50human cases have been diagnosed on the southeast shores of lake Vänern.Here, we performed whole genome sequencing and phylogeny of 11 TBEstrains collected from human samples, and from ticks and rodents, on aneast westerly axis from coast to coast along waterways through southernSweden. All 6 strains retrieved from the Lake Vänern region (three fromrodent M. Glareolus and three from tick I. Ricinus, all collected close tolocations of human cases of TBE) showed high genetic homology despitethat these viruses were found 100 km apart. The results are compatiblewith a clonal expansion of TBEV from a common viral ancestor introducedin the lake Vänern region. Surprisingly, this cluster could not be linkedspecifically to any one of the other five viruses collected from the east towest coast of southern Sweden. These findings argue against a continuousand consecutive spread westward in Sweden of a single strain of TBEV.Instead, the phylogenetic analyses are compatible with an introduction ofmultiple TBEV strains to Western Sweden, either from the east of Swedenor from abroad.REF O42Novel insights into the defence response in ticks against tick borneencephalitis virusSabine WEISHEIT 1,2 , Margarita VILLAR 4 , Marina POPARA 4 , HanaTYKALOVÁ 3 , Jan ERHART 3 , Daniel RUŽEK 3 , LiborGRUBHOFFER 3 , Jose DE LA FUENTE 4,5 , Mick WATSON 2 , LesleyBELL SAKYI 1 , John K. FAZAKERLEY 11 The Pirbright Institute, Pirbright, UK; 2 The Roslin Institute and Royal(Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh,UK; 3 Biology Centre, Institute of Parasitology and Faculty of Science, Universityof South Bohemia, Ceske Budejovice, CZECH REPUBLIC; 4 SaBio.Instituto de Investigación en Recursos Cinegéticos IREC (CSIC UCLMJCCM)s, Ciudad Real, SPAIN; 5 Department of Veterinary Pathobiology,Center for Veterinary Health Sciences, Oklahoma State University, Stillwater,USATicks are second only to mosquitoes in their importance in transmittinga wide variety of different pathogens including bacteria, protozoa andviruses. Many arboviruses of medical or veterinary importance, includingtick borne encephalitis virus (TBEV), Crimean Congo haemorrhagicfever virus, African swine fever virus and Nairobi sheep disease virusare transmitted by ticks. Although several studies have identified antiviralmechanisms in insects including Toll, IMD, JAK/STAT, melanisationand RNA interference (RNAi), little is known about the response of ticksto virus infection. RNAi is currently the only antiviral defence mechanismshown to be effective in ticks. To identify tick genes with a putativeantiviral role we compared the transcriptome and proteome of Ixodes sppcell lines and Ixodes ricinus ticks mock infected or infected with TBEV.Homologues of several genes known to be effective components of vertebrateinnate immunity, including ferritin binding (e.g. a homologue ofRattus norvegicus Fth1), calmodulin, heat shock proteins (e.g. HSP70)and components of the prophenoloxidase system (e.g. peroxinectin) andthe complement system (e.g. C4b binding protein and complement factorD), were differentially expressed. The roles of some of these moleculeswere investigated by gene silencing using dsRNA and a TBEV repliconexpressing a luciferase reporter protein. The results show that there is moreto the antiviral response of tick cells than just RNAi.Virologie, Vol 17, supplément 2, septembre 2013S53

5 th European Congress of VirologyThursday 12 th September 2013, 8h30 – 10h30WORKSHOP 25: “RESPIRATORY VIRUS INFECTIONS”KEYNOTE:Chairpersons: Fausto BALDANTI (Pavia, ITALY)& Astrid VABRET (Caen, FRANCE)AmphitheaterRespiratory virus infections: from pathogenesis to pandemicsPeter OPENSHAWProfessor of Experimental Medicine, Imperial College London W2 1PG,UNITED KINGDOMDespite concerns over population growth, water shortages, global warming,political and military strife, pandemic diseases remain major threatsto global prosperity and stability. In addition, health and wellbeing areconstantly eroded by common colds which act as co-factors in manycommon diseases.Our understanding the mechanism of disease induction by respiratorypathogens has undergone radical change over the past two decades. Respiratorysyncytial virus used to be thought of a cause of serious diseaseonly in infants but is increasingly recognized as a cause of morbidity andmortality in the elderly and immunocompromised. Natural infections aredifficult to study and animal models have limitations.Human experimental challenge is uniquely able to provide detailedinsights into the mechanisms of disease in the natural host. We have infectedadult volunteers with Memphis 37 strain of RSV, causing reproducibledisease without serious adverse events. We have found that viral load isrelated to and severity of symptoms, strengthening the case for the developmentof antiviral drugs. Importantly, there we found only an indirect andweak association between baseline antiviral antibody and susceptibilityto viral challenge. These studies need to be complemented by observationalstudies of natural infections, but are providing novel insights that areleading to improved understanding of viral disease.Studies of individual responses to viral infection are also helpful in planningfor outbreaks of respiratory disease. The UK made detailed pandemicplans in response to avian influenza; these plans were tested by the 2009-10 influenza pandemic. Although generally mild, there were over 8,000hospitalizations in the UK and probably some 250,000 deaths worldwide(to August 2010). The disease was especially severe in some patients withasthma, in pregnant women and in children under 5 years of age. However,many of those admitted to hospital had no known risk factors and wereotherwise young and healthy.We established two research consortia to study disease in UK hospitals: aclinical information network based on case audit (Flu-CIN) and an in-depthstudy of the pathogenesis of influenza with intensive collection and analysisof samples from the respiratory tract and periphery during diseaseand recovery. This second study (the Mechanisms Of Severe InfluenzaConsortium, MOSAIC) involved 45 co-investigators and recruited 255patients with influenza-like illness from 11 hospitals (total 4800 beds).Of these, 170 patients had confirmed influenza (87% pH1N1); the causeof illness was determined in all cases. Rooted in extensive clinical information(>4000 data fields per patient), various research teams focused onhost (cellular immunology, soluble mediator responses, transcriptomicsand genomics), pathogen (influenza virus genetic and antigenic characteristics,viral shedding and viral load) and co-pathogen (co-infecting orsecondarily-infecting bacteria and viruses), with the aim of explaining thevariable response to pH1N1 infection.This unique study is allowing a series of related research questions to beexamined, linking the information on a case-by-case basis from patientsand controls. The consortium has identified a human gene deletion (witha mouse homologue) linked to defective host responses in about 6% ofour cases but is rarely present in normal individuals. This has been confirmedon other populations in the Far East. The success of this consortiumdemonstrates that it is possible to conduct high-quality research during anunexpected pandemic, but emphasizes the need to maintain infrastructureand preparedness.ORAL COMMUNICATIONSREF O43Growth and characterization of different HRV C types in 3D humanairway epithelia reconstituted in vitroKomla SOBO 1,2 , Caroline TAPPAREL 1,2 , Laurent KAISER 1,2 , SandraVAN BELLE 2 , Samuel CONSTANT 3 , Song HUANG 31 University of Geneva, Geneva, SWITZERLAND; 2 Laboratory of Virology,Division of Infectious Diseases and Division of Laboratory Medicine, Universityof Geneva Hospitals, Geneva, SWITZERLAND; 3 Epithelix sárl, 14Chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland, Geneva,SWITZERLANDThe development of new molecular diagnostic tools has recently allowedthe discovery of human rhinovirus species C (HRV C). Several reportssuggest that HRV Cs may be overrepresented in children with pneumoniaor acute wheezing and exacerbation of asthma. HRV Cs cannot be propagatedin conventional immortalized cell lines, hence unlike HRV As andHRV Bs, the biological properties of these species C rhinoviruses havebeen difficult to study. However three groups have recently described thesuccessful amplification of HRV C15, untyped HRV C (W23), HRV C11and HRV C41 in sinus mucosal organ cultures {Bochkov, 2011 #1009}and in fully differentiated human airways epithelial cells (nasal or bronchialepithelial) {Hao, 2012 #1006; Ashraf, 2012 #1010}. Consistent withthese recent works, we report here that a panel of clinical HRV C specimensincluding HRV C2, HRV C7, HRV C12, HRV C15 and HRV C29types were all capable of mediating productive infection in reconstituted3D human primary upper airway epithelial tissues and that the virionsenter and exit preferentially through the apical surface. Similar to HRVA and HRV B, our data furthermore confirm the acid sensitivity of HRVCs. Finally, we show that the optimum temperature requirement duringthe growth of HRV Cs may be type dependant.REF O44Replication of Human Rhinovirus C in a Standard Cell LineAnna GÖRANSSON 1 , Nina JONSSON 1 , Fredrik LYSHOLM 2 , TobiasALLANDER 2 , A Michael LINDBERG 1 , Björn ANDERSSON 21 Linnaeus University, Kalmar, SWEDEN; 2 Karolinska Institutet, Stockholm,SWEDENHuman rhinovirus C (HRV C), a recently discovered picornavirus, is frequentlydetected during lower respiratory tract infections, primarily inchildren. HRV C is often associated with more severe infections than thoseof HRV A and –B. Studies of HRV C has so far been hampered due to theabsence of simple and rapid methods to propagate this virus in standard celllines. Recently Hao et al. reported replicating HRV C after transfection offully differentiated airway epithelial cells with RNA derived from a HRVC cDNA clone. Although this is a step forward it requires the access toprimary epithelial cells. Here we present data that support the replicationof a strain of HRV C in the standard cell line; human rhabdomyosarcoma(RD) cells. In vitro transcribed viral RNA from a cDNA clone of HRV C34was transfected into RD cells and viral replication was verified by methodsdetecting accumulation of viral genomes. Further characterization of thereplicating HRV C34 strain will be presented.Hao W, Bernard K, Patel N, Ulbrandt N, Feng H, Svabek C, Wilson S,Stracener C, Wang K, Suzich J, Blair W, Zhu Q. Infection and Propagationof Human Rhinovirus C in Human Airway Epithelial Cells. J Virol 2012;86 (24): 13524-32.S54 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF O45Novel system for analysis of virus host interactions in primary humanairway epitheliumHulda R. JONSDOTTIR 1 , Ronald DIJKMAN 1 , Regulo RODRIGUEZ 2 ,Volker THIEL 11 Institute of Immunobiology, Kantonal Hospital St.Gallen, St.Gallen,SWITZERLAND; 2 Institute of Pathology, Kantonal Hospital St.Gallen,St.Gallen, SWITZERLANDHuman airway epithelium represents the entry port of respiratoryviruses.Advances in isolation and cultivation of primary human lungepithelial cells have enabled careful studies of these viruses in airliquid interface cultures(HAE cultures) that morphologically resemblethe conducting airways in vivo.After induced differentiation thecultures contain different cell types such as basal, ciliated and gobletcells.Coronaviruses(CoVs) are common pathogens that infect a broadrange of vertebrate species and are associated with respiratory and entericdisease.Four common human CoVs(HCoV 229E,OC43,NL63 and HKU1)are detected worldwide and believed to be responsible for 6% of allrespiratory tract infections.Despite high incidence,knowledge of fundamentalCoV host interactions at the primary entry port is limited.HAEcultures have been shown to be a universal platform to study CoV infection.However,to characterize virus host interactions HAE cultures mustbe made amenable to genetic modification with lentiviral vectors for transgeneexpression and knock down of host gene expression.Therefore,amethod of transduction must be developed.Differentiated HAE culturesare refractory to apical and basolateral transduction with lentiviral vectors.However,undifferentiatedcells are readily transduced in suspensionwith 20 30% efficacy.Transgene expression remains stable during differentiationand does not affect cellular composition of the cultures.ModifiedHAE cultures will enable us to study host responses to HCoV infectionand gain insight into the biology of human respiratory viruses.REF O46In vitro and in vivo characterization of A(H1N1)pdm09 influenzavirus with HA 222 polymorphismJean Sebastien CASALEGNO 1,2 , Olivier FERRARIS 3 , Laetitia LINES 1 ,Maude BOUSCAMBERT 2 , Vanessa ESCURET 1,2 , MartineVALETTE 2 , Corinne BERGERON 1 , Sylvie PILLET 4 , BrunoPOZZETTO 4 , Bruno LINA 1,2 , Michele OTTMANN 11 Laboratoire de virologie et pathologie humaine, EA4610, UniversitéLyon 1, Lyon, FRANCE; 2 CNR grippe France Sud, CBPE, HCL, Bron,FRANCE; 3 IRBA, Lyon, FRANCE; 4 Laboratoire GIMAP, Université JeanMonnet, Saint Etienne, FRANCEEpidemiological surveillance of influenza A(H1N1)pdm09 during the pandemicrevealed a high number of deaths (7.1%) due to a D222G substitutionin the hemagglutinin (HA) of viruses (WHO, 2010). Biochemical andreplicative properties of A(H1N1)pdm09 viruses isolated from patientswere studied to identify factors that may increase the pathogenicity.Seven A(H1N1)pdm09 viruses isolated by the CNR Influenza South ofFrance were selected with a polymorphism HA 222D/G/N or E. Infectedpatients presented mild flu severe symptoms or died. Two distinct phylogeneticgroups were identified according to sequences (WHO 2012).Substitutions at antigenic sites of the HA affected poorly the antigenicityas showed by hemagglutination inhibition. No major difference in affinitywas detected for the seven NA in comparison with other A(H1N1)pdm09viruses. The affinity of HA for sialic acids a 2,6Gal and a 2,3Gal allowedto distinguish three groups of viruses. The infectivity was studied in vivoin BALB/c mice. The seven isolates exhibited various MID50 withoutincrease of pathogenicity (LD50>10e6).Most studies analysed viruses with HA 222G substitution. We have extendedour study to other N and E substitutions. These substitutions are ableto increase the spectrum of infection to type II pneumocytes and alveolarmacrophages, and thus increase pathogenicity. The analysis of the completegenome is in progress in order to provide additional insights of theseviruses.REF O47Genetic variation in influenza A virus: mutations associated withimmune reactions and severity of diseaseLaura KAKKOLA, Oxana DENISOVA, Denis KAINOVInstitute for Molecular Medicine Finland, Helsinki, FINLANDInfluenza A virus infection leads either to mild or severe disease. Underlyingmechanisms and risk factors for the severe disease are not known –probably both viral and host genetic factors affect the outcome of infection.High virus virulence may be associated with reassortments or specificmutations in viral genes located on eight segments of negative sense RNAgenome. Also, mutations in the host genome may affect host antiviralresponses resulting in the lack of efficient antiviral response or excessiveinflammatory response.Our aim is to identify viral determinants in influenza A virus genomesthat influence the severity of infection. The whole genomes of influenzaA virus isolates from last four epidemics in Finland were obtained withIllumina sequencing technology. Viral sequences were compared from nonhospitalized, hospitalized and intensive care treatment required patients.The effect of identified mutations on infection and host cell responseswas further characterized. One nonsense mutation leading to truncatedNS1 protein of influenza A(H1N1)pdm09 strain resulted in differences incytokine responses of infected macrophages.Analysis of whole genomes of viruses allows finding of novel mutationsaffecting drug resistance and virulence, providing new insights into virushost interactions.REF O48Analyses of Influenza Vaccine Break through Infections and Estimationof Influenza Vaccine Effectiveness based on a Sentinel PlatformMonika REDLBERGER FRITZ 1 , Michael KUNDI 2 , Therese POPOWKRAUPP 11 Medical University Vienna, Department of Virology, Vienna, AUSTRIA;2 Medical University Vienna, Institute of Environmental Health, Vienna,AUSTRIABackground: Influenza vaccine effectiveness (VE) is influenced by theantigenic similarity between vaccine and circulating strains. Therefore theAustrian sentinel surveillance system for monitoring virus evolution andVE was assessed.Methods: Viruses obtained from vaccinated and nonvaccinated sentinelpatients during six influenza epidemic seasons (2005/06 to 2008/09 and2010/11 to 2011/12) were characterized by detailed genetic and antigenicanalyses. Their relatedness to the vaccine strains of the respective seasonwas determined by phylogenetic analysis and antigenic mapping. Basedon these data the VE (overall and in different age groups) was estimated.Results: Of the 2733 sentinel patients of the six seasons investigated only7% were vaccinated. Of 1550 laboratory confirmed influenza virus infections90 occurred in vaccinated patients. Detailed comparative geneticand antigenic analyses of the viruses from vaccinated and nonvaccinatedpatients revealed a significantly higher frequency of driftvariants in vaccinatedpatients. In addition it was found that the driftvariant evolvingduring one season regularly represented the dominant strain of the followingseason. Depending on the match between circulating strains andvaccine strains the adjusted VE ranged from 43% to 74%. Age specificVE estimates ranged in mean from 63% in children = 14 years to 54% inadults aged = 65 years.Conclusions: Detailed antigenic and genetic analysis of virus strains derivedfrom vaccinated patients provides valuable information on evolvingdrift variants of epidemiologic relevance.Virologie, Vol 17, supplément 2, septembre 2013S55

5 th European Congress of VirologyThursday 12 th September 2013,11h00 – 13h00PLENARY SESSION 2: “INTERACTION OF VIRUSES WITHPATHOGENS OR ENDOSYMBRIONTS“Chairpersons: Roberto CATTANEO (Rochester, USA) &Bernhard FLECKENSTEIN (Erlangen, GERMANY)Amphitheater11:00 – 11:30How gut microbes enhance enteric virus infectivityJulie PFEIFFERDepartment of Microbiology, University of Texas Southwestern MedicalCenter, Dallas, TX 75390, USAEnteric viruses encounter a vast microbial community in the mammaliandigestive tract. We recently found that gut microbes are required forreplication and pathogenesis of two unrelated enteric viruses, poliovirusand reovirus (Kuss et al., Science, 2011). Similarly, Tanya Golovkina’slab demonstrated that the mouse retrovirus MMTV relies on intestinalmicrobiota for transmission (Kane et al., Science, 2011). Therefore, entericviruses from three different families (Picornaviridae, Reoviridae, andRetroviridae) benefit from intestinal bacteria, suggesting that other entericviruses may also require bacteria for optimal infection. The microbiotamediatedenhanced replication and pathogenesis of these viruses couldoccur through mechanisms affecting the host and/or virus. Although we areexamining host and virion effects for poliovirus and reovirus, we initiallyexplored potential microbe-dependent poliovirus virion alteration. Usingin vitro assays, we found that exposure to bacterial surface polysaccharides,including lipopolysaccharide (LPS) and peptidoglycan, enhancedpoliovirus stability and cell attachment/viral receptor binding, providingone mechanism by which intestinal microbiota promote enteric picornavirusinfection. Using LPS as a model bacterial polysaccharide, we foundthat poliovirus binds LPS and that the virion stabilization enhancementrequires higher LPS concentrations than the virion cell attachment enhancement.Additionally, we have identified a poliovirus mutant, VP1-T99K,with reduced LPS binding. We determined that VP1-T99K poliovirusbinds enough LPS to enhance cell attachment, but not enough to stabilizeVP1-T99K virions. Therefore, cell attachment enhancement and virionstability enhancement by bacterial polysaccharides are separable by thepolysaccharide concentration required and by a viral mutant. VP1-T99Kpoliovirus does not have replication or pathogenesis defects in vitro orin vivo, likely because virion stability is not a major selective pressureover the short time interval preceding the first cycle of viral replicationin the gut. However, due to virion instability in feces from lack ofmicrobiota-mediated stabilization, VP1-T99K has a transmission defectin mice. Taken together, these data suggest a model where picornavirusvirions bind bacterial surface polysaccharides, enhancing cell attachmentto promote infection and enhancing environmental stability to promotetransmission to a new host.11:30 – 12:00Ecology of viruses of endophytic fungiMarilyn ROOSSINCKThe Pennsylvania State University, Department of Plant Pathology andEnvironmental Microbiology, Center for Infectious Disease Dynamics,University Park, PA 16802, USAFungi that colonize plants (endophytes) often confer important benefits toplants. When plants grow in extreme environments fungi can be criticalfor plant survival. However, fungi don’t always confer these benefits bythemselves, sometimes viruses are required. We have found that a virus isrequired in a three-way mutualistic symbiosis (virus-fungus-plant) fromYellowstone National Park (YNP), where plants grow in high temperaturegeothermal soils. The virus, Curvularia thermal tolerance virus (CThTV),was isolated from a number of hot spring areas in YNP, and the diversityof the virus suggests that this relationship is longstanding in this environment.In other extreme environments, such as tidal zones along the Pacificocean, plants also survive with the help of endophytic fungi. In these systemswe also find viruses that are correlated with extreme adaptation. Theextreme diversity of viruses (largely unexplored), may be exploited by viralhosts to provide novel genetic information that is critical in environmentalchanges.12:00 – 12:30Leishmania RNA virus—a backseat driver to metastatic leishmaniasisNicolas FASELDepartment of Biochemistry, University of Lausanne, Epalinges, Vaud,SWITZERLANDLeishmaniases are diverse group of protozoan parasite infections rangingfrom cutaneous (CL) to visceralizing disease (VL) as well as complicatedmetastatic forms such as mucocutaneous (MCL) and disseminated leishmaniasis(DCL). These clinical variations are associated to leishmanialspeciation but the parasitic determinants of these outcomes are poorlydescribed. Our lab has linked the occurrence of metastatic disease to thepresence of a cytoplasmic dsRNA virus (LRV) in a subgenus of Leishmania.Here, viral RNA potently activates the host innate immune system viaToll-like Receptor 3 (TLR3), triggering a destructive hyper-inflammatorycascade. Using L. guyanensis clones that are either naturally infected byLRV (LRV+) or depleted in it (LRV-), we characterized this chronic inflammationin a murine model. Through TLR3 in macrophages, LRV+ potentlyinduced the secretion of IFN-beta and using mice deficient in type-I IFNreceptor, we showed that IFN-beta has a deleterious role in establishingthe severity of an infection caused by virus-carrying parasites. Together,these data confirm the importance of IFN-beta in TLR3-dependentpathogenesis.We also investigated other innate immune response modulators, amongstwhich, we demonstrated that both MyD88 and TLR9 play a crucialrole in the development of protective responses to Leishmania infectionregardless of their LRV status. Given the potential application forTLR-ligands to act as adjuvants in vaccination strategies, our studies providemuch-needed information to tailor the therapeutic use of adjuvants inleishmaniasis.12:30 – 13:00Wolbachia and arbovirus inhibition in mosquitoesSteven SINKINS 1 , Marcus BLAGROVE 1 , Sofia PINTO 1 , KirstySTAINTON 1 , Jenny MOLLOY 1 , Joshua BLIGHT 1 , Camilo AriasGOETA 2 , Anna-Bella FAILLOUX 21 University of Oxford, Peter Medawar Building for Pathogen Research,UK; 2 Institut Pasteur, Paris, FRANCEThe maternally inherited intracellular bacterium Wolbachia pipientis iscapable of rapid spread through insect populations using cytoplasmicincompatibility (CI), crossing sterilities that can give a reproductive advantageto females carrying the bacterium. Several studies have demonstratedthat the presence of certain strains of Wolbachia in mosquitoes can stronglyinhibit the transmission of several important arboviruses of humans. Inthe invasive mosquito Aedes albopictus, we have created a transinfectionS56 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologywith the wMel strain of Wolbachia from Drosophila, and shown that it canblock the transmission of dengue and chikungunya viruses. BidirectionalCI is generated with wildtype populations, which provides a mechanismfor introducing this strain to high frequency in natural populations, andthere were no major fitness costs detected in lab assays. The effects ofintroduced and naturally-occurring Wolbachia strains on the transcriptionof various mosquito genes will be presented, in stable mosquito lines andin several mosquito cell lines transinfected with various strains of Wolbachia,focusing on interactions with the mosquito immune system andseveral other pathways. A correlation between Wolbachia strain densitydifferences and virus inhibition will be discussed. These studies may helpunravel the mechanisms of virus inhibition in mosquitoes.Virologie, Vol 17, supplément 2, septembre 2013S57

5 th European Congress of VirologyThursday 12 th September 2013,13h30 – 14h45SYMPOSIUM BIOMERIEUX Molecular ScientificChair: Christine C. GINOCCHIO (Lake Success, USA)Room Prestige Gratte-CielErythrovirus B19 infections in Immunocompromised patientsMarianne LERUEZParis, FRANCEEvaluation of Parvovirus B19 R-gene qPCR assay for ErythrovirusB19 detection and quantification in Blood and Bone Marrow samplesAurélie SCHNURIGERParis, FRANCEHow molecular methods have improved Adenoviruses diagnosis.Monitoring in bone marrow or stem cell transplant recipientsMarcela ECHAVARRIABuenos Aires, ARGENTINAGoals and Potential impact of a complete Molecular Laboratory Automationfor virus detectionFlorence MORFINLyon, FRANCES58 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyThursday 12 th September 2013,15h00 – 17h15WORKSHOP 4: “ADAPTIVE IMMUNITY AND VACCINES”Chairpersons:Ben BERKHOUT (Amsterdam, THE NETHERLAND)& Daniel PINSCHEWER (Geneva, SWITZERLAND)Room Prestige Gratte-CielKEYNOTE:Increasing vaccine immunogenicity via manipulation of vaccinia virusBcl-2-like immunomodulatory proteinsGeoffrey L. SMITH 1 , Hongwei REN 1 , Rebecca P. SUMNER 1 , CarlosMALUQUER DE MOTES 1 , Daniel S. MANSUR 2 , Camilla T.O.BENFIELD 1 , Brian J. FERGUSON 11 Department of Pathology, University of Cambridge, Tennis Court Road,Cambridge CB2 1QP, UNITED KINGDOM; 2 Department of Virology,Faculty of Medicine, Imperial College London, St Mary’s Campus, NorfolkPlace, London W2 1PG, UNITED KINGDOMVaccinia virus (VACV) is the live vaccine used to eradicate smallpox.VACV replicates in the cytoplasm, has a dsDNA genome and encodesmany proteins that block the innate immune response to virus infection.These proteins include a family of proteins with structural similarity to B-cell lymphoma protein 2 (Bcl-2): proteins N1, B14, A52, K7 and F1 havehad their structures solved and proteins N2, A46, C6 and B22/C16 arepredicted to adopt a Bcl-2-like fold. Only proteins F1 and N1 have beendescribed as anti-apoptotic, and instead this family of proteins inhibitssignalling cascades that activate innate immunity. By deleting or mutatingthese proteins, the virulence and immunogenicity of VACV can be altered.Evidence will be presented showing that deletion or mutation of proteinsN1, C6 and K7 causes a reduction in virulence but an increase in virusimmunogenicity. This results in better protection against VACV challengethat is mediated by VACV-specific CD8+ cytotoxic T-cell or natural killer(NK) cell activity.show that CPXV012 inhibits ATP binding to TAP, thereby removing theenergy necessary to drive peptide transport. Using CPXV012 mutants,we identified the key amino acids of CPXV012 that are crucial for bothbinding to and inhibition of TAP. These residues have a strong affinity forphospholipids mimicking the ER membrane, and this property correlates toTAP inhibition. These findings provide insights into protein characteristicsrequired for TAP inhibition by CPXV012, the first viral TAP inhibitoridentified outside the herpesvirus family.REF O50The role of antigen avidity in memory inflation upon MCMV infectionLisa BORKNER 1 , Iryna DEKHTIARENKO 1 , Barbara ADLER 2 , LukaCICIN SAIN 11 Helmholtz Centre for Infection Research, Braunschweig, GERMANY;2 Max von Pettenkofer Institute, München, GERMANYHuman cytomegalovirus (HCMV) induces a strong immune response, withT cells specific for certain epitopes accumulating over time. As CMV isstrictly species specific, murine cytomegalovirus (MCMV) provides anin vivo model for HCMV. MCMV elicits two types of CD8 responses:(1) the conventional response with expansion followed by contractionand memory phase, or (2) the inflationary response, which consists ofan ongoing expansion of effector memory T cells which never contract. Itis not clear whether the difference in immune kinetics for certain epitopesis due to the context of their expression, or due to the nature of the peptide.We recently showed that introducing the herpes simplex virus 1 epitopeSSIEFARL (SL) in context of ie2 (memory inflation) and M45 (conventionalresponse) in MCMV resulted in divergent immune kinetics of the SLspecific CD8 T cell response arguing that inflationary responses depend onthe context of gene expression [I. Dekhtiarenko et al., J Immunol 190 (7)].We address here the question whether the nature of the peptide also playsa role. MCMV mutants containing the low avidity Y chromosome peptideKCSRNRQYL (KNL) in the context of ie2 and M45 have been createdand their CD8 response was compared to the MCMV mutants carrying thehigh avidity peptide SL. While the initial response against KNL was lowerthan that against SL, we still observed an accumulation of KNL specificCD8 T cells when the peptide was inserted in the inflationary epitope.Hence, our data argue that peptide avidity may also contribute to the sizeof the inflationary response.ORAL COMMUNICATIONSREF O49Cowpox virus protein CPXV012 avoids MHC I antigen presentationby blocking ATP binding to TAPRutger LUTEIJN 1 , Wouter VAN LEEUWEN 1 , Daniëlle HORST 1 ,Hanneke HOELEN 1 , Robert Jan LEBBINK 1 , Klaus FRÜH 2 , JacquesNEEFJES 3 , Maaike RESSING 1 , Emmanuel WIERTZ 11 University Medical Center, Utrecht, THE NETHERLANDS; 2 Vaccineand Gene Therapy Institute, Beaverton, UNITED STATES OF AMERICA;3 The Netherlands Cancer Institute, Amsterdam, THE NETHERLANDSViral antigen presentation by MHC class I molecules is crucial for theactivation of antiviral immune responses by patrolling cytotoxic T cells. Toavoid T cell detection, the MHC I antigen presentation pathway is targetedby many DNA viruses. For example, various herpesviruses interfere withthe supply of peptides necessary for the assembly of MHC I by inhibitingthe Transporter associated with Antigen Processing (TAP). TAP is also atarget of the recently identified cowpox viral protein CPXV12. This 9kDatype II transmembrane protein inhibits TAP mediated peptide transportthrough an unidentified mechanism. Here, we investigated the mechanismof TAP inhibition and the protein domains responsible for this effect. WeREF O51Specificity and predictability of CD4+ T cell responses after tick borneencephalitis virus infection and vaccination are strongly influenced bystructural features of viral proteinsJulia SCHWAIGER 1 , Judith H. ABERLE 1 , Karin STIASNY 1 , BernhardKNAPP 2 , Wolfgang SCHREINER 2 , Gottfried FISCHER 3 , OndrejSCHEINOST 4 , Vaclav CHMELIK 5 , Franz X. HEINZ 11 Department of Virology, Medical University of Vienna, Vienna, AUS-TRIA; 2 Section of Biosimulation and Bioinformatics, Medical Universityof Vienna, Vienna, AUSTRIA; 3 Department of Blood Group Serology andTransfusion Medicine, Medical University of Vienna, Vienna, AUSTRIA;4 Laboratory of Molecular Biology and Genetics, Hospital Ceske Budejovice,Ceske Budejovice, CZECH REPUBLIC; 5 Department of InfectiousDiseases, Hospital Ceske Budejovice, Ceske Budejovice, CZECH REPU-BLICStrong antibody responses by B cells require direct interaction with CD4+T cells which recognize MHCII associated epitopes of proteins internalizedby the B cell after Ig receptor recognition. We determined the helperT cell response after tick borne encephalitis (TBE) virus infection andvaccination of humans by analyzing overlapping peptides of the structuralproteins (C – capsid, prM/M – precursor/membrane,E–envelope) which– since the whole virus particle is internalized by the B cell – can allVirologie, Vol 17, supplément 2, septembre 2013S59

5 th European Congress of Virologypotentially contribute to the formation of neutralizing antibodies directedto E.The specificity of CD4+ T cells was analyzed using an IL 2 ELISpot assayand these experimental results were compared with epitope predictions forthe HLA class II alleles (DR, DP, DQ) of each individual.Our results show that the CD4+ T cell response to peptides of the C proteinis overrepresented after both, infection and vaccination. Despite strongindividual variation, patterns of immunodominance were identified whichare linked to structural features of the proteins (e.g. alpha helices or betasheets). The correlation between predicted and experimentally determinedepitopes differed strongly between the structural proteins. As many as 80%of the experimental epitopes in C but only less than 40% in prM/M and Ewere predicted by algorithms.In conclusion, there is evidence that protein specific structural featureshave a strong influence on epitope usage and on the predictability of suchepitopes by computer algorithms.REF O52A new lentiviral vector tool to evaluate measles virus escape frominfection- or vaccine-induced antibodiesCamille LEVY, Fouzia AMIRACHE, Caroline COSTA, CeciliaFRECHA, Claude P. MULLER, Hasan KWEDER, Robin BUCKLAND,François-Loïc COSSET, Els VERHOEYENCIRI, EVIR team, 69007, Lyon, FRANCELive measles virus (MV) vaccines are currently used worldwide and arehighly successful but MV still causes 4% of all child deaths worldwide.Importantly, circulating MVs give rize to MV strains mutated in differentepitopes of the MV envelope glycoproteins (gps), which is underlined byreoccuring MV outbreaks in the last years. Thus the risk exists that thevirus acquires multiple mutations allowing it to escape from vaccination.To evaluate this risk we engineered a lentiviral vector (LV) displayingat its surface the MV envelope gps H and F (H/F-LVs). These H/F-LVsretained the same target cell specificity as MV for human T, B and dendriticcells. Since these H/F-LVs encode for GFP, their neutralization by MVantibodies is detected by reduction of % of GFP+ target cells. Indeed,insertion of mutations in the Noose and NE major epitopes of the Hgp inthe H/F-LVs confirmed escape from NE and Noose specific antibodies butnot from antibodies in sera from MV vaccinated donors.Other emerging strains such as the MV-D genotypes are less sensitive toMV antibodies induced by vaccination. We attributed this to epitope shieldingby an extra glycosylation site, D416N, present in these MV D-cladestrains since introduction of this mutation into the H/F-LVs revealed lessneutralization by MV-specific antibodies. Finally, when this glycosylationsite was introduced into the H/F-LVs already mutated for the major epitopes,escape from MV-antibody neutralization was increased. Thus, thisvector tool might allow epitope-monitoring, invaluable in the surveillanceof measles epidemics.REF O53Design of Single Round Infection Alphaviruses: New Approaches ofVaccine DevelopmentIlya FROLOV 1 , Dal Young KIM 1 , Svetlana ATASHEVA 1 , EryuWANG 2 , Scott WEAVER 2 , Elena FROLOVA 11 University of Alabama at Birmingham, Birmingham, USA; 2 Universityof Texas Medical Branch, Galveston, USAThe Alphavirus genus in the Togaviridae family contains a number ofhuman and animal pathogens. The importance of alphaviruses has beenstrongly underappreciated; however, recent epidemics of chikungunyavirus has raised their profile. In spite of a continuous public health threat,to date no licensed vaccines have been developed to any alphavirus infections.The currently available experimental live vaccines and inactivatedviruses suffer from traditional draw backs, such as reactogenicity or lowefficiency and short lived immune response. We have applied an accumulatedknowledge about the mechanism of alphavirus replication and proteinfunction in virus host interactions to introduce a number of new approachesin designing attenuated variants. The artificially designed alphavirusesare constructed from genes derived from different, geographically isolatedviruses. They lack the important contributors to viral pathogenesis:proteins functioning in inhibition of the innate immune response, causeonly a single round infection in vivo and do not induce detectable diseaseeven in immunocompromized animals. Nevertheless, these mutants closelymimic natural viral infection in terms of RNA replication, productionof viral proteins and release of viral particles. Such alphaviruses alsoinduce a highly efficient, protective immune response. An additionalimportant characteristic of the recombinant viruses is their inability toreplicate in mosquito vectors. These new approaches are applicable for theconstruction of alphaviruses with a programmed, irreversibly attenuatedphenotype.REF O54Respiratory vaccination with live attenuated measles virus: studiestowards identification of the optimal site of vaccine deliveryRik DE SWART 1 , Rory D. DE VRIES 1 , Linda J. RENNICK 2 , GeertVAN AMERONGEN 1 , Stephen MCQUAID 3 , Selma YÜKSEL 1 ,R.Joyce VERBURGH 1 , Martin LUDLOW 2 , Albert D.M.E.OSTERHAUS 1 , W. Paul DUPREX 21 Erasmus MC, dept. Viroscience, Rotterdam, THE NETHERLANDS;2 Boston University, dept. Microbiology, Boston, USA; 3 Queen’s Universityof Belfast, Tissue Pathology, Belfast, NORTHERN IRELANDThe standard route of measles virus (MV) vaccination is subcutaneousinjection, which is far from the natural route of wild type MV transmission.Programs evaluating alternative vaccination routes have been hampered bya lack of knowledge about the primary cells that should be targeted. Theaim of our study was to determine whether MV vaccination via the respiratoryroute should target the upper or lower respiratory tract (LRT). Wegenerated a recombinant virus based on the Edmonston Zagreb (EZ) strain,expressing enhanced green fluorescent protein (EGFP) from an additionaltranscription unit in position 3 of the genome. The virus was grownin MRC 5 cells and formulated with the same stabilizers and excipientsused in the MV EZ vaccine produced by the Serum Institute of India.Four groups of twelve macaques were immunized with a dose of 10ˆ4TCID50 rMV EZ EGFP(3) via different routes of administration: injection,intra tracheal inoculation, intra nasal instillation or aerosol inhalation.In each group six animals were euthanized at early time points, whereasthe other six were followed up to assess immunogenicity. Infected cellswere detected in the muscle, nose and lungs, but systemic MV replicationwas virtually absent. Macrophages and dendritic cells were the predominanttarget cells in all cases. Animals vaccinated via the respiratory routehad the highest specific serum antibody when the virus was delivered tothe LRT. This study sheds new light on the tropism of live attenuatedMV vaccine, and identifies the LRT as the optimal target for respiratorydelivery.S60 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF O55Comparison of neutralizing and cross neutralizing antibodies inducedby Cervarix TM and Gardasil ® Human Papillomavirus ProphylacticVaccines: an independent studyLaura SQUARZON 1 , Monia PACENTI 2 , Serena MASIERO 1 , GiorgiaMARCATI 2 , Barbara MANTELLI 1 , Lorena GOTTARDELLO 3 , LilianaGABRIELLI 4 , Tiziana LAZZAROTTO 4 , Antonella CAPUTO 1,2 , LuisaBARZON 1,2 , Giorgio PALÙ 1,21 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Clinical Microbiology and Virology, Padova University Hospital,Padova, ITALY; 3 Department of Hygiene and Public Health, ULSS 16,Padova, ITALY; 4 Deaprtment of Specialty, Diagnostic and ExperimentalMedicine, University of Bologna, Bologna, ITALYAim of this independent study was to compare neutralizing antibodies(NAbs) and cross NAbs induced by Gardasil ® and Cervarix TM HPV prophylacticvaccines. The study population included consecutive adolescentgirls from Veneto and Emilia Romagna Regions, Italy, where the quadrivalentHPV16/18/6/11 vaccine Gardasil ® and the bivalent HPV16/18 vaccineCervarix TM , respectively, were offered by the public health system. HPVtype specific NAbs were measured at 1 6 months after completion ofthe third dose of vaccine by using the pseudovirion based neutralizationassay (PBNA) with HPV type 16, 18, 31, 45, 52, 58 pseudovirions. Thepresence of NAbs was defined by a titer of 1:40 or higher, according toWHO guidelines. Preliminary results were obtained from a group of 146girls aged 11 13 years vaccinated with Gardasil ® (n=90) or Cervarix TM(n=56). At 1 6 months after vaccination, 100% of Gardasil ® vaccinees hadHPV16 NAbs and 98.9% had HPV18 NAbs, while 100% of Cervarix TMvaccinees had both HPV16 and HPV18 NAbs. The titer of HPV16 andHPV18 NAbs was significantly higher (ten fold difference) in girls vaccinatedwith Cervarix TM than in girls vaccinated with Gardasil ® . Crossneutralization assays were performed in a subgroup of vaccinees. Lowlevel (titer 1:40) HPV31 NAbs were detected in 50% of subjects vaccinatedwith Gardasil ® , while 100% of subjects vaccinated with Cervarix TMhad HPV31 NAbs against (titer 1:40 to 1:2,560). Some girls vaccinatedwith Cervarix TM had low titer HPV45 and HPV58 NAbs. In conclusion,Cervarix TM induces higher titers of NAbs and cross NAbs than Gardasil ® .Virologie, Vol 17, supplément 2, septembre 2013S61

5 th European Congress of VirologyThursday 12 th September 2013,15h00 – 17h15WORKSHOP 14: “VIRUS STRUCTURE, DYNAMICIMAGING AND TRAFFICKING”KEYNOTE:Chairpersons: Paul DUPREX (Boston, USA)& François PENIN (Lyon, FRANCE)Room Tête d’OrParamyxoviral ultrastructures revealed by electron cryotomographyLassi LILJEROOS 1 , Juha T. HUISKONEN 2 , Magdalena A.KRZYZANIAK 4 , Ari ORA 1 , Petri SUSI 3 , Ari HELENIUS 4 and SarahJ. BUTCHER 11 Institute of Biotechnology, University of Helsinki, 00790, Helsinki, FIN-LAND; 2 Oxford Particle Imaging Centre, Division of Structural Biology,Wellcome Trust Centre for Human Genetics, University of Oxford, OxfordOX3 7BN, UNITED KINGDOM; 3 Department of Virology, University ofTurku, 20520, Turku, Finland; 4 Institute of Biochemistry, ETH Zurich,CH-8092 Zurich, SWITZERLANDParamyxoviruses include several significant human pathogens, likemeasles, mumps and respiratory syncytial viruses. Due to the highly pleomorphicnature of these viruses, three-dimensional whole-virus structureshave been missing until recently. Electron cryotomography is a suitablemethod to undertake such structural studies and reveal details of the componentsconstructing the virion. We have applied electron cryotomographytogether with subtomographic alignment and averaging to characterize theultrastructures of measles and respiratory syncytial viruses. We show thatthese two related viruses have significant differences in the way the matrixprotein is organized within the virion. We also discuss the implications ofthese differences on virion assembly.ORAL COMMUNICATIONSREF O56Real time cell biological analysis of vaccinia replicationN. Bishara MARZOOK, Timothy NEWSOME, ChristopherMCKENZIE, Helena LYNN, Dean PROCTER, JacquelynHORSINGTONSchool of Molecular Bioscience/University of Sydney, Sydney, AUSTRA-LIAThe fluorescent labeling of viruses is an important tool in the study of hostpathogen relationships. Vaccinia virus, the live vaccine used in the eradicationof smallpox, has proven particularly amenable to live cell microscopyowing to the ease with which it able to be engineered at the genome level.We report here a novel method that greatly reduces the number of stepsrequired in the process of creating fluorescent recombinant viruses. Thistechnique combines the ability of successful homologous recombinationoccurring using relatively short regions of homology with the principle oftransient dominant selection (TDS) employing selectable metabolic andfluorescent markers. We believe this technique provides a relatively rapid,robust and reliable method of fluorescently labeling viral proteins of interestor different cellular structures in a modular manner. Double or triplelabeled recombinant viruses can also be generated by co infection andpurification based on fluorescence. We will present our latest fluorescentviruses that can be used to visualise a range of host and viral structuresduring virus replication.REF O57Intracytoplasmic transport of hepatitis B virus capsids and their dissociationfrom microtubuleQuentin OSSEMAN 1 , Birgit RABE 2 , Aurelia CASSANY 1 , HaraldWODRICH 1 , Elodie BERDANCE 1 , Dominique BEGU 1 , IrinaSOWINSKEYA 3 , Paul PUMPENS 3 , Andris DISEHLER 3 , MichaelKANN 11 institute of Fundamental Microbiology and Pathgenicity (CNRS UMR5234), Bordeaux, FRANCE; 2 Institute of Medical Virology, Giessen, GER-MANY; 3 Latvian Biomedical Research and Study Centre, Riga, LATVIAHepatitis B virus (HBV) is a pararetrovirus which needs the transcriptionmachinery of the cell nucleus for replication. The virus thus depends on thetransport its genome from the cell periphery to the nuclear envelope. Theretrograde intracytoplasmic trafficking is facilitated along microtubules(MT) using motor protein complexes of the dynein family, which composesof 14 protein chains.We showed by lipofection of capsids that they transport the HBV genometo the nuclear pore leading to release of the genome exclusively into thenucleus. Intracytoplasmic transport was dependent upon intact MT.Using the different dynein light and intermediate chains expressed inE. coli we identified one light chain (DYNLL1, member of the LC8 family)specifically interacting with mature capsids only. DYNLL1 differs fromDYNLL2, which did not interact with the capsids, by six amino acids only.Single directed mutagenesis on each amino acid show that in fact L29 andH41 were important for direct interaction between capsids and DYNLL1.DYNLL1, also called hub protein, is known to interact with 66 cellularpartners via a common short linear motif which also interacts with thedynein intermediate chains (DYNIC). The binding of these interactionpartners is thus incompatible with a function in intracytoplasmic transportas their binding is competitive to dynein binding. We thus propose thatHBV capsid binding to DYNLL1 occurs in addition to DYNLL1 DYNDICinteraction, which would be the first example of a cargo attachment todynein via DYNLL1.REF O58Tracking genomes of DNA viruses in the cytosol and nucleus by clickchemistry and super resolution microscopyI. Hsuan WANG 1 , Maarit SUOMALAINEN 1 , Samuel KILCHER 2 ,Jason MERCER 2 , Anne NEEF 3 , Nathan LUEDTKE 3 , Urs F. GREBER 11 Institute of Molecular Life Sciences, University of Zurich, Zurich,SWITZERLAND; 2 Institute of Biochemistry, ETH Zurich, Zurich, SWIT-ZERLAND; 3 Institute of Organic Chemistry, University of Zurich, Zurich,SWITZERLANDHow viral genomes access the cytosol or nucleus, or are destroyed ispoorly known, largely because they have been difficult to track. Herewe used metabolic labelling and click chemistry to track the genomesof three families of DNA viruses, adenovirus, vaccinia virus and herpesvirus. The nucleosides 7 deaza 7 ethynyl 2 ′ deoxyadenosine (EdA) and2 ′ deoxy 5 ethynylcytidine (EdC) incorporated into adenovirus and herpesvirusreplication sites, as demonstrated by copper(I) catalyzed azide –alkyne (click) reactions with fluorescent azides, and gave normal adenovirusyields, unlike 5 ethynyl 2’ deoxyuridine (EdU). EdA and EdC alsointegrated into adenovirus particles. EdU but not (2 ′ 5) 2 ′ deoxy 2 ′ fluoro5 ethynyluridine (F ara EdU) was readily incorporated into vaccinia virusreplication centers and virions. In extracellular adenovirus, ethynyl taggedDNA was fully protected, but accessible to azide fluorophores in cytosolicviruses, as shown by g STED super resolution microscopy. Aftervirus disassembly at the nuclear pore complex, capsid free viral DNAwas found in the nucleus, but also in the cytosol suggesting that nuclearimport of viral DNA is a bottleneck. Our results show that click chemistrycompatible nucleoside analogues give fully infectious virus particles, andsuperior resolution and sensitivity in detecting single viral DNA genomesS62 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologythan in situ hybridization with fluorescent probes. Unlike in situ hybridization,it is fully compatible with immune cytochemistry procedures forantigen detection, and useful for studies of anti viral innate immunity.REF O59Dengue Virus Requires KDEL Receptor to Exit From the ERPei Gang WANG 1 , Ming Yuan LI 1 , Kevin KWOK 1 , Lewis SIU 1 ,Mateusz KUDELKO 1,2 , Roberto BRUZZONE 11 HKU Pasteur Research Pole, School of Public Health, The University ofHong Kong, HONG KONG; 2 Department of Biochemistry, The Universityof Hong Kong, HONG KONGNascent dengue viruses (DENV) assemble at the endoplasmic reticulum(ER) and traffic through the secretory pathway for maturation before beingreleased from infected cells. It was thought that intracellular DENV transportationfollowed the constitutive pathway. However, our recent work hasrevealed that DENV secretion is a more complex process that can be disruptedby depleting cells of class II ADP ribosylation factors (Arfs) withoutaffecting constitutive secretion (Kudelko et al., JBC 2012). To furthercharacterize the mechanism by which class II Arfs are involved, we nowdemonstrate using dengue recombinant subviral particles (RSPs), whichhave been shown to be a safe and convenient tool to mimic the secretionand maturation of DENV, that secretion requires the involvement of KDELreceptor (KDELR), a transmembrane protein cycling between ER andGolgi apparatus. Thus, depletion of KDELR by siRNA reduces secretion ofdengue RSPs and, using co immunoprecipitation, we have found evidencethat DENV prM binds to KDELR. This interaction occurs in the ER, as it isabolished by disruption of KDELR re cycling, which leads to its accumulationin stacks of cis Golgi. We have further identified that three positivelycharged amino acids located in the N terminus of prM are key residues forinteraction with KDELR. Using a KDELR binding deficient prME mutant,in which these residues were mutated, we demonstrate that the interactionbetween prM and KDELR does not affect either the budding process ofdengue virus but significantly reduces RSPs accumulation in post ER compartments,such as cis and trans Golgi. In conclusion, our results show thatdengue virus requires KDELR as a receptor to exit from the ER.REF O60Recruitment of the host plant heat shock protein 70 by Tomato yellowleaf curl virus coat protein is required for virus infectionRena GOROVITS, Adi MOSHE, Henryk CZOSNEKInstitute of Plant Sciences and Genetics in Agriculture, Rehovot, ISRAELA functional capsid protein (CP) is essential for host plant infection andinsect transmission of Tomato yellow leaf curl virus (TYLCV) and othermonopartite begomoviruses. We have previously shown that TYLCV CPspecifically interacts with the heat shock protein (HSP70) of the virusinsect vector Bemisia tabaci. Here we demonstrate that during TYLCVinfection of plants, CP and HSP70 co localize in aggregates, first in thecytoplasm, then in the nucleus of cells associated with the vascular system.The shift of HSP70 from a soluble protein into insoluble aggregateswas related to TYLCV associated stress. Interaction between HSP70 andCP was found in leaf and sap total protein extracts, as well as in cytoplasmicbut not in nuclear extracts. Inhibition of HSP70 expression byQuercetin caused a decrease in the amount of nuclear CP aggregates andre localization of fusion protein GFP CP from the nucleus to the cytoplasm.The current study reveals for the first time the involvement of hostplant HSP70 in TYLCV CP intracellular movement. As shown earlier,nuclear aggregates contained TYLCV DNA CP complexes and infectiousvirions were required for successful viral accumulation in plants. HSP70localization in TYLCV large nuclear aggregates supports the definitionof these structures as nuclear virus factories. HSP70 inactivation resultedin decrease of TYLCV DNA levels, demonstrating the role of HSP70 inTYLCV multiplication in planta.Virologie, Vol 17, supplément 2, septembre 2013REF 061Murine leukemia virus targets innate like B 1 B cells to establishinfection in miceXaver SEWALD, Christin HERRMANN, Fei LI, Walther MOTHESYale University School of Medicine, Department of Microbial Pathogenesis,New Haven, CT 06536, USARetroviruses are believed to efficiently spread via sites of cell cell contactdesignated virological synapses. This model was developed based onin vitro evidence in which infected cells establish cell cell contact withuninfected cells. Applying intravital microscopy, we recently providedsupport for the existence of virological synapses in vivo. Visualizing cellsinfected with fluorescently labeled murine leukemia virus (MLV) we identifiedB cells that were able to form long lived virological synapses withuninfected lymphocytes (1). In vivo virological synapses were, like theirin vitro counterpart, dependent on the expression of the viral envelopeglycoprotein (Env) and characterized by a prolonged polarization of viralcapsid to the cell – cell interface. B cells were among the first cells tobecome infected by incoming MLV. However, the specific subtype of Bcells that is susceptible to MLV had remained unknown. Here we presentevidence for a critical role of innate like B1 B cells in the establishment ofMLV infection in mice. Adoptively transferred B1 B cells are selectivelytargeted by MLV. Mice lacking B1 B cells are resistant to MLV infection.In addition, using knockout mice we provide evidence for the contributionof adhesion factors expressed by B1 B cells in spreading of retrovirusesin vivo. Thus, our work reveals the critical importance of a distinct Bcell subset in the susceptibility to retroviral infections under physiologicalconditions in vivo.Sewald X, et al. In vivo imaging of virological synapses. Nature Communications2012; 3: 1320.REF O62Biophysical analyses of the central region of HSV1 pUL36Stephane ROCHE, Nathalie SCRIMA, Stephane BRESSANELLILaboratoire de Virologie Moléculaire et Structurale, Gif sur Yvette,FRANCEHerpesvirus such as the type 1 herpes simplex virus (HSV1) have a characteristicfour layer organization. The viral genome is contained in anicosahedral capsid, itself enclosed in a proteinaceous tegument and finallyin a lipid envelope. The tegument probably is the least understood layer sofar. It can be subdivided in two components: the inner tegument comprisesall tegument proteins that strongly interact with the capsid while the outertegument protein can easily be detached from the capsids.Two of the inner tegument protein, pUL36 and pUL37, constitute a complexwith an unknown stoichiometry. They perform multiple functionduring the viral cycle. They are involved in the viral DNA ejection intothe nucleus, in the capsid transport or in the viral assembly. They bindmultiple cellular and viral proteins. The cofactors of pUL36 include thepenton protein pUL25 and the outer tegument protein pUL48, which suggestthat pUL36 could cross over the entire tegument. pUL36 is a giantprotein (337 kDa) and structural data are only available for its N terminaldeubiquitinase domain. As a consequence, the mechanisms by whichpUL36 fulfill its many function remain unknown.We could express an purify in large amounts several fragments of pUL36corresponding to about thousand consecutive aminoacids. Size exclusionand small-angle X-ray scattering analyses suggest that these fragmentsare extremely elongated, which suggest that this segment could constitutean elongated stalk, with the capsid binding domain at one end andthe outer tegument binding domain at the other end. We could alsosolve the crystal structure of one of these fragments. This fragment isan extremely elongated dimer, suggesting that pUL36 itself is at least adimer.S63

5 th European Congress of VirologyThursday 12 th September 2013,15h00 – 17h15WORKSHOPS 18: “EMERGING VIRAL DISEASES INVETERINARY VIROLOGY”Partnership with European Society For Veterinary Virology(ESVV)Chairpersons: Thierry VAN DEN BERG (Uccle, BELGIUM) &Wim VAN DER POEL (Lelystad, THE NETHERLANDS)Room Gratte-Ciel 1, 2, 3KEYNOTE:Emerging diseases in veterinary virologyWim H.M. VAN DER POELCentral Veterinary Institute of Wageningen University and ResearchCentre, Lelystad, THE NETHERLANDSGlobal changes promote the emergence of viruses, in the human field aswell as in the veterinary field. Environmental changes, increased traveland transport of animals and their produce provide increasing opportunitiesfor the rapid spread of viruses. Infections acquired from animals,known as zoonoses, are a major source of infectious disease and pose arisk to public health. Two very different examples of emerging virusesin the veterinary field are hepatitis E virus, family Hepeviruses, whichhas recently been identified as a zoonotic virus and now is detected in anincreasing number of animal species, and Schmallenberg virus, a vectorborne Orthobunyavirus which was identified in cattle and sheep in 2011and spread over Europe and eastwards subsequently. Introductions of previouslyunknown viruses can be an important threat to animal health andpublic health, and it is likely that the trend for increased viral disease emergencewill continue. Strategies to improve veterinary and public healthprotection with regard to emerging viruses have focused towards improvedsurveillance, improved detection of viruses in reservoirs, early diseaseoutbreak detection, and broadly based research to clarify important factorsthat favour (re-)emergence. National and International cooperation isneeded to early identify and control new and emerging viruses. The OneHealth approach, involving inclusive collaborations between physicians,veterinarians and other health and environmental professionals, will bemore and more important to combat emerging viral disease outbreaks.ORAL COMMUNICATIONSREF O63Avian Influenza Virus Hemagglutinins H2, H4, H8, and H14 supporta highly pathogenic phenotypeJuergen STECH, Olga STECH, Jutta VEITS, El Sayed ABDELWHAB,Ute WESSELS, Siegfried WEBER, Angele BREITHAUPT, MarcusGRÄBER, Sandra GOHRBANDT, Jessica BOGS, Jens P. TEIFKE,Thomas C. METTENLEITERFriedrich Loeffler Institut, Greifswald Insel Riems, GERMANYHighly pathogenic avian influenza viruses (HPAIV) evolve from lowpathogenic precursors with hemagglutinin (HA) serotypes H5 or H7 byacquisition of a polybasic HA cleavage site (HACS). Since the reason ofthis serotype restriction is still unclear, we aimed to distinguish betweencompatibility of a polybasic HACS with H5/H7 HA only and unique predispositionof H5/H7 for insertion mutations. Engineered polybasic HACSmutants from several low pathogenic avian strains (LPAIV) of serotypesH5N1, H3N8, H9N2, and H4N6 did not exhibit high virulence in chicken.Therefore, we generated HA reassortants by introducing a polybasicHACS into the HA of several LPAIV with serotypes H1, H2, H3, H4, H6,H8, H10, H11, H14 or H15, and co transfection with either H9N2 LPAIVor H5N1 HPAIV genes. Reassortants containing the engineered H2, H4,H8 or H14 in the HPAIV background were lethal and exhibited intravenouspathogenicity indices of 2.79, 2.37, 2.85, and 2.61 respectively, equivalentto natural HPAIV. Thus, in case of a polybasic HACS, nonH5/H7HA can enable high virulence in the HPAIV background. Then, we furthermapped those HPAIV virulence determinants by generating severalreassortants from a H5N1 LPAIV and a HPAIV strain and found that theHPAIV HA and NA alone enable 100% lethality and efficient transmissionto contact chickens. Thus, beside an HA with polybasic HACS, the NAis a second essential virulence determinant of H5N1 HPAIV in chicken.Overall, the restriction of natural HPAIV to H5 and H7 is likely due to aunique predisposition for acquisition of a polybasic HACS.REF O64Vaccination of chickens with neuraminidase recombinant virus repliconparticles confers protection against avian influenza virusGert ZIMMER, Meret RICKLIN, Stefan HALBHERRInstitute of Virology and Immunology (IVI), Mittelhäusern, SWITZER-LANDThe immune response to common influenza virus vaccines is dominatedby antibodies directed to hemagglutinin (HA), the major antigen of theviral envelope. In contrast, the role of the neuraminidase (NA) antigen inimmune protection is not well understood. NA acts as receptor destroyingenzyme and plays an important role in virus release from infected cells.In addition, NA may play a role in virus entry. Yet, the potential of NA asvaccine antigen has not been fully elucidated.In order to study the role of NA antibodies in immune protection, chickenswere immunized with recombinant RNA replicon particles expressing NAof subtypes N1 or N7. The immune sera specifically bound to NA and inhibitedhemagglutination by influenza viruses in a subtype specific manner.In addition, NA specific antibodies neutralized virus and protected cellsfrom infection in vitro. NA antibodies also inhibited spread of highlypathogenic avian influenza virus in a plaque size reduction assay more efficientlythan HA antibodies. Following challenge of immunized chickenswith low pathogenic avian influenza virus, serum markers of inflammationwere significantly reduced and virus shedding via the cloacal routewas completely prevented. These data suggest that NA antibodies protectchickens against avian influenza viruses by interfering with both virusentry and release. NA should therefore be considered as vaccine antigen infuture. The capacity of NA recombinant virus replicon particles to protectchickens against highly pathogenic avian influenza viruses is currentlyunder investigation.REF O65Influenza A virus infection dynamics in permanently infected pigfarms: evidence of recurrent infections, co circulations of differentvirus subtypes and reassortment eventsGaëlle SIMON 1,3 , Nicolas ROSE 2,3 , Séverine HERVÉ 1,3 , EricEVENO 2,3 , Nicolas BARBIER 1,3 , Florent EONO 2,3 , VirginieDORENLOR 2,3 , Mathieu ANDRAUD 2,3 , Claire CAMSUSOU 2,3 ,François MADEC 2,31 Anses, Laboratoire de Ploufragan Plouzané, Unité Virologie ImmunologiePorcines, Ploufragan, FRANCE; 2 Unité Epidémiologie et BienEtre Porcin, Ploufragan, FRANCE; 3 Université Européenne de Bretagne,FRANCES64 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyRepeated infections by different Influenza A virus subtypes within pigfarms increase the risk of emergence of novel reassortant viruses. Theobjectives of this study were to characterise the epidemiology of recurrentinfections with swine influenza viruses for identification of main determinants.A follow up study was carried out in 3 selected farms known tobe affected by repeated Influenza infections. Three batches of pigs werefollowed within each farm from birth to slaughter, through a representativesample of 40 piglets per batch. Piglets were monitored individuallyon a monthly basis for serology and clinical parameters. Daily virologicaland clinical investigations took place for two weeks when a fluoutbreak occurred. Influenza outbreaks, confirmed by Influenza A virusdetection, were reported at least once in each followed batch. These outbreaksoccurred at constant ages within farms and were correlated withan increase in the frequency of sneezing and coughing fits. H1N1 andH1N2 viruses from European enzootic subtypes as well as reassortantviruses between these lineages were identified consecutively and sometimessimultaneously according to studied batches, suggesting virus cocirculations at the farm, batch and sometimes individual levels. The estimatedreproduction rate R of Influenza outbreaks ranged between 2.5 [1.92.9] and 6.9 [4.1 10.5] according to age at infection time and serologicalstatus of infected piglets. Duration of shedding was also influenced bythe age at infection time, the serological status of the dam and minglingpractices. An impaired humoral response was identified in piglets infectedat a time they still presented maternally derived antibodies.REF O66Different strategies of host infection by bovine respiratory virusesJana KIRCHHOFF 1 , Sabine UHLENBRUCK 1 , Günther KEIL 2 , GeorgHERRLER 11 Institute of Virology/University of Veterinary Medicine Hannover, Hannover,GERMANY; 2 Friedrich Loeffler Institut, Riems, GERMANYBovine respiratory disease complex (BRDC) is the major cause of seriousrespiratory tract infections in calves. The disease is multifactorial, witheither stress or reduced immunity allowing several pathogens to emerge.We investigated the susceptibility of bovine airway epithelial cells (BAEC)to infection by different viruses belonging to the BRDC: bovine respiratorysyncytial virus (BRSV), bovine herpes virus type 1 (BHV 1) and bovineparainfluenza virus type 3 (BPIV 3). For this purpose, two culture systemsfor well differentiated BAEC were established: the air liquid interface(ALI) system, where filter grown BAEC differentiate into a pseudostratifiedrespiratory epithelium and precision cut lung slices (PCLS) whereBAEC are maintained in the original tissue organisation. Comparativeinfection studies demonstrated that BPIV 3 specifically infected ciliatedcells at the apical surface of the epithelium whereas BHV 1 infection wasrestricted to basal cells. In case of ALI cultures, this was achieved bypretreatment of cells with EGTA or injury of the cell layer with a sterileneedle. In contrast, overall infection level of BRSV was low in well differentiatedBAEC. In PCLS, infected cells were predominantly present insubepithelial regions and primary observations suggest that dendritic cellsmay play a role in viral spread. Altogether, these results indicate that thethree viruses of the bovine respiratory disease complex follow differentstrategies to enter primary target cells of the respiratory epithelium.some time in 2011 and subsequently spread across the Netherlands, Belgiumand France into the UK – most likely via midges blown across theChannel, similar to the incursion of Bluetongue in 2007. While SBV isgenetically most closely related to Douglasvirus, Sathuperivirus and Shamondaviruskey features in the pathogenesis from Akabanevirus, such asfetal malformations in ruminants also apply.In January 2012 the first malformed fetuses were observed in sheep, soonfollowed by cattle with cases rapidly spreading across Southern England.Analysis of weather conditions, serology results and sheep gestation times,suggest one or two parallel incursions into England before the end ofSeptember 2011. However, molecular analysis of the M and S gene segmentsfrom both sheep and cattle over the first year was unable to showa pattern consistent with incursion or spread of the virus demonstratinga random nature of changes instead that may reflect the multiple hostchanges.As cases did not cease over summer of 2012 it became evident that SBVremained in the UK, spreading further across England and Wales. Bothserological results of animals naturally infected and the analysis of bullsused for breeding demonstrated that SBV may persist in some animalsat least, which in turn may shed the virus via semen for several monthsfollowing infection.REF O68Modulation of the interferon signalling pathway by bluetongue virusVirginie DOCEUL, Emilie CHAUVEAU, Estelle LARA, StéphanZIENTARA, Damien VITOURUMR 1161 Virologie ANSES INRA ENVA, Maisons Alfort, FRANCEBluetongue virus (BTV) is a double stranded RNA virus belonging to theOrbivirus genus and the Reoviridae family that affects domestic and wildruminants including sheep, cattle and goats. In 2006, a strain of serotype8 of BTV has emerged in the Netherlands and spread to Central and WesternEuropean countries, causing significant economical losses. BTV isa strong inducer of type I interferon (IFN I) both in vivo and in vitro inmultiple cell types derived from various tissues and species. Recently, weidentified RIG I and MDA5 helicases as sensors of BTV infection in nonhematopoietic cells and showed that these molecules displayed antiviralactivities against the virus. As IFN I is detrimental for viral replication,most viruses have evolved mechanisms to counteract the host antiviralresponse triggered by the infection. In this study, we aimed to investigatethe ability of BTV to modulate the induction of IFN I. Using a reporterassay, we found that BTV inhibits IFN promoter activity after stimulationof the RIG I like receptor (RLR) pathway. We identified the BTV nonstructural protein NS3 as a potent inhibitor of IFN synthesis and showedthat the viral protein interferes with the RLR pathway downstreamRIG I and upstream IKKe activation. We are now undertaking furtherstudies to characterise the mechanisms involved in this inhibition. We arealso currently investigating the ability of BTV to interfere with the IFNI response pathway and particularly the JAK/STAT signalling cascade. Abetter understanding of the strategies evolved by BTV to counteract thehost antiviral responses is essential to design new prophylactic agents andcontrol the virus.REF O67Schmallenberg virus – two years after the incursion into the UKFalko STEINBACH, Anna LA ROCCA, Akbar DASTJERDI, TrevorDREW, Christopher FINNEGAN, Julie PEAKE, Sarah MCGOWAN,Sylvia GRIERSON, Bhudipa CHOUDHURYVirology Dept., AHVLA, Addlestone, UNITED KINGDOMSchmallenberg virus (SBV) is a novel Orthobunyavirus first reported inGermany at the end of 2011. The virus incurred into continental EuropeREF O69Study of the virulence of Serotypes 4 and 9 of the Orbivirus Africanhorse sickness virus in two mouse modelsMaria Ana DE LA GRANDIÈRE 1 , William ZONTA 1 ,AxelMAUROY 1 , Fabiana DAL POZZO 2 , Etienne THIRY 11 University of Liège, Faculty of Veterinary Medicine, Veterinary Virologyand Viral Animal Diseases, Liège, BELGIUM; 2 University of Liège,Faculty of Veterinary Medicine,Epidemiology and risk analysis applied toveterinary sciences, Liège, BELGIUMVirologie, Vol 17, supplément 2, septembre 2013S65

5 th European Congress of VirologyAfrican horse sickness (AHSV) is an infectious disease caused by a doublestranded positive RNA virus which belongs to the family Reoviridae, genusOrbivirus. The virus has nine serotypes and is transmitted by a culicoidesbiting midge, principally Culicoides imicola. The establishment of anexperimental model is needed for the investigation of the pathogenesisof this infection.Two mouse models, interferon a receptor knock out mice (A129 KOor IFNAR -/-) and immunocompetent mice (A129 WT), were tested.The viruses used for mice inoculations belonged to the two serotypeswhich caused epidemics in Europe, serotypes 4 and 9. The virus wasinoculated by subcutaneous (SC) route and/or by intra nasal (IN) route.Whole blood samples were taken from each mouse at regular intervals.The organs (liver, spleen, kidney, lung and brain) were taken at theend of the experiment or when the most affected mice were euthanized.All these samples were tested by a qRT PCR targeting AHSV genomesegment 7.Both serotypes of AHSV were detected by qRT PCR until three weeks postinfection in blood of IFNAR / mice and A129 WT mice infected by SCroute. Serotype 4 shows a higher peak of viremia than serotype 9. The peakof viremia was measured between day 2 and day 4 post infection. Theseresults demonstrate the potential of the immunodeficient mouse model forboth clinical and biological features.The setting up of this mouse model has developed a tool for efficient in vivostudy of AHSV.Research supported by the Belgium Federal Public Service, Health, FoodChain Safety and Environment.S66 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyThursday 12 th September 2013,15h00 – 17h15WORKSHOP 23: “VIRAL DIAGNOSIS IN CHRONICINFECTION AND DURING PREGNANCY”Chairpersons: Peter COYLE (UNITED KINGDOM)& Hubert NIESTERS (Groningen, THE NETHERLANDS)AmphitheaterKEYNOTE:Diagnosis and prognosis of human cytomegalovirus (HCMV) infectionin pregnancyMaria GRAZIA REVELLOSC Ostetricia e Ginecologia, Fondazione IRCCS Policlinico San Matteo,Pavia and Fondazione Carlo Denegri, Torino, ITALYHuman cytomegalovirus (HCMV) is still the leading infectious agent causingmental retardation and sensorineural deafness in neonates. In theabsence of a vaccine, accurate diagnosis of primary infection in the mother,and of congenital infection in the fetus remain key points in the managementof pregnancies complicated by HCMV infection. The combinationof standard and new serological assays together with virological and clinicalfindings allow diagnosis and dating of primary HCMV infection in themother in the majority of cases. Maternal correlates of vertical transmissionremain elusive. As for diagnosis of HCMV infection in the fetus, thetechnique employed for HCMV detection, time interval between maternalinfection and the procedure as well as delayed transmission in utero aremajor variables affecting sensitivity of prenatal diagnosis. A high viral loadin amniotic fluid is a poor predictor of clinical outcome. Some promisingprognostic markers of congenital disease are currently being investigatedin fetal blood. In conclusion, reliable diagnosis of primary maternalinfection is the first issue to be addressed. Then, prenatal diagnosis can beoffered, and the woman, properly counselled, can decide how to proceedwith her pregnancy.ORAL COMMUNICATIONSREF O70Human Cytomegalovirus specific hyperimmune globulin for preventionof congenital infection following primary infection in pregnancyMilena FURIONEFondazione Carlo Denegri, Torino, ITALYBackground: Primary HCMV infection in pregnancy carries the highestrisk of vertical transmission and congenital disease. A non randomizedstudy (Nigro et al, NEJM 2005) reported that HCMV specific hyperimmuneglobulin (HIG) significantly reduced the risk of congenital infectionfrom 40% to 16% in pregnant women with primary infection.Objective: To verify HIG efficacy under controlled conditions.Patients and Methods: An independent, phase IIB, double blind, drug vsplacebo multicenter trial was conducted. Primary objective was to assessdrug efficacy by comparing the number of infected fetuses/newborns in thetwo arms of treatment. Pregnant women with primary HCMV infection at5 26 weeks’ gestation were randomized to receive, within 6 weeks after thepresumed onset of infection, HIg or placebo every 4 weeks until 36 weeks’gestation or HCMV positive amniocentesis. Sixty women/arm were neededin order to show a reduction from 40% to 16% vertical transmission(80% power, 5% type I error, 10% attrition).Results: Transmission rate was 44% (27/62) in the placebo arm and 30%(18/61) in the drug arm (95% CI 31.7 – 2.2; p=0.13). No significant differencewas noted between the two study groups for virus specific antibodyand T cell response. Similarly, no significant difference was noted betweentransmitter and nontransmitter mothers in each arm for the same parameters.An higher number of serious adverse events was reported in the HIGarm compared to the placebo arm.Conclusions: Safety and efficacy of HIG need to be assessed in largerphase III studies.REF O71Phylogenetic analysis of human parvovirus B19 in pregnant womenin Lyon (FRANCE)Yahia MEKKI 1 , Clément LABOIS 1 , Jean Sébastien CASALEGNO 1 ,Daniel EIBACH 1 , Genevieve BILLAUD 1 , Annabelle SERVANTDELMAS 2 , Jerome MASSARDIER 2 , Mona MASSOUD 2 , CyrilHUISSOUD 2 , Axel FICHEZ 2 , Pascal GAUCHERAND 3 , Rene CharlesRUDIGUOZ 4 , Frederique LEBRETON 5 , Veronique TARDY 6 , BrunoLINA 71 Centre hospitalo universsitaire Lyon/Virologie/HFME, Lyon, FRANCE;2 Laboratoire de virologie/INTS/Hôpital Armand Trousseau, PARIS,France; 3 Centre hospitalo universsitaire Lyon/Gynecologie obstétrique/HFME, Lyon, FRANCE; 4 Centre hospitalo universsitaire Lyon/Gynecologie obstétrique/Croix Rousse, Lyon, FRANCE; 5 Centre hospitalouniverssitaire Lyon/Anatomie pathologie/HFME, Lyon, FRANCE;6 Centre hospitalo universsitaire Lyon/Biochimie, Lyon, FRANCE; 7 Centrehospitalo universsitaire Lyon/Virologie, Lyon, FRANCEParvovirus infection during pregnancy is an important cause of hydropsfetalis. It is attributed to anemia caused by viral induced destruction ofred blood cells. Other organs have been reported to be affected includingthe heart, liver and lungs. Parvovirus B19 is a human virus consisting ofthree distinct genotypes (1, 2 and 3). In our hospital laboratory we detectedseveral epidemics of Parvovirus touching pregnant women. Most of themare deadly for the embryos.We retrospectively performed sequencing and phylogenic classificationof ninety six laboratory samples currently detected by the diagnosticvirology laboratory (RT PCR Abbott ® ) were analyzed. The ninety sixsamples were composed of 4 placenta, 12 necropsies, 15 amniotic fluidand 65 blood samples. Sixty four samples were identified as genotype 1by genotype specific PCR (analysis NS1 VP1U region), and thirty onesamples were negative. Seventy laboratory samples undetected by RTPCR Abbott ® resulted in 4 samples identified as genotype 1 and 66 asnegative.In conclusion during a 6 years period (2005 to 2011), only Parvovirus B19genotype 1 has been detected in pregnant women and fetal infections inour hospital.REF O72Determination of Rubella Virus Specific Humoral and Cell MediatedImmunity in pregnant women with negative of equivocal rubellaspecific IgGChristelle VAULOUP FELLOUS 2 , Olivier PICONE 1 , Yousra BEJAOUIOLHMANN 2 , Anne Gaël CORDIER 3 , Sophie NEDELLEC 3 ,PDEVILLIER 4 , Nicolas GAIDOT 2 , Liliane GRANGEOT KEROS 21 Department of Gynecology and Obstetrics, Foch Hospital, Suresnes,FRANCE; 2 AP HP, Laboratory of Virology, Rubella Materno foetalinfection National Reference Center, Paul Brousse Hospital, Villejuif,FRANCE; 3 AP HP, Department of Gynecology and Obstetrics, AntoineBéclère Hospital, Clamart, FRANCE; 4 Department of Clinical Research,Foch Hospital, Suresnes, FRANCEVirologie, Vol 17, supplément 2, septembre 2013S67

5 th European Congress of VirologyObjectives: Immunity to Rubella virus is commonly determined by measuringspecific IgG (RV IgG). However, RV IgG results may be verydifferent and even discordant, depending on the assay used. Cell mediatedimmunity plays also a role in resistance to infection but is not routinelyinvestigated for diagnostic purposes. Our aim was to investigateboth humoral and cellular immunity of pregnant women with negativeor equivocal RV IgG titers before and after the recommended post partumvaccination.Methods: 86 patients were included in the study. In the pre vaccinationperiod, humoral immunity was investigated with 4 RV IgG ELISA assays, awestern blot and a cell mediated immunity test (based on the − interferonresponse after stimulation by rubella antigens). In the post vaccinal period,measuring rubella specific IgM and RV IgG avidity allowed us to determinewhether a primary or a secondary immune response occurred.Results: Patients, who initially had negative RV IgG but above half thecut off of the assay, had positive RV IgG E1 antibodies, positive cellmediated immunity and experienced a secondary immune response aftervaccination (44/86). Patients with RV IgG below half the cut off of theassay had negative RV IgG E1 antibodies, negative cell mediated immunityand experienced a primary immune response after vaccination (42/86).Conclusion: These results indicate that several individuals may beimmune despite negative RV IgG. This could lead us to reduce the sensitivitythreshold of the rubella serologic assays, with respect to theirdiagnostic accuracy.REF O73A delay in the maternal antibody response to human cytomegalovirus(HCMV) gH/gL/pUL128 130 131 complex is associated with virustransmission to the fetusDaniele LILLERI 1,2 , Anna KABANOVA 2 , Maria Grazia REVELLO 1 ,Elena PERCIVALLE 1 , Antonella SARASINI 1 , Emilia GENINI 1 ,Federica SALLUSTO 2 , Antonio LANZAVECCHIA 2 , Davide CORTI 2 ,Giuseppe GERNA 11 Fondazione IRCCS Policlinico San Matteo, Pavia, ITALY; 2 Institute forResearch in Biomedicine, Bellinzona, SWITZERLANDPrimary HCMV infection during pregnancy is associated with a high riskof virus transmission to the fetus. Serial serum samples from 43 HCMVtransmitter (T) and non transmitter (NT) pregnant women with primaryHCMV infection were analyzed for neutralizing antibodies against HCMVglycoprotein complexes to identify correlates of HCMV transmission. Inthis study, we report that early detection of HCMV neutralizing antibodiesdirected against the pentameric complex gH/gL/pUL128 131 and, in particular,to the UL128 131 gene products appears to be associated with alower rate of HCMV transmission from the mother to the fetus. This wasshown by the finding that NT mothers exhibit an earlier antibody responseto different antigenic sites of the pentamer as compared to T mothers. Thisassociation was found to be consistent with the lower number of neutralizationsites recognized by T as compared to NT women at the 1st andthe 2nd month post infection onset. Furthermore, anti pentamer antibodiesdisplay an in vitro inhibition of cell to cell spreading and virus transferto leukocytes. The biological relevance of anti pentamer antibodies hasbeen in parallel highlighted by the finding that antibodies to HCMV gBare produced by NT and T mothers with the same kinetics and in comparableamounts. Taken together, these findings indicate that: i) the pentamercomplex is a major target of the antibody mediated maternal immunity;ii) T women elicit a lower neutralizing antibody response in the first 2months after onset of infection as compared to NT women.REF 074Development of a multiplex PCR approach for detection of dual infectionsand recombinants involving HIV variantsPierre CAPPY, Fabienne DE OLIVEIRA, Marie GUEUDIN, JeanChristophe PLANTIERRouen University Hospital, Rouen, FRANCEBackground: The high genetic diversity of HIV necessitates consistentattention to diagnostic tools and patient follow up. Due to the co circulationof different HIV types and groups, dual infections (DIs) and recombinants(Rec) have been described, which may hinder diagnosis and therapeuticmanagement.Methods: We designed 2 multiplex PCRs (mPCRs) coupled to capillaryelectrophoresis: MMO2, targeting 2 genes for the screening of HIV M+Oand M+2 DIs, and MMO, targeting 5 genes for the detection of HIV M+ODIs and HIV MO Rec. mPCRs were assessed on DNA and RNA extractsfrom HIV M, O and 2 virus cultures, then on DNA and RNA mixtures,simulating DIs. They were finally assessed on material from clinical monoinfection and from patients presenting with an HIV M+O or an HIV MOinfection.Results: In vitro samples. For MMO2, the overall sensitivity was 88% onDNA and 100% on RNA, along with a specificity of 100%. For MMO,the sensitivity was 100%, along with a specificity of 94% on DNA and100% on RNA. On simulated DIs, both mPCRs correctly detected 90%and 100% of DNA and RNA samples respectively. Clinical samples. ThemPCRs specificity was 100%, with a global sensitivity of 87% and 91%for MMO2, and 60% and 81% for MMO, on DNA and RNA respectively.Finally, both mPCRs were able to detect 4 out of 6 DIs on RNA,2 out of 2 DIs on DNA for MMO2, and 2 out of 2 MO Rec on RNAfor MMO.Conclusions: Altogether, on in vitro and clinical samples, MMO2 turnsout to be an excellent screening tool to detect M+2 and M+O DIs, andMMO to detect both MO DIs and MO mosaic patterns.REF O75Clinical Evaluation of BioPlex 2200 HIV Ag Ab: An AutomatedScreening Method Providing Discreet Detection of HIV 1 p24, HIV1 Antibody, and HIV 2 AntibodyFrançois SIMON 1 , Maud SALMONA 1 , Sarah MAYLIN 1 , LucGUERRIER 2 , Bill LINK 31 AP HP Hôpital Saint Louis, Laboratoire de virologie, Paris, FRANCE;2 Bio Rad Laboratories, Inc., Marnes La Coquette, FRANCE; 3 Bio RadLaboratories, Inc., Hercules, USABackground/Purpose: To assess the clinical sensitivity/specificity of theBioPlex 2200 HIV Ag Ab assay. The study was performed at the laboratoryof Virology of St. Louis Hospital, Paris.Methods: The BioPlex 2200 HIV Ag Ab assay uses multiplex flow IA todetect p24 Ag, HIV 1 Ab (Groups M/O) and HIV 2 Ab in a single reactionvessel using a mixture of 4 dyed bead populations. Each populationis coated with a different HIV Ag or p24 Ab, and results can be reportedindividually. Ab reactive specimens can be typed as HIV 1 or HIV 2. Specimenswith similar levels of HIV 1/2 Ab reactivity are reported as reactiveundifferentiated. The study included 1505 prospective fresh samples fromhospitalized patients, 420 stored samples from infected patients with HIV1 (different B or non B/CRF subtypes incl. 8 HIV 1 Grp O), a panel of 15samples collected during Primary HIV infection (PHI)/86 HIV 2 samples.S68 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyAll samples were tested on BioPlex and its values were compared to AbbottArchitect 4th Gen.Results: 1470 of 1505 prospective samples were negative on BioPlex.Specificity on first intent was 99.4% & 99.5% on retest. 7/8 false positiveswere initially reactive on Architect. BioPlex detected all of the following:35 HIV 1 positives, including 4 early infection samples, 420 known HIV1+ samples, incl.158 of known genotypes across 28 different genotypes,15 retrospective PI/86 HIV 2 samples & 7 known co infected HIV 1/2samples.Conclusion: The BioPlex HIV Ag Ab, with HIV 1/2 Ab & Ag differentiation,showed excellent detection of PHI/mulitple genotypes and equalvalues to Architect.REF O76Impact of the coexistence of anti HBs antibodies on HBsAg quantificationin chronic hepatitis B carriersVincent THIBAULT 1 , Marie PANCHER 1 , Nathalie DESIRE 1,2 , RaphaelCROSNIER 1 , Coralie PALLIER 3 , Sepideh AKHAVAN 11 GH Pitie Salpetriere/AP HP/Virology, Paris, FRANCE; 2 UniversitePierre et Marie Curie/UPMC, Paris, FRANCE; 3 GH Kremlin Bicêtre/APHP/Virology, KREMLIN BICETRE, FRANCEPresence at the same time of HBsAg and anti HBs antibodies (HBsAg/Ab)is a rare entity in chronic hepatitis B (CHB) carriers. This study wasdesigned to characterize such serological profiles using three HBsAg quantification(qHBsAg) systems and to analyze the impact of HBsAg variantsin their performances.From 2578 CHB identified patients, 129 (5%) had an HBsAg/Ab profile.After exclusion of co infections (HIV, HCV, HDV), HBV reactivation orHBIg treatment, 101 samples from 62 (48%) patients were tested for qHBsAgand HBsAb in parallel using Abbott Architect (screening method),DiaSorin LiaisonXL and Roche Modular Cobas. HBsAg coding sequenceswere also generated from samples with an HBV viral load above 100IU/mL (n=32).HBsAg detection was confirmed with the 3 techniques for 98% (99) ofthe samples. 65% of the HBsAg/Ab profiles were confirmed on the 3techniques. The median HBsAg concentration was comparable for the 99samples whatever the technique used. A bias of 0.11 and 0.02 logIU/mLwere noticed for DiaSorin and Roche compared to Abbott, respectively.The overall correlation between the 3 qHBsAg techniques was good (r2:0.94 0.97). Substitutions within the ”a“ determinant did not have a majorinfluence on qHBsAg quantification. Major discrepancies were observedfor HBsAb quantification between the 3 techniques.The double HBsAg/Ab profile is not an analytical artifact and is confirmedon different techniques. HBsAg quantification is not influenced by thepresence of HBsAb or HBsAg substitution and is similar whatever theassessed technique.Virologie, Vol 17, supplément 2, septembre 2013S69

5 th European Congress of VirologyFriday 13 th September 2013, 8h30 – 10h30WORKSHOP 6: “RESTRICTION FACTORS OF VIRALINFECTION, INTERFERING RNA & IMMUNE RESPONSE”KEYNOTE:Chairpersons: Santo LANDOLFO (Turin, ITALY)& Soren PALUDAN (Aarhus, DENMARK)Room Prestige Gratte-CielRestriction Factors are key determinants of viral susceptibilityMonsef BENKIRANEInstitut de Génétique Humaine (CNRS-UPR1142), Montpellier, FRANCEThe outcome of viral infection results from complex interactions betweenviral proteins and host cell factors. Viruses have evolved to establish acomplex network of interactions with host facilitating factors required forthe achievement of the viral life cycle and to evade intracellular defensemechanisms which otherwise sense the virus and establish an antiviralstate. Indeed, upon entry in the human host, viruses are confronted withnumerous blocks that oppose their replication. The first line of defenseto be triggered is the so-called “intrinsic” immune system. The intrinsicimmune system includes proteins that detect the presence of the assailant(Pattern Recognition Receptors [PRRs]) and initiate a subsequentimmune response, as well as proteins referred to as restriction factors thatare directly devoted to arresting the replication cycle of the virus. We willuse primate lentiviruses and host restriction factors that block their replicationas examples to highlight their role in determining host susceptibilityand disease.ORAL COMMUNICATIONSREF 077Defining the relationship between miRNAs and cytoplasmic virusesSimone BACKES, Ryan LANGLOIS, Benjamin TENOEVERIcahn School of Medicine at Mount Sinai, New York, USAMicroRNAs (miRNAs) regulate gene expression through sequence specificbinding to target mRNAs, resulting in translational repression andsubsequent deadenylation. We have previously shown that poxvirusesinduce the overall degradation of host miRNAs through the virally encodedpoly(A) polymerase VP55 subunit. The discovery of a miRNA antagonizingfunction encoded by poxviruses suggests that miRNAs may aid inrestricting virus replication. In an effort to expand our knowledge aboutthe impact of host miRNAs on gene expression and replication of differentviruses, we are utilizing poxviral VP55 as a tool to investigatethe interplay between viruses, host miRNAs and the cellular responseto infection. To this end, we engineered Vesicular stomatitis virus (VSV)to express poxvirus VP55 (VSV VP55) in an effort to determine whetherloss of host miRNAs confered a replicative advantage. VSV VP55displayed high expression levels of poxvirus VP55 and was also capableof inducing polyadenylation of miRNAs. Currently, we are performingparallel infections both ex vivo and in vivo in the context of either wildtype and interferon defective genotypes in order to compare viral mRNAlevels, protein expression capacity and viral replication kinetics. As VP55induces overall degradation of host miRNAs in infected cells, this newtool will allow studying the impact of host miRNAs on the pathogenesisof different viruses.REF O78Regulation of virus endocytosis by miRNAsPierre Yves LOZACH 1 , Roger MEIER 2 , Andrea FRANCESCHINI 3 ,Peter HORVATH 2 , Marilou TETARD 1 , Christian VON MERING 3 , AriHELENIUS 21 INRS Institut Armand Frappier, Laval, CANADA; 2 Swiss Federal Instituteof Technology (ETH), Zurich, SWITZERLAND; 3 University of Zurich,Zurich, SWITZERLANDThe Bunyaviridae constitute a large family of enveloped animal viruses,many of which cause serious diseases in humans. Currently there areno available vaccines or treatments approved for human use. The strategiesemployed by these viruses for transmission and infection remainpoorly understood. To investigate into early bunyavirus host interactions,we screened siRNA libraries from two providers against the whole humangenome for their ability to block infection. These screens revealed morethan 500 cellular proteins with a potential role in bunyavirus entry andreplication. Our data support the view that some siRNAs act like miRNAsto down regulate gene expression. Therefore, we assessed miRNAs, withsequences identical to those of siRNA seed regions found in the screens(6 7 nucleotides), for their capacity to inhibit infection. Within these miR-NAs, we identified the miRNA 142.3p as blocking infection at the levelof virus entry. A DNA microarray screen shown that many genes regulatedby this miRNA have functions related to endocytosis. Within thissymposium, the confirmation of hits will be illustrated with the analysisof the cellular factor v SNARE VAMP 3 by state of the art fluorescencebased techniques in fixed and living cells. The prospects and challengesof future work on the cell biology of bunyavirus entry will be thendiscussed.REF O79Host microRNA molecular signatures associated with humaninfluenza A virus infections reveal an unanticipated antiviral activityfor miR 146aOlivier TERRIER 1 , Julien TEXTORIS 2 , Coralie CARRON 1 , VirginieMARCEL 3 , Jean Christophe BOURDON 3 , Manuel ROSACALATRAVA 11 Laboratoire de Virologie et Pathologie Humaine VirPath, Equipe VirCell,Université Claude Bernard Lyon 1, Université de Lyon, Lyon, FRANCE;2 Laboratoire d’Immunologie, UMR CNRS 7278, INSERM U1095, Facultéde Médecine Timone, Marseille, FRANCE; 3 Division of Medical Sciences,Centre for Oncology and Molecular Medicine, University of Dundee,Ninewells Hospital, Dundee, UNITED KINGDOMWhile post transcriptional regulation of gene expression by miRNAs havebeen shown to be involved in influenza virus replication cycle, only afew studies have further investigated this aspect in a human cellular modelinfected with human influenza viruses. In this study, we performed miRNAglobal profiling in human lung epithelial cells (A549) infected by differentsubtypes of human influenza A viruses. We identified a common miRNAsignature in response to infection by the different strains, highlighting apool of five miRNAs commonly deregulated, which are known to be involvedin the innate immune response or apoptosis. Among the five miRNAhits, the only up regulated miRNA in response to influenza infection correspondedto miR 146a. Based on a previously published gene expressiondataset, we extracted inversely correlated miR 146a target genes and determinedtheir first level interactants. This functional analysis revealed 8distinct biological processes strongly associated with these interactants:TLR pathway, innate immune response, cytokine production and apoptosis.To better understand the biological significance of miR 146a upregulation, using a reporter assay and a specific anti miR 146a inhibitor,we confirmed that infection increases the endogenous miR 146a promoteractivity and that inhibition of miR 146a significantly increased viralS70 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologypropagation. Altogether, our results suggest a functional role of miR 146ain the outcome of influenza infection, at the crossroads of several biologicalprocesses.REF O80Requirement of the autophagy initiating protein kinase Ulk1 for latestages of human cytomegalovirus replicationSabine FEICHTINGER, Thomas STAMMINGERInstitute for Clinical and Molecular Virology, University Erlangen Nuremberg,Erlangen, GERMANYAutophagy, a highly conserved cellular process sequestering portions ofthe cytoplasm into double membrane vesicles for lysosome dependentdegradation, constitutes an important effector mechanism of the host responseto viral infections. In our efforts to explore the role of the autophagyinitiating protein kinase Ulk1 for human cytomegalovirus (HCMV) replication,we detected a distinct mobility shift at late time points of infectionwhich corresponded to highly phosphorylated Ulk1. One of the phosphorylationsites has been described as target for AMP activated protein kinase(AMPK), a metabolic stress response kinase that commonly activatesUlk1. Moreover, stimulation of AMPK for generating an environmentconducive to HCMV replication has been demonstrated whereas the underlyingmechanism is still unclear. Subsequent inhibition of AMPK mediatedUlk1 phosphorylation not only reversed the HCMV induced modulationof Ulk1 but also resulted in an impairment of progeny virions, suggestingan HCMV induced Ulk1 activation via AMPK dependent phosphorylationthat contributes to viral assembly. Additional support for a proviralrole of Ulk1 was obtained by generating primary human fibroblasts witha stable Ulk1 knockdown. Comparative analyses revealed a profoundgrowth defect of HCMV in the absence of Ulk1 along with changes inthe tegument composition of viral particles. Altogether, our data pointto a crucial role of Ulk1 for late steps of an efficient HCMV replicationcycle.REF O81Hantavirus infection confers resistance to Natural Killer cell mediatedkilling and hantavirus nucleocapsid protein inhibits granzyme B andcaspase 3Shawon GUPTA 1,2 , Monika BRAUN 3 , Nicole TISCHLER 4 , MalinSTOLTZ 2 , Karin SUNDSTRÖM 1,2 , Niklas BJÖRKSTRÖM 3,5 , HansGustaf LJUNGGREN 3 , Jonas KLINGSTRÖM 1,2,31 Karolinska Institutet, Department of Microbiology, Tumor and CellBiology, Stockholm, SWEDEN; 2 Swedish Institute for CommunicableDisease Control, Solna, SWEDEN; 3 Karolinska Institutet, Center forInfectious Medicine, Department of Medicine, Karolinska UniversityHospital, Stockholm, SWEDEN; 4 Fundación Ciencia & Vida, Santiago,CHILE; 5 Karolinska Institutet, Liver Immunology Laboratory, Division ofGastroenterology and Hepatology, Department of Medicine, Stockholm,SWEDENHantaviruses cause hemorrhagic fever with renal syndrome (HFRS) andhantavirus cardio pulmonary syndrome (HCPS), two human diseases withhigh case fatality rates. Endothelial cells are the main targets for hantavirusesand vascular permeability is a hallmark of HFRS/HCPS. Anintriguing observation in patients with HFRS/HCPS is that the virus infectionleads to strong activation of cytotoxic lymphocytes, CD8 T cells andNatural Killer cells, but no obvious destruction of infected endothelialcells. Here, we provide a possible explanation for this dichotomy by showingthat hantavirus infected endothelial cells are protected from cytotoxiclymphocyte mediated killing. Hantaviruses were also able to inhibit chemicallyinduced apoptosis in both endothelial and epithelial cells. Whendissecting potential mechanisms behind this phenomenon, we discoveredthat the hantavirus nucleocapsid (N) protein contains multiple granzymeB sites and at least one caspase 3 site and that the N protein inhibits theenzymatic activity of both granzyme B and caspase 3. Transfection experimentsshowed that the N protein by itself was able to inhibit apoptosis.In a closer evaluation of the inhibitory caspase 3 site in the N proteinwe could conclude that the site is DLID285. Alanin replacements ofDLID285 to DLIA285 rendered N protein transfected cells susceptibleto apoptotic stimuli. Our findings provide a tentative explanation for thehantavirus mediated block of cytotoxic granule mediated killing, and hencethe protection of infected cells from cytotoxic lymphocytes.REF O82Inflammatory cytokines promote viral infection of polarized cellsNicola FLETCHER 1 , Rupesh SUTARIA 1 , Juandy JO 2 , AmyBARNES 1 , Miroslava BLAHOVA 1 , David ADAMS 1 , StuartCURBISHLEY 1 , Antonio BERTOLETTI 2 , Jane MCKEATING 11 University of Birmingham, Birmingham, UNITED KINGDOM; 2 Agencyof Science Technology and Research (A*STAR), Singapore, SINGAPOREBarrier sites of the body provide the first line of defense against pathogensand it is well recognised that intact epithelia are resistant to virus infection.These barriers comprise a network of polarized cells that adhere to eachother at cell junctions including: tight and gap junction proteins, cadherinsand junction adhesion molecules. Inflammatory mediators released by arange of activated immune cells perturb epithelial integrity; however, theirrole in viral infection is poorly understood. We demonstrate a new pathwaywhere hepatitis C virus (HCV) utilises the inflammatory cytokinesIL 1 and TNF a to promote particle entry into polarised hepatoma andblood brain barrier endothelial cells. These cytokines disrupt tight junctionintegrity and increase viral receptor dynamics to facilitate infection.Importantly, this effect is not limited to HCV, as TNF a also increased thepermissivity of cells to infection by Lassa, Measles and Vesicular Stomatitispseudoviruses. Stimulation of peripheral and liver derived macrophagesvia a wide range of toll like receptors and HCV particles induced TNFa and promoted infection of polarized epithelia. This study highlights anovel mechanism for viruses to exploit host inflammatory responses thatare classically considered to be anti viral to promote infection of polarizedcells. These observations are likely to have broad impact for a widerange of pathogens and will inform the design of future therapeutic agentstargeting conserved host pathways to prevent infection.Virologie, Vol 17, supplément 2, septembre 2013S71

5 th European Congress of VirologyFriday 13 th September 2013, 8h30 – 10h30WORKSHOP 10: “VIRUS ENTRY: ENVELOPEGLYCOPROTEINS, RECEPTORS, ENDOCYTOSIS”KEYNOTE:Chairpersons: Niklas ARNBERG (Umea, SWEDEN)& Urs GREBER (Zürich, SWITZERLAND)Room Gratte-Ciel 1, 2, 3Intermediate structures in the fusion associated transition of vesiculovirusglycoproteinEduard BAQUERO 1 , Aurélie A. ALBERTINI 1 , Malika OULDALI 1 ,Linda BUONOCORE 2 , John K. ROSE 2 , Jean LEPAULT 1 , StéphaneBRESSANELLI 1 , Yves GAUDIN 11 Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296),Avenue de la Terrasse, 91198, Gif sur Yvette Cedex, FRANCE; 2 YaleUniversity School of Medicine, 310 Cedar St., New Haven, CT 06510,USAEnveloped viruses enter cells through a membrane fusion reaction drivenby conformational changes of viral fusion glycoproteins. Three differentclasses of viral fusion proteins have been identified to date based on theircommon structural motifs. Crystal structures have provided atomic, staticpictures of pre- and post-fusion conformations for several of these glycoproteins;however, no atomic structure of a transitional intermediate thatcould form the bridge between the viral and cellular membranes is known.VSV G is the prototype of the third class and forms different trimers inits pre- and post-fusion conformations. We report here a crystal structurefor G of Chandipura virus, another vesiculovirus responsible fordeadly encephalopathies. In this single crystal, two distinct conformationscorresponding to early and late refolding states of G, arranged in aflat tetrameric assembly exposing fusion loops. Consistent with these data,electron microscopy and tomography show different intermediates at theviral surface depending on experimental conditions.This work reveals the chronological order of the structural changes in theprotein and offers a detailed pathway for the conformational transition.Particularly, our data confirm that the conformational change involvesmonomeric intermediates and that it likely proceeds to an elongated hairpinmonomer before subsequent collapse into the post-fusion trimer.Furthermore, our data and previously published mutagenesis analysis indicatethat after dissociation of the pre-fusion trimer into monomers, vesiculovirusfusion glycoprotein could re-associate not only into trimers but alsointo a dimeric (and even tetrameric) assembly which is optimally organizedto forming the initial bridge between the target and viral membranes.ORAL COMUNICATIONSREF O83Dipeptidyl peptidase 4: Entry portal for the emerging human coronavirusEMCBerend Jan BOSCH 1 , Huihui MOU 1 , Stalin RAJ 2 , Saskia SMITS 2,7 ,Dick DEKKERS 3 , Marcel MÜLLER 4 , Ronald DIJKMAN 6 , JeroenDEMMERS 3 , Ali ZAKI 5 , Ron FOUCHIER 2 , Volker THIEL 6,8 ,Christian DROSTEN 4, Albert OSTERHAUS 2 , Peter ROTTIER 1 , BartHAAGMANS 21 Virology Division, Department of Infectious Diseases & Immunology,Faculty of Veterinary Medicine, Utrecht University, Utrecht, THENETHERLANDS; 2 Viroscience Lab, Erasmus Medical Center, Rotterdam,THE NETHERLANDS; 3 Proteomics Department, Erasmus Medical Center,Rotterdam, THE NETHERLANDS; 4 Institute of Virology, Universityof Bonn Medical Centre, Bonn, GERMANY; 5 Virology Laboratory, DrSoliman Fakeeh Hospital, Jeddah, KINGDOM OF SAUDI ARABIA;6 Institute of Immunobiology, Kantonal Hospital St. Gallen, St. Gallen,SWITZERLAND; 7 Viroclinics Biosciences BV, Rotterdam, THE NETHER-LANDS; 8 Vetsuisse Faculty, University of Zürich, Zürich, SWITZERLANDCoronaviruses generally cause respiratory and enteric infections and arefound in many mammalian and avian species. They have a marked potentialfor host species switching by adapting to receptors of novel host species.Recently a novel coronavirus (hCoV EMC) was identified in patients withsevere and sometimes lethal lower respiratory tract infection. The emergingvirus is most closely related to CoVs found in bats. We recently haveidentified dipeptidyl peptidase 4 (DPP4) - also known as CD26 - as a functionalreceptor for hCoV EMC. DPP4, an exopeptidase found to occur onhuman lung surfaces, specifically co purified with the receptor binding S1subunit of the hCoV EMC spike protein from lysates of susceptible Huh7 cells. Antibodies directed against DPP4 inhibited hCoV EMC infectionof primary human bronchial epithelial cells and Huh 7 cells. We showthat non susceptible cells become infectable upon transient expression ofthe DPP4 receptor. Moreover, we found that the virus was able to use theevolutionary conserved DPP4 of other species, most notably bats. The peptidaseactivity of DPP4 was not necessary for virus entry. Furthermore wedefined the interacting domains within the S1 subunit as well as the receptor.Ongoing studies focus on the spike promiscuity for DPP4 proteins ofother species and the DPP4 receptor usage by related bat coronaviruses.The discovery of DPP4 as a functional receptor will help us understand thepathogenesis and epidemiology of this emerging virus and may facilitatethe development of intervention strategies.REF O84Human hepatitis B and D Viruses exploit sodium taurocholate cotransporting polypeptide (NTCP) to bind and enter hepatocytes ina species specific mannerYi NI 1 , Florian LEMPP 1 , Stefan MEHRLE 1 , Holger SÜLTMANN 2 ,Stephan URBAN 11 Department of Infectious Diseases, Molecular Virology, University HospitalHeidelberg, Heidelberg, GERMANY; 2 Cancer Genome Research(B063), Deutsches Krebsforschungszentrum/NCT, Heidelberg, GER-MANYHepatitis B (HBV) and Hepatitis D virus (HDV) binding to a hepatic receptorrequires a myristoylated N terminal preS domain of the large HBVenvelope protein. Accordingly, a synthetic lipopeptide comprising the respectivepreS sequence (Myrcludex B) specifically binds this receptor andblocks infection. In vivo pharmacokinetic studies of Myrcludex B in severalspecies revealed a hepatocyte specific receptor expression in even nonHBV susceptible hosts (e.g. mouse). Using a screening approach we identifiedsodium taurocholate cotransporting polypeptide (NTCP) as a possiblecellular target of Myrcludex B. In a biochemical approach this moleculehas also been identified recently as a functional HBV/HDV receptor.We here demonstrate that constitutive expression of mouse (m) or human(h) NTCP in human (HepG2, HuH 7, HepaRG cells) and mouse hepaticcells lines (Hepa1 6, Hep56D) render them competent for binding withMyrcludex B. However, hNTCP but not mNTCP expressing cell lines ofboth species became susceptible to HDV infection, indicating that mNTCPalthough competent in preS binding cannot support complete virus entry.Using a set of chimeras of hNTCP and mNTCP we mapped an extracellularloop domain which determines the susceptibility. In contrast to HDV, HBVinfection is completely abolished in the mouse cell lines, suggesting a hostspecific restriction during HBV infection.In conclusion we demonstrate the crucial role of NTCP as the specificHBV/HDV receptor and mapped an essential NTCP domain required forhost specificity.S72 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF O85LDL Receptor and its family members are the cellular receptors ofVSV and VSV G pseudotyped viral vectorsMenachem RUBINSTEIN, Danit FINKELSHTEIN, Daniela NOVICK,Sara BARAKWeizmann Istitue of Science, Rehovot, ISRAELVesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropicinfectivity, mediated by its coat protein, VSV G. Utilizing this property,recombinant forms of VSV and VSV G pseudotyped viral vectors arebeing developed for gene therapy, vaccination and viral oncolysis, and areextensively employed for gene transduction in vivo and in vitro. The broadtropism of VSV suggests that it enters cells through a highly ubiquitousreceptor whose identity has so far remained elusive. Here, we show thatthe low density lipoprotein receptor (LDLR) serves as the major entryport of VSV and of VSV G pseudotyped lentiviral vectors in human andmouse cells, whereas other LDLR family members serve as alternativereceptors. The widespread expression of LDLR family members accountsfor the pantropism of VSV, and for the broad applicability of VSV Gpseudotyped viral vectors for gene transduction. (PNAS, 2013, in press)REF O86Changes in SV40 binding to its receptor GM1 affects vacuolizationand plaque formation in CV1 monkey cellsNasim MOTAMEDI 1 , Thomas G. MAGALDI 1,2 , Xiaochu MA 1 , DanielDIMAIO 1,4,51 Department of Genetics, Yale School of Medicine, New Haven, CT, USA;2 Tumor Virus Molecular Biology Section, Laboratory of Cellular Oncology,National Cancer Institute, Bethesda, MD, USA; 3 Department ofTherapeutic Radiology, Yale School of Medicine, New Haven, CT, USA;4 Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA;5 Department of Molecular Biophysics and Biochemistry, Yale School ofMedicine, New Haven, CT, USAPolyomaviruses are small non enveloped DNA viruses with the capsid surfacecomposed entirely of the structural protein VP1. Polyomavirus VP1 sbind to oligosaccharide residues on their cell surface receptors. Simianvirus 40 (SV40), a monkey polyomavirus, binds to the terminal sialic acidresidue of the ganglioside GM1 to successfully infect cells. A hallmark ofSV40 infection in monkey cells is the dramatic vacuolization of cells priorto lysis and the subsequent formation of plaques in cultured cells. We haveintroduced mutations in the GM1 binding site of VP1 that inhibit SV40 bindingto GM1. However, these mutant viruses can still infect monkey cells,albeit at a lower efficiency. Interestingly, these mutant viruses fail to inducethe formation of cellular vacuoles or form plaques. Vacuolization is alsoinhibited by modulation of GM1 levels by siRNA knockdown of enzymesinvolved in ganglioside synthesis. Introducing additional mutations intothe GM1 binding site of VP1 rescues the phenotype of defective vacuolizationand plaque formation in monkey cells without restoring capsidbinding to GM1. These mutants presumably bind to a different oligosaccharide.Taken together, these experiments indicate that the ability of VP1to bind gangliosides, most likely GM1, is essential for plaque formationand vacuolization in CV1 cells and that gangliosides are important forstages of infection in addition to cell entry.REF O87Identification of functional networks in E1E2 glycoproteins unveilsHepatitis C virus fusion mechanisms and new therapeutic opportunitiesFlorian DOUAM 1,2 , Linda DIB 3,4 , Viet Loan DAO THI 1 , GuillemetteMAURIN 1 , Loic SCHWALLER 1,5 , Judith FRESQUET 1 , DimitriMOMPELAT 1 , François Loic COSSET 1 , Alessandra CARBONE 3 ,Dimitri LAVILLETTE 1,21 CIRI, International Center for Infectiology Research, Inserm U1111,Ecole Normale Supérieure de Lyon, Université Lyon 1, CNRS, Lyon,FRANCE; 2 Microbial Dynamics and Viral Transmission,CNRS, UMR5557 Ecologie Microbienne, Université Lyon 1, Villeurbanne, FRANCE;3 Laboratoire de Génomique des Microorganismes, UMR7238 CNRSUPMC, Paris, FRANCE; 4 Molecular Phylogenetics and Speciation,Département d’écologie et évolution, Université de Lausanne, Lausanne,SUISSE; 5 Statistique & Génome, AgroParisTech, INRA MIA, UMR 518,Paris, FRANCEChronic Hepatitis C virus (HCV) infection is a health threat affectingmore than 130 million people worldwide. Since cell entry is the firststep of virus host interaction, it represents a promising target for antiviraltherapies. However, the HCV entry process at the molecular levelis still poorly understood. Important domains of HCV E1 and E2 glycoproteinsinvolved in this process remain to be discovered and especiallytheir inter relationship during the entry process must be defined. In orderto identify such domains and interactions, we used two complementarystrategies: on the one hand, a classical molecular virology approach basedon the functional comparison of properties of two different strains, andon the other hand, a structural modeling and a combinatorial analysisof many strains of different genotypes helped to reconstitute networksof coevolving residues within and between viral glycoproteins. Differentchimeras and point mutants were analyzed in assays addressing assembly,binding and fusion using both, pseudotyped retroviral particles andcell cultured derived viruses. By these approaches, we were able to identifyparticular dialogs in different genotypes between residues in E1 andthe domain III of E2 which are critical for membrane fusion. Our resultshighlight that HCV has developed an original fusion mechanism, likelyconcealing some properties of both flavi and pesti virus fusion processes.We are currently developing strategies to exploit the potential therapeuticopportunity represented by critical E1E2 conformational changes duringentry.REF O88Targeted antiviral siRNA screen identifies the poxvirus genome uncoatingfactorSamuel KILCHER, Florian Ingo SCHMIDT, Christoph SCHNEIDER,Manfred KOPF, Ari HELENIUS, Jason MERCER1 Institute of Biochemistry ETH Zürich, Zürich, SWITZERLAND; 2 Instituteof Molecular Biomedicine ETH Zürich, Zürich, SWITZERLANDSince its discovery, RNA interference (RNAi) has been established asthe method of choice for large scale loss of function studies. Host factorrequirements of a variety of pathogens have been addressed using genomewide siRNA screens. In addition, viral genomes or mRNAs have beenVirologie, Vol 17, supplément 2, septembre 2013S73

5 th European Congress of Virologytargeted by RNAi during infection. The use of siRNA to study viral proteinfunction on the genome scale holds great potential but has not been appliedon a virome wide level.Here we present the first large scale RNAi screen directed against a singlevirus. A siRNA library targeting 80 conserved early viral genes was generatedfor the model poxvirus, vaccinia virus (VACV). As proof of conceptfor this strategy we set out to identify the poxvirus genome uncoatingfactor, a protein which has eluded identification for over 40 years. Highthroughput screening of the VACV RNAi library using EGFP expressingreporter viruses resulted in the unambiguous identification of this viralgene. Depletion of the uncoating factor led to the stabilization of viralcores and inhibition of virus DNA release. The protein localized to incomingcores, suggesting that it acts directly on these robust structures tofacilitate release of the viral DNA.In addition, RNAi mediated down regulation of the uncoating factor in amouse infection model resulted in reduced viral replication and spread invivo. These results indicate that virome RNAi libraries can be a powerfultool for the investigation of large DNA virus gene function, and simultaneousidentification of promising therapeutic targets and effective antiviralRNAs.S74 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyFriday 13 th September 2013, 8h30 – 10h30WORKSHOP 15: “HIGHLY PATHOGENIC VIRUSES”Chairpersons: Ake LUNDKVIST (Stockholm, SWEDEN) &Viktor VOLCHKOV (Lyon, FRANCE)AmphitheaterKEYNOTE:Transport and assembly of filovirusesLarissa KOLESNIKOVA, Gordian SCHUDT, Olga DOLNIK, andStephan BECKERInstitut für Virologie, Philipps-Universität Marburg, Hans-Meerwein-Str.2, 35043 Marburg, GERMANYThe family of Filoviridae comprises Marburg and Ebola virus which bothcause severe life-threatening diseases characterized by high fever, rash,thrombocytopenia and hemorrhagic diathesis. The pathogenesis of the syndromeis not completely understood; probably the dynamic replication offiloviruses in the infected host leads to an uncoordinated immune response.Detailed understanding of the basic mechanisms of filoviral assembly andinteraction with host cells is key to identify targets of antiviral intervention.The first sign of Filovirus replication that can be detected microscopicallyin the infected cell is the formation of inclusions in the perinuclear region.Inclusions contain all filoviral nucleocapsid proteins (NP, VP35, VP30,VP24, and L) but also the matrix protein VP40 and a number of cellularproteins. Viral nucleocapsids are formed within the inclusions by specificinteractions among the viral proteins. Mature nucleocapsids are thentransported across the cytoplasm to the plasma membrane with the helpof the actin cytoskeleton. The matrix protein accumulates at the plasmamembrane in a compartment that contains markers of the late endosomeand is probably of endosomal origin. In presence of the matrix protein,the only glycoprotein GP is recruited via the classical secretory pathwayto the peripheral matrix protein containing compartment. In the cell peripherynucleocapsids are associated with the matrix protein and channeledinto filopodia, the site of filoviral release. Nucleocapsids inside filopodiaare cotransported together with the unconventional motor protein myosin10. Transport of nucleocapsids and release of viral particles at filopodia issupported by the cellular ESCRT machinery.ORAL COMMUNICATIONSREF O89Mopeia virus and recombinant Lassa virus containing mutations intothe exonuclease of the nucleoprotein, but not wild type Lassa virus,induce a strong release of CXC and CC chemokines by human antigenpresenting cellsSylvain BAIZE 1,2 , Delphine PANNETIER 1,3 , Stéphanie REYNARD 1,2 ,Marion RUSSIER 1,21 Unité de Biologie des Infections Virales Emergentes, Institut Pasteur,Lyon, FRANCE; 2 International Center for Infectiology Research, Universitéde Lyon, Lyon, FRANCE; 3 Laboratoire P4 Inserm Jean Mérieux,Inserm US003, Lyon, FRANCEThe pathogenesis of Lassa fever, a hemorrhagic fever endemic in WestAfrica, is still unclear. We previously compared Lassa virus (LASV) withits genetically close but non pathogenic homolog Mopeia virus (MOPV)and demonstrated that the strong activation of antigen presenting cells(APC), including type I IFN production, observed in response to MOPVprobably plays a crucial role in controlling infection. Here, we show thathuman macrophages (MP) produced strong levels of CC and CXC chemokinesin response to MOPV infection, while dendritic cells (DC) releasedonly moderate levels of CXC chemokines. However, in the presence ofautologous T cells, DC were able to produce CC and CXC chemokines.Production of chemokines resulted from type I IFN synthesis, as levelsof both mediators were strongly correlated and as neutralization of typeI IFN led to inhibition of chemokine production. In contrast, only weaklevels of CXCL 10 and CXCL 11 were observed in response to LASV.Strikingly, the recombinant LASV harbouring mutations abrogating theability of NP to inhibit type I IFN response induces a massive synthesisof CC and CXC chemokines in both DC and MP. These resultsconfirm the crucial role of arenavirus NP in immunosuppression andpathogenicity. In addition, these differences in chemokine production mayprofoundly impact on the generation of virus specific T cell responses andthus, could be involved in the difference of pathogenicity between bothviruses.REF O90Soluble proteins of Ebola virus bind and activate dendritic cells andmacrophages causing release of pro and anti inflammatory cytokinesBeatriz ESCUDERO PÉREZ, Philip LAWRENCE, Viktor VOLCHKOVCIRI, INSERM U1111, Lyon, FRANCEEbola virus (EBOV) is a member of Filoviridae family and causes hemorrhagicfever with high fatality rates. Through RNA editing the EBOV GPgene codes for both the highly glycosylated surface protein (GP) and thesecreted glycoprotein sGP. Surface GP is responsible for virus attachmentand membrane fusion, but is also released from cells in a soluble form(shed GP) due to cleavage by cellular metalloprotease TACE.Both dendritic cells (DCs) and macrophages serve as early targets of EBOVand play a prominent role during infection. No cellular target has yetbeen identified for EBOV soluble proteins. Here, for the first time wedemonstrate that EBOV soluble proteins bind to human monocyte derivedDCs and macrophages. Importantly, we demonstrate that binding of shedGP but not sGP causes an activation of CD40, CD80 and CD86 expressionand an increase in levels of mRNA encoding TNFa, IL6, IL10 and IL12p40.Treatment of DCs and macrophages with shed GP resulted in release ofboth pro and anti inflammatory cytokines, as demonstrated by multiplexELISA. We have also revealed that the glycosylation pattern of shed GPis crucial in mediating this activation.Overall, this study suggests that shed GP but not sGP is responsible forthe early stimulation of human DCs and macrophages and thus may playa role in the dysregulation of host immune responses that, combined withmassive virus replication and virus induced cell damage, leads to a septicshock like syndrome and high mortality.REF O91Crimean Congo Hemorrhagic Fever Virus infected human monocytederived dendritic cells basolaterally transmits infection to humanepithelial cellsCecilia ANDERSSON 1,2 , Ali MIRAZIMI 1,2,31 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute,Stockholm, SWEDEN; 2 Department of Preparedness, SwedishInstitute for Communicable Disease, Solna, SWEDEN; 3 National VeterinaryInstitute, SVA, Uppsala, SWEDENCrimean Congo hemorrhagic fever virus (CCHFV) is an arthropod bornepathogen that in humans causes a severe disease with high case fatalityrates, Crimean Congo Hemorrhagic Fever characterized by hemorrhageand vascular leakage. The mechanisms determining the pathogenesis ofthis virus is however largely unknown. We have previously shown thatCCHFV entry and release take place basolaterally and that the infectionVirologie, Vol 17, supplément 2, septembre 2013S75

5 th European Congress of Virologyper se does not affect the permeability of polarized canine kidney epithelialcells. Several studies have also shown that CCHFV infection ofDCs and macrophages can induce release of pro inflammatory cytokines.We therefore set up an in vitro model system to study the effect of proinflammatory cytokines on tight and adherens junctions during CCHFVinfection. Human epithelial cells, Caco2, were grown on transwell membranesand transepithelial resistance measurements were made to ensureconfluence. Human monocyte derived dendritic cells (DCs) were isolatedfrom peripheral blood monocytes from healthy donors, and checked forpurity by CD14 positive flow cytometry. First, we infected Caco2 cellswith CCHFV. Uninfected or CCHFV infected DCs were then added touninfected Caco2 cells. Direct CCHFV infection of Caco2 cells occurredwere mainly through the basolateral side and the infection per se did notaffect cell layer permeability. We also discovered that CCHFV infectedDCs transmitted the infection to the Caco2 cells baselaterally. Currently,we are investigating the role of DC produced pro inflammatory cytokineson tight and adherens junctions.REF O92Pathogenesis of emergent Henipavirus infectionBranka HORVAT 1 , Cyrille MATHIEU 1 , Kévin DHONDT 1 , MarieCHALONS 1 , Romain VIVÈS 21 INSERM U1111, CNRS UMR5308, ENS Lyon, University of Lyon 1,Lyon, FRANCE; 2 Institut de Biologie Structurale Jean Pierre Ebel, UnitéMixte de Recherche (UMR) 5075, CNRS CEA Université Joseph Fourier,Grenoble, FRANCENipah virus (NiV) and Hendra virus (HeV) are closely related, recentlyemerged paramyxoviruses, belonging to Henipavirus genus. These zoonoticviruses are capable of causing considerable morbidity and mortalityin a number of mammalian species, including humans. We found that incontrast to closely related, highly lymphotropic Morbillivirus, human lymphocytesand monocytes were not permissive for NiV infection and a lowlevel of virus replication was detected only in dendritic cells. Interestingly,despite the absence of infection, human lymphocytes could efficientlybind NiV and transfer the virus to permissive cells. The transinfectionis mediated by an attachment receptor, which we have identified here asheparan sulphate. NiV infection in hamsters closely reproduces symptomsseen in humans and allowed us to analyze the importance of NiV transinfecitonin vivo. We show that circulating leukocytes captured and carriedNiV after intraperitoneal infection, without themselves being productivelyinfected and were able to transmit virus to permissive cells ex vivo. Use ofheparin efficiently blocked NiV transinfection and significantly improvedsurvival of NiV infected hamsters. Altogether, these results reveal a remarkablecapacity of NiV to hijack lymphocytes in order to transinfect hostcells, representing a rapid and potent way for Henipavirus disseminationthroughout the organism, which may contribute to its high pathogenicity.They open also novel perspectives for the development of new therapeuticapproaches against these emergent infections.REF O93A virus like particle system for Crimean Congo Hemorrhagic FevervirusStéphanie DEVIGNOT 1 , Michaela WEBER 1 , Eric BERGERON 2 , AliMIRAZIMI 3 , Stuart NICHOL 2 , Friedemann WEBER 11 Philipps University Marburg, Institute of Virology, Marburg, GER-MANY; 2 Centers for Disease Control and Prevention, Atlanta, USA;3 Smittskyddsinstitutet, Solna, SWEDENCrimean Congo Hemorrhagic Fever virus (CCHFV) is a bunyavirus responsiblefor outbreaks of CCHF disease. Mortality rates are ∼30%, and novaccine nor any treatment is available. Little is known about the virus/hostinteraction, since studies with the live virus require a Bio Safety Level(BSL) 4 facility.Virus like particles (VLPs) are structurally close to the native viral particle(but cannot replicate), and would facilitate the study of CCHFV underBSL 2 conditions. Here, we report the establishment of a VLP system forCCHF. Those VLPs express on their enveloped surface the two antigenicviral glycoproteins (Gc, Gn), and contain the viral polymerase L, as wellas a ribonucleoprotein complex (CCHFV specific minigenome and viralnucleoprotein N). The VLP minigenome can be transcribed and replicatedby a co expressed viral polymerase, thus allowing measurement of VLPinfection via reporter assay. Using this technique, we showed that the VLPscan be neutralized by CCHFV specific anti serum.The 12 kb gene for the polymerase L of CCHFV contains an OvarianTumor (OTU) protease domain. In vitro studies showed that over expressionof the ectopic OTU domain has anti IFN activity and can counteractthe antiviral effect of ISG15 (Frias Staheli et al, CHM, 2007). Here, weset out to study the role of OTU domain in the context of a full length,active CCHFV polymerase, using our VLP system. We were able to showthat both wild type and OTU inactive polymerases are sensitive to type IIFN, and ongoing experiments seem to revisit the role of ISG15 duringCCHFV infection.REF 094Marburg and Ebola Virus Vaccines: Antibodies are necessary forProtectionAndrea MARZI 1 , Flora ENGELMANN 2 , Friederike FELDMANN 3 ,Heinz FELDMANN 1 , Ilhem MESSAOUDI 21 Laboratory of Virology/Division of Intramural Research/National Instituteof Allergy and Infectious Diseases/NIH, Hamilton, MT, USA;2 Division of Biomedical Sciences/School of Medicine/University of CaliforniaRiverside, Riverside, CA, USA; 3 Operations Management/Divisionof Intramural Research/National Institute of Allergy and InfectiousDiseases/NIH, Hamilton, MT, USAMarburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagicdisease with high case fatality rates. These viruses pose a significant healthconcern due to the lack of approved therapeutics and vaccines as well astheir potential misuse as bioterrorism agents. Although not licensed forhuman use, recombinant vesicular stomatitis virus (rVSV) expressing thefilovirus glycoprotein (GP) has been shown to protect macaques fromMARV and EBOV infections, both prophylactically and therapeuticallyin a homologous challenge setting. However, the immune mechanisms ofprotection conferred by this vaccine platform remain poorly understood.We set out to investigate the role of humoral versus cellular immunity inrVSV vaccine mediated protection against lethal MARV or EBOV challenge.Groups of macaques were either depleted of CD4+ or CD8+ T cellsprior to and during rVSV vaccination and subsequently challenged withfiloviruses or regularly vaccinated and depleted of CD4+ or CD8+ T cellsduring filovirus challenge. Animals depleted of CD8+ T cells at any timeduring the experiment survived, suggesting a minimal role for CD8+ Tcells in rVSV mediated protection. Depletion of CD4+ T cells during vaccinationcaused a complete loss of GP specific antibodies and abrogatedvaccine protection. In contrast, depletion of CD4+ T cell during challengeresulted in survival of all the animals, indicating a minimal role for CD4+T cell immunity in vaccine mediated protection. Our results strongly indicatethat antibodies play a critical role in rVSV mediated protection againstfilovirus infections.S76 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyFriday 13 th September 2013, 8h30 – 10h30KEYNOTE:WORKSHOP 28: “GASTROINTESTINAL VIRALINFECTIONS”Chairpersons: Julie PFEIFFER (Dallas, USA)& Lennart SVENSSON (Linköping, SWEDEN)Room Tête d’OrGenotypic and Epidemiologic Trends of Viral Gastroenteritis InfectionsJan VINJEHead National Calicivirus Laboratory, Director CaliciNet, Division ofViral Diseases, Centers for Disease Control and Prevention (CDC),Atlanta, USAAmong the gastroenteritis viruses, noroviruses are the leading cause ofacute gastroenteritis outbreaks in humans worldwide and they recentlysurpassed rotavirus as most important cause of medically-attended gastroenteritisin children < 5 years of age. Sapoviruses, astroviruses andadenovirus type 40/41 are primarily associated with disease in young children.An estimated 71,000 hospitalizations and 800 deaths due to norovirusoccur in the US each year and a significant proportion of all foodborneoutbreaks can be attributed to norovirus. Food may become contaminatedat the source (i.e., irrigation, shellfish) or at point of service (e.g. infectedfoodhandler). Recent advances in standardized virus detection and typingmethods integrated in comprehensive surveillance networks (CaliciNet,NVSN, FBVE, NoroNet) have let to a better understanding of the impactof norovirus gastroenteritis among different risk groups as well as trendsin strain distribution over the years. Worldwide, the majority of norovirusoutbreaks are caused by genotype GII.4, of which new strains (GII.42006b, GII.4 2009 New Orleans, GII.4 2012 Sydney) emerge every 3years. An interesting trend which is not completely understood is that certainnon-GII.4 genotypes (GI.6, GI.7, GII.6, GII.7, and GII.12) are morelikely associated with foodborne outbreaks. Recent research on specificintervention therapies, such as antivirals or vaccines, have demonstratedthat these measures may become available to reduce disease burden in themost vulnerable groups such as the elderly and healthcare workers.ORAL COMMUINCATIONSREF O95Lewis negative phenotype is a strong restriction factor for genotypeP[8] rotavirus infectionsJohan NORDGREN 1 , Filemon BUCARDO 2 , Sumit SHARMA 1 , GökçeGÜNAYDIN 3 , Waqas NASIR 4 , Djeneba OUERMI 5 , León NITIEMA 5 ,Sylvia BECKER DREPS 6 , Margarita PANIAGUA 2 , JacquesSIMPORE 5 , Göran LARSON 4 , Lennart HAMMARSTRÖM 3 , LennartSVENSSON 11 Division of Molecular Virology, Linköping University, Linköping,SWEDEN; 2 Department of Microbiology, University of León, León,NICARAGUA; 3 Division of Clinical Immunology, Karolinska UniversityHospital Huddinge, Stockholm, SWEDEN; 4 Department of Clinical Chemistryand Transfusion Medicine, University of Gothenburg, Gothenburg,SWEDEN; 5 Centre de Recherche Biomoléculaire Pietro Annigoni SaintCamille CERBA/LABIOGENE, Université de Ouagadougou, Ouagadougou,BURKINA FASO; 6 Department of Family Medicine, University ofNorth Carolina School of Medicine, Chapel Hill, USAThe rotavirus (RV) surface protein VP4, is responsible for viral attachmentand entry. Two VP4 genotypes P[4] and P[8] are common worldwide,while genotype P[6] is more common in Africa. A recent in vitro studyshowed binding of these P genotypes to human histo blood group antigens(HBGAs), suggesting them as putative receptors.Here, we show that the Lewis HBGAs are strong restriction factors forcertain RV P genotypes. In Burkina Faso, we investigated HBGA salivaphenotypes (Lewis a and b) in children with diarrhoea (n=208). The Lewisnegative phenotype (Lea b), was unusually common in Burkina Faso(32%). Notably, P[8] RV strains (n=28) infected only Lewis positive children,mainly Lewis b (Lea b+, p

5 th European Congress of VirologyREF O97Emerging and common genotypes of Rotavirus among children withsevere diarrhea in Italy, 2007 2012Franco Maria RUGGERI 1 , Roberto DELOGU 2 , Giovanni IANIRO 2 ,Lucia FIORE 2 , ROTANET ITALY STUDY GROUP 21 Dept. of Veterinary Public Health & Food Safety, Istituto Superiore diSanità, Rome, ITALY; 2 National Center for Immunobiologicals Researchand Evaluation, Istituto Superiore di Sanità, Rome, ITALYRotavirus causes most acute diarrhea cases in infants worldwide. Despitemost of the 450,000 annual deaths are in developing world areas, morbidityis high also in industrialized countries, calling for universal use ofvaccines. Rotavirus has a segmented RNA genome, and reassortment betweenthe 11 RNA segments during dual strain infections is a major driveof virus evolution. Some animal strains adapt to infect man after zoonotictransmission and reassortment. Although human disease is mostly relatedto rotavirus genotypes G1 4, or G9, and P[4] or P[8], uncommon strainssometimes emerge and spread, raising concerns on vaccine herd immunity.Thus, molecular surveillance is useful to early detect emerging rotavirusesof animal or exotic origin. The RotaNet Italy surveillance networkwas established in Italy in 2007, and is linked to the EuroRotaNet network,including 17 European partners. Between 2007 2012, approximately 7,000rotaviruses from pediatric patients throughout Italy were genotyped. Mostviral strains belonged to common G and P types, but approximately 1.2%of rotaviruses were defined as uncommon, with respect to one or boththe G and P genotype. In particular, strains with rare G6P[6], G6P[9],G8P[4] and G12P[8] genotypes were subjected to detailed genome characterizationto explore their origin and evolution. In all cases, eight of the11 genome segments were sequenced, including genes for capsid proteinsVP4, VP7, VP6, and non structural proteins NSP1 5. Phylogenetic analysisconfirmed either exotic origin or partial reassortment with autochthonousrotaviruses.REF O98Unexplained diarrhoea in HIV 1 infected individualsBas OUDE MUNNINK 1 , Marta CANUTI 1 , Martin DEIJS 1 , Michel DEVRIES 1 , Maarten JEBBINK 1 , Sjoerd REBERS 1 , RichardMOLENKAMP 1 , Formijn VAN HEMERT 1 , Fransje SNIJDERS 2 , LiaVAN DER HOEK 1 , Cees SOL 11 Amsterdam Medical Center, Amsterdam, THE NETHERLANDS; 2 RivasBeatrix Hospital, Gorichem, THE NETHERLANDSGastrointestinal symptoms, in particular diarrhoea, are common in nontreated HIV infected persons. Although various enteric pathogens havebeen implicated, the etiology of diarrhoea remains unexplained in a largeproportion of HIV 1 infected patients. The hypothesis is that unexplaineddiarrhoea could be caused by yet unidentified pathogens, or by HIV 1itself. In this study we investigated stool samples of 196 HIV 1 infected persons,of whom 44 had diarrhoea. In 52% of the diarrhoea cases a causativeagent could be identified via sensitive detection assays (mostly norovirus),yet 48% remained enteropathogen negative. Among these enteropathogennegative cases, HIV 1 shedding in stool was frequently observed (67%),and there was more frequently HIV 1 RNA in diarrhoea stool samplesthan in non diarrhoea (p=0.05). A search for unknown or unexpectedpathogens was performed using virus discovery cDNA AFLP combinedwith Roche 454 sequencing (VIDISCA 454). Although a range of newor recently identified pathogens was identified (e.g. cosavirus, aichivirus,human gyrovirus, NANB 1 virus and a previously unknown virus:IASV (immunodeficiency associated virus)), investigation of diarrhoeaand control samples revealed that no association with disease is present.In conclusion, unexplained diarrhoea in HIV 1 infected patients is probablynot caused by yet unknown pathogens, but it seems more likelythat HIV 1 itself causes intestinal mucosal abnormalities which leads todiarrhoea.REF O99Rotavirus infections in young children in Ho Chi Minh City, VietnamPhan VU TRA MY 1,2 , Celeste DONATO 3 , Daniel COWLEY 3 , CorinneTHOMPSON 1,4 , Maia RABAA 1,4 , Hoang Le PHUC 5 , Pham Thi NgocTUYET 6 , Ha VINH 2 , Nguyen Tran CHINH 2 , Tang Chi THUONG 5 ,HaManh TUAN 6 , James CAMPBELL 1,4 , Jeremy FARRAR 1,4 , CarlKIRKWOOD 3 , Stephen BAKER 1,4,71 Oxford University Clinical Research Unit, Ho Chi Minh, VIETNAM;2 Hospital for Tropical Diseases, Ho Chi Minh, VIETNAM; 3 Murdoch ChildrensResearch Institute, Melbourne, AUSTRALIA; 4 Centre for TropicalMedicine, Oxford, UNITED KINGDOM; 5 Children’s Hospital 1, Ho ChiMinh, VIETNAM; 6 Children’s Hospital 2, Ho Chi Minh, VIETNAM; 7 TheLondon School for Hygiene and Tropical Medicine, London, UNITEDKINGDOMDespite the introduction of rotavirus (RV) vaccine, this enteric pathogenremains the principal cause of acute childhood diarrhea globally. RVinfections are particularly problematic in industrializing countries wherevaccine uptake has been limited and as a consequence of a lack of routinediagnosis, there is little data on the diversity of circulating RV genotypes.We performed a prospective hospital based study of RV induced diarrheabetween May 2009 and April 2010 to investigate the distribution of RVcausing diarrheal disease in children under the age of five years in Ho ChiMinh City, Vietnam. RV was detected in 46.8% of hospitalized diarrhealpatients (664/1,419), among which, 11.6% (74/664) were co infected withan additional viral and/or a bacterial pathogen. Genotyping of 664 RVisolates demonstrated a preponderance of G1 (81.9%) and P[8] (96.4%)genotypes with other genotypes detected at lower frequencies. Within therarer genotypes, we identified a novel G26 (0.6%) and GP combinationsof G3P[19] (0.2%), G9P[19] (1.4%) and G26P[19] (0.6%). The G26 RVhas been reported in a piglet before, but not reported in humans, leadingto our hypothesis that this novel G26P[19] RV may be a reassortment ofhuman and porcine RV strains. We performed whole genome sequencingfor a representative G26P[19] strain, and identified genome constellationof human derived and porcine related segments (G26 P[19] I5 R1 C1 M1A8 N1 T1 E1 H1). These data show that diverse RV genotypes causechildhood diarrhea and highlights the potential of zoonotic transfer of RVstrains from pigs to human.REF O100Molecular dating in the evolution of primate bocavirusesIgor BABKIN 1 , Irina BABKINA 1 , Aleksandr TUMENTSEV 2 , ArtemTIKUNOV 1,2 , Aleksander KURILSHIKOV 1 , Nina TIKUNOVA 11 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk,RUSSIA; 2 Novosibirsk State University, Novosibirsk, RUSSIAHuman bocavirus (HBoV) is associated with acute gastroenteritis inhumans, occurring mostly in young children and elderly people. Fourbocavirus genotypes have been found so far. Since there were no data onthe contribution of HBoV to gastroenteritis in Russia, 1781 fecal samplesS78 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologycollected from infants hospitalized with acute gastroenteritis in Novosibirsk,Russia during one year were tested for the presence of nucleicacids from HBoV and three major gastrointestinal viruses (rotavirus A,norovirus II, and astrovirus). HBoV was detected only in 1.9% of thesamples. Complete genome sequencing of three Novosibirsk isolates wasperformed. An evolutionary analysis of these sequences and the availablesequences of human and great apes bocaviruses demonstrated that thecurrent HBoV genotypes diverged comparatively recently, about 60–300years ago. The independent evolution of bocaviruses from chimpanzeesand gorillas commenced at the same time period. This suggests that theseisolates of great apes bocaviruses belong to separate genotypes within thespecies of human bocavirus, which is actually the primate bocavirus. Therate of mutation accumulation in the genome of primate bocaviruses hasbeen estimated. It has been demonstrated that HBoV1 diverged from theancestor common with chimpanzee bocavirus approximately 60–80 yearsago, while HBoV4 separated from great apes bocaviruses about 200–300years ago. The hypothesis postulating independent evolution of HBoV1and HBoV4 genotypes from primate bocaviruses has been proposed.Virologie, Vol 17, supplément 2, septembre 2013S79

5 th European Congress of VirologyFriday 13 th September 2013, 11h00 – 13h15PLENARY SESSION 3: “HOST RESTRICTION ANDBARRIERS TO VIRAL INFECTION”Chairpersons: Gabriela CAMPADELLI-FUIME (Bologna,ITALY) & Otto HALLER (Freiburg, GERMANY)Amphitheater11:00 – 11:30Innate sensing and IFN induction during infection with DNA virusesSoren PALUDANDepartment of Medical Microbiology and Immunology, Aarhus University,Bartholin Building, DK-8000 Aarhus C, DENMARKSeveral important human pathogenic viruses carry their genetic informationin the form of DNA in all or parts of the viral replication cycle.Recently it has emerged that microbial DNA is a potent stimulator ofinnate immune responses including interferon (IFN) production and aseries of intracellular and endosomal DNA sensors have been identified.Despite this, there is still limited knowledge on how and where inthe cell viral DNA is sensed by the innate immune system, how downstreamsignaling is activated, how viruses seek to evade these responses,and also the impact of DNA sensing on antiviral responses in vivo. Inthis talk, I will present the current knowledge on how DNA virusesstimulate IFN responses and discuss how this translates to antiviral responses,including the interaction with other innate immunological sensingsystems.11:30 – 12:00RNA virus persistence in Drosophila is controlled by siRNAs producedfrom de novo synthesized virus cDNAMaria-Carla SALEHInstitut Pasteur, Viruses and RNA Interference, Centre National de laRecherche Scientifique URA3015, Paris, FRANCEThe establishment and maintenance of persistent viral infections is widelydebated and remains largely misunderstood. We show that in Drosophila,persistence is achieved through a mechanism involving reverse transcriptionof non-retroviral RNA virus and the RNA interference pathway.Fragments of diverse RNA viruses are reverse transcribed early duringinfection, resulting in DNA forms embedded within LTR-retrotransposonsequences. Inhibition of reverse transcription hinders the appearance ofthese DNA forms and is accompanied by increased viral titers and celldeath. These viral/retrotransposon-DNA chimeras produce transcripts thatare processed by the RNAi machinery. Knocking down RNAi componentsin persistently infected cells shifts the equilibrium from persistent to acuteinfection. Our results reveal an unanticipated physiological function forretrotransposons and propose a role in RNAi-mediated immune protectionfor parasitic viral insertions into host genomes.12:00 – 12:30Viral adaptations preceding the AIDS pandemicFrank KIRCHHOFFInstitute of Molecular Virology, Ulm University Medical Center, Ulm,GERMANYCross-species transmissions of primate lentiviruses from monkey to apesand from apes to humans preceded the emergence of AIDS. Here, I willsummarize some of our current knowledge on the evolution and specificproperties of pandemic HIV-1 strains that most likely contributed toits effective spread and high virulence. It will also be discussed why therestriction factor tetherin, that inhibits virion release, poses a significanthurdle to cross-species transmissions of SIVs to humans. We found thatonly pandemic HIV-1 M (major) strains mastered this barrier perfectlyby “regaining” efficient tetherin activity by Vpu following the four independentcross-species transmissions that resulted in HIV-1 groups M, O,N, and P. This may explain why HIV-1 M it is almost entirely responsiblefor the HIV/AIDS pandemic. Furthermore, evidence will be presentedthat adaptation of rare or non-pandemic group N and O viruses to humansis still ongoing and that the multi-functionality of the accessory proteinsmay help primate lentiviruses to regain “lost” activities after cross-speciestransmission.12:30 – 13:15AWARD of the European Society of VirologyPathways of animal virus entryAri HELENIUSInstitute of Biochemistry, ETH Zurich, Schafmattstrasse 18, Zurich, CH-8093, SWITZERLANDWhen viruses enter animal cells most of them follow a stepwise programthat involves attachment to the cell surface, activation of signaling pathways,endocytosis, penetration into the cytosol, intracellular transport, andfinally uncoating of the viral genome. Since all these steps depend on cellularfunctions, hundreds of cellular proteins inadvertently end up assistingthe viruses. We have analyzed the entry into tissue culture cells of differentviruses using high-end light microscopy, automated siRNA and drugscreening, as well as biochemical and biophysical approaches. The virusesinclude members of alpha-, rhabdo, myxo-, paramyxo-, polyoma, herpes-,arena-, and poxvirus families. Our goal has been to trace the endocytosisand trafficking pathways taken by the incoming virus particles, definethe changes that trigger penetration and uncoating, and identify host cellfactors involved in each step. We find five distinct locations where penetrationcan occur; the plasma membrane, early endosomes, maturing andlate endosomes, macropinosomes, and the ER. I will review the generalconcepts, and describe in some detail the entry and uncoating strategiesused by influenza A and vaccinia virus.S80 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyFriday 13 th September 2013, 13h30 – 14h45SYMPOSIUM QIAGEN“ACHIEVING CLINICAL AND OPERATIONALEXCELLENCE THROUGH WORKFLOW OPTIMISATION”Chairpersons:Martin SCHUTTEN (Rotterdam, THE NETHERLANDS),David BOUTOLLEAU (Paris, FRANCE)Room Prestige Gratte-CielKEYNOTE: Modernizing in house developed molecular assays, theneed for automationMartin SCHUTTENRotterdam, The NetherlandsDetection and quantification of CMV DNA in peripheral blood andamniotic fluid in pregnancy complicated by primary CMV infectionFausto BALDANTIPavia, ItalyImproving laboratory efficiency and output: Comparison of artus QS-RGQ and established in-house workflowsTim CONIBEARLondon, United KingdomVirologie, Vol 17, supplément 2, septembre 2013S81

5 th European Congress of VirologyFriday 13 th September 2013, 13h30 – 14h45SYMPOSIUM SANOFI PASTEUR“NEW PARADIGM FOR DENGUE PREVENTION”Chairpersons: Nicholas JACKSON (FRANCE)& Patrick MAVINGUI (FRANCE)Room Tête d’OrCharacterization of the Sanofi Pasteur dengue vaccine candidate:hypotheses and investigations to explain results of the Phase IIb proofof-conceptefficacy trial in RatchaburiBruno GUYSanofi Pasteur, Lyon, FRANCEThe Sanofi Pasteur CYD tetravalent dengue vaccine candidate has beenextensively characterized at the microbiological and immunological levels,both in preclinical models and in clinical trials. The results from theseinvestigations showed that the vaccine induces broad and significantimmune response. However, the proof-of-concept efficacy trial of thisvaccine—a Phase IIb trial conducted in Thailand between 2009–2012(ClinicalTrials.gov: NCT00842530)—showed that this does not necessarilytranslate into protection against disease caused by all viruses of all 4serotypes. Indeed, despite high PRNT50 titers against DENV2, protectionwas not observed against the circulating DENV2 viruses. The researchprogram initiated to understand this unexpected result was built assumingthat the underlying explanation involves one or more of four key factorsinfluencing vaccine-induced immunity: the circulating viruses, the vaccine,the mosquito vector, and host immunity. Ongoing investigations, inpartnership with academic laboratories, are exploring the contributionsof: interference between vaccine serotypes, host immunity prior to vaccination,quantitative and qualitative immune responses (e.g., heterotypicand homotypic responses; enhancing and neutralizing responses), and cellmediated immunity.Role of the vector in dengue epidemic dynamicPatrick MAVINGUIMicrobial Dynamics and Virus Transmission Team, Microbial EcologyLaboratory, UMR CNRS 5557, USC INRA 1364, VetAgro Sup, UniversityLyon 1, Lyon, FRANCEMosquitoes are major vectors of arboviruses (arthropod-borne viruses) tohuman and animals. The species Aedes aegypti is recognized as the primaryepidemic vector of dengue virus (DENV). Recently, the sister taxon Aedesalbopictus, known as a secondary DENV vector, has been involved indengue outbreaks in several countries in Africa and Asia. These outbreakscoincided with a high increase in reports on the presence of Aedes albopictusthat seems to outcompete Aedes aegypti. Indeed, the last twenty years,Aedes albopictus originated from South-East Asia has become one of themost invasive mosquito species with an extending worldwide distribution,including in temperate regions such as Europe where it was incriminatedin the transmission of dengue cases. We will report the role of the twoAedes species in relation to recent data on spatiotemporal dynamics ofdengue epidemics.Arbovirus emergence caused by small genetic changes in the viralgenomeAnna-Bella FAILLOUXInstitut Pasteur, Arboviruses and Insect Vectors Laboratory, Departmentof Virology, 25 rue du Dr Roux, 75724 Paris cedex 15, Paris, FRANCEEmergence of arboviruses could result from their ability to exploit newenvironments, for example a new host. This ability is facilitated by the highmutation rate occurring during viral genome replication. The last emergenceof chikungunya (CHIK) in the Indian Ocean region corroboratesthis statement since a single amino acid change at the position 226 in theE1 glycoprotein in a region predicted to interact with the target membrane,has been selected for efficient transmission by an unusual mosquito speciesAe. albopictus, mostly in areas where the main vector Ae. aegypti wasabsent or less abundant. The variant harbouring the amino acid change hasmediated enhanced transmission by Ae. albopictus which is able to deliverinfectious viral particles by its saliva two days after ingesting an infectiousblood-meal. We demonstrated that the midgut barrier plays a key role inselecting this emerging CHIK variant. Thus, some point mutations in theviral genome may contribute to facilitate the adaptation to a particular vector.We are testing using experimental transmission if dengue virus can beselected for an enhanced transmission potential by the secondary vector,Ae. albopictus.Functional genomic study of asymptomatic versus symptomaticdengue viral infectionAnavaj SAKUNTABHAIFunctional Genetics of Infectious Diseases Unit, Department of Genomeand Genetics, Institut Pasteur, Paris, FRANCEA recent study of the global dengue burden estimated 390 million dengueviral (DENV) infection per year of which 96 million were apparent(symptomatic and come to medical attention). The key immunoprotectionagainst DENV could be identified from these inapparent DENVinfections who can clear the virus without developing symptoms. Weperformed a household investigation of patients with apparent DENVinfection in Cambodia and Cuba to detect individuals who had dengue viremiawithout clinical symptoms. We performed a whole genome study andtranscriptomic analysis of peripheral blood mononuclear cells comparingasymptomatic and symptomatic DENV infection. We identified naturalkiller cells and cytotoxic T cells activated in individuals with asymptomaticinfection whereas patients with dengue disease showed T helper 2 immunologicalresponses and activation of neutrophils. In addition, we foundevidence of genetic susceptibility to severe dengue in the European descentin Cuba.S82 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyFriday 13 th September 2013, 15H00 – 17h15WORKSHOP 7: “CELLULAR FACTORS CONTROLINGVIRAL INFECTION AND ROLE OF HOST GENETICS”Chairpersons: Frank KIRCHHOFF (Ulm, GERMANY)& Aine McKNIGHT (London, UNITED KINGDOM)Room Prestige Gratte-CielKEYNOTE:Host cell factors and pathways promoting and restricting hepatitis Cvirus replicationRalf BARTENSCHLAGERDepartment of Infectious Diseases, Molecular Virology, University of Heidelberg,Im Neuenheimer Feld 345, Heidelberg, GERMANYHepatitis C viruses (HCV) comprise a group of positive-strand RNAviruses belonging to the Flaviviridae family. As a major cause of acuteand chronic liver disease worldwide, HCV has received much attention.With the advent of highly efficient and robust cell culture systems forHCV, the principles of the viral replication cycle have been unravelled. Asurprisingly high number of molecules that are essential for or that promoteHCV entry into hepatocytes has been identified. Moreover, importantinsights into the biogenesis and architecture of the membranous replicationcompartment induced upon viral infection have been gained. Severalhost cell factors required for formation or activity of the viral replicationmachinery, such as cyclophilin A and phosphatidyl inositol-4-kinase IIIalpha,have been identified and they represent attractive targets for hostfactor-targeting antiviral therapy. Finally, it was found that HCV assemblyoccurs in close association with cytosolic and luminal lipid dropletsexplaining, at least in part, why HCV particles are tightly associated withhost cell lipoproteins and lipids.A hallmark of HCV infection is the high rate of persistence (∼80%)arguing that this virus has developed efficient strategies to overcome innateand adaptive immune responses. It is well established that the NS3 serinetypeprotease blocks the induction of antiviral cytokines by proteolyticcleavage of adaptor proteins involved in RIG-I and TLR-3 dependentsignalling. Paradoxically however, in infected patients interferon-inducedgenes are activated and the degree of activation negatively correlates withoutcome of interferon-based therapy. While the underlying mechanismsremain to be determined, it is obvious that HCV has developed strategiesto sustain persistence in spite of an antiviral program.ORAL COMMUNICATIONSREF O101Hepatitis C virus infection modulates the central carbon metabolismof the cellOlivier DIAZ 1,2 , Christophe RAMIÈRE 1,2,3 , Jonathan RODRIGUEZ 1,2 ,Aurélie BADILLO 4 , Jean Charles PORTAIS 5 , Vincent LOTTEAU 1,2 ,Patrice ANDRÉ 1,2,31 Université de Lyon, Lyon, FRANCE; 2 INSERM U1111 – CNRSUMR5308 Université Lyon 1, ENS de Lyon, Lyon, FRANCE; 3 HospicesCivils de Lyon, Lyon, FRANCE; 4 UMR 5086 CNRS Université Lyon 1,Lyon, FRANCE; 5 UMR5504, UMR792 Ingénierie des Systèmes Biologiqueset des Procédés CNRS, INRA, INSA, Toulouse, FRANCEPerturbations of glucido lipidic metabolism, induced by Hepatitis C virus(HCV) infection in the hepatocyte, are not completely elucidated. Recentlyseveral viruses were described as capable to subvert central carbon metabolism(CCM) to support their replication and assembly. We thus investigatedhow HCV modulates CCM. We monitored uptake and release of key metabolitesin supernatants of HCV infected Huh7.5 cells during 72 h postinfection. Glucose and glutamine uptake and lactate excretion were increasedindicating glycolysis modulations in infected cells. We identified theprotein protein interaction of NS5A with hexokinase (HK), the first andrate limiting enzyme of glycolysis. Enzymatic HK assay performed withcytosolic fraction of cells replicating the HCV subgenomic replicon (Huh913) revealed an activity increase. The same observation was performed incells overexpressing only NS5A. Moreover, addition of purified NS5A inin vitro HK assay was sufficient to increase HK activity. To further definefunctionally how HCV modulate CCM, 42 different metabolic moleculesreferred as intermediates or connected to glycolysis and citrate cycle metabolismwere analyzed in HCV infected Huh7.5 cells using HPAEC andIDMS. Data revealed that beside the apparent global increased glycolysisin infected cells, nucleotide precursor’s synthesis appeared enhancedwhereas no changes were observed within cell energy status. All togetherthese observations indicate a direct interaction between non structuralHCV proteins and glycolysis enzymes at the origin of CCM modulation byHCV.REF O102Coxsackievirus Hijacks Host miRNA 126 to Mediate Cross talkof ERK and Wnt/beta catenin Signal Pathways for Its ReplicationDecheng YANG, Xin YE, Maged HEMIDA, Ye QIU, Paul HANSON,Huifang Mary ZHANGUniversity of British Columbia, Vancouver, CANADACoxsackievirus B3 (CVB3), a positive single stranded RNA virus, is amember of the Enterovirus genus in the Picornaviridae family. It is the primarypathogen of viral myocarditis. CVB3 infection causes directed injuryof the heart by rapid replication in the myocardium. However, the molecularpathways leading to myocarditis remain unclear. microRNA (miRNA),as a novel regulator of gene expression, may play an essential role in CVB3pathogenesis. To study the functional role of miRNAs in modulating CVB3replication, we selected a heart abundant miRNA, miR 126, to test ourhypothesis. Here we describe a novel mechanism by which miR 126 regulatestwo signal pathways essential for CVB3 replication. We found thatCVB3 induced ERK1/2 activation triggered the phosphorylation of ETS1/2 transcription factors, which induced miR 126 upregulation. By usingboth miR 126 mimics and its inhibitors, we proved that the upregulatedmiR 126 suppressed SPRED1(sprout related, EVH1 domain containing 1)expression and in turn enhanced ERK1/2 activation. This positive feedbackloop of ERK1/2 miR 126 ERK1/2 promoted CVB3 replication at earlyphase of infection. Meanwhile, miR 126 expression stimulated the GSK3b activity and induced degradation of beta catenin via suppressing LRP6(low density lipoprotein receptor related protein 6) and WRCH1(Wnt responsiveCdc42 homolog 1), two newly identified targets in the Wnt/catenin pathway, which sensitized the cells to virus induced cell deathand increased viral progeny release to initiate new infections. Our resultsdemonstrate for the first time that dynamically upregulated miR 126 uponCVB3 infection targets SPRED1, LRP6 and WRCH1 genes, mediatingcross talk between ERK1/2 and Wnt/beta catenin signal pathways, andthus promotes viral replication and contributes to the viral cytopathogenicity.Virologie, Vol 17, supplément 2, septembre 2013S83

5 th European Congress of VirologyREF O103Human respiratory syncytial virus infection is modulated by StreptococcuspneumoniaeDuy Tien NGUYEN 1 , Rogier LOUWEN 2 , Geert VAN AMERONGEN 1 ,Ken LEMON 3 , Ad LUIJENDIJK 2 , Karin ELBERSE 4 , AlbertD.M.E. OSTERHAUS 1 , W. Paul DUPREX 5 , Rik L. DE SWART 11 Department of Viroscience, Erasmus MC, Rotterdam, THE NETHER-LANDS; 2 Department of Medical Microbiology and Infectious Diseases,Erasmus MC, Rotterdam, THE NETHERLANDS; 3 Department of Microbiology,Queen’s University of Belfast, Belfast, UNITED KINGDOM;4 National Institute of Public Health and the Environment, Bilthoven, THENETHERLANDS; 5 Department of Microbiology, Boston University, Boston,USAHuman respiratory syncytial virus (HRSV) and Streptococcus pneumoniaeare important causative agents of respiratory infections around the world.The burden for HRSV is highest in the pediatric population. Almost allinfants are infected by HRSV before the age of two years, but only asmall proportion develops severe disease. Bacterial pathogens such as S.pneumoniae can often be cultured from children hospitalized with HRSV.Interactions between viral and bacterial pathogens have been describedboth in vitro and in vivo, but causal relationships are poorly understood.S. pneumoniae has the ability to colonize humans without manifestationof disease. We have investigated whether S. pneumoniae colonizationmodulates HRSV infection, using a new recombinant (r) HRSV fromsubgroup B that expresses enhanced green fluorescent protein (EGFP),rHRSVB05EGFP. Ten S. pneumoniae serotypes obtained from colonizedchildren were obtained from a large cohort study. We have used humanB lymphocytes, primary well differentiated normal human bronchial epithelial(wd NHBE) cells with tight junctions and cilia for our in vitrostudies. Five of the ten S. pneumoniae serotypes enhanced HRSV infectionin B cells, and four of these also enhanced HRSV infections of wdNHBE cells. In ex vivo lung slice cultures obtained from uninfected cottonrats six S. pneumoniae serotypes enhanced HRSV infection. In conclusionwe show that S. pneumoniae colonization can modify HRSV infection invitro. Studies designed to investigate whether S. pneumoniae modulatesHRSV infection in cotton rats are ongoing.REF O104Gene loss and adaptation to hominoids underlie the ancient originof HIV 1Lucie ETIENNE 1 , Beatrice H. HAHN 4 , Frederick A. MATSEN 3 ,Michael EMERMAN 1,21 Division of Human Biology, Fred Hutchinson Cancer Research Center,Seattle, USA; 2 Division of Basic Sciences, Fred Hutchinson CancerResearch Center, Seattle, USA; 3 Division of Public Health Sciences,Fred Hutchinson Cancer Research Center, Seattle, USA; 4 Department ofMicrobiology, Perelman School of Medicine, University of Pennsylvania,Philadelphia, USAHIV 1 resulted from the cross species transmission of SIVcpz, a simianimmunodeficiency virus that naturally infects chimpanzees. SIVcpz, inturn, arose from the species jump and recombination of two SIV lineagesfrom old world monkeys. Lentiviral cross species transmissions are partlydriven by the capacity of viral accessory genes to adapt to antagonize hostantiviral factors. Therefore, to understand the ultimate origins of HIV 1,we analyzed the mechanisms by which the recombinant SIVcpz adaptedto hominoids.Phylogenetic analyses showed that the birth of SIVcpz was accompaniedby the complete loss of the accessory gene vpx, which, in other SIVs,is used to antagonize the host protein SAMHD1. Remarkably, the geneloss was associated with the reconstruction of the overlapping vif gene by“overprinting” using a cryptic stop codon in an alternate reading frame.The novel Vif notably acquired the conserved PPLP motif that plays a rolein HIV 1 Vif function. Overall, these events and subsequent mutationsresulted in a Vif that was more potent at restricting the hominoid versionof the antiviral protein APOBEC3G, but also led to the inability of SIVcpzto counteract the restriction factor SAMHD1. Lastly, we show that adaptationof SIV to chimpanzees facilitated the ability of Vif to antagonizehuman APOBEC3G, which was in favor of a successful viral emergencein humans.Thus, a complex set of adaptive events in response to selective pressurefrom hominoid restriction factors led to the origin of SIVcpz, andcontributed to lowering the species barrier to human infection.REF O105A novel mechanism of human cytomegalovirus evasion from restrictionfactor IFI16Valentina DELL’OSTE 1 , Deborah GATTI 1 , Francesca GUGLIESI 1 ,Marco DE ANDREA 1,2 , Matteo BIOLATTI 1 , Marta VALLINO 3 ,Manfred MARSCHALL 4 , Marisa GARIGLIO 2 , Santo LANDOLFO 11 Department of Public Health and Pediatric Sciences, University of Turin,Turin, ITALY; 2 Department of Translational Medicine, University of PiemonteOrientale “A. Avogadro”, Novara, ITALY; 3 Department of LifeSciences and Systems Biology, University of Turin, Turin, ITALY; 4 Institutefor Clinical and Molecular Virology, University of Erlangen Nuremberg,Erlangen, GERMANYIntrinsic immune mechanisms constitute an antiviral frontline defensemediated by constitutively expressed proteins termed “restriction factors”.Human Cytomegalovirus (HCMV) has evolved mechanisms to efficientlyevade the antiviral state. We have demonstrated that the DNA sensorIFI16 restricts HCMV replication by down regulating viral early and latemRNAs as well as their protein expression. Here, we show that duringearly stages of infection, HCMV induces IFI16 overexpression in primaryhuman fibroblasts (HELF). Despite this induction, continuous virus replicationindicates that somehow the virus is able to escape IFI16 restrictionactivity. Confocal microscopy revealed that, starting from 24 hours postinfection, IFI16 relocalizes from the nucleus to the virus assembly complex(AC), where it colocalizes with viral structural proteins such as gBand pp65. We demonstrate that the viral kinase pUL97 binds and phosphorylatesIFI16 thus triggering its egression from the nucleus of infectedcells. Then, the IFI16 AC complex allows IFI16 carriage in the newlyassembled virions where it is embedded in the matrix superficial layersas demonstrated by electron microscopy analysis. Altogether, these datashows a new mechanism through which HCMV is able to neutralize IFI16restriction activity by inducing first its delocalization and then its trappinginto egressing virions.REF O106Use of exome sequencing and RNAi to understand the influenceof host factors on the outcome of influenza infectionRachael WASH 1 , Deepti GURDASANI 1 , Stephanie FRANZ 1 , CarmenDIAZ SORIA 1 , Manj SANDHU 1,3 , Paul KELLAM 1,21 Wellcome Trust Sanger Institute, Cambridge, UNITED KINGDOM;2 University College London, London, UK; 3 University of Cambridge,Cambridge, UNITED KINGDOMDuring the influenza A H1N1/09 pandemic hospitalisation rates in Englandwere estimated at 4.7/100,000 population. Although many patients hadone or more pre existing comorbidities, up to 40% of patients did not andwere previously healthy. Whole virus genome sequencing of H1N1/09showed the extent of virus variation. Data suggests that whilst genomevariation occurs, and can subtly alter replication properties of the virus,it is not sufficient to account for severe or fatal influenza infection. Alterationsin human genes, such as IFITM3, that interact with the virus arehowever associated with severe influenza pathogenesis. To understand theinfluence of host genetics on virus infection we have analysed in detailS84 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologythe whole human exome sequence of patients who were severely infectedwith influenza during the pandemic that did not have severe comorbidities.Through this approach we have identified potential SNP variantsthat may affect susceptibility to influenza infection. We have used RNAiscreening in A549 cells to knockdown genes where SNP variants havebeen identified, followed by infection with influenza to validate this datain vitro. To gain more insight into the effect of these variants we have alsocompared influenza infection of human lymphoblastoid cell lines, whichhave either the ‘reference/wild type’ genotype or the SNP variant identifiedin our exome analysis associated with severe influenza. The results ofthis powerful strategy to identify host factors that affect the outcome ofinfluenza infections will be discussed.REF O107Characterization of one novel interferon stimulated gene participatingin the type I interferon block to HIV 1 infectionCaroline GOUJON 1 , Olivier MONCORGÉ 2 , Christopher WARD 1 ,Tomas DOYLE 1 , Hélène BAUBY 1 , Stéphane HUÉ 3 , WendyS. BARCLAY 2 , Reiner SCHULTZ 4 , Michael H. MALIM 11 Department of Infectious Diseases, King’s College London, London,UNITED KINGDOM; 2 Division of Infectious Diseases, Imperial CollegeLondon, London, UNITED KINGDOM; 3 Department of Infection, UniversityCollege London, London, UNITED KINGDOM; 4 Department ofMedical & Molecular Genetics, King’s College London, London, UNITEDKINGDOMType I interferon (IFN) induces a potent block to the early steps of HIV1 infection in some cell types, including primary CD4+ T cells, macrophages,THP 1 and U87 MG cells. We have previously shown that othercell types, such as T cell lines, were unable to block HIV 1 infection followingIFN stimulation despite normal IFN responsiveness. Consequently,we used a comparative transcriptomic analysis in permissive versus restrictivecells following IFN treatment to generate a list of interferon stimulatedgenes (ISGs) with potential anti HIV 1 activity. We screened a series of candidateISGs using a lenviral vector containing an antisense and inducibleexpression cassette.The overexpression in U87 MG cells of one of these ISGs, previously notknown to be anti viral, conferred a potent block to HIV 1 infection (up to10 50 fold decrease in infection efficiency). The block was observed withseveral strains of HIV 1, and both with wild type and VSV G pseudotypedviruses. This gene was highly induced by type 1 IFN in primary cells as wellas U87 MG and THP 1 cells, but its expression was almost undetectablein T cell lines. Importantly, the silencing of this gene partially rescuedthe IFN induced block to HIV 1 infection, both in U87 MG and THP 1cells (7 10 fold rescue out of 35 50 fold decrease in infection with IFN).Ongoing efforts include determining the range of anti viral specificityof this ISG and identifying the specific step at which it blocks HIV 1infection.Taken together, we have identified one new and potent effector of the typeI IFN response against HIV 1.Virologie, Vol 17, supplément 2, septembre 2013S85

5 th European Congress of VirologyFriday 13 th September 2013, 15h00 – 17h15WORKSHOP 9: “ANTIVIRAL THERAPY ANDRESISTANCE TO CHRONIC VIRAL INFECTION”KEYNOTE:Chairpersons: Henri AGUT (Paris, France)& Christine C. GINOCCHIO (Lake Success, USA)AmphitheaterCell-cell transmission governs spread and immune escape of HIV-1Alexandra TRKOLAInstitute of Medical Virology, University of Zurich, SWITZERLANDHIV enters and infects cells via two distinct routes either as cell-free virionor via cell-to cell transmission. While dependency on primary and coreceptorare identical in both pathways, cell-cell transmission is distinctivelymore effective than free virus infection in vitro. To what extent this reflectson the relative importance of the cell-cell transmission mode in naturalinfection has not yet been resolved. Recent findings highlight that, besidesoffering a more efficacious entry route for HIV, cell-cell transmission providesan essential strategy for the virus to evade the host’s neutralizingantibody response.ORAL COMMUNICATIONSREF O108Simultaneous Hepatitis C virus (HCV) NS3 protease and NS5B polymeraseresistance testing by an alternative error correction 454 deepsequencing pipelineF. Xavier LÓPEZ LABRADOR 1,4 , Karina SALVATIERRA 1 , AmbarGRIJALVA 2 , Elisa MARTRÓ 3,4 , Veronica ORTIZ 2 , AlejandroARTACHO 1 , Nancy LÓPEZ 2 , Ferràn PALERO 1 , John ARCHER 5 ,Marina BERENGUER 6,71 Joint Unit in Genomics and Health, Center for Public Health Research(CSISP)/Institut Cavanilles, University of Valencia. Publ, Valencia,SPAIN; 2 Minority Health and Health Disparities International ResearchTraining Program (MHIRT). National Institutes of Health/School of,Irvine, U.S.A.; 3 Microbiology Service, Hospital Universitari GermansTrias i Pujol, Autonomous University of Barcelona, Badalona, SPAIN;4 CIBER ESP (Centro de Investigación Biomédica en Epidemiología ySalud Publica), ISCIII, SPAIN; 5 Faculty of Life Sciences, Universityof Manchester, Manchester, UNITED KINGDOM; 6 Hepatology – Livertransplantation Section, Hospital Universitari La Fe, Valencia, SPAIN;7 CIBER EHD (Centro de Investigación Biomédica en Hepatologia yGastroentelogía), ISCIII, SPAIN; 8 Department Medicine, University ofValencia, Valencia, SPAINResistance mutations (RAVs) for several specific antivirals against HCV(STAT C) can be detected with deep sequencing to multiplex severalsamples in a single assay and to detect minority variants.Methods: The entire HCV NS3 protease and NS5b polymerase geneswere sequenced by Sanger chemistry in isolates from 60 patients (HCV1b). In ten selected patients, PCR products were cloned and sequenced(average 25 clones per patient/region); and simultaneous analysis of theprotease and polymerase was done by deep sequencing multiplexing (454Roche). Deep sequencing was tuned with error filtering algorithms, andmolecular clones as calibrators.Results: Conventional Sanger sequencing detected variations betweenpatients in several NS3 protease and/or NS5B sites, including RAVs(mostly to non nucleosidic inhibitors, NNI). No major resistance mutationsto boceprevir or telaprevir or nucleosidic(NI) NS5B inhibitors weredetected by Sanger, or by clonal sequencing. In contrast, RAVs not detectedby Sanger were detected by cloning, and 454 deep sequencing identifiedall variations found by cloning and also new variant sites, including new“real” and “noisy” RAVs.Conclusions: (I) Sanger sequencing is appropriate to detect major RAVs(mainly to NNI), but the “gold standard” cloning analysis is more sensitive;(II) We developed a multiplexed 454 deep sequencing pipelineallowing for simultaneous analysis of the HCV protease and polymerase;(III) Deep sequencing may be useful for detecting RAVs, but appropriateerror filtering algorithms are needed to avoid false positive results.REF O109Experience of HCV resistance after 2 years antiproteases clinicalpractice; detection of resistance mutations after long period of undetectabilityChristophe RAMIERE 1,2,3 , Mary Anne TRABAUD 1 , Vinca ICARD 1 ,Marianne MAYNARD MUET 4 , Fabien ZOULIM 4,2 , PatriceANDRÉ 1,2,3 , Caroline SCHOLTES 1,2,31 Hospices Civils de Lyon, Hôpital de la Croix Rousse, Laboratoire deVirologie, Lyon, FRANCE; 2 Université de Lyon, université Lyon 1, Lyon,FRANCE; 3 Inserm, U1111, Lyon, FRANCE; 4 Hospices Civils de Lyon,Hôpital de la Croix Rousse, Service d’hépatogastroenterologie, Lyon,FRANCENS3/4A protease inhibitors were recently approved for treatment of HepatitisC Virus (HCV) genotype 1 infection. During clinical trials resistancemutations were selected in patients with treatment failure. Their detectionbefore treatment or when virological breakthrough occur may be importantfor patient follow up and still needs to be explored. We evaluated theuse of NS3 genotyping for HCV resistance monitoring after 2 years ofclinical practice. Resistance genotyping was assessed by sanger populationsequencing of the NS3 gene in 116 genotype 1 (half 1a and half 1b)patients upon physician demand.60% of the patients were sequenced upon treatement failure, 40% beforetreatment. The majority of the patients with resistance associated variants(RAV) were of genotype 1a (63%) and only 32% of genotype 1b. Repartitionof the different RAVs will be discussed. Noteworthy RAVs weredetected 3 patients relapsing after completion of the whole therapy processand >40 weeks HCV RNA below quantification threshold. One livertransplanted patient relapsed with a low level A156S detected, althoughHCV RNA was undetectable after triple therapy on the transplantation day.Real life is in accordance with data observed in clinical trials regarding thehigher rate of selection of resistance with linear antiproteases for patientsinfected with subtype 1a versus 1b.Particular cases suggest the existence of reservoirs that may currently notbe reached with sufficient concentration by current treatments. HCV canreplicate in these sanctuary sites without contributing to plasma viral loads.REF O110Mutagenesis of HIV 1 reverse transcriptase ribonuclease H domaindefines residues contributing to ribonuclease H activity inhibition bydiketo acidsAngela CORONA 1 , Francesco Saverio DI LEVA 2 , FrancescaESPOSITO 1 , Roberto DI SANTO 3 , Roberta COSTI 3 , LucaPESCATORI 3 , Sandro COSCONATI 4 , Ettore NOVELLINO 5 , EnzoTRAMONTANO 11 Department of Life and Environmental Sciences, University of Cagliari,Cagliari, ITALY; 2 Department of Drug Discovery and Development, IstitutoItaliano di Tecnologia, Genoa, ITALY; 3 Istituto Pasteur FondazioneCenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco,“Sapienza” Università di Roma, Roma, ITALY; 4 DiSTABiF, Seconda Universitàdi Napoli, Caserta, ITALY; 5 Dipartimento di Farmacia, Universitàdegli Studi di Napoli “Federico II”, Napoli, ITALYS86 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyHIV 1 reverse transcriptase (RT) performs the viral RNA genome replicationcombining two synthetic and hydrolytic functions: DNA polymeraseand ribonuclease H (RNase H), both validated targets for drug development.HIV 1 RT inhibitors (RTI) are a primary resource for AIDStreatment, but the rapid selection of drug resistance strains moves forwardthe identification of novel RTI classes. Currently, no RNase H functioninhibitor has been approved. Diketoacid derivatives (DKA) have beenshown to inhibit HIV 1 RNase H activity by chelating Mg2+ cofactor in theactive site and to inhibit viral replication in cell culture. The scaffold of theprototype DKA, RDS1643, has been further developed to achieve compoundswith selectivity indexes >100, identifying derivatives RDS1711,RDS2291 and RDS1759. A molecular docking model suggested interactionsbetween DKA and amino acid residues, N474, R448, R557 andY501, located in the RNase H domain. Interestingly, N474 and Y501 arepart of the RNase H primer grip and well preserved for their functionalrole. To test the model, we constructed mutant RTs with single amino acidchanges in these positions and assessed the DKA effects showing that theyare inactive on mutant N474A and Y501A RTs. These results demonstratefor the first time that RNase H inhibition by DKAs is related not only totheir chelating properties, but also to the interaction with conserved residues.Therefore, they provide important insights for a more rational leadoptimization, laying the foundation for non toxic and highly selective HIV1 RNase H inhibitors.REF O111New anti CMV drugs alone or in combination: efficacy in cell cultureand placental explantsDeborah ANDOUARD 1 , Lucie MORERE 1 , Deborah ANDOUARD 1 ,Fadi SAADE 1 , William RAWLINSON 1,2 , Sébastien COTIN 1 , ClaudeCALLISTE 3 , François LABROUSSE 4 , Yves AUBARD 5 , Marie CécilePLOY 1 , Sophie ALAIN 11 Faculty of Medicine/University of Limoges/Inserm U1092 and CNRCytomegalovirus, Limoges, FRANCE; 2 Virology, SEALS MicrobiologyPrince of Wales Hospital, Randwick, AUSTRALIA; 3 Faculty ofPharmacy/University of Limoges/EA4021, Limoges, FRANCE; 4 CHUde Limoges/Pathology department, Limoges, FRANCE; 5 CHU deLimoges/Gynecology obstetrics department, Limoges, FRANCEIntroduction: Cytomegalovirus (CMV) is the first cause of congenitalviral infection. In this process, placenta acts as a gateway and inhibition ofCMV replication in placenta is essential. Current antivirals are toxic for thefetus or the mother. We thus tested the inhibitory effect of foscarnet (PFA)and less toxic new drugs Maribavir (MBV), Baïcalein (BAI), Artesunate(ART) alone or combined.Methods: laboratory strain AD169 and a clinical isolate P*from congenitallyinfected newborn were used. In vitro: Foci reduction assays in humanembryonic lung fibroblasts (MRC 5) were revealed at D5. Ex vivo: placentalvilli from 1st trimester abortion floating on gelfoam sponges wereinfected by capillarity from infected MRC 5 cells in presence of variousconcentrations of drugs. At various times post infection, explants wereremoved for immunohistochemistry and viral load measurement.Results: In vitro: MBV/PFA were synergistic with 66% inhibition at0,25 M MBV+12,5 M PFA (IC50 1 M and 50 M) as were BAI/PFA(42, 72 and 82% inhibition for BAI, PFA, and BAI 0,275 M+PFA50 M)and ART/MBV (26, 34 and 60% inhibition for ART, MBV and ART+MBV0,25 M each). The combination ART/BAI was antagonist. Ex vivo: Viabilityof explants was validated by persistent bHCG secretion and CMVinfection of syncitiotrophoblasts and fibroblasts was maximal at D18. Alldrugs show an inhibitory effect with IC50 close to that observed in vitro.Synergistic and antagonist effects were confirmed.Conclusion: Combination therapy with low toxicity compounds could bean option for prevention of CMV transmission.REF O112Generation and Characterization of defined HCMV UL97 and UL54mutations associated with drug resistanceLena FISCHER 1 , Kerstin LAIB SAMPAIO 2 , Gerhard JAHN 1 , KlausHAMPRECHT 1 , Katharina GÖHRING 1 , Else-Kröner-FreseniusFoundation1 Institute of Medical Virology, Tübingen, GERMANY; 2 Institute of Virology,Ulm, GERMANYIn HSCT recipients, drug resistant human cytomegalovirus (HCMV) infectionremains a serious problem. For characterization of newly emergingmutations marker transfer analysis is performed. Three new HCMV mutations,the UL54 mutations D515E and V715A and the UL97 mutationE596Y as well as a combination of three UL54 mutations (D515E, L516 M,I521T) were characterized.For the generation of HCMV mutants each point mutation as well asthe combination of the three UL54 mutations was introduced into theTB40/E derived bacterial artificial chromosome (BAC) Bac7 UL32 EGFPby en passant mutagenesis (Tischer et al., 2006). After reconstitution ofmutant virus, phenotypic characterization was performed by a modifiedplaque reduction assay using a HFF/ARPE co culture method. Resistancewas defined as a 2 fold decrease in drug sensitivity in comparison to thereference strain HCMV Bac7 UL32 EGFP.The UL97 mutation E596Y showed an IC50 ratio of 6 for ganciclovir(GCV) and was therefore classified as drug resistant. Both UL54 mutationscould also be classified as GCV resistant due to IC50 ratios of >2(D515E: 2,5; V715A: 3,9). Using Foscarnet (PFA), mutation D515E hadan IC50 ratio of 1,7 while mutation V715A had a ratio of 1,8 indicatingPFA sensitivity.The triple UL54 mutant had a IC50 ratio of 9,8 for GCV which is higherthan the ratios described for the single mutations I521T (ratio 3,1 Lurainet al., 2010), L516 M (ratio 1,3 data not published) and D515E (ratio2,5). Therefore the combination of the two resistance associated mutationsI521T and D515E seems to have an additive effect.REF O113Herpes simplex virus infection control with novel small interferingRNA poolsHenrik PAAVILAINEN 1 , Alesia ROMANOVSKAYA 2 , MichaelaNYGÅRDAS 1 , Minna PORANEN 2 , Dennis BAMFORD 2 , VeijoHUKKANEN 11 University of Turku, Turku, FINLAND; 2 University of Helsinki, Helsinki,FINLANDHerpes simplex virus (HSV) is a common pathogen of humans, causinginfections such as cold sores, herpes keratitis, genital herpes and the severeHSV encephalitis. There are antivirals available against HSV, but resistantstrains exist and in some cases current therapy is inadequate. We set outto test a novel RNA interference (RNAi) based approach to control HSVinfection.We elucidated the antiviral effects of pools of small interfering RNAs (siR-NAs), and studied the host responses induced by the RNA molecules, incells of neuronal and epithelial origin. We used swarms of anti HSV dsR-NAs, and commercial single site siRNAs, targeting the essential HSVUL29 gene. The anti HSV dsRNA was synthesized from HSV UL29sequence using the phage phi6 RNA dependent RNA polymerase. Thelong dsRNA was then cleaved into siRNAs with different dicers, creatingsiRNA pools targeting a large portion of the target gene. In neuronal cellsthe inhibition of HSV shedding with siRNA pools reached 99.9% (p

5 th European Congress of Virologybut clearly less than those observed using an 88 nt control dsRNA. Theseslight responses correlated little to antiviral efficacy.Escape mutants against pools would require drastic genomic alterationswhereas single site siRNAs can be rendered ineffective with subtle mutationin the virus genome. Our results are promising in developing a topicalRNAi approach for HSV infection control, for example against ocularinfections such as HSV keratitis.REF O114Biophysical characterization and antiviral effects of non canonicaldna structures in the herpes simplex virus type 1 genomeSara ARTUSI 1 , Maria Angela BIASOLO 1 , Arianna CALISTRI 1 ,Giorgio PALÙ 1 , Sara N. RICHTER 11 University of Padova, Department of Molecular Medicine, Padova, ViaGabelli 63, 35121, ITALYG quadruplexes (G4 s) are regulatory secondary conformations that formin guanine (G) rich nucleic acids. G4 s have been observed in humans,vertebrates, yeasts and bacteria, while little information on G4 s in virusesis available to date.Here we assessed the presence of putative G4 forming sequences inthe genome of the Herpes simplex virus type 1 (HSV 1). Nine highlyrepeated and conserved sequences were identified both in the codingand non coding strands. Six of them were found in the UL36 region,the largest HSV 1 gene encoding a glycoprotein with a crucial roleduring secondary envelopment. Other three sequences were located inthe genome terminal repeats, likely implicated in HSV 1 latency in neuronalcells. Biophysical studies, in the presence/absence of G4 stabilizingcations and ligands, demonstrated that the selected sequences fold in verystable G4 s. Two G4 ligands tested on HSV 1 infected cells exhibitedefficient antiviral properties without affecting cell viability. Real timePCR confirmed viral gene expression decrease upon G4 ligand treatment.Taq polymerase stop assays on purified HSV 1 DNA treated with G4ligands assessed enzyme inhibition exclusively in G4 forming regions,indicating that G4 s were very likely implicated in the observed antiviralactivity.The present data indicate the HSV 1 G4 s may be successfully exploitedas new targets for antiviral treatment. In addition, the abundance,stability and conservation of G4 regions within the genome of severalHSV 1 strains suggest the potential role of these sequences in key viralprocesses.S88 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyFriday 13 th September 2013, 15h00 – 17h15WORKSHOP 13: “VIRAL REPLICATION STRATEGIES”Chairpersons: Luis ENJUANES (Madrid, SPAIN)& Juan ORTIN (Madrid, SPAIN)Room Gratte-Ciel 1, 2, 3for virus viability was also demonstrated by reverse genetics, showed thatD99A, P116A and R190A mutations severely impaired or even abolishednsp12/nsp8 interaction and consequently polymerase activity. Moreover,conserved residues in nsp8 (K58) and nsp7 (K7 and N37) modulate RNAtemplate recognition by nsp7/nsp8/nsp12. Nsp14 is the first description ofa mismatch excision enzyme bound to a viral RNA polymerase, illustratingthe integration of RNA polymerization, repair and capping activitiesinto a single multi active complex.KEYNOTE:Influenza polymerase host adaptationAnna CAULDWELL, Jason LONG, Hongbo ZHOU, OlivierMONCORGE, Wendy BARCLAYSection of Virology, Imperial College London, UNITED KINGDOMTypical avian influenza A viruses do not replicate efficiently in mammals.This host range block is recapitulated in the laboratory in a cellbased polymerase activity assay in which viral polymerases reconstitutedfrom an avian virus do not amplify minigenomes inside human cells. Wepreviously showed that avian/human heterokaryons could support avianvirus polymerase activity suggesting there is an avian host factor lackingin human cells that could complement avian virus polymerase. Several distincthost adaptive mutations map to the polymerase complex (PB1, PB2and PA), and in addition it was recently reported that the nuclear exportprotein NEP encoded on RNA segment 8 can also alleviate the host rangeblock in polymerase activity. Some of these viral mutations increase polymerasefunction in both human and avian cells whereas others are humancell specific, suggesting the mechanisms by which they act and the hostfactors involved may be different. Some mutations which enhance activityin the reconstituted polymerase assay are not selected for in nature. Thismay be because they lead to attenuated virus replication, for example byincreasing interferon activation. Taken together these analyses can tell usmore about how influenza polymerase functions within the infected celland by which mechanisms one of the several host range barriers that limitavian influenza viruses in humans might be breached.ORAL COMMUNICATIONSREF O115Processive RNA synthesis, repair and capping activities embedded ina SARS Coronavirus multi protein complexLorenzo SUBISSI 1 , Clara C. POSTHUMA 2 , Jessika ZEVENHOVEN 2 ,Axelle COLLET 1 , Alexander E. GORBALENYA 2 , Eric J. SNIJDER 2 ,Etienne DECROLY 1 , Bruno CANARD 1 , Isabelle IMBERT 11 AFMB AMU, Marseille, France; 2 LUMC, Leiden, THE NETHERLANDSCoronaviruses include lethal human pathogens like the recently emergedHuman Coronavirus EMC (HCoV EMC) and Severe Acute RespiratorySyndrome Coronavirus (SARS CoV). They carry largest known viral RNAgenomes encoding replication/transcription machineries of unusual complexityand composed of, at least, 16 viral non structural proteins (nsps).The RNA dependent RNA polymerase (RdRp) nsp12 is poorly active invitro, but supposedly plays a central role in the efficient replication of thelarge (>30 kb) coronavirus genome. Here, we report that SARS CoV nsp7and nsp8, previously co crystallized as a hexadecamer reminding a slidingclamp, confer highly efficient and processive RNA synthesis properties tonsp12. The nsp7/nsp8/nsp12 complex further interacts in a stable fashionwith nsp14, a bi functional enzyme bearing 3 ′ 5 ′ exoribonuclease (ExoN)and RNA cap N7 guanine methyltransferase activities. The processiveRNA synthesis of nsp7/nsp8/nsp12 is not affected by nsp14 ExoN activity.Mutational analysis of conserved nsp8 residues, whose importanceREF O116Crystal structure of the N0 P complex of Nipah virus and of VSVprovide new insights into the encapsidation mechanism of the Paramyxoviridaeand RhabdoviridaeFilip YABUKARSKI 1 , Cédric LEYRAT 1 , Nicolas TARBOURIECH 1 ,Malene RINGKJØBING JENSEN 2 , Martin BLACKLEDGE 2 , RobRUIGROK 1 , Marc JAMIN 11 UMI3265 UJF EMBL CNRS UVHCI, Grenoble, FRANCE; 2 UMR5075CEA CNRS UJF Institut de Biologie Structurale, Grenoble, FRANCEEncapsidation of newly synthesized viral RNA genomes and antigenomesin a helical homopolymer of nucleoprotein (N) is an essential step in thereplication of non segmented negative strand RNA viruses. For Rhabdoviridaeand Paramyxoviridae, encapsidation relies on the continuousproduction of soluble RNA free nucleoprotein (N0) in the form of acomplex with the phosphoprotein (P), named the N0 P complex. In bothfamilies, this complex is formed by the interaction between the N terminalregion of P and the core of the N protein.We developed a strategy to reconstruct the N0 P complex from purifiedcomponents using an N mutant deleted of its N terminal arm (∼20 aa) anda peptide of P containing the N0 binding site. For vesicular stomatitis virus(VSV), a rhabdovirus, and for Nipah virus (NiV), a paramyxovirus, wecharacterized the structure of the N terminal region of P alone in solutionand we solved the crystal structure of the reconstructed N0 P complex. Thiswork demonstrates that the N terminal region of P is globally disorderedbut contains transiently populated a helices, and that it folds upon bindingto the N0. The structure of the reconstituted N0 P complexes revealedhow P prevents N0 from polymerizing and thereby from binding RNA.These structures also suggest mechanisms by which P stimulates initiationof RNA synthesis and controls encapsidation of the RNA genome. Thestructure of the NiV complex provides the first glimpse at the structure ofthe nucleoprotein of a Paramyxovirinae.REF O117Role of host factors in enterovirus RNA replicationFrank VAN KUPPEVELD, Hilde VAN TONGEREN, CristinaDOROBANTU, Lonneke VAN DER LINDEN, Lucian ALBULESCU,Jeroen STRATINGDivision of Virology, Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine, Utrecht University, Utrecht, THENETHERLANDSEnteroviruses (poliovirus, coxsackievirus, rhinovirus, enterovirus 71)extensively modify host cell membranes to create a scaffold for genomicRNA replication. As yet little is about the structure and biogenesis ofthese viral replication organelles and the identity of the host factors thatare involved in their formation. Previously, we showed that the enterovirus3A protein of enteroviruses recruits GBF1, a GEF of Arf1, to membranesand demonstrated a critical role of this protein in RNA replication(Wessels et al, 2006, Dev Cell). Recently, Golgi localized PI4KIII, whichis an effector of Arf1, was also recognized to be recruited to membranes by3A and to be important for viral RNA replication (Hsu et al., 2012, Cell).Here, evidence will be presented that PI4KIII is recruited to membranesVirologie, Vol 17, supplément 2, septembre 2013S89

5 th European Congress of Virologyindependent of GBF1 and Arf1 as well as of ACBD3, another 3A bindingprotein that was postulated to be important for PI4KIII recruitment tomembranes. Furthermore, we present evidence that oxysterol binding protein(OSBP), a PI4P binding protein that is believed to mediate transportof sterols between intracellular membrane compartments, is important forenterovirus RNA replication. Finally, we report the identification of novelPI4KIII inhibitor and show that this small molecule inhibits replicationof all enteroviruses and has excellent antiviral activity in a coxsackievirusinduced pancreatitis mouse model.REF O118Phosphatidylinositol 4 kinase III beta enhances human rhinovirusreplication by recruiting the oxysterol binding protein to ER Golgiderived membranesPascal S ROULIN 1 , Mark LÖTZERICH 1 , Federico T. TORTA 2 , LukasB. TANNER 2 , Frank J. VAN KUPPEVELD 3 , Markus R. WENK 2 , UrsF. GREBER 11 Institute of Molecular Life Sciences, University of Zurich, Zurich, SWIT-ZERLAND; 2 Singapore Lipidomics Incubator, Life Sciences Institute,National University of Singapore, Singapore, SINGAPORE; 3 Departmentof Infectious Diseases & Immunology, University of Utrecht, Utrecht,NETHERLANDSPositive strand RNA viruses form cytosolic membrane associated replicationcomplexes composed of host and viral proteins, viral RNA andcellular membranes of various origins. Like other picornaviruses, humanrhinoviruses (HRVs) rearrange Golgi membranes including the ER Golgiintermediate compartment and trans Golgi network. The picornaviral 2B,2C and 3A proteins had been implicated in reorganizing ER Golgi membranes.They are, however, not sufficient to give rise to the endomembranenetwork observed during replication, implying that additional viral and/orhost factors are involved. These factors include phosphatidylinositol 4kinases (PI4Ks), as shown in earlier studies with coxsackievirus, poliovirusor hepatitis C virus. PI4Ks catalyze the production of phosphatidylinositol4 phosphate (PI4P), which controls vesicular traffic and membrane biogenesis.Chemical inhibitors and knockdown approaches for PI4Ks nowshow that the activity of PI4KIII is crucial for rhinovirus replication incultured cells and primary human airway cells. PI4KIII and PI4P lipidsare enriched at sites of rhinovirus replication on Golgi membranes. Moreover,RNAi mediated knock down of the oxysterol binding protein (OSBP)and drug induced cholesterol depletions impair HRV replication. OSBPshuttles cholesterol from the ER to the Golgi, and binds PI4P dependingon its PH domain. Collectively, our data suggest a model where a balancedcomposition of PI4P and cholesterol on Golgi derived membranes supportthe establishment and maintenance of HRV replication domains.REF O119A Structural Basis for the Oligomer Assembly and Host ChromatinInteraction of Two Gamma2 Herpesviral LANA Proteins and TheirRole in Viral PersistenceMagdalena WEIDNER GLUNDE 1,3 , Jan HELLERT 1,2 , JoernKRAUSZE 2 , Ulrike RICHTER 3 , Heiko ADLER 4 , Roman FEDOROV 5 ,Marcel PIETREK 3 , Jessica RÜCKERT 3 , Christiane RITTER 2 , ThomasSCHULZ 3 , Thorsten LUEHRS 21 these authors contributed equally to this work; 2 Dept. of Structural Biology,Helmholtz Centre for Infection Biology, Braunschweig, GERMANY;3 Institute of Virology, Hannover Medical School, Hannover, GERMANY;4 Dept. of Gene Vectors, Helmholtz Centre Munich, Munich, GERMANY;5 Departmetn of Biophysical Chemistry, Hannover, Germany, Hannover,GERMANYKaposi’s sarcoma associated herpesvirus (KSHV) establishes a lifelonglatent infection in humans and causes Kaposi’s Sarcoma and several othermalignancies. Its latency associated nuclear antigen (kLANA) is requiredfor viral persistence, replication of latent viral episomes and for their mitoticdistribution to daughter cells. We solved the 3D X ray crystal structureof the C terminal DNA binding domains (CTD) of kLANA, and of thecorresponding protein, mLANA, of murine herpesvirus 68 at resolutionsof 2.60 Å, and of 2.14 Å, respectively. We could allocate functional propertiesof LANA to three distinct LANA faces. Native kLANA CTD dimersundergo self oligomerization, which is required for the formation of LANAcontaining nuclear microdomains (LANA speckles) and replication of thelatent KSHV origin in vitro. A specific binding site in kLANA for the ETdomain of BRD2/4 was identified by NMR spectroscopy, overlaps withthe area contacting viral DNA and is also required for latent episomalreplication. A positively charged surface patch of LANA enhances Brd2/4binding and LANA oligomerization, and thereby influences latent replicationof KSHV in vitro, and latent persistence of MHV68, in vivo. Our datasuggest that the oligomerisation of LANA dimers to form nuclear specklescreates multivalent LANA complexes, which integrate viral episome binding,host chromatin association and thereby ensure the replication andmitotic stability of latent gamma 2 herpesviruses.REF O120Unscheduled mitotic entry of HCMV infected cells leads to chromosomaldamage and mitotic cell deathMartin EIFLER, Ralf UECKER, Christian HAGEMEIER, LüderWIEBUSCHLabor für Pädiatrische Molekularbiologie/Charité UniversitätsmedizinBerlin, Berlin, GERMANYHuman cytomegalovirus (HCMV) is known to severely deregulate thehost’s cell cycle. HCMV stops cell cycle progression at the G1/S transition,thereby preventing competing cellular DNA synthesis and mitoticentry. Here, we show that cellular mitosis is indeed incompatible with productiveHCMV infection. We made use of a pUL21a point mutant unableto block cell cycle progression due to a defective cyclin A2 binding site.Confluent fibroblasts infected with this mutant entered mitosis between1 and 2 days post infection (dpi). The mitotic cells accumulated at themetaphase anaphase transition displaying the following features: histoneH3(Ser 10) phosphorylation, chromatin condensation, disassembly of thenuclear lamina and formation of a spindle apparatus. Moreover, IE1/IE2protein level decreased upon mitotic entry, suggesting an inherent instabilityof these proteins under mitotic conditions. The pUL44 containing viralreplication compartments were dislocated towards the spindle poles andshowed strongly reduced BrdU incorporation rates pointing to impairedviral DNA synthesis. The observed metaphase arrest was further characterizedby progressive fragmentation and pulverization of the chromosomalmaterial. Finally, the metaphase arrested cells underwent mitotic cell deathbetween 3 and 5 dpi. We conclude that the cyclin A2 binding function ofpUL21a is required for the coordinated progression of the HCMV lyticcycle. Unscheduled mitotic entry during the course of the HCMV replicationhas fatal consequences, leading to mitotic catastrophe and abortiveinfection.REF O121BET proteins as cofactors for gammaretroviral integrationSaumyashree GUPTA 1 , Peter CHEREPANOV 2 , Tobias MAETZIG 3 ,Axel SCHAMBACH 3 , Goedele MAERTENS 4 , Thomas SCHULZ 11 Institute of Virology, Hannover Medical school, Hannover, GERMANY;2 Chromatin Structure and Mobile DNA Lab, Cancer Research UK, London,UK; 3 Department of Experimental Haematology,Hannover MedicalSchool, Hannover, GERMANY; 4 Department of Infectious diseases, ImperialCollege London, London, UNITED KINGDOMS90 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyRetroviral replication proceeds through a stable proviral DNA intermediate,whose integration into cellular DNA is mediated by the viralintegrase with the help of mostly uncharacterized cellular cofactors. Thecellular transcriptional co activator, LEDGF (Lens epithelium derivedgrowth factor) interacts with lentiviral integrases (e.g. HIV 1), is a cofactorin lentiviral integration and determines the preferential integration of HIV1 into active transcription units. In contrast to HIV 1, gammaretroviruseslike Murine leukemia virus (MLV), prefer to integrate near transcriptionstart sites, CpG islands and promoter regions of cellular genes. The cellularfactors determining this integration pattern are not known. Here we showthat the cellular chromatin binding proteins, BET (bromodomain and ETdomain) proteins, Brd2 and Brd4, strongly interact with gammaretroviralintegrases. We mapped the interaction sites to a characteristic structuralfeature in the ET domain of Brd2 and to three amino acids in MLVintegrase. We observed an increase in gammaretroviral integration in thepresence of BET proteins both in vitro and in vivo. Furthermore, the BETbromodomain inhibitors, I BET and JQ1, which inhibit the binding to acetylatedhistones, decreased MLV integration in MLV infected cells. Thus,BET proteins might act as chromatin acceptors for gammaretroviruses andstimulate integration, thereby playing a similar role as LEDGF does forHIV. Using gammaretroviral vectors for gene therapy carries a stronglyincreased risk of insertional mutagenesis. The identification of a cellularcofactor for gammaretroviral integration will therefore provide the basisfor new strategies to direct gene therapy vectors into safe landing sites.Virologie, Vol 17, supplément 2, septembre 2013S91

5 th European Congress of VirologyFriday 13 th September 2013, 15h00 – 17h15KEYNOTE:WORKSHOP 16: “ZOONOTIC VIRUSES”Partnership with the FP7-EU programs ANTIGONE& PREDEMICSChairpersons: Thijs KUIKEN (Rotterdam, THENETHERLANDS) & Sylvie VAN DER WERF(Paris, FRANCE)Room Tête d’OrCoronaviruses: designed to emerge?Eric J. SNIJDERDepartment of Medical Microbiology, Leiden University Medical Center,THE NETHERLANDThe emergence – less than a decade apart – of the SARS- and MERSvirusesemphasizes the significant pandemic potential of the coronavirus(CoV) family. They are respiratory and enteric pathogens of humans anddomesticated animals, and also appear to be omnipresent in wild-life,in particular in bats. The crossing of species barriers is a common CoVfeature, since all endemic human CoVs appear to have been transmittedfrom animal hosts.At the genomic level, CoVs stand out for having the largest known RNAgenomes (up to 32 kilobases), which is replicated by an RNA-synthesizingmachinery of unprecedented complexity in the RNA virus world. CoVenzymes include both “standard enzymes” and less common (or unique)functions, like an exoribonuclease implicated in a primitive form of proofreadingthat may push back the CoV error threshold and thus secure thesurvival of the large CoV genome. RNA recombination, potentially linkedto the mechanism producing subgenomic mRNAs, appears to have shapedthe evolution of various CoV genes, including those encoding replicaseand spike protein. Moreover, different CoV lineages have acquired specificsets of “accessory protein genes”, which are commonly dispensable forcell culture replication but important in pathogenesis and immunity on thelevel of the infected organism. The genetic heterogeneity of CoVs and theunderlying molecular mechanisms are key factors in host switching andadaptation, and this poses a special challenge for the design of antiviralstrategies aiming to combat zoonotic CoV infections at large.ORAL COMMUNICATIONSREF O122Phylodynamics and dispersal of domestic dog rabies in an African cityHerve BOURHY 1 , Emmanuel NAKOUNÉ 2 , Matthew HALL 3 , AnthonyLE PELLETIER 1 , Chiraz TALBI 1 , Laurence WATIER 4,5 , Edward C.HOLMES 6 , Philippe LEMEY 7 , Andrew RAMBAUT 3,81 Institut Pasteur, Unit Lyssavirus Dynamics and Host Adaptation, WHOCollaborating centre for Reference and Research on Rabies, Paris,FRANCE; 2 Institut Pasteur de Bangui, Bangui, RÉPUBLIQUE CENTRA-FRICAINE; 3 Institute of Evolutionary Biology, University of Edinburgh,Ashworth Laboratories, Edinburgh, UNITED KINGDOM; 4 INSERM,U657 and Institut Pasteur, PhEMI, Paris, France; 5 Faculté de MédecineParis Ile de France Ouest, Université de Versailles – Saint Quentin,Versailles, France; 6 Sydney Emerging Infections & Biosecurity Institute,School of Biological Sciences and Sydney Medical School, Sidney,AUSTRALIA; 7 Rega Institute, KU Leuven, Leuven, BELGIUM; 8 FogartyInternational Center, National Institutes of Health, Bethesda, USALittle is known about the spatiotemporal dynamics of rabies in dogs andthe processes responsible for its maintenance in specific geographic localities.In a large scale gene sequence study, we previously demonstratedhow anthropogenic and evolutionary dynamics shape the spatial distributionand spread of RABV in North Africa (Talbi et al., Plos Pathogens2010). In the present study we inferred the evolutionary history of 172RABV sequences (5050 nucleotides) collected in Bangui, and from otherneighbouring cities. Bangui is the capital of Central African Republic andlacks a specific program to control dog rabies. The timescale of the evolutionaryhistory of RABV in Bangui was estimated using a Markov chainMonte Carlo approach. Our data indicate that the epidemiology of RABVin this large city is characterized by periodic introduction of new lineagesof RABV into the city. The introductions and spread of the correspondingviruses are responsible for small outbreaks that all seem to contributeto a succession of epidemic waves, the pattern of which was determinedusing epidemiological data (periods=7.4 and 4.5 years). These waves wereseparated by unexpected extinctions of the transmission chains followedby the introduction and spread of new lineages not circulating before. Inconclusion, our data indicate that rabies is not sustainably maintained ina large city over relatively long time scales, suggesting a more importantrole of sparsely connected versus large homogenous dog populations inthe maintenance of RABV at local geographical scales.REF O123Arboviruses in Europe, an increasing threatNatalie CLETON 1,2 , Marion KOOPMANS 1,2 , Chantal REUSKEN 11 National Institute for Public Health and the Environment, Bilthoven, THENETHERLANDS; 2 Erasmus Medical Center, Rotterdam, THE NETHER-LANDSBackground: Globally the number of travelers has risen from 450 millionin 1990 to nearly 950 million in 2010. 5 10% of travelers report to a medicalcaretaker after travel. Consequently, doctors are increasingly confrontedwith travel related diseases.Objective: The magnitude of travel within Europe and beyond and theconstantly changing dynamics of arbovirus diseases across the globedemands up to date information about arbovirus threats to travelers andthe countries they visit.Method: We systematically reviewed current knowledge on medicallyimportant travel related arboviruses in Europe, and ranked the arbovirusesby geographic region, clinical syndrome, and probability of occurrence.Results: The majority of clinically important arboviruses belong to theFlaviviridae, Bunyaviridae or Togaviridae families. In Northern and EasternEurope, tick borne encephalitis is one of the most important endemicarboviruses, but West Nile virus and Sandfly fever viruses may producecomparable clinical symptoms. Febrile disease, arthritis and rash may becaused by a range of arboviruses, including Sindbis, Tahyna and Chikungunyaand Dengue. Additionally, outside of Europe there are a large variety ofarboviruses that overlap in geographical region as well as primary clinicalsyndrome with European arboviruses.Conclusion: Diagnosing endemic or travel associated arboviral diseaserequires knowledge about geographic distribution, travel and exposurehistory. Differential diagnosis requires testing for multiple arboviruses asclinical syndromes and geographic distributions overlap.REF O124A charge dependent interaction is necessary for Rift Valley fever viruslike particle entry into its host cellMaria BAUDIN 1 , Delowar HOSSAIN 1 , Friedemann WEBER 2 , MagnusEVANDER 11 Department of Clinical Microbiology, Unit of Virology, Umeå University,Umeå, SWEDEN; 2 Institute for Virology, Philipps University Marburg,Marburg, GERMANYS92 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyRift Valley fever virus (RVFV) is mosquito borne and highly virulent tohumans. The virus is able to infect many different animals and mosquitoesas well as various cell types in vitro but not much is known about the entrymechanisms.In this study we have used Rift Valley fever virus like particles (VLPs)containing a reporter gene to study the virus cell interaction. Cells orRVF VLPs were treated with various compounds to characterize theireffect on RVFV binding to its host cell. We also analysed the net surfacecharge of the RVFV glycoproteins since this can affect the cellularbinding.Binding of RVF VLPs to cells was dependent on charge and divalentions. Binding was inhibited by the highly negatively charged heparin.The RVFV glycoprotein Gn had a predicted isoelectric point (pI) of 7.6and a net positive charge of +1.7 at pH 7.4, suggesting interaction betweenthe Gn ectodomain and the negatively charged cell surface. RVFVGc on the other hand, was highly negatively charged, 14.2 at pH 7.4,most probably reflecting that Gc is not exposed until after binding. Sialicacid, 3 integrins and heparan sulfate was not important for RVF VLPbinding.Conclusions: The binding of RVFV to host cells was dependent on chargeinteractions between the cell surface and, most probably, the ectodomainof the RVFV Gn glycoprotein. Divalent ions were also important, whilesialic acid, 3 integrins and heparan sulfate had no involvement in theRVFV cellular binding.REF O125Functional comparison of the SARS CoV and bat borne SARS likeCoronavirus spike glycoproteins: entry process and interplay withchiropteran cellsMarkus HOFFMANN 1 , Tim GÜTZKOW 1 , Nadine KRÜGER 1 ,Christoph LOSEMANN 1 , Marcel Alexander MÜLLER 2 , Jan FelixDREXLER 2 , Karl Heinz ESSER 3 , Christian DROSTEN 2 , GeorgHERRLER 11 Institute of Virology/University of Veterinary Medicine Hannover, Hannover,GERMANY; 2 Institute of Virology/Bonn Medical Centre, Bonn,GERMANY; 3 Institute of Zoology/University of Veterinary Medicine Hannover,Hannover, GERMANYThe severe acute respiratory syndrome associated coronavirus (SARSCoV) uses the angiotensin converting enzyme 2 (ACE2) as a receptormolecule and horseshoe bats (genus Rhinolophus) are believed to serveas a natural reservoir. The detection of SARS CoV reactive antibodies inAfrican bats and RNA of SARS like coronaviruses (SL CoV) worldwide,lead to the question whether another zoonotic spillover can occur, but thefailure to isolate such a SL CoV makes it necessary to use alternativemethods to answer this question.We employed a pseudotype infection system, based on the vesicular stomatitisvirus (VSV) to analyze whether SL CoV S can mediate entry intomammalian (chiropteran) cell lines and established a cell based fusionapproach involving co expression of CoV S and human or chiropteranACE2. Furthermore, we used soluble CoV S to elucidate the presence ofan appropriate receptor molecule.VSV pseudotyped with SL CoV S was unable to infect any of the tested celllines; parallel infection experiments with filoviral glycoproteins, as well asinfections by paramyxo and influenza viruses confirmed the susceptibilityof chiropteran cells to viral infection. SARS CoV S mediated entry intochiropteran cell lines required human ACE2 expression in trans and thefusion assay revealed that only trypsin activated SARS CoV S was capableof syncytia formation. Finally, we show that soluble SL CoV S can bindto cells expressing human ACE2, leading us to the assumption that a postbinding step is responsible for the observed restrictions.REF O126Efficient replication of the novel human betacoronavirus EMC onprimary human epithelium highlights its zoonotic potentialRonald DIJKMAN 1 , Eveline KINDLER 1 , Hulda JONSDOTTIR 1 ,Doreen MUTH 2 , Ole HAMMING 3 , Rune HARTMANN 3 , ReguloRODRIGUEZ 4 , Robert GEFFERS 5 , Ron FOUCHIER 6 , ChristianDROSTEN 2 , Marcel MÜLLER 2 , Volker THIEL 1,71 Inst. of Immunobiology, Kantonal Hospital St.Gallen, St. Gallen, SWIT-ZERLAND; 2 Inst. of Virology, Univ. of Bonn Medical Center, Bonn,GERMANY; 3 Centre for Structural Biology, Univ. of Aarhus, Aarhus,DENMARK; 4 Inst. of Pathology, Kantonal Hospital St.Gallen, St. Gallen,SWITZERLAND; 5 Helmholtz Center for Infection Research, Braunschweig,GERMANY; 6 Viroscience Lab, Erasmus Medical Center, Rotterdam,THE NETHERLANDS; 7 Vetsuisse Faculty, Univ. of Zürich, Zürich, SWIT-ZERLANDThe recent emergence of a novel human coronavirus (HCoV EMC) in theMiddle East raised considerable concerns as it is associated with severeacute pneumonia, renal failure and fatal outcome, and thus resembles theclinical presentation of severe acute respiratory syndrome (SARS) observedin 2002/2003. Like SARS CoV, HCoV EMC is of zoonotic origin andclosely related to bat coronaviruses. The human airway epithelium (HAE)represents the entry point and primary target tissue for respiratory virusesand is highly relevant for assessing the zoonotic potential of emergingrespiratory viruses, such as HCoV EMC. Here we show that pseudostratifiedHAE cultures derived from different donors are highly permissiveto HCoV EMC infection, and by using RT PCR and RNAseq data weexperimentally determined the identity of seven HCoV EMC subgenomicmRNAs. Although, the HAE cells were readily responsive to type I andtype III interferon (IFN), we observed neither a pronounced inflammatorycytokine nor any detectable IFN responses following HCoV EMC, SARSCoV, or HCoV 229E infection, suggesting that innate immune evasionmechanisms and putative IFN antagonists of HCoV EMC are operationalin the new host. Importantly, however, we demonstrate that both type Iand type III IFN can efficiently reduce HCoV EMC replication in HAEcultures, providing a possible treatment option in cases of suspected HCoVEMC infection.REF O127Identification of cellular factors involved in HEV replication using anin vivo model of HEV infection in swine and a proteomic approachSophie ROGEE 1,2,3 , Jérome BOUQUET 1,2,3 , Elodie BARNAUD 1,2,3 ,Marine DUMAREST 1,2,3 , Philippe CHAFFEY 4,5,6 , Nicole PAVIO 1,2,31 UMR 1161 INRA, Maisons Alfort, FRANCE; 2 UMR virologie ANSES,Maisons Alfort, FRANCE; 3 ENVA, Maisons Alfort, FRANCE; 4 Institutcochin Inserm U1016, Paris, FRANCE; 5 Institut cochin CNRS UMR8104,Paris, FRANCE; 6 Université Paris Descarte, Paris, FRANCEHepatitis E virus (HEV) is a zoonotic virus and in industrialized countriespigs are the major source of HEV infections. In France, strains of HEVpresent in human and swine populations are of the same genotype andbelong to subtype 3f, 3e and 3c. Mechanisms involved in replication andvirulence of HEV are still unknown. To characterize the pathogenesis andvirulence effectors of different HEV subtypes, a study of the cellular factorsmodulated during HEV infection was carried out using a differentialproteomic approach. This study was performed in a model of experimentalinfection of pigs, with the three subtypes of HEV present in France.The kinetics of HEV fecal excretion and viral RNA detection in the liverand bile of each infected animal did not show significant difference in termsof duration or quantity, whatever the strain used. Differentially expressedVirologie, Vol 17, supplément 2, septembre 2013S93

5 th European Congress of Virologyproteins were defined by abundance ratio upper than +1.5 or smaller than1.5 (p value

5 th European Congress of VirologySaturday 14 th September 2013, 8h30 – 10h30WORKSHOP 2: “ONCOLYTIC VIRUSES AND GENETHERAPY”Chairpersons: Penelope MAVROMARA (Athens, GREECE) &Dirk NETTELBECK (Heidelberg, GERMANY)Room Tête d’OrKEYNOTE:New lentiviral vector pseudotypes offer exciting perspectives for T, B,DC and HSC mediated immuno and gene therapyEls VERHOEYENCIRI, EVIR team, INSERM U1111, Lyon, FRANCEImportant target cells for immuno- and gene therapy are human T cells, Bcells, dendritic cells (DCs) and hematopoietic stem cells (HSCs). We haveengineered two new lentiviral vector (LV) pseudotypes that allow highlevel transduction of these targets treatment of several genetic dysfunctionsof the hematopoietic system. One LV displaying the measles virusglycoproteins at their surface (MV-LVs), which was able to transduce efficientlystimulated and resting human T, B cells and DCs and thymocytes.A second LV displaying the Baboon retrovirus envelope (BAEV-LVs)allowed high level transduction of T and B cells and thymocytes. Forboth LVs this characteristic is unique since VSVG-LVs fail to transducethese resting target cells. Each of these LV pseudotypes demonstrates adifferent transduction pattern of thymocyte subpopulations. Even morestriking was that these new pseudotypes allowed unexpectedly at lowvector doses efficient transduction of HSCs. Importantly, reconstitutionof NOD/SCID gamma-c-/- mice with MV-LV or BAEVTR transducedpre-stimulated or resting hCD34+ cells resulted reproducibly in transductionlevels close to 100% or 50-70%, respectively, of all analyzedmyeloid and lymphoid engrafted lineages in all hematopoietic tissues,including CD34+ lineage negative cells in the bone marrow. Moreover,these high transduction levels were confirmed in secondary miceengraftments.In conclusion, these new LV technologies offer exciting perspectivesfor multiple Tgene and immuno therapy applications in thefuture.ORAL COMMUNICATIONSinfected cells to concentrate iodide enabling non invasive imaging andcombination radiovirotherapy. Through high resolution in vivo and exvivo imaging we documented homogeneous virus seeding in xenograftsand subsequent spread in perfused tissue. Infection of metastases wasmore rapid and intense than infection of primary tumors, achieving isotopeuptake within nearly 3 fold the efficiency of the thyroid. Virotherapy combinedwith systemic 131 I resulted in more rapid disease regression thaneither therapy alone. We then asked if oncolytic efficacy was dependenton the primary MV receptor signaling lymphocyte activation molecule(SLAM) or the MV attenuation receptor CD46. Strikingly, only SLAMdependent entry sustained efficient viral spread, tumor regression, and prolongedsurvival. While virus entry into cells can occur through multiplereceptors, the efficacy of oncolytic protocols may depend on the concurrentactivation of cellular responses that support efficient viral replication.Functioning as both an entry receptor and signaling molecule, SLAMexpression on hematological tumors represents a targetable vulnerabilityfor MV based oncolysis in future clinical trials.REF 0130Cell surface receptor targeting expanded: from oncolytic measlesviruses to lentiviral and AAV vectorsAnke RASBACH, Christian BUCHHOLZPaul Ehrlich Institut, Langen, GERMANYRestricting gene transfer to the relevant cell type of choice at the level ofcell entry is among the main challenges for vector improvement in geneand virotherapy. Vectors with a restricted tropism, ideally highly specificjust for the cell type of interest for the given therapeutic application,are expected to improve safety and efficacy of gene therapy and facilitatein vivo gene transfer strategies. Altering receptor usage of a viral vectoris a complex protein engineering task, which was first accomplished foroncolytic measles viruses (MVs) by fusing a tumor antigen specific singlechain antibody fragment (scFv) to the virus attachment protein hemagglutinin(H) and abolishing natural receptor usage through point mutations.Cytoplasmic tail truncations in H and the MV fusion protein (F) thenallowed transfer of this approach to lentiviral vectors (LVs). Recently,this strategy has been extended to AAV vectors by mutating the heparansulfate proteoglycan (HPSG) binding site in the capsid proteins andsimultaneously fusing a designed ankyrin repeat protein (DARPin) withhigh affinity for the tumor antigen Her2/neu to the VP2 capsid protein.Thus, three different vector types can now be manipulated in a flexibleand rationally based approach to enter cells via a free chosen cell surfacemolecule. Data on the ex vivo and in vivo application of these vectortypes targeted to different cell types such as endothelial cells, lymphocytes,hematopoietic stem cells, tumor cells and putative tumor stem cells will bediscussed.REF 0129Mantle cell lymphoma radiovirotherapy: high resolution in vivo imagingdocuments critical role of measles virus entry through thesignaling lymphocyte activation moleculeRoberto CATTANEO, Tanner MIEST, Marie FRENZKEMayo Clinic, Rochester MN, USAWe developed here a vaccine identical measles virus (MV) as an oncolyticagent against mantle cell lymphoma, which is incurable but radiosensitive.We armed the virus with the sodium iodide symporter, which causesREF 0131Preclinical therapy of disseminated carcinomas and of Gliomas withretargeted oncolytic herpesvirusesGrabriella CAMPADELLI-FIUME 1 , Valentina GATTA 1 , PatriziaNANNI 1 , Laura MENOTTI 2 , Carla DE GIOVANNI 1 , PaoloMALATESTA 3 , Pier Luigi LOLLINI 11 Department of Experimental, Diagnostic and Specialty Medicine Universityof Bologna, Bologna, ITALY; 2 Department of Pharmacy andBiotechnology University of Bologna, Bologna, ITALY; 3 IRCCS San MartinoIST University of Genoa, Genoa, ITALYVirologie, Vol 17, supplément 2, septembre 2013S95

5 th European Congress of VirologyOncolytic viruses aim to selectively kill cancer cells. To generate cancerspecific oncolytic Herpes simplex viruses (oHSVs), we retargeted HSVto Human Epidermal growth factor Receptor 2 (HER2), overexpressed inovary and breast cancers. The HER2 retargeted HSV (R LM249) exclusivelyinfects cells expressing HER2, and exerts antitumor activity whenadministered i.t. Ablation of disseminated cancer cells in vivo remains amajor hurdle. Here, we report the efficacy of systemically (i.p.) administeredR LM249 against disseminated tumors in mouse models thatrecapitulate tumor spread in ovarian or breast carcinoma patients. Humanovarian carcinoma SK OV 3 cells were implanted i.p. or breast tumor MDAMB 453 cells were injected i.v. in immunodeficient Rag2/;Il2rg/ mice.Repeated i.p. administrations of R LM249 caused a 95% reduction in thetotal weight of peritoneal neoplastic nodules; 60% of treated mice werefree from tumor peritoneal diffusion. R LM249 exhibited also anti metastaticactivity against ovarian metastases and reduced brain metastases.The i.p. administration of the HER 2 retargeted oncolytic HSV effectivelyreduced i.p. ovarian carcinomatosis and inhibited both visceral and distantmetastases.HER2 is expressed also by high grade gliomas (HGGs). HER2 retargetedHSV R LM113 was effective against HGG and lengthened the medial survivaltime of NOD/SCID or immunocompetent mice carrying orthotopicHGGs.Cumulatively, HER2 retargeted HSVs exerted anticancer activity againstperitoneally diffuse breast and ovary cancers, and against gliomas.REF 0132Gene therapy with a replicative HSV vector expressing LIF limits lossof oligodendrocytes and modulates autoimmunity during experimentaldemyelinating diseaseMichaela NYGÅRDAS 1 , Henrik PAAVILAINEN 1 , Nadine MÜTHER 2 ,Claus Henning NAGEL 2 , Matias RÖYTTÄ 3 , Beate SODEIK 2 ,Hukkanen VEIJO 11 Department of Virology, University of Turku, Turku, FINLAND;2 Institute of Virology, Hannover Medical School, Hannover, GERMANY;3 Department of Pathology, Turku, FINLANDHerpes simplex viruses (HSV) have several features making them goodgene therapy vector candidates, especially for the treatment of central nervoussystem (CNS) disease. Experimental autoimmune encephalomyelitis(EAE) is an inflammatory autoimmune disease of the CNS and is theprimary disease model for multiple sclerosis (MS). Demyelination andinflammation are hallmarks of these diseases. Leukemia inhibitory factor(LIF) has neuroprotective properties and limits demyelination and oligodendrocyteloss. Here, we studied the effect on EAE of an HSV 1 vectorexpressing LIF.We constructed BAC derived HSV 1 vectors, deleted of the neurovirulencegene gamma34.5, encoding LIF or a control gene ZeoR. Female SJL miceinduced for EAE, were treated with 10E7 PFU vector intracranially. ClinicalEAE scores were observed daily. Brain, spinal cord and trigeminalganglion (TG) samples were collected on days 9, 14 and 21 p.i. for microscopicalanalysis and for the study of viral and host gene expression withquantitative RT PCR.The HSV LIF significantly alleviated the clinical scores from day 15 p.i.when compared to untreated mice. Virus replication was detected in brain,TGs and spinal cords by virus culture, PCR and IVIS luminometry. Wedetected an increase in oligodendrocytes in the brains of HSV LIF treatedmice during recovery. The HSV LIF therapy also lowered IL 17 and inducedIL 10 and TGF beta mRNA expression in the brains, indicating a shiftfrom Th17 towardsaTregulatory cell response. We acknowledge MartinMesserle and Eva Maria Borst for their advice on BAC mutagenesis.REF 0133Efficient generation gf Human Natural Killer Cells by viral transformationBernhard FLECKENSTEIN 1 , Armin ENSSER 1 , Benjamin VOGEL 1 ,Karin TENNERT 1 , Florian FULL 1,21 Institut für Klinische und Molekulare Virologie, Universitätsklinikum,Friedrich Alexander Universität, Erlangen, GERMANY; 2 Dept. of Microbiologyand Immunobiology, Harvard Medical School, Boston, MA, USAThe short lifespan of human natural killer (NK) cells is one of the majorobstacles in human NK cell research. Few continuous NK like cell linesexist and all except one derive from NK cell leukemia or lymphomapatients.Here we describe the in vitro growth transformation of functional humanNK cells by Herpesvirus saimiri (HVS), a primate rhadinovirus. Infectionof human NK cells was revealed by using a selectable recombinant HVS H2Kk marker virus. This led us to the discovery that HVS infection resultsin continuous expansion of NK cells; these growth transformed NK celllines have a strict requirement for exogenous IL 2, they express effectormolecules and exert cytolytic activity similar to freshly isolated NK cells.HVS transformation of human NK cells can provide large numbers andspecific populations of NK cells for biochemical and functional analysis.Further studies will have to address an eventual therapeutic potential andtheir value in generating cell lines of NK cell deficiencies and in studyingvirus interactions with human innate immunity.REF 0134Lentiviral delivery of multiple short hairpin RNAS: A gene therapyapproach for HIV 1 infectionFrancesca SPANEVELLO 1 , Arianna CALISTRI 1 , Claudia DELVECCHIO 1 , Barbara MANTELLI 1 , Saverio Giuseppe PARISI 1 , GiorgioPALÙ 1 , Marina CAVAZZANA CALVO 2,3 , Cristina PAROLIN 41 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 René Descartes University of Paris, Paris, FRANCE; 3 NeckerChildren’s Hospital, Paris, FRANCE; 4 Department of Biology, Universityof Padova, Padova, ITALYHIV gene therapy holds considerable promise as an alternative or complementarystrategy to drug based antiretroviral therapy for the treatmentof HIV 1 infection. Among the antiviral genes designed to inhibit HIV 1replication, short hairpin RNAs (shRNAs) mediating knockdown of viralgenes or cellular co factors have proven to be effective. In this study, weobtained lentiviral vectors expressing shRNAs targeting the viral genesvif, tat/rev and the cellular gene CCR5. To account for HIV 1 variability,we adopted combinatorial RNA interference approaches based onthe simultaneous expression of the different shRNAs either from distinctpromoters or as an extended shRNA. We tested and compared three differentpromoters for shRNA expression: the human U6, 7SK and H1polymerase III promoters. The safety and the biological activity of thedeveloped vectors were investigated both in cell lines and in human primarycells. Our results indicate that shRNA efficacy is strictly dependenton the employed promoter. Moreover, single promoter activity can differconsiderably among the selected combinatorial RNAi platforms. Ourfindings show that the insertion of up to three promoter/shRNA cassettesinto a single vector did not negatively affect the inhibitory effect of eachindividual shRNA. These results gain insights into the design constrainsand the promoter selection required to express a combination of antiviralshRNAs and confirmed the potential of RNAi to inhibit HIV 1replication.S96 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologySaturday 14 th September 2013, 8h30 – 10h30WORKSHOP 11: “VIRUS EXIT: ASSEMBLY, MATURATIONAND RELEASE”Chairpersons: Ari HELENIUS (Zurich, SWITZERLAND) &Delphine MURIAUX (Montpellier, FRANCE)Room Prestige Gratte-CielKEYNOTE:ESCRTing enveloped viruses out of cellsG. EFFANTIN 1 , A. DORDOR 1 , G. SCHOEHN 1,2 , H. GÖTTLINGER 4 ,R. SADOUL 3 , V. SANDRIN 5 , W. SUNDQUIST 5 , W. WEISSENHORN 11 UVHCI, UMI 3265 University Joseph Fourier-EMBL-CNRS, Grenoble;2 IBS UMR 5075 CEA-CNRS-UJF, Grenoble; 3 Institut des Neurosciencesand University Joseph Fourier, Grenoble; 4 University of MassachusettsMedical School, Worcester, MA; USA; 5 Department of Biochemistry, Universityof Utah School of Medicine, Salt Lake City, UT; USAEndosomal sorting complexes required for transport (ESCRTs) catalyzemultivesicular body biogenesis, distinct steps in cell division and buddingof some enveloped viruses including HIV-1. Out of the five complexesconstituting the ESCRT machinery (ESCRT-0, I, II, III and VPS4) onlymembers of ESCRT-III and the VPS4 complex are essential for all ESCRTdependentcellular processes. Enveloped viruses such as HIV-1 employlate domains to recruit the ESCRT machinery to the sites of budding inorder to mediate virion release via a final membrane scission step catayzedby ESCRT-III and VPS4. HIV-1 obtains access to ESCRT-III either viaESCRT-I or Alix. ESCRT-III proteins (CHMP1, 2, 3, 4, 5, 7 and IST1) arecytosolic and transiently recruited to cellular membranes upon activation,which induces their polymerization leading to membrane remodeling. Wepresent cryo electron microscopy data on helical polymers of ESCRT-IIICHMP2A-CHMP3 assembled in the absence of membrane and CHMP2Bpolymers assembled inside cellular membrane tubes. We define the basicbuilding block of the ESCRT-III polymer and show that ESCRT-III proteinsbecome protease resistant and thermostable upon higher order assembly.An interaction map of ESCRT-III proteins will be presented and discussedwith respect to ESCRT-III recruitment and synergism between CHMP3and CHMP2A during HIV-1 budding.ORAL COMMUNICATIONSREF O135Intermolecular RNA interactions and the selective packaging ofinfluenza A genomic segmentsRoland ROLAND MARQUET 1 , Cyrille GAVAZZI 1 , Matthieu YVER 2 ,Emilie FOURNIER 1 , Boris ESSERE 2 , Jean Christophe PAILLART 1 ,Annie CAVALIER 3 , Jean Paul ROLLAND 3 , Daniel THOMAS 3 , BrunoLINA 2,4 , Manuel ROSA CALATRAVA 2 , Catherine ISEL 1 , VincentMOULES 51 Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRS,IBMC, Strasbourg, France; 2 Virologie et Pathologie Humaine, UniversitéLyon 1, Faculté de Médecine RTH Laennec Lyon est, Lyon, FRANCE;3 Interactions Cellulaires et Moléculaires, Université Rennes 1, CNRS,Rennes, FRANCE; 4 Laboratoire de Virologie, CNR des virus influenzae(Sud France), CBPE, HCL, Groupement Hospitalier Est, Rennes,FRANCE; 5 VirNext, Lyon, FRANCE; 6 Illkirch Graffenstaden, FRANCEThe genome of influenza A viruses (IAV) is split into 8 viral RNAs(vRNAs) that are packaged as viral ribonucleoproteins (vRNPs). Themolecular basis of the vRNA packaging mechanism remains elusive: itwas hypothesized that packaging could be mediated by direct interactionsbetween the vRNA packaging regions, but such interactions have neverbeen demonstrated in virions. We showed that the 8 vRNAs of a humanH3N2 IAV (MO) and of an avian H5N2 IAV (EN) form complex interactionnetworks in vitro, but these networks and the regions of the vRNAsthey involve are different. Electron tomography also revealed significantdifferences in the interactions between vRNPs in EN and MO virions, suggestingthat the packaging signals might not be conserved between IAVs.We focused on the interaction between the PB1 and NS EN segments,which is inhibited in vitro by silent mutations in either vRNA, and restoredwhen combining the two mutated segments. Using reverse genetics,we produced the wild type, the two single mutant, and the double mutantviruses. Quantification of the vRNAs by Q RT PCR and observation ofthe viral particles by electron microscopy indicated that this interactionalso exists in infected cells and/or in viral particles and revealed a globaldefect in vRNA packaging in single mutant viruses. In addition, competitionexperiments between wild type and mutant vRNAs for packagingsupported a role for this interaction in the selective packaging of thesegenomic RNA segments. These results constitute the first direct evidenceof a functional vRNA/vRNA interaction.REF O136Characterization of a 3D in vitro model for assembly of very lowdensity HCVUrsula ANDREO 1 , Margaret A. SCULL 1 , Brenna R. FLATLEY 1 , VyasRAMANAN 2 , Shengyong NG 2 , Linda ANDRUS 1 , Alice A. CHEN 2 ,Edward A. FISHER 3 , Sangeeta N. BHATIA 2 , Charles M. RICE 11 Center for the Study of Hepatitis C, Laboratory of Virology and InfectiousDisease, The Rockefeller University, NY, USA; 2 Harvard MIT Division ofHealth Sciences and Technology, Massachusetts Institute of Technology,Cambridge, USA; 3 Department of Medicine (Cardiology) and the Marcand Ruti Bell Program in Vascular Biology; and the Department of CellBiology, NY, USAHepatitis C is a major public health problem with over 130 millionpeople infected worldwide. Hepatitis C virus (HCV) derived from infectedpatients is characterized by a lower buoyant density and higher specificinfectivity than in vitro derived virus. This discrepancy is likely due toHCV association in vivo with host lipoproteins. HCV association withlipoprotein of very low density (VLDL) is thought to occur during virusassembly and likely impacts virus susceptibility to neutralization andreceptor usage. The Huh7 derived human liver cell lines used to produceHCV particles in vitro do not properly reproduce VLDL secretion and donot make HCV particles of very low density. Thus, a human in vitro modelsystem is needed to study HCV association with VLDL. We have developed3D liver ‘organoids’ consisting of Huh7.5 human hepatoma cellsin coculture with fibroblastic stromal cells, encapsulated in a fully definedpoly(ethylene glycol) (PEG) based matrix. Here we show that this 3Dmodel is able to produce apoB containing lipoproteins of VLDL density.Furthermore, 3D organoids are susceptible to HCV infection, yield hightiters in the supernatants over prolonged periods of time and secrete HCVparticles of very low density as demonstrated by sucrose density ultracentrifugation.Thus, we believe that 3D organoids constitute a better in vitromodel to study HCV assembly and interaction with host lipoproteins.REF O137Infectivity of an alphacoronavirus depends on the tyrosine based sortingmotif in its S protein cytoplasmic domainChristel SCHWEGMANN WESSELS 1 , Katarina SHAHWAN 1 , SandraSIEWERT 1 , Luis ENJUANES 2 , Georg HERRLER 11 University of Veterinary Medicine Hannover, Institute for Virology, Hannover,GERMANY; 2 Centro Nacional de Biotecnología, CSIC, Departmentof Molecular and Cell Biology, Madrid, SPAINVirologie, Vol 17, supplément 2, septembre 2013S97

5 th European Congress of VirologyThe tyrosine based sorting motif in the cytoplasmic tail of the TGEV Sprotein is important for its retention in the ERGIC, the site of coronavirusbudding. This tyrosine based motif is well conserved in S proteins ofalphacoronaviruses. To study the role of this sorting motif (1440YXXI) ininfectivity, in incorporation of S protein into virus particles and in interactionwith the coronavirus M protein, the motif was disrupted with a tyrosineto alanine mutation (Y/A). A lysine to methionine mutation (K/M) at position1444 transformed the tyrosine based retention signal into a tyrosinebased endocytosis signal.Recombinant TGEV were constructed and analyzed for infectivity. Thevirus with a disrupted tyrosine motif (YI/AA) in the S protein was not ableto infect ST cells whereas the wildtype virus did. A complementation ofthe non infectious rTGEV with authentic S protein or the K/M mutant ledto the production of infectious virus. We analyzed whether the tyrosinebased motif is important for interaction with the M protein. The authenticS as well as as both mutants (Y/A, K/M) could interact with M as shown byimmunofluorescence and western blot analysis. In a VLP assay we coulddemonstrate that the Y/A and YI/AA mutant was not incorporated intoparticles while authentic S and K/M could be found together with M inVLPs.We conclude that the tyrosine based sorting signal in the TGEV S proteinis essential for incorporation of S into virions. The interaction with so farunknown cellular proteins via this motif seems to be one key factor for theassembly of infectious virus.REF O139Essential role of the Fusion protein during Sendai virus particle formationManel ESSAIDI LAZIOSI 1 , Anastasia SHEVTSOVA 1 , DenisGERLIER 2 , Laurent ROUX 11 Department of Microbiology and Molecular Medicine, Faculty of Medicine,University of Geneva, Geneva, SWITZERLAND; 2 Ecole NormaleSupérieure de Lyon, INSERM U1111, CNRS UMR 5308, Lyon, FRANCEPrevious studies have shown the importance of the fusion protein (F)during Sendai virus particle production. The absence of the TYTLE motifin the cytoplasmic domain of F has the same deleterious effect on VPproduction as the suppression of the entire F protein (Fouillot Coriou, N.and Roux, L. 2000). The mechanism by which this motif is involved inthis process is not understood. We demonstrate by confocal microscopyand immunoprecipitation that a F protein harboring 5 alanines instead ofthe TYTLE motif is not express at the cell surface although it is still ableto interact with the matrix protein (M). Interestingly, the absence of themutated F at the cell surface correlates with a lower representation of HNand M at cell surface. In addition, these two proteins show a more diffusedand disorganized pattern, both at the cell surface and in the cytoplasm. Inthe end, the mutated F retains M in the cytoplasm and prevents the processof assembly. These results highlight the important role of the fusionprotein during virus particle formation.REF O138Association of apolipoprotein B with Hepatitis C virus is involvedin viral infectivity and is dependent on the liver enriched proteinCidebChristophe RAMIÈRE 1,2,3 , Liviu Sorin ENACHE 4 , MarinePORCHEROT 2,3 , Olivier DIAZ 2,3 , Laure PERRIN COCON 2,3 , VincaICARD 1 , Caroline SCHOLTÈS 1,2,3 , Vincent LOTTEAU 2,3 , PatriceANDRÉ 1,2,31 Hospices Civils de Lyon, Lyon, FRANCE; 2 INSERM U1111, CIRI, Lyon,FRANCE; 3 Université de Lyon, Lyon, FRANCE; 4 University of Medicineand Pharmacy, TârguMure, ROMANIAIn patients’ blood, Hepatitis C Virus (HCV) circulates as lipo viral particles(LVP), hybrid particles associating viral and lipoprotein components, inparticular apolipoprotein B (apoB). Contrary to natural LVPs, HCV particlesproduced in vitro in Huh7.5 cells (HCVcc) are characterized bypoor association with apoB and low specific infectivity. We thus hypothesizedthat apoB could be involved in HCV infectivity and examinedthe mechanisms responsible for its association with HCV virions. We firstdemonstrated by neutralization experiments that 2 monoclonal anti apoBantibodies, recognizing epitopes in the Low–Density Lipoproteins Receptorbinding domain of apoB, were able to strongly neutralize (ca 80 to90%) infection of Huh7.5 cells by HCVcc, contrary to antibodies targetingepitopes outside this domain. We also identified Cideb, a liver enrichedprotein involved in lipoprotein assembly, as an essential factor for associationof HCV with apoB. We showed that Cideb directly interacted withHCV E2 envelope glycoprotein. We also observed that Cideb expressionwas low in Huh7.5 cells and that Cideb overexpression in this cell line wassufficient to induce secretion of E2 in association with apoB. Moreover,differentiation of Huh7.5 cells in presence of human serum and DMSO ledto a dramatic increase of Cideb expression and further secretion of HCVparticles characterized by higher association with apoB and improved specificinfectivity. Together these results suggest that apoB plays an importantrole in HCV entry and that Cideb is essential for HCV association withapoB.REF O140A comparative functional RNA interference screen identifies factorsdifferentially required for lipid droplet homeostasis and life cycle ofFlaviviridae membersGualtiero ALVISI 1,2 , Ina Karen STOECK 2 , Sandeep AMBERKAR 3 ,Narsis Aftab KIANI 3 , Christoph SOMMER 4 , Wolfgang FISCHL 2 ,Marion POENISCH 2 , Fred HAMPRECHT 4 , Giorgio PALÙ 1 , LarsKADERALI 4 , Ralf BARTENSCHLAGER 21 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Department of Molecular Virology, University of Heidelberg, Heidelberg,GERMANY; 3 Institute of Medical Informatics & Biometry, Dresden Universityof Technology, Dresden, GERMANY; 4 Heidelberg Collaboratoryfor Image Processing, University of Heidelberg, Heidelberg, GERMANYLipid Droplets (LDs) play a central role in storage and mobilization oflipids and are involved in lipid metabolism related diseases, as well as inthe replication cycle of several viruses, including the hepatotropic hepatitisC virus (HCV) and the Dengue virus (DENV). However, in spiteof their importance, the pathways and factors regulating LD homeostasisin human liver cells are poorly characterized, and their suitability asantiviral targets has not been explored. To overcome this limitation, weassembled a siRNA library targeting 230 genes potentially regulating LDhomeostasis. This library was used to perform a comparative functionalRNA interference screen to identify key factors of LD homeostasis waswell as factors promoting or restricting HCV and DENV replication. Wecould identify 59 genes as important factors for LD homeostasis, most ofthem also playing key roles for viral replication. Bioinformatic analysisof hits identified biological processes regulating both viral life cycle andLD homeostasis. These include COPI coated vesicle budding, RNA splicingor proteasomal and ubiquitin dependent catabolic processes. Uponconfirmation by a secondary deconvolution screen, the DDX3 dead boxhelicase and the calcium independent phospholipase A2 iPLA2 wereselected for follow up studies. Our results suggest a novel role for DDX3in HCV assembly/release, which appears to be independent from its interactionwith core protein. Moreover, pharmacological ablation of iPLAactivity selectively impaired HCV particle production, but did not affectthe DENV life cycleS98 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologySaturday 14 th September 2013, 8h30 – 10h30WORKSHOP 19: “VIRAL EVOLUTION ANDQUASISPECIES”Chairpersons: Esteban DOMINGO (Madrid, SPAIN) &Alexander PLYUSNIN (Helsinki, FINLAND)Room Gratte-Ciel 1, 2, 3KEYNOTE:Monitoring short-term virus evolution in vivo by deep sequencingtransmitted viral subpopulationsK.A. STAPLEFORD 1 , L. COFFEY 1 ,S.LAY 4 , V. DUONG 4 ,O. ISAKOV 5 , C. ARIAS-GOETA 2 , H. BLANC 1 , A. BORDERIA 1 ,S. BEAUCOURT 1 , T. HALILOĞLU 6 , C. SCHMITT 3 , I. BONNE 3 ,N. BEN TAL 7 , N. SHOMRON 5 , A.B. FAILLOUX 2 , P. BUCHY 4 ,M. VIGNUZZI 11 Viral Populations and Pathogenesis Unit, Institut Pasteur, Paris,FRANCE; 2 Arboviruses and Insect Vectors Lab, Institut Pasteur, Paris,FRANCE; 3 Ultrastructural Microscopy Platform, Institut Pasteur, Paris,FRANCE; 4 Institut Pasteur de Cambodge, Phnom Penh, CAMBODIA;5 Department of Cell and Developmental Biology, Tel Aviv University,ISRAEL; 6 Dept of Chemical Engineering, Boğaziçi University, Istanbul,TURKEY; 7 Department of Biochemistry and Molecular Biology, Tel AvivUniversity, ISRAELThe emergence of new strains with epidemic potential is often associatedwith the accumulation of adaptive mutations that are generally identifiedretrospectively. Although it may be impossible to predict the long-termevolution of currently circulating strains, we hypothesized that by investigatingthe relative frequencies of minority variants in viral subpopulationsin vivo, we could identify variants with the potential to emergence asnew epidemic strains. To examine this, we performed natural infections ofmosquitoes with chikungunya virus and examined the spatial and temporaldynamics of virus populations in different compartments. We first infectedindividual Aedes mosquitoes with the pre-epidemic E1 V226 strain andidentified the emergence of the epidemic mutation A226 V within 7 days ofinfection. We then performed new infections with the A226 V strain andidentified two novel E1 mutations emerging in the saliva compartment.These mutations increased virus fitness in both mosquitoes and mammals,and were found to naturally arise and transmit in insect-to-mammal transmissionexperiments. In vitro studies suggest that these mutations increaseextra-cellular particle stability providing a possible mechanism for theirselection. These studies illustrate how deep sequence monitoring of viruspopulations during natural infection can be a powerful tool for surveillanceand short-term prediction.ORAL COMMUNICATIONSREF O141Low fidelity strains of alphaviruses with fitness defects in vitro andin vivoKathryn ROZEN GAGNON, Kenneth STAPLEFORD, Hervé BLANC,Marco VIGNUZZIInstitut Pasteur, Paris, FRANCELittle is known about the molecular mechanisms or determinants of replicationfidelity in RNA genome replication. Chikungunya virus (CHIKV)is an arthropod borne alphavirus transmitted by Aedes mosquitoes and isre emerging as a major human pathogen. We previously isolated a highfidelity CHIKV polymerase variant, C483Y, which has decreased titers inmammalian and insect hosts [1]. To further investigate effects of position483 on polymerase fidelity and CHIKV pathogenesis, we substituted everyamino acid at this position. Among the 12 stable variants, we isolated thefirst arbovirus low fidelity variants. Because residue C483 is conservedacross the genus, we substituted several amino acids at analogous position482 in the distantly related alphavirus Sindbis virus (SINV). We againfound low fidelity variants with higher mutation frequencies than WT.Together, these findings suggest that position C483 is a general determinantof intrinsic polymerase fidelity. In addition, these novel low fidelitystrains allow us to examine the fitness of error prone variants with higherlevels of diversity. CHIK low fidelity strains have lower specific infectivityin mammalian and mosquito cells, as well as severe replication defects inmosquito cells. In vivo studies in mice show reduced titers in key organsand altered tropism, and analogous studies are underway in mosquitoes.Pending our results, low fidelity variants could be valuable candidates forlive attenuated vaccines that are non transmissible by mosquitoes.Coffey LL, et al. Proc Natl Acad Sci USA2011; 108: 16038-43.REF O142Factors Influencing the Fixation of Beneficial Mutations in BacteriophageQbeta Evolved at Increased Error RateLaura CABANILLAS, María ARRIBAS, Ester LÁZAROCentro de Astrobiología, Torrejón de Ardoz, SPAINRNA virus populations constitute a huge reservoir of mutants which providemultiple adaptive possibilities. The increase of the replication errorrate by artificial means is generally associated to higher numbers of deleteriousmutations per genome, which can lead the virus population toextinction. Under increased mutagenic conditions beneficial mutations arealso generated, although they usually have difficulties to fix due to theirpresence in deleterious backgrounds. Another factor that can delay thefixation of beneficial mutations is competition among them, when theyare present in different genomes that spread simultaneously. In the currentstudy we have analyzed the propagation of beneficial mutations inbacteriophage Q, evolved in the presence of the mutagenic nucleosideanalogue 5 azacytidine (AZC). The analysis of the mutant spectra of thevirus populations isolated at different points of the transfer series showeda large number of polymorphic mutations, some of them with demonstratedselective value. Polymorphisms distributed into several evolutionarylines that can compete among them, making it difficult the emergence ofa defined consensus sequence (Cabanillas et al., 2013). The presence ofaccompanying deleterious mutations, the high degree of recurrence of thepolymorphic mutations and the occurrence of epistatic interactions contributeto generate a complex dynamics of interaction among mutations. Thissituation permits the coexistence of multiple adaptive pathways that canprovide selective advantages by different molecular mechanisms.REF O143Response of hepatitis C virus to long term passage in the presenceof interferon alpha. Multiple mutations and a common phenotypeCelia PERALES 1,2 , Celia PERALES 1,2 , Nathan N. BEACH 1 , IsabelGALLEGO 1,2 , Maria Eugenia SORIA 1 , Josep QUER 2,3,4 , Juan IgnacioESTEBAN 2,3,4 , Charles M. RICE 5 , Esteban DOMINGO 1,2 , JulieSHELDON 11 Centro de Biología Molecular Severo Ochoa (CSIC UAM), Madrid,SPAIN; 2 Centro de Investigación Biomédica en Red de EnfermedadesHepáticas y Digestivas (CIBERehd), Barcelona, SPAIN; 3 Liver Unit,Internal Medicine Hospital Universitari Vall d’Hebrón, Vall d’HebrónInstitut de Recerca (VHIR), Barcelona, SPAIN; 4 Universitat Autónomade Barcelona, Barcelona, SPAIN; 5 Center for the Study of Hepatitis C,Laboratory of Virology and Infectious Disease, Rockefeller University,New York, USAVirologie, Vol 17, supplément 2, septembre 2013S99

5 th European Congress of VirologyThe molecular basis, biological effects, and consequences for therapy ofinterferon a (IFN a) resistance in hepatitis C virus (HCV) are largely unknown.We have explored the evolutionary dynamics of HCV in cell culturein response to IFN a by adapting the HCVcc Huh 7.5 virus cell systemto show a robust, sustained viral replication in the absence or presence ofIFN a. A gradual exposure to IFN a allowed sustained HCV replicationfor at least 100 passages. Virus survival, fitness levels, genetic changes,and phenotypic traits were examined. The virus passaged in the presenceof IFN a acquired IFN a resistance as evidenced by enhanced progenyproduction and viral protein expression in an IFN a environment, and adecreased sensitivity to a combination of IFN a and ribavirin. Consensusgenomic nucleotide sequences of the viral populations after 30, 45, and100 passages revealed some adaptative mutations, and multiple, non synonymousmutations scattered throughout the genome associated with IFNa resistance. A phenotypic trait common to all passaged viral populationswas an increased shut off of host cell protein synthesis, accentuated ininfections with IFN a selected populations carried out in the presence ofIFN a. The trait was associated with enhanced phosphorylation of PKR andeIF2a, although other contributing factors are likely. The results suggestthat multiple, independent mutational pathways can confer IFN a resistanceto HCV, and might explain why no unified picture has been obtainedregarding IFN a resistance in vivo.REF O144Codon specific positive selection and atypical nucleotide substitutionpatterns in prolonged poliovirus infectionTapani HOVI 1 , Carita SAVOLAINEN KOPRA 1 , Teemu SMURA 1 ,Soile BLOMQVIST 1 , Haider AL HELLO 1 , Merja ROIVAINEN 1National Institute for Health and Welfare, Helsinki, FINLANDPoliovirus (Family PIcornaviridae, genus Enterovirus, species Humanenterovirus C) usually causes an acute, a few weeks long infection inhumans. A prolonged infection may be established In immune deficientindividuals immunized with the attenuated oral poliovirus vaccine, resultingin years long shedding of the virus in stools and subsequently intosewage. Judging from sequence analysis of virus strains isolated from paralyticpatients, polioviruses evolve very rapidly reaching rates of the orderof 0.01 substitutions/site/year, with most of the fixed mutations being synonymoustransitions. We analyzed capsid protein VP1 coding sequences of102 type 2 vaccine derived poliovirus strains (VDPV) isolated from sewagein Slovakia during 22 months in 2003 2005. Most likely, all the viruses hadbeen shed by one unidentified person, though occasional spreading to andabortive infection in close contacts cannot be excluded. The sequencesdiffered from that of the parental Sabin 2 by 12.5 15.6% with a maximumof 9.6% between the VDPV strains. Unlike the data from epidemictransmission, the nucleotide substitution pattern among the VDPV strainsshowed relatively low overall transition/transversion (Ts/Tv) bias (5.6)and Ts/Tv rate ratios for both purines and pyrimidines hundreds fold lowerthan those found in sequence sets based on strains derived from poliovirustransmission. Codon wise analysis of synonymous versus non synonymoussubstitution rates suggested occurrence of positive selection at threeseparate locations on capsid wall during the 22 months observation period.REF O145Evolutionary implications of distinct recombination patterns observedwithin Human enterovirus A speciesAlexander LUKASHEV 1 , Elena SHUMILINA 1 , Ilya BELALOV 1 , OlgaIVANOVA 1 , Tatiana EREMEEVA 1 , Olga TROTSENKO 2 , VadimREZNIK 3 , Jan Felix DREXLER 41 Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow,RUSSIA; 2 Center of Hygiene and Epidemiology in Khabarovsk Region,Khabarovsk, RUSSIA; 3 Khabarovsk Institute of Epidemiology and Microbiology,Khabarovsk, RUSSIA; 4 Institute of Virology, University of BonnMedical Centre, Bonn, GERMANYHuman enteroviruses are amongst the best studied positive strand viruses.In particular, natural recombination among circulating enteroviruses hasbeen explored in detail. It has been shown that they recombine every fewyears within a species, and recombination frequency can differ several foldbetween serotypes. However, most studies have been centered on one orfew serotypes, while a species may comprise up to 40 serotypes. We studiednatural recombination among 79 isolates representing most of serotypes ofHuman enterovirus A species. Recombination was very frequent amongstcoxsackieviruses. Median half life of a non recombinant tree node betweendifferent genome regions was 4 6 years (and never more than 11years), and all viruses that differed by more than 6% nt sequence wererecombinant. Enterovirus 71, member of the same species, on contrary,was much less involved in recombination. Three lineages of EV71 circulatedglobally for 20 40 years almost without recombination. Five outof over 150 EV71 genomes had alien non structural genes, but there wasnot a single case of EV71 non structural proteins combined with capsidproteins of another serotype. We speculate that these contrasting recombinationprofiles of EV71 and coxsackieviruses represent two evolutionarystrategies of non structural genes, extreme promiscuity vs. adaptation tospecific capsid genes or proteins, and together with a mutator phenotypemay be termed an evolver genotype. Despite dynamic evolution on a scaleof years, circulating lineages of enterovirus genes were stable on a scaleof decades.REF O146Evolution of the hemagglutinin of pandemic H1N1 (2009): maintainingoptimal receptor binding by compensatory substitutionsErik DE VRIES 1 , Robert DE VRIES 1,2 , Carles MARTÍNEZROMERO 3 , Ryan MCBRIDE 2 , Peter ROTTIER 1 , Adolfo GARCÍASASTRE 3 , James PAULSON 2 , Xander DE HAAN 11 Virology Division, Faculty of Veterinary Medicine, Utrecht University,Utrecht, THE NETHERLANDS; 2 Department of Chemical Physiology,The Scripps Research Institute, San Diego, USA; 3 Department of Microbiologyand Department of Medicine, Division of Infectious Diseases,Icahn School of Medicine Mount Sinai, New York, USAPandemic influenza A virus was introduced in 2009. In 4 years it has spreadin the population and genetic changes that counteract adaptive immunityare expected. However no major antigenic differences have been reportedyet. Recent publications showed that changes in virus binding aviditycould provide the first step in counteracting immune pressure. We thereforeexamined the effect of the most frequently occurring amino substitutionsin the hemagglutinin (HA) of pdmH1N1 (in NCBI flu database before 110 2012) on the receptor binding properties of recombinant soluble HAtrimers. Only two changes (S186P and S188T) increased binding aviditywhereas substitutions A137T and A200T decreased binding avidity.Construction and analysis of an HA protein tree reveals the worldwideemergence of several HA variants in the latest seasons. The consecutivesubstitutions that formed these variants were reconstructed stepwise inthe recombinant soluble HA trimers. The two major variants both harboredcombinations of substitutions (S186P/A137T and S188T/A200Trespectively) with opposite individual effects on binding. Strikingly, thecombination of these substitutions restores binding avidity and bindingspecificity (glycan array analysis) to the same level and specificity asobserved for the original virus. The results strongly suggest that the HAof pdmH1N1 was already optimally adapted to the human host upon itsemergence in april 2009. Moreover, as predicted, transient increases in bindingavidity might be one of the first adaptations involved in counteractingantibody neutralization.S100 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologySaturday 14 th September 2013, 8h30 – 10h30WORKSHOP 26: “VIRAL INFECTIONS IN TRANSPLANTAND IMMUNOCOMPROMISED PATIENTS”Chairpersons: Paulo PAIXAO (Lisbon, PORTUGAL)& Thomas SCHULZ (Hannover, GERMANY)AmphitheaterKEYNOTE: AWARD of the Italian Society of Virology: “Excellence inVirology”Virologic and immunologic monitoring of human cytomegalovirusinfections in the immunocompetent and immunocompromised host:perspectives for the development of a subunit glycoprotein pentamericvaccineGiuseppe GERNALaboratori Sperimentali di Ricerca, Area Trapiantologica, FondazioneIRCCS Policlinico San Matteo, 27100 Pavia, ITALYPCR analysis of hundreds of immunocompetent healthy subjects with andwithout reactivated HCMV infections never revealed presence in wholeblood of HCMV DNA at a measurable level (>100 copies/ml blood). Asimilar approach in the immunocompromised host allowed to define onsetof clinical symptoms at 1,000,000 DNA copies/ml in SOTR, while inHSCTR caution suggested that severe clinical syndromes, such as pneumonia,could start at a viral load of ≤ 100,000 copies/ml. This level ofVL allowed to fix blood levels for preemptive therapy at 300,000 HCMVDNA copies for SOTR, and 30,000 HCMV DNA copies for HSCTR.Levels of HCMV-specific CD4+ and CD8+ T-cell responses showing protectionagainst HCMV reactivation were 0.4 T-cells/l blood for boththe immunocompetent and SOTR patients, and 1.0 CD4+ and 3.0 CD8+T-cells/l for HSCTR. In pregnant women with primary HCMV infectionlevels of VL never overcame 1,000 DNA copies/ml blood, due tothe prompt T-cell response. Neutralizing antibody titers, as determinedin ARPE-19 epithelial cells, appeared very early during primary infection,reaching extraordinarily high titers in the immunocompromised host(both primary and reactivated infections). B-T-cells interactions must beinvestigated.On the vaccine side, the gH/gL/pUL128L pentamer complex elicits a verystrong antibody response to its components and to the whole complex,both in natural infection of humans and experimentally inoculated mice.The T-cell response to the pentamer is now under study.ORAL COMMUNICATIONSREF O147Hepatitis E virus: an underestimated opportunistic pathogen in recipientsof allogeneic hematopoietic stem cell transplantationSuzan PAS 1 , Jurjen VERSLUIS 2 , Erik AGTERESCH 2 , Robert DEMAN 3 , Jolanda MAASKANT 1 , Marguerite SCHIPPER 4 , AlbertOSTERHAUS 1 , Jan CORNELISSEN 3 , Annemiek VAN DER EIJK 11 Department of Viroscience, ErasmusMC, Rotterdam, THE NETHER-LANDS; 2 Department of Hematology, ErasmusMC, Rotterdam, THENETHERLANDS; 3 Department of Hepatogastroenterology, ErasmusMC,Rotterdam, THE NETHERLANDS; 4 Department of Pathology, ErasmusMC,Rotterdam, THE NETHERLANDSHepatitis E virus (HEV) is an emerging health issue in industrializedcountries, particularly in the immunocompromised. Since littleis known of HEV infections in allogeneic hematopoietic stem celltransplantation recipients (alloHSCT), we studied HEV infection inalloHSCT.Patients receiving alloHSCT between January 2006 July 2011 were included.Anti HEV serostatus (Wantai assay) before alloHSCT and presence ofHEV RNA after alloHSCT and during episodes of liver function abnormalitieswere assessed. The course of HEV infection and clinical implicationswere studied from confirmed cases.328 Patients were included in the study. In total, eight HEV genotype3 infected cases (2.4%) were identified, of which five developed chronicHEV. These were misdiagnosed before as hepatic graft versus host disease(n=5) or drug toxicity (n=3). Seroprevalence prior to alloHSCT was 12.9%,2 patients (0.6%) were anti HEV IgM positive, though HEV RNA was notdetected.Four of eight cases died with HEV viremia, signs of ongoing hepatitis(n=4)and neurologic disease(n=1). The four living patients cleared HEV supportedby ribavirin treatment (n=1) and reduction of immune suppression(n=3). Two of four living patients were diagnosed with chronic hepatitisand fibrosis by liver biopsy. One HEV patient presented with viralreactivation.We conclude that a differential diagnosis including HEV is mandatorygiven the clinical impact. Future alloHSCT recipients should be screenedfor HEV RNA prior to transplantation and should be monitored afteralloHSCT, especially if liver abnormalities occur.REF O148Prolonged rhinovirus infection in hematological immunocompromisedpatients with impaired T cell immunityAntonio PIRALLA 1 , Marco ZECCA 2 , Anna Amelia COLOMBO 3 ,Alessia GIRELLO 1 , Patrizia COMOLI 2 , Rita MACCARIO 2 , FaustoBALDANTI 11 Molecular Virology Unit, Virology and Microbiology Department,Fondazione IRCCS Policlinico San Matteo, Pavia, ITALY; 2 PediatricHematology Oncology and Research Laboratories Fondazione IRCCSPoliclinico San Matteo, Pavia, ITALY; 3 Institute of Hematology, FondazionePoliclinico San Matteo IRCCS, University of Pavia, Pavia, ITALYRespiratory samples from patients hospitalized with acute respiratory syndromein the period 2006 2012 were tested with a panel of respiratoryviruses. In 12 hematopoietic stem cell transplant recipients (HSCTR) and3 patients with hematologic diseases a prolonged rhinovirus (HRV) shedding(>30 days) was observed. In these 15 patients, HRV was quantifiedby real time RT PCR and molecular typing was performed by sequencingthe 5 ′ NCR VP4 region in sequential respiratory samples. In detail:11/15 (73.3%) patients were pediatric (10 HSCTR, and 1 acute myeloidleukemia) and 4/15 (26.7%) were adults (2 HSCTR and 2 acute lymphocyticleukemia). The median duration of HRV positivity was 64 days(range 30 174 days). Phylogenetic analysis showed the persistence ofa single HRV type in all patients except one. In 9/12 (75.0%) HSCTRpatients, HRV infection occurred across the transplant aplastic period(either during induction or prior to engraftment). Low level (

5 th European Congress of VirologyREF O149Factors for Adenovirus Infection and Disease in Pediatric HematopoieticStem Cell Transplant patientsLinda FEGHOUL 1 , Jean Hugues DALLE 2 , Sylvie CHEVRET 1 , MarieOUACHÉE 2 , André BARUCHEL 2 , Mony FAHD 2 , Valérie GUÉRIN ELKHOUROUJ 2 , Ghislaine STERKERS 2 , François SIMON 1 , JérômeLEGOFF 11 Université Paris Diderot, Hopital Saint Louis, Paris, FRANCE;2 Université Paris Diderot, Hopital Robert Debré, Paris, FRANCEAdenovirus (Adv) infections are critical in hematopoietic stem cells transplantation(HSCT). The aim was to determine risk factors for Adv infectionand disease in pediatric HSCT patients. Between September 2010 andDecember 2011, 73 pediatric HSCT patients were prospectively followedfor 12 months and weekly tested for adenovirus in blood and stool. Sheddingwas defined as a positive detection in stool, systemic infection aspositive detection in blood, and disease as systemic infection with Advsymptoms. Multivariable Cox models were used to define predictive factorsfor shedding in stool, systemic infection and disease with estimatedhazard ratio (HR) of event as a measure of association. Cumulative incidenceof shedding, infection and disease at 3 months post HSCT were 40%,23% and 17% respectively. Based on multivariable models, cord bloodtransplant was associated with shedding in stool (HR=4.29) and systemicinfection (HR=3.01). Acute GvHD was predictive of systemic infection(HR=1.46) and Adv disease (HR=2.55). For the patients with intestinalshedding, the occurrence of systemic infection was increased in case ofhigh viral load detected in stool (p=0.02). The sensitivity and specificity tohave a systemic infection were respectively 93.33% and 62.5% for loadsin stool above 5.36 log copies/ml. Conclusions: Cord blood transplant andacute GvHD appear the most appealing risk factors for AdV infection andshould be considered to initiate a molecular screening for Adv. Patientswith high AdV viral load in stool must be considered for early anti Advintervention.REF O150Level and kinetics of Plasma Torque Teno Virus DNA after LungTransplantation as a Marker to Guide Immunosuppressive TherapyIrene GOERZER 1 , Mats HALOSCHAN 1 , Peter JAKSCH 2 , IsabellaBEJVL 1 , Robert STRASSL 3 , Walter KLEPETKO 2 , ElisabethPUCHHAMMER STOECKL 11 Department of Virology, Medical University of Vienna, Vienna, AUSTRIA;2 Division of Thoracic Surgery, Medical University of Vienna, Vienna,AUSTRIA; 3 Clinical Institute of Virology, Medical University of Vienna,Vienna, AUSTRIATorque teno virus (TTV), widely spread among humans, causes persistentviremia in healthy persons probably without clinical disease. Transplantrecipients receive immunosuppressive therapy. Its optimization is criticalto avoid rejection or infection, but it is still a challenge as appropriate markersreflecting the state of immunosuppression are lacking. In this study,we investigated whether level and kinetics of TTV DNAemia could reflectthe patient’s immunosuppression after lung transplantation. Plasma TTVDNA kinetics was analyzed by quantitative PCR in 31 lung transplant recipients(LTRs) over 2 years posttransplantation. Its relation to tacrolimus(Tac) versus cyclosporine (CsA), to infection events and to developmentof bronchiolitis obliterans syndrome (BOS) was assessed. In all LTRs,TTV DNA significantly increased up to day 90 posttransplantation andsustained high TTV levels thereafter. The extent of TTV increase wasassociated with mean drug concentrations (p=0.012) and mean TTV levelsthereafter were lower in CsA treated than in Tac treated patients (p

5 th European Congress of VirologyA total of 111 FFPE blocks from 79 skin lesions were collected from17 organ transplant recipients. HPV DNA detection was performed byPM PCR RHA. Viral infection was visualized by using a combinationof antibodies raised against the E4 and L1 proteins of the genotypes,alongside FISH to identify sites of viral genome amplification.Out of the 111 FFPE blocks analysed,94 were positive by PCR analysiswith universal primers. The commonest genotypes were HPV5 and8. All the tissue samples were probed for the viral markers. Six lesionsfrom 4 patients showed expression of cytoplasmic E4 overlapping withFISH positive nuclei and L1 in the more superficial layers.Positive areaswere detected in precancerous lesions or at the borders of more malignanttumours.Here,we demonstrate that HPV transcription is occurring at sites ofskin transformation in the OTR setting.Virus activation in sun exposedbody sites may cause UV exposures to be more deleterious to host cell,potentially increasing the likelihood that these cells become cancerous.Virologie, Vol 17, supplément 2, septembre 2013S103

5 th European Congress of VirologySaturday 14 th September 2013, 11h00 – 13h00PLENARY SESSION 4: “THE UNIVERSE OF VIRUSES”Chairpersons:Marion KOOPMANS (Bilthoven, THE NETHERLAND)& Geoffrey SMITH (London, UNITED KINGDOM)Amphitheater11:00 – 11:30Order to the Viral UniverseDennis BAMFORDInstitute of Biotechnology, University of Helsinki, PO Box 56, Viikinkaari5, Helsinki 00014, FINLANDThe virosphere is enormous as well as the viral genomic diversity.However, the virion, the hallmark of a virus, can be constructed onlyusing the available protein folds. The number of unique protein folds isvery limited (current estimates being less than 1500) and only a smallsubset of these has properties suitable to assemble the virion. This wouldmean that the virion structure space is very limited and may even bedefined. We have set out to test this hypothesis by comparing virionand virus coat protein structures and globally isolating new viruses andcategorizing them to currently known virion morphotypes. It appears thatonly very seldom novel virion architectures are discovered supportingthe hypothesis put forward. It will be discussed how these observationsaffect the higher order classification of viruses.11:30 – 12:00Viral reservoirs and the promise of pandemic preparednessChristian DROSTENInstitute of Virology, University of Bonn Medical Center, Bonn, GERMANYTen years after the SARS epidemic, zoonotic and emerging viruses havebecome a growing field of research. Some remarkable novel virus descriptionsin animals have demonstrated how ignorant we are of the diversityof viruses around us. In our efforts to delineate viral origins we may haveto re-asess our concept of reservoir. In many instances, we are mixing upecological and epidemiological implications of viral evolution. Among thebiggest challenges in this field is the integration of the concepts of virushostcodivergence, and host switching (as well as the challenge to infer thelatter from phylogenies). In addition, assessments of viral reservoirs withthe intention to predict future pandemic threats would have to take intoaccount important host and virus traits which cannot be predicted merelyfrom virus genes. For example, we need to know whether there are hostswhich have a higher protensity to carry broader spectra or higher concentrationsof viruses, potentially without being affected. Among the virusesborne in such reservoirs, there may be some that are more promiscuous intheir choice of hosts, potentially due to the conservedness of their receptorstructures or the way they interfere with conserved- or not-so-conservedimmune properties. A synopsis of available approaches demonstrates howmuch work needs to be done before we will be able to assess functional,rather than genetic diversity of reservoir-borne viruses. However, there isno reason for pessimism as on our way there, we will come across surprisingand promising new insights which, for instance, may teach us how tobetter model viral diseases in the laboratory.12:00 – 12:30Emerging bunyavirusesRichard ELLIOTTMRC - University of Glasgow Centre for Virus Research, Institute of Infection,Immunity and Inflammation, Glasgow G61 1QH, Scotland, UNITEDKINGDOMThe family Bunyaviridae currently contains more than 350 named virusisolates characterised by possession of a tri-partite, single stranded RNAgenome that encodes proteins using a negative- or ambi-sense strategy.Viruses replicate in the cytoplasm and assemble at the Golgi. The familyis divided into 5 genera, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirusand Tospovirus. Tospoviruses infect plants and are transmitted byvarious species of thrips. Hantaviruses are maintained in nature as persistentinfections of rodents and are transmitted to humans in aerosolisedrodent excretions. Orthobunyaviruses, nairoviruses and phleboviruses areall arboviruses and are transmitted by mosquitoes, sandflies, midges, ticksand other blood-feeding arthropods. They cause a range of disease syndromesin man ranging from self-limiting fevers, through encephalitis andhepatitis, to fatal haemorrhagic fever, and include pathogens such as LaCrosse, Oropouche, Crimean-Congo haemorrhagic fever and Rift Valleyfever viruses. Bunyaviruses provide many examples of emerging viruses,in part due to climate change, though many other factors are involved.Recent examples include the human pathogen SFTSV in China, Japanand Korea, and the animal pathogen Schmallenberg virus that has spreadrapidly throughout Europe. Current knowledge on the molecular biologyof these viruses will be discussed.12:30 – 13:00Emerging respiratory virusesRon FOUCHIERDepartment of Virology and National Influenza Center, Erasmus MedicalCenter, Rotterdam, THE NETHERLANDSBackground: Since the start of the avian influenza A(H5N1) epizooticsin Southeast Asia in 1997 and the outbreak of SARS in 2003, numerousprograms to improve preparedness for emerging infectious diseaseswere initiated. In this context, we discovered a novel coronavirus associatedwith acute respiratory disease and renal failure with a fatal outcome(N Engl J Med. 2012 367:1814-20) in the summer of 2012, after whichnew detections have been reported continuously. In addition, Chinesehealth authorities reported the detection of human cases of avian influenzaA(H7N9) virus infection since the end of March 2013 (N Engl J Med.2013 368:1888-97). These two newly emerging respiratory viruses are ofconcern to public health.Objective: I will review the current situation with respect to the novelcoronavirus emerging in the Middle East and avian A(H7N9) influenzavirus emerging in China. As of June 21, 2013, the novel coronavirus wasreported in 64 laboratory-confirmed cases of infection, including 38 fatalities.Also as of June 21, a total of 132 laboratory-confirmed cases ofhuman infection with avian influenza A(H7N9) virus including 37 deathshave been reported to the WHO. So far, there is no evidence for sustainedhuman-to-human transmission for both virusesConclusion: These discoveries and the current period of low levels ofvirus circulation in (probably) animal hosts, provides an opportunity tointervene and prevent more widespread outbreaks or pandemics. However,this will require strong commitments from the countries where the casesso far have occurred. The current state of scientific investigation will bediscussedS104 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology13h15 – 14h30 SYMPOSIUM FINOVI“VIRAL HEPATITIS”Chairpersons: Birke BARTOSCH (Lyon, FRANCE), RalfBARTENSCHLAGER (Heidelberg, GERMANY)Room Prestige Gratte-CielEntry and entry inhibition of Hepatitis B VirusStephan URBANDepartment of Infectious Diseases, Molecular Virology, University HospitalHeidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg,Heidelberg, GERMANYFor almost three decades after the discovery of Hepatitis B Virus (HBV)the early events of infection (attachment, receptor binding and fusion)remained entirely unresolved. Although the extraordinary hepatotropismof HBV and its peculiar host specificity were supposed to be linked to specificreceptor binding, it stayed unclear which domain(s) within the viralenvelope protein(s) are essential for mediating entry and which cellularreceptor(s) are addressed. One reason was the lack of suitable in vitro infectionsystems. Following the establishment of the susceptible HepaRG cellline and primary hepatocytes from humans (PHH) and tupaia belangeri(PTH) as reliable in vitro infection systems, systematic analyses allowedthe identification of a myristoylated preS1-subdomain as essentialfor specific hepatocyte binding. Using a different approaches sodium taurocholateco-transporting polypeptide (NTCP/SCL10A1) could recentlybe identified as this highly specific hepatic receptor for the myristoylatedpreS1 subdomain. The development of peptidic ligands of this receptoras potent inhibitors of viral entry opened a novel therapeutic option totreat acute and chronically infected patients. The exclusive targeting ofthe lead substance of such lipopeptides (Myrcludex B) to the receptor inthe liver and the remarkable inhibitory potential at picomolar concentrationsmakes it a promising novel drug which successfully passed a phase Iaclinical trial and recently entered into a phase IIa efficacy trial. Moreover,the identification of NTCP as the long sought receptor for HBV allows theestablishment of robust cell culture systems and animal models for futureapproaches aiming at the identification of host factors, the identificationof novel drugs and the development of immune competent small animalmodels.Keywords: Hepatitis B Virus entry, Myrcludex B, Hepatitis B virus Receptor,NTCP, SCL10A1A role for SIGMA-1 receptor in early steps of viral RNA replicationat the onset of Hepatitis C virus infectionMartina FRIESLAND 1 , Lidia MINGORANCE 1 , Gema CALVO 1 , JosanCHUNG 2 , Francis V. CHISARI 2 , Pablo GASTAMINZA 1 *1 Departamento de Biología Molecular y Celular; Centro Nacional deBiotecnología-CSIC-Madrid; SPAIN; 2 Department of Immunology andMicrobial Science; The Scripps Research Institute-La Jolla, CA; USAHepatitis C virus genome replication is thought to occur in a membranouscellular compartment derived from the endoplasmic reticulum (ER). Themolecular mechanisms by which these membrane-associated replicationcomplexes are formed during HCV infection are only starting to be unraveledand both viral and cellular factors contribute to their formation. Inthis study, we describe the discovery of sigma-1 receptor (S1R) as a cellularfactor that mediates early steps of viral RNA replication. S1R is acholesterol-binding protein that resides in lipid-rich areas of the ER and inmitochondria-associated ER membranes (MAMs). Several functions havebeen ascribed to this ER-resident chaperone, many of which are related toCa2+ signaling at the MAMs and lipid storage and trafficking. Downregulationof S1R expression by RNAi in Huh-7 cells leads to a proportionaldecrease in susceptibility to HCV infection. Similar RNAi studies in persistentlyinfected cells indicate that S1R-expression is not rate-limiting forpersistent HCV RNA replication, as marked reduction in S1R in these cellsdoes not lead to any decrease in HCV RNA or viral protein expression.However, subgenomic replicon transfection experiments indicate that S1Rexpression is rate-limiting for HCV RNA replication, without impairingprimary translation. Overall, our data indicate that initial steps of HCVinfection are regulated by S1R, a key component of MAMs, suggestingthat these structures could serve as platforms for initial RNA replicationduring HCV infection.Cell entry process of in vitro and in vivo produced hepatitis C virus(HCV)S. CALATTINI, L. DAO-THI, F. FUSIL, J. MANCIP, D. LAVILLETTE,F.L. COSSET*, M. DREUX**equal contributionsCIRI, International Center for Infectiology Research, EVIR Team, Lyon,FranceLabEx EcofectUniversité de Lyon, Lyon, FRANCEHCV particles assemble along the very-low-density lipoprotein pathwayand are released from hepatocytes as entities varying in their degreeof association to lipoprotein components ie, lipid and apolipoprotein,as well as buoyant densities. Little is known about the cell entry pathwayof these viral sub-populations, which involves several cell surfacemolecules, including the scavenger receptor BI (SR-BI), a receptor forhigh-density lipoprotein that binds the viral envelope protein E2. Usingin vitro-produced HCV, we show that virus sub-populations differing intheir buoyant densities utilize SR-BI in a manifold manner. First, SR-BImediates primary cell attachment of HCV particles of intermediate densitythrough interactions involving apolipoproteins, such as apoE incorporatedin virions, but not the E2 glycoprotein. Second, we found that SR-BImediates cell entry of HCV particles independent of their buoyant density.This function does not depend on E2/SR-BI interaction, but relieson its lipid transfer activity and the other HCV entry co-factors. Finally,our results underscored a third function of SR-BI governed by the hypervariableregion 1 of E2 leading to enhanced cell entry and dependingon SR-BI ability to bind to E2. Since our results indicated that virionassociatedlipoprotein components act as host-derived ligands for entryfactors such as SR-BI lipid transporter, we sought to analyze how lipoproteinmetabolism modulates viral forms and entry properties of invivo-produced HCV, using the Fah-/-/Rag2-/-/IL2Rgc-/- (FRG) mousemodel that allows robust repopulation with primary human hepatocytesand human liver functions. We demonstrate that HCV particles recoveredfrom the serum of infected mice display higher and uniform specificinfectivity along all buoyant densities, as compared to in vitro-producedHCV particles. Thus, this model constitutes a valuable tool to define theinterplay between lipoprotein metabolism and HCV entry route.Beyond the “stealthy” character: HBV is a weak inducer of innateimmunity, but also a potent inhibitorDavid DURANTELCancer Research Center of Lyon (CRCL), UMR INSERM U1052/CNRS5286, University of Lyon (UCBL), and LabEx DEVWECAN, FRANCEHBV chronically infects around 250 millions of individuals worldwide.First line therapies consist of the treatment with various nucleoside analogues(NUC), which are very efficient at reducing blood viral load, but notat eliminating the persistent form of viral nucleic acid, the cccDNA, frominfected hepatocytes. To complement NUC-based therapy, it would beuseful to develop new types of antiviral, including immune-modulators.In this respect it is important to understand how the virus escapes hostVirologie, Vol 17, supplément 2, septembre 2013S105

5 th European Congress of Virologyimmune system. Indeed to establish itself as a chronic infection, HBV hasevolved many strategies to evade both innate and adaptive immunity.Focusing on the interplay between HBV and liver innate immunity, wewill give a state of the art overview, based on literature-reported and ourown results, of the strategy used by HBV to inhibit the function of professionalliver innate cells (Kupffer cells, pDC, NK/NKT, and LSEC),as well as innate immunity within infected hepatocytes. We will showthat, beside the fact HBV is a rather poor inducer of innate responses,including IFN response, HBV is also a potent inhibitor of these responses invarious cell types. Inhibitory strategies involve many highly secreted viralproteins/antigens, as well as virion-associated proteins. In more details,we will show that the HBV-mediated blockade of IFN response involvesthe capsid protein (HBc) located within the nucleus. Targeting HBcnuclear functions may help restoring innate immune function in infectedcells and therefore represents a novel immunotherapeutic option againstHBV.S106 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology13h15 – 14h30 SYMPOSIUM INSTITUTPASTEUR – FONDATION MERIEUXNETWORKS“FROM PUBLIC HEALTH AND SURVEILLANCE TO DRUGDISCOVERY”Chairpersons: Florence PRADEL (Lyon, FRANCE)& Kathleen VICTOR (Paris, FRANCE)Room Gratte-Ciel 1, 2, 3Pilot pneumonia multicentric study in the GABRIEL Networkof Fondation MÉRIEUX: The relevance of the respiratory virusesValentina PICOT 1 ; Melina MESSAOUDI 1 , Jean-Noel TELLES 1 ,Florence KOMURIAN-PRADEL 1 , GABRIEL members 2 , GláuciaPARANHOS-BACCALA 11 Emerging Pathogens Laboratory, Fondation Mérieux, International Centerfor Infectiology Research (CIRI), Lyon, FRANCE; 2 GABRIEL membersThe Pilot Multi-centric Pneumonia Study was launched in 2010 in theframework of Fondation Mérieux’s research activities coordinated bythe Emerging Pathogens Laboratory (LPE) and the GABRIEL network*.Designed as a prospective multi-centric case-control study, this researchproject is carried out in 9 countries: Brazil, Cambodia, China, Haiti, India(2 sites), Madagascar, Mali, Mongolia, and Paraguay in close collaborationwith hospitals and partners from local research institutions. Thispilot study follows a harmonized protocol (total sample size: 1000 casesand 1000 controls), ethical approval and is carried out under good clinicalpractices compliance. The final purpose of this study is to provide informationon the pneumonia etiology (viruses and bacterial) to improve casemanagement of the pneumonia ill child.*www.fondation-merieux.org*www.gabriel-network.orgFactors associated with severity of influenza virus infections in Africaand Asia: the Institut Pasteur International Network Research ProjectRichard NJOUOM 1 , Soatiana RAJATONIRINA 2 , ArnaudTARANTOLA 3 , Patrice TCHENDJOU 1 , Jean Michel HERRAUD 2 ,Philippe BUCHY 3 , Magali HERRANT 41 Centre Pasteur Cameroon, Yaounde, CAMEROON; 2 Institut PasteurMadagascar, Antananarivo, MADAGASCAR; 3 Institut Pasteur Cambodia,Phnom Penh, CAMBODIA; 4 Institut Pasteur International Network,Paris, FRANCEWhile some factors have been identified to be potentially associated withsevere forms of influenza in the industrialized world, very little informationis available from the developing world. Our hypothesis is that the severityof influenza might be higher in developing countries where health caresystems are less performant and co-infections are prominent compared tothe industrialized countries. In order to identify risk factors associated withsevere influenza infections in developing countries, a prospective multicentercase-control study of patients with laboratory-confirmed influenzainfection is currently going on in the Institut Pasteur International Networkin Africa (Cameroon and Madagascar) and Asia (Cambodia). Cases(severe forms of influenza requiring hospitalisation) are age-matched (± 5years) to controls (patients with mild forms of influenza treated as outpatients).The inclusion of subjects started in 2011 and will end in December2013. The results of this study will help to better understand the burdenof influenza in developing countries.Identification of viral pathogens and host-biomarkers in overlappingfebrile childhood illnesses in a malaria-endemic region ofMadagascarJonathan HOFFMANN 1,2 ; Muriel MAEDER 1 , HenintsoaRABEZANAHARY 1 , Martin RANDRIAMAROTIA 3 , FlorenceKOMURIAN-PRADEL 2 , Eustache PARAMITHIOTIS 4 , ValentinaPICOT 2 , Bénédicte CONTAMIN 1 and GláuciaPARANHOS-BACCALÀ 21 Centre d’Infectiologie Charles Mérieux (CICM), Faculté de Médecine,Université d’Ankatso, Antananarivo, MADAGASCAR; 2 EmergingPathogens Laboratory, Fondation Mérieux, International Center forInfectiology Research (CIRI), Lyon, FRANCE; 3 Fondation Médicaled’Ampasimanjeva (FMA), MADAGASCAR; 4 Caprion Proteomics, Montréal,Québec, CANADAInstitut Pasteur and Fondation Mérieux in Madagascar are centered theirresearch efforts understanding the respiratory viral diversity. Childrenliving in malaria-endemic regions need access to high-quality diagnosisfitting biomedical treatment for malaria illnesses and acute respiratoryinfections (ARIs). In the absence of appropriate diagnostic tools, managementof children illnesses is presumptive and symptom-based. Malariais essentially defined by fever while cough and/or difficult breathing asARIs. Both Institutions recently described the viral etiology of ARIs inchildren under 5 years-old in Madagascar. Another ongoing study aimedto identify candidate biomarkers linked to the well-known viral pathogensand Plasmodium. This is a promising approach to develop accurate andrapid diagnostics tools and overcome anti-malarial drug resistance in thesepediatric febrile illnesses.*www.fondation-merieux.orgEnterovirus 71 Epidemic in CambodiaChanna MEY 1 , Thérèse COUDERC 2 , Francis DELPEYROUX 2 , VeasnaDUONG 1 , Marc ELOIT 2 , Jeremy FARRAR 3 , Zhou GANG 4 , SothyHENG 5 , Denis LAURENT 5 , Marc LECUIT 2 , Jean-ClaudeMANUGUERRA 2 , Peijun REN 4 , Beat RICHNER 5 , Rogier VANDOORN 3 , Huang ZHONG 4 , Huachen ZHU 6 , Arnaud TARANTOLA 1 ,Sok TOUCH 7 , Guan YI 6 , Ralf ALTMEYER 4 , Philippe BUCHY 11 Institut Pasteur Cambodia, Phnom Penh, CAMBODIA; 2 Institut Pasteur,Paris, FRANCE; 3 Oxford University Clinical Research Unit, Ho ChiMinh city, VIETNAM; 4 Institut Pasteur in Shanghai/Chinese Academyof Science, Shanghai, CHINA; 5 Kantha Bopha hospital, Phnom Penh,CAMBODIA; 6 State Key Laboratory of Emerging Infectious Diseases,The University of Hong Kong, SAR CHINA; 7 Communicable DiseaseDepartment, Ministry of Health, CAMBODIAFrom April to December 2012, over 100 children, mostly under 3 years,died from acute febrile encephalitis associated with respiratory distress.Human Enterovirus 71 (EV71) was detected in 82% of severe cases. SeveralEV71 strains were fully or partially sequenced, demonstrating that theCambodian isolates belonged to the C4 subgenotype with close phylogenicrelationships with strains circulating in China between 2009 and2011 and in Vietnam in 2012. No recombination was observed in the fullgenome sequences analyzed. In less severe cases other human enterovirusesand coxsackie species were detected. Seroepidemiological studiesare currently being conducted to better estimate the burden of the diseaseand the history of the virus circulation in country.Virologie, Vol 17, supplément 2, septembre 2013S107

5 th European Congress of VirologySuramin inhibits Enterovirus 71 replication in vitro and in vivo by blockingvirus entry into target cells through binding of the naphthalenetrisulfonic acid group to the viral capsidREN Peijun 1,2,3 , ZOU Gang 1 , Benjamin BAILLY 1,4 , XU Shanshan 1 ,ZENG Mei 5 , CHEN Xinsheng 6 , Patrice GUILLON 4 , AndreaMAGGIONI 4 , Thomas HASELHORST 4 , FernandoARENZANA-SEISDEDOS 3 , LI Jian 6 , Mark VON ITZSTEIN 4 ,LI Qihan 7 , Ralf ALTMEYER 11 Unit of Anti-infection research, Institut Pasteur of Shanghai, ChineseAcademy of Sciences, No. 411 Hefei Road, 200025, Shanghai, China;2 Doctoral School of Biochemistry, Biotherapy, Molecular Biology, Infectiology,the University of Paris Diderot-Paris 7, 35 rue Hélène Brion,75013, Paris, France; 3 Viral Pathogenesis Laboratory, Institut Pasteur, 28,Rue Dr.Roux, 75724, Paris, France; 4 Institute for Glycomics, Gold CoastCampus, Griffith University, 4222, Queensland, Australia; 5 Departmentof Infectious Diseases, Children’s Hospital of Fudan University, 399Wanyuan Road, 201102, Shanghai, China; 6 WuXi AppTec, Co., Ltd.,200131, Shanghai, China; 7 Institute of Medical Biology, Chinese Academyof Medicine Science, 650118, Kunming, ChinaEnterovirus 71 (EV71) is the causative of severe infections of centralnervous system leading to cardiopulmonary complications and death inchildren under the age of 5. Using a library of FDA-approved drugswe identified that the anti-parasitic pediatric drug Suramin as inhibitorof EV71 replication in vitro (IC 50 0.08 M, selectivity index >10,000).Time of addition and virus binding assays demonstrated that Suraminblocks virus entry into susceptible target cells. Structure-activity relationshipanalysis and Saturation Transfer Difference Nuclear MagneticResonance identified that Suramin binds to the virus surface capsid proteinsvia the naphthalene tri-sulfonic groups. Suramin reduces mortalityin mice and EV71 peak viral load in adult rhesus monkeys when administerediv at the highest human dose allometrically scaled to the monkey.Our data show that the approved pediatric drug Suramin inhibits EV71replication by neutralizing virus particles prior to virus attachment. Ourdata suggest that Suramin is a candidate for further therapeutic developmentfor a safe and efficacious therapy or prophylaxis of severe EV71infections.S108 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologySaturday 14 th September 2013, 14h45 – 16h45WORKSHOP 1: “ONCOGENIC MECHANISMS OFVIRUSES”Chairpersons: Massimo TOMMASINO (Lyon, FRANCE) &Fabien ZOULIM (Lyon, FRANCE)Room Tête d’OrKEYNOTE:How does Kaposi Sarcoma-associated Herpes virus cause a vasculartumour?Thomas F. SCHULZInstitute of Virology, Hannover Medical School, Hannover, GERMANYKaposi Sarcoma-associated Herpesvirus (KSHV), aka HHV 8, is a classI carcinogen as defined by the International Agency for Research againstCancer (IARC/WHO). This classification is based on epidemiological findingsas well as laboratory data suggesting that KSHV, or some of itsproteins and miRNAs, have oncogenic properties in experimental settings.However, the risk for a KSHV-infected individual to develop Kaposi Sarcoma(KS) is low (incidence of classic KS is 1 -3/100 000 of a populationwith a KSHV seroprevalence of 20-30%), unless additional conditions,such as immune suppression or inflammation, are present (> 1000 foldexcess risk of KS in HIV-infection and approx. 200 fold excess risk intransplant recipients). KSHV thus differs markedly from some humanpapillomaviruses (risk of a persistent HPV-16 infection to lead to cervicalcancer in the 10-15% range) or Human T-cell tropic virus I (life time riskof an infected individual to develop Adult T-cell leukaemia in the rangeof 1 – 3%). These epidemiological data suggest the need for a carefulinterpretation of experimental findings that suggest the rapid appearanceof tumours in animal models of KS.On the other hand, the strong accelerating role of immune suppression andinflammation in the pathogenesis of Kaposi Sarcoma suggest that KSHVprovides an interesting model to study the interplay of inflammation anda carcinogenic virus. In this talk I will review experimental findings thatpoint to a role of KSHV in the aberrant angiogenesis seen in KS lesionsand the role of inflammatory stimuli.ORAL COMMUNICATIONSREF O153Identification of novel target genes of Epstein Barr Virus miRNAsMarjolein HOOYKAAS 1 , Lione WILLEMS 1 , Sander VAN HOOFF 2 ,Maaike RESSING 1 , Marian GROOT KOERKAMP 2 , FrankHOLSTEGE 2 , Robert Jan LEBBINK 1 , Emmanuel WIERTZ 11 Department of Medical Microbiology, UMC Utrecht, Utrecht, THENETHERLANDS; 2 Microarray Facility, Department of Molecular CancerResearch, UMC Utrecht, Utrecht, THE NETHERLANDSMicroRNAs (miRNAs) are posttranscriptional gene regulators that playimportant roles in many cellular processes. These small non coding RNAmolecules bind to complementary target mRNAs, thereby inducing RNAdestabilization and inhibition of translation. Several DNA viruses hijackthe cellular miRNA machinery to produce their own miRNAs, which offersopportunities to regulate both cellular and viral gene expression. However,the targets and functions of viral miRNAs remain largely unknown. Membersof the herpesvirus family encode the highest numbers of miRNAs.Epstein Barr Virus (EBV), for example, expresses only 1 9 viral proteinsduring latency, but this repertoire is complemented by more than 40 differentmiRNAs. Their lack of immunogenicity offers a specific advantageover protein coding genes. EBV miRNAs are abundantly expressed inEBV associated tumors, including nasopharyngeal carcinoma, indicatingthat they may play a role in tumorigenesis.Using a newly developed lentivirus based miRNA expression system, EBVmiRNAs were expressed in the nasopharyngeal cell line HK1. Transcriptomeprofiling revealed numerous genes differentially expressed betweenmiRNA positive and negative cells, including previously identified bonafide EBV miRNA targets such as IPO7 and DAZAP2. Amongst the potentialmRNA targets are regulators of cell growth and apoptosis and genesthat may impact adaptive and innate immunity. We validated a set of thenewly identified genes by using miRNA reporter constructs and confirmedthat they are direct targets of specific EBV miRNAs. The role of these newmiRNA target interactions in EBV biology is being established, a.o. usinga series of newly developed miRNA inhibitors. These inhibitors effectivelycounteract EBV miRNA function and offer new perspectives for thetreatment of EBV associated malignancies.REF O154Alternative splicing signatures discriminate ATL cells from untransformedCD4+ counterparts deriving from HTLV 1 infectedindividualsMorgan THENOZ 1 , Céline VERNIN 1 , Christiane PINATEL 2 , NicolasNAZARET 3 , Joel LACHUER 3 , Antoine GESSAIN 4 , DidierAUBOEUF 5 , Eric WATTEL 1 , Franck MORTREUX 11 Oncovirologie et Biotherapies, UMR5239 CNRS/ENS Lyon/UCBL/HCL,Hopital Pierre Benite, Lyon, FRANCE; 2 Oncovirologie et Biothérapies,Centre Léon Bérard, Lyon, FRANCE; 3 ProfileXpert, Neurobiotec Service,Bron, FRANCE; 4 Unit of Epidemiology and Physiopathology of OncogenicViruses, Department of Virology, Institut Pasteur, Paris, FRANCE;5 Institut National de Santé et de Recherche Médicale U590, Centre LéonBérard, Lyon, FRANCEThe clonal expansion and malignant transformation of HTLV 1 infectedCD4+ T cells has been linked to the reprogramming effects of HTLV 1on host transcriptional profile. Coupled to transcription, alternative splicing(AS) is a post transcriptional process that plays critical role in thecomplexity of transcriptome and splicing abnormalities frequently occurin cancer. To examine whether AS modifications associate with HTLV1 associated leukemogenesis, we compared the exon expression profilesof ATL cells with that of CD4+ T cell clones obtained by limited dilutioncloning of PBMC deriving from HTLV 1 carriers. 3 ATL cells and12 untransformed infected clones clustering in infected, uninfected, PHAstimulated or unstimulated CD4+ T cells were compared for exon RNAcontent using Exon Chip Human microarray. Hierarchical clustering analysisof data identified 12516 alternative spliced events (3642 genes) thatclearly separated ATL samples from the 4 untransformed phenotypes mentionedabove. Microarray data were confirmed for 18 AS events using exonspecific RT PCR analysis. Pathway analysis of alternatively spliced genesin ATL cells revealed new AS based pathways for p53 signaling, cell cycleand DNA replication while those of untransformed infected CD4+ T cellswere enriched in pathways for cellular movement and DNA repair. Thesefindings unveil a new layer of complexity in the interplay between HTLV 1and host cell gene expression machinery in which AS might play a centralrole in tumor initiation and promotion.Virologie, Vol 17, supplément 2, septembre 2013S109

5 th European Congress of VirologyREF O155High risk human papillomavirus E6 induces expression of the histonedemethylase KDM2B by repressing miR 146a in human keratinocytesElektra PETA 1 , Alessandro SINIGAGLIA 1,2 , Valentina MILITELLO 1 ,Angela GRASSI 3 , Barbara DI CAMILLO 3 , Marta TREVISAN 1 ,Giorgio PALÙ 1 , Luisa BARZON 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 IOV Istituto Oncologico Veneto, Padova, ITALY; 3 Departmentof Information Engineering, University of Padova, Padova, ITALYHigh risk HPV infection modulates expression of cellular microRNAs(miRNAs) involved in cell proliferation and tumorigenesis. MiRNAmicroarray analyses were used to identify alterations in miRNA expressionprofile in primary cultures of human foreskin keratinocytes inducedby expression of E6 and E7 of HPV16 or HPV6. While HPV6 E6 andE7 expression did not significantly change cellular miRNA expressionprofile, E6 and E7 of HPV16 significantly altered expression of some cellularmiRNAs, often with opposing effects, such as in the case of miR 34miRNAs and miR 146a, that were downregulated by E6 and upregulatedby E7. Among the significantly modulated miRNAs, miR 146a, that hasbeen shown to inhibit innate immune response and interferon pathway,was selected for further studies. Overexpression of miR 146a in humankeratinocytes and cervical carcinoma cell lines led to decreased cell proliferationand invasiveness. In promoter/luciferase reporter assays in humankeratinocytes, inactivation of NF kB trascription factor binding site in themiR 146a promoter abolished luciferase reporter activity independentlyfrom HPV16 E6 and E7 expression, while inactivation of IRF3/IRF7 andc Myc binding sites inhibited and induced, respectively, the miR 146a promoterin the presence of HPV16 E6. Repression of miR 146a promoter by cMyc was confirmed in HPV positive cancer cell lines. KDM2B/FBXL10,a H3K36 specific histone demethylase that is highly expressed in embryonicstem cells and induces pluripotency, was predicted to be novel directtarget for miR 146a and validated experimentally.invasive capacity of LMP1 expressing cells through extracellular matrix.Taken together, our data suggest that LMP1 mediated upregulation ofFascin contributes to EBV associated pathogenesis.REF O157Cell cycle modulation by Marek’s Disease Virus: the tegument proteinVP22 triggers S Phase arrest and DNA damage in proliferating cellsLaëtitia TRAPP FRAGNET 1 , Danièle VAUTHEROT 1 ,YvesLEVERN 1 , Sylvie REMY 1 , Elisa BOUTET ROBINET 2 , Gladys MIREY 2 ,Jean François VAUTHEROT 1 , Caroline DENESVRE 11 INRA, UMR1282, NOUZILLY, France; 2 INRA/INP, Toxalim, UMR1331,TOULOUSE, FRANCEMany viruses modulate cell cycle progression to enhance their replicationand persistence in the host cell. In the case of oncogenic viruses, this processmay ultimately lead to cellular transformation and oncogenesis. Inthe present study, we demonstrate that Marek’s disease virus (MDV), analphaherpesvirus responsible of T lymphoma in chickens, is also able tosubvert the cell cycle progression. Infection of primary chicken embryoskin cells with MDV triggered the re entry of quiescent cells into the cellcycle and delayed the progression of the cell cycle into S phase. We couldalso identified the tegument protein VP22 as a major MDV encoded cellcycle regulator since its over expression in proliferating cells led to a dramaticcell cycle arrest in S phase. This striking functional feature of VP22appears to be conserved among members of the Alphaherpesvirinae andis depended on its nuclear localization and histones binding capacity. Themechanism underlying the VP22 mediated S phase arrest might rely on theability of VP22 to generate DNA double strand breaks. Taken together,our results shed a new light on the mechanisms of MDV induced lymphomagenesisby describing a new role for the VP22 tegument protein inviral reprogramming of the cell cycle of the host cell associated to DNAdamage induction.REF O156Induction of the tumor marker Fascin by latent membrane protein 1(LMP1) depends on NF KB and could contribute to invasionAndrea K. KRESS 1 , Martina KALMER 1 , Caroline F. MOHR 1 ,Christine GROSS 1 , Melanie C. MANN 1 , Arnd KIESER 2 ,Bernhard FLECKENSTEIN 11 Institute of Clinical and Molecular Virology, Friedrich Alexander UniversitätErlangen Nürnberg, Erlangen, GERMANY; 2 Research Unit GeneVectors, Helmholtz Zentrum München German Research Center for EnvironmentalHealth, Munich, GERMANYThe actin bundling protein Fascin (FSCN1) is a tumor marker that is highlyexpressed in numerous types of cancer including lymphomas and is importantfor migration and metastasis of tumor cells. Fascin has also beendetected in B lymphocytes that are freshly infected with Epstein Barrvirus (EBV), however, both the inducers and the mechanisms of Fascinupregulation are still unclear. Here we show that the EBV encoded oncoproteinlatent membrane protein 1 (LMP1), a potent regulator of cellularsignaling and transformation, is sufficient to induce both Fascin mRNAand protein in lymphocytes. Fascin colocalizes with actin in the cytoplasmof LMP 1 transfected Jurkat cells and in lymphoblastoid cell lines. Fascinexpression is mainly regulated by LMP1 via the C terminal activationregion 2 (CTAR2). Block of canonical NF KB signaling using a chemicalinhibitor of IKB kinase (IKK ) or cotransfection of a dominant negativeinhibitor of IKBa (NFKBIA) reduces LMP1 induced Fascin expression.Beyond that, chemical inhibition of IKK reduces Fascin mRNA levels inEBV transformed lymphoblastoid cell lines, indicating that canonical NFKB signaling is required for LMP1 mediated regulation of Fascin both intransfected and transformed lymphocytes. Use of specific shRNAs leadsto knockdown of LMP1 induced Fascin expression and could influence theREF O158The human bocavirus is associated with lung and colorectal cancersand persists in solid tumoursOliver SCHILDGEN, Verena SCHILDGEN, Monika MALECKI,Ramona Liza TILLMANN, Michael BROCKMANNKliniken der Stadt Köln, Cologne, GERMANYThe human bocavirus (HBoV) is the second human pathogenic parvovirus.It causes respiratory infections and gastroenteritis. Some autonomousanimal parvoviruses and also some human non autonomous parvovirusesare known to persist and even integrate into the host genome resulting intransformation of the infected cells and eventually contribute to the multistep development of cancer. Also HBoV persists in a so far unknown percentageof patients without causing clinical symptoms beyond those of theprimary infection. The aim of the present study was to analyze the role ofHBoV in lung and colorectal cancers. Therefore, formalin fixed paraffinembedded archived tumor samples were screened for HBoV DNA by PCR,Southern blotting, and sequencing. Positive tissues were further subjectedto FISH analyses specifically detecting HBoV DNA in the infected cells.In total, 11 of 60 (18.3%) lung and 9 of 44 (20.5%) colo rectal tumorswere tested positive for HBoV DNA, confirmed by sequencing and FISHanalyses. HBoV DNA thereby is present in the nuclei of infected cells,either in single or multiple copies, and appears also to form filaments.Moreover the FISH patterns give rise to the hypothesis that HBoV DNApersists either as cccDNA or integrates into the host genome. This supportsthe further hypothesis that the virus plays an active role in cancer byinteractions with the host genome, or contributes to cancer developmentindirectly by inducing a persisting inflammation, as other DNA viruses likethe human hepatitis B virus do. The occurrence of HBoV DNA filamentscould confirm the postulated? or rolling hairpin replication mechanism.S110 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologySaturday 14 th September 2013, 14h45 – 16h45KEYNOTE:WORKSHOP 20: “VIRUS EPIDEMIOLOGY”Chairpersons: Tatjana AVSIC-ZUPANC(Ljubljana, SLOVENIA)& Bruno POZZETTO (Saint Etienne, FRANCE)Room Gratte Ciel 1, 2, 3HCMV maternal-fetal transmission: epidemiology and experimentalmodelingDana WOLFClinical Virology Unit, Hadassah Hebrew University Medical Center,Jerusalem, ISRAELHuman cytomegalovirus (HCMV) is the leading cause of congenitalinfection, associated with severe birth defects, placental damage, andintrauterine growth retardation. The risk of congenital infection is higherin the case of primary maternal infection, yet the contribution of recurrentinfection to the burden of congenital infection is increasingly recognized.I will describe our studies, in which we combined prospective neonatalscreening with maternal serological, demographic, and socioeconomicdata, to determine the birth prevalence and distribution of congenital infectionin Israel, according to the type of maternal infection. We have furtherdefined the protective effect of prior maternal immunity, and identifiedprenatal risk factors for congenital infection.We are also interested in the mechanism of transmission in the maternalfetalinterface. To gain insight into the initial events of transmission, wehave established an ex vivo model of HCMV infection in first-trimesterdecidual organ cultures. Using both laboratory-derived and clinical HCMVstrains, we demonstrated the broad viral target-cell range. Antiviral drugsand neutralizing HCMV antibodies inhibited viral spread in the decidua.We have further revealed a profound tissue innate response to HCMVinfection, with formation of proinflammatory environment in the infecteddecidua. The ex vivo-infected decidual cultures can serve as a surrogatehuman model to address viral and tissue determinants of damage, and facilitateevaluation of antiviral interventions at the maternal-fetal interface.ORAL COMMUNICATIONSREF O159HCMV reactivation of breastfeeding mothers of preterm infants: fromtransmission to preventionKlaus HAMPRECHT 1 , Rangmar GOELZ 2 , Teresa SCHUETTE 1 ,Christian F. POETS 2 , Gerhard JAHN 11 Institute of Medical Virology, Tuebingen, GERMANY; 2 Department ofNeonatology, Tuebingen, GERMANYSeropositive breastfeeding mothers may transfer HCMV postnatally totheir infants via breast milk (Hamprecht et al., 2008). Mothers of preterminfants under potential risk for transmission with birth weight

5 th European Congress of VirologyEylul University, Faculty of Medicine, Department of Public Health, Izmir,TURKEYThe majority of HCV infections are due to type 1b viruses in Turkey.However, the distribution of HCV genotypes in Kayseri region is differentfrom the rest of Turkey in where genotype 4 viruses are detected in onethird of HCV infections. In this study, we analyzed the demographic andlaboratory data of 218 HCV RNA positive treatment naïve patients admittedto Kayseri Training and Research Hospital between 2010 and 2011.The distribution of sex, age and viral loads of these patients with respectto HCV genotypes were analyzed. Randomly selected sera from 32 of the72 genotype 4 infected patients from this cohort were subjected to PCRamplification in the NS5B region and further characterized by sequencing,phylogenetic and molecular clock analysis.Types 1, 4, 2 and 3 HCV were detected in 62.4%, 33.0%, 4.1% and 0.5% ofthe samples, respectively. Most of the patients infected with types 1 and 4are over the age of 40 and approximately two thirds of them are female. Incontrast, type 2 patients exhibit male predominance and most are under theage of 40. Viral loads of type 4 HCV infected patients were significantlylower than those of type 1. The NS5B sequences of 32 Kayseri genotype 4isolates were closely related with type 4d sequences, but formed a separatecluster. The introduction of type 4d HCV to Kayseri region probably tookplace 30 75 years ago as predicted by molecular clock analysis. The exactnature of this outbreak awaits to be revealed by field epidemiology andmolecular analysis of longer sub genomic regions of the isolates.REF O162Factors Challenging Epidemiology of Poliomyelitis in the Polio FreeCountriesNatalya ROMANENKOVA 1 , Maina BICHURINA 1 , NadezdaROZAEVA 1 , Olga IVANOVA 2 , Tatyana EREMEEVA 2 , MaryaYAKOVENKO 21 Saint Petersburg Pasteur Institute, Saint Petersburg, RUSSIA; 2 M.P.Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow,RUSSIAThe spread of revertant vaccine poliovirus of type 2 from a paralytic patientto four healthy contacts in a hospital illustrated the emergence of vaccinederived polioviruses (VDPV) with increased transmissibility. The isolationof vaccine type 3 poliovirus displaying 1,1% nucleotide substitutions in thegenomic region VP1 from a patient with acute flaccid paralysis confirmedthe possibility of spread of VDPV among well immunized population.The ongoing risk of importation of wild polioviruses (WPV) into polio freecountries remains till poliomyelitis is eradicated. In 2010 an importantoutbreak of poliomyelitis with 458 cases caused by imported wild type1 poliovirus (WPV1) occurred in Tajikistan which had previously beenpolio free. Sequencing of the VP1 region of WPV1 isolates revealed thatthe causative agent of the outbreak in Tajikistan was closely related toWPV1 previously isolated in India.During the last years 27 poliovirus strains were isolated from 572 healthymigrants’ children in Saint Petersburg. The majority of polioviruses wereclassified as vaccine like. However four PV1 isolated from Tajik childrenin 2010 were confirmed by sequencing as wild type 1 polioviruses closelyrelated to the lineage isolated in Tajikistan. The WPV1 of this lineage werealso found in poliomyelitis cases and their contacts among migrants fromTajikistan and Uzbekistan. These data showed that polio free countrieswith a great number of migrants, like in Russia, can be threatened bysecondary importation of a wild poliovirus even if they have traditionallymaintained high poliovirus vaccine coverage.The systematic surveillance of patients with AFP and migrants’ childrenand adequate epidemiological measures are indispensable in orderto prevent transmission and indigenous circulation of wild polioviruses inpolio free countries.REF O163Seroepidemiological and phylogenetic characterisation of measlesoutbreaks in Ireland, 2004 to 2012Bernadette O. RIORDAN, Michael CARR, Linda DUNFORD, AllisonWATERS, Jeff CONNELL, William HALL, Jaythoon HASSANNational Virus Reference Laboratory, University College Dublin, Dublin,IRELANDMeasles is a vaccine preventable disease. A World Health OrganisationEuropean Region (WHO ER) strategic plan exists for measles eliminationby 2015 in Europe; however, outbreaks continue to occur despite childhoodimmunisation programmes. Ireland is classified as an area of highincidence.We have retrospectively investigated the serology and molecular epidemiologyof measles cases identified in Ireland from 2004 2012. Threemeasles outbreaks occurred over the study period: 2004, 2009/2010 and2011. Measles IgM ranged from 22 29% in outbreak years to 5 10% in theintervening years. Age profile analysis revealed that whereas individuals>10 years accounted for only 8% of cases in the 2004 outbreak, measlescases in this age group increased to 33% and 29% in the 2009/2010 and2011outbreaks respectively. Notably, the

5 th European Congress of VirologySaturday 14 th September 2013, 14h45 – 16h45WORKSHOP 21: “VIRUS DISCOVERY ANDMETAGENOMICS”Partnership with the FP7-EU Program EMPIRIEChairpersons: Albert OSTERHAUS (Rotterdam,THE NETHERLAND) & Veronika VON MESSLING(Langen, GERMANY)Room Prestige Gratte-CielKEYNOTES:EMPERIE: where do we go?Albert D.M.E. OSTERHAUSDepartment of Viroscience, Erasmus Medical Center, Rotterdam, THENETHERLANDSFP7 project EMPERIE can best be characterized by the rapid response ofits partners towards the novel emerging virus influenza A/pH1N1 (emergedin 2009), Schmallenberg virus (emerged in 2011), and the MiddleEast Respiratory Syndrome Coronavirus (MERS-CoV, emerged in 2012).The latter two were discovered within EMPERIE using its discovery platform.For influenza A/pH1N1, studies on the epidemiology, antigenicand genetic characteristics, replication-kinetics, reassortment potential,pathogenesis and transmission of this pandemic virus were conducted,providing valuable information for risk assessment and containment strategies.Novel serological and sequencing methods were developed for rapiddetection, and characterization of this novel virus. Upon identification ofthe Schmallenberg virus EMPERIE partners have characterized the virus,studied its epidemiology, developed a method for detection of the virus,distributed it to other laboratories and provided training in diagnostics.Following the discovery of MERS-CoV by the EMPERIE partners, noveldeep genome sequencing methods and real-time RT-PCR were developed.These methods enabled rapid detection and are employed to examineMERS-CoV evolution. They are also important in identification of animalreservoirs and implementing public health measures. WHO recommendedthe use of the assays developed in EMPERIE for screening of patientsfulfilling the MERS-CoV case definition?Identification and characterization of MERS-CoVV. Stalin RAJ 1 , Huihui MOU 2 , Saskia L. SMITS 1 , RonA.M. FOUCHIER 1 , Albert D.M.E. OSTERHAUS 1 , Berend JanBOSCH 2 , Bart L. HAAGMANS 11 Department of Viroscience, Erasmus Medical Center, Rotterdam, THENETHERLANDS; 2 Virology Division, Department of Infectious Diseases& Immunology, Faculty of Veterinary Medicine, Utrecht University,Utrecht, THE NETHERLANDSMost human coronaviruses (CoVs) cause mild upper respiratory tractdisease but may be associated with more severe pulmonary disease inimmunocompromised individuals. SARS-CoV on the other hand causedsevere lower respiratory disease with nearly 10% mortality and evidence ofsystemic spread. Recently, another coronavirus (HCoV-EMC) was identifiedin patients with severe and sometimes lethal lower respiratory tractinfection. Genetically the virus bears resemblance to bat CoVs HKU4 and5 and - based on phylogenetic analysis using a small fragment of the virus– to a bat CoV found in Pipistrellus pipistrellus in the Netherlands. Coronaviruseshave zoonotic potential due the adaptability of their S proteinto receptors of other species, most notably demonstrated by SARS-CoV.We identified dipeptidyl peptidase 4 (DPP4) - also known as CD26 - asa functional receptor for HCoV-EMC. DPP4 specifically co-purified withthe receptor binding S1 domain of the HCoV-EMC spike protein fromlysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibitedHCoV-EMC infection of primary human bronchial epithelial cellsand Huh-7 cells. Expression of human and bat DPP4 in non-susceptibleCOS-7 cells enabled infection by HCoV-EMC. The use of the evolutionaryconserved DPP4 protein from different species as a functional receptor providesclues about HCoV-EMC ′ s host range potential. In addition, it willcontribute critically to our understanding of the pathogenesis and epidemiologyof this emerging human CoV, and may facilitate the developmentof intervention strategies.ORAL COMMUNICATIONSREF O165Novel pathogenic viruses and their zoonotic potentialMarta CANUTI, Martin DEIJS, Michel DE VRIES, Bas B. OUDEMUNNINK, Seyed Mohammad JAZAERI FARSANI, Maarten FJEBBINK, Lia VAN DER HOEKDepartment of Medical Microbiology, Academic Medical Center (AMC),Amsterdam, THE NETHERLANDSIn an increasingly globalized World the speed at which viruses diffuseworldwide is escalating quickly: viruses have the possibility tospread in different environment and evolve divergently in different hostpopulation, leading to the emergence of new variants/types (also viarecombination/reassortment). Besides, new viruses, often characterized bypandemic potential, are continuously introduced from the animal reservoirto humans.We developed a virus discovery technique (VIDISCA 454, Virus DiscoverycDNA AFLP combined with Roche high throughput sequencing) anda culture system with primary human airways epithelial (HAE) cells whichcan be incorporated into early warning and surveillance systems to preventviral spreading and zoonosis and which can be used in risk assessment.VIDISCA 454 is a sequence independent virus discovery techniquethat allows the amplification of any virus without prior knowledge ofthe genome sequence. We are currently able to provide roughly 10000sequences per samples with reduced costs and to detect known and unknownviruses from different clinical and laboratory material. HAE cellscan be used to culture viruses which cannot be cultured on commercial celllines and evaluate their replication/zoonotic potential, study the evolutionof a virus in vitro and study the immuno response to new viruses.The virus discovery assay we optimized has become a sensitive methodto identify novel viruses directly in clinical samples and HAE culturingsystem is a convenient tool to culture/characterize novel and previouslyidentified human respiratory viruses.REF 0166Discovery of rodent coronaviruses related to major human and animalpathogensJan Felix DREXLER 1 , Victor Max CORMAN 1 , Jonas SCHMIDTCHANASIT 2 , Matthias SCHLEGEL 3 , René KALLIES 1 , Eric M.LEROY 4,5 , Alvaro AGUILAR SETIÉN 6 , Sonja MATTHEE 7 , ChantalREUSKEN 8 , Rainer G. ULRICH 3 , Christian DROSTEN 11 Institute of Virology, University of Bonn Medical Centre, Bonn, GER-MANY; 2 Bernhard Nocht Institute for Tropical Medicine, Departmentof Virology, Hamburg, GERMANY; 3 Friedrich Loeffler Institut, Institutefor Novel and Emerging Infectious Diseases, Greifswald–InselRiems, GERMANY; 4 Centre International de Recherches Médicales deFranceville, Franceville, GABON; 5 Institut de Recherche pour le Développement,UMR 224 (MIVEGEC), IRD/CNRS/UM1, Montpellier, FRANCE;6 Unidad de Investigación Médica en Inmunología, Hospital de Pediatría,México DF, MÉXICO; 7 Department of Conservation Ecology andEntomology, Stellenbosch University, Stellenbosch, SOUTH AFRICA;8 Netherlands Center for Infectious Disease Control, Bilthoven, THENETHERLANDSVirologie, Vol 17, supplément 2, septembre 2013S113

5 th European Congress of VirologyCoronaviruses (CoVs) are classified into four genera termed Alpha, Beta,Gamma and Deltacoronavirus. In the aftermath of the SARS CoV pandemic,numerous novel bat CoVs were described, suggesting an originof mammalian alpha and betacoronaviruses in bats. The Betacoronavirus2a clade contains major animal and human CoVs (hCoV), includinghCoV OC43, hCoV HKU1, bovine CoV (BCoV) and Mouse hepatitisvirus (MHV). No bat ancestors have ever been detected for this CoVclade. We screened 4,820 rodent sera from Mexico, Germany, the Netherlands,Gabon, South Africa and Thailand by nested RT PCR for CoVs.CoV RNA could be detected in 56 specimens (1.2%) from all samplingsites. Partial RNA dependent RNA polymerase sequences indicated theseviruses corresponded to at least 8 novel CoVs that phylogenetically clusteredacross the entire 2a CoV clade. Some viruses were closely related tohCoV OC43, BCoV and MHV. Taxonomic classification was confirmedby full genome sequencing of two representative rodent viruses directlyfrom clinical material. Recombination events were detected between therodent viruses and other 2a CoVs. These data suggested an origin of theentire 2a CoV clade in rodents.REF O167Virome analysis of French bat species in contact with humans: identificationof a large majority of novel mammalian related virusesLaurent DACHEUX 1 , Minerva CERVANTES GONZALEZ 1 , JeanMichel THIBERGE 2 , Mathias VANDENBOGAERT 2 , GhislaineGUIGON 2 , Corinne MAUFRAIS 3 , Valérie CARO 2 , Hervé BOURHY 11 Institut Pasteur, Lyssavirus Dynamics and Host Adaptation Unit, NationalReference Centre for Rabies, Paris, FRANCE; 2 Institut Pasteur,Genotyping of Pathogens and Public Health Platform, Paris, FRANCE;3 Institut Pasteur, Computer Center for Biologists, Paris, FRANCEThe ability to predict viral zoonotic epidemics has become a major publichealth issue. In depth understanding of the viral population in key animalspecies acting as reservoir represents an important step towards thisgoal. Among them, bats are of special interest as they can harbour diverseviruses, some of which causing severe human diseases. However, dataconcerning the whole diversity of their viral population (virome) remainlimited. In this study, the virome of 9 French insectivorous bat specimens (5different species) in close contact with humans was determined. Sequenceindependent amplification and high throughput sequencing, together witha dedicated bioinformatics analysis pipeline, were applied to pooled tissues(brain, liver and lungs). Determination of the sequence homology ofcontigs and unassembled reads provided a global taxonomic distributionof viral related sequences for each sample, and highlighted differencesbetween bat species, but also within a same bat species. The spectrum ofviral families represented was large, including viruses infecting bacteria,plants/fungi, insects or vertebrates, with the most abundant being virusesinfecting mammals. In particular, we detected a selected panel of newmammalian viruses, including bornaviruses, rotaviruses, gammaretrovirusesand bunyaviruses with the identification of the first bat nairovirus.These results confirmed that bats constitute a major reservoir of viral diversity,which need to be carefully analyzed in an attempt to identify theirpotential role in the spread of zoonotic viral infections.REF O168Identification of novel cetacean poxviruses in animals stranded inSouthern EnglandFalko STEINBACH 1 , James BARNETT 2,6 , David EVEREST 3 , NickDAVISON 2,4 , Paul JEPSON 5 , Rob DEAVILLE 5 , ChristopherFINNEGAN 1 , Akbar DASTJERDI 11 Virology Dept., AHVLA, Addlestone, UNITED KINGDOM; 2 AHVLA,Truro, UNITED KINGDOM; 3 Specialised Scientific Support, AHVLA,Addlestone, UNITED KINGDOM; 4 Scottish Marine Animal StrandingScheme, SRUC Veterinary Services, Inverness, UNITED KINGDOM;5 Institute of Zoology, ZSL, London, UNITED KINGDOM; 6 Environmentand Sustainability Institute, Univ Exeter, Penryn, UNITED KINGDOMPoxvirus infections in marine mammals have been mainly reportedthrough their clinical lesions and electron microscopy. In cetaceans, acutaneous manifestation of reported poxvirus infections has been hyperpigmentedskin lesions, also described as ‘tattoo’ lesions. Poxvirusparticles in association with such lesions have been demonstrated andsome strains were characterised resulting in two new viruses, cetaceanpoxvirus 1 (CePV 1) and CePV 2 being suggested. In this study, poxlesions of cetaceans stranded in Southwest England (Cornwall) between2008 and 2012 were investigated by electron microscopy and molecularanalysis.We analysed eight samples where poxvirus particles had been identified byelectron microscopy, resembling parapox viruses in shape, but orthopoxviruses in their surface morphology. Subsequent phylogenetic analysisof 360 nucleotides of a highly conserved region within the DNA polymerasegene clustered the sequences with those previously described asCePV 1 and 2 and ruled out both parapox and orthopoxviruses. However,taking the close genetic distance of this gene fragment within other generainto account it is reasonable to postulate further, novel cetaceanpox virusspecies. The nucleotide similarity within each cluster (tentative species)detected ranged from 98.6% to 100% whilst the similarity between theclusters was no more than 95%.The detection of several species of poxvirus in cetaceans of different animalspecies confirms the notion to consider a cetaceanpox virus genuswith members comparable in heterogeneity to other poxvirus genera.S114 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologySaturday 14 th September 2013, 14h45 – 16h45WORKSHOP 22: “INNOVATIVE METHODS IN VIRALDAIGNOSIS”Chairpersons: Xavier de LAMBALLERIE (Marseille,FRANCE) & Elisabeth PUCHHAMMER (Vienna, AUSTRIA)AmphitheaterKEYNOTE:Molecular diagnostics in the future: how will it look like and what arethe challenges?Hubert G.M. NIESTERSUniversity Medical Center Groningen, Department of Medical Microbiology,Division of Clinical Virology. Hanzeplein 1, 9713 GZ Groningen,THE NETHERLANDSWe all agree that the introduction of molecular diagnostic technologieshas changed clinical virology tremendously over the last few decades.It has been beneficial for patient care and diagnostics, as well assistedthe laboratories in dealing with the rapid detection of new viruses whichcontinuously seem to appear.The expectations and demands for the laboratories has also changed. Regulationsare demanding that the laboratories should be accredited, in whichprocesses around quality control (external and internal) as well as validationand verification of methods and processes are properly documented.Also, the availability of rapid molecular testing of relevant targets and therapid characterization of viruses (type, strain) by sequence analysis, doesbecome more accessible. A short turn-around-time with a lot of clinicallyimportant information does become a trend; not only the quantitative detectionof the virus, but also its sequence characterization. Important in thisturmoil of information will be the communication possibilities between thedifferent technologies within the laboratory (whether this is quality controlrelated or diagnostic result related) as well as between the laboratory andthe clinicians and/or patients.An overview of the new and existing technologies will be presented togetherwith the challenges we still have to overcome.ORAL COMMUNICATIONSResults: Target antigens were selected for the genus flavivirus, and spottedonto nitrocellulose pads on a microarray using a non contact array spotter.Serum samples (N=60) from patients with virological and serologicalconfirmed arboviral infections and 40 control sera of non exposed individualswere incubated in serial 2 fold dilutions followed by incubation withan IgG or IgM specific Cy5 labeled conjugate. After quantifying signalsusing a scanarray scanner, data were analyzed in ‘R’.Profiling of IgG antibodies from the patients confirmed the diagnosis for92% of cases (95% dengue, 82% west Nile, 100% Japanese encephalitisand 100% Usutu virus). Four patients showed reactivities suggesting(co)infections with another virus within the same genus. Specificity wasbetween 92% (dengue) and 100% (West Nile, Japanese encephalitis andUsutu virus) for IgG, and 100% for IgM. Profiling of antibodies in patientsexposed to flaviviruses showed highly discriminatory patterns of reactivityin most patient.REF O170Current mumps outbreak in Belgium: is laboratory confirmation byIgM useful?Liesbeth SEAUX, Elizaveta PADALKOGhent University Hospital, Ghent, BELGIUMNonetheless the vaccination state of the Belgian population is high, we arecurrently facing an outbreak of mumps. This current outbreak started in2012 among Ghent university students. The genotype G variant, not endemicto our regions, was the culprit. Fully immunised patients were infectedbecause current vaccines only contain mumps genotype A antigens as wellas currently available laboratory tests. The aim of this study was to evaluatethe influence of currently circulating genotype G on the performanceof routine mumps IgM test (Enzygnost ® Anti Parotis Virus/IgM, BehringElisa Processor III, Siemens) at the Ghent University Hospital.Based on laboratory information system query for 2012, only patients witha serological request form consistent with the possible clinical pictureof mumps were included. Secondly, the electronic patient records of allwithhold cases were carefully examined.From 234 analysed cases by now, 47 presented with a suspicious clinicalpicture for mumps. Thirty two patients (68%) were between 14 and 28years old. In 35 out of 47 patients (74%) mumps was actually clinicallydiagnosed. Only 13 of these 35 clinically clear cases (37%) showed positivemumps IgM. In total 16 of 47 cases (34%) tested mumps IgM positive.In conclusion, in the current outbreak the routine IgM mumps test was notfitted to confirm the clinical diagnosis. Our study emphasizes that mumpsdiagnosis should be made on clinical basis.REF O169Validation of a flavivirus protein microarray for simultaneous detectionof and differentiation between west Nile, Japanese encephalitis,Usutu and dengue virus immunoglobin G and M antibodiesNatalie CLETON 1,2 , Chantal REUSKEN 1 , Gert Jan GODEKE 1 , JohanREIMERINK 1 , Marion KOOPMANS 1,21 National Institute for Public Health and the Environment, Bilthoven, THENETHERLANDS; 2 Erasmus Medical Center, Rotterdam, THE NETHER-LANDSBackground: The flavivirus genus holds the world’s most widespreadarboviral travel related diseases, causing meningoencephomyelitis, rash,althralgia and hemorrhagic fever. Diagnosis is based mostly on serology,as viremia is short lived. A diagnostic problem is that clinical syndromescaused by different flaviviruses and their geographical distribution overlap,and antibodies cross react extensively in common serological tests.Objective: To assess the potential added value for diagnosis and surveillanceof protein microarray based profiling of antibodies to 4flaviviruses.REF O171Human Papillomavirus Genotyping: Comparison of the seegene AnyplexII HPV28 with the PGMY CHUV AssayRoland SAHLI 1,2 , Christine ESTRADE 1,21 Institute of Microbiology, Centre Hospitalier Universitaire Vaudois andUniversity of Lausanne, Lausanne, SWITZERLAND; 2 WHO HPV LabNetRegional Reference Laboratory for Europe, Lausanne, SWITZERLANDGenotyping of human papillomaviruses (HPV) is essential for surveillanceof HPV vaccines and cervical cancer screening. The PGMY CHUV (PG)assay from the WHO Laboratory Manual allows genotyping of 31 HPV byPCR and reverse blotting hybridization. The Seegene Anyplex II HPV28(H28) is a new real time PCR assay for genotyping and semi quantifying28 HPV. H28 was compared with PG as a reference restricted on their26 common HPV, either retrospectively (309 DNA) or prospectively (84samples) to also evaluate H28 automation (NIMBUS IVD).H28 and PG were fully concordant on 246 (79.6%; 27 neg, 219 pos) out ofthe 309 retrospective DNA. The other 63 (20.4%) cases were either fullyVirologie, Vol 17, supplément 2, septembre 2013S115

5 th European Congress of Virology(11, 3.5%) or partially discordant (52, 16.8%). While genotype specificagreement overall was very good (Kappa=0.903), HPV51 was significantlymore often detected by PG and the converse was true with HPV40,42, 54, and 68.Sixty five (77.4%) out of the 84 prospective samples were fully concordant(47 neg, 18 pos). Re evaluation of the 19 discordant cases after exchangingthe source of DNA was consistent with a higher sensitivity of the H28 assaydue mainly to the NIMBUS extraction procedure.H28 is a very sensitive and specific HPV assay for cervical cancerscreening as an adjunct to cytology and for epidemiological studies. Itsautomation capacity renders it particularly well suited for high workload.The additional benefit of its semi quantitative readout ought to be furtherevaluated for use as a tool in primary screening of cervical cancer.REF O172MxA: a diagnostic marker of viral infection in childrenIlka ENGELMANN 1 , Francois DUBOS 2 , Pierre Emmanuel LOBERT 1 ,Anne SARDET 3 , Anne DECOSTER 4 , Alain MARTINOT 2 , DidierHOBER 11 Université Lille 2, Faculté de Médecine, CHRU Lille, Laboratoire de virologieEA3610, Lille, France; 2 Université Lille Nord de France, UDSL,Faculté de Médecine, CHRU Lille, Urgences pédiatriques et maladiesinfectieuses, EA2694, Lille, France; 3 Service de Pédiatrie, Centre HospitalierDr Schaffner, Lens, FRANCE; 4 Laboratoire de l’Hôpital St Philibert,GHICL, Lomme, FRANCEObjective: The protein MxA is induced during viral infection and its bloodvalues could help to differentiate between viral and bacterial infection inchildren.Patients/Methods: We performed a prospective multicenter study includingchildren admitted at pediatric emergency departments. MxA bloodvalues were measured in children with confirmed viral infections, confirmedbacterial infections, uninfected controls and children with differentinfectious syndromes without microbiologic confirmation. The MxA thresholdindicative of viral infection and the diagnostic performance of MxAwere determined using ROC analysis.Results: MxA was measured in the blood of 556 children. The MxAthreshold for differentiating patients with viral infection from uninfectedpatients was determined using the first 44 uninfected controls and 77confirmed viral infections. The threshold was determined at 200 ng/mL.The other patients (n=435) formed the validation population. MxA levelswere higher in patients with viral infections than in those with bacterialinfections and uninfected controls (p

Virologie 2013, 17 (supplément 2) : 117-284Poster presentations summaryp. S118 CATEGORY 01: Oncogenic mechanisms of virusesReferences from 001 to 008p. S121 CATEGORY 02: Oncolytic viruses and gene therapyReferences from 009 to 015p. S123 CATEGORY 03: Innate immunity against virusesReferences from 016 to 036p. S129 CATEGORY 04: Adaptive immunity and VaccinesReferences from 037 to 061p. S136 CATEGORY 05: Viral (immuno) pathogenesisReferences from 062 to 076p. S141 CATEGORY 06: Restriction factors of viral infection, interferingRNA & immune responseReferences from 077 to 088p. S144 CATEGORY 07: Cellular factors controling viral infectionand role of host geneticsReferences from 089 to 104p. S148 CATEGORY 08: Antiviral therapy and Resistance to acuteviral infectionReferences from 105 to 126p. S155 CATEGORY 09: Antiviral therapy and Resistance to chronicviral infectionReferences from 130 to 146p. S160 CATEGORY 10: Virus entry: envelope glycoproteins, receptors,endocytosisReferences from147 to 168p. S166 CATEGORY 11: Virus exit: assembly, maturation and releaseReferences from 169 to 180p. S170 CATEGORY 12: Viral gene expression – transcription, translationReferences from 181 to 197p. S175 CATEGORY 13: Viral replication strategiesReferences from 198 to 215p. S180 CATEGORY 14: Virus structure, dynamic imaging and traffickingReferences from 216 to 229p. S184 CATEGORY 15: Highly pathogenic virusesReferences from 230 to 251p. S190 CATEGORY 16: Zoonotic viruses (Antigone, Predemics)References from 252 to 271p. S196 CATEGORY 17: Vector borne viral infections (EUROWN,VECTORIE, WINGS)References from 272 to 293p. S203 CATEGORY 18: Emerging diseases in veterinary virology(ESVV)References from 294 to 344p. S216 CATEGORY 19: Viral evolution and quasispeciesReferences from 346 to 359p. S220 CATEGORY 20: Virus EpidemiologyReferences from 360 to 396p. S231 CATEGORY 21: Virus discovery and metagenomics(EMPERIE)References from 397 to 416p. S236 CATEGORY 22: Innovative methods in viral diagnosisReferences from 417 to 460p. S248 CATEGORY 23: Viral diagnosis in chronic infection andduring pregnancyReferences from 461 to 491p. S257 CATEGORY 24: Typing of viruses (molecular epidemiology,taxonomy)References from 492 to 507p. S262 CATEGORY 25: Respiratory virus infectionsReferences from 508 to 538p. S270 CATEGORY 26: Viral infections in transplant and immunocompromisedpatientsReferences from 539 to 562p. S277 CATEGORY 27: Neurotropic virusesReferences from 563 to 577p. S281 CATEGORY 28: Gastrointestinal viral infectionsReferences from 578 to 589doi:10.1684/vir.2013.0527Virologie, Vol 17, supplément 2, septembre 2013S117

5 th European Congress of VirologyPOSTERS01. ONCOGENIC MECHANISMS OFVIRUSESPosters: REF 001 to REF 008deleted at the expected sites. The EBER 1 deletion mutants have beencloned into EBER expression plasmids carrying the EBV oriP and hygromycin/ampicillinresistance genes. These plasmids have now been stablytransfected into 293T cells and a Burkitt’s lymphoma cell line (BL30) andtheir effect on cell proliferation and apoptosis will be presented.Understanding the function of EBERs may shed light on the molecularmechanism(s) by which EBV induces tumorigenesis.REF 001Selective activation of non canonical NF kappaB through a uniqueTRAF 3 binding motif in the viral oncoprotein TioBrigitte BIESINGER 1 , Sarah Jill DE JONG 1,4 , Jens ChristianALBRECHT 1 , Fabian GIEHLER 2 , Arnd KIESER 2 , Heinrich STICHT 31 Virologisches Institut, Friedrich Alexander Universitaet, Erlangen,GERMANY; 2 Abteilung Genvektoren, Helmholtz Zentrum Muenchen,Muenchen, GERMANY; 3 Institut fuer Biochemie, Friedrich AlexanderUniversiaet, Erlangen, GERMANY; 4 St. Giles Laboratory of Human Geneticsof Infectious Diseases, The Rockefeller University, New York, USAHerpesvirus ateles is capable to transform human T cells to long termgrowth in culture. Its oncoprotein Tio (Two in one) transfers this phenotypeto a transformation deficient recombinant herpesvirus saimiri. Oligomeric,membrane anchored Tio constitutes a signaling platform that recruitsSrc family kinases to activate STAT factors and TRAF6 to activate canonicalNF kappaB. Independent of these interaction partners, Tio inducesNIK stabilization and p100 processing, the hallmarks of non canonicalNF kappaB activation, through an as yet unidentified mechanism. We nowidentified a distinct sequence motif in Tio that specifically recruits TRAF3,a negative regulator of non canonical NF kappaB signaling. Through thisunique motif, Tio triggers a ubiquitin independent depletion of TRAF3from the cytosol and thereby releases the intrinsic activity of the non canonicalNF kappaB signaling cascade leading to p100 processing. Notably,mutation of the TRAF3 binding site does not affect canonical signalingintermediates and target gene regulation. Thus, Tio targets TRAF3 toinduce non canonical NF kappaB independent of cross talk into the canonicalcascade. In summary, we have identified a new and unique type ofTRAF3 binding motif that selectively mediates non canonical NF kappaBactivation and discloses studies into the specific functions of this pathwayand its role in T cell growth regulation.REF 002Creation of Epstein Barr virus encoded small RNA (EBER) mutantsand analysis of their functionGulfaraz KHAN, Pretty PHILIP, Waqar AHMEDUAE University, College of Medicine, Al Ain, UAEEpstein Barr virus (EBV), an oncogenic lymphotropic herpesvirus is implicatedin the pathogenesis of a number of human malignancies. Althoughthe mechanism by which EBV leads to cell transformation is not clear, anumber of viral latent gene products, including non protein coding smallRNAs have been implicated in the transformation/proliferation process.EBER 1 and EBER 2 are two such RNA molecules which are abundantlyexpressed in all EBV infected cells, but their function is poorly understood.Both of these RNA molecules have a secondary structure and are boundto cellular proteins. We set out to investigate the functions of EBER 1 bycreating EBER 1 mutants. We used spliced overlap extension PCR (SOEPCR) approach to create EBER 1 mutants with deletions in regions normallybound to cellular proteins. Using this approach, we have successfullydeleted stem loops (SL) 1, 3 and 4 of EBER 1. The subsequent mutantswere cloned and sequenced to verify that the intended target regions wereREF 003Human cytomegalovirus activates pro oncogenic pathways in primaryhuman hepatocytes and HepG2 cellsAmit KUMAR, Wasim ABBAS, Quentin LEPILLER, GeorgesHERBEINUniversity of Franche Comté, Besançon, FRANCEBackground: In the recent past, there has been increased concern in thepotential involvement of human cytomegalovirus (HCMV) in carcinogenesis.HCMV seroprevalence was enhanced in patients with hepatocellularcarcinoma (HCC) however, a possible link between HCC and HCMVinfection remains to be assessed. The aim of this work was to investigatethe protumor influence of HCMV on primary human hepatocytes (PHH)and HepG2 cells. Methods: Following infection of PHH and HepG2 cellsby two different strains of HCMV, we measured the production of IL 6 inculture supernatants by ELISA and the protein levels of STAT3, pSTAT3,JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western blotting ininfected and uninfected cells. Ki67Ag expression was measured as a proliferationmarker whereas transformation was probed using soft agar colonyformation and tumorsphere assay. Results: PHH and HepG2 cells infectedwith HCMV resulted in the production of IL 6 and the subsequent activationof the IL 6R JAK STAT3 pathway. Expression of Cyclin D1, Survivinand Ki67Ag was upregulated by HCMV despite a paradoxical overexpressionof p53 and p21. Notably, we observed the formation of coloniesin soft agar seeded with PHH infected with HCMV and 2.5 fold moretumorspheres in HCMV infected HepG2 cultures than in uninfected ones.Conclusion: HCMV activated the IL 6 JAK STAT3 pathway in PHH andHepG2 cells, favored cellular proliferation, induced PHH transformationand enhanced HepG2 tumorsphere formation. Our observations raise thepossibility that HCMV infection might be involved in the onset of HCC.REF 004MicroRNA profiling in E6/E7 HPV transformed human keratinocytesGiorgio MANGINO 1 , Maria Vincenza CHIANTORE 2 , MarcoIULIANO 1 , Maria Simona ZANGRILLO 1 , Gabriele VACCARI 3 , RositaACCARDI 4 , Massimo TOMMASINO 4 , Gianna FIORUCCI 2,5 ,Giovanna ROMEO 1,51 Sapienza University of Rome/Dept. of Medico Surgical Sciences andBiotechnologies, Latina, ITALY; 2 Istituto Superiore di Sanità/Dept. ofInfectious, Parasitic and Immune mediated Diseases, Rome, ITALY;3 Istituto Superiore di Sanità/Dept. of Veterinary Public Health andFood Safety, Rome, ITALY; 4 International Agency for Research onCancer WHO/Infections and Cancer Biology Group, Lyon, FRANCE;5 CNR/Institute of Molecular Biology and Pathology, Rome, ITALYMicroRNAs (miRNAs) are naturally occurring noncoding RNAs that playa key role in gene regulation. They are highly conserved single strandedRNAs (∼22 nucleotides) that are cleaved from larger precursor transcripts.miRNAs exert their regulatory effects by cleaving the mRNAtargets or repressing their translation and are predicted to regulate at leastone third of all human genes. This posttranscriptional gene regulationhas been demonstrated to be active in fundamental cellular processes ascell proliferation, differentiation, and death. miRNAs are also involvedin the development and progression of human diseases, such as cancer.S118 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyAccumulating evidence indicates a role of miRNAs in tumorigenesis andtumor cells bear a specific and altered pattern of miRNA expression. Weperformed a high throughput screening using a TaqMan MicroRNA Assayfor the quantitation of 384 miRNAs in normal primary human keratinocytescompared to keratinocytes transformed by both E6 and E7 derivedfrom mucosal HPV 16 or cutaneous HPV 38 (K16 and K38 cells, respectively).Our analysis identified 18 miRNAs mainly deregulated in eitherK16 and/or K38 cells. Eight deregulated miRNAs (i.e. miR 10a, 18a, 19a,19b, 34a, 99a, 579 and 590) were further tested using miRNA single assayto confirm the results obtained in the screening. Since IFN may interferewith E6 and/or E7 dependent transformation, we characterized the miRNAexpression in K16 and K38 cells treated with this cytokine. Studies on thebiological significance of interesting specific targets are also in progress.REF 005The HTLV 1 encoded bZIP factor promotes cell proliferation andgenetic instability through activation of oncogenic microRNAsFranck MORTREUX 1 , Céline VERNIN 1 , Christiane PINATEL 5 ,Antoine GESSAIN 2 , Olivier GOUT 3 , Marie Hélène DELFAU LARUE 4 ,Catherine LEGRAS LACHUER 6 , Eric WATTEL 7,11 Université de Lyon 1, CNRS UMR5239, Oncovirologie et Biothérapies,Laboratoire de Biologie Moléculaire de la Cellule, Lyon, FRANCE;2 Institut Pasteur, Unité d’Epidémiologie et Physiopathologie des VirusOncogènes, Paris, FRANCE; 3 Fondation Rothschild, Service de Neurologie,Paris,France; 4 CHU Henri Mondor, Laboratoire d’Immunologie,Créteil, FRANCE; 5 Centre de Recherche sur le Cancer de Lyon, CentreLéon Bérard, Lyon, FRANCE; 6 Université Lyon I, Faculté de Médecineet de Pharmacie de Lyon, ISPBL ProfileXpert LCMT, Lyon, FRANCE;7 Université Lyon I, Service d’Hématologie, Pavillon Marcel Bérard,Centre Hospitalier Lyon Sud, Pierre Bénite, FRANCEViruses disrupt their host cells microRNAs (miRNAs) network for facilitatingtheir replication. That of HTLV 1 relies on the clonal expansionof its host CD4+ and CD8+ T cells yet the virus causes adult T cellleukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype.Infected cells express Tax and HBZ viral oncoproteins. Tax is expressedin untransformed cells where it promotes cell proliferation, genetic instabilityand miRNAs deregulation whereas in contrast, HBZ is expressedby untransformed and malignant T cells where hitherto, it is consideredto promote cell proliferation and to silence virus expression. Here weshow that an HBZ/miRNAs axis promotes cell proliferation and geneticinstability. Infected CD4+ but not CD8+ T cells were found to overexpressoncogenic miRNAs such as miR 17 and miR 21. HBZ activated these miR-NAs via a posttranscriptional mechanism while in addition to promotingcellular growth; HBZ decreased DNA stability. These effects were alleviatedby either miR 21/miR 17 knockdown or by the ectopic expression ofOBFC2A, a factor that protects genome stability and that we found targetedby miR 17 and miR 21 in HTLV 1 infected CD4+ T cells. This considerablyextends the oncogenic potential of HBZ and suggests that viral expressionmight be involved in the remarkable genetic instability of ATLL cells.REF 006Molecular signatures of tumorigenesis in penile squamous cell carcinomaand their association with high risk human papillomavirusinfectionElektra PETA 1 , Rocco CAPPELLESSO 2 , Valentina MILITELLO 1 ,Alessandro SINIGAGLIA 1,3 , Giulia MASI 1 , Matteo FASSAN 2 ,Ambrogio FASSINA 2 , Luisa BARZON 1 , Giorgio PALÙ 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Department of Medicine, University of Padova, Padova, ITALY;3 IOV Istituto Oncologico Veneto, Padova, ITALYPenile squamous cell carcinoma (PSCC) is a rare tumor associated withhigh risk (HR) HPV infection in 30 60% of cases. Altered expressionof miRNAs has been reported in HPV related cervical and in head andneck cancers, but there are no studies on PSCC. The presence of HPVinfection and its relationship with p53 and p16INK4a expression and withexpression of a panel of cellular miRNAs involved in HPV related cancerwas investigated in 59 PSCCs and 8 condylomas. HR HPV DNA(HPV16 in most cases) was detected in 2/5 in situ PSCCs and in 16/54invasive PSCCs. All penile condylomas were positive for low risk HPV6 orHPV11. Immunohistochemical analysis showed that HPV positive PSCCswere typically p16INK4a positive and p53 negative. Among investigatedmiRNAs, miR 218 was significantly down regulated in PSCCs with highrisk HPV infection. In addition, miR 218 was markedly silenced in HRHPV negative PSCCs with apparently functional p53. Hypermethylationof the promoter of the SLIT2 gene, which contains miR 218 in an intronof its transcript, has been described as a mechanism of miR 218 downregulationin several cancers. Thus, methylation of SLIT2 promoter andits association with HR HPV infection and p53 status was investigatedin PSCCs. Methylation of the SLIT2 promoter was frequently observedin PSCCs and was inversely correlated with miR 218 expression levelsespecially in HR HPV negative and p53 negative PSCCs. In conclusion,this study describes different molecular signatures of tumorigenesis (i.e.,HR HPV infection, p53 inactivation, and miR 218 downregulation) inPSCCs.REF 007Tax promotes the expression of the alternative spliced isoformCD44v10 by slowing down the elongating RNA polymerase IIMorgan THENOZ 1 , Céline VERNIN 1 , Christiane PINATEL 2 , NicolasNAZARET 3 , Joel LACHUER 3 , Antoine GESSAIN 4 , DidierAUBOEUF 5 , Eric WATTEL 1 , Franck MORTREUX 11 Oncovirologie et Biotherapies, UMR5239 CNRS/ENS Lyon/UCBL/HCL,Hopital Pierre Benite, Lyon, FRANCE; 2 Oncovirologie et Biothérapies,Centre Léon Bérard, Lyon, FRANCE; 3 ProfileXpert, Neurobiotec Service,Bron, FRANCE; 4 Unit of Epidemiology and Physiopathology of OncogenicViruses, Department of Virology, Institut Pasteur, Paris, FRANCE;5 Institut National de Santé et de Recherche Médicale U590, Centre LéonBérard, Lyon, FRANCEDeregulation of gene expression is the hallmark of HTLV 1 infection,involving a complex interplay between the viral oncoprotein Tax and hostcell transcription machinery. It has been recently shown that changes inpromoter structure and occupation impinge on both transcription and alternativesplicing (AS) by modulating the RNApolII elongation rate. By usingExon microarrays, we identified here that ectopic Tax expression induceschanges in the exon expression patterns. Overall, 857 were found differentiallyspliced upon Tax expression. Of these, 309 genes were changed intheir transcriptional levels. AS events were validated by exon specific RTPCR assays. More particularly, we identified in that Tax induced NF kBdependent transcription of CD44 results in the inclusion of variable exonv10 (CD44v10) in mature transcripts. The protein expression of CD44v10at the plasma membrane of Tax+293T cells was proved by FACS analysisand CD44v10 mRNA was also detected in infected CD4+ T cells invivo. CD44 variants are described in a wide variety of cellular functionsincluding lymphocyte activation, migration, and tumor metastasis. At themechanistic level, qChIP analysis of CD44 gene occupancy showed thatTax induced splicing of CD44v10 coincides with a significant enrichmentof both RNApolII and Tax at the region encoding variable exons, indicatingthat Tax interferes with the elongation rate of RNApolII. Together, theseresults unmask new mechanisms that connect Tax, transcription and alternativesplicing with the HTLV 1 induced transcriptome reprogramming ofCD4+ T cells.Virologie, Vol 17, supplément 2, septembre 2013S119

5 th European Congress of VirologyREF 008A humanized mouse model of human T cell leukemia virus, type 1associated diseasesEleonore PERES 1 , Eleonore PERES 1,2 , Julien VILLAUDY 3 , LouisGAZZOLO 1,2 , Madeleine DUC DODON 1,21 Laboratoire de Biologie Moléculaire de la Cellule, UMR 5239, CNRS,Lyon, FRANCE; 2 Laboratoire de Biologie Moléculaire de la Cellule, UMR5239, CNRS, Lyon, FRANCE; 3 Department of Medical Microbiology,Laboratory of Experimental Virology, University of Amsterdam, Amsterdam,NETHERLANDSHuman T cell Leukemia virus, type 1 (HTLV 1) is the etiological agentof adult T cell leukemia/lymphoma (ATLL) and of several inflammatorydiseases including HTLV 1 Associated Myelopathy/Tropical Spastic Paraparesis(HAM/TSP). HTLV 1 infected T cells have been hypothesized tocontribute to the development of these disorders, although the precisemechanisms are not well understood in part due to the lack of faithfulanimal model. The emergence of “humanized” mice that are sustainablyengrafted with a human immune system (HIS), have significantly contributedin delineating and recapitulating the immuno pathogenesis of humaninfectious diseases. Recently, we have shown that these HIS mice are efficientlyand persistently infected by HTLV 1, and able to develop CD4+ Tcell lymphomas with characteristics of ATLL. Consequently, this animalmodel of ATLL is providing insights of the development of the leukemogenicprocess and the design of a therapeutic strategy. Furthermore, wehave observed that the proliferation of activated human CD4+ T lymphocytesin the peripheral blood of HIS mice correlates with HTLV 1 proviralintegration, Tax expression as well as the detection of soluble CD25 (alphachain of Interleukin 2 receptor) in the blood of infected animals. Clearly,such a correlation provides a convenient approach to follow the progressionof the early phase of the infection process, before the apparition ofclinical signs. Using that approach, we are currently studying the effect ofHTLV 1 infectionS120 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology02. ONCOLYTIC VIRUSES AND GENETHERAPYPosters: REF 009 to REF 015REF 009Capsid mutations adapt coxsackievirus A9 to the A549 lung carcinomacell line by multiple mechanismsNaseebh BAESHEN, Çigdem WILLIAMS, Glyn STANWAYUniversity of Essex, Colchester, Essex, UNITED KINGDOMOncolytic virotherapy is a novel treatment for human cancer by usingviruses that are specially designed to infect and destroy malignant cells,while leaving normal cells unharmed. It is important to understand howviruses can be targeted more effectively at cancer cells. We have adaptedCAV9 to more efficient growth in the A549 cell line. Mutations were foundin the virus capsid and fall into 2 classes: those that bring about heparansulphate binding, achieved by the generation of linear and conformationalbinding sites, and mutations at a striking position immediately at the 3 foldaxis. We are currently studying how these mutations facilitate infection,particularly their effect in terms of cell entry mechanisms, and adaptingthe virus to other cancer cells to determine if common mechanisms areinvolved. In this way we hope to understand the basis of tropism towardscancer cells and enhance the prospects of using enteroviruses such asCAV9 for cancer therapy.REF 011Evaluation of oncolytic properties of Influenza A virus in humanpancreatic adenocarcinoma cellsMatteo Samuele PIZZUTO 1,3 , Samantha KASLOFF 1 , MicolBENUSSI 2 , Ilaria CAPUA 1 , Wendy BARCLAY 31 Istituto Zooprofilattico Sperimentale delle Venezie, Padua, ITALY;2 Istituto Oncologico Veneto, Padua, ITALY; 3 Imperial College London,London, UNITED KINGDOMPancreatic ductal adenocarcinoma (PDA) is the most common and aggressiveform of pancreatic cancer. Therapies proven successful in other tumortypes have shown little efficacy in treating PDA, and the need for alternativetreatments is crucial. Virotherapy uses natural or engineered oncolyticviruses (OVs) to selectively kill tumor cells. PDA cells are highly heterogeneousin their permissiveness to various OVs (Moerdyk Schauweckeret al., 2012). Recently Capua et al., (2013) demonstrated the ability ofinfluenza viruses (IVs) to infect human origin pancreatic beta cells in vitroas well as pancreatic islets ex vivo. The fact that IVs have such tropismfor the pancreas led us to investigate their oncolytic potential againstPDA cells. Like many cancer cells, PDA cells are often IFN deficient(Ghadimi et al., 1999). OV selectivity can thus be enhanced by creatingviruses deficient in IFN antagonism (Muster et al., 2004). Using reversegenetics, recombinant influenza viruses (rgIVs) truncated in NS1 or mutatedin the Mitochondrial Targeting Sequence (MTS) of PB1 F2 (Vargaet al., 2012) were generated. These mutations compromised the virus abilityto control IFN induction. The rgIVs grew in different pancreatic celllines particularly those that are IFN deficient. Furthermore, the rgIVs inducedhigh levels of apoptosis in BXPC 3 cells, a PDA line previously shown“resistant” to other OV candidates (Murphy et al., 2012). So far few studieshave examined IV as an OV, yet our preliminary results indicate thatit should be taken into account for further studies on treatment of PDA.REF 010Effect of pseudorabies virus immediate early IE180 protein andherpes simplex virus type 1 ICP4 protein on the activity of the hTERTand CEA tumor specific promotersLaura LERMA 1 , Carlos PIÑERO 1 , María TORRES 1 , BeatrizMARTÍN 1 , Filip LIM 2 , Enrique TABARÉS 11 Autonoma University of Madrid, Madrid, SPAIN; 2 Severo Ochoa MolecularBiology Center, Madrid, SPAINPseudorabies virus (PRV) and herpes simplex virus type 1 (HSV 1) havebeen proposed and used in oncolytic virotherapy for human cancer. Inthe present study, we try to analyze the activity of immediate early (IE)transcription factors of these viruses (IE180 and ICP4, respectively) onthe human tumor specific telomerase reverse transcriptase (hTERT) andcarcinoembryonic antigen (CEA) promoters which have shown potentialfor transcriptional targeting in cancer gene therapy. The approach used wasto simultaneously co transfect into HeLa cells plasmids containing bothviral IE genes with other plasmids containing either the hTERT promoter orCEA promoter linked to the EGFP gene. The results show that ICP4 alonecan activate the hTERT and CEA promoters, whereas IE180 reduces theactivity of the hTERT promoter and induces the activity of CEA promoter.This property might be used as one more advantage in the use of PRV ingene therapy compared to HSV 1 since might eliminate the potential riskof oncogenesis through deregulation of host gene expression, such as thetelomerase by viral vectors in normal cells.REF 012Generation of induced pluripotent stem cells by integrating and nonintegrating viral vectors: Comparison of different reprogrammingmethodsMarta TREVISAN 1 , Giulia COSTANZI 1 , Valeria MARTINELLO 2 ,Giorgio PALÙ 1 , Luisa BARZON 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Clinical Microbiology and Virology, Padova University Hospital,Padova, ITALYInduced pluripotent stem cells (iPSCs) can be generated by ectopic expressionof the transcription factors Oct4, Sox2, Klf4, and c Myc in somaticcells. iPSCs share many features with embryonic stem cells such as pluripotencyand self renewal and hold great promise for the future of celltherapy. Reprogramming by using retroviral vectors (RV) is the first widelyused method but might lead to tumorigenesis rendering iPSCs not suitablefor clinical application. Aim of this study was to optimize and comparemethods for reprogramming fibroblasts to generate iPSCs. Methods werebased on the use of RV, adenoviral (AdV) and Sendai virus (SV) vectorsfor OCT4, SOX2, KLF4, and c MYC gene transfer. RVs and AdVs wereproduced in house, while the SV system was a commercial kit. Differenttransduction and reprogramming conditions were tested. Reprogrammingof fibroblasts with RV was less efficient than the method based on SVvectors (0.01 0.05% and 0.1% of transduced cells, respectively), whileVirologie, Vol 17, supplément 2, septembre 2013S121

5 th European Congress of Virologyreprogramming with AdV was inefficient. In our experience, RV systemrepresented an affordable in house method for reprogramming, leading toiPSCs generation with sufficient efficiency although with the risk of mutagenesisdue to vector integration. The SV based protocol was an efficient,virus free method, with consistent capacity of reprogramming, but with thedisadvantage of being more expensive than the other methods. AdV couldrepresent the method of choice, being integration free and easy to produce,but more efforts should be done to improve reprogramming conditions.REF 013Lentiviral and fluorescent reporter vectors for genome editing usingzinc finger nucleasesNadia GUETTARI, Monika MINNER, Qiaohua KANG, YongmeiFENG, Gregory D. DAVISSigma Aldrich Biotechnology, St. Louis, MO, USAZinc finger nuclease (ZFN) design and manufacturing at Sigma Aldrichhas successfully addressed a wide variety of genome editing applicationsranging from gene knockout to more challenging, site restrictedprojects requiring targeted double strand breaks near disease SNPs andother small genetic elements. Despite major advances in ZFN engineering,ZFN delivery and expression remain challenging for particular celltypes. Genetic tools such as shRNA and microRNA have benefited greatlyfrom lentiviral gene expression, enabling manipulation of primary cells,non dividing cells, and a variety of other difficult cell types. Lentiviraltiters are known to be negatively impacted by large genetic payloads, andsubcloning of repetitive sequences into lentiviral vectors has often resultedin vector recombination and instability (Holkers et al., 2013). Due to theircompact protein structure and low content of repetitive sequence, ZFNshave been successfully expressed and implemented using lentiviral vectors(Lombardo et al., 2007). Here we present data showing the use of lentiviraldelivery methods in genome editing applications using CompoZr ZFNs. Asan additional alternative approach to overcoming low delivery and expressionlevels in particular cell types, we have created vectors in which ZFNexpression is linked to fluorescent reporters. This reporter linked expressionformat enables enrichment of cell populations that have undergoneboth efficient transduction and subsequent high level expression of ZFNtransgenes and can substantially increase genome editing frequenciesREF 014Measles virus glycoprotein pseudotyped lentiviral vectors transduceprestimulated and resting long-term repopulation hCD34+ cells at anefficiency without precedentCamille LEVY, Anais GIRARD, Fouzia AMIRACHE, CeciliaFRECHA, Caroline COSTA, Didier NEGRE, François-Loïc COSSETand Els VERHOEYENCIRI, EVIR team, Lyon, FRANCEHematopoietic stem cell (HSC) based gene therapy holds promise for thecure of many inherited and acquired diseases. The field is now movingtowards the use of lentiviral vectors (LVs): clinical trials now use VSV-G-LVs at high doses combined with strong cytokine-cocktails to obtaintherapeutic transduction levels with the risk of compromising ‘HSC’ character.Previously, we have shown that measles virus (MV) glycoproteindisplaying LVs (MV-LVs) were able to transduce efficiently stimulatedand resting T and B cells. We now evaluated these MV-LVs for hCD34+cell transduction after SCF+TPO prestimulation in order to better preservethe ’HSC’ characteristics. After a single application at a low vector doses,MV-LVs stably transduced 100% of stimulated hCD34+ cells, where VSV-G-LVs reached 5-10%. Moreover, these MV-LVs allowed also efficienttransduction of 50-70% quiescent hCD34+-cells, an efficacy without precedent.We found that this high transduction level was correlated withthe expression of the MV receptor, CD46. Importantly, reconstitution ofNSG mice with MV-LV transduced hCD34+ cells resulted in transductionlevels close to 100% for all analyzed myeloid and lymphoid cell lineagesincluding CD34+ cells in the bone marrow. Moreover, this transductionpattern was confirmed in secondary mice engraftments. Together, theseresults strongly suggest that the MV-LVs efficiently transduce true HSCsat extremely high levels. This paves the way to HSC-based gene therapyof multiple diseases such as Fanconi Anemia, for which high level HSCcorrection is needed to be successful.REF 015Baboon retrovirus envelope pseudotyped LVs allow efficient transductionof progenitor T cells, thymocytes and adult T and B cellsAnais GIRARD, Fouzia AMIRACHE, Caroline COSTA, Camille LEVY,Dimitri LAVILLETTE, Didier NEGRE, François-Loïc COSSET and ElsVERHOEYENCIRI EVIR team, Lyon, FRANCEEfficient gene transfer into quiescent T and B lymphocytes for gene therapyor immunotherapy purposes may allow the treatment of severalgenetic dysfunctions of the hematopoietic system, such as immunodeficiencies,and the development of novel therapeutic strategies for cancersand acquired diseases. Previously, we have shown that measles virus gpsLVs (MV-LVs) were able to transduce efficiently stimulated or resting Tand B cells. We now showed that the newly engineered Baboon retrovirusenvelope pseudotyped LVs (BAEVgp-LVs) allowed in addition to hematopoieticstem cell transduction, high level transduction of activated T andB cells and resting B cells, where VSVG-LVs fail. Moreover, both naiveand memory cells were efficiently transduced and their phenotypes conserved.Since the thymus plays a key role in the induction of self-toleranceand by intra-thymus injection of an LV in situ correction of a geneticimmunodeficiency might be envisaged, it is important to develop vectorsfor efficient thymocyte transduction. BAEVgp-LVs allowed high leveltransduction of the thymocytes in the early stages of development with preferencefor early thymic progenitor T cells while MVgp-LVs, showed anequivalent transduction level of all thymic subpopulations. BAEVgp-LVsare especially superior over VSVG-LVs for the transduction of the veryimmature T cell progenitor subpopulation, which is long-lived in vivoand might confer a permanent therapeutic effect. Currently, the vectorsare tested for intra-thymic transduction in human immune system micemodel.S122 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology03. INNATE IMMUNITY AGAINSTVIRUSESPosters: REF 016 to REF 036REF 016Characterization of human papillomavirus (HPV) integration sitesand somatic mutations in STK11 gene in penile cancerMaria Lina TORNESELLO 1 , Clorinda ANNUNZIATA 1 , LuigiBUONAGURO 1 , Simona LOSITO 2 , Franco BUONAGURO 11 Molecular Biology and Viral Oncology Unit, Istituto Tumori FondazionePascale, Napoli, ITALY; 2 Pathology Unit, Istituto Tumori Fondazione Pascale,Napoli, ITALYBackground: HPV infection accounts for 40 50% of all cases of penilecarcinoma (PC) suggesting that, besides the virus, host genetic alterationsare relevant for neoplastic transformation. In invasive cervical cancer twofactors have been shown to perturb human genes: 1) HPV integration intoloci relevant for cancer pathogenesis; and 2) mutations/deletions of tumoursuppressor genes. In this perspective we have analysed HPV integrationsites and STK11 mutational status in HPV positive and HPV negative PC.Methods: Genomic DNA was extracted from 26 PC cases. Integrationsites of HPV were characterized by means of Alu HPV based PCR, cloningand sequencing. Genetic alterations in the exons 1 to 9 of STK11gene were analysed by PCR and sequencing, and by quantitative real timePCR. Ratios of potentially deleted and non deleted exons were indicativeof specific loss of STK11 coding regions. Results: Viral–human DNAjunctions showed that in two cases HPV integration occurred within thechromosome 8q21.3 region, in which the FAM92A1 gene is mapped, andinside the chromosome 16p13.3, within TRAP1 gene. Heterozygous deletionsof STK11 exon 1and 2 were identified in 14.3% HPV positive andin 8.3%) of HPV negative cases (8.3%). Complete nucleotide sequencinganalysis of exons 1 9 showed only a single nucleotide change upstreamthe exon 2 coding region in one sample. Conclusions: The present resultssuggest that HPV integration events occur in gene loci relevant for celltransformation and that single nucleotide mutations and/or deletions ofSTK11 gene are rare events in PC.REF 017Breast cancer and virus: Molecular homology between Human MammaryTumor Virus 3 ′ orf and scorpion toxin, a ligand of voltage gatedsodium Na+ channel. Omega 3, a Na+ channel modifier, reduces therisk of metastatic breast cancerGuy Mong Ky TRAN 1,4 , Adrien CAPRANI 2,31 Retired, Public Health, European AIDS Clinical Society (EACS) ScientificCouncil member, Chatenay Malabry, FRANCE; 2 CNRS Jussieu, Paris,FRANCE; 3 Association Positifs, Paris, FRANCE; 4 Association Positifs,Scientific Advisor, Paris, FRANCEHuman Mammary Tumor Virus (HMTV) was found in Breast Cancer(BC), in variable % cases: Tunisia 74%, Australia 42%, Italy 38%, UnitedStates 36%, Argentina 31%, Vietnam 0.8% (Levine PH, 2004). HTMV+(Wang Y, 1995) MCF 7 and MDA MB 231 cell lines injected in nude miceinduce metastatic breast cancer (Shafie SM, 1980; Price JE, 1990). In 123treated BCs, Bougnoux P (1995) found after 48 months 70% metastasisif the breast fat alpha linolenic acid [omega 3 (w 3)] level was low (9% ifit was high): Thus, w 3 protects against metastasis. As w 3 modifies thevoltage gated Na+ channel (NaCh) (Xiao YF, 2001; Banu I, 2006), welooked for a NaCh ligand (scorpion toxin) in HMTV. By amino acid (aa)sequence comparison, we found a molecular homology between HMTV3 ′ orf and scorpion toxin chimera (Possani LD, 2000)/Euscorpius Flavicaudus(AAT76439) (58 71) implicated in development: HMTV 124 AKNYIFTNKT 133 109 KYSSTPQPG 101 (in retro inverso)Toxin chim 1 AKNGYILNKST 11 13 KYSCSTRPG 21HMTV 282 YIYLGTGMHFWGKI 295 265 LTQKEKDDMKQQVH 278Toxin 29 YVAESGYCQ(F,W)GKI 42 58 IVQKEKDQVRQHI H 71The toxin active site (El Ayeb M, 1986, Kharrat R, 1989): N terminus(K2/Y5)/Y14/W38 loop corresponds to HMTV: K125/Y127/Y108/W292.HTMV 3 ′ orf contains a scorpion toxin, explaining the metastasis protectionconferred by w 3, a NaCh blocker; the w 3 rich soya diet in Asia,added to the low HTMV frequency in Asia (Japan/China/Vietnam), mayexplain the low asian BC prevalence. We suggest to use w 3 and otherNaCh ligands (Tran GMK, Allostery EMBO Conf 2013; Djamgoz MBA,2006) with BC chemotherapy.REF 018Toscana virus inhibits the interferon beta response in cell culturesNadege BRISBARRE 1 , Sebastien PLUMET 2 , Philippe DE MICCO 1 ,Isabelle LEPARC GOFFART 2 , Sebastien EMONET 3,21 UMR 7268/Aix Marseille université CNRS/EFS, marseille, FRANCE;2 IRBA Antenne Marseille, marseille, FRANCE; 3 IRBA, lyon, FRANCETOSV is an arthropod borne virus (family Bunyaviridae, genus Phlebovirus)transmitted to humans by sandflies. It is an emerging pathogen inthe Mediterranean basin where it causes outbreaks of acute meningitisand meningoencephalitis in native or foreign (tourists) populations duringsummertime when the vector population reaches its maximum density.The innate mammalian immune system can establish a first line of defenseagainst invading viruses; cells synthesize and secrete beta interferon (IFN) (type I IFN) which prompt cells to an antiviral state. Previous studiesdemonstrated that some viruses belonging to the genus Phlebovirus maydevelop anti IFN strategies, but information regarding TOSV is widelylacking. We explored the relationships between TOSV and type I interferon(IFN) response. Our main findings were as follows: i) TOSV growthin Vero cells is sensitive to an antiviral state induced by low dose additionof exogenous IFN beta; ii) no IFN mRNA or IFN were detectableafter infection of HeLa and 293T cells (mammalian cells) by TOSV fromtwo different lineages; iii) TOSV inhibits IFN production induced bySendaï virus, a well known inducer of IFN production. These experimentsdemonstrate the ability of TOSV to inhibit the IFN production.The existence of an active anti IFN system against the mammalian IFNdefense mechanism suggests that a mammalian host exists probably duringthe natural life cycle of TOSV; this anti IFN capability is maintained bymeans of regular contact with this mammalian host.REF 019Type III interferon (IFN lambda) antagonizes the antiviral activityof interferon alpha in vitro against west nile virusDaniele LAPA, Maria Rosaria CAPOBIANCHI, Licia BORDI, EleonoraLALLE, Claudia CAGLIOTI, Serena QUARTU, Antonino DI CARO,Ippolito GIUSEPPE, Concetta CASTILLETTNational Institute for Infectious Disease L. Spallanzani, Rome, ITALYType III interferons (IFN lambda 1 to 4 also known as IL28/29), are themost recently discovered members of IFN family. The influence of naturalgenetic polymorphisms on spontaneous and therapy driven recoveryfrom HCV infection suggests that this novel IFN type is involved in innateantiviral defence mechanisms. Synergism between different IFN types iswell established, but for type I and type III IFNs no conclusive evidencehas been reported so far. The aim of the study was to evaluate the possiblesynergism/antagonism between IFN alpha and IFN lambda1 andVirologie, Vol 17, supplément 2, septembre 2013S123

5 th European Congress of Virologylambda2 in the inhibition of replication of WNV (lineages 1 and 2) andin the activation of intracellular pathways of IFN response in differentcell lines. To this aim, A549 and Vero E6 cells were treated with increasingamounts of IFN lambda either IFN lambda1a or lambda2a or IFNalpha, used alone or in combination, then infected with WNV 1 or 2; theextent of virus yield inhibition was established; mRNA levels of MxA and2 ′ 5 ′ OAS were measured as well. The antiviral potency of IFN lambda1and lambda2 was lower than that of IFN alpha. When IFN alpha andlambda were used together, the Combination Index (CI) for virus inhibitionwas >1 (although more pronounced in Vero E6 cells infected with WNVlineage 2), indicating antagonistic effect. The same results were obtainedusing other viruses (Chikungunya virus, EMCV and HSV 1). Antagonismbetween IFN alpha and lambda was also observed for the inductionof mRNA for both MxA and 2 ′ 5 ′ OAS. Elucidating the interplay betweenIFN alpha and lambda may help to better understand innate defencemechanisms against viral infections, including the molecular mechanismsunderlying the possible influence of IL-28B polymorphisms on the naturalhistory of flavivirus infections. This work was partially supported by theEuropean Union Seventh Framework Programme [FP7/2007-2013] underGrant Agreement n ◦ 278433-PREDEMICS.REF 020The measles virus phosphoprotein interacts with the linker domainof STAT1Patricia DEVAUX, Lauren PRINISKI, Roberto CATTANEOMayo Clinic, Rochester, MN, USAThe measles virus (MV) phosphoprotein (P) and V proteins block the interferon(IFN) response by impeding phosphorylation of the signal transducerand activator of transcription 1 (STAT1) by the Janus kinases (JAKs). Wesought to characterize here how STAT1 interacts with MV P. Based onsequence and function conservation between MV and Nipah virus (NiV)P proteins, we postulated that the region of STAT1 interacting with NiV P(residues 509 to 712) also interacts with MV P. Within this region, sinceMV P does not interact with STAT2, we mutated segments that have nohomology to it. These segments cover the linker domain, the Src homology2 (SH2) domain and the transactivation domain. We initially producedmutants of two or three residues within these segments, and then alsomutants of individual residues. The expression and effect of these mutantswere tested in STAT1 deficient cells. Certain mutants of the linker, theSrc homology 2 domain (SH2), or the transactivation domain had reducedor abolished phosphorylation through JAKs after IFN treatment. Othermutants, mainly localized in the linker, failed to interact with P as documentedby the lack of interference with nuclear translocation. Thus, thefunctional footprint of P on STAT1 localizes mainly to the linker domain;there is also some overlap with the STAT1 phosphorylation functionalfootprint on the SH2 domain. Based on these observations, we propose amodel of how the MV P and V proteins may operate in concert to inhibitthe JAK/STAT pathway.REF 021Mechanism of inhibition of the innate immune response by alphavirusnsP2 proteinElena FROLOVA 1 , Ivan AKHRYMUK 1 , Sergey KULEMZIN 11 University of Alabama at Birmingham, Birmingham, USAOutcome of viral infection at the cellular level depends on competitionbetween cellular antiviral response and ability of the virus to inhibit itsactivation. The antiviral response induces pathways mediating non cytopathicclearance of the virus from infected cells and release of interferon andother cytokines, which establish the antiviral state in yet uninfected cells.Numerous viruses promote infection spread and viremia development by atleast partially inhibiting cellular antiviral response. It has been previouslydemonstrated that the Old World alphaviruses, including Sindbis, SemlikiForest and chikungunya viruses, evade the innate immune responseby inducing rapid and global inhibition of cellular transcription, whichprevents activation of cellular antiviral genes. We have defined the mechanismof transcriptional shutoff utilized by the Old World alphaviruses.During virus replication, the nonstructural protein nsP2 is transported intothe nucleus of the infected cells, where it induces rapid degradation ofthe catalytic subunit of the DNA dependent RNA polymerase Rpb1. Wefound that degradation of Rpb1 does not depend on the nsP2 associatedprotease activity. Instead, nsP2 imitates activation of the transcriptioncoupled repair mechanism, which leads to complete, ubiquitin dependentdegradation of Rpb1 within 4 6 h post infection. Interestingly, the NTPaseactivity of nsP2 helicase domain is critical for Rpb1 degradation. Thus,the Old World alphaviruses employ a previously unknown and uniquemechanism for robust inhibition of cellular antiviral response.REF 022Characterization of the effect of antiviral enzyme RNase L on differentpathogenic strains of influenza A virusWai Yip LAM 1 , Denis Pc CHAN 2 , Paul Ks CHAN 2,3 , Stephen KwTSUI 11 School of Biomedical Sciences, Faculty of Medicine, The Chinese Universityof Hong Kong, Shatin, HONG KONG; 2 Stanley Ho Centre forEmerging Infectious Diseases, Faculty of Medicine, The Chinese Universityof Hong Kong, Shatin, HONG KONG; 3 Department of Microbiology,Faculty of Medicine, The Chinese University of Hong Kong, Shatin, HONGKONGInfluenza A viruses cause a highly contagious respiratory disease inhumans and are responsible for the periodic widespread pandemics, thathave caused high mortality rates. Recently, the World Health Organization(WHO) has recorded a mortality rate of greater than 60% in thehighly pathogenic avian influenza A (H5N1) virus infection. The fatal outcomeof human influenza A H5N1 is found to be associated with reactivehaemophagocytic syndrome and multi organ failure mediated by hypercytokinemia.Ribonuclease L (RNase L) is a latent endoribonuclease in theinterferon regulated double strand RNA (dsRNA) activated antiviral 2 ′ 5 ′oligoadenylate synthetase (OAS)/RNase L pathway. Activated RNase Lwill cleave viral single strand RNA (ssRNA) predominantly at single strandedUA and UU dinucleotides position(s), resulting in small, often duplexRNA cleavage products. These small RNA cleavage products can furtheractivate the interferon (IFN) as well as other cytokines responses via specificpathogen recognition receptors, such as retinoic acid inducible gene I(RIG I), melanoma differentiation associated gene 5 (MDA5) and toll likereceptors (TLR). We postulate that different viral RNA sequences fromdifferent pathogenic strains of influenza A virus will be cleaved by RNaseL in a sequence specific manner and the resulting amount of cleavage RNAproducts will thus be different. Bioinformatic data demonstrating a quantitativedifference between the number of UA/UU sites between highlypathogenic and low pathogenic viruses. The results were further verifiedusing an in vitro model.REF 023Mononegavirales leader RNA as agonist of RIG IJade LOUBER 1 , Louis Marie BLOYET 1 , Eva KOWALINSKI 2,3 ,Stephen CUSACK 2,3 , Denis GERLIER 11 Centre International de Recherche en Infectiologie, CIRI, INSERMU111, CNRS UMR5308, Université Lyon 1, ENS Lyon, Lyon, FRANCE;2 European Laboratory of Molecular Biology, Grenoble, FRANCE; 3 VirusCell host Interactions Unit, UJF EMBL CNRS, UMI 3265, Grenoble,FRANCES124 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyHost defence against viruses depends on the rapid triggering of innateimmunity through the induction of type I interferon (IFN) response. Tothis end, dedicated receptors are able to detect microbe associated molecularpatterns. RIG I can sense viral RNA into the cytoplasm and theninduce the production of IFN. Studies performed with in vitro synthesizedRNA indicate that RIG I responds to double strand RNA (dsRNA)with a free triphosphorylated 5 ′ end (5 ′ ppp). However, the identity of viralRNA(s) recognized by RIG I during an infection remains unclear. We showthat synthetic leader RNA of a filoviridae (Ebola virus), a rhabdoviridae(Rabies virus) and a paramyxoviridae (Measles virus) can bind RIG I invitro. In Huh7.5 cells that express an inactive RIG I mutant, the transfectionof the three synthetic leader RNAs activates the IFN beta promoteronly when an active RIG I is expressed. Binding of synthetic leader RNAof Ebola, Rabies and Measles viruses by RIG I was detected in cellula.Indeed, synthetic leader RNAs were selectively immunoprecipitated withRIG I. Furthermore, during activation of RIG I by a synthetic 5 ′ ppp dsRNAor by polyinosinic: polycytidylic acid, a dsRNA analogue, no oligomerizationof RIG I could be observed by co immunoprecipitation. These resultssuggest that leader RNA of Mononegavirales can be good RIG I agonistswithout evidence of intracellular oligomerization of RIG I.REF 024Influenza B Virus Induces Interferon Expression Directly upon EntrySanna MÄKELÄ 1 , Pamela ÖSTERLUND 1 , Veera ARILAHTI 1 , IlkkaJULKUNEN 1National Institute for Health and Welfare, Helsinki, FINLANDInfluenza A (IAV) and B (IBV) viruses cause annual epidemics worldwide.IAV has broad host specificity whereas IBV infects only humans, causingmilder symptoms than IAV. We have previously shown that IAV escapesearly host recognition, while IBV induces interferon (IFN) expressiondirectly upon entry, without any replication of the virus. IFN expressionwas mediated by IFN regulatory factor 3 (IRF3) and the kinetic differencebetween the viruses was seen also in the phosphorylation of mitogen activatedprotein kinase (MAPK) p38. The IAV evasion of early immunerecognition did not depend on viral NS1 protein. The goal of our ongoingresearch is to investigate whether the endosomal trafficking differs betweenIAV and IBV, using human monocyte derived dendritic cells (moDCs).Disturbances of endosomal acidification by infecting moDCs with IBVlacking its ion channel protein NB decreased the IFN lambda1 expression,but did not affect its time kinetics. Our preliminary results show thatendosomal maturation and the release of viral material to the cytosol areneeded for the IBV induced early IFN expression as evidenced by treatmentwith different inhibitors of endosomal acidification. This suggeststhat the early IBV induced IFN expression is initiated after virus recognitionin the cytosol, probably via retinoic acid inducible gene 1 (RIG I)receptor, thus excluding the endosomal Toll like receptors 3, 7 and 8.REF 025Regulation of interferon Beta gene expression by TCF/Beta cateninecomplexesVasco MARCATO 1 , Zeyni MANSUROGLU 1 , Michèle BOULOY 2 ,Marie FLAMAND 2 , Eliette BONNEFOY 11 Université Paris Descartes, Paris, FRANCE; 2 Pasteur, Paris, FRANCEThe expression of the gene coding for interferon beta (IFN B) proteinthat plays an essential role during the establishment of the innate antiviralresponse is finely regulated. In the absence of infection, the IFN Bgene is maintained in a constitutively silent state. After virus infection,the degree of induction of IFN B expression that varies from one cell toanother depends on the coordinated recruitment over the IFN B promoterof transcription and chromatin remodeling factors. We have recentlyidentified two binding sites for the T cell transcription factor (TCF) on theIFN B promoter. These factors regulate the expression of numerous genesacting either as repressors, in association with Groucho and histone deacetylases,or as transcriptional activators when associated with beta catenin.Using inhibitors of kinase GSK3, that induce TCF/beta catenin complexformation, we have revealed the capacity of TCF/beta catenin complexesto activate the expression of the IFN B gene in the absence as well as duringviral infection. Our results obtained with IFN B promoters deleted on theTCF binding region show that TCF/beta catenin complexes act directly onthe IFN B promoter requiring the presence of the region containing theTCF binding sites where catenin interacts with the promoter as shownusing chromatin immunoprecipitation technique. The regulation of IFN Bgene expression by TCF/beta catenin complexes open up interesting newpossibilities for the modulation of the expression of IFN B expression byWnt and AKT pathways.REF 026Activation of Type III Interferon Inhibits Dengue Virus Replicationin an Epithelial Cells ModelHelen Kenia PALMA OCAMPO 1,2 , Gerardo SANTOS LOPEZ 2 , JulioRoberto REYES LEYVA 2 , Veronica VALLEJO RUIZ 2 , Nora HildaROSAS MURRIETA 11 Laboratorio de Bioquímica y Biología Molecular, Instituto de Ciencias,Benemérita Universidad Autónoma de Puebla, Puebla, MÉXICO;2 Laboratorio de Virología, Centro de Investigación Biomédica de Oriente,Instituto Mexicano del Seguro Social, Puebla, MÉXICOInterferons (IFNs) are key cytokines in the innate immune response andthe first line of defense against viral infection. The newly identified typeIII interferon (IFN ) shares common biological functions with IFN a,but exert their action through different receptors. Because the ability ofdengue virus to escape from the IFN a response during infection and theantiviral activity of IFN against a large number of viruses, our goal wasto examine whether interferon has the ability to inhibit dengue virusinfection in cells expressing IFN receptors as viral infection targets. Inthis study, we showed that the C33 A epithelial cell line was permissibleto dengue virus infection using the plaque forming assay. Also, IFN transcripts were detected by RT PCR assays during viral infection. Additionally,an inhibition of viral replication was shown as the plaque formingunits decreased in a dose dependent manner when IFN a or IFN treatedcells were infected with dengue virus. Subsequently, we performedkinetics of viral replication inhibition under different infectious doses ofinterferon treated cells. The results showed a similar pattern, but the IFN treatment has a greater inhibitory effect of viral replication than IFN a.This IFN mediated antiviral activity could inhibit dengue virus infectionby activation of various signaling pathways that trigger the cellularantiviral state. The data provides evidence indicating that IFN , throughintracellular or extracellular antiviral mechanisms, inhibits dengue virusreplication in cultured cells lines.REF 027Cytokine profile in West Nile virus infectionsAnna PAPA 1 , Afroditi ANASTASIADOU 2 , Katerina TSERGOULI 1 ,Dimitrios BOUTEL 2 , Ioannis KAKOULIDIS 21 Aristotle University of Thessaloniki, Thessaloniki, GREECE; 2 GiannitsaGeneral Hospital, Giannitsa, GREECEBackground: West Nile virus is a mosquito borne flavivirus causing tohumans a variety of symptoms, from asymptomatic or mild infection (WestNile fever, WNF), to severe, and often fatal, infection of the central nervoussystem (West Nile neuroinvasive disease (WNND). The current knowledgeabout the immune response and pathogenesis of the disease is limited. WeVirologie, Vol 17, supplément 2, septembre 2013S125

5 th European Congress of Virologyestimated the serum levels of various cytokines, chemokines and growthfactors in acute WNV human cases and correlate them with the clinicalsyndrome and the outcome.Materials and Methods: Serum levels of 27 cytokines (IL-1, IL-1ra,IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17,basic FGF, eotaxin, G-CSF, GM-CSF, IFN-g, IP-10, MCP-1, MIP-1a,MIP-1b, PDGF-BB, RANTES, TNF-a, and VEGF) were measuredsimultaneously in 24 Greek patients (13 male), 20 with WNND and4 with WNF. Their age was 30 87 years (median 75). Ten cases werefatal. The Bioplex suspension array system was used, and the statisticalanalysis was performed by SPSS. Results: Levels of 23 cytokines wereaffected, with IL-1b, IL-6, IL-15, IFN-g, and VEGF being affectedat highest. IL-6, IL-10 and MCP-1 concentrations were significantlyhigher in fatal WNND cases than the non fatal WNND and WNF cases.Cytokines levels differ also according to the day of illness. Conclusions:Many cytokines play a catalytic role in the course and outcome of WNVinfections. Understanding the host immune response to WNV will enablefuture therapeutic approaches for the disease.REF 028The measles virus C protein interferes with double stranded RNAaccumulation that leads to protein kinase R activationChristian PFALLER 1 , Monte J. RADEKE 2 , Roberto CATTANEO 3 ,Charles E. SAMUEL 1,21 Department of Molecular, Cellular and Developmental Biology, Universityof California Santa Barbara, Santa Barbara, CA 93106, USA;2 Neuroscience Research Institute, University of California Santa Barbara,Santa Barbara, CA 93106, USA; 3 Department of Molecular Medicine,Mayo Clinic, Rochester, MN 55902, USARecombinant Moraten measles virus (MVvac) lacking expression of Cprotein (CKO) is a potent activator of the double stranded RNA (dsRNA)dependent protein kinase (PKR). Activation of PKR mediates downstreamsignaling events including eIF2a phosphorylation, IFN inductionand stress granule formation. Here we show that significant amounts ofdsRNA accumulate during MVvac CKO infection, but not during infectionwith isogenic MVvac expressing C protein. The dsRNA accumulatedat late stages of infection and localized with viral inclusion bodies (IBs)containing N and P proteins. Sensitivity tests with actinomycin D andcycloheximide suggest that the dsRNA is a viral replication product. PKRautophosphorylation and stress granule formation correlated with the kineticsof appearance of dsRNA. Protein and RNA quantification revealed thatthe CKO mutation did not affect the steady state balance of viral proteincompared to viral RNA. RNA deep sequencing established that neitherthe viral transcription gradient nor the ratio of plus strand vs. minus strandviral RNA was altered in MVvac CKO compared to MVvac infected cells.Furthermore, defective interfering viral genomes were not detected. Aprotocol for isolation of PKR was established to obtain RNAs bound toPKR during MV infection. Analysis of selected RNAs by qPCR suggestedthat PKR predominantly bound viral mRNAs but not full length RNA. Wehypothesize that the lack of C expression alters the amount and subcellularlocalization of PKR activator RNA in MVvac CKO infected cells.REF 029Phosphatidylinositol 3 kinase (PI3K) Akt pathway: which role inJcDNV infection?Fanny SALASC 1,2 , Anne Sophie GOSSELIN GRENET 2,3 , ThierryDUPRESSOIR 1,2 , Mylène OGLIASTRO 21 EPHE, Laboratoire de Pathologie comparée des invertébrés, Montpellier,FRANCE; 2 INRA, UMR 1333, Diversité, Génomes & InteractionsMicroorganismes Insectes, Montpellier, FRANCE; 3 Université Montpellier2, Sciences et Techniques du Languedoc, Montpellier, FRANCEJunonia coenia densovirus (JcDNV) is a parvovirus infecting lethallySpodoptera frugiperda (a lepidopteran pest) larvae. During ingestion ofcontaminated food, virus particles cross the midgut epithelium withoutreplicating to reach and replicate in the underlying trachea, epidermis thenhaemocytes [1]. In insect cells, JcDNV particles are internalized into clathrincoated vesicles and reach the nucleus using the endosomal pathway.Wortmannin inhibits PI3K activity and blocks JcDNV infection [2]. Thepresent study analyses the implication of PI3K/Akt pathway in JcDNVinfection. We used rapamycin and torin 1, two inhibitors of the kinaseTOR (target of rapamycin), a key downstream mediator of Akt that inhibitsautophagy and regulates protein synthesis. Our results show that bothdrugs significantly affect viral replication. We will focus on the role ofboth processes during infection. First, autophagy is a lysosomal degradationpathway, implicated in innate and adaptive immunity and oftensubverted by viruses. To assess the potential role of autophagy in JcDNVinfection, we will use RNA interference, targeting three Atg (autophagyrelated genes) proteins: Atg4, Atg6, Atg7 and the kinase Vps34. Second,the protein synthesis pathway is dependent on TOR modulating 4EBP andS6K activities. We will measure the level of protein phosphorylation alongthis pathway (TOR, S6K and rpS6) during infection. Expression levels ofS6K, 4EBP and TOR will be also evaluated.1. Mutuel D, et al.Virology 2010; 403: 137-44.2. Vendeville A, et al. J Virol 2009; 83: 4678-89.REF 030Mapping Rig I Like Receptors protein partners upon Chinkungunyavirus infectionRaul Yusef SANCHEZ DAVID 1 , Chantal COMBREDET 1 , PhilippeDESPRES 2 , Anastasia KOMAROVA 1 , Fréderic TANGY 11 Viral Genomics and Vaccination Unit, Pasteur Institut, Paris, FRANCE;2 Unité des Interactions moléculaires Flavivirus Hôtes, Pasteur Institut,Paris, FRANCEThe host innate immune response to Chikungunya virus is type I interferondependant. Today, our knowledge of the molecular players recognizing thisvirus during infection is still a matter of research. There are two knowndifferent mechanisms of recognition, one is IPS 1 (Interferon PromoterStimulator 1) dependent and the other is IPS 1 independent (Schilte et al,2012). Here, we studied the IPS 1 dependant pathway for Chikungunyavirus recognition via the cytosolic Rig I Like Receptors (RLR): Rig I,Mda5 and Lgp2. The role of these cytosolic receptors in Chikungunyavirus detection is currently controversial. To understand the mechanismsof action of RIG I, MDA5 and LGP2 during infection, we have establishedHEK293 cell lines that express these RLRs fused with a tag allowingaffinity complex purification. These cell lines were characterized for theircapacity to overexpress the respective tagged RLR’s and to produce typeI interferon upon in vitro transfection of synthetic RNA’s. Infection ofthese cells with Chikungunya virus was efficient and viral replication wasassessed. Tagged RLRs were purified by affinity chromatography frominfected cells lysates. The aim is now to map the protein partners of RLRsupon viral infection using mass spectrometry analysis. The results of thishigh throughput study will be presented.REF 031Requirements for the binding of human cytomegalovirus immediateearly protein 1 (IE1) to the cellular restriction factor PMLEva Maria SCHILLING 1 , Myriam SCHERER 1 , Victoria OTTO 1 ,Joachim STUMP 2 , Nina REUTER 1 , Heinrich STICHT 2 , ThomasSTAMMINGER 11 Institute for Clinical and Molecular Virology, University of ErlangenNürnberg, Erlangen, GERMANY; 2 Institute of Biochemistry, Universityof Erlangen Nürnberg, Erlangen, GERMANYNuclear domain 10 (ND10) components PML, Sp100, hDaxx and ATRXwere shown to act as cellular restriction factors constituting the first lineof innate antiviral defense against human cytomegalovirus (HCMV) byS126 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologysilencing IE gene expression. This ND10 mediated defense mechanism,however, is counteracted by the immediate early protein (IE1) of HCMV.During infection, IE1 targets ND10 by binding to PML. Subsequently, itinduces PML de SUMOylation and mediates disruption of ND10. However,the exact mode of PML binding and ND10 disruption remains unclear.The aim of our study was to clarify which motifs or regions of IE1 arerequired for binding to the cellular restriction factor PML. Since SUMOinteracting motifs (SIMs) are involved in protein protein interactions, weperformed bioinformatic analyses to identify putative SIMs in IE1. FourSIMs were detected which were subsequently mutated to determine theinfluence on PML binding and ND10 disruption. The mutations had nosignificant effect concerning the ability to disrupt ND10, suggesting thatthe interaction between IE1 and PML is not mediated by SIMs. In addition,we generated C terminal IE1 truncations to better define which region ofIE1 is crucial for PML interaction. Interestingly, the deletion of an alphahelical region of IE1 led to the complete loss of PML binding, therebyhighlighting the role of this domain for PML binding. Taken together,these findings provide new insights into structural requirements for theND10 antagonistic activity of IE1.REF 033Immune recognition of picornavirus RNAs and viral evasion strategiesFrank VAN KUPPEVELD, Qian FENG, Martijn LANGEREISDivision of Virology, Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine, Utrecht University, Utrecht, THENETHERLANDSRIG I and MDA5 are cytosolic RNA sensors that play critical roles ininnate antiviral responses. Major advances have been made in identifyingRIG I ligands, but our knowledge of the ligands for MDA5 remains restrictedto data from transfection experiments mostly using poly(I:C). Here, wedetermined the MDA5 stimulatory activity of various viral RNAs producedduring picornavirus infection (i.e., the ss virion RNA, which containsa VPg peptide at the 5 ′ end, the VPg lacking viral ssRNA, and two replicationintermediates – the perfectly duplexed replicative form (RF) andthe partially ds replicative intermediate (RI). Besides transfection studiesusing purified viral RNAs, we also studied RNA recognition during a normalinfection (using mengovirus Zn in which the known IFN antagonistis inactivated) by using inhibitors that interfere with specific steps of viralRNA replication. Our results show that viral ssRNAs (with or without5 ′ VPg) do not activate MDA5, whereas RF is a highly potent MDA5ligand. Moreover, picornaviruses actively suppress IFN a/ response togain replication advantage. While the IFN antagonist of Cardioviruses hasbeen identified, the strategy of Enteroviruses to interfere with IFN a/induction remains unclear. Previous studies demonstrated that the viralproteinase 3C cleaves crucial factors of the IFN a/ pathways. Our preliminarydata show a yet unappreciated role of another viral proteinase, 2A,in antagonizing MDA5 mediated IFN a/ induction.REF 034Study of the effect of CD40 ligand on the lytic cycle of Herpes SimplexVirus type 1 (HSV 1)Virginia Maria VLAHAVA 1 , Eftychia STAVRAKAKI 1 , AristeidisELIOPOULOS 1,2 , Georgios SOURVINOS 11 University of Crete, Medical School, Heraklion, GREECE; 2 Instituteof Molecular Biology and Biotechnology Foundation for Research andTechnology, Heraklion, GREECEPrevious studies have shown that CD40 ligand (CD40L) has the ability todirectly confer antiviral protection in cells expressing the CD40 receptor.On that ground, we sought to investigate how CD40L exerts its protectiveeffect on cells infected by HSV 1. For that reason, we generated a stablytransfected U2OS cell line that expresses the CD40 receptor. After testingthat the CD40 CD40L interaction does not lead to cell death, we wenton to investigate whether there is an effect on the production of progenyvirus following treatment with CD40L. We concluded that indeed thereis a reduction of progeny virus in the presence of CD40L. Growth curvesalso showed that the cells treated with CD40L produced lower viral yieldsduring the course of the infection. The antiviral effect was specific, asthe expression of a mutant CD40 receptor which lacks the ability to bindTRAFs, resulted in similar titers of progeny virus, regardless the presenceof CD40L. In order to examine whether the binding ability of HSV 1virions is compromised by the CD40 CD40L interaction, a binding assaywas performed and confocal microscopy verified the efficient attachmentof the virions. Subsequently, we monitored the distribution of the capsidprotein VP16 after treatment with CD40L and observed that nuclear entryof the virus is delayed. Additionally, replication of the virus was stalledin the presence of CD40L as this was measured by the DNA copies of theEarly ICP8 gene 2, 12 and 24 hpi. Our data, suggest that CD40L poses ablockade on HSV 1 from the very early stages of the infection.REF 035Human Coronavirus EMC/2012 accessory protein 4a functions as atype I interferon antagonistDaniela NIEMEYER 1 , Doreen MUTH 1 , Tasnim SULIMAN 1 , GaborHORVATH 2 , Ron A.M. FOUCHIER 3 , Friedemann WEBER 4 , ChristianDROSTEN 1 , Marcel A. MÜLLER 11 Institute of Virology, University of Bonn Medical Centre, Bonn, GER-MANY; 2 Institute of Innate Immunity, University of Bonn Medical Centre,Bonn, GERMANY; 3 Viroscience Lab, Erasmus MC, Rotterdam, THENETHERLANDS; 4 Institute for Virology, Philipps University Marburg,Marburg, GERMANYA novel human coronavirus, HCoV EMC, emerged recently in the MiddleEast causing severe respiratory tract infections similar to severe acute respiratorysyndrome (SARS). The interferon (IFN) system is a major part ofthe innate immunity controlling viral replication. It can be triggered uponrecognition of double stranded (ds)RNA by RIG I like helicases (RIG I andMDA5). Whereas several SARS CoV accessory proteins were shown toinhibit the IFN response, functions of HCoV EMC accessory proteins areunknown. In the presented study we focused on putative anti IFN mechanismsof the HCoV EMC accessory proteins p3, p4a, p4b and p5. Theaccessory genes were cloned into eukaryotic vectors for overexpressionanalyses in human and primate cells. In an IFN beta promoter reporterassay only p4a inhibited the activation of the IFN response. In confirmatoryassays p4a blocked the nuclear translocation of a co expressed GFPIRF 3 and the secretion of bioactive IFN bioassay. An in silico analysispredicted a dsRNA binding motif for p4a. In HCoV EMC infected cellsp4a co localized with viral dsRNA molecules suggesting direct interaction.An IFN beta promoter assay with targeted IFN stimulation at the levelof the RIG I helicases revealed that p4a inhibited MDA5 but not RIG Iactivation.In conclusion, p4a co localization with dsRNA and its inhibitory effecton MDA5 activation support the idea that p4a is a functional IFN antagonistinhibiting the IFN induction pathway. Direct interaction studiesbetween p4a, dsRNA and MDA5 will be necessary to unravel the detailedmechanism.Virologie, Vol 17, supplément 2, septembre 2013S127

5 th European Congress of VirologyREF 036Virion-associated Hepatitis B core (HBc) protein is a very early negativeregulator of the IFN responseMarion GRUFFAZ 1,2 , Barbara TESTONI 1,2 , SouphaloneLUANGSAY 1,2 , Floriane FUSIL 3 , Malika AITGOUGHOULTE 1,2 ,Jimmy MANCIP 3 , Marie Anne PETIT 1,2 , Hassan JAVANBAKHT 4 ,Klaus KLUMPP 4 , François-Loïc COSSET 3 , Fabien ZOULIM 1,2,4,5 ,David DURANTEL 1,21 INSERM U1052, Centre de Recherche en Cancérologie (CRCL), Lyon,FRANCE; 2 Université de Lyon, UMR S1052, CRCL, Lyon, FRANCE;3 INSERM U1111, Centre International de Recherche en Infectiologie(CIRI), Lyon, FRANCE; 4 Hoffmann-La Roche Inc, Nutley, USA;5 Hospices Civils de Lyon (HCL), Lyon, FRANCE; 6 Institut Universitairede France (IUF), Paris, FRANCEThe hepatitis B virus (HBV) does not induce any innate responses duringacute infection. This lack of induction could be due to a lack of detection byinnate sensors or result from an active evasion via the prompt establishmentof inhibitory mechanisms. To substantiate the latter our aim was to identifynew mechanisms of inhibition of the IFN response. Using a set of relevantcell culture models (HepaRG cell lines and primary human hepatocytes), aswell as HBV-infected liver humanized mice, we found that HBV is capableto inhibit dsRNA-mediated IFN response within minutes/hours after theonset of infection without the need of de-novo synthesis. Using engineeredcell lines, as well as transfection of purified nucleocapsids (NCs) or recombinantHBc, we demonstrate that HBc is necessary and sufficient for thisinhibition. HBc needs to be located in the nucleus to inhibit the transcriptionof targeted genes, as inhibition of its trafficking with anti-capsid drugs,revert the phenotype. ChIP-qPCR analyses revealed that HBc is capableto bind to target promoters and recruit epigenome-modifying enzymes toestablish repressive marks on these genes. Our results demonstrate thatHBc is responsible for the very early inhibition of IFN response by HBV.The precocity of this inhibition is instrumental for the establishment ofa persistent infection and is possible because virion-associated HBc iscapable, after cytosol trafficking of NCs and HBc nuclear translocation, toearly inhibit IFN gene expression by recruiting histone methyltransferasesleading to epigenetic repressive marks.S128 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology04. ADAPTIVE IMMUNITY ANDVACCINESPosters: REF 037 to REF 061REF 037Nanocomplexes as delivery system for vaccine preparationsMadina ABITAYEVA 1 , Pavel ALEXUYK 1 , AizhanTURMAGAMBETOVA 1 , Irina ZAITSEVA 1 , AndreyBOGOYAVLENSKIY 1 , Vladimir BEREZIN 1Institute of microbiology and virology, Almaty, KAZAKHSTANIn recent years to increase the efficiency subunit vaccines have startedthe development using the principles of nanotechnology. The inclusion ofadjuvants with different structures (liposomes, nanosomes, sferosomes,protein complexes, etc.) in compound of subunit vaccine can greatlyincrease the immunogenicity of antigens, balanced induce componentsof cellular and humoral immune response. The most perspective is use ofthe virus like nanoparticles derived from plant saponins, with low toxicityand high immunogenic activity. In this research, has been tasked to studythe immunogenic and protective activity at animal immunization of wholevirion inactivated virus in combination with the immunostimulatory nanocomplexesobtained on the basis of saponins extracted from the plants ofthe flora of Kazakhstan.To study the immunogenic and protective activity of received preparationanimals were immunized with different strains of inactivated influenzavirus in combination with immunostimulatory nanoparticles obtained onthe basis of plant saponins. Immunogenicity was evaluated by titer ofimmunoglobulins and interleukins in the blood serum one week aftersecond immunization. The protective activity of the preparations was evaluatedby the number of surviving animals after experimental infection.Studies have shown that immunization of mice with immunostimulatorynanocomplexes in the presence of viral antigen leads to the induction ofhumoral and cellular immune responses. Stimulation of the immune responseoccurs even with whole virione vaccines based on influenza virusesH5N1 and H1N1, derived by reverse genetics. Were established the highprotective activity of immunostimulating complex against virus infectionsin vaccinated animals of different ages, capable of vertical transmission.The need to improve immunogenicity of current influenza vaccines,especially in elderly people, have led to the licensure of two new “implementedvaccines” for people aged =65 years: Fluad (MF59 adjuvantedsubunit vaccine) and Intanza 15 g (split non adjuvanted intradermal vaccine).The aim of our study was to compare the immunogenicity of thetwo implemented vaccines commercially available for the Winter season2012/13 (A/California/7/09 (H1N1), A/Victoria/361/11 (H3N2) andB/Wisconsin/1/10). A total of 172 elderly (mean age 86 years) chronicallyill volunteers living in nursing homes located in Umbria, Italy, wererandomly assigned to receive one dose of either Fluad (N. 92) or Intanza(N. 80). Haemagglutination inhibiting (HI) antibody titers were determinedin blood samples collected before and 1 month after vaccination. Theresults obtained showed that both vaccines were able to induce increases inthe amount of HI antibodies against all the three vaccine strains, althoughthe three immunogenicity criteria of European Medicine Agency werenot always satisfied. Non inferiority of Intanza was demonstrated forall strains (ratios [Fluad/Intanza] of post vaccination Geometric MeanTiters were always

5 th European Congress of Virologyfirst expanded by DNA vaccination of the recipient mice. We characterizedspleen derived T lymphocyte cytokine responses to different peptides.The WNV structural proteins were expressed as a series of overlapping 20mer peptides fused to a carrier protein. Cytokine based ELISPOT assaysrevealed that peptides are recognized by T cells recovered directly ex vivofrom vaccinated animals.REF 041High hydrostatic pressure effect on the epitope mapping of the tobaccomosaic virus coat proteinDaniel FERREIRA DE LIMA NETO 1,2 , Clarice WEIS ARNS 1 , CarlosFRANCISCO SAMPAIO BONAFÉ 21 Instituto de Biologia/Departamento de Microbiologia/UniversidadeEstadual de Campinas, Campinas, BRASIL; 2 Instituto de Biologia/Departamentode Bioquímica/Universidade Estadual de Campinas,Campinas, BRASILThis study was aimed to investigate the effect of high hydrostatic pressureon Tobacco Mosaic Virus (TMV), a virus model for immunologyand one of the most studied virus to date. When subjected to treatmentwith pressures it was observed a significant change in the recognized epitopesin comparison to sera from immunized mice with the native formof the virus. These alterations were further studied by combining the highpressure treatment with urea or low temperatures and inoculation of thesealtered virions in Balb C mice, the collected sera titers were determined byELISA and cross referenced between the groups tested. The titter obtainedshowed that the antigenicity of viral particles was maintained after treatmentsusing monoclonal antibodies against the native form. The epitopeprediction algorithms could not infer the some observed changes in theepitope profile suggesting conformational changes in the protein structure.The antigenicity of canonical epitopes was maintained, although bindingintensities were different among the treatments used. Patterns of recognitionfrom the epitope mapping were then cross checked with the predictionalgorithms for the TMVcp amino acid sequence to infer which alterationsmight have occurred. Our findings suggest that different cleavages siteswere exposed after the treatments; this was verified via epitope mappingusing sera from mice immunized with the virus after the high pressurecondition was imposed.REF 042Surface Display of Foot and Mouth Disease Virus Recombinant Proteinin Escherichia coliSoheil HADJI KHODADAD 1 , Gholamreza AHMADIAN 1 , MehdiSHAMS ARA 1 , Mona KARBALAYI 2 , Sahar SEDDIGHZADEH 11 NIGEB, Department of Biotechnology, Tehran, IRAN; 2 Tarbiat ModarresUniversity, Department of Medical Bacteriology, Tehran, IRANEmergency vaccines designed to protect livestock against FMDV are quiteeffective; however, they do little to induce mucosal immunity. The absenceof mucosal immunity allows for the establishment of sub clinical infections,the generation of carrier animals, and ultimately in the emergenceof escape mutants. This project used synthetic peptides, administeredorally to induce secretory immunity in the gastrointestinal tract of cattle.Methods: Several different FMDV peptides containing the immunogenicregion of VP1 were expressed on the surface of E.coli. These peptidesthen administered orally. Systemic and mucosal antibody production wasevaluated. Results: The immunogenicity of the VP1 epitopes expressed inthe recombinant E.coli was tested by immunizing cattle. Ten days after thelast inoculation, animals were bled and the sera analyzed for the presenceof anti FMDV antibodies by ELISA and Western blot. The results indicatedthat all the sera from the VP1 immunized cattle presented a specificantibody response against FMDV VP1 antigen. The anti VP1 specificityof the antibody response in the sera of the vaccinated cattle was furtherconfirmed by Western blot using purified inactivated FMDV as antigen.Conclusions: These results demonstrated the possibility of using a noveland simple, methodology for obtaining high levels of foreign immunogenicepitopes which could be directly applied in the development ofveterinary medicine vaccine. Additional studies will be performed usingnovel synthetic peptides designed to block the attachment of the virus toGIT epithelial cells.REF 043Variation in the fine specificity of the human antibody response afternatural infection or vaccination with tick borne encephalitis virus(TBEV)Johanna JARMER 1 , Jürgen ZLATKOVIC 1 , GeorgiosTSOUCHNIKAS 1 , Judith H. ABERLE 1 , Vaclav CHMELIK 2 , KarinSTIASNY 1 , Franz X. HEINZ 11 Department of Virology/Medical University of Vienna, Vienna, AUSTRIA;2 Department of Infectious Diseases/Hospital Ceske Budejovice, CeskeBudejovice, CZECH REPUBLICIndividual specific variations in the fine specificity of polyclonal serumantibody (Ab) responses are incompletely understood aspects of humoralimmunity that can have a strong influence on functional activity. Weaddressed this issue in the context of TBEV, one of the major humanpathogenic flaviviruses, both after natural infection and vaccination withthe purified formalin inactivated whole virus vaccine. The envelopeprotein E is the major target of neutralizing Abs and contains 3 structuraldomains: DI, DII and DIII. We made use of the modular organization ofE and established immunoassays with recombinant proteins that allowedus the quantification of Abs to each of the domains of E, as well as prM(found in immature virions) and broadly flavivirus cross reactive epitopes.Both after infection and vaccination, extensive individual differenceswere observed in the composition of domain specific Abs. However, inboth groups the humoral response is dominated by Abs to DII, whereasthe more strongly neutralizing DIII reactive Abs are underrepresented.Post infection sera showed a significantly higher neutralization potentialand a stronger prM response, while those after vaccination containedmore broadly flavivirus cross reactive Abs. Our results suggest that serumAb fine specificity is not only subject to strong individual variation, butalso differs when the virus is presented to the immune system in thecourse of replication or in an inactivated form.REF 044Identification of linear human B cell epitopes of tick borne encephalitisvirusSuvi KUIVANEN 1 , Jussi HEPOJOKI 1 , Antti VAHERI 1,3 , OlliVAPALAHTI 1,2,31 Haartman Institute, Helsinki, FINLAND; 2 Department of Basic VeterinarySciences, Helsinki, FINLAND; 3 HUSLAB, Helsinki, FINLANDTick borne encephalitis (TBE) is a neurological disease transmitted tohumans by ticks. It is endemic in many European countries, Russia andChina. The causative agent, tick borne encephalitis virus (TBEV), belongsto the family Flaviviridae, a group of globally important arthropod borneviruses, such as dengue, Yellow fever, Japanese encephalitis and West Nilevirus. The flavivirus genome is approximately 11 kilobases long singlestranded positive sense RNA. The virion consists of the three structuralproteins (C, prM and E), the positive sense RNA genome and a lipid membranederived from the host cell. The seven nonstructural proteins (NS1,NS2A, NS2B, NS3, NS4A, NS4B and NS5) are found in the infected celland most of them function at least in genome replication. The structuralprotein E is known to raise protective neutralizing antibody responses. Ithas been shown with other flaviviruses that also NS1 can elicit protectiveimmune responses. Linear epitope mapping has been carried out with otherS130 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyflaviviruses targeting the glycoprotein part of the genome. In the presentstudy, our aim is to identify antigenic regions useful for assays to distinguishbetween vaccinations and natural infections. We synthesized TBEVgenome in 567 peptides onto amino PEG membrane. The membrane isprobed with sera of individuals with acute TBE patients, TBE vaccinationor old TBE immunity. Using the Pepspot assay, we have identifiednovel linear epitopes in the nonstructural region, particularly in NS2Band NS4B. These novel epitopes may further be developed to differentialdiagnostics.in a cohort of 12 years old girls screened for HPV memory B cells 1 6months after vaccination; 2) screen a cohort of Gardasil vaccinees thathave been vaccinated starting from year 2008 (12 years old girls versus25 45 years old women), to evaluate the long lasting response of memoryB cells 4 5 years after vaccination. In the cohort of Gardasil vaccinees weobserve a change in the number of specific memory B cells after 4 5 yearsfrom vaccination compared to the group tested 1 6 months after vaccination.Moreover preliminary data on the comparison between Cervarix andGardasil immunogenicity will be presented.REF 045Induction of cytolytic CD4+ T cell immune responses by ParvovirusB19 VP2 virus like particlesArun KUMAR 1 , Anu KANTELE 2,3 , Lea HEDMAN 1 , KlausHEDMAN 1,4 , Rauli FRANSSILA 11 Departments of Virology, Haartman Institute, University of Helsinki,Helsinki, FINLAND; 2 Department of Bacteriology and Immunology,Haartman Institute, University of Helsinki, Helsinki, FINLAND; 3 Divisionof Infectious Diseases, Helsinki University Central Hospital, Helsinki,FINLAND; 4 Helsinki University Central Hospital Laboratory Division,Helsinki, FINLANDBackground: A novel conception of CD4+ T cells with cytolytic potential(CD4+ CTL) is emerging. These cells appear to play a part in controllingmalignancies and chronic infections. Human parvovirus B19 can cause apersistent infection, yet no data have been reported that would show thepresence of B19 specific CD4+ CTLs. Such cells could have a role in thepathogenesis of autoimmune disorders and in some thyroid malignanciesassociated with B19.Methods: The cytolytic potential of human parvovirus B19 specific T cellswas explored by stimulating PBMC with recombinant B19 VP2 virus likeparticles. Cytolytic potentintial was evaluated by EIA based quantitationof Granzyme B (GrB) from tissue culture supernatants.Results: GrB responses with the B19 antigen were readily detectable inB19 seropositive individuals. The responses proved stronger than thosewith the HBoV control antigens. T cell depletion and HLA blocking experimentsshowed GrB to be secreted by CD4+ T cells. Interestingly, someB19 seronegative subjects also had GrB responses with B19 VLPs.Conclusions: A vigorous B19 specific GrB response was found in seropositiveindividuals which suggests a role for CD4+ CTL also in B19immunity. Such cells could function within, immune regulation and in thetriggering off autoimmune phenomena such as SLE or rheumatoid arthritis.REF 046Evaluation of B Cell immunity after HPV vaccinationBarbara MANTELLI 1 , Valentina TELATIN 1 , Laura SQUARZON 1 ,Tiziana LAZZAROTTO 2 , Liliana GABRIELLI 2 , LorenaGOTTARDELLO 3 , Ivana SIMONCELLO 3 , Luisa BARZON 1 , GiorgioPALÙ 1 , Antonella CAPUTO 11 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Policlinico S. Orsola Malpighi, Bologna, ITALY; 3 Department of PublicHealth, Azienda Ospedaliera di Padova, Padua, ITALYCervical cancer is one of the most common malignancies in womenworld wide and more than 95% of all cervical tumors are associated withhuman papillomavirus (HPV) infection. To date, two prophylactic vaccinesagainst HPV infections are available: a bivalent HPV16/18 L1 VLPvaccine (Cervarix) and a tetravalent HPV 6/11/16/18 L1 VLP vaccine(Gardasil). Both vaccines are safe, highly immunogenic and protective;nevertheless, several key issues are still open regarding the duration ofmemory B cell responses after HPV vaccination. We designed an independentstudy to 1) compare the immunogenicity of the two vaccinesREF 047A Novel Vaccine Strategy is Effective Against Congenital Cytomegalovirusin a Pre Clinical Animal ModelAlistair MCGREGOR 1 , Matthew ROOT 2 , Yeon CHOI 11 Texas A&M Health Science Center College of Medicine, College Station,USA; 2 University of Minnesota, Minneapolis, USACongenital CMV is a leading cause of mental retardation and deafness.The guinea pig is the only small animal model for congenital CMV. Weinvestigated a disabled infectious single cycle (DISC) vaccine strategy,where the virus lacks the ability to express an essential gene, except whengrown on a complementing cell line. The minor capsid gene (UL85) wasselected as it is essential for virus capsid assembly. A series of guinea pigCMV (GPCMV) BAC mutants were generated. In the DISC strain, theGP85 late promoter was removed and a tet off promoter and an upstreamSV40 polyA cassette were substituted to place GP85 expression understrict control of a tet off system. Transfection of mutant BACs onto tet offguinea pig fibroblast cells only resulted in virus from the mutant containingthe tet off promoter (vDISCGP85). The DISC vaccine immune responsewas investigated. Animals (n=6) induced an anti GPCMV antibody response(ELISA titer >1:1260) and a cell mediated response to the pp65 andIE1 homologs. In a congenital protection study, female guinea pigs (n=15)were vaccinated and mated. At late second trimester of pregnancy animalswere challenged (10 5 pfu wild type GPCMV). A control group, non vaccinatedpregnant animals (n=15) were similarly challenged. Animals wentto term and viral load in target organs of pups was analyzed. Based onlive pup numbers in the vaccinated and non vaccinated groups (94.4%vs 63.6%) the vaccine strategy was successful (P=0.002). Pups from thevaccinated group had zero or reduced viral load in target organs comparedto control pups. The vaccine failed to prevent brain infection in all pups(11% vs 50% in control). However, the outcome was statistically significant.The merits of modifying the vaccine to include neutralizing antibodytargets to a novel endocytic complex for virus infection of epi/endothelialcells will be discussed.REF 048Unusual features of vaccinia virus Extracellular Virion form (EV)neutralization resistance revealed in human antibody responses to thesmallpox vaccineMohammed RAFII EL IDRISSI BENHNIA 1 , Lilia KORIAZOVA 3 ,Shinichiro KATO 3 , Philip L. FELGNER 6 , Dirk ZAJONC 2 , Dennis R.BURTON 4 , Yan XIANG 6 , James E. CROWE JR 5 , Bjoern PETERS 1 ,Shane CROTTY 11 Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology(LIAI), LA JOLLA, USA; 2 Division of Cell Biology, LIAI, LA JOLLA,USA; 3 Kiowa Hakko Kirin California, LA JOLLA, USA; 4 Departmentof Immunology and Microbial Science and IAVI Neutralizing AntibodyCenter, LA JOLLA, USA; 5 Vanderbilt Vaccine Center, University MedicalCenter, Nashville, USA; 6 Division of Infectious Diseases, Department ofMedicine, University of California, Irvine, USA; 7 Department of Microbiologyand Immunology, University of Texas Health Science Center, SanAntonio, USAVirologie, Vol 17, supplément 2, septembre 2013S131

5 th European Congress of VirologyThe EV of vaccinia virus (VACV) is essential for viral pathogenesis andis difficult to neutralize with antibodies (Abs). We previously showed thathigh concentrations of anti B5 Abs are insufficient on their own to directlyneutralize VACV EV. This allowed for at least two possible interpretations:covering the virion surface is insufficient for neutralization, or there areinsufficient copies of B5 to allow anti B5 IgG to fully cover the surface ofthe virion and another viral receptor protein remains active. We endeavoredto test these possibilities, focusing on the Ab responses elicited immunizationagainst smallpox. We tested whether human monoclonal antibodies(MAbs) against the three major EV antigens, B5+A33+A56 could individuallyor together neutralize EV. While anti B5 or anti A33 (but notanti A56) MAbs of appropriate isotypes were capable of neutralizing EVin the presence of complement, a mixture of anti B5, anti A33, and antiA56 MAbs was incapable of directly neutralizing EV. This remained truewhen neutralizing the IHD J strain, which lacks a functional version ofthe fourth and final known EV surface protein, A34. These immunologicaldata are consistent with viral proteins not being the active component ofthe EV surface for target cell binding and infectivity. We conclude thatthe EV virion evades direct neutralization by human Ab responses to thesmallpox vaccine, and the protection afforded by the smallpox vaccineanti EV response is predominantly mediated not by direct neutralizationbut by isotype dependent effector functions.REF 049Kinetics of antibody response in heifers vaccinated with inactivatedgE negative BHV 1 marker vaccine by enzyme linked immunosorbentassayAtilla SIMSEK 2 , Orhan YAPICI 1 , Oguzhan AVCI 2 , Oya BULUT 2 ,Kamil ATLI 2 , Irmak DIK 2 , Sibel YAVRU 21 Kyrgzstan Turkey Manas University, School of Veterinary Science,Department of Virology, Bishkek, KYRGYZSTAN; 2 Selçuk University,School of Veterinary Science, Department of Virology, Konya, TURKEYIn countries with high prevalence of infection, the control of Bovine Herpesvirustype 1 (BHV 1) is associated with the vaccination of cattle withglycoprotein E (gE) negative marker vaccines. These marker vaccines,either inactivated or live attenuated, are deleted in the gene coding for thenon essential gE of BHV 1 in order to allow serological differentiation betweenvaccinated and infected cattle. The aim of the study was the assessmentof rise and persistence of antibodies to BHV 1 after two step vaccinationmarker inactivated vaccine with an interval of 21 days. Five heiferswere serologically surveyed regularly for one year period after vaccination.The kinetic of immunoglobulin in serum samples from heifers were monitoredby enzyme linked immunosorbent assay (ELISA). BHV 1 antibodiesin serum samples were first detected on average at day 8 after first vaccination.Maximum titres were obtained eight weeks after first vaccination.REF 050Monitoring HPV type specific neutralizing antibodies induced by aprophylactic quadrivalent human papillomavirus types 6/11/16/18 l1virus like particle vaccineLaura SQUARZON 1 , Monia PACENTI 2 , Serena MASIERO 1 , GiorgiaMARCATI 2 , Barbara MANTELLI 1 , Lorena GOTTARDELLO 3 ,Antonella CAPUTO 1,2 , Luisa BARZON 1,2 , Giorgio PALÙ 1,21 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Clinical Microbiology and Virology, Padova University Hospital,Padova, ITALY; 3 Department of Hygiene and Public Health, ULSS16, Padova, ITALYVaccination programs with prophylactic human papillomavirus (HPV)vaccines are being widely implemented, targeting adolescent girls prior tosexual debut. Since the risk of HPV exposure persists throughout sexuallife, the duration of protection is critical to vaccine effectiveness. Aim ofthis independent study was to evaluate vaccine induced neutralizing antibodies(NAbs) in girls and young women vaccinated with the prophylacticquadrivalent HPV types 6/11/16/18 vaccine Gardasil ® (Merck) at differenttime points from the last vaccine dose. The study population included agroup of 200 females who attended vaccination centers of the Departmentof Public Health in Padova, Italy. NAbs specific for HPV16/18/6/11 weremeasured at 1 to 6 months, 2 years, 3 years and 4 years after completionof the third dose of vaccine by using pseudovirion based neutralizationassays (PBNAs). The presence of NAbs was defined by a titer of 1:40or higher, according to WHO guidelines. Seropositivity rates were 100%for HPV16 at all time points after vaccination, while 98.4% for HPV18at 1 6 months and 80% at 4 years after vaccination. Seropositivity rateswere lower (92%) for HPV6 and HPV11 at 1 6 months and declined withtime (80% and 60% at 4 years, respectively). Peak geometric mean titers(GMT) of HPV16 NAb was significantly higher than HPV18/6/11 NAbsand in adolescents than in young women. A significant decline of NAbtiters for all four HPV types was observed over time. Monitoring NAbsmight be useful to define the duration of protection and immunologicalcorrelates of protection.REF 051Equal protection against influenza viruses A/H5N1 and A/H1N1 inducedby flagellin delivering conserved consensus human and A/H5N1M2eLudmila STEPANOVA 1 , Anna KOVALEVA 1 , Marina POTAPCHUK 1 ,Alexander KOROTKOV 1 , Roman KOTLYAROV 2 , Nikolai RAVIN 2 ,Ludmila TSYBALOVA 1 , Oleg KISELEV 11 Research Institute of Influenza, St. Petersburg, RUSSIA; 2 Center ’Bioengineering’Russian Academy of Science, Moscow, RUSSIAWe developed a recombinant fusion protein (Flg 2M2eh2M2ek) containing2 consensus sequences of M2e human influenza A (M2eh) and 2copies of M2e/Chicken/Kurgan/5/05 NS R.G. 5:3 H5N1 (M2ek) linkedto the C terminal end of full length flagellin. Balb/c mice were immunizedintranasal 3 times at 2 weeks intervals with 7,5 g M2e. Antibodytiters (IgG, IgG1, IgG2a) in serum and sIgA, IgG in bronchoalveolarlavage (BAL) against synthetic M2e (M2eh, M2ek) were determined byELISA. Sera of immunized mice were tested for reactivity with MDCKcells infected by influenza A in MDCK whole cell ELISA assay. Weestimated the proliferative activity and intracellular cytokine staining inspleen lymphocytes of immunized mice after activation of M2ek. Immunizedmice were challenged with 5 LD50 adapted to mice A/PR/8/34(H1N1) and A/Chicken/Kurgan/5/05 NS R.G. 5:3 (H5N1). Immunizationwith Flg 2M2eh2M2ek induced high titers of IgG in serum and BAL,sIgA in BAL and titers against M2eh and M2ek were practically equal.Intranasal administration of Flg 2M2eh2M2ek lead to dominating IgG1response and formation of specific Tx2 memory cells producing IL 4.It was shown that sera of immunized mice bound specifically to PR8infected and A/Chicken/Kurgan infected MDCK cells, indicating that antiM2e antibodies recognize native M2e on the surface of influenza infectedcells. Mice were protected from lethal challenge of A/PR8/34 (70%survival) and Chicken/Kurgan/5/05 NS R.G. 5:3 (H5N1) (90% survival).Our results demonstrate the possibility of developing candidate influenzavaccine protecting as against human so avian influenza viruses.REF 052Improvement of Immunogenicity of Influenza Virus M2e ProteinLiudmila TSYBALOVA 1 , Liudmila STEPANOVA 1 , VictorKUPRIYANOV 2 , Marina POTAPCHUK 1 , Alexander KOROTKOV 1 ,Nikolay RAVIN 21 Research Institute of Influenza, Saint Petersburg, RUSSIA; 2 Centtre‘Bioengineering’ Russian Academy of Science, Moscow, RUSSIAS132 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyEctodomain of matrix 2 protein (M2e) of influenza virus forms part ofmany vaccines (currently under development) with widespread protectionagainst various influenza A strains. The need to increase the immunogenicityof this protein is faced by the developers. This study is noted forenhanced design of recombinant protein with M2e and HBc (core antigenof hepatitis B) both included in it. Authors demonstrate that M2e insertionin immunodominant loop region (at the top of surface “spikes” on HBc particle)gives rise to immunogenicity, as compared to proteins where M2e isconnected to N termination of HBc particle. The immune response furtherincreases with the number of M2e copies at the insertion point. Level of IgGin blood determined by ELISA after twofold boost immunization of micewith 20 g of M2e HBc with one copy of M2e in each loop was 1:134000,while in case of four copies the level was 1:532480 (p

5 th European Congress of VirologyREF 056Evaluation of “In Line” tools for viruses characterization during Vaccineproduction processMarc FIORUCCI 1 , Alexandra BEAUFRÈRE 1 , Cyril PAYA 1 , SteveCRUSSARD 1 , Blandine DE SAINT VIS 1 , Jean REYES 1 , ChristopheGAILLARD 1Merial, Lyon, FRANCEMonitoring the process and optimizing production yields of viruses isa key step for vaccine industry. As a consequence a characterization ofviruses in a real time manner would be an advantage for manufacturers.In this study, three different technics have been evaluated allowing a rapidcharacterization of viruses: 2 virus counters (Flow cytometry and qViro)and a molecular amplification test (qPCR). This study shows that flowcytometry (FCM), with a specific staining, is able to qualitatively characterizeviral particles but relation between FCM titers and infectious titeris not obvious. qViro, is a fast and nonspecific technic based on coulterprinciple allowing a direct counting of viral particle. Although, qVirowas easy to setup and handle, relation between qViro titer and infectioustiter has not been yet demonstrated probably due to the complexity of thesample. Finally, qPCR has been evaluated and a strong relation is shownwith infectious titer. Interestingly qPCR titers were not impacted by thenature of the sample allowing the use of this test throughout the productionprocess.In conclusion, this study describes the ability of new tools to characterizeviruses particles in a real time manner. A bioequivalence has been shownbetween qPCR titre and infectious titer whereas viral counters are moreadapted for qualitative characterization of virus particles.REF 057Obtaining recombinant properly folded neuraminidase NA ofInfluenza Virus using a novel system for stable, high level expressionfrom the T7 promoter in Escherichia coli cellsAgnieszka ROMANIK 1 , Malgorzata KESIK BRODACKA 1 , ViolettaSACZYNSKA 1 , Violetta CECUDA ADAMCZEWSKA 1 , KatarzynaFLORYS 1 , Boguslaw SZEWCZYK 2 , Grazyna PLUCIENNICZAK 1 ,Andrzej PLUCIENNICZAK 11 The Institute of Biotechnology and Antibiotics, Warsaw, POLAND;2 University of Gdansk, Gdansk, POLANDNeuraminidase NA is a second immunodominant glikoprotein of InfluenzaVirus. NA is an enzyme that removes sialic acids from the surface of thecells, hence newly formed virions can be released from infected cells.Production of the recombinant NA (rNA) protein is crucial in developingnew influenza subunit vaccines or as an antigen in NA antibody testsaccording to DIVA (Differentiating Infected from Aaccinated Animals)strategy. Recombinant NA is produced mainly in insect or mammaliancells. Using bacterial expression system to produce this protein is not incommon usage. Main problem in using the prokaryotic expression systemis obtaining of stable recombinant protein with high level of expression.Here we report obtaining of the recombinant NA of avian H5N1Influenza Virus in Escherichia coli cells using a new system for stableexpression of protein from T7 promoter. In order to provide expressionin bacterial cells we deleted first 33 amino acid of NA protein (wholetransmembrane domain) and added six histidine to N terminus of theprotein. The recombinant protein was expressed in bacterial cells as inclusionbodies. We purified and renaturated protein on column packed withNiNTA resin. We received pure, properly folded rNA, which was confirmedby SDS PAGE and ELISA test. rNA protein obtained with describedmethod can be directly used in immunology test and vaccines for influenzavirus.REF 058AIDS vaccine Thai RV 144 correlate of protection: Envelope gp120V2 loop, which induces protective neutralizing IgG antibodies, is amarine Conus mu conotoxin binding to the voltage gated Na+ sodiumchannelGuy Mong Ky TRAN 1,4 , Adrien CAPRANI 2,31 Retired, ex Public Health resident, ex European AIDS ClinicalSociety (EACS) Scientific Council member, Chatenay Malabry, FRANCE;2 Association Positifs, Paris, FRANCE; 3 CNRS Jussieu, Paris, FRANCE;4 Association Positifs.org, Scientific Advisor, Paris, FRANCEThai RV 144 vaccine efficacy is 31,2%; protective IgG target the gp120V1 V2 loops. We analyse the V2 loop (Zolla Pazner S, 2013) by aminoacid (AA) sequences comparison by BLASTP with visual search and threedimensional (3D) structure (Xue T, 2003; Pallaghy PK, 1997). The 2 Thaivaccine strains V2 loops were screened on toxins binding to the voltagegated Na+channel (NaCh). Result: 1) 3 mu conotoxin active site AAs(K13, Q14, K16) (Conus Geographicus, Kinoshitai, Striatus, Betulinuschimera) (Ekberg J, 2008) are found in the Thai V2 loop (V172 crucial):Thai V2 166 RDKKQ KVHALFY R 178conotoxin 10 RDKKQCKVHALCCGR 242) The vaccine MN strain V2 loop mimics the scorpion toxin N terminusactive site (Kharrat R, 1989); its deletion abolishes the toxicity (El AyebM, 1986). Interestingly antibodies against N terminus induce broad crossreactive protection (Devaux C, 1999). The toxin precursor (Cn7, AaH, BotIX chimera) (Possani LD, 2000) is included.MN strain 157 CSFQMTGLEDKVKKEYALLYK 178Scorpion 13 CLFMTGVEAEIKVKKEGYALQYK 103) V2/V3 loops of HIV 2/SIV PBJ14 (fatal AIDS) were 3D superimposedon spider atratoxin (Atx)/versustoxin, 2 NaCh ligands. V2YxxxWYxxDxxC is conserved in HIV 2. V3 is SGLVFH: Atx 4 KR MKY AWYNQQ C TGLFKKC 42HIV 2 KK MK Y AWY QD C SGLVFHThe scorpion venom concept of AIDS (Tran GMK,1989) is confirmed bythe homology between the Thai V2 loop and mu conotoxin, a NaCh ligand.Omega 3, a NaCh modifier, is efficient in AIDS (Caprani A, 2012). AIDSvaccine should target V2/V3, avoid mitigating IgA (Haynes BF, 2012) andadd Mannose vaccine. ACK: No.REF 059Thai AIDS vaccine RV 144: Molecular homology between HIV 1 gp120 envelope first conserved region C1, which induces IgA antibodiesincreasing AIDS risk, and the complement receptor CR1Guy Mong Ky TRAN 1,4 , Adrien CAPRANI 2,31 Retired, ex Public Health resident, ex European AIDS Clinical Society(EACS) Scientific Council, 31 Av du Bois, 92290 Chatenay Malabry,FRANCE; 2 CNRS Jussieu, Paris, FRANCE; 3 Association Positifs, Paris,FRANCE; 4 Association Positifs, Scientific Advisor, Paris, FRANCEThai RV 144 vaccine, despite gp120 V1/V2 loops neutralizing IgG, has alow 31% efficacy, mitigated by IgA antibodies against the 1st conservedregion C1 (odds ratio 3.15) (Haynes BF, 2012). We analyse the biologicalsignificance of C1 by BLASTP and visual amino acid (AA) sequencescomparison: We screened C1 of the vaccine 2 Thai strains and all HIV1 strains (Los Alamos, Kuiken C, 2012) on Homo Sapiens and founda molecular homology between C1 (112 122) and complement receptorCR1 (2089 2099):C1 WDQSLKPCVKL, C1 WGPKLKPCVKL (strain37 cpx) (Powell RL, 2007), CR1 WGPKL(H,P)CSRV.The tip of the CR1 loop (Proline P2095) is in retro inverso: 2096 (H,P)2095, instead of 2095 PH 2096. Such tip inversion exists: EpidermalGrowth Factor (EGF) and Transforming Growth Factor alpha (TGF a) bindS134 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyto the same EGF Receptor, despite different tip linear sequence, but in 3Dhave superimposed loop tip AAs (E and V):Rat EGF 24 ESV 26TGF a 26(E,Q,V) 24 Anti C1 IgA harmfulness is explained by Wagner C (2006):Anti CR1 antibodies profoundly inhibit T cell proliferation by inhibitinginterleukin 2 (IL 2)/interferon g synthesis and IL 2 efficacy, explainingIL 2 clinical trials failure despite T4 cell rise (Pett SL, 2010). CR1 isdecreased in lung tuberculosis (Senbagavalli P, 2008). It acts on macrophagesand dendritic cells. Conclusion: An AIDS vaccine must avoid antiC1 IgA antibodies, because C1 is a molecular mimetic of CR1 and thereforethe cross reactive anti C1 IgA are anti CR1 auto antibodies whichprofoundly inhibit T cell proliferation. They could promote tuberculosis.ACK: No.REF 060Construction of potencial melanoma vaccines using chimeric mousepolyomavirus virus-like particlesMartina KOJZAROVA, Alena MORAVKOVA, Martin FRAIBERK,Jitka FORSTOVACharles University in Prague, Faculty of Science, Prague, CZECH REPU-BLICPolyomaviruses are small dsDNA viruses with icosahedral capsids composedof three structural proteins. The major capsid protein, VP1, is ableto self-assemble into virus-like particles (VLPs) not only in presence butalso in absence of the minor structural proteins, VP2 and VP3. Using differentapproaches, sequence of VP1 can be modified, so that the resultingprotein and subsequent VLPs carry one or more foreign epitopes. Whilepapilloma vaccines based on VLP technology are broadly used, applicationof chimeric VLPs based on polyomaviruses is fully open for utilizationin vaccination, immunotherapy or foreign molecule delivery. In our laboratory,three types of chimeric mouse polyomavirus (MPyV) VLPs wererecently prepared, carrying epitopes derived from i) human or ii) mousevariants of melanoma marker protein Melan-A/MART-1 (VLPs-Melan-A/MART-1) or iii) control epitope of chicken ovalbumin (OVA-VLPs).In all three cases, we obtained structurally stable VLPs correspondingin shape to wild-type ones. All tested chimeric VLPs successfully enterinto mouse cells. Evaluation of the immune response of mice immunizedwith the two types VLPs-Melan-A/MART-1 and control VLPs-OVAshowed considerable potential of chimeric VLPs in induction of humoralresponse not only against the carrier protein but also against the carriedepitope.REF 061Recombinant vaccines against Porcine Circovirus 2 based on MousePolymavirus structuresMartin FRAIBERK, Martina KOJZAROVA, Alena MORAVKOVA,Jitka FORSTOVACharles University in Prague, Faculty of Science, PRAGUE, CZECHREPUBLICPorcine circovirus type2 (PCV2) is a member of Circoviridae family thatinvades pigs. It is a small non-enveloped virus with the ssDNA circulargenome. The PCV2 particles, 16 -18 nm in diameter display icosahedralsymmetry and are composed of one capsid protein (Cap) only. PCV2is a causative agent of Postweaning Multisystemic Wasting Syndrome(PMWS) which causes significant economic waste in swine breeding.The aim of this study is to develop a recombinant vaccine against PCV2.Although the PCV2 cap protein is able to form virus like particle (VLPs),their production is insufficient in baculovirus production system. Therefore,the structures of the major capsid protein, VP1, of the MousePolyomavirus (MPyV) has been used as a carrier for exposure of PCV2 -cap epitopes. MPyV VP1 protein is able to self assemble into VLPs or alsointo pentamers when its C-terminus is shortened or fused with additionalsequence. We employed a strategy based on insertion of selected immunogenicepitopes of PCV2 on the surface of MPyV VP1 VLPs or insertionof entire Cap protein inside the MPyV VP1 VLPs. Another strategy usedis to prepare giant chimeric pentamers from MPyV VP1 connected in itsC- terminus with Cap protein sequences.Virologie, Vol 17, supplément 2, septembre 2013S135

5 th European Congress of Virology05. VIRAL (IMMUNO)PATHOGENESISPosters: REF 062 to REF 076REF 062Japanese encephalitis virus modulates the cross talk between dendriticcells and T helper cells: A mechanism of immune evasionNimesh GUPTA 1,2,3 , Pushpa HEGDE 1,4 , Sudhanshu VRATI 5,6 ,Jagadeesh BAYRY 1,2,3 , Sebastien LACROIX DESMAZES 1,2,3 , SriniKAVERI 1,2,31 Institut National de la Santé et de la Recherche Médicale, Unité872, Paris, FRANCE; 2 Centre de Recherche des Cordeliers, Equipe16 Immunopathology and therapeutic immunointervention, UniversitéPierre et Marie Cu, Paris, FRANCE; 3 Université Paris Descartes,Paris, FRANCE; 4 Université de Technologie de Compiègne, Compiègne,FRANCE; 5 Vaccine and Infectious Disease Research Centre, TranslationalHealth Science and Technology Institute, Gurgaon, INDIA; 6 NationalInstitute of Immunology, Aruna Asaf Ali Marg, New Delhi, INDIAThe mechanisms underlying Japanese encephalitis virus (JEV) pathogenesisneed to be explored to delineate appropriate therapeutic approaches.It is believed that JEV manipulates the innate and adaptive compartmentsof the host’s immune system to evade immune response and cross theblood brain barrier. The present study was designed to explore the functionalmodulations of dendritic cells after exposure to JEV and to assessthe consequences on CD4+ T lymphocyte proliferation and polarization.Human monocyte derived dendritic cells (DCs) were either infected with1 MOI of virus or mock infected. JEV induced a significant increase in theexpression of maturation markers 48 hours post infection, along with thatof the PD L1 ligand. Importantly, the JEV EDIII domain alone reproducedthe increased expression of PD L1 by DCs. JEV infected DCs induced thegeneration of regulatory T cells (Tregs) in allogenic mixed lymphocytereactions. The induction of Tregs by JEV infected DCs was significantlyreduced upon blocking PD L1 using an antagonist. In addition, JEV infectedDCs significantly altered the proliferation and reduced the polarizationof T helper cells towards the Th1 phenotype. The results, for the first time,suggest that JEV evades the host’s immune system by conferring tolerogenicproperties to DCs, thus inducing Tregs and reducing, via PD L1 axis,the polarization of T helper cells towards the Th1 phenotype. Immuneevasion by JEV implicates the EDIII domain of JEV envelope.REF 063Exogenous Nef Induces Proinflammatory Signaling Events in MurineMacrophages and Microglial CellsElisabetta AFFABRIS 1 , Giorgio MANGINO 1,2 , MarylindaFAMIGLIETTI 1 , Valeria SERRA 1 , Caterina CAPONE 1 , ZulemaAntonia PERCARIO 1 , Stefano LEONE 1 , Gianna FIORUCCI 3 ,Sebastian LULF 4 , Giovanna ROMEO 2 , Cristina AGRESTI 5 , TizianaPERSICHINI 1 , Matthias GEYER 6,41 Department of Science, University Roma Tre, Rome, ITALY; 2 Departmentof Medico Surgical Sciences and Biotechnologies, University La Sapienza,Rome, ITALY; 3 Institut of Molecular Biology and Pathology, CNR,Rome, ITALY; 4 MPI für Molekulare Physiologie, Abteilung PhysikalischeBiochemie, Dortmund, GERMANY; 5 Department of Cell Biology and Neuroscience,Ist. Superiore di Sanità, Rome, ITALY; 6 Center of AdvancedEuropean Studies and Research, Group Physical Biochemistry, Bonn,GERMANYMurine cells are not permissive for Human Immunodeficiency Virus 1(HIV 1), but transgenic mice expressing selected HIV 1 genes revealedthat Nef is a major disease determinant. Nef is a molecular adapter thatinteracts with several cellular partners inducing regulation of cell surfacereceptors expression and activation signals. In hu macrophages Nefexpression induces the production of extracellular factors that recruit T cellfavouring their infection. In addition Nef induces its transfer to uninfectedcells via exosomes, cellular protusions or cell to cell contacts and Nef treatmentof human monocyte derived macrophages induces internalization ofthe protein and production of proinflammatory molecules. Recently wehave treated murine macrophages and microglial cells with myristoylatedNefSF2. Like hu cells murine macrophages responded to Nef treatmentactivating IKK and specific MAPKs. Activation of NF kB is mandatoryfor producing inflammatory molecules, that induce tyrosine phosphorylationof the signal transducer and activator of transcription (STAT) 1, 2,and 3. Therefore murine macrophages respond to Nef similarly to humanones. Nef treatment of murine microglial cells leads to nitric oxide productionand upregulates the expression of inducible nitric oxide synthase(iNOS). Nef regulates iNOS expression through NF KB activation andIFN release. Both myristoylation and a highly conserved acidic clusterof the viral protein are required for all those effects. Finally we report thepresence of neurotoxic factors in the supernatants of Nef treated microglia.REF 064HLA G expression is increased in mice infected with influenza A H1N1pdm09 virusBlanca Lilia BARRÓN 1 , Adriana RODRÍGUEZ 1,2 , Ma. EugeniaMANJARREZ 21 Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas,Instituto Politécnico Nacional; 2 Instituto Nacional de EnfermedadesRespiratorias, Secretaria de SaludIt is known that influenza A viruses have developed several strategies forsubverting host defenses, facilitating their spread; such as, inhibition ofthe differentiation of monocytes into dendritic cells, the cytotoxic activityof natural killer cells as well as the interferon pathway. Another strategyused by viruses to escape from immunosurveillance is downregulation ofclassical HLA molecules; but also, it was demonstrated that there is an upregulation of non classical immunotolerant molecules such as HLA G inseveral viral infections. HLA G is a non classical HLA class I moleculethat have immunotolerance properties, and Qa 2 is the murine homolog ofHLA G. We analyzed the expression of this molecule by immunohistochemistryin trachea and lung tissue samples from Balb/c mice infected withinfluenza A virus A H1N1 pdm09. We also, determined the TH1 (IFN and IL 2) and TH2 (IL 4 and IL 10) cytokines profile in bronchial alveolarlavages. Compared to mock infected animals, the HLA G was present inthe lung and trachea tissues from infected mice, 3 days after infection (dai),reaching its highest expression 6 dai. Besides that, we found a reduction inthe TH1/TH2 cytokine expression, during the infection, and an importantcellular infiltrate was observed in the lung tissues. These results showedthat influenza A H1N1 pdm09 virus infection in Balb/c mice increasedthe expression of HLA G molecules in the lung and trachea tissues, witha reduction in TH1/TH2 cytokines expression.REF 065Interstitial lung disease in horses infected by EIAV (Equine InfectiousAnemia Virus)Caroline LEROUX 3 , Pompei BOLFA 1 , Marie NOLF 2,3 , Jean LucCADORÉ 2,3 , Christine DOLMAZON 3 , Cornel CATOI 1 , Jean FrançoisMORNEX 3,41 Pathology Department, University of Agricultural Sciences and VeterinaryMedicine, Faculty of Veterinary Medicine, Cluj Napoca, ROMANIA;2 Internal Medicine Department, VetAgro Sup, Marcy L’étoile, FRANCE;3 UMR754 INRA University Lyon 1, Retrovirus and Comparative Pathology,Lyon, FRANCE; 4 Hôpital Louis Pradel, Hospices Civils de Lyon,Lyon, FRANCES136 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyAlthough restricted to members of the Equidae, Equine Infectious AnemiaVirus (EIAV) has an almost worldwide distribution. EIAV belongs to theRetroviridae family, from the lentivirus genus related to HIV in humans;the induced EIA (Equine Infectious Anemia) is one of the eleven notifiableequine diseases listed by World Animal Health Organization (OIE).EIA is widely considered a blood borne disease primarily transmitted byhaematophagous insects or iatrogenically. Other routes of transmissionhave not been explored although there is evidence that EIA was spreadvia aerosolized particles during the 2006 EIAV outbreak in Ireland. Weexplored the potential of EIAV to induce pulmonary lesions in naturallyinfected equids. Lungs from 77 EIAV seropositive horses have been collectedin Romania in the context of the EIAV wide range National surveyamong farm horses. Three types of lesions have been scored on paraffinembedded lungs: lymphocyte infiltration, (peri)bronchiolar inflammation,and thickness of the alveolar septa. Expression of the p26 EIAV capsid(CA) protein has been evaluated by immunostaining. Compared toEIAV negative horses, 52% of the EIAV positive horses displayed a mildinflammation around the bronchi, 22% had a moderate inflammation withinflammatory cells inside the wall and epithelial bronchiolar hyperplasiaand 6.5% had a moderate to severe inflammation, with destruction of thebronchiolar epithelium and accumulation of smooth muscle cells withinthe pulmonary parenchyma. Changes in the thickness of the alveolar septawere also present. Interestingly, EIAV p26 was expressed in the cytoplasmof cells compatible by their morphology and localization with alveolar andbronchiolar epithelial cells. To summarize, we found lesions compatiblewith interstitial diffuse pneumonia similar to that observed during otherlentiviral infections such as FIV (Feline Immunodeficiency Virus) in cats,SRLV (Small Ruminant LentiVirus) in sheep and goat or HIV in youngchild. The presence of EIAV capsid in lung epithelial cells suggests thatEIAV might be responsible for the bronchointerstitial damages observed.Confirmation of this pulmonary route will have profound implications forunderstanding the epidemiology and pathogenesis of this disease, as EIAVis currently thought to be transmitted by blood and its tropism restrictedto cells of the monocyte/macrophage lineage.REF 066Synergistic Interactions Between the NS3hel and E Proteins Contributeto the Virulence of Dengue Virus Type 1Daisy STROTTMANN 1 , Luana DE BORBA 1 , Peter W. MASON 2 ,Claudia N. DUARTE DOS SANTOS 11 Instituto Carlos Chagas FIOCRUZ/PR, Curitiba, BRAZIL; 2 NovartisVaccines and Diagnostics, Cambridge, USADengue virus (DENV) constitutes a significant public health problem intropical regions of the world. Despite major advances in DENV biology,many aspects of dengue pathogenesis remain largely unknown. Some possibleviral genetic determinants of the intrinsic virulence of DENV inthe host have been identified; nevertheless, no conclusive evidence of acorrelation between viral genotype and virus transmissibility and pathogenicityhas been obtained. We use reverse genetics technology to engineerDENV 1 with subsets of mutations previous identified in highly neurovirulentstrains to provide insights into the molecular mechanisms underlyingdengue neuropathogenesis. We found that single mutations affecting theenvelope protein (E) and the helicase domain of the NS3 (NS3hel) protein,introduced in a different genetic context, had a synergistic effect increasingDENV replication capacity in human and mosquito derived cells. E mutationsalone generated no detectable virulence in the mouse model, however,the combination of these mutations with NS3hel mutations, which weremildly virulent on their own, resulted in a highly neurovirulent phenotype.We also demonstrated correlations between the presence of these mutationsand viral replication efficiency, viral loads, the induction of innateimmune response genes and pathogenesis in a mouse model. Given currentlimitations to our understanding of the molecular basis of dengue pathogenesis,these results could contribute to provide insights into virus/hostinteractions and new information about the mechanisms of basic denguebiology.REF 067Toll like receptor 2, 4 and 9 single nucleotide polymorphisms in cytomegalovirusinfectionMiroslawa STUDZINSKA 1 , Agnieszka JABLONSKA 1 , KatarzynaRUDNICKA 2 , Patrycja SUSKI 1 , Edyta PARADOWSKA 11 Laboratory of Molecular Virology and Biological Chemistry Instituteof Medical Biology, Polish Academy of Sciences, Lodz, Lodz, POLAND;2 Student of the University of Lodz, Faculty of Biology and EnvironmentalProtection, Division of Microbial Genetics, Lodz, POLANDToll like receptors (TLRs) play an important role in the subsequent initiationof innate and adaptive immune responses to microbial pathogens.Several single nucleotide polymorphisms (SNPs) within TLR genes seemto be associated with host pathogen defense mechanisms. Little is knownabout the role of TLR polymorphisms in the pathogenesis of human cytomegalovirus(HCMV) infection. In the present study, we investigated thepossible association between the polymorphisms of TLR2 (Arg677Trpand Arg753Gln), TLR4 (Asp299Gly) and TLR9 (T 1237C, T 1486C)with HCMV infection. Blood samples obtained from children and adultsdiagnosed with HCMV, and healthy control subjects were included in thisstudy. The SNPs were genotyping by PCR RFLP method. Digested PCRproducts were analysed by capillary electrophoresis.We did not observed differences in the frequencies of TLR2 (Arg753Gln),TLR4 and TLR9 genotypes among control and HCMV infected groups.Nonetheless, it is interesting that we found heterozygosity for the TLR2Arg677Trp SNP in 72% health adults. In contrast, none of the HCMVinfected patients and children showed a heterozygous TLR2 polymorphism.In conclusion, our study demonstrated that SNPs in the TLR2(Arg753Gln), TLR4 and TLR9 were not associated with HCMV infection.Acknowledgements: Project financed by the European Regional DevelopmentFound under the Operational Programme Innovative Economy,Grant No. POIG.01.01.02 10 107/09.REF 068Andes virus (ANDV) infection of an in vitro 3 dimensional organotypichuman lung modelKarin SUNDSTROM 1,2 , Anh Thu NGUYEN HOANG 3 , ShawonGUPTA 1,2 , Puran CHEN 3 , Clas AHLM 4 , Mattias SVENSSON 3 , JonasKLINGSTRÖM 1,2,31 Department of Microbiology, Tumor and Cell Biology/Karolinska Institute,Stockholm, SWEDEN; 2 Department of Preparedness/SwedishInstitute for Cummmunicable Disease Control, Solna, SWEDEN; 3 Centerfor Infectious Medicine/Karolinska Institute, Stockholm, SWEDEN;4 Department of Clinical Microbiology/Umeå University, Umeå, SWEDENAndes virus (ANDV) is a rodent borne hantavirus that causes hantaviruspulmonary syndrome (HPS) with a case fatality rate of 40%. Infectionsoccur via inhalation of virus contaminated excreta, and are followed byan approximately 18 days long incubation period. As of today, it is notwell known why hantaviruses cause disease in humans. In particular, verylittle is known regarding the effect hantaviruses may have at the initial siteof infection during the incubation period, and why infection change fromasymptomatic to a life threatening disease in humans. In this study weused a 3 dimensional organotypic model of human lung tissue to investigateearly and long term effects of ANDV infection. Increased progenyVirologie, Vol 17, supplément 2, septembre 2013S137

5 th European Congress of Virologyvirus production was observed approximately 10 15 days after infection,coinciding in time with induction of a short lived type I and type III interferonresponse. Furthermore, the peak in viral production was followedby increased levels of extracellular IP 10, IL 6, IL 8 and VEGF A, anddecreased levels of RANTES in infected models. Caspase 3 was not activatedby infection, suggesting that ANDV does not induce apoptosis. Lateafter infection lower levels of extracellular cytokeratin 18, a marker forepithelial cell death, were observed in infected models indicating thatANDV might impact epithelial cell survival. This indicates that directANDV induced pro inflammatory cytokine responses and VEGF A productionin the lungs might be involved in HPS pathogenesis. We alsoobserved that dentritic cells in the model had an antiviral effect on ANDVinfection.REF 069Marburg virus structural protein VP24 acts as an activator of Nrf2pathway by targeting cellular Keap1Viktor VOLCHKOV 1 , Audrey PAGE 1 , Saint Patrick REID 2 , MathieuMATEO 1 , Valentina A. VOLCHKOVA 1 , Kirill NEMIROV 1 , A.CSHURTLEFF 2 , Philip LAWRENCE 1 , Audrey BAULE 1 , OlivierREYNARD 1 , Michele OTTMANN 1 , Vincent LOTTEAU 3 , R.K.THIMMULAPPA 4 , S. BISWAL 4 ,S.BAVARI 21 CIRI, INSERM U1111, Molecular basis of viral pathogenicity, Universitéde Lyon, Lyon, FRANCE; 2 United States Army Medical Research Instituteof Infectious Diseases, Fort Detrick, Frederick, USA; 3 IMAP team, CIRI,INSERM U1111, Lyon, FRANCE; 4 Department of Environmental HealthSciences, Bloomberg School of Public Health, Johns Hopkins University,Baltimore, USAMarburg virus (MARV) is the causative agent of severe hemorrhagic feverin humans and nonhuman primates characterized by massive dysregulatedinflammation and an extremely high fatality rate. In this study we demonstratethat the structural protein VP24 of MARV targets Keap1 (Kelch likeECH associated protein 1) and acts as an activator of the Nrf2 (Nuclearfactor erythroid derived 2) antioxidant response pathway causing enhancedexpression of Nrf2 dependent proteins upon expression of VP24 aloneand during MARV infection. Keap1 mutants defective for Nrf2 bindingwere impaired in their ability to interact with VP24, suggesting that theNrf2 and VP24 binding sites to Keap1 are overlap. Using Nrf2 knockoutmice, we observed higher resistance to infection compared to wild typeanimals and lower MARV replication. Persistent activation of the cellularNrf2 response pathway would favor better survival of MARV infected cellsand may contribute to disorganization of cellular inflammatory responsesduring infection.REF 070Human Cytomegalovirus: An amazing orchestra conductor directingthe cell cycle in different cell typesMaria Cristina ARCANGELETTI 1 , Diego GERMINI 1 , IsabellaRODIGHIERO 1 , Prisco MIRANDOLA 2 , Flora DE CONTO 1 , MariaCristina MEDICI 1 , Carlo CHEZZI 1 , Adriana CALDERARO 11 Department of Clinical and Experimental Medicine University of Parma,Parma, ITALY; 2 Department of Biomedical, Biotechnological and TranslationalSciences University of Parma, Parma, ITALYHuman cytomegalovirus (HCMV) is a ubiquitous agent that infects themajority of the population and then establishes a life long latency. Thebroad range of pathologies caused by HCMV in ‘at risk’ individuals isstrictly connected to its ability to infect many cell types in vivo, includingproliferating and resting cells. Several literature data show that HCMV canalter the cell cycle to its own advantage. In the present study three cellularmodels mimicking important HCMV targets in vivo, have been used toinvestigate the virus induced cell cycle perturbation by cytofluorimetryand molecular approaches.Human embryo fibroblasts (MRC5) and human umbilical vein endothelialcells (HUVEC) are proliferating cells, permissive to HCMV infection:while the former support a classic lytic cycle, the latter sustain a persistentinfection, with a slower release of viral particles. THP 1 monocytesare replicating cells and important HCMV latency sites; following theirdifferentiation into macrophages, they enter a quiescent condition but,paradoxically, they become permissive to HCMV lytic infection. Ourresults show that HCMV is able to differentially alter the cell cycle ineach of these models, pushing the resting THP 1 macrophages to re enterthe cell cycle and the slowly cycling HUVEC to a higher cycling activity;on the other hand, the actively replicating MRC5 fibroblasts are arrestedin G1 G1/S phases of cell cycle.Understanding this HCMV differential behaviour may be relevant toexplain peculiar pathogenic aspects of virus induced diseases involvingthese cell types in vivo.REF 071Rubella virus increases the activity of the mitochondrial respiratorychain with marginal induction of oxidative stressClaudia CLAUS 1 , Kristin SCHÖNEFELD 1,2 , Denise HÜBNER 1 , UweGerd LIEBERT 11 Institute of Virology, University of Leipzig, Leipzig, GERMANY; 2 Instituteof Biochemistry, University of Leipzig, Leipzig, GERMANYMitochondria provide most of the energy required for viral replication andassembly in eukaryotic cells. The+ssRNA rubella virus (RV) exerts profoundalterations on mitochondrial function and metabolic activity. Thisincludes an increase of the intracellular ATP content and the mitochondrialmembrane potential. In this study, three cell lines with a different metabolicbackground showed a strong increase in the activity of mitochondrialrespiratory enzyme succinate:ubiquinone oxidoreductase (complex II ofelectron transport chain) and a moderate increase of ubiquinol:cytochromec oxidoreductase (complex III). This ability was lost upon mutation of twoknown key functions of the capsid protein, namely inhibition of proteinimport into mitochondria and inhibition of apoptosis induction (Ilkowet al., PLoS Pathog. 2011). Moreover, the RV mitochondria interactionsappear to be specific as they were absent in control infections with measlesvirus, which reportedly does not associate with mitochondria. While oxidativestress markers were only marginally increased during RV infection,a higher protein and/or mRNA level of transcriptional activators of mitochondrialbiogenesis and activity were detected. The results presentedhere provide a comprehensive analysis of the activity of mitochondrialrespiratory chain complexes and show that RV exerts an overall positiveinfluence on mitochondrial activity and biogenesis. These results establishnew insights into the regulation of mitochondrial functions by viruses,which usually cause mitochondrial dysfunctions.REF 072Plasminogen activator inhibitor influences susceptibility to influenzavirus infection in miceDai Lun SHIN 1,2 , Bastian HATESUER 1 , Ruth STRICKER 1 , KlausSCHUGHART 1,2,31 Helmholtz Center for Infection Research, Department of InfectionGenetics, Braunschweig, GERMANY; 2 University of Veterinary MedicineHannover, Hannover, GERMANY; 3 University of Tennessee, HealthScience Center, Tennessee, USACleavage of the hemagglutinin (HA) precursor protein is crucial for infectivityand pathogenesis of influenza A virus (IAV). The HA of influenzasubtypes with a monobasic cleavage site can be processed by several serineS138 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyproteases, such as HAT, TMPRSS2, and TMPRSS4. The A/WSN/33 H1N1virus expresses a unique neuraminidase (NA) protein which is able toconvert plasminogen into plasmin and thus allows cleavage of HA in theabsence of serine proteases. Recent studies show that A/Puero Rico/8/34H1N1 virus induces the conversion of plasminogen via the annexin IIpathway, even if the NA is unable to bind with plasminogen. Therefore,processed monobasic HA can lead to infection and hyperfibrinolysis whichinitiates a strong cytokine response in mouse models. However, other hostplasminogen activators and inhibitors may also play an active role in processingHA. In this study, we investigated the possible involvement ofSerpinE1 (plasminogen activator inhibitor type 1) for the susceptibility ofthe host to IAV infections. Our results showed that SerpinE1 knock outmutants were more susceptible to H1N1 infections compared to C57BL/6Jwild type mice. The increase in susceptibility might be caused by theabsence of the inhibiting factor of fibrinolysis which may influence thepathogenesis of IAV in vivo.REF 073Search for the necrosis inducing determinants in the sequence of Peanutstunt virus strainsAleksandra OBREPALSKA STEPLOWSKA, PrzemyslawWIECZOREK, Barbara WRZESINSKA, Henryk POSPIESZNYInstitute of Plant Protection National Research Institute, Poznan,POLANDPeanut stunt virus (PSV) is a serious pathogen of legumes, belonging to theCucumovirus genus in the family Bromoviridae. PSV strains infect highrange of plant species causing different local or/and systemic symptomson them. The pathogenesis and host range depends on the strain molecularcharacteristics. Necrosis formation might constitute hypersensitiveresponse of the host aiming at isolation of the virus within adjacent cellsand its further elimination. Among PSV strains, three PSV P, Ag, andG, related to the I subgroup, are characterized on molecular level. Firsttwo are associated with subviral element satellite RNA (satRNA). Thosestrains cause in some hosts local necrotic spots, which are particularlyevident in Chenopodium quinoa. The molecular determinant(s) of necrosisformation remains still undetermined in the case of this virus. Data frompreviously characterized viruses indicate that even single point mutationwithin the whole genome, both coding and non coding can reverse theproperties of the virus in this context. In this study we prepared series ofpseudorecombinants of aforementioned strains which consist of variouscombinations of three viral genomic strands, in satRNA free and satRNAassociated versions. We tested their infectivity and virulence and hostreaction on inoculation. Our results indicate that sequence(s) responsiblefor local necrosis inductions in C. quinoa is/are located within genomicstrands and not subviral RNAs. Aknowledgement. This study was supportedby the Polish Ministry of Higher Education and Science grant NoNN310117537.REF 074Human Endogenous retrovirus Type W (HERV W) detected in immaturedendritic cells of patients with psychiatric disordersMarion SCHNEIDER 1 , Alexandra ALTMANN 1 , Karl BECHTER 2 ,Hervé PERRON 31 Experimental Anaesthesiology, University Hospital Ulm, 89081 Ulm,GERMANY; 2 Clinic for Psychiatry and Psychotherapy II, BKK Guenzburg,89312 Guenzburg, GERMANY; 3 Geneuro Innovation, 69008 Lyon,FRANCEHuman Endogenous retrovirus Type W (HERV W) has now consistentlybeen associated with Multiple Sclerosis through MSRV Env expression,but may also play a role in major psychoses and neuroinflammatory signaturesof bipolar disorders (BD) and schizophrenia (SZ). HERV W envgene encodes a protein associated with systemic inflammation and neurotoxicity,but still, neurotoxicity can be also mediated by several proinflammatory cytokines of innate immunity. As another characteristic,inflammatory diseases give rise to high amounts of circulating immaturedendritic (iDC) cells. We here asked whether these iDC also occur inpatients with major depression, BD and SZ. Primary cultures were set upfrom blood mononuclear cells (PBMC). After 3 weeks, the characteristic,highly pleomorphic and non polarized cell type occurred, and ∼1 × 105iDC were enriched per 2 × 106 PBMC originally seeded. Using flow cytometry,all cultures expressed CD11c, CD178, CD64, HLA DR and themigratory antigen CD11b, but CD14 and co stimulatory molecules CD80,CD86 were only weakly expressed. In 7 13% of these iDC, we found theHERV W Env protein by a specific monoclonal antibody. The stainingpattern of Env antigen was characteristic: asymmetric, vesicular type andmainly in the Golgi region. Env positive cytoplasmic vesicles appearedto be eventually released as microparticles or ectosomes as confirmed byflow cytometry. The current culture strategy may be relevant to isolateHERV W Env positive iDC from different patients for further studying theretroviral genome in neuroinflammation.REF 075Plasminogen Controls Inflammation and Pathogenesis of InfluenzaVirus Infections via FibrinolysisFatma BERRI 1 , Guus F. RIMMELZWAAN 2 , Michel HANSS 3 ,Emmanuel ALBINA 4 , Marie Laure FOUCAULT GRUNENWALD 1 ,Vuong B. LE 1 , Stella E. VOGELZANG VAN TRIERUM 2 , Patrica GIL 4 ,Eric CAMERER 5,6 , Dominique MARTINEZ 4 , Bruno LINA 1 , RogerLIJNEN 7 , Peter CARMELIET 8,9 , Béatrice RITEAU 1,101 VirPath, EA4610 Virologie et Pathologie Humaine, Faculté de MédecineRTH Laennec, Université Claude Bernard Lyon 1, Lyon, France;2 Department of Virology, Erasmus Medical Center, Rotterdam, NETHER-LANDS; 3 Laboratoire d’Hematologie, CBPE, Hospices Civils de Lyon,Lyon, FRANCE; 4 CIRAD, UMR CMAEE, Montpellier, France INRA,UMR1309 CMAEE, Montpellier, FRANCE; 5 INSERM U970, Paris CardiovascularCentre, Paris, FRANCE; 6 Université Paris Descartes, Paris,FRANCE; 7 Center for Molecular and Vascular Biology, KU Leuven, Leuven,BELGIUM; 8 Laboratory of Angiogenesis & Neurovascular Link,Vesalius Research Center, VIB, Leuven, BELGIUM; 9 Laboratory of Angiogenesis& Neurovascular Link, Vesalius Research Center, KU Leuven,Leuven, Belgium; 10 INRA, Nouzilly, Indre et Loire, FRANCEDetrimental inflammation of the lungs is a hallmark of severe influenzavirus infections. Endothelial cells are the source of cytokine amplification,although mechanisms underlying this process are unknown. Here, usingcombined pharmacological and gene deletion approaches, we show thatplasminogen controls lung inflammation and pathogenesis of infectionswith influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemicH1N1 viruses. Reduction of virus replication was not responsible for theobserved effect. However, pharmacological depletion of fibrinogen, themain target of plasminogen reversed disease resistance of plasminogendeficient mice or mice treated with an inhibitor of plasminogen mediatedfibrinolysis. Therefore, plasminogen contributes to the deleterious inflammationof the lungs and local fibrin clot formation may be implicatedin host defense against influenza virus infections. Our studies suggestthat the hemostatic system might be explored for novel treatments againstinfluenza.Virologie, Vol 17, supplément 2, septembre 2013S139

5 th European Congress of VirologyREF 076Up Regulation of TRP and ASICS Receptor Expression on HumanNeuronal Cells and Primary Bronchial Epithelial Cells Following RespiratoryVirus InfectionSara Louise COSBY, Haniah ABDULLAH, Shadia OMAR, JeremyPARKER, Michael SHIELDS, Ultan POWER, Liam HEANEY, LorcanMCGARVEYQueen’s University Belfast, Centre for infection and Immunity, Belfast,UKAirway nerves control crucial reflexes such as cough and bronchoconstriction.In asthma and other respiratory conditions these reflexesbecome hyperactive causing bouts of cough and wheeze typically triggeredby exposure to environmental irritants and exacerbated during viralinfections. Currently there are no treatments that adequately ‘reset’ thishypersensitive state to normal levels. The novel transient receptor potential(TRP) channel proteins and acid sensing ion channels (ASICS), whichare found on the surface of many cell types including airway sensorynerves and epithelial cells represent potential candidate receptors. Rhinovirus(RV) and Respiratory Syncytial Virus (RSV), known to causecommon cold symptoms, are frequently detected in patients with chronicasthma. Measles virus (MV) also infects the airways and causes acharacteristic hacking cough. Bronchial epithelial cells (PBEC) were isolatedby bronchial brushing from the lung of healthy donors and asthmaticpatients. Differentiated neuroblastoma cells were used as models for airwaynerves. Cells were infected with RV, RSV or MV and levels of selectedTRP and ASIC proteins/mRNA measured. TRP channel levels increasedafter RV infection. Virus induced soluble factors up regulated TRPA1 butnot TRPM8. Similar results for TRP and ASICS channels were foundwith RSV and MV infection. PBEC from 50% of asthmatic subjects hada higher response than those from non asthmatics. Our results indicatethat strategies could be used to inhibit up regulation of TRP and ASICSreceptors during virus induced asthma exacerbation.S140 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology06. RESTRICTION FACTORS OF VIRALINFECTION, INTERFERING RNA &IMMUNE RESPONSEPosters: REF 077 to REF 088as regulators of innate immune pathways such as miR 155, miR 146a, miR21 and members of the let 7 family among others. We have selected fetal aswell as adult human primary fibroblasts and peripheral blood mononuclearcell derived macrophages as cellular models for this study in view of theirprevious extensive use in the investigation of the activation and evasion ofthe innate immune response by HSV 1. Furthermore, we present analysesof the microRNA responses induced by HSV 1 deletion mutants that lackfunctions associated with the immune evasion by the virus.REF 077Modulation of miR 34 miRNAs and related pluripotency genes inhuman keratinocytes by human papillomavirus oncogenesMarta TREVISAN 1 , Elektra PETA 1 , Alessandro SINIGAGLIA 1,2 ,Valentina MILITELLO 1 , Angela GRASSI 3 , Barbara DI CAMILLO 3 ,Luisa BARZON 1 , Giorgio PALÙ 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 IOV Istituto Oncologico Veneto, Padova, ITALY; 3 Departmentof Information Engineering, University of Padova, Padova, ITALYThe onset of cervical cancer is due to the interplay between human papillomavirus(HPV) oncogenes and cellular alterations, including deregulationof microRNA (miRNA) expression, that are finally responsible for tumorinitiation. In this study, the effect of high risk HPV and low risk HPVoncoproteins on miRNA expression profile was analyzed by microarrayanalysis in primary cultures of human foreskin keratinocytes stably expressingE6 and E7 of HPV6 or HPV16. Low risk HPV6 E6 and E7 expressiondid not significantly affect cellular miRNA expression profile. On the otherhand, high risk HPV16 oncoproteins modulated expression of several miR-NAs among which, members of the miR 34 family ranked the maximumfold change variation. Mir34b/c and mir34a resulted, respectively, upregulatedby HPV16 E7 and downregulated by HPV16 E6 expression. MiR34 miRNAs are master regulators of tumor suppression, antagonizing processesthat are necessary for basic cancer cell viability. In addition, miR 34miRNAs have been implicated in governing stemness and differentiationbeing thus key regulators of cell fate decision. Thus, a panel of pluripotencygenes associated with a more undifferentiated phenotype wereinvestigated in keratinocytes positive for E6, E7, and E6/E7 of HPV6 andHPV16. OCT4 and nanog, key players in stemness maintenance, and, to alesser extent, other pluripotency markers resulted upregulated in HPV16E6 and E6/E7 but not in E7 positive cells, thus undelaying the role of E6oncogene in inducing cell reprogramming and E7 protein in sustaining amore differentiating phenotype.REF 078Regulation of cellular microRNAs involved in innate immune responsesby HSV 1Eliza TSITOURA 1 , Chrysa KOKKINAKI 1 , Katerina ANTONIOU 2 ,Demetrios SPANDIDOS 1 , George SOURVINOS 11 Laboratory of Virology, Medical School, University of Crete, Heraklion,GREECE; 2 Department of Thoracic Medicine, University Hospital,Medical School, University of Crete, Heraklion, GREECEIn recent years, an additional level of complexity in the regulation of hostresponses to viruses has been unveiled with the discovery of microRNAs.In particular, microRNAs which regulate the expression of pathogen recognitionreceptors as well as downstream activator and suppressor molecules,participate in complex positive and negative feedback loops of the innateimmune and inflammatory pathways. To date, very few studies exist thataddress the subject of host microRNA regulation by HSV 1 whilst a wealthof evidence has accumulated regarding the mechanisms of blockade of hostresponses by the virus. We have focused therefore on the effects of HSV 1infection on the expression of selected microRNAs previously identifiedREF 079A Kaposi Sarcoma Herpesvirus Interferon Regulatory Factor promotesestablishment of LatencyModester DAMAS, Semra KATI, Magdalena WEIDNER GLUNDE,Thomas SCHULZInstitute of Virology, Hannover Medical School, Hannover, GERMANYKaposi Sarcoma Herpervirus (KSHV), also known as human herpevirus 8(HHV8), is a human oncogenic virus. It is the infectious agent of Kaposi’ssarcoma (KS) and is associated with two B cell lymphoproliferativediseases, primary effusion lymphoma (PEL) and Multicentric Castleman’sdisease. Following infection of primary endothelia cells, KSHV rapidlyestablishes a latent gene expression pattern and switches off the expressionof immediate early, early and late viral proteins. Establishment of latencyis accompanied by epigenetic (histone methylation) modifications of viralthe genome. However, the mechanisms initiating these events are unknown.Promyelocytic leukemia nuclear bodies (PML NBs) are known torestrict the permissive replication of several DNA viruses. In order to overcomethis restriction, several DNA viruses have developed mechanisms todisrupt PML NBs. Here we show that during infection of primary endothelialcells with KSHV, PML NBs stay intact suggesting that they mightbe involved in restricting permissive KSHV replication.We identified one of KSHV interferon regulatory factors (vIRFs) as a viralprotein that stabilizes PML NBs by increasing PML expression levels andarresting cell cycle. Silencing of this vIRF by siRNA during the infectionof primary endothelial cells resulted in the increased expression of earlyviral proteins and delayed establishment of latency. Reactivation of KSHVfrom latency is enhanced in the absence of this vIRF. These observationssuggest that one of the KSHV vIRFs may be involved the establishmentof KSHV latency in endothelia.REF 080KSHV and B cells Using recombinant virus to address the role of vIRF3Martin Stefan SCHMIDT 1 , Armin ENSSER 1 , Frank NEIPEL 1FAU Erlangen Nuremberg Institute for Virology, Erlangen, GERMANYKaposi’s Sarcoma associated herpesvirus (KSHV), also known as humanherpesvirus 8 (HHV 8), is associated with three diseases: 1) Kaposi’sSarcoma 2) multicentric Castleman’s disease and 3) Primary EffusionLymphoma (PEL). KSHV codes for four viral interferon regulatory factors(vIRFs), homologues of cellular IRFs. One of these, vIRF 3, is latentlyexpressed in all PEL cells and is essential for their continuous proliferationand downregulates MHC II expression. BAC16 RFP coding for the fulllength KSHV genome and BAC16 RFP vIRF 3, which contains the KSHVgenome with a deleted vIRF 3 gene, were used to produce recombinantvirus in iSLK cells. BJAB cells were infected with recombinant KSHV andselected for latently infected cells. BAC16 RFP vIRF 3 infected cells exhibiteda reduced growth rate compared to BAC16 RFP infected. This pointsto a role of vIRF 3 in cell growth in the BJAB/BAC16 model. Microarrayanalysis revealed 479 genes regulated more than two fold only in BAC16RFP infected cells, 298 genes regulated only in BAC16 RFP vIRF 3 infectedcells and 233 genes regulated in both types of KSHV infected cellsif compared to uninfected cells. Genes known to play a role in immuneVirologie, Vol 17, supplément 2, septembre 2013S141

5 th European Congress of Virologyresponse were overrepresented amongst the genes regulated in infectedcells. One of the highly upregulated genes in BAC16 RFPvIRF 3 infectedcells was TL1A. TL1A plays a role in costimulation of T cells and theTL1A/DR3 pathway is essential for the development of antiviral immunity,pointing to a novel mechanism of viral immune evasion by vIRF 3.Real time PCR targeting these miRNAs have been performed and confirmedthe differential expression pattern of these miRNAs at different timepoints following Coxsackie B4 virus infection. As a result, knowledge oftissue specific miRNAs expression during Coxsackie B virus replicationis essential to understand the aetiological role of virus induced T1D.REF 082Role of short telomeric repeat regions of Marek’s disease virus inreplication and lymphomagenesisAnnachiara GRECO, Nadine FESTER, Nikolaus OSTERRIEDER,Benedikt B. KAUFERInstitut für Virologie/Freie Universität Berlin, Berlin, GERMANYMarek’s disease virus (MDV) is a cell associated alphaherpesvirus thatcauses generalized polyneuritis and visceral lymphoma in chickens. MDVis able to integrate its genome into host telomeres. The MDV genome harborstwo telomeric regions at both termini: multiple telomeric repeats(mTMR), with a variable length, and a short telomeric repeat region(sTMR), with a fixed number of 6 repeats in all known MDV strains. Wepreviously demonstrated that mTMRs are dispensable for MDV replicationin vitro but play an important role in integration and tumor formationin vivo. To address the function of sTMRs in MDV replication and pathogenesis,we first deleted the entire sTMR sequence (vs TMR). vs TMRwas unable to replicate in vitro, indicating an essential role of the geneticelement. Furthermore, the minimal length of sTMR repeats necessary forMDV replication was determined using a set of sTMR truncation mutants.To address the role of the sTMR in MDV pathogenesis, we substituted theTMRs with a scrambled repeat sequence (vsTMRmut). vsTMRmut replicatedwith kinetics comparable to parental and revertant viruses in vitroand in vivo; however, disease and tumor incidence in vivo were severelyimpaired, confirming a function of sTMR in MDV pathogenesis. Takentogether our data suggest a central role of sTMR in MDV replication, likelyacting as spacer between the proximal packaging signal pac 1 and the DR 1cleavage site, as well as in MDV genome integration and tumor formation.REF 083Expression profile of microRNAs during Coxsackie B virus infectionin pancreatic cellsWai Yip LAM 1 , Apple Cm YEUNG 2 , Karry Lk NGAI 2 , Paul KsCHAN 2,3 , Stephen Kw TSUI 11 School of Biomedical Sceinces, Faculty of Medicine, The Chinese Universityof Hong Kong, Shatin, HONG KONG; 2 Department of Microbiology,Faculty of Medicine, The Chinese University of Hong Kong, Shatin, HONGKONG; 3 Stanley Ho Centre for Emerging Infectious Diseases, Faculty ofMedicine, The Chinese University of Hong Kong, Shatin, HONG KONGType 1 diabetes (T1D) is a disease characterized by the loss of insulinproducing beta cells in the pancreatic islets of Langerhans. It has beenreported that the incidence of T1D is increasing worldwide. MicroRNAs(miRNAs) are 21 to 23 nucleotides tissue specific non coding RNAs. MiR-NAs can bind to target sites in messenger RNAs with imperfect base pairingand, significantly reduce translational efficiency of the target proteins. Wehypothesize that Coxsackie B virus infection of beta cells may be inducingtowards the development of T1D. Furthermore, intact miRNA response inbeta cells are critical for the survival of beta cells and protection fromdiabetes subsequent to Coxsackie B infection. Pancreatic cells INS 1Eand RIN 5F cells were infected with Coxsackie B4 virus, respectively.MiRNAs were extracted and labeled using the Agilent miRNA labelingreagent. MiRNA microarray was used to delineate the expression profileof the miRNAs. Among the profiles of differentially regulated miRNA, itwas found that rno miR 375 and rno miR 382 were highly up regulatedor down regulated (> 3 fold) in both cell lines: RIN 5 and INS 1E, infectionwith Coxsackie B4 virus as compared with non infection controls.REF 084Cross talk between autophagic and apoptotic modulators in CCHFVinfected hepatocytesMarie MOROSO 1 , Olivier FERRARIS 2 , Raquel RODRIGUES 1 ,Christophe PEYREFITTE 2 , Glaucia PARANHOS BACCALA 11 Fondation Mérieux, Lyon, France; 2 Institut de Recherche Biomédicaledes Armées, Lyon, FRANCECrimean Congo hemorrhagic fever virus (CCHFV) is a tri segmentednegative stranded RNA virus belonging to the genus Nairovirus (Bunyaviridaefamily). It is transmitted to humans by ticks or direct contact withinfected blood and causes severe or even fatal hemorrhagic diseases inhumans. We demonstrated that hepatocytes are the main target cells ofCCHFV infection. To define the liver involvement in the pathogenesis,we analysed host responses induced by CCHFV in HuH7 and HepG2 celllines infected in vitro. HuH7 and HepG2 were shown to be permissiveto the virus and replicate it at a high viral load. In HuH7 infected cells,CCHFV elicited a cytopathogenic effect, but not in infected HepG2.Both CCHFV infected hepatocytes displayed common secreted IL 8. Notany other inflammatory cytokines such as TNF and IL 1 nor type I IFNwere detected. In addition, we observed a pro apoptotic CCHFV for bothhepatocytes but at different times.We found that CCHFV induced apoptosis in hepatocytes cells which wasmediated by the ER stress pathway, more characteristically the over expressionof CHOP and PERK mRNAs. Interestingly, CCHFV infected cellshad up regulated LC3B and p62 autophagic genes expression suggestingan activation of the autophagic machinery. We further decided to investigateat the protein level the autophagic markers. Detection of autophagicvesicles and LC3 protein conversion induced by CCHFV in the cross talkbetween apoptosis and autophagy will then be explained.REF 085Investigation of herpes simplex virus influence on male fertility: differentapproaches for different challengesVictor NAUMENKO 1 , Yurii TYULENEV 1 , Ekatherina MALOLINA 1 ,Andrey KULIBIN 2 , Elena GUSHCHINA 1 , Alla KUSHCH 11 Ivanovsky Institute of Virology, Moscow, RUSSIA; 2 Koltsov Institute ofDevelopmental Biology, Moscow, RUSSIAIntroduction: several studies have provided data for HSV impact in malesterility. At the same time it is not clear whether HSV is able to infectmature spermatozoa and what are pathogenic mechanisms of virus associatedsterility. The aim of this work was to develop several experimentalmodels for investigation of HSV influence on spermatogenesis and malefertility.Materials and methods: following objects were used for modeling ofHSV infection: human spermatozoa infected in vitro (model I), humantestis organotypic culture (model II), intraperitoneally infected prepuberalmice (model III), mature mice infected in rete testis (model IV). Rapidculture method, qPCR, electron and confocal microscopy were performedfor HSV detection and investigation of viral effects on spermatogenesis.Results: model I allowed to yield infected human spermatozoa in vitroand to reveal viral proteins in cytoplasm and acrosome. Using modelII it was shown that HSV was able to infect spermatogonia, spermatocytes,spermatides and it led to dramatic decrease of immature germ cellspopulation. Model III was useful to demonstrate that HSV could achievetestis before blood testis barrier formation. Mature mice that had beenS142 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyinfected in prebuberal period appeared to be infertile. Ascending infectionwas simulated in model IV and it was shown that HSV infectionresulted in leukocyte infiltration of interstitium and phagocytosis of malegametes. Conclusion: models obtained can be used to develop strategiesfor prophylaxis, diagnostics and treatment of HSV induced infertility.REF 086mTOR independent autophagy counteracts apoptosis in herpes simplexvirus type 1 infected U251 glioma cellsBiljana RISTIC 1 , Gordana TOVILOVIC 2 , Marina SILJIC 1 , ValentinaNIKOLIC 1 , Tamara KRAVIC STEVOVIC 3 , Marija DULOVIC 4 , MarinaMILENKOVIC 1 , Mihajlo BOSNJAK 3 , Vladimir BUMBASIREVIC 3 ,Maja STANOJEVIC 1 , Vladimir TRAJKOVIC 11 Institute of Microbiology and Immunology, School of Medicine, Universityof Belgrade, Belgrade, SERBIA; 2 Institute for Biological Research,University of Belgrade, Belgrade, SERBIA; 3 Institute of Histology andEmbriology, School of Medicine, University of Belgrade, Belgrade, SER-BIA; 4 Institute of Medical and Clinical Biochemistry, School of Medicine,University of Belgrade, Belgrade, SERBIAIn the present study, we investigated the role of autophagy, a process ofintracellular degradation of unnecessary or dysfunctional cellular components,in U251 human glioma cell death induced by herpes simplexvirus type 1 (HSV 1). HSV 1 induced apoptotic death in U251 cells, characterizedby phosphatidylserine externalization, caspase activation andDNA fragmentation. HSV 1 triggered autophagy was demonstrated bythe acridine orange staining of intracellular acidic vesicles, elcctron microscopyanalysis of autophagic vacuoles, and immunoblot assessment of theconversion of LC3 I to autophagosome associated LC3 II and degradationof the selective autophagic target p62. The phosphorylation of the majorautophagy repressor mTOR and its downstream target p70S6K was paradoxicallyincreased upon exposure to HSV 1, which was in agreementwith reduced phosphorylation of the mTOR negative regulator AMPKand augmented phosphorylation of mTOR activator Akt. HSV 1 triggeredapoptotic changes and cell death were accelerated in U251 cells treatedwith pharmacological autophagy inhibitors bafilomycin A1 or 3 methyladenine,as well as in cells in which the expression of autophagy essentialLC3 was downregulated by RNA interference. Taken together, these dataindicate that mTOR independent autophagy counteracts apoptotic deathof HSV 1 infected U251 glioma cells.REF 087p53 protein isoforms: key regulators in the front line of pathogeninfections? The example of influenza virusesOlivier TERRIER 1 , Jean Christophe BOURDON 2 , Bruno LINA 1 ,Manuel ROSA CALATRAVA 11 Laboratoire de Virologie et Pathologie Humaine VirPath, Equipe Vir-Cell, Université Claude Bernard Lyon 1, Université de Lyon, Lyon,France; 2 Division of Medical Sciences, Centre for Oncology and MolecularMedicine, University of Dundee, Ninewells Hospital, Dundee, UNITEDKINGDOMPrevious studies have described the role of p53 isoforms, including p53and 133p53a, in the modulation of activity of the full length p53,which regulates the cell fate outcome. Although several studies reporton the suppressive function of p53 isoforms and related deregulation oftheir expression in human cancers, investigations into the putative roleof p53 isoforms and their regulation in pathogen infections have onlyrecently begun. In the context of influenza infection, an interplay betweeninfluenza viruses and p53 has been previously described, p53 beinginvolved in the antiviral response. However, the role of physiological p53isoforms has never been explored in this context. We have demonstratedthat p53 isoforms play a role in influenza A virus infection, usingsilencing and transient expression strategies in human lung epithelialcells. In addition, with the help of a panel of different influenza virusesfrom different subtypes, we have also shown that the infection differentiallyregulates the expression of p53 and 133p53a. Altogether, ourresults highlight the role of p53 isoforms in the viral cycle of influenzaA viruses, acting as regulators of viral production in a p53 dependentmanner. Recent reports have recently highlighted the role of p53 isoformsin epithelial cells infected by other pathogens. Despite major differencesin term of models and experimental strategies, these studies share someinteresting preliminary conclusions regarding a new facet of p53 isoformbiology and will help to provide new insights into the roles of p53 inpathogenesis.REF 088Potential role of autophagy during Wolbachia antiviral interferenceagainst chikungunya virus in mosquito cellsVincent RAQUIN 1 *, Claire VALIENTE MORO 1 , ClaireBERNARDIN 2 , Florence TRAN 1 , Van TRAN VAN 1 , Patrick POTIER 1 ,Dimitri LAVILLETTE 1 , Patrick MAVINGUI 11 University of Lyon; Microbial Dynamics and Viral Transmission group,UMR 5557 Microbial Ecology, University Lyon 1, CNRS, Lyon, FRANCE;2 University of Lyon; Opportunistic Pathogenic Bacteria and Environmentgroup, UMR 5557 Microbial Ecology, University Lyon 1, CNRS, Lyon,FRANCEArthropod-borne virus (Arbovirus), such as dengue virus (DENV) andchikungunya virus (CHIKV), are major threats to public health. Eachyear, they are responsible for millions infections worldwide. Arbovirusare mainly transmitted by mosquitoes, whose vector competence relies onenvironmental, genetic and immune factors. Recently, it was demonstratedthat mosquito, like bacteria and fungi, could also modulate the vectorcompetence. For instance Wolbachia, an intracellular alphaproteobacterium,was shown to decrease the transmission rate of DENV, CHIKV,and Plasmodium, in some mosquito species. However, the cellular andmolecular mechanisms of this interference remain poorly understood.To improve our knowledge on the mechanisms involved, we used theC6/36 cell line derived from Aedes albopictus. We first established chroniccell infection with Wolbachia strain wAlbB, naturally infecting thismosquito species, and then performed viral infections. Results showedthat replication and infectiosity of CHIKV decrease in the presence ofWolbachia in comparison to aposymbiotic cells. In light of recent studiessuggesting the importance of autophagy during microbial infection,we further explored the contribution of such process in the modulationof co-infection Wolbachia-CHIKV in mosquito cells. The results willbe discussed in regard with the antiviral effect of Wolbachia againstCHIKV. We will speculate on the role autophagy may play in the bacterialinterference against mosquito-borne pathogens and, to a large extent, insymbiosis.Virologie, Vol 17, supplément 2, septembre 2013S143

5 th European Congress of Virology07. CELLULAR FACTORS CONTROLINGVIRAL INFECTION AND ROLE OF HOSTGENETICSPosters: REF 089 to REF 104REF 089Structural and functional analyses of the innate immune receptortetherinNicolas ASCHMAN 1 , Nolwenn MIGUET 1 , Andreas HINZ 1 , PatriciaRENESTO 1 , Yoshiko USAMI 2 , Heinrich GÖTTLINGER 2 , WinfriedWEISSENHORN 11 Unit of Virus Host Cell Interactions, UMI 3265, Universite Joseph FourierEMBL CNRS, Grenoble, FRANCE; 2 Program in Molecular Medicine,University of Massachusetts Medical School, Worcester, USATetherin is an interferon inducible host factor that inhibits the release ofsome enveloped viruses including HIV 1, from the surface of infectedcells. Many susceptible viruses have evolved proteins that specificallycounteract this activity, such as the accessory protein Vpu in the case ofHIV 1. Tetherin is a type II transmembrane protein with a 170 Å long rodshaped extracellular region, modified with a C terminal glycosyl phosphatidylinositol(GPI) anchor. Crystal structures further reveal a dimericassembly mediated through a disulfide linked coiled coil domain. Althoughthe mechanism of virus particle retention is not fully understood, tetherin’sunique double membrane anchored topology suggests that it insertsinto both cellular and viral membranes during the budding process. Inorder to better characterise the antiviral mechanism of tetherin, we studiedthe role of various determinants such as irregular coiled coil residues,dimer stabilising disulfides and glycosylation by site directed mutagenesis.We propose that the protein’s inherent conformational flexibility isrequired for efficient retention of virions. Furthermore, it has been previouslyobserved that even in the presence of the antagonist Vpu, sometetherin is still incorporated into cell free virions. This led us to speculatethat the antiviral mechanism depends on its cell surface concentration andpossible organisation of tetherin into higher order assemblies at HIV 1budding sites. Molecular details of tetherin structure and function will bepresented.localized on the left arm of the third chromosome, affect the resistance toDCV infection (Magwire et al, 2012). We are now deciphering the roleof this uncharacterized gene in the control of DCV infection. Our loss offunction and gain of function experiments indicate that pastrel encodes amolecule opposing DCV infection, raising the question of the mechanisminvolved. Co localization experiments indicate that Pastrel is localized inlipid droplets. All together, our data suggest a connection between lipiddroplets and restriction of viral infection in Drosophila, as already describedin mammals between the restriction factor Viperin, present on lipiddroplets, and the replication of the human pathogen Hepatitis C Virus(Helbig et al, 2011).REF 092Host dependence of the thermosensibility of measles virus vaccinestrains and critical function of HSP90 in viral replicationLouis Marie BLOYET 1 , Jérémy WELSH 1 , Jessica RABILLOUD 1 ,Branka HORVAT 1 , Denis GERLIER 1 , Boyan GRIGOROV 1CIRI, INSERM U1111, CNRS, Université Lyon 1, ENS Lyon, Lyon,FRANCEAttenuation of measles virus (MeV) that gave rise to a well toleratedvaccine was ultimately reached after adaptation to grow into chickenembryonic fibroblasts (CEF). Moraten and Schwarz vaccines have beenselected for their ability to grow in CEF at 32 ◦ C but not at 37 ◦ C. Thisthermosensibility is not intrinsic to the viruses as they displayed a normalgrowth at 37 ◦ C in human or simian cells. A correlation between the temperaturerestriction of viral growth and a decreased expression of HSP90in CEF lead us to investigate the role of HSP90 in MeV replication cycle.In these cells, HSP90 appears to be a limiting factor since overexpressionof HSP90 in CEF enhanced MeV growth. Furthermore, inhibition ofHSP90 either by siRNA or by addition of a chemical inhibitor decreasedor abrogated the viral RNA synthesis and viral production not only in CEFbut also in other cell types such as Vero cells. Kinetics experiments andreversibility of the inhibition upon drug retrieval pointed to the polymerasecomplex as a likely target of HSP90 activity. Upon blocking HSP90, theamount of MeV L protein detectable by western blot was strongly decreased,while P expression remained unaffected. Treatment of the cells witha proteasome inhibitor did not prevent the decrease induced by the HSP90inhibitor. HSP90 was detected in viral particles suggesting a direct interactionbetween L and HSP90 and ongoing experiments aim at investigatingthis interaction.REF 091Pastrel: a new Drosophila gene restricting infection by the picornalike virus DCVVincent BARBIER, Akira GOTO, Carine MEIGNIN, KarimMAJZOUB, Jean Luc IMLERUPR9022 Institut de Biologie Moléculaire et Cellulaire (I.B.M.C.), Strasbourg,FRANCESince the discovery of the evolutionarily conserved TOLL and IMD pathways,involved in anti fungal and anti bacterial immune responses, thefruit fly Drosophila melanogaster is used as a model to study the molecularmechanisms of innate immunity. To defend against viral pathogens,Drosophila relies on two main facets: the RNA interference (RNAi) pathway,which plays a broad role in the control of viruses, and virus specificinducible responses (Kemp et al, 2013). We also observed that the fly geneticbackground can modulate the resistance to infection by Drosophila CVirus (DCV), a natural pathogen of Drosophila. A genome wide associationstudy recently showed that polymorphisms in the gene named pastrel,REF 093Restriction of rAAV mediated transgene expression in dystrophicmuscles through epigenetic silencing mechanismsJean Baptiste DUPONT 1 , Benoît TOURNAIRE 1 , BéatriceMAROLLEAU 2 , Laetitia VAN WITTENBERGHE 2 , ChristopheGEORGER 2 , Laurence DUBREIL 3 , Emilie LECOMTE 1 , LaurenceJEANSON LEH 2 , Bernard GJATA 2 , Fulvio MAVILIO 2 , Richard O.SNYDER 1,4,5 , Philippe MOULLIER 1,4 , Adrien LÉGER 11 INSERM UMR 1089, Nantes, FRANCE; 2 GENETHON, Evry, FRANCE;3 INRA UMR 703, Nantes, FRANCE; 4 Department of Molecular Geneticsand Microbiology, University of Florida, Gainesville, USA; 5 CERHB,University of Florida, Gainesville, USARepressive epigenetic modifications such as DNA methylation or HistonePost Translational Modifications (HPTMs) can restrict viral genomeexpression and participate in the establishment of latency. During theirlong coevolution with host cells, viruses developed molecular strategiesto counteract epigenetic restriction. However, due to their lack ofviral genes, retro/lentivectors and adenoviral vectors have lost this abilityS144 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyresulting in complete transgene silencing. So far, this has never been shownfor recombinant Adeno Associated Virus (rAAV) vectors, although the episomalrAAV genome is known to associate with cellular histones in vivo.Importantly, in pathological situations where cells are epigenetically affected,rAAV genome expression may be hampered together with treatmentefficacy. Duchenne Muscle Dystrophy (DMD) is one such example, as theabsence of dystrophin in murine (mdx) muscles results in the deregulationof several histone deacetylases. Here we looked at potential restrictionof the rAAV genome in dystrophic muscles through epigenetic silencingmechanisms. We injected mdx mice and Wild Type (WT) controlswith a rAAV vector. Our results show that rAAV genomes are not onlyless stable in mdx muscles compared to WT controls, but are also lesstranscriptionally active and enriched with HPTM specific of repressedheterochromatin. Our data so far indicate that rAAV transduction is specificallyrestricted in dystrophic muscles, lowering its therapeutic index.Therefore, combining rAAV vectors and epigenetic drugs could be relevantin the context of DMD.REF 094Kaposi’s sarcoma associated herpesvirus (kshv) tegument protein orf75 is essential for viral lytic replication and plays a critical role in theantagonization of nuclear domain 10 instituted intrinsic immunityArmin ENSSER 1 , Thomas STAMMINGER 1 , Doris JUNGNICKL 1 ,Nina REUTER 1 , Elke BOGNER 3 , Kevin BRULOIS 4 , BrigitteSCHOLZ 1 , Michael STÜRZL 2 , Jinjong MYOUNG 5 , Jae Ung JUNG 4 ,Full FLORIAN 1,61 Institut für Klinische und Molekulare Virologie, Universitätsklinikum,Friedrich Alexander Universität, Erlangen, GERMANY; 2 Division ofMolecular and Experimental Surgery, Universitätsklinikum, FriedrichAlexander Universität, Erlangen, GERMANY; 3 Institut für MedizinischeVirologie, Charité Universitätsmedizin, Berlin, GERMANY; 4 Departmentof Molecular Microbiology and Immunology, Keck School of Medicine,University of Southern California, Los Angeles, CA, USA; 5 Novartis Institutesfor Biomedical Research, Emeryville, CA, USA; 6 Department ofMicrobiology and Immunobiology, Harvard Medical School, Boston, MA,USAKnock down experiments in primary human fibroblasts show that KSHVinfection is restricted by nuclear domain 10 (ND10) components PMLand SP100, while ATRX is degraded and Daxx is dispersed from ND10after KSHV infection. The ORF75 tegument protein, a viral FGARAT,was identified as the viral factor that induces the degradation of ATRXand relocalization of Daxx. Isolated expression of ORF75 in primarycells also induces a relocalization of PML and of Sp100, indicating thatthis viral effector protein is able to influence multiple ND10 components.ORF75 dose dependent degradation of ATRX, but not of Daxxor SP100 was observed. siRNA knockdown of ORF75 reduced virus particleproduction and lytic gene expression. Moreover, by constructing aKSHV mutant harboring a stop codon at the beginning of ORF75, wecould demonstrate that ORF75 is absolutely essential for viral replicationand the initiation of viral immediate early gene expression. Doxycyclininduced expression of ORF75 complemented the growth defect of theORF75 Stop virus. Using recombinant viruses, carrying either Flag orYFP tagged variants of ORF75, we could further corroborate the roleof ORF75 in the antagonization of ND10 mediated intrinsic immunity,and show that it is independent of the previously described PML antagonismby the vIRF3. We assume that overcoming ND10 intrinsic defenseconstitutes a critical event in the replication of all herpesviruses; on theother hand, restriction of herpesviral replication by ND10 componentsmay also promote gammaherpesvirus latency as the default outcome ofinfection.REF 095Regulation of HIV 1 integration by chromatin structure and cellularproteinsMarc LAVIGNE 1,2 , Johan XAVIER 1 , Mehdi MORCHIKH 1,2 , MonicaNAUGHTIN 1 , Francesca DI NUNZIO 2 , Zofia HAFTEK TERREAU 1 ,Elodie CHATRE 1 , Camille MARTEL 1 , Cédric VAILLANT 1 , PierreCHARNEAU 2 ,YvesJACOB 21 Ecole Normale Supérieure, Lyon, FRANCE; 2 Institut Pasteur, Paris,FRANCEA viral enzyme called integrase catalyzes integration, a critical step ofretroviral replication. In the case of HIV 1, this enzyme is a promisingantiviral target. However, viruses resistant to catalytic inhibitors of integrasehave already emerged and other integrase properties need to bestudied as antiviral targets. In this report, we present data concerning twoof these properties. First, using new in vitro integration assays into chromatintemplates, we have characterized elements of the chromatin structure,at both DNA and nucleosome levels, involved in integration site selectivity.Second, HIV 1 integrase interacts with cellular proteins and someof them, like the LEDGF/p75 protein, are essential for integration. Thisprotein tethers integrase to chromatin. We have recently identified twocellular proteins interacting with the LEDGF/p75 PWWP chromatin bindingdomain: the transcription activator TOX4 and the splicing cofactorNOVA1. Using various approaches (mutagenesis, interaction and co localization),we have confirmed the specificity of these interactions. We havealso observed an inhibition of HIV 1 replication in cells overexpressingthe PWWP interacting region of these proteins. In the case of NOVA1, thisinhibition corresponds to a defect of integration. We are now studying theeffects of TOX4 and NOVA1 proteins on HIV 1 integration, using cellularand in vitro approaches. We propose a regulation of LEDGF chromatininteraction by TOX4 and NOVA1 proteins. This regulation could play arole during HIV 1 integration and could define a new antiviral target.REF 096Genetic control is an important factor influencing the clinical courseof tick borne encephalitis virus infectionMartin PALUS 1,2 , Jarmila VOJTÍŠKOVÁ 3 , Jirí SALÁT 1,4 , JanKOPECKÝ 1,2 , Libor GRUBHOFFER 1,2 , Marie LIPOLDOVÁ 3 , PeterDEMANT 5 , Daniel RUŽEK 1,41 Institute of Parasitology, Biology Centre of the Czech Academy ofSciences, Ceské Budejovice, CZECH REPUBLIC; 2 Faculty of Science,University of South Bohemia, Ceské Budejovice, CZECH REPUBLIC;3 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,Prague, CZECH REPUBLIC; 4 Department of Virology, VeterinaryResearch Institute, Brno, CZECH REPUBLIC; 5 Roswell Park CancerInstitute, New York, USATick borne encephalitis (TBE) is caused by one of the most prevalentarboviruses in Europe and in north eastern Asia. Clinical course ofTBE, a disease caused by TBE virus, ranges from asymptomatic or mildinfluenza like infection to severe debilitating encephalitis or encephalomyelitis.Despite the importance of this disease, the underlying factorsinvolved in development of disease remains uncovered. To address thiscritical issue we developed an animal model of TBE based on BALB/cc STS/A (CcS/Dem) recombinant congenic mouse strains showing differentseverities of the infection in relation to the host genetic background.Recombinant congenic strains of mice differring in genetic backgroundexhibit different patterns of immune related genes expression followingTBE virus infection. We observed not only differences in the geneVirologie, Vol 17, supplément 2, septembre 2013S145

5 th European Congress of Virologyexpression and clinical forms after subcutaneous infection but even thesame patterns after intracerebral infection. After inoculation of TBE virus,BALB/c mice showed medium susceptibility to the infection, STS micewere highly resistant, and CcS 11 mice were highly susceptible. The resistantSTS mice showed lower and delayed viremia, lower virus productionin the brain, and the lowest cytokine/chemokine mRNA production, buthad the strongest neutralizing antibody response. These data indicate thatthe genetic control is an important factor influencing the clinical course ofTBE. Combining functional and clinical information with genetic informationmay improve our ability to predict the progression of the diseaseand to optimize the treatment.REF 097Insights into structural determinants for nuclear domain 10 disruptionby the major immediate early protein IE1 of humancytomegalovirusThomas STAMMINGER 1 , Myriam SCHERER 1 , Stefan KLINGL 2 ,Madhumati SEVVANA 2 , Victoria OTTO 1 , Joachim STUMP 3 , HeinrichSTICHT 3 , Yves MULLER 21 Institute for Clinical and Molecular Virology, Erlangen, GERMANY;2 Division of Biotechnology, Erlangen, GERMANY; 3 Division of Bioinformatics,Erlangen, GERMANYRecent research identified the nuclear domain 10 (ND10) componentsPML, hDaxx, and Sp100 as components of an intrinsic immune responsethat suppress the initiation of human cytomegalovirus (HCMV) replication.This antiviral defense, however, is efficiently counteracted by the viralimmediate early protein IE1. To gain further insights into the mechanismby which IE1 disrupts ND10, we set out to determine the three dimensionalstructure of IE1. Here we present the crystal structure of a central, 45 kDasubdomain of the rhesus macaque cytomegalovirus IE1 protein (RhIE1)at 2.3 A resolution. RhIE1 exhibits an elongated all alpha helical structurethat displays no obvious homology to known protein folds. In vitro andin vivo approaches revealed that function and structure are conserved betweenIE1 proteins of cytomegaloviruses. Consistently, a reliable model forIE1 of HCMV was generated, based on the crystal structure of the RhIE1subdomain. In order to investigate the functional importance of the IE1subdomain for ND10 disruption, we engineered a recombinant HCMVexpressing this truncated IE1 variant. Analogous to full length IE1, theIE1 subdomain localized to the dot like ND10 structure and was sufficientto induce de SUMOylation of PML, Intriguingly, this event resulted in therelease of ND10 associated proteins into the nucleoplasm, e.g. Sp100, butnot of PML itself. We propose that ND10 disruption by IE1 is an at least twostep process involving PML de SUMOylation and disassembly of PMLaggregates which are mediated by distinct domains of the IE1 protein.REF 098HCV induced dedifferentiation of hepatocytes and metabolic consequencesJulia GRAF 1 , Ralf BARTENSCHLAGER 2 , Ulrike PROTZER 11 Institute of Virology,Technische Universität München,Helmholtz ZentrumMünchen, Munich, GERMANY; 2 Department of Infectious Diseases,Molecular Virology, Heidelberg University, Heidelberg, GERMANYChronic HCV infection is associated with a 2 fold increased risk todevelop insulin resistance and as a consequence type II diabetes. We useda cell culture model for chronic HCV infection in differentiated hepatomacells to study HCV induced alterations of hepatic insulin signaling. UponHCV infection, expression of the hepatocyte specific glucose transporterGLUT2 and glucose uptake into the cells were reduced. In addition, wefound reduced insulin receptor (IR) expression as well as diminishedinsulin induced Akt activation. When Akt activation is missing, Akttarget transcription factor Foxo1 is expected to be retained in the nucleus,where it maintains the expression of gluconeogenesis key enzymesPEPCK and G6Pase. In HCV infected cells unlike in insulin resistance,Foxo1 located to the cytoplasm and PEPCK and G6Pase expressionlevels were very low despite reduced Akt signalling. Reduced expressionof IR, GLUT2 and especially hepatocyte specific gluconeogenesis keyregulators PEPCK and G6Pase despite lacking Akt activation, led us tothe hypothesis that HCV may interfere with hepatocyte differentiation.Indeed, several hepatocyte differentiation markers were down regulated,and we observed a re entry of HCV infected cells into the cell cycle. Highnumbers of cells were in S phase supporting HCV RNA replication byenhancing the availability of nucleotides. Taken together, we found thatHCV lead to hepatocyte dedifferentiation. As a consequence, the capacityof hepatocytes to take up glucose was reduced, contributing to elevatedglucose levels in HCV infected patients.REF 099Mutual ubiquitination of Herpes Simplex Virus ICP0 and the ubiquitinligase SIAH 1Claus Henning NAGEL 1 , Anja POHLMANN 2 , Beate SODEIK 2 ,Joachim HAUBER 11 Heinrich Pette Institute Leibniz Institute for Experimental Virology, Hamburg,GERMANY; 2 Hannover Medical School, Institute for Virology,Hannover, GERMANYUpon de novo infection with Herpes Simplex Virus (HSV), the regulatoryICP0 is one of the first viral genes to be expressed from incoming viralDNA. Prominent features of ICP0 include the degradation of componentsof PML nuclear bodies and enabling the onset of low multiplicity infection.Several cellular interaction partners of ICP0 are subject to ubiquitinationand proteasomal degradation. We previously showed an interactionof ICP0 with the E3 ubiquitin ligase SIAH 1 resulting in ubiquitinationand proteasomal degradation of ICP0 itself. We demonstrate a specificinteraction via a conserved Val X Pro (VxP) binding motif in HSV 1ICP0. Mutation of this motif to Asn X Asn (NxN) abrogated binding toSIAH 1. Notably, ICP0 of HSV 2 contains two functional VxP motifs.ICP0 carrying the NxN mutations was no longer ubiquitinated by SIAH1. Whereas our previous results showed a stabilisation of SIAH 1 by bindingto ICP0, we also discovered a superimposed ubiquitination of SIAH1 by ICP0; again this functional interaction depended on the integrity ofthe VxP motifs in ICP0. In order to study the ICP0/SIAH 1 interactionduring the course of infection, HSV mutants with NxN mutations or deletionsin the RING domain were constructed by BAC mutagenesis. Theresulting viruses were viable and in the case of NxN exhibited no attenuation,although virally encoded ICP0 carrying the NxN mutation no longerbound to SIAH 1. This novel virus host interaction within a cellular ubiquitinationnetwork reveals connections of HSV infection to DNA damageresponse or hypoxia signalling pathways.REF 100The Human Herpes Virus 8 infection alters lipid synthesis and inducesneutral lipid accumulation in primary endothelial cellsRaffaello POMPEI 1 , Sabrina UDA 1 , Enrica PIRAS 1 , FabrizioANGIUS 1 , Barbara BATETTA 1 , Angela INGIANNI 1Department of biomedical sciences, Cagliari, ItalyHuman Herpes Virus 8 (HHV8), the causative agent of Kaposi’s sarcoma(KS), induces an intense transcriptional reprogramming of endothelial(HUVEC) cells in vitro, prolonging their life span and augmenting cellsurvival in the presence of apoptotic inducers. In this work, lipid synthesisand metabolism were studied in HHV8 infected HUVEC cells. Cholesteroland triglyceride synthesis were analysed by the use of 14C acetate and14C oleate during lytic and latent HHV8 infection from days +3 to +24.A quantitative analysis of neutral lipids in infected cells was performedS146 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyin situ using Nile Red dye, which displayed a red emission for polar lipidsand a yellow emission for neutral lipids. The results showed that during thelytic phase (1 4 days after HHV8 infection) a depression of cholesterol andcholesterol ester synthesis (about 26%) was observed, while triglyceridesappeared to be enhanced up to 45% as compared to controls. After 14 24days, during the latent phase of infection, there was an increased synthesisof cholesterol (up to +44%) and cholesterol esters (up to +58%) in infectedcells, whereas triglycerides were progressively lowered. Imaging analysisshowed that the lipid droplets and total neutral lipid content were definitelyhigher (up to +85%) in HHV8 infected cells compared to the controls.Lipid synthesis inhibitors Sigma C75 and Sandoz 58035 were used toexamine the importance of lipid synthesis modification and neutral lipidaccumulation in the pathogenesis of KS and neo angiogenesis. This workwas financed by the Fondazione Banco di Sardegna 2012.REF 101Effect of Nitric Oxide on Mitochondrial Function in HepG2 cellsInfected by Dengue virusLeandro SILVA DA COSTA 1 , Holmes DE SOUZA CAMPOS 1 , ThaisMORAES DA CONCEIÇÃO 1 , Andrea THOMPSON DA POIAN 11 Universidade Federal do Rio de Janeiro Instituto de Bioquímica Médica,Rio de Janeiro, BRAZILDengue virus (DENV) infection causes dengue fever life threateningdengue hemorrhagic fever and dengue shock syndrome. Experimentalevidences suggest that liver is an important site of virus replication anddamage of this organ has been found in severe cases of DENV infection.It has been shown that human hepatocyte cell line, HepG2 producescytokines, and nitric oxide (NO) upon infection, which may be involvedin disease manifestations. In this work, we evaluated the effect of NOon DENV replication and mitochondria function in HepG2 cells. DENVreplication was evaluated by plaque assay and flow cytometry. NO productionwas investigated by intracellular NO quantification and inducibleNitric Oxide Synthase (iNOS) expression. Mitochondrial function wasmeasured in intact cells using high resolution respirometry. These resultsshowed an increase in NO production and in the expression iNOS and proapoptotic genes (BAX, Caspase 9) during the course of infection. Additionally,we demonstrated that infected cells present a significantly decreasein basal respiration and a 45% decrease in FCCP induced maximum electrontransport system capacity followed by apoptosis. When cells weretreated with NO inhibitor 1400w, these effects were abolished, suggestingNO involvement in mitochondrial alteration and apoptosis. We proposethat NO modulates mitochondria bioenergetics and dysfunction in DENVinfected cells. We believe that this study contribute to elucidate the molecularmechanisms underlying the role of NO in liver dysfunction causedby DENV. Financial Support: CNPq, FAPERJ.REF 103The Impact of Genetic Variations in IL28B Region on Response toTreatment of Chronic Hepatitis CIvana LAZAREVIC 1,2 , Jelena DJORDJEVIC 1,3 , Maja CUPIC 1,2 ,Danijela KARALIC 1,2 , Dragan DELIC 1,4 , Neda SVIRTLIH 1,4 , JasminaSIMONOVIC 1,4 , Petar SVORCAN 1,3 , Natasa MILIC 1,5 , TanjaJOVANOVIC 1,21 University of Belgrade, Faculty of Medicine, Belgrade, SERBIA;2 Institute of Microbiology and Immunology, Belgrade, SERBIA;3 Departement of Gastroenterology and Hepatology, Clinical Center“Zvezdara”, Belgrade, SERBIA; 4 Clinics of Infectious and TropicalDiseases, Clinical Center of Serbia, Belgrade, SERBIA; 5 Institute forMedical Statistics and Informatics, Belgrade, SERBIAThe three single nucleotide polymorphisms (SNPs) near IL28B gene wereshown to be highly predictive of sustained virological response (SVR)in patients with chronic hepatitis C virus (HCV) infection. The studyattempted to demonstrate the role of single and combined IL28B polymorphisms(rs8099917, rs12979860 and rs12980275) and other host andviral factors in predicting response to treatment, in Caucasian patientsinfected with HCV genotype 1. The study comprised 106 patients whounderwent standard 48 week therapy and out of which 55.7% achievedSVR. IL28B genotypes were determined by SSP PCR (Sequencespecific primer PCR). Patients carrying genotypes CCrs12979860 orAArs12980275 were 3.5 and 3 times more likely to achieve SVR, respectively.Genotypes GGrs8099917 and TTrs12979860 were identifiedas predictors of treatment failure. The presence of IL28B profile includingat least one of the favorable genotypes was identified as the mostimportant factor associated with SVR, followed by younger age andlower grade of histological activity. The strongest PPV of single SNPs forachieving SVR was observed for CCrs12979860 (76.9%). The presenceof GGrs8099917 showed the strongest NPV of 85.7%. The correlationof SNPs with other host and viral factors revealed association ofTTrs8099917 and lower AST levels. Results of this study confirm thatall investigated IL28B polymorphisms are associated with treatment responseand that presence of any of the favorable IL28B genotypes can beconsidered independent pretreatment determinant of the effectiveness oftherapy.REF 104Treatment outcome prediction model based on baseline viral geneticfactors and clinical variables in patients with chronic hepatitis CElisa MARTRÓ 1,2 , Verónica SALUDES 1,2 , Elisabet BASCUÑANA 1 ,Elena JORDANA LLUCH 1 , Sònia CASANOVAS 1 , Mercè ARDÈVOL 3 ,Esther SOLER 4,5 , Ramón PLANAS 4,5 , Vicente AUSINA 11 Microbiology Service, Hospital Germans Trias i Pujol, Badalona, SPAIN;2 CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, SPAIN.,Badalona, SPAIN; 3 Hospital Pharmacy, Hospital Universitari GermansTrias i Pujol, Badalona, SPAIN; 4 Liver Unit, Hospital UniversitariGermans Trias i Pujol; 5 CIBER Enfermedades Hepáticas y Digestivas(CIBEREHD), Barcelona, SPAINOnly about 50% of patients chronically infected with HCV genotype 1respond to treatment with pegylated interferon alfa (PegIFN a) and ribavirin(RBV), and protease inhibitors have to be administered together withthese drugs increasing costs and side effects. We aimed to develop a predictivemodel of treatment response based on a combination of baselineclinical and viral parameters. Seventy four patients chronically infectedwith HCV 1b and treated with PegIFN a and RBV were studied. Hostand viral related factors (viral load, and genetic variability in the E1 E2,core and ISDR regions) were assessed. Multivariate discriminant analysiswas used to develop a predictive model on the training group (N=53),which was then validated in the validation group (N=21). The predictivemodel generated included the following variables in decreasing order ofsignificance: number of viral variants in the E1 E2 region, an amino acidsubstitution pattern in the viral core region, the IL28B polymorphism,serum GGT and ALT levels, and viral load. Treatment outcome was accuratelypredicted in the training group (AUROC=0.9444; 96.3% specificity,94.7% PPV, 75% sensitivity, 81% NPV), and the accuracy remained highin the validation group (AUROC=0.8148, 88.9% specificity, 90.0% PPV,75.0% sensitivity, 72.7% NPV). The model obtained including host andviral factors had a high PPV in our population of Spanish HCV 1b treatmentnaïve patients. Accurately identifying those patients responding tothe dual therapy could help reducing implementation costs and side effectsof new treatment regimens.Virologie, Vol 17, supplément 2, septembre 2013S147

5 th European Congress of Virology08. ANTIVIRAL THERAPY ANDRESISTANCE TO ACUTE VIRALINFECTIONPosters: REF 105 to REF 126REF 105Viperin vs. Tick Borne Encephalitis Virus: Mechanisms of a PotentAntiviral ProteinKirstin VONDERSTEIN 1 , Arunkumar UPADHYAY 1 , Ju Tao GUO 2 ,Friedemann WEBER 3,4,5 , Anna K ÖVERBY 1,31 Virology, Department of Clinical Microbiology, Molecular InfectionMedicine Sweden (MIMS), Umeå University, Umeå, SWEDEN;2 Department of Microbiology and Immunology, Drexel University Collegeof Medicine, Doylestown, Pennsylvania, USA; 3 Department of Virology,University Freiburg, Freiburg, GERMANY; 4 Institute for Virology, PhilippsUniversity Marburg, Marburg, GERMANY; 5 Centre for BiologicalSignalling Studies (BIOSS), Albert Ludwigs University Freiburg, Freiburg,GERMANYTick borne encephalitis virus (TBEV; family Flaviviridae), a positivesense, single stranded RNA virus, is the medical most important arbovirusin Europe and Russia. TBEV has a significant impact on publichealth as it causes ∼10,000 annual cases of encephalitis and meningitis,and no antiviral treatment is available. We aim to investigate differentantiviral approaches against TBEV, starting with its interactions with theinterferon (IFN) system. Therefore, three different Western TBEV strainswere tested for their sensitivity to IFN alpha pre treatment, measuringvirus replication in cells and virus load in the supernatant. TBEV turnedout to be very sensitive to IFN, as viral RNA and titres were reduced byup to three logs after treatment with 100 units IFN alpha. To identify thecellular protein mediating the anti TBEV effect of IFN alpha, a screen ofinducible cell lines expressing different ISGs was performed. This screenidentified viperin (virus inhibitory protein, endoplasmic reticulum associated,interferon inducible) as a very strong inhibitor of virus replication.Viperin is known to have antiviral activity against many different viruses,however, the mechanism of action is not clear. Since TBEV is extremelyviperin sensitive it is a perfect model virus for investigating the antiviralmechanism of viperin. We have set up a number of assays to determinewhich step of the virus lifecycle that viperin is targeting and screened a setof mutants to determine which domains are responsible for the antiviralactivity of viperin.REF 107Synthetic RIG I Activator Double Stranded Triphosphorylated RNAProvides an Antiviral State in Yellow Fever Replicating Hepatic CellsMohammad Intakhab ALAM, Debby Van Den BOORNKONIJNENBERG, Gunther HARTMANNInstitute for Clinical Chemistry and Clinical Pharmacology, Bonn,GERMANYCytoplasmic helicase RIG I is a viral sensor which recognizes incomingviral RNA genomes containing 5’triphosphate dsRNA structure. Flaviviruesincluding Yellow Fever Virus (YFV) exhibits a mechanism toovercome innate immunity by inhibiting RIG I. Here we first time examinedthe potent antiviral activity of immunostimulatory RIG I ligand5 ′ triphosphorylated RNA (3p RNA) against YFV and possibly for severaldiverse flaviviruses. We used renilla reporter expressing subgenomicyellow fever virus replicon (YFVR) model and vaccine strain 17D forour study. In HepG2 cell culture, 3p RNA inhibited one log replicationof YFVR and 2.5 logs of vaccine strain 17D at non toxic concentration(0.8 g/ml) in dose dependent manner. In contrast to 3p RNA, Poly ARNA a negative control did not inhibit YFV replication that supports theexclusive antiviral activity of 3p RNA. Consequently, 3p RNA inducedIFN showed superior antiviral effect than positive control, rec IFN. Ourfurther analysis demonstrates that 3p RNA potentially enhances RIG Isignaling pathway for the induction of type 1 IFN by upregulating ZAPs,RIG I and IRF3. Interestingly, IFN signaling pathway was also found upregulatedby 3p RNA leading to the upregulation of IRF9, mainly STAT2and several antiviral genes 2 ′ 5 ′ OAS, RNAseL, MxA, ISG15, IFIT1 andIFIT2 to provide an antiviral state in YFVR replicating cells. Virus inhibitionwas not observed by 3p RNA in Vero cells, RIG I/MEFs and flagtagged RIG I over expressing HepG2 cells. RIG I/MEFs showed enhancedreplication of YFVR compared to WT MEFs. 3p RNA strongly inhibitedNS4B and renilla luciferase protein of YFVR. Altogether, these findingsraise the possibility that RIG I Ligand, a potent immune stimulator maybe a promising antiviral therapeutic RNA molecule for YFV.REF 108Anticancer compound ABT 263 accelerates apoptosis and imbalancescytokine production in virus infected cells and lowers survival ratesof infected miceOxana DENISOVA 1 , Laura KAKKOLA 1 , Xavier SAELENS 2 , DenisKAINOV 11 Institute for Molecular Medicine Finland, FIMM, Helsinki, FINLAND;2 Department for Molecular Biomedical Research, VIB and Departmentof Biomedical Molecular Biology, Ghent University, Ghent, BELGIUMABT 263 is an orally administered small molecule that inhibits anti apoptoticfunction of Bcl xL protein and triggers cancer cell death. Here weshow that at non cytotoxic concentrations ABT 263 also enhances apoptosisof non cancerous influenza virus infected human cells. Moreover, ABT263 disarms immune responses and facilitates death of influenza virusinfected tumor free mice without affecting non infected animals. Theseresults indicate that phase I/II ABT 263 could be hazardous for cancerpatients with viral infections. The strategy we developed here using ABT263 could be implemented for studying effects of other anticancer agentson infection and immunity.REF 109Development of a new anti viral medication for the grippeValentina DIVOCHA, Anatoly GOZHENKOState Enterprise, Odessa, UKRAINEThe grippe till now is a mass disease resulted in hospitalization of thousandsof sick persons and high mortality rate.In Ukraine some 10-14 million of people fell ill annually which constitute25-30% of the general morbidity rate. Number of fatalities doesnot decrease, on the contrary it has a tendency to stabilization and evenincrease. The objective: to excrete inhibitor of trypsin – like proteinasesof industrial wastes of human gamma globulin manufacturing as this antienzyme takes part in blocking of hemagglutinin of virus grippe cleavage.Materials and methods: we used the wastes of the first stage of gammaglobulin manufacture (II+III) from donors’ blood which contained a greatamount of this inhibitor and the latter was excreted by ion change chromatographyon Diethylaminoethyl (DEAE) cellulose. Results: derivationthe pure inhibitor included the following stages: the enzyme excretion,ultrasonic disintegration of cells, ion change chromatography on DEAE –cellulose, dialysis, lyophilic drying. The method described allows to obtainfive isoforms with inhibitory activity. The greatest amount on trypsin likeS148 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyproteinases was registered in the isoform of the V th fraction. Conclusion:the V th isoform with high inhibitory activity was used for the study oftherapeutical properties and the experimental grippe A at white mice. Theisoform mentioned showed a promoted protective effect.REF 110A Rapid In Situ Enzyme Linked Immunosorbent Assay for DengueVirus Antiviral Marine Seaweed ScreeningClaudia DUARTE DOS SANTOS 1 , Andrea Cristine KOISHI 1 , PaulaRODRIGUES ZANELLO 1 , Éverson Miguel BIANCO 2 , JulianoBORDIGNON 11 Instituto Carlos Chagas/Fiocruz PR, Curitiba, BRASIL; 2 UniversidadeFederal de Santa Catarina, Florianópolis, BRASILDengue (DEN) is the most important mosquito borne viral disease in theworld. The total economic burden of dengue illness in the Americas wasestimated to cost US$ 2.1 billion per year on average. In Brazil it wasreported a total of 204,650 notifications of dengue and 324 severe caseswith 33 deaths in the first seven weeks of 2013. There is no vaccine orspecific antiviral therapy for prevention and treatment of DENV infection.Hence, drug discovery research for dengue is of utmost importance. Herewe propose a target free approach for dengue drug discovery based on anovel, rapid, and economic 96 well format in situ enzyme linked immunosorbentassay which can be adapted for high throughput screening. Thein situ ELISA was standardized for Huh 7.5 cell line and infection withall four serotypes of DENV, being DENV 1, 2 and 3 clinical isolates fromBrazil and DENV 4 a laboratory strain. The in situ ELISA was comparedto the foci forming assay and the results presented a high correlation (averager2 = 0.95), furthermore statistical analysis showed an average S/B of7.2 and Z factor of 0.62; demonstrating assay consistency and reliability.The assay was used to screen the antiviral activity of a panel of fifteen seaweedextracts at the maximum non toxic dose previously determined bythe MTT and neutral red cytotoxic assays. Eight seaweed extracts showeda dengue infection inhibition in the post infection treatment when comparedto the controls, with some variations depending on the virus serotype.Among these, four extracts were chosen for further evaluation. The preinfectiontreatment showed no significant inhibition of virus infection;on the other hand, treatment during the infection was highly effective,indicating that these extracts must interfere in early steps of the virusinfection cycle. Financial support: Fiocruz, CNPq, Fundação Araucaria,CAPES.REF 111Antiviral and cytoprotective activities of fullerenols with the differentcontent of Hydroxyl groupsMikhail EROPKIN 1 , Elena MELENEVSKAYA 2 , TatyanaBRYAZHIKOVA 1 , Elena EROPKINA 1 , Daria DANILENKO 11 Reserch Institute of Influenza, Saint Petersburg, RUSSIA; 2 Institute ofHigh Molecular Compounds, Saint Petersburg, RUSSIAWe have synthesized polyhydroxy fullerenes – fullerenols with thedifferent compound of hydroxyl groups – C60(OH)12 14, C60(OH)1824 and C60(OH)30 38. The first of them was non soluble in water andhad no biological activity when it was applied in cell cultures in formof a suspension. The two other variants of fullerenol possessed a broadspectrum of antiviral activity in vitro against the actual strains of humaninfluenza virus A(H1N1), A(H3N2) and avian influenza A(H5N1),human herpes simplex virus, adenovirus and respiratory syncitial virus.Water soluble variants of fullerenol were non toxic in vitro towardshuman and animal cells of the different tissue origin. Besides the antiviralactivity fullerenols demonstrated a protective effect against the UVAinduced phototoxicity. The maximum biological actitivy for all testedindices was shown in the fullerenol variant C60(OH)18 24. The watersoluble biologically active variants of fullerenol could be useful in thepharmacology since they could serve the basis for new effective and nontoxic antiviral and cytoprotective formulations.REF 112Consecutive Alternating Administration of Antiviral Combinationsas a Highly Effective Chemotherapy of Coxsackievirus B3 InfectionsAngel S. GALABOV, Ralitsa VASSILEVA PENCHEVAThe Stephan Angeloff Institute of Microbiology, Bulgarian Academy ofSciences, Sofia, BULGARIAEnteroviruses cause a great variety of diseases – from mild respiratoryand enteric infections to severe diseases of the CNS and the heart. Due toa rapid development of drug resistance of initially drug sensitive virusesanti enteroviral chemotherapeutics for clinical use are not registered sofar. One of the possible approaches to overcome this problem is by usingcombined chemotherapy. However, the application of a standard combinationtherapy, consisting of simultaneously given drugs could lead to thedevelopment of multiple resestance. The aim of the study, described in thecurrent paper is to apply a novel approach for combined administration ofanti enteroviral compounds, consisting of a consecutive application of thesubstances in experimental infection with Coxsackieviruses B3 in vivo.The used in vivo experimental models were neurotropic and cardiotropicCVB3 infection in newborn mice. Compounds partnering in combinationswere selected as specific inhibitors of enteroviral replication with differentmode of action disoxaril, guanidine HCl and oxoglaucine. The administrationof consecutively applied triple combination resulted in 40 60%survival rate of the treated with the combination mice, infected with lethalCVB3 infections. The effectiveness of the combination is also proven bysignificant lengthening of the mean survival time. Data presented in thecurrent study prove the effectiveness of previously chosen triple consecutivelyapplied combination of anti enteroviral inhibitors. By using suchcombination the occurence of drug resistance is totally avoided.REF 11310 23 DNAzyme targeted against the M2 gene transcript of influenzaA virus significantly inhibits viral RNA translation and replicationBinod KUMAR 1 , Prashant KUMAR 1 , Roopali RAJPUT 1 , LatikaSAXENA 1 , Dibyaranjan PATI 1 , Mradul DAGA 2 , Madhu KHANNA 11 Department of Respiratory Virology, Vallabhbhai Patel Chest Institute,University of Delhi, Delhi, INDIA; 2 Department of Medicine, MaulanaAzad Medical College, New Delhi, INDIAThe influenza A virus M2 ion channel protein is highly conserved amongdifferent viral strains and is essentially required during the trafficking,assembly and budding processes of virus, thus an attractive target fordesigning antiviral drugs. We designed several 10 23 DNAzymes (Dz)targeting different regions of the matrix gene (M2) of influenza A virusesand analyzed their ability to specifically cleave the target RNA in bothcell free systems as well as in cell culture using transient transfections.The Dz that worked best was further standardized with MgCl2 in a dosedependent manner. RT PCR and real time RT PCR assays showed significant75% inhibition of M2 gene of influenza A viruses upon specific Dztreatment. The transfection of MDCK cells with Dz considerably reducedthe cytopathic effect caused by influenza A virus (A/PR/8/34 H1N1) andconsiderably reduced the M2 protein expression. As expected, the mutantDz did not hinder in virus replication showing high level of specificity ofdesigned Dz towards the target RNA. Our results demonstrate substantialreduction in whole virus replication thereby paving new dimensionsin antiviral therapy. Our study, for the first time, has documented antiviralpotential of Dz against M2 transcript of influenza A virus. Thus, wepropose that the 10 23 DNAzymes may be used as selective and effectiveinhibitor of viral RNA replication, and can be explored further fordevelopment of a potent therapeutic agent against influenza infection.Virologie, Vol 17, supplément 2, septembre 2013S149

5 th European Congress of VirologyREF 114Inhibition of Influenza A viruses by synthetic peptides derived fromthe HA steam regionRogelio LÓPEZ MARTÍNEZ 1 , Gema RAMÍREZ SALINAS 2 , JoséCORREA BASURTO 2 , Blanca L BARRÓN 11 Escuela Nacional de Ciencias Biológicas/Instituto Politécnico Naciona,Mexico City, MEXICO; 2 Escuela Superior de Medicina/Instituto PolitécnicoNacional, Mexico City, MEXICOInfluenza A viruses are, capable to cause severe human respiratory infections.Currently there are only two types of drugs to treat influenza Aviral infections, the M2 H + ion channel blockers and the neuraminidase(NAI) inhibitors. Moreover the emergence of drug resistant influenza virusstrains has catalyzed the search for new antiviral agents to complementor replace the existing drugs. The influenza surface glycoprotein hemagglutinin(HA) is a potential target for antiviral drugs development due toits key properties in the initial stage of infection: receptor binding andfusion activities of viral and endosome membranes. For that reason, wedesigned nine antiviral peptides (AVPs) against influenza A viruses, usingseveral bioinformatics tools. The AVPs were chemically synthetized andtheir antiviral activity and cytotoxic activities were tested in vitro. Wefound that AVPs derived from highly conserved sequences of HA1 andHA2 subunits of the HA steam region were capable to inhibit influenzaA viruses of human, avian and porcine origin, either H1 or H5 subtypes,without showing any cytotoxic effect. The protection of the cells againstviral infection was directly related to the AVP concentration, showing to bea dose response effect. To elucidate the probable antiviral mechanism the2D and 3D structures of each AVP was predicted and used for a dockinganalysis. The results suggest that influenza virus infection was probablyblocked by a direct interaction of the AVPs with the viral HA, interferingand preventing the HA conformational changes required to carry out itsfunction in the fusion event to allow viral entry into the cell. This strategyis a potential successful alternative for designing new anti influenza drugswhich could be developed into very effective antiviral medicaments.REF 115FK506 Binding Proteins (FKBPs) are Necessary for ReplicationHuman Coronaviruses SARS CoV, HCoV NL63 and HCoV 229E asindicated by their inhibition by the drug FK506YueMALAUER 1 , Javier CARBAJO LOZOYA 1 , Marcel A. MÜLLER 2 ,Stefan KALLIES 2 , Volker THIEL 3 , Christian DROSTEN 2 , AlbrechtVON BRUNN 11 Max von Pettenkofer Institute, Ludwig Maximilians Universität München,München, GERMANY; 2 Institute of Virology, University of Bonn,Bonn, GERMANY; 3 Institute of Immunobiology/Kantonsspital St. Gallen,St. Gallen, SWITZERLANDFK506 binding proteins represent a subset of the immunophilin proteinfamily including the structurally unrelated cyclophilins. They are expressedat high levels in almost all tissues, and they are characterized by thepresence of at least one prolyl cis/trans isomerase domain (PPIase) excertingregulatory or chaperone functions. FK506 (Tacrolimus) is a bacterialmacrolide that binds to FKBPs. It excerts two independent functions: a)The FK506 FKBP complex binds to the cellular phosphatase Calcineurin(CaN) thus inactivating the important immunologic CaN/NFAT (NuclearFactor of Activated T cells) pathway. B) Independently, FK506 inhibitsPPIase and chaperone activities of FKBPs resulting in misfolding andinactivity of cellular and also certain viral proteins in virus infected cells.We have recently shown the dependence of Coronavirus (CoV) replicationon active cyclophilin pathways (Pfefferle et al., 2011, PLoS Pathog7(10): e1002331). Here we report that the drug FK506 inhibits stronglythe growth of human coronaviruses SARS CoV, HCoV NL63 andHCoV 229E at low, non cytotoxic concentrations in cell culture (Carbajoet al., 2012, VirRes 165, 112–117). As shown by plaque titration,qPCR, Luciferase and green fluorescent (GFP) reporter gene expression,replication is diminished by several orders of magnitude. Knockdown ofthe cellular FK506 binding proteins FKBP1A (=FKBP12) and FKBP1B(=FKBP1.6) in CaCo2 cells prevents replication of HCoV NL63, suggestingthe requirement of these members of the immunophilin family forvirus growth.REF 116PLA2 CB isolated from Crotalus durissus terrificus has high directactivity against enveloped virusesVanessa MULLER 1 , Mohd Jaseem KHAN 1 , Adélia CINTRA 2 , LuizFIGUEIREDO 3 , Suely SAMPAIO 2 , Victor AQUINO 11 Laboratory of Virology Faculty of Pharmaceutical Science from RibeirãoPreto, Ribeirão Preto, BRAZIL; 2 Laboratory of Toxinology of Faculty ofPharmaceutical Science from Ribeirão Preto, Ribeirão Preto, BRAZIL;3 Virology Research Center of Faculty of Medicine from Ribeirão Preto,Ribeirão Preto, BRAZILPhospholipases represent a versatile class of enzymes that occur ubiquitouslyin nature catalyzing the hydrolysis of the glycerophospholipids atthe sn 2 position releasing free fatty acids and lysophospholipids, playinga key role in various biological activities. We have shown that PLA2 CB,isolated from Crotalus durissus terrificus, has a direct activity on dengueand yellow fever viruses (DENV and YFV). Here, we evaluated the directantiviral activity of PLA2 CB on other enveloped viruses [Rocio virus(ROCV), Oropouche virus (OROV) and Mayaro virus (MV)]. The effectof PLA2 CB was evaluated by replication inhibition, which was measuredby plaque reduction test using a virucidal strategy. The viruses (25100 PFU) were incubated with serial decimal dilutions of PLA2 CB (50to 0.00005 ng/L) for 1 h at room temperature. Subsequently, these mixtureswere used to infect the vero cells. After 1 h, the overlay medium wasadded and the plate was incubated for 5 to 10 days at 37 ◦ C. The cellswere stained with naphtol blue black for plaque counting to calculated theeffective concentration that inhibit 50% of cell infection (EC50). Consideringthe cytotoxicity concentration that kills 50% of cells (CC50) to be100 ng/L, the SI (SI=CC50/EC50) was calculated for each viruses andfound to be 3.1X104, 9.6X103 and 3.8X104 for ROCV, OROV and MV,respectively. The high SIs observed against ROCV, OROV and MV weresimilar to the SI found previously for DENV and YFV, therefore, suggestingthe potential use of PLA2 CB as a model for development of a broadantiviral drug.REF 117Novel small molecule inhibitors of the RNA Polymerase of InfluenzaA and B VirusesGiulio NANNETTI 1 , Giulia MURATORE 1 , Beatrice MERCORELLI 1 ,Serena MASSARI 2 , Laura GORACCI 3 , Gabriele CRUCIANI 3 , OrianaTABARRINI 2 , Giorgio PALÙ 1 , Arianna LOREGIAN 11 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Department of Chemistry and Technology of Drugs, University of Perugia,Perugia, ITALY; 3 Department of Chemistry, University of Perugia,Perugia, ITALYInfluenza viruses are responsible for yearly epidemics and pandemics thatcan have high mortality rates. There is an urgent need for new anti influenzadrugs, due especially to incomplete protection by vaccines and the emergenceof resistance to the current antivirals. We previously performedan in silico screening of small molecule libraries to search for inhibitorsof the protein protein interactions between the PA and PB1 subunits ofinfluenza A virus RNA polymerase and we identified a few compoundsthat are able to specifically interfere with such interactions (Muratore et al.,PNAS 2012, 109:6247 52). Among these compounds, the hits 5, 10, andS150 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology31 were selected for the design and synthesis of analogues, with the aimof increasing the potency of the compounds. Some of the analogues werenon cytotoxic in a panel of cell lines and more potent than the originalcompounds, exhibiting a strong inhibition of the physical interactionbetween the PA and PB1 subunits of influenza virus RNA polymerasecomplex, both in vitro and in cells. Moreover, the most promising analogueswere able to inhibit the replication of a panel of influenza A and Bvirus strains, including an oseltamivir resistant isolate, at low micromolarconcentrations. In addition, these compounds did not inhibit the replicationof other RNA or DNA viruses, demonstrating to possess specific antiviralactivity against influenza viruses. Taken together, our results suggest thatthese compounds could provide the basis for the development of a newgeneration of therapeutic agents against influenza A and B viruses.REF 118Changes of fermental system in organism of white mice at ExperimentalFlu under the influence of Piler lightTatyana SOVA 1 , Oksana LAGODA 1 , Valentina DIVOCHA 1Ukrainian Research Institute for Medicine of Transport, Odessa,UKRAINENormal functioning of both a human and an animal depends on variousvital power sources: light, air, waters, food and electromagnetic wavesarriving from the environment.Objective: to study the PILER (Polarized, Incoherent, Low Energy Radiation)light action on proteinase, infectious, hemagglutinating activity in anorganism of white mice at experimental flu.Materials and methods: 40 white mice (the line Balb/c, weight 13 14 gr),A/PR/8/34(H1N1) the flu virus, the device with PILER light. Infection ofmice with the flu virus has been carried out intranasal under anesthesia.The animals have been randomized into 4 groups, 10 animals in everygroup. The first group was the control one for the animals. The secondgroup of animals was exposed to the influence PILER – light (control).The third group of animals was infected with a lethal dose of the flu Avirus. The fourth group also caught a lethal dose of the flu A virus andwas exposed to the influence PILER light (treatment). Each mouse of thegroups under experiment received 11 sessions of PILER light influence.Results: proteinase, infectious, hemagglutinating activity and protein inthe lungs and blood serum of healthy mice did not differ much from thosein the group of healthy animals irradiated with PILER light. The treatmentof the animals previously infected with a lethal dose of virus A flu withPILER light detained the virus’s reproduction for a day. Infectious andhemagglutinating activity was lower in comparison with those in controlgroup for virus. 20% of animals, having been treated with PILER light,were alive by the 14th days after infection, while 100% fatality of animalsin the control group took place by the sixth days after infection.Conclusions: PILER light strengthens protective forces of an organism atflu.REF 119The crystal structure of the foot and mouth disease virus leader proteasein complex with E 64 R P NH2 provides new insights on itssubstrate specificityJutta STEINBERGER 1 , Irina GRISHKOVSKAYA 2 , Tim SKERN 11 Department of Medical Biochemistry, Max F. Perutz Laboratories, MedicalUniversity of Vienna, Vienna, AUSTRIA; 2 Department of Structuraland Computational Biology, Max F. Perutz Laboratories, University ofVienna, Vienna, AUSTRIAThe Leader protease (Lbpro) of the Foot and mouth disease virus is essentialfor the successful replication of the virus. Leaderless virus was shownto be attenuated and not able to spread within the infected host. Therefore,Lbpro represents a promising target for the development of antiviralcompounds. Though being one of the smallest members of papain likecysteine proteases, Lbpro shows great substrate specificity ranging fromP7 to P5 ′ . Moreover, Lbpro only accepts a positively charged residue ateither the P1 or the P1 ′ position; substrates containing basic residues atboth positions are inhibitory. Recent data on the specificity determinantsat the P’ side inspired the extension of E64, a well known epoxide basedinhibitor of cysteine proteases, by the dipeptides Gly Pro NH2 or Arg ProNH2. Both modified inhibitors, termed E 64 G P NH2 and E 64 R P NH2,could improve the Ki of E64 (3.4 M) to 1.3 M and 0.03 M, respectively.These findings suggest an important role for Arg at the P1 ′ position.However, the crystal structure of Lbpro in complex with the inhibitor E 64R P NH2 is lacking electron density for the side chain of the P1 ′ Arg fromthe Cgamma atom onwards, despite being in close proximity to D49 andE147. Consequently, we aim to investigate the importance of the residuesD49 and E147 for the interaction with Arg at position P1 ′ .Our findingsshould provide further knowledge to improve inhibitor design and maylead to the development of a more potent and specific inhibitor againstFMDV infections.REF 120Antiviral properties of edible berriesAizhan TURMAGAMBETOVA, Nadezhda SOKOLOVA, IrinaZAITCEVA, Andrey BOGOYAVLENSKIY, Vladimir BEREZINInstitute of microbiology and virology, Almaty, KAZAKHSTANVariety therapeutic properties of plants are caused by the presence of alarge in their composition and structure chemicals (alkaloids, glycosides,saponins, vitamins, tannins, essential oils, etc.), which have a pharmacologicaleffect on the human organism, and the cause of the disease.Therefore the study of the possibility of using of herbal remedies forthe treatment of viral infections is the actual problem of Pharmacognosyand virology. The aim of research was to study the antiviral effect ofextracts obtained from elderberry, lingonberry, barberry, bird cherry and2 grapes (“Isabella” and “Muscat”) on the activity of the influenza virusstrain A/Alma Ata/8/98 (H3N2). Choice of plants was due to the presenceof polyphenolic compounds belonging to the different chemical nature.The antiviral activity of the research berries in the dose range of 1 to100 mg/kg body weight was studied for their ability to inhibit the reproductionof 100 infectious doses of influenza virus. It is shown that in dose of100 mg per kg extracts derives from berries of elderberry, barberry, lingonberryand bird cherry exhibit high virus inhibitory and virucidal properties,enabling their use as additional antiviral therapy. Extracts obtained fromdifferent grape varieties, in the tested range of doses did not show antiviralactivity.REF 121Virucidal activity of Morus spp. plant extracts on Caliciviridae: theimpact on a human norovirus surrogateMihayl VARBANOV 1,2 , Ines THABTI 1,2,3 , Thomas KASSAB 1,2 , AliAFERCHICHI 3 , Raphael E. DUVAL 1,21 CNRS, UMR 7565, SRSMC, Nancy, FRANCE; 2 Université de Lorraine,Faculté de Pharmacie, Nancy, FRANCE; 3 Laboratoire d’Aridoculture etCultures Oasiennes, Medenine, TUNISIAHuman noroviruses (HNoV) are highly pathogenic viruses belonging tothe Caliciviridae family, responsible for worldwide epidemic outbreaksof gastroenteritis, and thus representing a heavy burden to infants andimmunologically disadvantaged hosts. To date, there is still no specifictreatment for HNoV. In this aspect, natural products offer an alternativein terms of innovative drug therapies. The object of this study was todetermine the antiviral properties of leaves and stem bark of the mulberryVirologie, Vol 17, supplément 2, septembre 2013S151

5 th European Congress of Virologytree (Morus spp.), known for their multiple biological activities. In ourapproach we used feline calicivirus F9 (FCV F9), a member of the familyof Caliciviridae, like a norovirus surrogate, as the culture of HNoV remainsdifficult in standard laboratory conditions. The antiviral activity of 12water and water: alcohol plant extracts of leaves and stem bark of threedifferent species of mulberry from southern Tunisia Morus alba var. alba,Morus alba var. rosa and Morus rubra were tested on FCV F9. The resultsshowed that two particular water: alcohol extracts, one of stem bark ofMorus rubra and one of leaves of Morus alba var. rosa were the mosteffective in reducing viral titer and cytopathogenic effects (CPE) inducedby the virus. Both extracts showed reduction factors of FCV F9 titers by6 and 4, respectively. Given their anti FCV F9 activity the extracts werealso tested for virucidal potential and both displayed important virucidaleffect leading to 1.8 and 2.7 fold reduction of the CPE respectively, thusoffering promising applications in antiviral strategies.REF 122Inhibition of dengue viruses by siRNAs targeting the NSB4 and NS5coding regionsPaula VILLEGAS, Elizabeth ORTEGA, Alfonso MÉNDEZ TENORIO,Blanca Lilia BARRÓNEscuela Nacional de Ciencias Biológicas/Instituto Politécnico Nacional,Mexico City, MEXICODengue viruses (DENV 1 4) represent the major arthropod borne viralinfection in the world. The main problem is that the infection with a secondserotype different to which infected the first time increases the risk todevelop hemorrhagic disease and until now, there is no vaccine or effectivedrug against the four DENV serotypes. For this reason, the objective of thiswork was to design a siRNA capable to block the four types of DENV to beproposed as therapy against all DENV infections. To design the siRNAs,we chose the most conserved regions of DENV genome, the NS4B andNS5 coding proteins. Three siRNAs were designed: siRNA 1 (NS4B) andsiRNA 2 and 3 (NS5), synthetized and tested against DENV. For this, Verocells were transfected separately with each one of the siRNAs; 6 h after,the cells were infected with 100 TCID50 of DENV 1, 3 or 4 and incubated.After five days, the viral protein NS1 was detected on the supernatant byusing Platelia kit. The siRNA 1 reduced the expression of the NS 1 proteinby 99.88% for DENV 1, 99.9% for DENV 3 and 99.98% for DENV4, similar results were found with the siRNA 3, for DENV 1 (99.9%),for DENV 3 (99.96%) and for DENV 4 (99.92%); however the siRNA2 induced protection only for DENV 3 (99.9%), and DENV 4 (99.9%)but not for DENV 1. All siRNAs are currently been tested for DENV 2inhibition. In conclusion, siRNAs targeting the NS4B (siRNA 1), and NS5(siRNA 3) coding regions were capable to silence the infection of threeDENV serotypes, and these siRNAs could be used to develop a specifictreatment for dengue viruses infection.REF 123From SARS Coronavirus (SARS CoV) Orfeome to Cellular Targets:Inhibition of Cyclophilins by Cyclosporine A (CsA) and Nonimmunosuppressive Derivatives Results in Pan coronaviral ReplicationInhibition, including the new HCoV EMCAlbrecht VON BRUNN 1 , Javier CARBAJO LOZOYA 1 , DoreenMUTH 2 , Yue MA LAUER 1 , Marisa KURZ 1 , Brigitte VON BRUNN 1 ,Mingxia SU 1 , Marcel A. MÜLLER 2 , Christel SCHWEGMANNWEELS 3 , Heinz Jürgen THIEL 4 , Miroslav MALEŠEVIC 5 , Thomas F.BAUMERT 6 , Gunter FISCHER 7 , Jürgen HAAS 8 , Christian DROSTEN 21 Max von Pettenkofer Institute/Ludwig Maximilians Universität München,München, GERMANY; 2 Institute of Virology,University of Bonn, Bonn,GERMANY; 3 Institute of Virology, Tierärztliche Hochschule Hannover,Hannover, GERMANY; 4 Institute of Virology, Veterinärmedizin/Justus LiebigUniversity Gieβen, Gieβen, GERMANY; 5 Institute of Biochemistry,Martin Luther University Halle Wittenberg, Halle, GERMANY; 6 InsermU1110, Institut de Virologie, Université de Strasbourg, Strasbourg,FRANCE; 7 Max Planck Institute for Biophysical Chemistry Goettingen,BO Halle, Halle, GERMANY; 8 Division of Pathway Medicine, Universityof Edinburgh, Edinburgh, UNITED KINGDOMCoronaviruses (CoV) are important human and animal pathogens thatinduce severe respiratory, gastrointestinal and neurological disease. Theoutbreak of the severe acute respiratory syndrome (SARS) in 2002/2003has demonstrated human vulnerability to CoV epidemics. Neither vaccinesnor therapeutics are currently available against zoonotic CoV. Knowledgeof host cell proteins that are involved in pivotal virus host interactionscould define novel broad spectrum antiviral targets. Using a systems biologyapproach, we identified prolyl cis/trans isomerases of the cyclophilinfamily as interaction partners of the CoV Nsp1 by genome wide yeasttwo hybrid interaction screening (Pfefferle et al., 2011, PLoS Pathog 7:e1002331). Nsp1 interaction with cylophilins modulated the calcineurin(CaN)/NFATpathway and cytokine secretion, a key feature of viralimmunopathogenesis. However, inhibition of the prolyl cis/trans isomeraseactivity of cyclophilins by CsA, might also contribute to inhibit thereplication of CoVs of all genera, including SARS CoV, HCoV 229Eand NL63, feline CoV (FCoV), porcine transmissible gastroenteritis virus(TGEV), mouse hepatitis virus (MHV) as well as avian infectious bronchitisvirus (IBV). Outbreak of a novel group C beta CoV (HCoV EMC) fromthe Arabian Peninsula, which recently has attained worldwide attention as“SARS like” CoV is inhibited by CsA. Consequently, CaN inactive CsAderivatives, which showed reduced immunosuppressive activity (CsD)or nonimmunosuppressive CsA derivatives might serve as broad rangecoronaviral inhibitors including HCoV EMC.REF 124Combinational antiviral therapy with Carragelose and Zanamivir issynergistically active against influenza A infectionMartina KURZ 1 , Christine GRAF 1 , Marielle KÖNIG SCHUSTER 1 ,Christiane KOLLER 1 , Benjamin REUTTERER 1 , Norman KIRCHOFF 1 ,Sabine NAKOWITSCH 1Marinomed Biotechnologie GmbH, Vienna, AUSTRIAAcute viral respiratory tract infections (ARTI) are a global burden to publichealth. ARTI are caused by single or mixed infection with various respiratoryviruses, including rhino (hRV), corona (hCoV), respiratory syncytial,and influenza viruses (IV). Therefore, a broad antiviral therapy is needed.A nasal spray containing Carragelose has shown antiviral effectivenessagainst a broad variety of respiratory viruses in several clinical trials.Zanamivir, a neuraminidase inhibitor, is effective against IV infectionsalso when applied intranasally. A potential synergistic antiviral activity ofa therapy consisting of Carragelose and Zanamivir was evaluated. In vitrothe antiviral effectiveness of Carragelose against hRV and hCoV is maintainedin the combination formulation. Compared to Zanamivir alone,the combination product is 50 times (H7N7), two (H1N1/09pdm), three(H5N1) or even five (H3N2) orders of magnitudes more potent in inhibitingIV infection. This synergistic effect was confirmed in vivo. Lethalmouse models were established for IV A H1N1/09pdm and H7N7 infections.Mice were treated intranasally two times a day for five days withCarragelose, Zanamivir or the combination product. For both IV subtypesthe survival rate of mice was significantly higher for the combination productcompared to Zanamivir alone. The therapy was effective even whenthe treatment started as late as 48 (H7N7) or 72 (H1N1/09pdm) hours afterinfection. We conclude that intranasal administration of the combinationproduct is a promising option for the prophylaxis and therapy of ARTI.S152 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF 125Resveratrol Inhibition of Human Rhinovirus ReplicationChiara NARDIS 1 , Chiara NARDIS 1 , Elena MATTIA 1 , Alessandra DELEO 1 , Antonio FRANCIOSO 2 , Luciana MOSCA 2 , PaolaMASTROMARINO 11 Department of Public Health and Infectious Diseases, Sapienza University,Rome, ITALY; 2 Department of Biochemical Sciences, SapienzaUniversity, Rome, ITALYRhinoviruses are the major etiological agents of common cold and asthmaexacerbations. However, no specific therapy is available. Resveratrol (RV)shows protective properties against several viral infections. We evaluatedthe antiviral effect of RV towards human rhinovirus 16 (HRV 16) in two differentcellular models: cell culture monolayer and human nasal epithelium,composed by basal, ciliated and mucus cells. Infection was established assingle or multiple cycles of virus replication. Viral yield in the presenceof different non cytopathic concentrations of RV was assessed by plaqueassay. In both cellular systems, RV showed a strong inhibitory effect onHRV 16 infective particles production, that appeared to be dose and timedependent. It has been reported that HRV 16 induces activation of NF kBwhich in turn promotes the transcription of ICAM 1, the major receptor forHRVs. We examined the effect of RV on the HRV 16 induced expressionof ICAM 1 and viral capsid proteins by immunofluorescence and westernblot assay. RV treatment inhibited viral protein synthesis and the HRV 16induced increase of ICAM 1 level. Since HRV 16 replication induces theproduction of pro inflammatory cytokines, we also evaluated the effects ofRV on IL 6, IL 8, and RANTES levels in human nasal epithelia by ELISAassay. HRV 16 induced increase of proinflammatory cytokines was reversedby RV treatment. These results suggest that RV may have a possibleclinical application in HRV infections, by reducing viral replication andthe related inflammatory symptoms.REF 126Antiviral Drug Design Platform (AD2P): screening of dengue virusinhibitorsJustine LETIENNE 1 , Cécilia EYDOUX 1 , Sabine MILHAS 1,3 , KarineALVAREZ 1 , Barbara SELISKO 1 , Xavier MORELLI 3 , GillesQUERAT 2 , Bruno CANARD 1 , Jean Claude GUILLEMOT 11 CNRS, AFMB, UMR 7257, ESIL, Marseille, FRANCE; 2 UMR 190,Unité des Virus Emergents, Faculté de Médecine de Marseille, Marseille,FRANCE; 3 Centre de Recherche en Cancérologie de Marseille (CRCM),CNRS UMR7258/INSERM U1068, Marseille, FRANCEWorldwide resurgence of dengue virus (DENV) infections is a majorconcern and few therapeutic treatments are available. The virus groupconsists of 4 serotypes and can induce similar symptoms ranging frommild febrile illness to a life threatening dengue hemorrhagic fever. Antiviralscreens have proved useful for the identification of novel humanemerging virus inhibitors. A screening workstation has been developed totest chemical compounds, based on the dynamic interface between threeplatforms: Interactions, Dynamics and Drug design platform (INT 3D);Screening platform Marseille Luminy (PCML) and Virology screeningplatform Marseille Timone (PCVMT). Our team (PCML) combines biochemicaland cell based screening assays to select efficient compounds(chemically synthesized or natural extracts). Our approach consists infocusing on non structural proteins of DENV that are essentials for viralreplication. A first strategy is to target non structural proteins NS5 and NS3to address the impact of drugs on enzymatic activities such as polymeraseor helicase activities. We are also interested in protein protein interactions(PPIs) virus network: viral (NS5/NS3) or cellular; in order to screenprotein protein interaction inhibitor (2P2I) library via HTRF or BRETassays. In addition, the data obtained in vitro are all validated in cellularcontext with a DENV replicon system and confirmed on cells infectedby dengue clinical isolates to assess inhibition effects and cytotoxicity ofcompounds. These strategies lead to the identification of promising DENVinhibitors.REF 127Polyclonal and monoclonal antibodies against human parechovirus 1and 3Brenda WESTERHUIS 1 , Ksm BENSCHOP 1 ,GKOEN 1 ,AqBAKKER 2 , Y CLAASSEN 2 , T BEAUMONT 2 , Kc WOLTHERS 11 Laboratory of Clinical Virology, Department of Medical Microbiology,Academic Medical Center, Amsterdam, THE NETHERLANDS; 2 AIMMTherapeutics, Academic Medical Center, Amsterdam, THE NETHER-LANDSThe immune response against picornaviruses is mainly humoral, andprotective neutralizing antibodies (Abs) are thought to be genotype specific.Human parechoviruses (HPeVs) are commonly detected picornavirusinfections in children. We observed that HPeV3 was difficult to neutralizein vitro, in disagreement with what is known for picornaviruses. The aimwas to study HPeV neutralization and develop neutralizing Abs againstHPeVs. Polyclonal Abs were obtained by rabbit immunization. Humanmonoclonal Abs were generated by generation of stable antibody producingB cells by genetic programming (AIMSelect). All Abs were testedfor neutralization in different cell lines. Polyclonal rabbit and monoclonalhuman antiHPeV1 Abs neutralized HPeV1 with high titers (>1:1024)and cross neutralization was found with HPeV2 (>512). In contrast, polyclonaland monoclonal aHPeV3 were not able to fully neutralize HPeV3infection in different cell lines. Although viral replication was inhibited tosome extent in the presence of Abs: the polyclonal aHPeV3 showed viralinhibition up to titer 1:256, and one monoclonal aHPeV3 inhibited replicationup to 1:32. In this study we generated highly neutralizing HPeV1Abs, unexpected both mAbs showed clear cross reactivity against HPeV2.Thus, cross neutralisation can occur in human picornaviruses, and therebypossibly cross protection in vivo as well. The generation of HPeV3 nAbswas difficult, and in line with our previous results. This finding needsfurther exploration.REF 128Mutation in the RNA-dependent RNA polymerase of Chikungunyavirus is responsible for resistance to T-705Nidya Segura GUERRERO 1,5 , Leen DEALNG 1 , Gilles QUERAT 2 ,Boris PASTORINO 2 , Mathy FROEYEN 6 , Kai DALLMEIER 1 , DirkJOCHMANS 1 , Felio BELLO 5 , Ali TAS 4 , Eric J. SNIJDER 4 , XavierDELAMBALLERIE 2 , Martina BRYON 3 , Johan NEYTS 1 , Martijn J.VAN HEMERT 4 , Pieter LEYSSEN 11 KU Leuven, Leuven, BELGIUM; 2 Université de la Méditerranée,Marseille, FRANCE; 3 Erasmus Medical Center, Rotterdam, THENETHERLANDS; 4 Leiden University Medical Center, Leiden, THENETHERLANDS; 5 Universidad del Rosario, Bogotá DC, COLOMBIAChikungunya virus (CHIKV) induced disease is an acute febrile illnesscharacterized by arthritis, arthralgia or severe joint pain that can persistfor several months up to years after the infection. There are no antiviraldrugs available for the treatment or prevention of CHIKV infections.Favipiravir, a nucleobase mimetic also known as T-705, is an effectiveand selective inhibitor of the replication of a broad spectrum of RNAviruses. We report here for the first time on the selective antiviral activityof T-705 on CHIKV replication and alphaviruses in general in tissueculture with median activities (EC50) in the range of 2-25 M observedfor lab as well as a panel of clinical strains of CHIKV. Additionally, firstproof-of-concept of the anti-CHIKV effect of T-705 is demonstrated invivo. In the interferon receptor-deficient (AG129) mice oral administrationof T-705 (300 mg/kg.day) reduced CHIKV induced mortality whenVirologie, Vol 17, supplément 2, septembre 2013S153

5 th European Congress of Virologyused in both a pre- and post-exposure scenario. Currently, the precisemechanism of action of T-705 has still to be unraveled. We provide firstexperimental proof that a single mutation in the RNA-dependent RNApolymerase (RdRP) is responsible for phenotypic resistance to T-705.To that end we generated CHIKV variants that were resistant to T-705and its analogue T-1105 and were found to bear mutations in viral nonstructuralproteins. Moreover, by reverse genetics a single K291R aminoacid change in the F1 motif of the viral RdRP could be shown to be responsiblefor full (∼2-fold) phenotypic resistance to the antiviral effect ofT-705.This research was funded by the European Union (FP7/2007-2013 SIL-VER n o 260644).REF 129Anti-H5N1 Specific Polyclonal Immunoglobulins are Efficient to Neutralizeand to Block H5N1 Infection: A New Approach for the ClinicalManagement of H5N1 Infected PatientsC.H. HERBRETEAU 1 , P. BUCHY 2 , F. JACQUOT 3 , C. DURAND 1 ,S. RITH 2 , L. VACHER 1 , M. JOLIVET 1 , V. LOTTEAU 1 , H. RAOUL 3 ,V. DEUBEL 2 , J.F. SALUZZO 1 , B. LÉPINE 11 Fab’entech, Lyon, FRANCE; 2 Institut Pasteur in Cambodia, Phnom Penh,CAMBODIA; 3 INSERM Jean Mérieux BSL4 Laboratory, Lyon, FRANCEHighly pathogenic avian influenza virus (H5N1) remains a major globalhealth concern, with over 630 human cases reported since 2003.In this context, Fab’entech has developed an anti-H5N1 post-exposuretreatment based on the use of highly purified specific polyclonal immunoglobulinsfragments (Fabenflu ® ). Microneutralization in vitro assay wasused to investigate the neutralization activity of anti-H5N1 immunoglobulinfragments derived from horse plasma immunized against H5N1A/Vietnam/1194/04. 100 TCID50 of 21 H5N1 strains belonging to variousclades were incubated with a range of dilution of immunoglobulins andthen transferred onto MDCK cells for neutralization analysis. The resultsshowed a high level of neutralization of all the strains, isolated in differentpart of the world and representative of 10 years evolution of the avianH5N1 virus, thus confirming the excellent cross-neutralization activityof this product against a large panel of H5N1 strains. Efficacy was alsoinvestigated in multiple in vivo experiments in BALB/c mouse intranasallyinfected at Day 0 with 1LD50 or 10LD50 of A/Vietnam/1194/04 strain.An administration protocol consisting of 5 consecutive injections followingvirus inoculation (Day1 to Day 5) and an optimal dose of 10 IU/kgin mice was demonstrated to be efficient in reducing and delaying animalmortality. These data, associated with safety and pharmacokinetics data ofa phase 1 clinical trial, demonstrate the excellent potential of Fabenflu ® tomarkedly reduce the mortality and morbidity associated with H5N1 avianinfluenza infection in human.S154 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology09. ANTIVIRAL THERAPY ANDRESISTANCE TO CHRONIC VIRALINFECTIONPosters: REF 130 to REF 146REF 130Humanized antibody targeting the envelope protein from HERV Wendogenous retrovirus in ongoing clinical trials for Multiple SclerosisHervé PERRON 1,2,3 , Jean Baptiste BERTRAND 2 , JaquesPORTOUKALIAN 3 , Reza FIROUZI 3 , Jack VANHORSSEN 4 , CorinneBERNARD 1 , Raphaëlle GERMI 5 , Patrice MORAND 5 , PatriceMARCHE 6 , Jean Louis TOURAINE 3 , Alois LANG 1 , FrançoisCURTIN 11 Geneuro, Geneva, SWITZERLAND; 2 Geneuro Innovation, Lyon,FRANCE; 3 Lyon1 Unviversity, Lyon, FRANCE; 4 VU University, Amsterdam,THE NETHERLANDS; 5 Joseph Fourier University & Hospital,Grenoble, FRANCE; 6 INSERM U823, Grenoble, FRANCEHERV W family retains elements expressing an envelope protein (Env),which activates inflammation through Toll Like receptor 4 (TLR4) onantigen presenting cells. HERV W/Env was evidenced by several independentRT PCR and immunohistological studies in MS brain lesions.About 75% of MS patients had Env antigenaemia in serum, as confirmedby quantitative RT PCR in blood lymphoid cells. Immunohistologyof MS brain lesions showed expression of HERV W/Env in perivascularmacrophages and microglia. The animal model for MS, ExperimentalAllergic Encephalomyelitis, was induced by HERV W/Env in mice. Importantinflammatory demyelination was revealed by MRI and by histology.Anti myelin autoimmunity was also demonstrated. A Humanized antiHERV W/Env monoclonal antibody preventing activation of TLR4 byHERV W/Env displayed therapeutic and preventive effects in HERVW/Env induced mouse model. The Phase I and IIa clinical trials havenow been validated and the first assessment of a therapeutic agent targetinga human endogenous retroviral protein is ongoing in MS patients.This antibody offers a strategic position in MS therapy, as (i) targetingan abnormally expressed immunopathogenic protein from an endogenousretrovirus, monitored by detection in patients, (ii) blocking the pathogeniceffect of this protein upstream the TLR 4 immune cascade activation, aswell as its direct effects on brain endothelial cells and on remyelinatingoligodendrocytes, and, (iii) without affecting the physiological functionsof the human immune system as the majority of present treatments.REF 131The Efficacy of Colloidal Silver “ASAP” In Ameliorating Hepatitis BSurface Antigen Inffection Among Clinic Antendees in Abuja NigeriaOlu Joseph AJOBIEWE 1 , Semiyu OLAGOLDEN 2 , O. ODUNZE 21 NATIONAL HOSPITAL, ABUJA F.C.T, NIGERIA; 2 IMOSTATE UNI-VERSITY, OWERRI, NIGERIAIntroduction: several antiviral agents had failed to significantly clearhepatitis B Surface antigen in the systems of infected “ in” and “out”patients from our past clinical experience. This prompted us to test theeffect of Silver solution (ASAP) for this purpose. Study aim: to test theefficacy of colloidal Silver “ASAP” in ameliorating Hepatitis B Surfaceantigen infection among our clinic attendees. Study design/methods: thesetting of this prospective study was in Lugbe Federal Housing Estate inAbuja Municipal Area Council (AMAC) in Abuja, the federal capital cityof Nigeria. The patients recruited for the study were mostly artisans andfew low cadre civil servants living in and around Lugbe and its environ.Sixty(60) people, making up of 40 men and 20 women were involved in thestudy who were highly reactive to the HbsAg serology test at the beginningof the study. They were also presenting with clinical signs and symptomsof hepatitis infection. Each of them was evaluated on various appointmentdays after silver solution (ASAP) 5mls per day was administered on them.Result: significant decline in reactivity to HbsAg serology test (p

5 th European Congress of Virologyof 17.3 per strain. At the protein level, 30 amino acid changes, correspondingto 22 different positions within pUS28, were identified, that is 6.2%of the total codons of the protein. Seven changes were located in putativefunctional motifs and 9 were unpreviously reported. All these polymorphismswere distributed evenly alongside pUS28. Overall, the frequency ofthese changes ranged from 2.4% to 56.1%. Among drug resistant viruses,22 out of the 30 polymorphisms found in drug sensitive viruses were evidencedtogether with 9 additional ones. Three novel polymorphisms werelocated in putative functional motifs. The distribution of US28 genotypeswas similar among drug sensitive and drug resistant viruses. In conclusion,the polymorphism of CMV US28 chemokine receptor is low and does notseem to be affected by CMV susceptibility to current antivirals.REF 134Genotypic characterization of human cytomegalovirus terminaseDavid BOUTOLLEAU 3 , Léa PILORGE 1 , Sonia BURREL 2,3 , ZaïnaAÏT ARKOUB 3 , Henri AGUT 2,31 Service de Virologie, Hôpital Pontchaillou, CHU de Rennes, Rennes,FRANCE; 2 UPMC Univ Paris 06, ER1 DETIV, Paris, FRANCE;3 Service de Virologie, Groupe Hospitalier Universitaire La Pitié Salpêtrière,CharlesFoix, Paris, FRANCEHuman cytomegalovirus (CMV) DNA packaging involves the CMV terminasecomposed of large UL56 and small UL89 subunits. Letermovir(AIC246), a novel anti CMV agent currently tested in phase II clinicaltrial, is able to interfere with CMV terminase. This work aimed to performthe genetic analysis of CMV terminase in genotypically characterized drugsensitive (n=33) and drug resistant (n=30) viruses. Full length CMV UL56and UL89 genes were amplified, sequenced, and compared to that of referencestrain AD169. Among drug sensitive viruses, interstrain identityvaried from 98 to 100% for both genes, with mean numbers of nucleotidemutations of 25.1 and 17.1 per strain for UL56 and UL89, respectively.At the amino acid level, 20 and 8 changes were identified in pUL56 andpUL89, respectively, that is 2.4% and 1.2% of the total codons of the proteins.Overall, the frequency of these changes ranged from 3% to 100%.Seven changes in pUL56, including 1 deletion, and 2 in pUL89 wereunpreviously reported. With regards to the putative functional domainsof CMV terminase subunits, all amino acid changes were located outsideconserved regions, except S345A in pUL89. Conversely to pUL89,polymorphisms in pUL56 clustered mainly within two variable regions.Among drug resistant viruses, 19 out of the 28 polymorphisms foundin drug sensitive viruses were evidenced. Three changes were located inconserved domains (G649D and H698Q in pUL56, A659 V in pUL89).In conclusion, CMV terminase exhibits a low polymorphism and does notseem to be likely impacted by resistance to current antivirals.REF 135Genetic analysis of herpes simplex virus type 1 UL5/UL52 helicaseprimase complexSonia BURREL 2 , Marianne COLLOT 1,2 , Zaïna AIT ARKOUB 2 , HenriAGUT 1,2 , David BOUTOLLEAU 1,21 UPMC Univ Paris 06, ER1 DETIV, Paris, FRANCE; 2 Service de Virologie,Hôpitaux Universitaires La Pitié Salpêtrière Charles Foix, AP HP,Paris, FRANCEAmenamevir (ASP2151, Astellas Pharma) is a non nucleoside drug thatinhibits herpes simplex virus type 1 (HSV 1) UL5/UL56 helicase primase(HP) complex involved in viral DNA replication. Previous data evidencedsome amino acid changes associated with reduced susceptibilities of HSV1 to amenamevir in both subunits of HP complex that might alter viralreplication and pathogenicity. This work aimed to perform the genotypiccharacterization of HSV 1 HP complex among 26 drug sensitive isolates,6 acyclovir resistant isolates and KOS laboratory strain. Full length UL5and UL52 genes were amplified, sequenced, and compared to that of HSV1 reference strain 17. Among drug sensitive isolates, interstrain identityvaried from 99.4% to 99.9% for both genes, with mean numbers of nucleotidemutations of 8.9 and 12.4 per strain for UL5 and UL52, respectively.At the amino acid level, 21 and 26 changes were identified in pUL5 andpUL52, respectively, that is 2.4% and 2.5% of the total codons of theproteins. Of note, 1 isolate harbored a deletion at codons 695 696 betweenpUL52 conserved domains III and IV. For both proteins, all changesidentified lied within nonconserved regions, except V347I in pUL5. Theanalysis HP complex among 6 acyclovir resistant isolates showed polymorphismspreviously reported in drug sensitive isolates together with 6undescribed changes that were located outside functional and conservedregions, except M766 V in pUL52. This work confirms that UL5/UL52HP complex is highly conserved in HSV 1 clinical isolates and constitutesa legitimate antiviral target.REF 136Resistance development in HIV 1 infected individuals failing ARVtherapy: investigation of Gag and Pol polyproteins functional roleIlaria CARLI 1 , Claudia DEL VECCHIO 1 , Saverio Giuseppe PARISI 1 ,Federico DAL BELLO 1 , Arianna CALISTRI 1 , Giorgio PALÙ 1 , CristinaPAROLIN 21 Department of Molecular Medicine, Padua, ITALY; 2 Department of Biology,Padua, ITALYThe introduction of Antiretroviral Therapy (ARV) in the cure of AIDShas decreased the morbidity and mortality rate of HIV 1 positive patients.Nevertheless, since therapy is required lifelong, a significant minority ofpatients are likely to develop resistance to at least one class of drugs. Themechanisms of resistance mainly involve mutations altering the interactionof viral enzymes and inhibitors. Recent studies reveal that, besidesthe enzymes encoding ones, other regions might contribute to the developmentof resistance. In particular, some specific cleavage and non cleavagesite mutations in Gag increase polyprotein processing, thus compensatingfor the catalytic loss of the viral Protease (PR) functional activity inducedby primary resistance mutations. In this context, we are interested instudying the functional role of HIV 1 Gag in the viral life cycle of ARVselected viruses. We analyzed clinical samples of HIV 1 infected patientsfailing PR Inhibitors (PIs) and RT Inhibitors (RTIs) among a cohort offive infectious diseases units located in Veneto in northeastern Italy. Weoptimized PCR amplification and sequencing conditions for gag and polgenes. The relative contribution of Gag, PR and/or RT mutations on virusreplication ability and their interdependence are now examined using HIV1 molecular clones carrying patient derived sequences. Our results wouldcontribute to better characterize the role of Gag and the relations with PRand RT in resistance development, their relevance in viral replication andevolution in the presence or in the absence of drugs.REF 137Design, Synthesis and Biological Evaluation of New 1,3 Diarylpropenonesas Single Site Dual Inhibitors of HIV 1 RTFrancesca ESPOSITO 1 , Rita MELEDDU 1 , Valeria CANNAS 1 , SimonaDISTINTO 1 , Claudia DEL VECCHIO 2 , Giulia BIANCO 1 , AngelaCORONA 1 , Filippo COTTIGLIA 1 , Stefano ALCARO 3 , CristinaPAROLIN 4 , Elias MACCIONI 1 , Enzo TRAMONTANO 11 Department of Life and Environmental Sciences, University of Cagliari,Cagliari, ITALY; 2 Department of Molecular Medicine, University ofPadova, Padova, ITALY; 3 Department “Salvatore Venuta”, Universityof Catanzaro, Catanzaro, ITALY; 4 Department of Biology, University ofPadova, Padova, ITALYA small library of 1,3 diaryl propenones has been designed, and synthesizedas dual inhibitors of both HIV 1 reverse transcriptase DNA polymeraseand ribonuclease H associated functions. Compounds were assayed onS156 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologythese enzyme activities and exhibited dual inhibition properties in the lowmicromolar range. Interestingly, amino acids mutations in the Non Nucleosideinhibitors binding pocket strongly affected the propenone derivativesRNase H inhibition, suggesting long range RT structural effects, withoutreducing their DNA polymerase inhibition ability. Thus we performedbiochemical and computational analyses in order to clarify their mode ofaction. Notably, these studies indicated that propenone derivatives are characterizedby a novel mechanism of action and bind to two different allostericinterdependent pockets. Hence, these results set the basis for the furtherdevelopment of RT inhibitors interacting with a novel RT binding site.REF 138Initial study on the mode of action of castalagin against herpes simplexvirusAngel S. GALABOV 1 , Neli VILHELMOVA 1 , Stephane QUIDEAU 21 The Stephan Angeloff Institute of Microbiology, Bulgarian Academyof Sciences, Sofia, BULGARIA; 2 Universite de Bordeaux, Institut desSciences Moleculaires, Pessak Cedex, FRANCEOur previous studies showed that castalagin, a nonahydroxyterphenoylbearing C glycosidic ellagitannin, manifests a significant antiviral activityagainst HSV 1 and HSV 2 strains both sensitive and resistant toACV. Moreover, the combination effect of ellagitannins with acyclovirwas shown as a markedly synergistic, especially the effect of this combinationtowards the replication of HSV 1 sensitive strain [Vilhelmova et al.,Antiviral Res. 89, 2011, 174 181; Antiviral Res. 90/2, 2011, N144, A64A65]. Here we present results of the initial step of a clearing up the modeof antiviral activity of castalagin on the replication of HSV 1 (Victoria,an ACV sensitive strain) in MDBK cells, including (i) timing of additionstudy in the one step growth cycle setup (MOI=1); (ii) the effect on theviral adsorption in MDBK cells, and (iii) the effect on the extracellularHSV virions. The susceptible to castalagin period of the virus groth cycleembraces markedly the initial first six hours post virus adsorption, as recordedat the 24th h: an inhibitory effect exceeding 3 logs when the compoundwas added immediately after or at the 1st and 2nd h post virus inoculation,and about 2 logs within the 3 h – 6 h time interval. The compound additionafter the 12th h manifested a negligible activity. Castalagin at the concentrationof 10 M (MTC) showed a marked effect on virus adsorption: log1.7 on the 30 min, 2.2 logs on the 45 min and 3.2 logs on the 60th min. Awell expressed inhibitory effect of 2 logs was registered by 1 M atthe60 min. At a concentration of 0.1 M effect the effect was clearly weaker(log 1) and at a concentration of 0.01 M a suppression of virus adsorptionwas not observed. The compound showed a marked direct inactivatingeffect on extracellular HSV 1, which is markedly temperature dependent.The effect was significantly higher at 37oC: castalagin 10 M decreasedthe infectious virus by log 1.6 at an exposure time of 30 min, 1.8 logs at60 min, 2 logs at 90 min, and 2.2 at the 120 min. This activity was stronglydose dependent and exposure time dependent: 30 min at the castalaginconcentrations of 10 M and 1 M, 60 min at 0.1 M and 0.01 M.REF 139Association study of IL28B: rs12979860 and rs8099917 polymorphismswith response/resistance to standard treatment of Moroccanspatients infected with hepatitis C virusNadia KANDOUSSI, Mohamed Rida TAGAJDID, BouchraBELFQUIH, Hicham ELANNAZ, Saâd MRANILaboratory of Virology, HMIM V, Faculty of Medicine and PharmacyMohamed V – Souissi University, Rabat, MOROCCOIntroduction: recently, four genome wide association studies (GWAS)have identified single nucleotide polymorphisms (SNPs) near the IL28Bgene (encoding IFN 3) to be strongly associated with spontaneous andtreatment induced clearance of HCV infection. The purpose of this studywas to investigate the association between two IL28B polymorphisms(rs8099917 and rs12979860) and response/resistance to standard treatmentof Moroccan patients infected with hepatitis C virus and allelic frequenciesof these polymorphisms answered most in a Moroccan population. Materialsand methods: this study was conducted on retrospective data of 265adult patients with chronic HCV infection who were diagnosed by antiHCV antibodies (ELISA). Data collection was performed with an Excelbased case report form laboratory, between January 2011 and December2012. All Patients had been treated with bitherapy IFN/RIBAVIRIN. 159patients had a sustained virologic response and 106 patients have persistentHCV viral load. Genotyping for the IL 28B rs12979860 C/T andrs8099917G/T polymorphisms was performed by High Resolution Meltingtechnical “HRM”. Early virological response (EVR) (12 weeks), endof treatment response (EOTR) (48 weeks), sustained virological response(SVR) (72 weeks) and relapse were evaluated using conventional andquantitative polymerase chain reaction (PCR) assays. Results and discussion:the frequency of the C allele in the population was 39%. Overall, 43%of patients experienced SVR. The IL28B CC genotype was significantlyassociated with higher treatment response rates and a lower relapse ratecompared to the other genotypes [84% vs. 58% EVR, 92% vs. 63% EOTR,76% vs. 38% SVR]. Thus, the IL28B genotype appears to be a strong predictorof SVR. This study, together with similar research examining otherSNPs, should help to define adequate protocols for the treatment of patientsinfected with HCV genotype 1, especially those with a poor prognosis.REF 140Prognostic role of Hepcidin level in Cchronic Hepatitis C virus patientson Interferon therapyNashwa KHADR, Hadier OKASHA, Hanan NOUH, Walaa SEMARYFaculty of Medicine University of Alexandria, Alexandria, EGYPTHCV is one of the most important health issues worldwide. Egypt has thehighest prevalence of hepatitis C in the world estimated about 15% 20%PEG–IFN plus ribavirin combination therapy became the gold standardin the treatment of HCV this treatment enhances sustained virologicalresponse (SVR)There are several host and viral factors aid in predictingresponse to treatment Hepcidine hormone is one of these host factors ithas a beneficial role in assessing treatment response during the courseof therapy Patients with chronic HCV often have increased liver iron, acondition associated with reduced response to antiviral therapy. The hepatichormone Hepcidine is the major negative regulator of iron release inthe circulation. The aim of the work is to assess the serum concentrationof hepcidin in chronic hepatitis C patients and evaluate any possibleassociation with the disease activity (viral load) as well as the efficacy ofpegylated IFN and Ribavirin therapy. This study was carried on 35 chronicHCV patients attending el kabary hospital to receive peg IFN/Ribavirintherapy and 15 chronic HCV patients not on IFN therapy as a control groupHepcidin hormone levels were measured in sera of 35 chronic hepatitis Cpatients before starting therapy (base line) at 12 and 24 weeks of therapyusing Hepcidine enzyme immuno assay (USCN life science Inc) Qualitativedetection of HCV RNA to asses sustained virologic response (SVR)using reverse transcriptase PCR at week 24 of antiviral therapy. Our casesshowed a male predominance (70%) over females (30%) and they showedthey showed a slight elevation of AST and Alkaline phosphatase while theother laboratory markers (ALT, Albumin, Bilirubin and Hb) were withinnormal values. The results of qualitative PCR for cases after 6 months oftherapy were in agreement with the RT-PCR results The level of hepcidinin all cases was very low before starting therapy and it showed a significantincrease during the course of therapy. This rise was detected earlyin responding cases. And it is significantly increased after 6 months oftherapy as compared to the control group. We found a negative correlationbetween baseline hepcidin level and baseline viral load of the cases thatshowed response after 6 months of therapy (undetectable viral RNA) wefound no significant relation between degree of fibrosis and viral load ofthe studied cases. Conclusion Chronic HCV infection is associated withVirologie, Vol 17, supplément 2, septembre 2013S157

5 th European Congress of Virologyreduced level of serum hepcidin hormone. The reduced serum hepcidinin chronic HCV patients is fully reversible after IFN/RBV therapy Initialrise in serum hepcidin concentration could be used as one of indicatorsof patient response to interferon therapy. We could not find a significantrelation between baseline viral load and degree of liver fibrosis or liverenzymes. No significant relation was found between hepcidin levels andneither degree of liver fibrosis nor liver enzymes.REF 141Determination of antiviral resistance mutations in chronic hepatitis Bsubjects used antiviral therapy by pyrosequencing methodSevin KIRDAR 1 , Sibel ÖZSU 2 , Alpay ARI 3 , Emine Deniz BAYRAM 21 Adnan Menderes University, Medicine Faculty, Department of MedicalMicrobiology, Aydin, TURKEY; 2 Medical Microbiology Laboratory, IzmirBozyaka Training and Research Hospital, Izmir, TURKEY; 3 Department ofInfectious Disease, Izmir Bozyaka Training and Research Hospital, Izmir,TURKEYIntroduction and aim: Chronic hepatitis B virus (HBV) infection is aserious public health problem in the worldwide. Antiviral therapy usingnucleos(t)ide analogues (NAs) is an effective treatment. However theyneed long term treatment. The existence of HBV variants with primaryantiviral resistance may be important for treatment choice. In this studywe aimed to show the HBV rt mutants in chronic hepatitis B patients whohave received nucleos(t)ide analogues therapy.Method: Fifty serum samples obtained from 50 chronic hepatitis Bpatients who used antiviral therapy from January 2010 to March 2013.The mean age of the patients (15 female, 35 male) was 45.75 (range:2873) years. Serum samples of the patients were analysed by pyrosequencingmethod and HBV DNA was quantitated with real time PCR.Results: HBV rt mutations were detected in 13 samples (26% of the cases)and the remaining 37 samples (74% of the cases) showed wild type (WT)isolates. The distribution of HBV RT mutations was detected as 204 V/I in8 cases(61.5%), L180 M in three cases (23.7), A181 V in one case (7.6%).Both of 204 V/I and 1 L180 M were found in one case (7.6%). The one ofthe cases detected L180 M mutation converted from wild type.Conclusion: Pyrosequencing is a rapid, specific, and sensitive tool thatmay be useful in detecting and quantifying subpopulations of resistantviruses. In this study, it was concluded that this method may be a convenienttool for monitoring HBV infected patients receiving nucleos(t)ideanalogues therapy.Key words: antiviral therapy, mutation, Hepatitis B virusREF 142Intracellular co factors for HCMV replication – novel targets forantiviral therapyIra NAPIERKOWSKI, Elke BOGNERInstitute of Virology, Charité Medical School, Berlin, GERMANYHuman cytomegalovirus (HCMV) is a Herpesvirus that establishes lifelong latency in the host after primary infection. While HCMV infection isusually asymptomatic in immunocompetent people, primary infection andreactivation in individuals with a compromised or undeveloped immunesystem can cause life threatening disease. Although antiviral compoundsfor treatment of HCMV infection and disease are available resistance andtoxicity are major problems. Therefore, the development of new antiviralcompounds with novel drug targets is imperative. One potential new antiviraltarget is the HCMV terminase complex consisting of a large subunit,pUL56, and a small subunit, pUL89. The terminase is required for cleavingand translocating concatemeric HCMV DNA and packaging unit lengthgenomes into procapsids – an essential process for viral replication andtherefore a promising target for new antivirals. This study aims to identifycellular proteins involved in viral replication and packaging using theYeast Two Hybrid System, followed by confirmation of found interactionsby other methods. Possible inhibitors based on the identified hostproteins will then be designed, synthesised, and characterised regardingtheir antiviral activity. So far, Yeast Two Hybrid library screens were performedfor the terminase subunits, the portal protein pUL104 and pUL77,which is also involved in DNA packaging. Potential cellular interactionpartners could be found for pUL77 and have been investigated further byretransformation into yeast and co immunoprecipitation.REF 143Transmission of drug resistant HIV 1 strains among injecting drugusers from the Greater Lisbon, PortugalJoão PIEDADE 1,2 , Sandra VIDEIRA E. CASTRO 1 , Carina SOUSA 1 ,Elizabeth PÁDUA 3 , Ricardo PARREIRA 1,2 , Aida ESTEVES 1,21 Grupo de Virologia, Unidade de Microbiologia Médica, Instituto deHigiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, POR-TUGAL; 2 Unidade de Parasitologia e Microbiologia Médicas (UPMM),Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,Lisbon, PORTUGAL; 3 Lab. Nac. Referência IST VIH/SIDA e Hepatites Be C, Dep. de Doenças Infecciosas, Instituto Nacional de Saúde Dr. RicardoJorge, Lisbon, PORTUGALHIV 1 drug resistance information is useful in the clinical setting, as itcan guide the choice of antiretrovirals, also providing data on the circulationof resistant strains. Viral RNA, purified from blood samples fromHIV 1 infected injecting drug users, was amplified by RT nested PCR.DNA sequencing of protease (PR), reverse transcriptase (RT), integrase(IN) and C2V3C3 gp120 amplicons originated fragments of 297, 598, 813and 516 bp, respectively, for resistance analysis. The Stanford GenotypicResistance Interpretation Algorithm was used for PR, RT and IN. Aftertranslation, C2V3C3 sequences were compared to a subtype B consensusto search for the presence of genetic polymorphisms associated tomaraviroc (MVC) resistance. A total of 123 PR, RT or IN sequences wasobtained. Overall, 15 resistance associated mutations were found, with adistribution of 1 3 mutations/sequence (4 minor in PR; 3 high level, 1 lowlevel and 2 polymorphisms in RT; 5 in IN, 1 major and 4 minor). Halfof the subjects infected with these strains (n=11) were drug naïve, showingtransmission of resistant viral strains. Based upon published data,we observed 12 different single mutations on V3 loop, as well as 2 mutationalpatterns, in 35 sequences, at variable numbers. The presence of thepatterns 11S+26 V and 20F+25D+26 V, in 3 sequences, is relevant, sincethey have been associated with MVC resistance in vivo. Our results willbe useful for therapy implementation and monitoring of infection in thispopulation, having also an impact on the clinical management of HIV 1drug experienced individuals.REF 144In silico approach of the influence of HBV envelope glycoproteinson HBs antigen clearance under anti HBV treatmentEvelyne SCHVOERER 1,2 , Aurelie VELAY 1,2 , FrançoisGOEHRINGER 1,3 , Hélène JEULIN 1,2 , Mouni BENSENANE 4 , ThierryMAY 3 , Fabien ZOULIM 5 , Jean Pol FRIPPIAT 1 , Jean PierreBRONOWICKI 41 Université de Lorraine, EA 7300 « Stress, IMmunité, PAthogènes », Vandoeuvreles Nancy, FRANCE; 2 Centre Hospitalier Universitaire de Nancy,Laboratoire de Virologie, Vandoeuvre les Nancy, FRANCE; 3 Centre HospitalierUniversitaire de Nancy, Service des Maladies Infectieuses etTropicales, Vandoeuvre les Nancy, FRANCE; 4 Centre Hospitalier Universitairede Nancy, Service d’Hépato Gastroentérologie, Vandoeuvreles Nancy, FRANCE; 5 Université de Lyon, Unité Inserm UI1052, Lyon,FRANCES158 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyBackground: Hepatitis B virus (HBV) related chronic infection remainsfrequent. A success in therapy consists of HBV DNA disappearance inblood followed by HBs antigen (Ag) clearance, difficult to achieve. Hostrelated and viral factors favouring HBs Ag clearance under antiviral treatmentby nucleos(t)idic analogues (NUCs) are not well known. Highlyvariable HBV envelope S proteins play a crucial role in viral entry andin host immunity. Our aim is to investigate how HBV envelope proteinvariability could lead to differential response to NUCs. Methods: Aminoacid sequences of HBV S protein (overlapping with polymerase ORF) areobtained from direct nucleotidic sequencing and S protein antigenicity predictedby bio informatics (Antheprot). The data are correlated to responseto treatment. Results: S/pol sequences were analyzed in two patients’groups according to HBs Ag clearance (‘resolvers’ or not) under NUCs:three ‘resolvers’, HBe Ag + (2/3), mean viral load when sequencing at3.5 log cp/mL and three non resolvers, HBe Ag + (2/3), mean viral loadat 4.6 log cp/mL. Our first results showed that the ‘resolvers’ exhibited ahigher S predicted antigenic peak (s119 s125) compared to non resolvers,with a single mutation (sT125 M) just after the KTC pattern crucial in theconformation of the loop 1 in HBs Ag “a” determinant. Conclusion: Eventhough preliminary, our data argue in favor of a possible influence of HBVS protein antigenicity on HBs Ag clearance under NUCs. These resultsdeserve to be explored on more numerous patients and in functional assayson S proteins.REF 145Prednisolone Increases CD4 T Cell Counts and Prevents AIDSdefining Conditions in HIV Patients in AfricaCarsten SCHELLER 1 , Christa KASANG 1 , Samuel KALLUVYA 2,3 ,Charles MAJINGE 2 , Gilbert KONGOLA 3 , Mathias MLEWA 1,2 , IreneMASSAWE 2 , Albrecht ULMER 6 , Hartwig KLINKER 5 , EleniKOUTSILIERI 1 , Wolfgang PREISER 7 , Johannes HAIN 8 , BenediktWEISSBRICH 1 , Axel RETHWILM 1 , August STICH 41 University of Würzburg, Institute of Virology and Immunobiology, Wuerzburg,GERMANY; 2 Bugando Medical Centre, Mwanza, TANZANIA;3 BUCHS, Mwanza, TANZANIA; 4 Medical Mission Institute, Wuerzburg,GERMANY; 5 University of Würzburg, Division of Infectious Diseases,Dept. of Internal Medicine, Wuerzburg, GERMANY; 6 HIV Intensive CareUnit, Stuttgart, GERMANY; 7 Division of Medical Virology, NationalHealth Laboratory Service/University of Stellenbosch, Tygerberg, SOUTHADRICA; 8 University of Würzburg, Institute of Mathematics and Informatics,Chair of Mathematics VIII (Statistics), Wuerzburg, GERMANYAntiretroviral therapy is the best treatment for HIV infection but still notavailable to millions of patients worldwide. We conducted a randomized,double blind, placebo controlled two year clinical study (ProCort1;clinicaltrials.gov NCT01299948) to assess the effect of prednisolone inHIV infection. A total of 326 HIV infected patients with initial CD4counts higher than 300 cells/l were randomly assigned to either 5 mgprednisolone per day or placebo. The primary end point was progressionto AIDS as defined by onset of an AIDS defining condition or adrop of CD4 counts below 200 cells per l, or death from any cause.Baseline CD4 counts were significantly lower in the prednisolone arm(512.14 cells/l ± S.E.M. 13.39) than in the placebo arm (554.40 ± 15.75;p=0.042). Average first year changes in CD4 counts were +20.1 cells/lfor prednisolone (P

5 th European Congress of Virology10. VIRUS ENTRY: ENVELOPEGLYCOPROTEINS, RECEPTORS,ENDOCYTOSISPosters: REF 147 to REF 168REF 147Candida albicans biofilm can retain and release Human HerpesSimplex Virus type 1 in vitroElham MAZAHERI, Arianna SALA, Carlotta Francesca ORSI,Elisabetta BLASI, Claudio CERMELLIDepartment of Diagnostic, Clinic and Public health medicine, Universityof Modena and Reggio Emilia, Modena, ITALYMicroorganisms universally attach to surfaces and produce extracellularpolymeric saccharides (EPS), resulting in the formation of a biofilm. Biofilmscan pose a serious problem for public health because of the increasedresistance of biofilm associated organisms to antimicrobial agents andthe potential for these organisms to cause infections in patients withindwelling medical devices. Moreover, involvement of enteric viruseswith a variety of biofilms has been reported, although very little is knownabout this phenomenon. The presence of some pathogenic viruses in waterbiofilms underlines the ability of viruses to attach and cling to biofilmsretaining their infectivity. No information is available so far on interactionsbetween pathogenic viruses and Candida albicans biofilm. This biofilm isresponsible for severe device related disseminated infections causing invasivecandidemias with a very high rate of mortality. The aim of this in vitrostudy was to ascertain whether Herpes Simplex Virus type 1 (HSV 1) canbe encompassed in Candida biofilm produced in cell culture plates and/oron silicone and PVC catheters. HSV 1 was added to mature biofilms andthe amount of infectious virus embedded in biofilm matrix detached bywashing and energetic scratching was titrated on VERO cells 24 48 h later.Experiments with planktonic Candida were carried out in parallel, as wellas in the absence of Candida. According to our results, free virus particlesof HSV 1, as well as HSV 1 infected cells, remain embedded in Candidabiofilm on tissue cell culture plates as well as on both types of catheter witha significantly higher load than in the presence of planktonic Candida orin the negative controls. These results provide the first evidence that infectiousviruses, after being entrapped in Candida biofilms, can retain theirinfectivity and be released posing a health risk for patients with implantedmedical devices. Interactions between HSV 1 embedded in Candidabiofilm and disinfectants as well as neutralizing antibodies and drugs arediscussed.Key words: pathogenic viruses, Candida albicans biofilm, Extracellularmatrix, infectious reservoirCurrent efforts to understand the basic processes of the replicative cycle ofhantaviruses have been in part hampered by the lack of a reverse geneticsystem andthe biosafety requirements. Recombinant Hantaan virus likeparticles (VLPs) have been previously described being prepared by coexpression of the viral Gn/Gc glycoproteins and the nucleoprotein. Ourcurrent research is focused on understanding the participation of viralproteins in the formation of Andes virus (ANDV) and other hantavirusparticles and on the hantavirus cell entry mechanism. ANDV VLPs wereobtained from purified supernatants of transfected cells and characterizedby Western blots, electron microscopy and dynamic light scattering. Theresults show that ANDV Gn and Gc are sufficient to self assemble intoVLPs without the need of other viral components. Further, we investigatedthe function of Gn and Gc during cell entry. Specifically, we discuss resultsrelated with cell binding, glycoprotein activation and multimerization,membrane interaction and fusion.Funding: FONDECYT 1100756 and CONICYT PFB 16. RA is supportedby a CONICYT doctoral fellowship.REF 149Thermodynamics Tune the Paramyxovirus Membrane FusionMachineryMislay AVILA SÁNCHEZ 1,2 , Lisa ALVES 1,2 , Mojtaba KHOSRAVI 1,2 ,Nadine ADER EBERT 1,2 , Andreas ZURBRIGGEN 1 , PhilippePLATTET 11 Division of Neurological Sciences, DCR VPH, Vetsuisse Faculty, Universityof Bern, Bern, SWITZERLAND; 2 School for Cellular and BiomedicalSciences, University of Bern, Bern, SWITZERLANDThe morbillivirus cell entry machinery consists of a fusion (F) trimer thatdrastically refolds to mediate membrane fusion following receptor inducedconformational changes in its binding partner, the tetrameric attachment(H) protein. To investigate the molecular determinants that control F refolding,we initially generated F chimera between measles virus (MeV) andcanine distemper virus (CDV) and identified a central pocket within themorbillivirus F’s globular head domain that regulates the intrinsic thermalenergy of the metastable, prefusion state. Most mutants of this “pocket”were de stabilized, a phenotype, which, depending on the mutant andtriggering system (receptor type and H’s origin) resulted in membranefusion activation or inhibition. Strikingly, under specific triggering conditions,some F mutants exhibited resistance to a broadly active morbilliviruscell entry inhibitor; a molecule known to enhance thermostability of prefusionF complexes. By exploring the intrinsic thermal energy of each Fmutants, in the presence and absence of the antiviral compound, we foundthat de stabilized F trimers were able to adapt to the fusion inhibitor asa result of low inherent thermal stabilities that could compensate for theprefusion state’s stabilizing effect exerted by 3 g. Finally, our data additionallyrevealed that the nature of the F triggering system was directlyimpacting the ability of F trimers to resist, or not, to the antiviral molecules.In summary, our results not only demonstrate how thermodynamicscontribute to antiviral drug adaptation but shed light on how the triggeringof the morbillivirus membrane fusion machinery is finely regulated.REF 148Function of Andes Hantavirus Gn and Gc glycoproteins in viral selfassembly and entry into the cellRodrigo ACUÑA 1,2 , Nicolás CIFUENTES 1 , Pierre Yves LozachLOZACH 3 , Nicole TISCHLER 1,21 Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago,CHILE; 2 Facultad de Ciencias Biológicas, Universidad Andrés Bello,Santiago, CHILE; 3 Laboratory for Cell Biology of Arboviral Infections,INRS Institut Armand Frappier, Université du Québec, Québec, CANADAREF 150Biophysical and structural characterization of Bunyavirus membranefusionDavid BITTO, Juha HUISKONENWellcome Trust Centre of Human Genetics, University of Oxford, Oxford,UNITED KINGDOMBunyaviruses are enveloped, mostly arthropod borne viruses that cancause infections in vertebrate hosts, including humans. Symptoms mayS160 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyrange from asymptomatic to hemorrhagic fevers, encephalitis and/or hepatitiswith high fatality rates in severe cases. Approved vaccines andefficient antiviral treatments are unavailable. Our work focuses on thecell entry of Bunyaviruses and we use Uukuniemi virus (UUKV, genusPhlebovirus) as a model system. Target cell penetration by UUKV islow pH dependent and believed to occur from within a late endosomalcompartment (Lozach et al. 2010 and 2011), presumably by membranefusion. To analyze membrane fusion in more detail, we developed anin vitro fusion assay using fluorescent UUKV particles and/or fluorescentliposomes. We show that low pH mediated UUKV/liposome lipidmixing and liposome content release is favoured by negatively chargedlipid head groups in target membranes. To complement the biophysicalassays, we use electron cryo tomography to study intermediate structuresarising during UUKV fusion. By combining functional and structural data,we wish to unravel mechanistic details of UUKV fusion. This will lead toa more detailed understanding of Bunyavirus entry and thus a more solidfoundation for vaccine and drug development.H stalks “opens” to trigger F, last models suggest that tetrameric H headsmay also move as “dimeric blocs” to transmit signals for F activation. Wecharacterized an anti CDV H mAb (1347), which strongly inhibited cellcell fusion. Epitope mapping, using soluble H forms, revealed a segmentin the H stalk, which, once transposed into a foreign protein, allowed forits recognition by 1347. 1347 did not cross react with MeV H, but substitutionof two residues in the epitope (by the corresponding ones in CDVH) enabled MeV H detection and cell cell fusion inhibition. Importantly,the epitope locates above the putative F contacting zone, which correlatedwith 1347 binding activity being unaffected by the presence of F. However,1347 bound to a membrane bound “headless” CDV H variant with a higherefficiency, suggesting that H heads, in a pre receptor bound state, impededfull 1347 recognition. Remarkably, a spontaneous basal level of F triggeringwas monitored in headless H/F coexpressing cells, and this, regardlessof the presence or absence of 1347. Overall, these findings suggest that1347 does not inhibit F activation by impairing H stalks dependent intrinsictriggering activity. Rather, we suggest that 1347 may alter putativereceptor induced H heads to stalks “un clamping” movements.REF 151Murine hepatitis coronavirus is a late penetrating virus that requiresa functional HOPS complex for fusionCornelis DE HAAN, Christine BURKARD, Hélène VERHEIJE, OliverWICHT, Peter ROTTIER, Berend Jan BOSCHUtrecht University, Utrecht, THE NETHERLANDSMany viruses that are taken up via endocytosis depend on endosomal maturationand the associated pH drop to induce virus cell fusion. Particularlylate penetrating viruses have been described as sensitive to perturbations ofendosomal maturation. Murine hepatitis virus (MHV), a prototypic coronavirus,contains furin cleaved spike fusion proteins. It does not appear torequire a low pH for activation of its fusion protein and is able to inducecell cell fusion. While these observations are indicative of cell entry byfusion at the plasma membrane, we and others have recently also shownthat MHV relies on clathrin mediated endocytosis for productive entry.To solve this apparent contradiction we have studied the entry pathway ofMHV in more detail using specific drugs, siRNAs and knock out cells incombination with virus replication dependent reporter and novel replicationindependent fusion assays. Our results confirm the important role ofclathrin mediated endocytosis and indicate the involvement of the actincytoskeleton in virus entry. In addition, MHV was shown to be criticallydependent on endosomal maturation for successful infection. Virions colocalizedwith Rab7 and LAMP1, markers of late endo lysosomal vesicles,while virus cell fusion was impaired in cells lacking Rab7 or a functionalHOPS complex. These results indicate that MHV, despite carrying a furincleaved fusion protein that induces cell cell fusion, is a late penetratingvirus that needs to be transported to the late endo lysosomal compartmentsfor virus cell fusion and productive entry to occur.REF 152Inhibition of Membrane Fusion by a mAb interacting with theMorbillivirus Attachment Protein Stalk DomainNadine EBERT ADER 1,2 , Philippe PLATTET 1 , AndreasZURBRIGGEN 11 Division of Neurological Sciences, DCR VPH, Vetsuisse faculty, Universityof Bern, Bern, SWITZERLAND; 2 Graduate School for Cellular andBiomedical Sciences, University of Bern, Bern, SWITZERLANDMeV and CDV entry systems rely on two envelope glycoproteins: theattachment (H) and the fusion (F) proteins that co operate to achieve plasmamembrane fusion. The H ectodomain consists of a stalk supporting a headthat contacts different receptors. While the central section of morbillivirusREF 153Characterization of the core structure of glycoprotein B of Herpessimplex virus 1Massimiliano GALDIERO 1,3 , Marco CANTISANI 2,3 , AnnaritaFALANGA 2 , Novella INCORONATO 1 , Luigi RUSSO 1 , Alfonso DESIMONE 4 , Rita BERISIO 5 , Stefania GALDIERO 2,3,51 Department of Experimental Medicine II University of Naples, Napoli,ITALY; 2 Department of Pharmacy University of Naples Federico II,Napoli, ITALY; 3 Centro Interuniversitario di Ricerca sui Peptidi Bioattivi,University of Naples Federico II, Napoli, ITALY; 4 Division of MolecularBiosciences, Imperial College, London, UNITED KINGDOM; 5 Istituto diBiostrutture e Bioimmagini, CNR, Napoli, ITALYEntry of enveloped viruses requires fusion of viral and cellular membranes,driven by conformational changes of viral glycoproteins. The crystallizedtrimeric form of glycoprotein B of Herpes simplex virus has been describedas a post fusion conformation and several studies prove that as other classIII fusion proteins gB undergoes a pH dependent switch between the preand post fusion conformation. Here, using peptides corresponding to thelong helix of the post fusion structure and several biophysical techniques,including fluorescence and circular dichroism spectroscopies, surfaceplasmon resonance, and molecular dynamic simulations, we provideevidence of the existence of a thoroughly different pre fusion conformation;during the conformational rearrangements, some epitopes are lost orformed, which supports the hypothesis for the search of a minimal peptidicsequence able to interact with the virus in its prefusogenic conformation.REF 154Receptor use of Lassa virus in human dendritic cellsAna Rita GONÇALVES 1 , Marie Laurence MORAZ 1 , AntonellaPASQUATO 1 , Ari HELENIUS 2 , Pierre Yves LOZACH 2 , Stefan KUNZ 11 Institute of Microbiology, Lausanne University Hospital and University ofLausanne, Lausanne, SWITZERLAND; 2 Institute of Biochemistry, FederalInstitute of Technology Zurich, Zurich, SWITZERLANDThe arenavirus Lassa (LASV) causes a severe hemorrhagic fever with highmortality in humans. Antigen presenting cells, in particular dendritic cells(DC), are early and preferred targets of LASV and their productive infectioncontributes to virus induced immunosuppression observed in fataldisease. Here we characterized the role of LASV candidate receptors inviral entry into primary human DCs using a chimera of the prototypicarenavirus lymphocytic choriomeningitis virus (LCMV) expressing theVirologie, Vol 17, supplément 2, septembre 2013S161

5 th European Congress of VirologyLASV glycoprotein (rLCMV LASVGP). Several cellular receptors havebeen described for LASV, including the ECM receptor dystroglycan (DG),the Tyro3/Axl/Mer (TAM) receptors Axl and Tyro3, and the C type lectinDC specific ICAM 3 grabbing nonintegrin (DC SIGN). We found thatprimary human monocytes and monocyte derived DCs (MDDC) expressonly low levels of functional DG and lack Axl and Dtk. Differentiationof monocytes into DCs enhanced virus attachment and entry, concomitantwith the up regulation of DC SIGN. The binding of LASV to DC SIGNwas mediated by mannose sugars located on the N terminal GP1 subunitof LASVGP. DC SIGN served as an attachment factor for LASV andaccelerated capturing of free virus by MDDCs, facilitating productiveinfection. However, in contrast to the Phlebovirus Uukuniemi virus, knownto use DC SIGN as an authentic entry receptor in DCs, infection withrLCMV LASVGP was less dependent on DC SIGN and showed strikinglydistinct kinetics. The data at hand provide evidence for a role of DCSIGN as an enhancing factor for LASV entry into human DCs and suggestthe existence of other yet unknown receptor(s) involved in productiveLASV infection in this crucial cell type.This research was supported by Swiss National Science Foundation grantFN 310030 132844 (S.K.).REF 155Characterization of binding properties of the IBV spike proteinMartina HESSE, Georg HERRLER, Christine WINTERInstitute for Virology, Hannover, GERMANYThe Infectious Bronchitis Virus (IBV) belongs to the family of Coronaviridae,which comprises a variety of animal and human pathogens. A crucialstep in the viral lifecycle is the entry into host cells, which begins withviral attachment. For IBV it is known that attachment is mediated by theS1 subunit of the spike protein. a2,3 linked sialic acids serve as its receptordeterminants, but the exact localization of the sialic acid binding domainwithin the S1 subunit is still not identified. It is the aim of our study tofurther analyse the binding characteristics of the IBV spike protein. As atool for our investigations we use soluble chimeric proteins which consistof the S1 subunit of different IBV spike proteins fused to a human IgG Fctag. The constructs allow analysing the attachment of spike proteins to primarychicken cells and tissues in different settings by detecting the Fc tagwith fluorochrome labelled antibodies. For identification of the IBV sialicacid binding site certain domains of the spike protein have been deletedand the mutant’s binding ability was addressed. First experiments revealedthat a deletion of 42 aminoacids at the N Terminus of the spike proteinreduces the binding to host cells clearly. Mutants with smaller deletionscould narrow the responsible site down to seven amino acids. An alaninscan is performed to identify the aminoacids important for binding. Toconfirm that the respective domain is responsible for sialic acid binding itis cloned into Coronavirus spike proteins which do not possess sialic acidbinding properties of their own.REF 156Molecular Basis of Canine Distemper Virus Cell Entry through theHuman CD150/SLAM ReceptorMojtaba KHOSRAVI 1,2 , Lisa ALVES 1,2 , Maria BIERINGER 3 , JürgenSCHNEIDER SCHAULIES 3 , Andreas ZURBRIGGEN 1 , PhilippePLATTET 11 Division of Neurological Sciences, DCR VPH, Vetsuisse faculty, Universityof Bern, Bern, SWITZERLAND; 2 Graduate School for Cellularand Biomedical Sciences, University of Bern, Bern, SWITZERLAND;3 Institute for Virology and Immunobiology, University of Würzburg, Würzburg,GERMANYThe measles virus (MeV) and canine distemper virus (CDV) entry systemsare regulated by two envelope glycoproteins: the attachment (H)and fusion (F) proteins, which tightly co operate to mediate membranefusion. While MeV is still associated with over 120 ′ 000 human deathsper year, CDV has an ever increasing host range with recently reportedsevere outbreaks in primates. Inefficient vaccine availability in developingcountries combined with the overarching increasing tendency of relaxedmeasles vaccination in developed countries may set the stage for swiftadaptation of CDV to humans with putative dramatic global health outcomes.We here refined the molecular basis of CDV adaptation to thehuman SLAM (hSLAM) receptor. Indeed, we recently reported that, initially,CDV poorly infects hSLAM expressing cells with the requirementof a single substitution in CDV H to enable efficient CDV infections.Here, we found that wt CDV H nevertheless engaged to hSLAM. However,the avidity of the CDV H/hSLAM interaction was rather low, whichprecluded productive membrane fusion triggering. We identified threeresidues in hSLAM locating at the binding interface which, when mutatedinto the canine SLAM corresponding amino acid (E71G, E75K andR130H), allowed wt CDV glycoproteins to promote fusion without improvingthe weak CDV H/receptor interaction. Remarkably, fusion was alsoachieved when the CDV H/hSLAM combination was coupled with destabilized CDV F variants. Overall, our data revealed that only minormodifications in hSLAM, CDV H or CDV F lead to efficient cell entrytrough the human receptor. The ease of CDV adaptation to heterotypicreceptors may hence contribute to its threatening, ever increasing, hostrange.REF 157Impact of nectin 1 during entry of Herpes simplex virus type 1 intoepidermis and keratinocytesDagmar KNEBEL MÖRSDORF 1,2 , Philipp PETERMANN 1 , KatharinaTHIER 1 , Elena RAHN 1 , Wilhelm BLOCH 3 , Martin J. BARRON 4 ,Michael J. DIXON 41 Center for Biochemistry, University of Cologne, Cologne, GER-MANY; 2 Department of Dermatology, University of Cologne, Cologne,GERMANY; 3 Department of Molecular and Cellular Sports Medicine,German Sports University, Cologne, GERMANY; 4 Faculty of LifeSciences, University of Manchester, Manchester, UNITED KINGDOMKeratinocytes of the skin and mucosa represent the primary entry sitefor herpes simplex virus type 1 (HSV 1) in vivo. We aim to identify themolecular mechanisms on which virus entry depends in keratinocytes tounderstand how HSV 1 overcomes the barrier function of the epithelia.Here, we used a mouse model to address the role of nectin 1, one ofthe major co receptors for HSV 1 in various cell lines. Virus entry wasstudied after ex vivo infection of murine epidermal sheets by visualizingexpression of the immediate early gene ICP0. The studies are based onour observation that infection initiates efficiently in the basal keratinocytesof the epidermis once the dermis is removed. EM studies suggestvirus internalization via fusion of the viral envelope with the basal surfaceof the epidermal sheets and endocytic uptake. Our results in nectin 1deficient epidermal sheets revealed a 70 90% reduction in the number ofinfected keratinocytes in comparison with wildtype epidermis. Interestingly,we observed nearly no infection in isolated nectin 1 deficient primarykeratinocytes. This observation correlated with the presence of HVEM, afurther HSV 1 co receptor, in murine epidermis and the lack of HVEMexpression in primary murine keratinocytes. Overexpression of HVEM innectin 1 deficient keratinocytes restored infectivity. Thus, we suggest thatHVEM supports entry into a subpopulation of cells in nectin 1 deficientepidermis while the absence of nectin 1 and HVEM in primary keratinocytesleads to a loss of entry. In summary, our results demonstrate a majorimpact of nectin 1 as co receptor for HSV 1 in murine epidermis and asupplementary role of HVEM.S162 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF 158Acid activated structural reorganization of the Rift Valley fever virusGc fusion proteinJeroen KORTEKAAS 1 , Matthijn DE BOER 2 , Lotte SPEL 2 , PeterROTTIER 2 , Rob MOORMANN 1 , Berend Jan BOSCH 21 Department of Virology, Central Veterinary Institute of WageningenUniversity and Research Centre, Lelystad, THE NETHERLANDS;2 Department of Infectious Diseases and Immunology, Virology Division,Faculty of Veterinary Medicine, Utrecht University, Utrecht, THENETHERLANDSEntry of Rift Valley fever virus (RVFV) into target cells is mediatedby the viral glycoproteins Gn and Gc. We investigated the RVFV entryprocess and, in particular, its pH dependent activation mechanism usingour recently developed RVFV replicon particle system. Entry of the virusinto the host cell was efficiently inhibited by lysosomotropic agents thatprevent endosomal acidification and by compounds that interfere withdynamin and clathrin dependent endocytosis. Exposure of plasma membranebound virions to an acidic pH (

5 th European Congress of Virologythree amino acids in the nectin 4 V domain F G loop crucial for functionalinteractions with MV H; in addition, residues in other two loops influencethese interactions. Thus MV appropriates the adhesive surface of nectinsto infect the respiratory epithelium.REF 162Different sensitivity of primary chicken oviduct epithelial cells for thecoronavirus IBV strains QX, B1648 and Beaudette is not related tothe binding properties of their spike proteinsAnn Kathrin MORK 1 , Sahar ABD EL RAHMANN 2 , GeorgHERRLER 1 , Christine WINTER 11 University of Veterinary Medicine, Hannover, Institute of Virology,Hannover, GERMANY; 2 Mansoura University, Department of Virology,Mansoura, EGYPTAs a member of the family Coronaviridae, Infectious bronchitis virus(IBV) is a one of the most important pathogens within the poultry industry.A characteristic of IBV is the occurrence of many different strainsbelonging to different serotypes, which makes a complete control of thedisease by vaccines challenging. Reasons for differences in the tissue tropismand pathogenicity of the IBV strains, as e.g. a predilection to thekidneys or the oviduct are still an open question. The QX strain has beena major pathogen in poultry flocks in Asia, Europe and other parts of theworld. It is the cause of severe problems with kidney disease and reproductivetract disorders. We analyzed infectivity and binding properties of theQX strain and compared it with the nephropathogenic IBV strain B1648and the apathogenic IBV Beaudette strain. We observed that the analyzedstrains infected primary oviduct epithelial cells with different intensity.Epithelial cells of the oviduct showed a high sensitivity to the QX strain, alower sensitivity to the Beaudette strain and were nearly not sensitive to thenephropathogenic B1648 strain. In contrast, binding tests on cryosectionsand FACS analysis with isolated primary oviduct epithelial cells and thecorresponding soluble spike proteins revealed that all tested spike proteinsbound with high affinity to these cells. Our results show that the differentsensitivity of primary chicken oviduct epithelial cells for the tested IBVstrains cannot be explained with a different affinity of the spike proteinsto host epithelial cells.presence of H. H specific monoclonal antibodies as well as a mutationin H interfering with H/F co operation, blocked cell entry. The particlesmediated stable and specific transfer of reporter genes into Her2 positivehuman tumor cells also in vivo, while exhibiting improved infectivity andhigher titers as compared to Her2 targeted vectors displaying the targetingdomain on H. Extending the current model of MV cell entry, the datasuggest that receptor binding of H is not required for its fusion helperfunction but particle cell contact in general may be sufficient to inducethe conformational changes in the H/F complex and activate membranefusion.REF 164Integrins modulate the entry efficiency of West Nile Virus into cellsKatja SCHMIDT 1 , Markus KELLER 1 , Martin H. GROSCHUP 1Friedrich Loeffler Institut, Greifswald Insel Riems, GERMANYThe underlying mechanisms allowing West Nile virus (WNV) to replicatein a large variety of different arthropod, mammal and bird speciesare largely unknown, but are believed to rely on highly conserved proteinsrelevant for viral entry and replication. A previous study had postulatedearlier that integrin av3 functions as the receptor for WNV; however,its involvement has been doubted recently. The present study was designedto clarify the involvement of integrins in WNV entry. A cell culturemodel was established based on specific integrin knock out cell lines with aspecific modification in the particular integrin subunit genomic sequence.Wild type and specifically integrin deficient mouse fibroblasts lackingthe integrin subunits av, 1 or3, respectively, allowed (i) studying theinvolvement of integrins, (ii) identifying the integrin subunit involved and(iii) addressing their function in WNV entry. Additional questions wereaddressed as to the extent to which they participate in virus entry and to possibledifferences in the entry efficiencies in distinct WNV strains. All celllines were permissive but differences between integrin expressing and nonexpressing cells were seen. Results clearly demonstrate that the expressionof 1 and 3 integrins positively affected virus yields significantly,as seen in integrin 3 rescue and integrin 1 floxed cells in comparison tothe corresponding integrin deficient cell line. However, integrins obviouslydo not function at the level of WNV binding to the cell surface but ratherdownstream during entry or in post entry stages.REF 163The receptor attachment function of measles virus hemagglutinincan be replaced with an autonomous protein that binds Her2 whilemaintaining its fusion helper functionAnke RASBACH 1 , Tobias ABEL 1 , Robert MÜNCH 1 , Klaus BOLLER 1 ,Jürgen SCHNEIDER SCHAULIES 2 , Christian BUCHHOLZ 11 Paul Ehrlich Institut, Langen, GERMANY; 2 University of Würzburg,Würzburg, GERMANYCell entry of enveloped viruses is initiated by attachment to the virusreceptor followed by fusion between the virus and host cell membranes.Measles virus (MV) attachment to its receptor is mediated by the hemagglutinin(H), which is thought to produce conformational changes in themembrane fusion protein (F) that trigger insertion of its fusion peptideinto the target cell membrane. Here, we uncoupled receptor attachmentand fusion helper function of H by introducing Y481A, R533A, S548L,and F549S mutations into the viral attachment protein that made it blindto its normal receptors. An artificial attachment protein specific for Her2was incorporated into pseudotyped lentivirus particles as a separate transmembraneprotein along with the F protein. Surprisingly, these particlesentered efficiently into Her2 positive SK OV 3 as well as CHO Her2 cells.Cell entry was independent of endocytosis, but strictly dependent on theREF 165Stem anchor interactions of the fusion protein E in flavivirusmembrane fusionKarin STIASNY, Janja BLAZEVIC, Iris MEDITS, Victoria BRADT,Franz X. HEINZMedical University of Vienna, Vienna, AUSTRIAViral membrane fusion proceeds through a sequence of steps that are drivenby structural changes of viral envelope proteins (fusion proteins). Weattempt to define intermediate stages of this process using the class IIfusion protein E of the flavivirus tick borne encephalitis virus as a model.After virus uptake by endocytosis, fusion is triggered by the acidic pHin endosomes which leads to a conversion of the E homodimers on thevirion surface into more stable trimers. The crystal structures of truncatedE proteins in their pre and post fusion forms lack the so called ‘stem’region and the double membrane anchor. Since these elements are hypothesizedto have crucial functions in fusion, we investigated their roleby mutagenesis of infectious viruses as well as recombinant E proteins.The stem connects the ectodomain to the membrane anchor and is predictedto ‘zipper’ along the trimer core during E conformational changes,thus providing part of the energy required for fusion. We identifiedS164 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyimportant interaction sites between the stem and the trimer core thatare involved in stabilizing the post fusion trimer. In addition, we showthat both transmembrane domains of E are not only essential for virusassembly, but also for efficient fusion, most likely contributing to thelate stages of membrane merger. Our data provide evidence for bothintra and inter trimeric E protein interactions mediated by the stemanchor region and thus extend the existing models of flavivirus membranefusion.REF 166Early mechanisms in the life cycle of ocular adenovirusesRickard STORM, Niklas ARNBERGUmeå University, Department of Clinical Microbiology, Division of Virology,Umeå, SWEDENEpidemic keratoconjunctivitis (EKC) is an ocular infection that is mainlycaused by adenovirus type 8 (Ad8), Ad19 and Ad37 and affects roughly20 30 million individuals every year, worldwide. EKC is a severe andcontagious disease, and is characterized by conjunctivitis, keratitis, pain,edema, lacrimation, hemorrhages and decreased vision that may last formonths or years and in rare cases can lead to blindness. EKC causing Adshave been shown to bind to the two terminal sialic acids of a branchedhexasaccharide that corresponds to the glycan in the GD1a ganglioside1.Viral attachment to ocular cells is mediated by the knob domain of thefiber protein, which is anchored to the viral capsid by the penton baseprotein. The penton bases are located in each of the twelve corners of theicosahedral capsid and contains integrin interacting motifs such as RGD(Arginine Glycine Aspartic acid), which are known to be used by multipleAds, including Ad37, for interaction with aV integrins. This interactionhas been shown to be important for efficient cellular entry and endosomalescape by multiple Ad types. Via homology modulation and bioinformaticanalysis we identified other potentially exposed integrin interacting motifs.We have also seen that specific integrin blocking antibodies as well assoluble peptides that mimick the integrin interacting motifs inhibited Ad37infection of corneal cells. In summary, our preliminary data suggest thatEKC causing Ads interact with additional integrins besides the previouslyidentified alphaV integrins.REF 168TIM and TAM receptors mediate dengue virus infectionLaurent MEERTENS 1,2,3 , Xavier CARNEC 1,2,3 , Manuel PERERALECOIN 1,2,3 , Rasika RAMDASI 1,2,3 , FlorenceGUIVEL-BENHASSINE 4 , Erin LEW 5 , Greg LEMKE 5 , OlivierSCHWARTZ 4 , Ali AMARA 1,2,31 INSERM U944, Laboratoire de Pathologie et Virologie Moléculaire,Hôpital Saint-Louis, Paris, FRANCE; 2 Institut Universitaired’Hématologie, Hôpital Saint-Louis, Paris, FRANCE; 3 University ParisDiderot, Sorbonne Paris Cité, Hôpital St., Paris, FRANCE; 4 Unité Viruset Immunité, Institut Pasteur, Paris, FRANCE; 5 Molecular NeurobiologyLaboratory, Immunobiology and Microbial Pathogenesis Laboratory, TheSalk Institute, La Jola, USADengue viruses (DV) are responsible for the most medically relevantarboviral diseases. Molecular interactions that occur between DV and thehost cell during virus entry are poorly understood, hampering the developmentof novel antiviral strategies. Here, we identify TIM and TAMproteins, which are receptors that mediate the phosphatidylserine (PtdSer)– dependent phagocytic engulfment and removal of apoptotic cells, as DVentry factors. Cells poorly susceptible to DV become strongly infectedwhen expressing either TIM-1 or TIM-4, from the TIM family, or AXL orTYRO3, from the TAM receptor tyrosine kinase family. Conversely, additionof anti-TIM or anti-TAM antibodies, or silencing the molecules insusceptible cells, inhibits DV infection. TIM receptors mediate DV entryby directly interacting with virion-associated PtdSer. TAM-mediated DVinfection relies on the indirect recognition of PtdSer by the TAM ligandGas6, which bridges virus to the target cell. This dual mode of virus recognitionby TIM and TAM receptors reveals how DV usurp the apoptotic cellclearance pathway for infectious entry.Virologie, Vol 17, supplément 2, septembre 2013S165

5 th European Congress of Virology11. VIRUS EXIT: ASSEMBLY,MATURATION AND RELEASEPosters: REF 169 to REF 180REF 169Nuclear export of herpesviral proteins and its role in tegumentationof infectious particlesSusanne M. BAILER 3,1 , Verena RASCHBICHLER 1 , Diana LIEBER 21 Max von Pettenkofer Institut, Ludwig Maximilians Universität München,München, GERMANY; 2 Institut für Virologie, Universitätsklinikum Ulm,Ulm, GERMANY; 3 Biological Interfacial Engineering, University of Stuttgart,Stuttgart, GERMANYTransport of proteins between cytoplasm and nucleus is mediated by transportfactors of the importin alpha and beta families and occurs along agradient of the small GTPase Ran. We have recently generated a novelbipartite assay called NEX TRAP (Nuclear EXport Trapped by RAPamycin)for in vivo analysis of protein nuclear export. The assay is based onthe rapamycin induced dimerization of the modules FRB (FK506 rapamycin(FR) binding domain) and FKBP (FK506 binding protein 12): Apotential nuclear export cargo is fused to FRB, to EYFP for direct visualizationas well as an SV40 derived nuclear localization sequence (NLS)for constitutive nuclear import. An integral membrane protein that residesat the trans Golgi network (TGN) is fused to a cytoplasmically exposedFKBP and serves as reporter. EYFP NLS FRB fusion proteins withexport activity accumulate in the nucleus at steady state but continuouslyshuttle between nucleus and cytoplasm. Rapamycin induced dimerizationof FRB and FKBP at the TGN traps the shuttling protein outside of thenucleus, making nuclear export permanent. Using the NEX TRAP thenuclear export activity of herpesviral proteins that form the tegument ofinfectious particles was determined. Mutational analysis is in progress toidentify and functionally characterize sequences that drive nuclear exportof herpesviral proteins. Our results will provide new insights into the spatiotemporal distribution of viral proteins in the course of infection. Thepotential role of nuclear export in primary and secondary tegumentationwill be discussed.REF 170Arenavirus envelope glycoprotein and cellular protease SKI 1/S1Pmaturations: Two events tightly interconnectedJoel DA PALMA 1 , Dominique BURRI 1 , Nabil G. SEIDAH 2 , StefanKUNZ 1 , Antonella PASQUATTO 11 Institute of Microbiology,University Hospital Center and University ofLausanne, Lausanne, SWITZERLAND; 2 Laboratory of Biochemical Neuroendocrinology,Clinical Research Institute of Montreal, Affiliated to theUniversité de Montréal, Montréal, CANADAA crucial step in the arenavirus life cycle is the maturation of the envelopeglycoprotein precursor GPC by the cellular enzyme SKI 1/S1P. Targetingits activity is an effective approach to block arenavirus cell to cell propagationand so far no SKI 1/S1P independent viral escape variants haveemerged. SKI 1/S1P is matured by sequential removal of its prodomainby auto processing, first at the B/B’ site, followed the by C site. We analyzedthe effect of mutations at these sites on the maturation and abilityof the enzyme to process arenavirus GPC. We found that that single pointmutations at B or B’ site do not affect SKI 1/S1P maturation or activity,indicating redundancy. However, combination of both mutations blocksenzyme maturation and specifically affects processing of arenaviral GPCs,but not cellular substrates. C site mutation revealed the existence of a novelauto processing site (C’) that may compensate and overcome the block atthe C site. In contrast to the B/B’ mutant, the C/C’ mutant exhibit enhancedactivity towards viral substrates, while cellular substrates remain unaffected,underscoring differential recognition of viral and cellular substrates.In conclusion, maturation of SKI 1/S1P prodomain is crucial to determinesubstrate specificity. Our findings are also important in the context of viraladaptation to the host, we speculate that arenaviruses have evolved to takeadvantage of immature ER resident forms of the enzyme that have selectiveenhanced activity towards viral glycoproteins, which uniquely mimicthe autoprocessing of SKI 1/S1P at the C site.REF 171Deciphering the role of Influenza M1 matrix protein in virus assemblyAdeline KERVIEL 1 , Baptiste PANTHU 2 , Didier DECIMO 2 , JensRADZIMANOWSKI 3 , Cendrine FAIVRE MOSKALENKO 4 , CyrilFAVARD 1 , Theophile OHLMANN 2 , Bruno LINA 5 , WinfriedWEISSENHORN 3 , Michele OTTMANN 5 , Delphine MURIAUX 11 CPBS CNRS UMR 5236, Montpellier, FRANCE; 2 Laboratoire de VirologieHumaine ENS UMR S 758, Lyon, FRANCE; 3 Unit for Virus HostCell Interactions UJF EMBL CNRS UMI 3265, Grenoble, FRANCE;4 Laboratoire de Physique ENS UMR 5672, Lyon, FRANCE; 5 Virologieet Pathologies Humaines EMR 4610 CNRS UMS 3453 Inserm US7, Lyon,FRANCEThe family of Orthomyxoviridae is defined by viruses that have a negativesense, single stranded, and segmented RNA genome. Influenza A viruspossesses a lipid membrane derived from the host cell, harboring HA,NA, and M2 proteins. The “core” of the virus consists of the vRNP complex,composed of eight viral RNA segments, the polymerase proteins(PB1, PB2, PA), the nucleoprotein (NP) and the nuclear export protein(NEP/NS2). The matrix protein M1 is lying beneath the lipid envelope.Different contradictory studies tried to explain how M1 was or not the“major driver” for viral assembly, as it associates the vRNP complex inthe nucleus and also binds the membrane, thus triggering the transport ofthe vRNP and virus assembly. However, the precise roles of M1 protein inviral assembly are not clear. In our laboratory, we set up a minimal VLP(Virus Like Particles) production system based on M1 in order to investigateif M1 alone (or in the presence of HA, NA and/or M2) is able to reachthe plasma membrane and form VLP in eukaryotic cells. We are also testingthe involvement of M1 basic residues in these processes by directed sitemutagenesis. Wild type M1 (influenza A H1N1) and mutants were testedfor their ability to bind membrane and produce VLP by membrane flotationassays, immunoblots and immunofluorescence localization: we observedthat M1 is able to bind membrane in vitro and in cellulo and seems to beable, alone, to produce VLP. Further investigations are ongoing to elucidateM1 membrane binding determinants during influenza A assembly.REF 172Study of factors affecting assembly and morphology of retroviralparticles in vitroRuzena PICHALOVA, Tibor FUZIK, Pavel ULBRICH, Tomas RUMLInstitute of Chemical Technology, Prague, Prague 6, CZECH REPUBLICAssembly of retroviral particles which is driven by oligomerization ofstructural polyprotein Gag is one of critical steps of retroviral life cycle andits inhibition would prevent virus from further spreading. We found thatMason Pfizer monkey virus (M PMV) Gag deletion mutant can be assembledin vitro into spherical as well as tubular virus like particles (VLPs)depending on reducing conditions during particle formation process. Wesuggest that the presence of reducing agents could disrupt disulfide (S S)bonds present in Gag and thus affect VLP morphology. In HIV 1 capsidprotein (CA) two cysteines (C198 and C218) were predicted to form S Sbond and thus stabilize the last two helices in its C terminal domain [1].S166 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyIn M PMV CA two cysteines (C193 and C213) correspond to their positionsin HIV 1 but there is also one additional cysteine (C82) that is notpresent in CA HIV 1. Our goal was to study the effect of mutations C82,C193 and C213 on in vitro formation and morphology of VLPs assembledfrom M PMV Gag deletion mutant. These mutations prevent cysteinesfrom S S bond formation and thus could mimic the presence of reducingagents during assembly. Obtained VLPs were analyzed by transmissionelectron microscopy and ultracentrifugation in sucrose gradient, followedby SDS PAGE. Our findings could be important for futher understandingof retroviral particle assembly process.Financial support from specific university research MSMT No 20/2013and GACR P302/12/1895.1. Worthylake DK, et al. Acta Crystallogr Sect D Biol Crystallogr 1999;55: 85.The great diversity, wide distribution, and mobility of bats represent riskfactors for zoonotic transmission. SARS Coronavirus (CoV) and the novelhuman Betacoronavirus EMC are closely related to bat CoV from thegenus Betacoronavirus. A variety of batCoV belong to the genus Alphacoronavirus.However, no infectious CoV’s could be isolated from batsso far. To analyze coronaviral replication in chiropteran cells, the transmissiblegastroenteritis virus (TGEV) was used as model for the genusAlphacoronavirus. By Western blot analysis, reverse transcription PCRand immunofluorescence analysis virus entry, replication, and virus releasewas examined. Cells, transfected with the specific receptor for TGEV,became susceptible for infection which was shown by intracellular detectionof viral spike and membrane proteins. Release of viral RNA wasobserved for all tested cell lines. Successful release of infectious virus particlesfrom cell lines of African flying foxes (genus: Eidolon, Epomops, andHypsignathus), the common pipistrelle (Pipistrellus pipistrellus) and theshort tailed fruit bat (Tadarida brasiliensis) was revealed after subsequentviral replication in inoculated swine testis cells. In contrast, supernatantsobtained from Daubenton’s bat did not contain any infectious virus. Collectively,these data lead to the assumption that beside the viral receptor,cellular factors could play a crucial role for species restriction.REF 173Association of apolipoprotein B with Hepatitis C virus is involved inviral infectivity and is dependent on the liver enriched protein CidebChristophe RAMIÈRE 1,2,3 , Liviu Sorin ENACHE 4 , MarinePORCHEROT 2,3 , Olivier DIAZ 2,3 , Laure PERRIN COCON 2,3 , VincaICARD 1 , Caroline SCHOLTÈS 1,2,3 , Vincent LOTTEAU 2,3 , PatriceANDRÉ 1,2,31 Hospices Civils de Lyon, Lyon, FRANCE; 2 INSERM U1111, CIRI, Lyon,FRANCE; 3 Université de Lyon, Lyon, FRANCE; 4 University of Medicineand Pharmacy, TârguMure, ROMANIAIn patients’ blood, Hepatitis C Virus (HCV) circulates as lipo viral particles(LVP), hybrid particles associating viral and lipoprotein components, inparticular apolipoprotein B (apoB). Contrary to natural LVPs, HCV particlesproduced in vitro in Huh7.5 cells (HCVcc) are characterized bypoor association with apoB and low specific infectivity. We thus hypothesizedthat apoB could be involved in HCV infectivity and examinedthe mechanisms responsible for its association with HCV virions. Wefirst demonstrated by neutralization experiments that 2 monoclonal antiapoB antibodies, recognizing epitopes in the Low–Density LipoproteinsReceptor binding domain of apoB, were able to strongly neutralize (ca80 to 90%) infection of Huh7.5 cells by HCVcc, contrary to antibodiestargeting epitopes outside this domain. We also identified Cideb, a liverenriched protein involved in lipoprotein assembly, as an essential factorfor association of HCV with apoB. We showed that Cideb directly interactedwith HCV E2 envelope glycoprotein. We also observed that Cidebexpression was low in Huh7.5 cells and that Cideb overexpression in thiscell line was sufficient to induce secretion of E2 in association with apoB.Moreover, differentiation of Huh7.5 cells in presence of human serumand DMSO led to a dramatic increase of Cideb expression and furthersecretion of HCV particles characterized by higher association with apoBand improved specific infectivity. Together these results suggest that apoBplays an important role in HCV entry and that Cideb is essential for HCVassociation with apoB.REF 174Production of infectious virus particles of Alphacoronaviruses differsin a variety of bat speciesAnna Theresa RUEDIGER, Christel SCHWEGMANN WEELSUniversity of veterinary medicine, Hannover, GERMANYREF 175The Epizootic Hemorrhagic Disease Virus (EHDV) Induces andBenefits from Cell Stress, Autophagy and ApoptosisBen SHAI 1 , Eran SCHMUKLER 1 , Roy YANIV 1 , Naomi ZIV 1 , GalitHORN 1 , Velizar BUMBAROV 2 , Hagai YADIN 2 , Nechama I.SMORODINSKY 1 , Eran BACHARACH 1 , Ronit PINKASKRAMARSKI 1 , Marcelo EHRLICH 1Tel Aviv University, Tel Aviv, ISRAEL; Kimron Veterinary Institute, BeitDagan, ISRAELThe mode and timing of virally induced cell death withhold the potentialof regulating viral yield, viral transmission and the severity of virally induceddisease. Orbiviruses such as the Epizootic Hemorrhagic Disease Virus(EHDV) are non enveloped and cytolytic. To date, the death of cells infectedwith EHDV, the signal transduction pathways involved in this processand the consequence of their inhibition have yet to be characterized. Here,we report that two EHDV isolates (EHDV2 Ibaraki and an Israeli isolate ofEHDV7) induce protein kinase R (PKR) activation, protein synthesis inhibition,c Jun N terminal kinase (JNK) activation, cell rounding, autophagyand apoptosis. The timing of these phenomena and the effects of specificchemical inhibitors of PKR, JNK and autophagy suggest they are interconnectedand sequential. Moreover, these inhibitors and a specific caspaseinhibitor reduced the yield of infectious virions, suggesting that the activitiesof PKR and JNK and the induction of autophagy and apoptosis arebeneficial to EHDV replication. Moreover, through live cell microscopy,we identified a mode of EHDV induced cell death involving the ruptureof a bubble like membrane protrusion, which results in spillage of thecellular cytoplasmic contents. This mode of cytolysis may have potentialimplications in viral spread and in the host response to EHDV infection.REF 176RhoB is important for Human Cytomegalovirus lytic replicationGeorge SOURVINOS, Demetrios SPANDIDOS, Nektaria GOULIDAKIMedical School, University of Crete, Heraklion, GREECEHuman Cytomegalovirus (HCMV), a ubiquitous herpesvirus, is a significantpathogen causing medically severe diseases to immunocompromisedindividuals. HCMV primary lytic infection is followed by long terms oflatency. During productive infection, HCMV has evolved a variety of interactionswith proteins of the host, facilitating its successful replication andspread. RhoB belongs to the family of Rho GTPases, which regulatesdiverse cellular processes. Rho proteins are implicated in the entry andegress from the host cell of mainly and herpesviruses, whereas herpesvirusesare the least studied in this regard. Here we set out to studythe role of RhoB GTPase during HCMV lytic infection. We examined thecellular distribution of RhoB during the course of the wild type HCMVAD169 virus infection in fixed cells and of the recombinant UL32 EGFPVirologie, Vol 17, supplément 2, septembre 2013S167

5 th European Congress of VirologyHCMV virus infection in live cells. We found that RhoB translocated tothe assembly complex (AC) of HCMV, a zone where many viral structuralproteins are known to accumulate. Furthermore, RhoB localized atthe AC even when the expression of the late HCMV proteins was inhibited.At the very late stages of infection, cellular projections were formed,containing either the endogenous or transiently expressed RhoB, potentiallycontributing to the viral spread. Interestingly, knockdown of RhoB inHCMV infected cells resulted in significant reduction of the virus titer andhad a dramatic effect on the cellular localization of many viral proteins.In conclusion, our results suggest a requirement of RhoB during HCMVlytic infection.REF 177a/b hydrolase domain containg protein (ABHD5/CGI 58), the causativeprotein for Chanarin Dorfman syndrome, supports hepatitis Cvirus productionGabrielle VIEYRES 1 , Kathrin WELSCH 1 , Lars KADERALI 2 , ThomasPIETSCHMANN 11 Institute of Experimental Virology, TWINCORE, Hannover, GERMANY;2 Institute for Medical Informatics and Biometry, Medical Faculty, Universityof Technology Dresden, Dresden, GERMANYHepatitis C virus (HCV) replication is tightly linked with the host lipidmetabolism. In particular, HCV assembly depends on cytosolic lipid droplets(LDs) and on lipoprotein synthesis, and virions are secreted as lipoviro particles. To elucidate how HCV usurps these pathways, we conducteda rational siRNA based screen by selecting host genes involved in LDbiology and secretion of very low density lipoproteins. The knockdownof nearly half of our candidates significantly inhibited HCV assembly orrelease. Among primary hits we focused on ABHD5 whose knockdownrepressed infectious HCV production similar to ApoE, a known HCVassembly factor. Importantly, this defect was rescued by expression ofan RNAi resistant ABHD5 variant. ABHD5 is associated with the ChanarinDorfman syndrome (CDS), a rare inherited lipid storage disease.Intriguingly, ABHD5 mutants causative of CDS did not support HCVproduction. ABHD5 is a ubiquitously expressed protein which binds LDsto activate lipases for triglyceride mobilization onto ER resident lipoproteinprecursors, thus resulting in lipoprotein maturation. Our preliminaryresults indicate that ABHD5 interacts with HCV core protein in infectedcells. We are currently investigating the localization of ABHD5 uponHCV infection. In parallel, we examine the influence of ABHD5 on distinctsteps of viral assembly and on the virion physicochemical propertiesto pinpoint the step(s) of HCV production controlled by ABHD5. Collectively,ABHD5 could be a key player in the intracellular maturation ofHCV particles and their loading with ApoE and lipids.REF 178Permissivity Of Primary Human Hepatocytes And DifferentHepatoma Cell Lines To Cell Culture Adapted Hepatitis C VirusFrancois HELLE 1 , Etienne BROCHOT 1 , Carole FOURNIER 1 ,Véronique DESCAMPS 1 , Laure IZQUIERDO 1 , ThomasHOFFMANN 1 , Yves Edouard HERPE 2 , Abderrahmane BENGRINE 2 ,Sandrine BELOUZARD 3 , Czeslaw WYCHOWSKI 3 , JeanDUBUISSON 3 , Catherine FRANCOIS 1 , Jean Marc REGIMBEAU 4 ,Sandrine CASTELAIN 1 , Gilles DUVERLIE 1,21 EA4294, Laboratoire de Virologie, Centre Hospitalier Universitaire etUniversité de Picardie Jules Verne, Amiens, FRANCE; 2 Biobanque dePicardie, Centre Hospitalier Universitaire et Université de Picardie Jules,Amiens, FRANCE; 3 Institut Pasteur de Lille; Center of Infection & Immunityof Lille (CIIL); Inserm U1019; CNRS UMR8204; Univ Lille Nordde France, Lille, FRANCE; 4 Département de Chirurgie Digestive, CentreHospitalier Universitaire et Université de Picardie Jules Verne, Amiens,FRANCEDeveloping efficient and physiologically relevant culture systems for allHepatitis C virus (HCV) genotypes remains an important goal. In thiswork, we performed successive infections and obtained a JFH1 strain derivedvirus reaching high titers. Six potential adaptive mutations were identifiedand their effect on HCV replication and infectious particle productionwas investigated. This cell culture adapted virus enabled us to efficientlyinfect primary human hepatocytes, as demonstrated using the RFP NLSIPS reporter protein and intracellular HCV RNA quantification. However,induction of a strong type III interferon response was responsible for HCVinhibition. The disruption of this innate immune response led to a stronginfection enhancement and permitted the detection of viral protein expressionby western blotting as well as progeny virus production. This cellculture adapted virus also enabled us to easily compare the permissivityof seven hepatoma cell lines. In particular, we demonstrated that HuH 7,HepG2 CD81, PLC/PRF/5 and Hep3B cells were permissive to HCV entry,replication and secretion even if the efficiency was very low in PLC/PRF/5and Hep3B cells. In contrast, we did not observe any infection of SNU 182,SNU 398 and SNU 449 hepatoma cells. Using iodixanol density gradients,we also demonstrated that the density profiles of HCV particles producedby PLC/PRF/5 and Hep3B cells were different from that of HuH 7 andHepG2 CD81 derived virions. These results will help the development ofa physiologically relevant culture system for HCV patient isolates.REF 179SARS CoV envelope protein palmitoylation or disulfide bondformation is dispensable for efficient virus like particle assemblyChin Tien WANG 1,2 , Ying Tzu TSENG 2 , Shiu Mei WANG 1 , Kuo JungHUANG 21 Institute of Clinical Medicine/National Yang Ming University, Taipei,TAIWAN; 2 Department of Medical Research and Education/Taipei VeteransGeneral Hospital, Taipei, TAIWANCoronavirus membrane (M) proteins are capable of interacting withnucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndromecoronavirus (SARS CoV) M co expression with N is sufficient forproducing virus like particles (VLPs), but the addition of E co expressionmarkedly enhances VLP yields. Whether E can release from cells orE/N interaction exists so as to contribute to enhanced VLP production isunknown. It also remains to be determined whether E palmitoylation ordisulfide bond formation plays a role in SARS CoV virus assembly. Inthis study we show that SARS CoV N is released from cells through anassociation with E protein containing vesicles. Further analysis suggeststhat domains involved in E/N interaction are largely located in both carboxylterminal regions. Changing all three E cysteine residues to alaninesdid not exert negative effects on E release, E association with N, or Eenhancement of VLP production, suggesting that E palmitoylation modificationor disulfide bond formation is not required for SARS CoV virusassembly. We found that removal of the last E carboxyl terminal residuemarkedly affected E release, N association, and VLP incorporation, butdid not significantly compromise the contribution of E to efficient VLPproduction. The independence of the SARS CoV E enhancement effecton VLP production from its viral packaging capacity suggests a distinctSARS CoV E role in virus assembly.REF 180Proteomic analysis of purified dengue virus from HepG2 infected cellsupernatantsFrederic BEDIN 1 , Romain FRAGNOUD 1 , Alexandre PACHOT 1 ,Glaucia PARANHOS BACCALA 21 BioMerieux SA, Lyon, FRANCE; 2 Fondation Merieux, Lyon, FRANCEDengue is the most common mosquito borne viral disease of public healthsignificance. Patients can develop life threatening dengue hemorrhagicS168 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyfever or dengue shock syndrome (DHF/DSS) due to endothelial cells andplatelets dysfunctions. Therapy is symptomatic and aims at controllingthe clinical manifestation of hemorrhages and shock. Prognosis of diseaseseverity is essential to adjust patient management. The pathogenesis of theDHF/DSS has not been fully elucidated but the nature of the virus hostinteractions has been identified as a potential risk factor. In order to analysevirion associated host protein composition, a method allowing the detailedanalyse of proteome of purified virions from HepG2 cells infected withdengue virus (DV) was developed. Five days after infection, virions werepurified from the culture supernatants by the combination of ultracentrifugationsand a water insoluble polyelectrolyte based technique. The purifiedparticles were controlled by immunoblots and electron microscopy. Afterin gel hydrolysis, peptides were analysed by nano liquid chromatographycoupled to ion trap mass spectrometer and identified on data libraries. Thelevel of viral NS1 protein excreted in the culture supernatants and thequantity of virus released were shown to be proportional to the viral input.Dengue virus structural proteins and more than 40 cellular proteins werespecifically and repeatedly identified on purified DV. The impact of theseproteins on disease outcome is discussed, as well as their potential use asfingerprint for dengue severity prognosis.Virologie, Vol 17, supplément 2, septembre 2013S169

5 th European Congress of Virology12. VIRAL GENE EXPRESSION –TRANSCRIPTION, TRANSLATIONPosters: REF 181 to REF 197REF 181In vitro Molecular Analysis of Ribosomal Initiation ComplexesAssembly during the Initiation of Translation of a Wild Strain anda Live Attenuated Coxsackievirus B3 RNAsJawhar GHARBI 1,2 , Amira SOUII 1 , Manel BEN M’HADHEBGHARBI 1,2 , Audrey BROSSARD 3 , Nathalie CHAMOND 3 , MahjoubAOUNI 1 , Bruno SARGUEIL 31 Laboratory LR99 ES 27, University of Monastir, Monastir, TUNISIA;2 Higher Institute of Biotechnology, Monastir, TUNISIA; 3 Laboratoire deCristallographie et RMN Biologiques (UMR 8015) faculté de Pharmacie,Université Paris Descartes, Paris, FRANCECoxsackievirus B3 (CVB3) is an Enterovirus of the family of Picornaviridae.The Group B coxsackieviruses include six serotypes (B1 to B6)that cause a variety of human diseases including myocarditis, meningitisand diabetes. Among the group B, the B3 strain is mostly studied forits cardiovirulence and its ability to cause acute and persistent infections.Translation initiation of CVB3 RNA has been shown to be mediated bya highly ordered structure of the 5 ′ untranslated region (5 ′ UTR), whichharbors an internal ribosome entry site (IRES). Translation initiation is acomplex process in which initiator tRNA, 40S and 60S ribosomal subunitsare assembled by eukaryotic initiation factors into an 80S ribosome atthe initiation codon of the mRNA. We have previously described that theSabin3 like mutation (U473 C) introduced in CVB3 genome led to a defectivemutant with a serious reduction in translation efficiency. In this study,we analyzed the efficiency of formation of ribosomal initiation complexes48S and 80S through 10 30% and 10 50% sucrose gradients using RabbitReticulocyte Lysates (RRLs) and stage specific translation inhibitors: 5’Guanylyl imidodiphosphate (GMP PNP) and Cycloheximide (CHX), respectively.We demonstrated that the interaction of 48S and 80S ribosomalcomplexes within the mutant CVB3 RNA was abolished compared withthe wild type RNA by ribosome assembly analysis. Taken together, it canbe possible that the mutant RNA was unable to interact with some transacting factors critical for enhanced IRES function.REF 182Molecular Characterization RNA Protein Interactions during the Initiationof Translation of wild type and a mutant Coxsackievirus B3strainsJawhar GHARBI 1,2 , Manel BEN M’HADHEB GHARBI 1,2 , AmiraSOUII 11 Laboratory LR99 ES27, University of Monastir, Monastir, TUNISIA;2 Higher Institute of Biotechnology, Monastir, TUNISIATranslation initiation of Coxsackievirus B3 (CVB3) RNA is directed byan internal ribosome entry site (IRES) within the 5 ′ untranslated region.Host cell factors involved in this process include some canonical translationfactors and additional RNA binding proteins. We have, previously,described that the Sabin3 like mutation (U473 >C) introduced in CVB3genome led to a defective mutant with a serious reduction in translationefficiency. With the aim to identify proteins interacting with CVB3 wildtype and Sabin3 like IRESes and to study interactions between either HeLacell or BHK 21 protein extracts and CVB3 RNAs, UV cross linking assayswere performed. We have observed a number of proteins that specificallyinteract with both RNAs. In particular, molecular weights of five of theseproteins resemble to those of the eukaryotic translation initiation factors4G, 3b, 4B and PTB. We have demonstrated a better affinity of CVB3RNA binding to BHK 21 proteins and a reduced interaction of the mutantRNA with almost cellular polypeptides compared to the wild type IRES.On the basis of phylogeny of some initiation factors and on the knowledgeof the initiation of translation process, we focused on the interaction ofboth IRESes with eIF3, p100 (eIF4G) and 40S ribosomal subunit by FilterBinding assays. We have demonstrated a better affinity of binding to thewild type CVB3 IRES. Thus, the reduction efficiency of the mutant RNAto bind to cellular proteins involved in the translation initiation could bethe reason behind inefficient IRES function.REF 183Characterization of human cytomegalovirus microRNA US25 2 3pCarlotta ALBONETTI 1 , Marta TREVISAN 1 , AlessandroSINIGAGLIA 1,2 , Enrico LAVEZZO 1 , Giulia MASI 1 , Luisa BARZON 1 ,Giorgio PALÙ 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 IOV Istituto Oncologico Veneto, Padova, ITALYThe activity of only few human cytomegalovirus (HCMV) microRNAs(miRNAs) has been investigated so far and mainly involved in viral lifecycle and evasion from host immune response. Aim of this study was tocharacterize HCMV miR US25 2 3p expression profile and to identifyits target genes. MiR US25 2 3p was one of the most expressed HCMVmiRNAs in MRC 5 cells infected with HCMV Towne strain; it was highlyexpressed since 4 hours post infection and continued to accumulateover time. Among targets for miR US25 2 3p that were predicted bya consensus of sequence based algorithms, PTBP1 (polypyrimidinetract binding protein 1), HDAC11 (histone deacetylase 11), and IFI30(INF inducible protein 30) were selected for experimental validation.Luciferase assays and western blot analysis demonstrated that the 3 ′UTR sequences of these genes were targeted by miR US25 2 3p and thatprotein expression levels were significantly inhibited by the miRNA ina dose dependent manner. In transfected cells, miR US25 2 3p inhibitedPTBP1 dependent regulation of alternative splicing of cellular andviral transcripts. Alternative splicing of HCMV major immediate earlygene IE1, that is known to be antagonized by PTBP1, was enhancedby miR US25 2 3p. Finally, the positive effect of miR US25 2 3p onHCMV replication was verified in a miR US25 2 3p knock out model.In conclusion, miR US25 2 3p is expressed in the early phases of HCMVreplication and targets genes involved in the epigenetic regulation of viraland host gene expression and in innate immune response.REF 184Identification of structural features regulating the expression ofKaposi’s sarcoma herpesvirus microRNAsMaud CONTRANT 1 , Aurélie FENDER 1 , Béatrice CHANE WOONMING 1 , Sébastien PFEFFER 11 IBMC UPR 9002, Strasbourg, FRANCERecently, micro (miRNAs) have been identified as crucial players in thepathogenesis and survival of some viruses. Kaposi’s sarcoma associatedherpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma and isinvolved in agressive B lymphomas. It encodes 12 precursor miRNAs (premiRNAs), expressed on the same long primary transcript (pri miRNA).KSHV miRNAs are encoded from the latency region and are important toinhibit apoptosis and to regulate the host cell cycle. Our goal is to showhow the accumulation of these small RNAs is regulated in KSHV infectedcells. The biogenesis of miRNA is a highly controlled process that involvessequential maturation of pri miRNA by two RNase III enzymes: Drosha inthe nucleus and Dicer in the cytoplasm. We used quantitative northern blotS170 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyanalysis and deep sequencing to investigate KSHV miRNA expression.Using two cellular contexts, a B cell line infected by KSHV and a stable cellline with a genomic integration of the KSHV miRNA cluster, we identifieddifferential accumulation of the KSHV miRNAs. Our results suggest thatthe expression of the viral miRNAs is post transcriptionally regulated. Wefocus on factors influencing the regulation, like structural features of thepri miRNA or accessory proteins important for the expression of miRNAs.We determined the secondary structure of KSHV pri miRNAs by SHAPE(Selective 2’ Hydroxyl Acylation analyzed by Primer Extension). Then,we verified if the pre miRNA stem loops were optimal Drosha substratesand we tried to identify regulatory elements required for the expression ofKSHV miRNAs.chemiluminescent (CL) detection have been developed to quantify viralDNA, NS and VP1 2 transcripts, and NS and VP1 2 proteins followingUT7/EpoS1 infection. CL microscope imaging methods allowed evaluatingthe fraction of infected cells and the amount of viral nucleic acidswithin each single cell. In our experimental conditions, B19 transcriptionalactivity, leading to the production of both NS and VP mRNAs, hasbeen detected early in viral cycle, in increasing fractions of cells from 12hpi, and before genome replication, evident from 24 hpi. NS protein hasbeen detected mainly at early stages of infection, while the capsid proteinsaccumulate within cells only at 48 72 hpi, suggesting an inhibitory effecton capsid protein translation as a post transcriptional regulation event.REF 185Characterization of T Antigens encoded by Trichodysplasia spinulosaassociated polyomavirus; Evidence for Middle T ExpressionMariet FELTKAMP, Christina DARGEL, Els VAN DER MEIJDENLeiden University Medical Center, Leiden, THE NETHERLANDSIn 2010 we identified the Trichodysplasia spinulosa associated polyomavirus(TSPyV) in a patient with Trichodysplasia spinulosa (TS), a follicularskin disease of immunocompromized patients. Like other polyomaviruses(PyVs), the genome of TSPyV can be divided in an early (T) and late (VP)coding region. T antigens play key roles in viral DNA replication andtranscription, and in some cases are responsible for cellular transformation.The T antigen mRNAs are alternatively spliced to encode at least twoproteins, Small T (ST) and Large T (LT), respectively. For some PyVs upto five T transcripts have been identified including one coding for MiddleT (MT), as shown for the murine and hamster PyV. To characterize theT antigens encoded by TSPyV, the complete early region of TSPyV wascloned into pcDNA3 and transiently expressed in HeLa and 293T cells.Sequencing of RT PCR products revealed five different transcripts: T1 (STlike), T2 (LT like), T3 (17kT/57kT like), T4 (MT like) and T5 (ST* like).Transcripts T2, T3 and T4 or T5 (similar expected size) were confirmedby northern blotting. Experiments to confirm protein expression of theidentified T antigen transcripts, in transfected cell lysates and in clinicalsamples, are in progress. In summary, for TSPyV a transcription patternwas found that largely overlaps with other human PyVs. In addition a MTtranscript was identified so far unique for human PyVs. To what extendthe identified T transcripts result in protein expression is the subject ofcurrent study. When obtained, these data will be presented as well.REF 186Analysis on Parvovirus B19 life cycle at single cell level by chemiluminescentimaging assaysGiorgio GALLINELLA 1 , Francesca BONVICINI 1 , Gloria BUA 1 ,Elisabetta MANARESI 1 , Mara MIRASOLI 2 , Martina ZANGHERI 2 ,Aldo RODA 21 Department of Pharmacy and Biotechnology, University of Bologna,Bologna, ITALY; 2 Department of Chemistry, University of Bologna, Bologna,ITALYHuman Parvovirus B19 (B19) is a single stranded DNA virus whosegenome encodes a multifunctional non structural protein (NS), two capsidproteins (VP1, VP2) and additional small non structural proteins (7.5, 9.0and 11 kDa). B19 genome expression has been extensively investigated bymeans of techniques, such as quantitative PCR based assays, that requireextracted nucleic acids from a large number of cells. As a consequence, thedata obtained represent a mean value within cell population. Consideringthat B19 replication process is highly restrictive within the same cell population,methodologies allowing analysis at single cell level could representpotent tools for the investigation of cell virus interactions. In the presentstudy, in situ hybridization and immunocytochemical assays exploitingREF 187Down regulation of HCV Ires dependent translation by nS5a ismediated by PkrEirini KARAMICHALI, Eliza TSITOURA, Peli FOKA, UraniaGEORGOPOULOU, Penelope MAVROMARAHellenic Pasteur Institute, Athens, GREECETranslation initiation of the HCV genome is cap independent and is drivenby an internal ribosome entry site (IRES), located mainly within the5 ′ non coding region. Different data suggested that different viral proteinscan regulate the activity of the HCV IRES as part of the virus’s strategies tocontrol its life cycle. Among them, HCV NS5A protein negatively modulatesthe HCV IRES activity in a specific specific and dose dependentmanner in HepG2 cells. PKR is the RNA activated protein kinase andmediates an anti viral response in humans. PKR plays multiple roles incells, in response to different stress situations. It is previously reportedthat HCV IRES can activate PKR through its binding at domain II ofthe HCV IRES. NS5A protein through the IFN sensitivity determiningregion (ISDR) may mediate IFN resistance and through a direct interactionwith the protein kinase catalytic domain seems to repress PKRactivity. The aim of this study is to investigate whether HCV NS5A downregulation of HCV dependent translation is mediated through PKR. Toaddress this issue, transient transfection was carried using different plasmidvectors either expressing a bicistronic transcriptional unit carrying thechloramphenicol acetyltransferase (CAT) and the firefly luciferase (LUC)genes separated by the HCV or EMCV IRESs or expressing separatelyHCV NS5A and PKR proteins. Induction of PKR was performed by RNAtransfection using polyI;C or interferon. Silencing of PKR was performedusing specific siRNA. We present data concerning the effect of PKR inHCV IRES dependent translation in hepatoma cells as well as, the effect ofNS5A on the modulation of PKR. We provide evidence that HCV NS5Aprotein represses HCV IRES activity through inactivation of the PKR.REF 188The analysis of the inhibition of human cytomegalovirus ImmediateEarly 2 protein by a small molecule reveals interference with DNAbinding and new target sites on a responsive promoterBeatrice MERCORELLI 1 , Anna LUGANINI 2 , Serena MASSARI 3 ,Oriana TABARRINI 3 , Santo LANDOLFO 2 , Giorgio GRIBAUDO 4 ,Giorgio PALÙ 1 , Arianna LOREGIAN 11 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Department of Public Health and Microbiology, University of Turin,Turin, ITALY; 3 Department of Chemistry and Technology of Drugs, Universityof Perugia, Perugia, ITALY; 4 Department of Life Sciences andSystems Biology, University of Turin, Turin, ITALYHuman cytomegalovirus (HCMV) Immediate Early 2 (IE2) protein is amulti tasking, essential protein which regulates both viral and cellulargene expression. By protein protein interactions and by direct bindingto specific DNA sequences, IE2 is able to transactivate responsiveVirologie, Vol 17, supplément 2, septembre 2013S171

5 th European Congress of Virologypromoters, to repress its own promoter, and to modulate the host cellcycle. Since the molecular aspects of its functioning still remain to befully elucidated, taking advantage of a specific IE2 inhibitor termedWC5, we investigated deeper the mechanisms of IE2 regulatory activityon responsive promoters. By mutagenesis, cell based assays, and electrophoreticmobility shift assays, we demonstrated that the abortive effectexerted by the compound on HCMV replication is due to the interferencewith the interaction between IE2 and different DNA sequences locatedwithin responsive viral promoters. Moreover, by focusing on a prototypicEarly promoter, i.e., the DNA polymerase gene promoter, we analysedthe contribution of different DNA elements in its transactivation andidentified two previously uncharacterised sequences bound by IE2 invitro, involved in UL54 promoter transactivation in transfected cells,and that may serve for the recruitment of IE2 at the promoter. Our studycontributes in shedding light on the molecular aspects of IE2 activity asan essential virus encoded transcription factor.REF 189DNA methylation: A key regulator of Marek’s disease virus life cycle?Carole MIGNION 1 , Isabelle GENNART 1 , Damien COUPEAU 1 , SylvieLAURENT 2 , Ginette DAMBRINNE 2 , Denis RASSCHAERT 2 , BenoîtMUYLKENS 11 Integrated Veterinary Research Unit (IVRU), Namur, BELGIUM;2 Transcription Lymphome Viro Induit (TLVI), Tours, FRANCEMarek’s disease is a lymphoproliferative disease, induced by an avianherpesvirus, Marek’s disease virus (MDV). MDV presents two alternativestages of infection: lytic and latent. During latency, only few transcripts,involved in the maintenance of the latency phase and/or in tumerogenesis,are expressed. During the lytic phases, latency transcripts are repressedwhile others genes involved in the lytic phase are reactivated. In this study,we investigated the role of DNA methylation in the regulation of the geneexpression switch observed in the alternative stages of MDV infection.Five genes related with lytic (pp38, ICP4 and ICP27) and latent (clustermicroRNA 9/4 and vTR) phases were selected. Firstly, the impact of DNAdemethylation was assessed on pp38, ICP27 and miR9/4 transcript levelsin latently infected cells. The viral genome demethylation induced a sharpincrease in the expression level of the three genes, showing strong associationbetween DNA methylation patterns and promoter activities. Then,the promoters were analyzed to determine their DNA methylation patternthrough Bisulfite Genomic Sequencing Analysis at relevant in vitro andin vivo conditions of MDV infection. For pp38, ICP4 and vTR promoters,the CpG methylation level is high (80%) in latency while it is lower (18%)during reactivation and presents 0% in lytic infection. On the contrary,miR4 5p promoter shows 0% in the three conditions. In vivo studies onperipheral blood leucocyte infected with MDV revealed that ICP4, ICP27and vTR promoters display a DNA methylation percentage close to 0%.REF 190Investigation of influenza A virus M segment splicing controlOlivier MONCORGÉ 1 , Anita ARTARINI 2 , Alexander KARLAS 2 ,Thomas MEYER 2 , Chad M SWANSON 3 , Michael H MALIM 3 , WendyS BARCLAY 11 Section of Virology, Department of Medicine, Imperial College London,London, UNITED KINGDOM; 2 Molecular Biology Department, MaxPlanck Institute for Infection Biology, Berlin, GERMANY; 3 Department ofInfectious Diseases, Kings College London School of Medicine, London,UNITED KINGDOMInfluenza virus, as an obligatory intracellular parasite, relies on host cellfunctions for various aspects of its replication cycle. Splicing is one ofthe key cellular functions essential for influenza virus replication. Humangenes typically include introns, which are removed by the splicing machinery.Two of the viral gene segments (M and NS) produce spliced andunspliced viral mRNAs. Regulation of M and NS genes splicing is currentlynot well understood. It is believed that both virus and cellular factorsare involved in the kinetics of influenza virus mRNA splicing, and inefficientsplicing control has been suggested to be responsible for abortivereplication in some models. We developed a cell based influenza polymerasedriven minireplicon assay that monitors expression of three mRNAsproduced from the M gene: M1 (unspliced) and M2 and m3 (spliced).Using this assay, we tested whether the nature of the viral polymerasethat directs viral mRNA transcription affects the efficiency of splicing andalso confirmed that the viral protein NS1 modulates M gene pre mRNAsplicing. Investigating a role for the host cell in influenza splicing control,we compared M gene splicing in cell types of mammalian and avian originsand investigated the role of some cellular factors on M gene splicingcontrol. We also tested in this system some known small molecule inhibitorsof the splicing machinery and concluded that this assay can also beused to screen for novel inhibitors of splicing that might control influenzareplication and from which the virus could not escape.REF 191The metabolic sensors FXRa, PGC 1a and SIRT1 cooperativelyregulate hepatitis B virus transcriptionChristophe RAMIÈRE 1,2,3 , Claire CURTIL 2,3 , Liviu SorinENACHE 2,3,4 , Pauline RADREAU 2,3 , Anne Gaëlle DRON 1,2,3 , CarolineSCHOLTÈS 1,2,3 , Alexandre DELOIRE 2,3 , Didier ROCHE 5 , VincentLOTTEAU 2,3 , Patrice ANDRÉ 1,2,31 Hospices Civils de Lyon, Lyon, FRANCE; 2 INSERM U1111, CIRI, Lyon,FRANCE; 3 Université de Lyon, Lyon, France; 4 University of Medicineand Pharmacy, TârguMure, ROMANIA; 5 Edelriss.a.s., Lyon, FRANCEHepatitis B virus (HBV) transcription is highly dependent on liver enrichednuclear receptors involved in metabolic regulation. Among thesenuclear receptors, the bile acid receptor farnesoid X receptor a (FXRa) hasbeen shown to enhance HBV core promoter activity and pregenomic RNAsynthesis. Interestingly, two FXRa modulators, peroxisome proliferatoractivated receptor coactivator 1 a (PGC 1a) and the deacetylase SIRT1have been detected associated to HBV genomes ex vivo. Moreover, PGC1a induction during fasting has been shown to increase HBV replication inmice liver. SIRT1 is also activated in the fasted state. However its impact onHBV life cycle is currently unknown. In this study, we investigated the roleof SIRT1 activation on the regulation of HBV transcription and replicationin vitro. Using a luciferase reporter system, we first demonstrated thatSIRT1 activation increases the transcription from the core promoter regionthrough an FXRa and PGC 1a dependent mechanism. Moreover, we showedin cells transfected with a full length HBV genome, that FXRa, PGC1a and SIRT1 cooperate to enhance pregenomic RNA synthesis and covalentlyclosed circular DNA accumulation. We thus identified a subnetwork,composed of the nuclear receptor FXRa, the transcriptional coactivatorPGC 1a and the energy sensor SIRT1, which increases HBV replication.Each of these proteins is a key regulator of physiological metabolism,strengthening the hypothesis that HBV replication is tightly controlled byenvironmental stimuli, very similarly to metabolic genes.REF 192In vivo transcriptomic study of 39 viral genes and 17 host genes inPacific oyster, Crassostrea gigas infected by ostreid herpesvirus 1:comparing two oyster families presenting different levels of susceptibilityAmelie SEGARRA, Nicole FAURY, Florian MAUDUIT, PhilippeHAFFNER, Jean François PEPIN, Delphine TOURBIEZ, SuzanneTRANCARD, Agnes TRAVERS, Pierrick MOREAU, TristanRENAULTIfremer LGPMM, La Tremblade, FRANCES172 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologySince 1991, high rates of mortality among Pacific oysters spat have beenassociated with the detection of a herpesvirus called ostreid herpesvirus1, (OsHV 1) and reported in different countries, including France. However,no study has been performed to understand and follow viral geneexpression during the viral infection whereas gene expression in otherherpesviruses was widely studied. An in vivo transcriptomic study wasperformed during an OsHV 1 infection in Crassostrea gigas spat in orderto better undrstand interactions between the host and its pathogen. In thiscontext, 39 viral genes and 17 host genes were selected and analysed byreal time PCR using two oyster families presenting different levels of susceptibiltyto the virus. First virus RNAm transcripts were detected at 8 hpost injection (hpi) in the most susceptible family whereas in the less susceptibleone first transcripts were observed at 12 hpi. After 12 hpi 39 viralgenes were detected in the most susceptible family. However, in the lesssusceptible family, RNA transcripts of all the 39 virus genes were detectedonly at 26 hpi. In addition, analysis of the relative expression of the hostgene encoding the Myeloid differentiation factor 88 showed an up expressionin the most susceptible family at 26 h pi. This study focused on thedetecion of RNAm transcripts in Crassostrea gigas spat challenged withOsHV 1. First results confirm the replication of the virus during infectionand therefore information on the viral life cycle of OsHV 1 and the defensemechanisms against the virus can be drawn.REF 193The effect of coxsackievirus A9 (CAV9) infection on the nucleusproteins PSF, SC35 and PML, located in the nuclear paraspeckles,speckles and PML bodies respectivelyAshjan SHAMI, Glyn STANWAY, Maysoon MUTABAGANIUniversity of Essex, Colchester, UNITED KINGDOMPicornaviuses replicate in the cytoplasm, but there is growing evidencethat the cell nucleus is affected by infection e.g. transcription factor cleavage,relocation of nuclear proteins and alteration to nucleo cytoplasmicshuttling. The nucleus has a number of sub domains and structures, someof which have been shown to be affected by virus infection. We have previouslyobserved that human parechoviruses affect the distributions of thenuclear paraspeckle protein PSF, but not proteins associated with PMLbodies or nuclear speckles. We are currently studying whether enteroviruseshave the same effect, using coxsackie virus A9 (CAV9), to determinewhether this effect on nuclear paraspeckles and lack of an effect on specklesand PML bodies are general features of picornavirus infections. Thisis being studied using EGFP PSF, EGFP SC35 and EGFP PML fusionsand antibodies to these nuclear proteins to investigate the timescale of relocalisation,how the virus achieves any change and the role these changesplay in enteroviruses and parechvirus replication.REF 194Characterization of the translational mechanism that controls thesynthesis of the short form of HCV core+1/ARFP proteinNiki VASSILAKI 1 , Ioly KOTTA LOIZOU 1 , PanagiotisSAKELLARIOU 1 , Ralf BARTENSCHLAGER 2 , PenelopeMAVROMARA 11 Hellenic Pasteur Institute, Molecular Virology, Athens, GREECE;2 University of Heidelberg, Department of Infectious Diseases, MolecularVirology, Heidelberg, GERMANYPrevious studies from our laboratory and others have shown that HepatitisC Virus (HCV) genome, in addition to the polyprotein precursor openreading frame (ORF), possesses a second functional ORF overlapping thecore gene that encodes a protein initiated by internal translation initiationat codons 85/87, known as core+1/ARFP/S (short). To investigate themolecular mechanism that controls core+1/S synthesis, we searched fora putative IRES like element within the core coding sequence. For this,different parts of the 1a/2a core coding sequences were tested for theirputative IRES activity after insertion in a dual luciferase reporter construct.In vitro transcribed RNAs derived from these cassettes were transfectedinto Huh7 cells. The results showed the presence of a functional regulatoryelement within the 5 ′ end of core/core+1 coding sequence that coulddirect internal translation initiation of the core+1/ARFP. This element iscomprised of the core nucleotides located upstream of the core+1 initiatorcodons 85/87. The integrity of the conserved core RNA structures stemloop SL47 and SL87 is essential for this IRES activity. Interestingly, inthe JFH1 replicon system, core+1/S is the predominant isoform of core+1protein at early hours of viral translation/replication cycle and is expressedindependently of a functional viral IRES or polyprotein initiator whereashighly correlated with intact stem loops SL47 and SL87. At late hours ofvirus replication cycle, core+1/short is coexpressed with a longer core+1isoform that is translated in a IRES dependent manner.REF 195Investigating the interactions between the FMDV Lbpro and hostfactor eIF4GIIMartina AUMAYR 1 , Sofiya FEDOSYUK 1 , Georg KONTAXIS 2 ,TimSKERN 11 Max F. Perutz Laboratories, Medical University of Vienna, Vienna,AUSTRIA; 2 Max F. Perutz Laboratories, University of Vienna, Vienna,AUSTRIAThe picornaviral foot and mouth disease virus (FMDV) is the causativeagent of a highly contagious disease affecting cloven hoofed animals.Upon infection, the single stranded RNA genome is released into thecytoplasm and interacts with the host cell translation machinery. In orderto achieve efficient replication of their own proteins, picornavirusesinterfere with cellular processes. Thus, FMDV hijacks the host celltranslation machinery by cleaving the eukaryotic initiation factor (eIF)4Gleading to the shut off of host cell protein production. eIF4G cleavage isperformed by the leader protease (Lbpro). Picornaviral cleavage of 4G isa determinant of virulence, but has not yet been elucidated structurally.We are therefore investigating how the Lbpro interacts with 4G. Weshowed that 4E enhances the cleavage of 4G by the Lbpro in an in vitroassay. A complex of 4G and Lbpro is not detectable on size exclusionchromatography. However, when 4E is present a complex of all threeproteins is stably formed. Experiments with surface plasmon resonanceshowed similar results and confirmed that the dissociation constant of the4G 4E complex with the Lbpro is about 10 fold lower than that for eachsingle protein to the Lbpro. The single mutation C133S near the LbproC terminus influences the 4G cleavage by reducing Lbpro binding tothe 4G/4E complex. We have started to elucidate the structure of the 4Gfragment by means of two and three dimensional NMR experiments.REF 196Interaction of tick borne encephalitis virus and host cell on proteinsynthesis levelHana TYKALOVÁ 1,2 , Zuzana VAVRUŠKOVÁ 2 , Jirí CERNÝ 1,2 , LiborGRUBHOFFER 1,21 Faculty of Science, University of South Bohemia, Ceské Budejovice,CZECH REPUBLIC; 2 Biology Centre, Institute of Parasitology, CzechAcademy of Sciences, Ceské Budejovice, CZECH REPUBLICTick borne encephalitis virus (TBEV) is a lurking menace that endangerslives of thousands of people annually. In order to accomplish their lifecycle successfully, viruses must compete with host cell at many levelsand need to hijack host cell machinery. To determine an unknown aspectof TBEV pathogenesis we focused on the virus interaction with host cellprotein synthesis pathway in human neural cells. Several aspects wereevaluated. We studied the onset of TBEV protein synthesis in the host cellsVirologie, Vol 17, supplément 2, septembre 2013S173

5 th European Congress of Virologyupon the infection as well as we wanted to explore its link to host proteinsynthesis. We used a novel metabolic labelling methodology utilizing theClick iT ® AHA chemistry, which is a sensitive, non toxic alternative tothe traditional radioactive labelling technique. We compared the amountof de novo synthesized proteins in infected versus non infected cells. Thenwe focused on involvement of mTOR and PKR pathways in the regulationof protein synthesis by monitoring the key regulation proteins, such asS6K1, 4EBP1, eIF2A. We also used a low molecular weight inhibitorwortmannin to elucidate ability of TBEV to synthesize its proteins whenclassical cap dependent translational pathway was blocked. We observedactive virus reproduction, which indicates that TBEV can use a differentcap independent mechanism of translation initiation. In conclusion, webrought a new insight to the complex interaction of TBEV and host cellon protein synthesis level.REF 197Study of alternative splicing of Cauliflower Mosaic Virus 35S RNAClément BOUTON, Angèle GELDREICH, Maria DIMITROVA, LyubovA. RYABOVA, Mario KELLERInstitut de Biologie Moléculaire des Plantes, CNRS Université de Strasbourg,Strasbourg, FRANCECauliflower Mosaic Virus (CaMV) is a plant pararetrovirus and the typemember of the family Caulimoviridae: its genome is a circular dsDNA,which is replicated via reverse transcription of a pregenomic RNA. CaMVgenome contains seven ORFs and is transcribed into two RNAs, a pregenomicpolycistronic 35S RNA and a subgenomic 19S RNA. The 35S RNAserves both as a replicative intermediate and as a mRNA for expressionof all viral proteins except the transactivator – viroplasmin (TAV) protein,which is mainly produced from the 19S RNA. Translation of the 35SRNA occurs via non canonical translation mechanisms mainly mediatedby TAV. In the Cabb S strain, the 35S RNA undergoes alternative splicingthat involves four donor sites and one acceptor site all located in the RNA5 ′ region (Kiss László et al., EMBO J. 1995). Splicing is essential forCaMV infectivity since mutations of the acceptor site make the virus noninfectious and downregulate expression of the viral protein P2 consideredas toxic (Froissart et al., J Gen Virol. 2004). Our results showed that thesplicing patterns of two CaMV strains Cabb S and Cabb B JI are morecomplex than previously described. By using a reverse transcription PCRbased approach we found several new splice sites that are used to generatemore than ten different spliced 35S RNAs in each strain. Some of theseRNAs can be reverse transcribed into dsDNA. Interestingly, almost allsplice sites are conserved among the sequenced CaMV strains. Howeverthe importance of multiple splice sites for CaMV is not yet understood.Our last data will be presented.S174 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology13. VIRAL REPLICATION STRATEGIESPosters: REF 198 to REF 215characterize the CHIKV RdRp and the molecular details of CHIKV RNAsynthesis in vitro. The in vitro system will be applied in mechanism ofaction studies on inhibitors of CHIKV replication. Determination of theCHIKV RdRp crystal structure should ultimately allow structure baseddrug design.REF 198Herpes Simplex Virus type 1 tegument protein VP11/12 causes disruptionof the Golgi apparatus in neuronsYennyfer ARANCIBIA 1 , Carolina MARTIN 1 , Gonzalo MARDONES 2 ,Carola OTTH 11 Instituto de microbiologia clinica, facultad de medicina, universidadAustral de Chile, Valdivia, CHILE; 2 Instituto de fisiologia, facultad demedicina, universidad Austral de Chile, Valdivia, CHILETegument proteins are layered between the capsid and the envelope ofherpesviruses, and they are delivered into the cytoplasm during viral infectionto manipulate host cell functions. VP11/12 is one of the most abundanttegument proteins of Herpes Simplex Virus type 1 (HSV 1), but its functionduring infection is not well established. During T cell infection VP11/12activates the lymphocyte specific Src family kinase (SFK) Lck, and istyrosine phosphorylated in an Lck dependent manner; however the downstreameffects are poorly understood. Recently, it has been shown that HSV1 has an effect on the dynamics of the Golgi, but it is unknown whethertegument proteins are involved. Considering that VP11/12 has been foundassociated to the trans Golgi network (TGN), we hypothesized that thisprotein plays a role in Golgi fragmentation. The aim of this study was toevaluate both morphological changes and the distribution of Golgi proteinsduring either infection or overexpression of VP11/12 in neurons inprimary culture. We found that infection with HSV 1 induces Golgi fragmentationand redistribution of Golgi proteins. These changes were alsoinduced by overexpression of VP11/12. Our results suggest that duringHSV 1 infection VP11/12 induces changes in the structure and functionof the Golgi apparatus, which may have consequences for neuronal survivaland functionality. Funding: FONDECYT 1120464 and CONICYT24121539.REF 200Functional fitness of ribonucleoprotein complexes reconstituted frominfluenza A H5N1, H1N1pdm09 and H3N2Paul CHAN, Karry NGAI, Martin CHANDepartment of Microbiology, Chinese University of Hong Kong, CHINABackground: Pandemic influenza strains are aroused from reassortmentbetween human and avian viruses. To understand cross species adaptation,we examined the functional fitness of hybrid ribonucleoproteincomplexes reconstituted from avian and human viruses. Methods: ViralRNA polymerase subunits PB1, PB2, PA and NP derived from influenza AH5N1, H1N1pdm09 and H3N2 were co expressed with pPolI vNP Luc inhuman cells, and with its function evaluated by luciferase reporter assay.A quantitative RT PCR was used to measure vRNA, cRNA, and mRNAlevels for assessing the replication and transcription efficiency. Mutationswere created to identify signature of increased adaptability. Results:H5N1 ribonucleoprotein complexes incorporated with PB2 derived fromH1N1pdm09 and H3N2 increased the polymerase activity in human cells.Furthermore, single amino acid substitutions at PB2 of H5N1 could affectpolymerase activity in a temperature dependent manner. Enhancement inreplication and transcription activities of ribonucleoproteins was observedafter introduction of lysine at residue 627 in the H5N1 PB2 subunit.Although less strongly in polymerase activity, E158G mutation appearedto alter the accumulation of H5N1 RNA levels in a temperature dependentmanner.Conclusions: H5N1 viruses can adapt to humans either by acquisition ofPB2 from circulating human adapted viruses through reassortment, or bymutations at critical sites in PB2. This information may help to predict thepandemic potential of newly emerged influenza strains.REF 199Characterization of the Chikungunya virus RNA dependent RNApolymeraseIrina ALBULESCU, Eric SNIJDER, Martijn VAN HEMERTLeiden University Medical Center, Leiden, THE NETHERLANDSChikungunya virus (CHIKV) is a mosquito borne alphavirus that causesfever and an incapacitating arthralgia that may persist for months. A registeredvaccine or specific antiviral therapy are currently lacking to preventor treat CHIKV infection. The objective of this study is to gain more insightinto the structure and function of the CHIKV RNA dependent RNA polymerase(RdRp) by purifying it from bacteria and characterizing it in anin vitro system. The CHIKV RdRp, nsP4, was expressed as a His taggedSUMO fusion protein and was purified from the soluble fraction of a bacteriallysate by immobilized metal affinity chromatography. The tag wasremoved by protease treatment yielding nsP4 with its native N terminaltyrosine residue. Preliminary results suggest that this protein preparationhas terminal adenylyl transferase activity. For the reconstitution of de novoRdRp activity in vitro, a membrane fraction from mammalian cells expressingCHIKV nsP123 is expected to be required. Therefore, nsP123 andvariants with defective nsP1 2 and/or nsP2 3 cleavage sites or an inactivatednsP2 protease domain were expressed in mammalian cells, to analyse theirrole in CHIKV positive and negative strand RNA synthesis. These proteinsin combination with nsP4 and various templates are currently used toREF 201Role of the ubiquitin proteasome system in porcine circovirus type 2replicationShuang CHENG, Qigai HEHuazhong agricultural university, Wuhan, CHINAPorcine circovirus type 2 (PCV2), which is the primary agent of porcinecircovirus associated diseases (PCVAD), resulted in swine immunosuppressivedisease. The primary target cells of PCV2 are mononuclearphagocytes lineages. To characterize the pathogenesis of PCV2 infection,the differential proteomes of porcine alveolar macrophages, with andwithout PCV2 infection, were analyzed at different time points with twodimensional gel electrophoresis (2 DE) followed by MALDI TOF/TOFidentification. Mass spectrometry identified ubiquitin proteasome systemassociated proteins were differential expressed. Then the role of ubiquitinproteasome system in PCV2 infection was further studied. The resultsshow that MG132, a proteasome inhibitor, influence the viral activity asdetermined by decrease in viral genomic copies at the early infectionstage, and decrease in viral protein expression and viral transcription.PCV2 infection led to an accumulation of ubiquitylated proteins, withno apparent change in chymotrypsin like activity. Using small interferingRNA, we demonstrate that gene silencing of ubiquitin reducesthe genomic copies of PCV2. Taken together, our data suggest that theubiquitin proteasome system is associated with effective replication ofPCV2.Virologie, Vol 17, supplément 2, septembre 2013S175

5 th European Congress of VirologyREF 202Molecular determinants of cap synthesis and RNA positioning inflavivirus NS5 methyltransferaseAxelle COLLET 1 , Gilles QUÉRAT 2 , Francoise DEBART 3 , BrunoCANARD 1 , Decroly ETIENNE 11 Architecture et Fonction des Macromolécules Biologiques, CNRS andAix Marseille Université, UMR 7257, polytech Case 925, 13288 Marseille,FRANCE; 2 IRD, UMR 190, Faculté de Médecine – Timone, 13385Marseille cedex 5, FRANCE; 3 IBMM, CNRS, UM1 UM2, UMR 5247,Université Montpellier 2, 34095 Montpellier cedex 05, FRANCEDengue virus (DV) and West Nile Virus (WNV) belong to the Flavivirusgenus. They are important human pathogen responsible of epidemicscausing tens of thousands of deaths each year worldwide. We lack antiviraltreatment blocking these arboviruses. Flaviviruses replicate in thecytoplasm of infected cells and add a cap 1 structure at the 5’ end oftheir genomic RNA. The N terminal domain of the protein NS5 carriesthree enzymatic activities involved in RNA capping: the 2’O and N7methyltransferase (MTase) and the guanylyltransferase (GTase), the latterremaining controversial. Since RNA capping is essential for viral replication,the NS5 MTase domain is a promising antiviral target. Although thestructure of the MTase domain of NS5 (NS5 MTase) has been resolved bycrystallography, the exact positioning of the RNA necessary during eachcatalytic step remains hypothetical. In this work, we performed structurefunction analysis of the MTase by alanine mutagenesis. The influence ofthe mutations on the N7 and 2 ′ O MTase activities and the RNA recruitmentin the cap binding site was determined in vitro. In addition, we also analysedthe mutants by reverse genetics. Our results suggest that i) the NS5MTase carries the GTase activity and ii) the different catalytic activities ofthe N55 MTase require a repositioning of the RNA to first methylate theguanosine cap at N7 position and then the first nucleotide at 2 ′ O positionof the RNA in the same catalytic pocket.REF 204A genetic system to study replication, transcription and persistenceof Merkel Cell Polyomavirus (MCPyV)Nicole FISCHER 1 , Juliane THEISS 1,2 , Malik ALAWI 3 , DanielaINDENBIRKEN 2 , Adam GRUNDHOFF 21 University Medical Center Hamburg Eppendorf/Institute for MedicalMicrobiology and Virology, Hamburg, GERMANY; 2 Heinrich Pette Institute,Leibniz Institute for experimental Virology, Hamburg, GERMANY;3 University Medical Center Hamburg Eppendorf/HEXT Bioinformaticsupport, Hamburg, GERMANYAt least 95% of Merkel cell carcinoma (MCC) harbor clonally integrated,defective genomes of the recently discovered Merkel Cell Polyomavirus(MCPyV). Due to the lack of a permissive replication system very littleis known about the natural biology of the virus. We have recently developeda semi permissive replication system based on a consensus MCPyVgenome that allows recapitulation of the early and most of the late phaseof the viral life cycle. Here, by introduction of a self cleaving GFP tag intothe viral backbone, we have generated a tractable genetic system that permitsidentification and isolation of live cells harboring replicating MCPyV.Using this system in conjunction with RNAseq, we for the first time haveanalyzed the transcription profile of replicating viral genomes and showthat the virus does not produce a functional VP3 transcript. Furthermore,sequencing of small RNAs indicates that MCVmiR M1, a microRNAencoded by MCPyV, differs from its annotated sequence when expressedfrom an authentic viral genome. Using a hairpin mutant unable to expressthe viral miRNA, we show that MCV miR M1 negatively regulates earlyantigen expression and viral DNA replication, but also stimulates lategene expression. Interestingly, we find that replicating wt as well as GFPtagged MCVSyn genomes are able to persist in tissue cultures for severalweeks, indicating that MCPyV may be able to establish quasi latentinfections. We hypothesize that MCVmiR M1 is an important factor thatregulates viral replication and thereby contributes to the establishment ofviral persistence.REF 203Multiplicity of infection determines cell fate in human monolayer cellcultures after influenza A virus infectionDaria DANILENKO, Tatyana D. SMIRNOVA, Mikhail Yu. EROPKINResearch Institute of Influenza, Saint Petersburg, RUSSIAInfluenza A viruses cause substantial morbidity and mortality. In vitromodeling of influenza infection permits to predict and evaluate possibleconsequences of in vivo infection. We performed a series of experimentsusing human cell cultures of epithelial (A549) and endothelial (ECV 304)origin infected with various influenza A strains (H1N1, H1N1pdm 2009,H2N2, H3N2 and H5N1). Two doses of infection were chosen: high dosethat corresponded to multiplicity of infection (MOI) =0,1 and low dosewith a MOI of 0,0001. Both cell cultures underwent gradual apoptosis inresponse to high doses of viruses 16 24 h post infection (p.i.) Low doses ofvirus didn’t lead to apoptosis but instead in some cases stimulated cellularproliferation in response to infection. Importantly, proliferation of epithelialcell line A549 was stimulated only by pandemic A(H1N1)pdm 2009virus and A(H2N2) virus. Endothelial line ECV 304 showed enhancedproliferative response to all influenza viruses tested except for A(H5N1).It’s worth noticing that ECV 304 cell line infected with low MOI didn’tproduce infective virus progeny as no virus could be detected by hemagglutinationtest at day 5. Still the cell line supported viral replication asit was confirmed by the presence of viral RNA (by RT PCR) and viralNP with the use of monoclonal antibodies. After three serial passagesof ECV 304 line viral RNA was still detected by RT PCR. These resultsdemonstrate the role of endothelial cells in persistent influenza virus infectionand the importance of viral dose on the subsequent fate of infectedcells.REF 205Functional insights into measles virus NTAIL PXD interaction usingthe biGene/biSilencing (biG/BiS) systemDenis GERLIER 1 , Joanna BRUNEL 1 , Antoine GRUET 2 , AndreaVASSENA 2 , Marion DOSNON 2 , Patricia DEVAUX 3 , RobertoCATTANEO 3 , Sonia LONGHI 21 CIRI, INSERM U1111, CNRS UMR5308, Lyon, FRANCE; 2 AFMB CNRSet Aix Marseille Université,UMR 7257, Marseille, FRANCE; 3 Mayo Clinic,Rochester MN, USAStudying the structure function relationship of individual components ofthe replication machinery of a Mononegavirales is not straightforward.The nucleocapsid NC is formed by a long N homopolymer that coversthe genome and allows the recruitment of the P cofactor bound to the Lpolymerase. The dynamic interaction of the C terminal X domain (PXD)of P with the MoRE located at the C terminal of N (NTAIL) of each Nsubunit is proposed to allow the P tetramer to cartwheel along the helicoidalNC of paramyxovirinae. This ensures the regular displacement of theP L polymerase complex during viral RNA synthesis. NTAIL mutationslocated both inside and outside the MoRE and possibly affecting the dynamicsof the PXD/NTAIL interaction were selected from a novel descriptivein vitro evolution system and tested in the virus infection context usinga novel bigene bisilencing system “biG biS”. This consists in buildingrecombinant virus with a duplicated N gene, N1 and N2, with each genecopy being targeted by siRNA si1 and si2, respectively, so as to get selectiveexpression of N1 or N2 in two independent cell lines constitutivelyexpressing si1 or si2 shRNA. By allowing selective expression of naturalN1, bi N viruses with a wild type phenotype were easily rescued andamplified in si2 cells. Infection of si1 and parental cells allowed studyingS176 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologythe functional impact of mutations introduced in N2 expressed alone or incombination with wild type N. Obtained results provided new informationon the cartwheeling model.REF 206Innate immunity activation is required to mediate an effective HumanCytomegalovirus Lytic infection in THP 1 macrophagesDiego GERMINI 1 , Maria Cristina ARCANGELETTI 1 , IsabellaRODIGHIERO 1 , Prisco MIRANDOLA 2 , Flora DE CONTO 1 , MariaCristina MEDICI 1 , Rita GATTI 2 , Carlo CHEZZI 1 , AdrianaCALDERARO 11 Department of Clinical and Experimental Medicine University of Parma,Parma, ITALY; 2 Department of Biomedical, Biotechnological and TranslationalSciences University of Parma, Parma, ITALYToll like receptors (TLR), the main class of immune sensor moleculestriggering the innate immunity pathways, are known to be involved inthe infection of different RNA and DNA viruses, including herpesviruses.Human cytomegalovirus (HCMV) has evolved several strategies to exploitcellular functions connected with the innate immunity activation. Specificviral gene products have an immunomodulatory effect, allowing the virusto evade host defenses and even improve the infection effectiveness. Theaim of the present study was to ascertain whether specific TLRs are exploitedby HCMV to sustain an efficient lytic cycle in THP 1 differentiatedcells, a macrophage like model mimicking one of the most important viraltargets in vivo. We show that TLR 3, 4 and 5 transcripts are stimulatedby HCMV infection of THP 1 macrophages but that only TLR4 expressionincreases in UV inactivated virus infected cells. Using two differentTLR4 antagonists, we analysed the pattern of HCMV immediate earlyviral transcripts and proteins, as well as the viral yield, demonstratingthat the inhibition of the TLR4 pathway induces a significant decreaseof HCMV infection. Furthermore, by confocal microscopy analysis wealso produced data supporting not only HCMV TLR4 interaction at theplasma membrane level but also in the cytoplasmic compartment. Thesedata support a key role for TLR4 in favouring an efficient HCMV productiveinfection in THP 1 macrophages; its involvement seems not onlyconfined to the very early steps but also to later infection stages.REF 207Direct interaction between cyclin T1 and the cyclin dependent kinaseortholog pUL97 of human cytomegalovirusLaura GRAF 1 , Rike WEBEL 1 , Stuart HAMILTON 2 , William D.RAWLINSON 2 , Heinrich STICHT 3 , Manfred MARSCHALL 11 Institute for Clinical and Molecular Virology, University of ErlangenNuremberg, Erlangen, GERMANY; 2 Virology Division, MicrobiologySEALS POWH, University of New South Wales, Sydney, AUSTRALIA;3 Division of Bioinformatics, Institute of Biochemistry, University of ErlangenNuremberg, Erlangen, GERMANYThe human cytomegalovirus encoded protein kinase pUL97 regulatesvirus replication at various levels, such as viral DNA replication, geneexpression and nuclear capsid egress, by phosphorylating cellular andviral proteins. Due to structural and functional similarities, pUL97 canbe considered as a cyclin dependent kinase (CDK) ortholog. The primarymechanism of CDK activation in normal human cells is the binding to correspondingcofactors termed cyclins. This study provides first evidence thatpUL97 interacts with cyclin T1 (cycT1), which is the regulatory subunitof CDK9. Indirect immunofluorescence analyses revealed partial colocalizationof pUL97 with cycT1 in subnuclear compartments which wasmost pronounced in viral replication centers. The distribution patterns ofpUL97 and cycT1 were independent of HCMV strain and host cell type.The interaction between pUL97 and cycT1 was demonstrated using yeasttwo hybrid and coimmunoprecipitation analyses. The sequence domain ofpUL97 responsible for the interaction with cycT1 was the region betweenamino acids 231 280, a region which does not contain a known cyclinrecognition motif. The role of the pUL97 cycT1 interaction in phosphorylationdependent regulatory processes and kinase activity of pUL97 iscurrently investigated. This is the first demonstration of interaction betweena herpesviral CDK like protein kinase and a cellular cyclin. Furtherinvestigation of this interaction will contribute to better understanding ofpUL97 function, especially with regard to the role of pUL97 as a CDKortholog.REF 208Upregulation of the transcription elongation factor ELL2 by Tax as anovel mechanism of HTLV 1 gene regulationAndrea K. KRESS, Melanie C. MANN, Sarah STROBEL, BernhardFLECKENSTEINInstitute of Clinical and Molecular Virology, Friedrich Alexander UniversitätErlangen Nürnberg, Erlangen, GERMANYThe viral oncoprotein Tax encoded by Human T cell lymphotropic virustype 1 is a potent modulator of host cell transcription. To test whetherTax could also affect transcriptional elongation, microarray analysis wasperformed. Among all known cellular elongation factors, the eleven nineteenlysine rich elongation factor 2 (ELL2) was the only one selectivelyupregulated in the presence of HTLV 1/Tax. ELL2 is known as the stoichiometricallylimiting factor of the super elongation complex. Furtheranalysis of various HTLV 1 transformed and patient derived cell lines byqPCR and immunoblot revealed ELL2 to be significantly upregulated inthe presence of Tax. Repression of Tax in Tax transformed Tesi cells leadsto reduced amounts of ELL2 mRNA and protein. Moreover, siRNA mediatedknockdown of Tax in MT 2 diminishes ELL2 expression. Transfectionof increasing amounts of Tax in 293T cells leads to an increase of ELL2transcripts, suggesting that ELL2 expression is dependent on Tax. Furthermore,we found that coexpression of ELL2 significantly increases Taxmediated transactivation of the HTLV 1 promotor in a dose dependent manner.The enhanced transactivation is likely due to a promotor independentand Tax specific enhancement of Tax expression by ELL2. Interestingly,siRNA mediated knockdown of ELL2 in MT 2 leads to strong reduction ofTax protein, suggesting a positive feedback loop between ELL2 and Taxin HTLV 1 infected cells. Taken together, we identified ELL2 as a newfactor which may play an important role in HTLV 1 transcription.REF 209Protein kinases determine intracellular localization and transportactivity of the human cytomegalovirus regulatory protein pUL69Manfred MARSCHALL 1 , Laura GRAF 1 , Zin NAING 2 , SabrinaWAGNER 1 , Rike WEBEL 1 , Corina HUTTERER 1 , Gillian M. SCOTT 2 ,William D. RAWLINSON 2 , Thomas STAMMINGER 1 , MarcoTHOMAS 1 , Sabine FEICHTINGER 11 Institute for Clinical and Molecular Virology, University of ErlangenNuremberg, Erlangen, GERMANY; 2 Virology Division, MicrobiologySEALS POWH, University of New South Wales, Sydney, AUSTRALIACyclin dependent protein kinases (CDKs) are important regulators of cellularprocesses and are functionally integrated in the replication of humancytomegalovirus (HCMV). We recently demonstrated a regulatory impactof CDK9 on the viral mRNA export factor pUL69. In addition, the HCMVCDK ortholog pUL97 also showed strong pUL69 interaction resulting inpUL69 phosphorylation and regulation of pUL69 nuclear mRNA exportactivity. In vitro kinase analyses demonstrated pUL69 phosphorylation byboth CDK9/cyclin T1 and pUL97 on multiple sites, as single site mutationsdid not prevent phosphorylation. Direct pUL69 cyclin T1 interaction wasdemonstrated using coimmunoprecipitation analyses with proteins fromVirologie, Vol 17, supplément 2, septembre 2013S177

5 th European Congress of Virologyprimary fibroblasts infected with HCMV AD169 or Merlin, as well asproteins from transiently transfected cells. Confocal microscopy showedthat selective inhibitors of CDK or pUL97 induced an accumulation ofpUL69 in fine speckled nuclear aggregates, which contained colocalizedCDK9, cyclin T1, RNAP II and to some extent pUL97. Speckled aggregationoccurred at the late phase of viral replication and was independentof viral strain (AD169, Merlin) and host cell type (HFF, MRC 5, TEV1, ARPE 19). Interestingly, speckled aggregation was also observed tooccur spontaneously in ∼1% of cells in the absence of kinase inhibitors.These findings point to a dependence of correct intranuclear localization ofpUL69 on distinct kinase activities. A model describing the phospho regulatedstate of pUL69 responsible for localization and transport function ispresented.REF 210The plant virus NSs protein of the Tomato spotted wilt virus enhancesbaculovirus replication and cell death in Lepidoptera and Diptera celllinesVirgínia Carla OLIVEIRA, Fabricio Da Silva MORGADO, DanielMENDES PEREIRA ARDISSON ARAÚJO, Daniele VITORIANOFREITAS, Bergmann MORAIS RIBEIRO, Renato OLIVEIRARESENDEDepartment of Cell Biology, Brasília, BRAZILWe have shown in a previous work that NSs protein of Tomato spottedwilt virus expressed by a recombinant Autographa californica multiplenucleopolyhedrovirus baculovirus (vAcNSs) can suppress gene silencingin lepidopteran insect cell lines. In this work, replication of the recombinantvirus in this cell line (velvetbean caterpillar derived, UFL AG 286)was shown to be higher than wild type virus (AcMNPV). Interestingly,although with lower absolute values of viral DNA, a difference of 6.75 x(12 h p.i.) and 2.2 x (24 h p.i.) was observed for AcMNPV and vAcNSsinfected nonpermissive C6/36 cells. A cytotoxicity assay resulted in adecrease of 2.65 (UFL AG 286) and 1.77 times (BM 5) in cell viability; ina dipteran cell line (C6/36) a reduction of 1.73 times in cell viability wasobserved; BTI Tn 5B1 4 vAcNSs infected cells showed 2.12 times lessviable cells in relation to AcMNPV infected cells. Furthermore, bioassayswere performed by intrahaemocoelic injection of the baculovirus buddedvirus form and oral infection with purified occluded viruses of the positiveocclusion recombinant virus vAcNSsocc+ and the wild type AcMNPV.For intrahaemocoelic infection, the vAcNSs LT50 values were significantlylower than those for AcMNPV on larvae of S. frugiperda [LT50 of4.82 and 7.52 days with 105 BVs] and A. gemmatalis [LT50 of 3.20 and7.34 days with 105 BVs). In conclusion, NSs is capable of boosting viralreplication in insect cells by probably suppressing gene silence machineryand could efficiently improve baculovirus replication and bioinsecticideaction.of the resulting cell cycle independent IE gene expression for the progressionof the HCMV lytic cycle. Infection of early S phase cells with thecell cycle independent HCMV mutant leads to a stable, p53 independentG2 arrest. Only a small minority of IE positive cells were able to entermitosis. These mitotic cells showed signs of an abortive infection whereasthe G2 arrested cells readily support both early and late viral geneexpression, and accordingly, also viral DNA replication. We hypothesizedthat the recently discovered HCMV pUL21a mediated degradation ofcyclin A2 is required to maintain the virus permissive G2 arrested state. Totest this hypothesis we constructed an HCMV double point mutant whereboth pp150 and pUL21a were disabled in cyclin A2 binding. Intriguingly,infection with this double mutant forced mitotic entry of up to 60% of IEpositive cells, leading to a significant retardation of virus growth. Takentogether, we uncovered the existence of two independent viral mechanismsthat cooperatively assist HCMV to avoid the negative cell cycle effects ofuncontrolled cyclin A2 CDK activity.REF 212PUL21a dependent degradation of Cyclin A2 is essential for the inhibitionof cellular DNA synthesis and mitotic entry during lytic HCMVinfectionLüder WIEBUSCH, Boris BOGDANOW, Martin EIFLER, EllenRICHTER, Ralf UECKER, Barbara VETTER, Christian HAGEMEIERCharité Universitätsmedizin, Berlin, GERMANYLytic human cytomegalovirus (HCMV) infection leads to a pronouncedcell cycle arrest at the G1/S transition. This arrest is characterized by highcyclin E1 but low cyclin A2 levels. Here we show that cyclin A2 downregulation is mediated by the UL21a gene product (pUL21a) of HCMVand represents the crucial step of the viral arrest mechanism. We identifieda short peptide motif within the pUL21a N terminus that is required forspecific pUL21A cyclin A2 binding. Transfection of pUL21a was foundto induce proteasomal degradation of cylin A2 in a cyclin A2 binding(Cy) motif dependent manner. Point mutation of the pUL21a Cy motifwithin the HCMV genome also relieves the block of cyclin A2 expressionduring HCMV infection. The resulting increase of cyclin A2 protein levelsproved to be sufficient to abrogate the viral inhibition of cellular DNA synthesis.Moreover, it triggers the accumulation and nuclear translocation ofcyclin B1 CDK1 complexes and accordingly, the mitotic entry of infectedcells. Deletion of the whole UL21a open reading frame, has only moderateeffects on cyclin A2 expression and cell cycle progression, suggestingthat the pUL21a Cy motif and the previously described pUL21a mediatedinhibition of the APC/C act antagonistically with respect to cyclin A2 proteinstability. Importantly, the cyclin A2 induced mitotic entry of infectedcells results in mitotic catastrophe and, consequently, an abortive infection.Thus, the pUL21a cyclin A2 interaction is essential for the maintenance ofa cell cycle state conducive for the completion of the HCMV replicationcycle.REF 211The cyclin A2 interaction motifs of pp150 and pUL21a cooperateto prevent HCMV from entering a non productive, mitotic state ofinfectionHenry WEISBACH, Christoph SCHABLOWSKY, ChristianHAGEMEIER, Lüder WIEBUSCHLabor für Pädiatrische Molekularbiologie, Charité UniversitätsmedizinBerlin, Berlin, GERMANYThe lytic replication cycle of human cytomegalovirus (HCMV) can onlybe started in the G0/G1 phase of the cell division cycle, due to a cyclinA2 CDK dependent block of immediate early (IE) gene expression inS/G2. This block can be prevented by mutating a cyclin A2 binding sitein the viral tegument protein pp150. Here we analyzed the consequencesREF 213Identification of protein synthesis as a regulatory node in EpizooticHemorrhagic Disease Virus (EHDV) infection and development of anovel imaging technique for its study in single cellsMarcelo EHRLICH 1 , Sima BARHOOM 1 , Ben SHAI 1 , EranSCHMUKLER 1 , Barry COOPERMAN 3 , Zeev SMILANSKY 2 , EranBACHARACH 1 , Ronit PINKAS KRAMARSKI 1 , Orna ELROY STEIN 11 Tel Aviv University, Tel Aviv, ISRAEL; 2 Anima Cell Metrology, Bernardsville,USA; 3 University of Pennsylvania, Philadelphia, USAManipulation of the cellular protein synthesis apparatus is a central regulatorymechanism of the replication cycle of different viruses. Suchmanipulation by orbiviruses, such as the Epizootic Hemorrhagic DiseaseS178 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyVirus (EHDV), has not been studied. To address this matter, we employeda broad range of techniques including quantitative fixed and live cellmicroscopy. Specifically, we developed a novel method, Fluorescent tRNATranslation Monitoring (FtTM), that enables single cell measurements ofprotein synthesis with sub micron resolution. In FtTM, fluorescently labeledtRNAs are transfected into cells and the FRET signals that are generatedwhen differently labeled tRNAs localize to the ribosome are quantified byconfocal microscopy. We report that EHDV ignites a complex signallingresponse involving the activation of the protein kinase PKR, the phosphorylationof the translation factor eIF2alpha, the activation of the c Jun Nterminal kinase (JNK) and the induction of autophagy, all of which affectthe levels of production of infective virions. Moreover, FtTM measurementsemploying labeled bulk tRNA revealed a spatial confinement ofprotein synthesis in infected cells. To further develop FtTM, we comparedthe codon usage of EHDV with that of a mammalian host and identifiedcodons highly enriched in the EHDV genome. FtTM measurements withspecific tRNAs directed at such codons allowed for the measurement ofthe synthesis levels of a viral protein (NS3) at single cell resolution. siRNAmediated knock down of NS3 reduced such FtTM signals and infectivevirion production.REF 214Biological role of HCV core+1/ARF protein: a computationalapproachIoly KOTTA LOIZOU, Penelope MAVROMARAHellenic Pasteur Institute, Athens, GREECEHepatitis C virus (HCV) infection is a major cause of chronic liver diseaseand hepatocellular carcinoma. HCV is an enveloped positive stranded RNAvirus, belonging to the Flaviviridae family. The HCV genome producesa polyprotein precursor which is processed by proteases and yields atleast 10 proteins. HCV possesses a second open reading frame (ORF)within the core gene, encoding an intrinsically disordered protein knownas core+1/ARFP. Two predominant isoforms of core+1/ARFP have beenreported: core+1/ARFP/L initiating from codon 26 and core+1/ARFP/Sinitiating from codons 85/87. In this study we aim to gain insight into thebiological role of core+1/ARFP, which still remains elusive. To this end,we constructed a dataset of 4428 core+1/ARF amino acid sequences from18 confirmed HCV subtypes. As shown before, the length of the core+1ORF is subtype dependent. Both core+1/ARFP isoforms are less conservedthan core but at least as conserved as the HCV non structural (NS)2 protein. Subsequently, 4 exemplar core+1/ARF amino acid sequencesderived from the well studied isolates 1a HCV 1 and H77, 1b Con1 and 2aJFH 1 were assessed using the Eukaryotic Linear Motif (ELM) databasefor the presence of binding motifs. They were found to contain 3 commonbinding motifs, associated with cell cycle control, cell proliferation andsignal transduction. Preliminary results also indicate that core+1/ARFPmay possess cysteine endopeptidase activity. In addition, hidden markovmodels (HMMs) were created for core+1/ARFP and are currently used insearch of homologous amino acid sequences.REF 215Analysis of genomes of hepatitis A virus strains with high replicationrate in cell cultureTatyana BONDARENKO 1,3 , V. SVYATCHENKO 1 , N. KISILEV 1 ,V. TERNOVOI 1 , A. KUSLII 2 , S. NETESOV 31 SRC VB “Vector”, Koltsovo, RUSSIA; 2“ Vector BiAlgam”, Koltsovo,RUSSIA; 3 Novosibirsk State University, Novosibirsk, RUSSIAThe aim of our study was analysis of several hepatitis A virus (HAV)strains adapted to cell cultures. The typical cultivation period of HAVstrains in cell cultures is 21 28 days. However several HAV strains suchas FG, HM175/43C and MB 7 were adapted successfully to FRhK 4 cellsand had a cultivation period of 7 10 days. Later MB 7 was adapted to cellculture 4647 and it was named MB 7/4647. Then MB 7/4647 was adaptedto cell culture HEK293 and it was named MB 7/293. This strain had ashort replication period (4 6 days) and high final virus yield compared toMB 7/4647. In our study we sequenced the full genome of MB 7/293 andcompared it with the parent MB 7/4647 and 12 other HAV strains fromGenBank. We have found that the genome MB 7/293 carries 3 substitutionsin 5’ UTR and 20 substitutions in the coding region that lead to 10 aminoacid replacements in VP3, VP1, 2B and 2C proteins in comparison withMB 7/4647. We assume that two mutations in 5’ UTR located in thedomain I can also affect the replication rate. We also suppose that themost important amino acid replacement is the change of Lys (839) to Asnlocated in 2A/2B proteolytic site. Thus viral strains with enhanced growingfeatures in vitro can be selected for the development of a vaccine againstHAV.Virologie, Vol 17, supplément 2, septembre 2013S179

5 th European Congress of Virology14. VIRUS STRUCTURE, DYNAMICIMAGING AND TRAFFICKINGPosters: REF 216 to REF 229REF 216Structural and functional studies of the N terminal dimerizationdomain of the Epstein Barr virus nuclear antigen 2Sybille THUMANN 1 , Anders FRIBERG 2 , Sybille THUMANN 1 , PeijianZOU 2 , Janosch HENNIG 2 , Michael SATTLER 2 , Bettina KEMPKES 11 Department of Gene Vectors, Helmholtz Center Munich, GermanResearch Center for Environmental Health, Munich, GERMANY;2 Institute of Structural Biology, Helmholtz Center Munich, GermanResearch Center for Environmental Health, Munich, GERMANYEpstein Barr virus (EBV) is a gammaherpesvirus that establishes lifelongasymptomatic infections in the majority of human adults. It is the causativeagent of infectious mononucleosis if primary infection is delayed toadolescence. Furthermore, epidemiological and molecular evidence linksEBV to the pathogenesis of endemic Burkitt ′ s lymphoma, nasopharyngealcarcinoma, a subset of Hodgkin ′ s disease, and other malignanciesof lymphoid and epithelial cell origin. EBV has the unique ability totransform resting B cells into permanently proliferating, latently infectedlymphoblastoid cell lines that express six EBV nuclear antigens and3 latent membrane proteins. One of the first nuclear antigens expressedpost infection is the Epstein Barr virus nuclear antigen 2 (EBNA2). It canform dimers and transactivates a set of viral and cellular genes. Here weanalyzed the EBNA2 N terminal dimerization domain (END). NMR spectraof the END domain were recorded and revealed that the dimerizationof the monomers is presumably driven by the formation of a hydrophobiccore. Based on this structure, interface and surface mutants of theEND domain were generated. They were tested for their capacity to selfassociate by co immunoprecipitation and for their ability to transactivateEBNA2 target genes in a Burkitt ′ s lymphoma cell line. Interestingly, notonly interface mutations that impair dimerization, but also surface mutationsof the END domain prevent biological activity of the transactivatorEBNA2.REF 217Structural virology of the murid Gamma Herpesvirus 4Bruno CORREIA 1 , Colin MCVEY 1 , Marta MIRANDA 21 Institute of Chemical and Biological Technology, Lisbon, PORTUGAL;2 Institute of Molecular Medicine, Lisbon, PORTUGALThe murid herpesvirus 4 (MuHV4) has been widely explored as a modelfor research on the human gamma herpesvirus pathogenesis due to itsability to infect mice via the nasal passages and also because it is geneticallyrelated to other malignancy associated pathogens such as the humanEpstain Barr virus (EBV) and Kaposi’s Sarcoma herpesvirus (KSHV).As for EBV, MuHV4 establishes a lifelong latency stage in the nucleusof host B cells as a multicopy episome and only a small fraction ofviral genes are expressed. Amongst this fraction of genes, an ORF protein,ORF73, was demonstrated to promote a deficit on the establishmentof latency in vivo when mutant viruses failed to express them. ORF73reveals remarkable sequence homology with KSHV latency associatednuclear antigen (LANA) and striking predicted structural homology withthe C terminal domain of the EBV nuclear antigen 1 (EBNA1), mutuallycrucial for viral latency by tethering the viral episome to host chromosomes,therefore ensuring replication and segregation of the viralgenome to daughter cells. C terminal LANA and EBNA1 were describedto bind terminal repeat (TR) DNA of the viral genome upon dimerization,suggesting that C terminal of ORF73 may also be involved inepisome maintenance functions. We crystallized and solved the first X raycrystallographic structure of the murid ORF73 protein unbound to viralDNA and highlighted important residues and structural motifs that mayexert functional relevance as therapeutic targets by manipulation of virallatency.REF 218Visualization of the pre integration complex of the murine leukemiavirus during early steps of infectionEran BACHARACH 1 , Efrat ELIS 1 , Marcelo EHRLICH 1 , Darren J.WIGHT 2 , Virginie C. BOUCHERIT 2 , Adi PRIZAN RAVID 1 , NihayLAHAM KARAM 1 , Kate N. BISHOP 21 Tel Aviv University, Tel Aviv, ISRAEL; 2 MRC National Institute for MedicalResearch, London, UNITED KINGDOMTo integrate, reverse transcribed retroviral genomes are imported fromthe cytoplasm to the chromosomes as part of a pre integration complex(PIC). Differences in retrovirus PIC trafficking influence their ability toinfect resting and/or dividing cells. Lentiviruses, including HIV, infectboth dividing and resting cells. In contrast, simple oncoretroviruses, suchas murine leukemia viruses (MLVs), are restricted to dividing cells. p12,a cleavage product of MLV Gag precursor, is thought to influence MLVintegration. Viruses with specific lethal mutations in p12 showed defectsin early stages of infection, with normal generation of linear genomicDNA, but no formation of circular DNA forms (which are thought to marknuclear entry of the viral DNA). In the past we demonstrated that p12is a functional component of the MLV PIC and recently we also revealeda role for p12 as a tethering factor, which connects this PIC to mitoticchromosomes. The fact that p12 escorts the MLV DNA throughoutthe early stages of infection promoted us to microscopically detect theincoming MLV PIC by labeling p12. This allowed us the visualization ofthe PIC both by immunofluorescence and by real time imaging. Here wedescribe the possible connection of PIC movements to the cytoskeleton,PIC trafficking in respect to changes in the cell cycle, the possible contributionof p12 to PIC stability and the gradual uncoating of the PIC duringits trafficking. Altogether these results highlight steps important for MLVintegration, which also contribute to the high tropism of MLV to dividingcells.REF 219Structural characterization of the Influenza virus matrix protein M1Jens RADZIMANOWSKI 1 , Marta BALLY 2 , Eric CONDAMINE 3 ,Stéphanie MANGENOT 2 , Patricia BASSEREAU 2 , WinfriedWEISSENHORN 11 Unit of Virus Host Cell Interactions (UVHCI), Grenoble, FRANCE;2 Institut Curie, Paris, FRANCE; 3 Institut de Biologie Structurale (IBS),Grenoble, FRANCEInfluenza viruses (genus A, B and C) are negative strand segmentedRNA viruses, which acquire their envelope from the plasma membrane.Although the matrix protein M1 is an integral part of infectious maturevirions and forms a helical protein layer underneath the viral membrane,its role in assembly and budding is poorly understood. Unlike othermatrix proteins from enveloped viruses M1 expression alone does notinduce VLP formation. We present structural data on M1 and in vitromembrane binding to better understand the role of M1 in the viral lifeS180 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologycycle. We determined the crystal structure of the N terminal domainfrom Influenza virus C M1 and produced low resolution models of fulllength M1 from influenza A and C viruses based on SAXS analyses.Because M1 has to at least partially dissociate from the membraneduring entry, we analyzed potential structural changes induced by theendosomal pH by NMR and tested M1 interaction with model membranesat physiological and acidic pHs. We will present an integratedstructural model of M1 function based on our structural and functionaldata.and is attenuated for propagation in cell culture. Importantly, we alsoshow that the efficient translation of viral mRNAs containing a translationenhancer sequence also contributes to the disassembly of SGs in infectedcells. Further, we show that the nsP3/G3BP interaction also blocksSG induction by other stresses than virus infection such as pateamineA and 2 deoxy d glucose treatment. This is one of few described viralmechanisms for SG disruption and underlines the role of SGs in anti viraldefence.REF 220Influence of the actin bundling protein Fascin on HTLV 1 transmissionChristine GROSS, Martina KALMER, Bernhard FLECKENSTEIN,Andrea K. KRESSInstitute of Clinical and Molecular Virology, Friedrich Alexander UniversitätErlangen Nürnberg, Erlangen, GERMANYHuman T cell lymphotropic virus type 1 (HTLV 1) preferentially infectsCD4+ T cells via cell to cell transmission, which requires both the viralTax protein and the host cell cytoskeleton. We previously reported Taxdependent upregulation of the actin bundling protein Fascin (FSCN 1)in HTLV 1 infected cells. Now, we asked whether Fascin contributes toHTLV 1 transmission. Using confocal microscopy, we found that Tax inducedFascin colocalized with Actin in transfected and chronically infectedT cells. Moreover, both Fascin and Actin accumulated at cell cell contactssuggesting recruitment of Actin by Fascin. Use of shRNAs led to specificand stable repression of Fascin in both Tax transfected and chronicallyinfected T cells without affecting cell vitality. Moreover, the accumulationof both Fascin and Actin at cell cell contacts was reverted in the presenceof Fascin specific shRNAs. In transmission assays, we found that repressionof Fascin in HTLV 1 infected cells led to reduced transactivation ofco cultured reporter T cells. However, a first series of experiments indicatedan increase of the viral core protein gag p19 in the supernatantsof HTLV 1 infected cells when Fascin was repressed, suggesting elevatedvirus release. In contrast, block of both actin and tubulin reduced therelease of gag p19 levels into the supernatants. Taken together, these datasuggest that repression of Fascin re routes HTLV 1 from cell to cell to cellto supernatant transmission.REF 221Sequestration of G3BP by Semliki Forest virus NsP3 inhibits theassembly of stress granules on viral mRNAsMarc PANAS 1 , Margus VARJAK 2 , Aleksei LULLA 2 , Kai Er ENG 1 ,Andres MERITS 2 , Gunilla B. KARLSSON HEDESTAM 1 , Gerald M.MCINERNEY 11 Department of Microbiology, Tumor and Cell Biology, Stockholm,SWEDEN; 2 Tartu University Institute of Technology, Tartu, ESTONIADynamic, mRNA containing stress granules (SGs) form in the cytoplasmof cells under environmental stresses including viral infection. Manyviruses appear to employ mechanisms to disrupt the formation of SGson their mRNAs, suggesting that they represent a cellular defence againstinfection. Here, we report that early in Semliki Forest virus infection, theC terminal domain of the viral non structural protein 3 (nsP3) forms acomplex with Ras GAP SH3 domain binding protein (G3BP) and sequestersit into cytoplasmic foci in a manner that inhibits the formation ofSGs on viral mRNAs. These nsP3+G3BP+ foci contain neither TIA 1/Rnor members of the 43S translation initiation complex indicating that theyare not SGs, but often contain dsRNA suggesting they are viral RNAreplication complexes or similar structures. A viral mutant carrying a Cterminal truncation of nsP3 induces more numerous and persistent SGsREF 222Architecture of respiratory syncytial virus revealed by electron cryotomographySarah BUTCHER 1 , Lassi LILJEROOS 1 , Magdalena KRZYZANIAK 2 ,Ari HELENIUS 21 University of Helsinki, Helsinki, FINLAND; 2 ETH Zurich, Zurich,SWITZERLANDRespiratory syncytial virus is a human pathogen that causes severe infectionof the respiratory tract. Current information about the structure ofthe virus and its interaction with host cells is limited. We carried out anelectron cryotomographic characterization of cell culture grown RSV todetermine the architecture of the virion. The particles ranged from 100 nmto 1000 nm in diameter and were spherical, filamentous, or a mixture ofthe two. The filamentous morphology correlated with the presence of acylindrical matrix protein layer linked to the inner leaflet of the viralenvelope and with local ordering of the glycoprotein spikes identified bysubvolume averaging. Recombinant viruses with only the fusion protein intheir envelope showed that this glycoprotein was predominantly in a formthat resembled the post fusion conformation. The ribonucleocapsids wereleft handed, randomly oriented, and curved inside the virions. In filamentousparticles, they were often adjacent to an intermediate layer of proteinassigned to M2 1. Our results indicate important differences in structurebetween the Paramyxovirinae and Pneumovirinae subfamilies within theParamyxoviridae, and provide new insight into host cell entry and exit ofa serious pathogen.REF 223Structures of Fibre Protein Head Domains from Two New AdenovirusesAbhimanyu Kumar SINGH, Mark VAN RAAIJCentro Nacional de Biotecnologia (CNB CSIC), Madrid, SPAINAdenoviruses are linear, non enveloped, dsDNA viruses which are icosahedralin shape. They are divided into five genera. The fibre protein, one ofthe major structural proteins of adenovirus is the principle determinant ofviral tropism and facilitates primary host cell recognition via its globularhead domain. We have determined crystal structures of fibre proteinhead domains of Turkey Hemorrhagic Enteritis Virus (THEV, TurkeyAdenovirus type 3) from the Siadenovirus genus and Snake Adenovirustype 1 (SnAdV1) from the Atadenovirus genus. THEV exists in virulentas well as in avirulent forms, the difference between them lies in twomutations located in fibre head. Our 2.2 Å structure of the fibre head froman avirulent strain localises these mutations. The secondary structuretopology resembles other adenovirus fibre heads, although a difference isthe presence of an extended beta hairpin, which contacts a neighbouringmonomer. The positively charged surface suggests that the receptor maybe acidic. Curiously, the closest structural homologues are reovirus fibressigmaC and sigma 1. SnAdV1 has one fibre per penton base, but itsreceptor is not known. Using site directed mutagenesis and multi wavelengthanomalous dispersion technique we have determined the structureof its fibre protein head domain at 1.7 Å. The eight beta sheet containingVirologie, Vol 17, supplément 2, septembre 2013S181

5 th European Congress of Virologysandwich structure is smaller and more compact when compared with otheradenovirus or reovirus fibre heads, mainly due to it having shorter loops.The DG loop of human adenovirus fibre head is replaced by a short alphahelix.REF 224Host Nucleolin/C23 is an essential partner for vRNPs nucleocytoplasmicexport during Influenza infectionCoralie CARRON 1 , Olivier TERRIER 1 , Aurélien TRAVERSIER 1 ,Gaëlle CARTET 1 , Sabine HACOT 2 , Bruno LINA 1 , Jean JacquesDIAZ 2 , Manuel ROSA CALATRAVA 11 VirCell Team, VirPath Laboratory, EA 4610, Lyon, FRANCE; 2 UMRInserm U1052 CNRS 5286, CRCL, Lyon, FRANCEInfluenza are nuclear replicating viruses that hijack host machineries inorder to replicate and interfere with host antiviral response. Influenzainfection induces an important remodelling of the host cell architecturewith marked modifications of the nucleolar ultrastructure (Terrieret al., 2012). Moreover, several proteomic studies have shown that thenucleolar proteome was strongly impacted during infection, suggestinga “nucleolar experience” for influenza viruses. In this context, we haveshown that subcellular distribution of the three major nucleolar componentswas differentially altered during infection. We then focused onnucleolin, a multifunctional protein implicated in ribosome synthesis andchromatin remodelling processes. We first observed that nucleolin wasdynamically relocated at the nuclear periphery upon infection, followingthe same dynamic pattern as vRNPs during their export towards thecytoplasm. Moreover, we demonstrated a vRNA dependant interactionbetween endogenous nucleolin and the viral nucleoprotein (NP) duringinfection. Finally, nucleolin silencing leads to a delay in vRNPs traffic anda loss of their association with chromatin compartment where they interactwith the host export machinery (Chase et al, 2011), and then reducedyields of viral production. Our results strongly indicate that influenza Aviruses exploit an unusual chromatin targeting strategy to achieve theirvRNP export process. We propose a model in which NP would hijacknucleolin functions for this process and the consecutive snatching of Crm1dependent export machinery.Rhinoviruses (HRVs) are the major cause of virus induced exacerbationsof asthma. Recent findings suggest that HRV pathology may involve disruptionof host cell nucleocytoplasmic trafficking mediated, at least inpart by 3C protease that localises to the nucleus of infected cells as 3CDand 3C. We have investigated nuclear targeting of 3C/3CD using HRVinfected cells and cells transfected to express HRV 3CD. Using 3C specificimmunofluorescence/confocal microscopy and western blotting ofHRV infected HeLa cells, we found that 3CD localises in the nucleusand cytoplasm of infected cells at 6 h post infection concomitant withdegradation of key molecules of the nuclear pore complex; 3C is observedlater, and predominantly in the cytoplasm. When expressed as a GFPfusion protein in transfected cells, 3CD was processed to 3C, localised tothe nucleus and resulted in degradation of specific nucleoporins and mislocalization of an endogenous nuclear protein. Uncleaved 3CD localizedprimarily in the cytoplasm, suggesting that the putative nuclear localizationsignal in 3D is not functional; GFP 3D was also cytoplasmic. GFP 3Cand its proteolyticaly inactive mutant localized in both nucleus and cytoplasmof transfected cells suggesting that 3C does not require cleavage ofnucleoporins to localize to the nucleus.In conclusion, 3C has an inherent ability to localize to the nucleus ofinfected cells; 3CD is unable to facilitate its nuclear localization. Thenuclear accumulation of 3CD in HRV infected cells probably requiresother viral proteins.REF 226Role of the small GTPase Rab27a during Herpes simplex virus infectionof oligodendrocytic cellsJosé Antonio LÓPEZ GUERRERO 1 , Antonio Jesús CRESPILLO 1 ,Alberto FRAILE RAMOS 2 , Enrique TABARÉS 3 , Antonio ALCINA 4 ,Raquel BELLO MORALES 11 Centro de Biología Molecular Severo Ochoa, UAM CSIC, Madrid,SPAIN; 2 Universidad Complutense de Madrid, Facultad de Medicina,Madrid, SPAIN; 3 Universidad Autónoma de Madrid, Facultad de Medicina,Madrid, SPAIN; 4 Instituto de Parasitología y Biomedicina LópezNeyra, CSIC, Granada, SPAINThe morphogenesis of herpes simplex virus type 1 (HSV 1) comprisesseveral events, of which some are not completely understood. It has beenshown that HSV 1 glycoproteins accumulate in the trans Golgi network(TGN) and in TGN derived vesicles. It is also accepted that HSV 1 acquiresits final morphology through a secondary envelopment by budding intoTGN derived vesicles coated with viral glycoproteins and tegument proteins.Nevertheless, several aspects of this process remain elusive. Thesmall GTPase Rab27a has been implicated in regulated exocytosis, and itseems to play a key role in certain membrane trafficking events. Rab27aalso seems to be required for human cytomegalovirus assembly. However,despite the involvement of various Rab GTPases in HSV 1 envelopment,there is, to date, no data reported on the role of Rab27a in HSV 1 infection.Herein, we show that Rab27a colocalized with GHSV UL46, a tegumenttagged green fluorescent protein HSV 1, in the TGN. In fact, thissmall GTPase colocalized with viral glycoproteins gH and gD in thatcompartment. Functional analysis through Rab27a depletion showed asignificant decrease in the number of infected cells and viral production inRab27a silenced cells. Altogether, our results indicate that Rab27a playsan important role in HSV 1 infection of oligodendrocytic cells.REF 225Nuclear Localisation of Rhinovirus 3C/3CDReena GHILDYAL 1 , Erin WALKER 1 , Lora JENSEN 1 , AlexFULCHER 2 , David JANS 21 University of Canberra, Canberra, AUSTRALIA; 2 Monash University,Melbourne, AUSTRALIAREF 227C terminal determinants of the cytomegalovirus GPCR pUS27 forendocytic receptor sortingIna NIEMANN 1 , Anna REICHEL 1 , Heinrich STICHT 2 , ThomasSTAMMINGER 11 Institute for Clinical and Molecular Virology, University of ErlangenNuremberg, Erlangen, GERMANY; 2 Emil Fischer Zentrum, Institute ofBiochemistry, University of Erlangen Nuremberg, Erlangen, GERMANYHuman cytomegalovirus (HCMV) encodes several G protein coupledreceptor (vGPCR) homologues, including pUS27, pUS28, pUL33, andpUL78. In order to fully understand vGPCR functions, it is necessaryto precisely define the mechanisms of receptor endocytosis. A rapid andconstitutive internalization of all four vGPCRs was demonstrated by antibodyfeeding experiments. While the well characterized receptors pUS28and pUL78 may play a role for chemokine sequestration, the knowledgeconcerning the sorting abilities of pUS27 is limited. To gain further insightinto the subcellular trafficking of HCMV encoded pUS27, we constructedvarious deletion mutants expressing an N terminal Flag or a C terminalGFP tag. Internalization assays were evaluated by confocal microscopyand FACS analysis. In contrast to recent publication, deletion of 46 aminoacids from the C terminus only partially disrupted endocytosis. Furthermore,we observed pUS27 sorting into different endosomal compartmentsS182 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologywhen comparing N or C terminally tagged versions. We assume that thiseffect is caused by several functionally important linear motifs of thepUS27 C terminus, which were identified by bioinformatic analysis: inaddition to a postulated Di leucine sorting motif, our data illustrate furtheressential sorting motifs for receptor internalization of pUS27. Moreover,coimmunoprecipitation experiments revealed that adaptor protein complex2 may be of importance for vGPCR sorting. Taken together, our data reveala sofar unrecognized complexity in the endocytic trafficking of pUS27.REF 228Characterisation of the HCoV NL63 nucleocapsid proteinKrzysztof PYRC 1,2 , Kaja ZUWALA 1 , Wojciech KABALA 1 , MichalBURMISTRZ 1 , Michal ZDZALIK 1 , Sylwia KEDRACKA KROK 1 ,Grzegorz DUBIN 1,2 , Jan POTEMPA 11 Faculty of Biochemistry, Biophysics and Biotechnology, JagiellonianUniversity, Krakow, POLAND; 2 Malopolska Centre of Biotechnology,Jagiellonian University, Krakow, POLANDHuman coronavirus NL63 was discovered in 2004 and since then numerousunique features of the virus have been identified. In the current workthe nucleocapsid (N) protein is characterized. This protein is known toshield the genomic RNA to form long helical ribonucleocapsid present inthe virus core. Both in vitro studies using primary human epithelial cellsand biochemical studies using expressed complete N protein and separateN and C terminal domains were carried on. We determined that the proteindoes not localize to the nucleus and that its overexpression results information of cytoplasmic granules, previously proposed to interfere withtranslation of eukaryotic proteins. We also determined that the N proteinforms oligomers and efficiently binds RNA and DNA. Proteins werecharacterized using differential scanning calorimetry, gel shift assays, sizeexclusion chromatography, mass spectroscopy and other relevant methods.REF 229Rhinovirus 3C protease effects specific nucleoporin cleavage and mislocalisationof nuclear proteins in infected host cellsErin WALKER 1 , Alex FULCHER 2 , Robert MCCUAIG 1 , BelindaTHOMAS 3 , Philip BARDIN 3 , David JANS 2 , Reena GHILDYAL 11 University of Canberra, Canberra, AUSTRALIA; 2 Monash University,Clayton, AUSTRALIA; 3 Monash Institute of Medical Research, Clayton,AUSTRALIAHuman Rhinovirus (HRV) infection results in shut down of essentialcellular processes, in part through disruption of nucleocytoplasmic transportby cleavage of the nucleoporin proteins (Nups) that make up thehost cell nuclear pore. Although the HRV genome encodes two proteases(2A and 3C) able to cleave host proteins such as Nup62, littleis known regarding the specific contribution of each. To further elucidatethe specific roles of 2A and 3C in HRV infection, we infectedcells with HRV 16 and also transfected cells with GFP tagged 3C, andperformed Western and immunofluorescence analysis. Western analysisof infected cell lysates revealed that Nup98 is cleaved before theappearance of 3C, while Nup153 is cleaved after the appearance of 3C,suggesting cleavage by 2A and 3C, respectively. Western analysis oftransfected cell lysates confirmed these results, demonstrating GFP 3Cis able to cleave Nup153 but not Nup98. Immunostaining analysis ofHRV16 infected cells revealed significant changes in the cellular localisationof nuclear proteins SC35 and hnRNP A1, as well as relocalisationof nucleolin. The relocalisation of SC35 and nucleolin could be reproducedby transfection with GFP 3C, suggesting 3C is the key mediatorof this effect. In contrast, relocalisation of hnRNP A1 that occurredduring HRV16 infection could not be replicated by transfection with 3Calone, suggesting that 2A is required together with 3C to achieve thecomplete disruption of nucleocytoplasmic transport as observed duringinfection.Virologie, Vol 17, supplément 2, septembre 2013S183

5 th European Congress of Virology15. HIGHLY PATHOGENIC VIRUSESPosters: REF 230 to REF 251REF 230The V Protein of Tioman Virus Is Incapable of Blocking Type I InterferonSignaling in Human CellsPierre Olivier VIDALAIN 1,2 , Grégory CAIGNARD 1,2 , MarianneLUCAS HOURANI 1,2 , Kevin P. DHONDT 3,4 , Jean LouisLABERNARDIÈRE 1,2 , Thierry PETIT 5 ,YvesJACOB 6 , BrankaHORVAT 3,4 , Frédéric TANGY 1,21 Unité de Génomique Virale et Vaccination, Institut Pasteur, Paris,FRANCE; 2 CNRS UMR 3569, Paris, FRANCE; 3 INSERM U758, EcoleNormale Supérieure de Lyon, Lyon, FRANCE; 4 University of Lyon 1, Lyon,FRANCE; 5 Zoo de La Palmyre, Les Mathes, FRANCE; 6 Unité de Génétique,Papillomavirus et Cancer Humain, Institut Pasteur, Paris, FRANCEThe capacity of a virus to cross species barriers is determined by thedevelopment of bona fide interactions with cellular components of newhosts, and in particular its ability to block IFN alpha/beta antiviral signaling.Tioman virus (TioV), a close relative of mumps virus (MuV), hasbeen isolated in giant fruit bats in Southeast Asia. Nipah and Hendraviruses, which are present in the same bat colonies, are highly pathogenicin human. Despite serological evidences of close contacts between TioVand human populations, whether TioV is associated to some human pathologyremains undetermined. Here we show that in contrast to the V proteinof MuV, the V protein of TioV (TioV V) hardly interacts with humanSTAT2, does not degrade STAT1, and cannot block IFN alpha/beta signalingin human cells. In contrast, TioV V properly binds to human STAT3and MDA5, and thus interferes with IL 6 signaling and IFN beta promoterinduction in human cells. Because STAT2 binding was previously identifiedas a host restriction factor for some Paramyxoviridae, we establishedSTAT2 sequence from giant fruit bats, and binding to TioV V was tested.Surprisingly, TioV V interaction with STAT2 from giant fruit bats isalso extremely weak and barely detectable. Altogether, our observationsquestion the capacity of TioV to appropriately control IFN alpha/betasignaling in both human and giant fruit bats that are considered as itsnatural host.REF 231Arenaviruses differentially subvert type I IFN induction and activationof mitochondrial apoptotic pathway via RIG I/MAVSChristelle PYTHOUD, Stefan KUNZUniversity of Lausanne, Institute of Microbiology, Lausanne,SWITZERLANDA hallmark of arenavirus infection is the virus ability to subvert host cellinnate immunity and to suppress the induction of type I interferons (IFNs)via the RIG I/MAVS pathway. The major IFN antagonist of arenavirusesis the viral nucleoprotein (NP), which blocks activation and nuclear translocationof IRF3 in response to virus infection. The RIG I/MAVS pathwayhas recently been linked to virus induced mitochondrial apoptosis via IRF3and Bax in addition to its prominent role in activation of the cellular typeI IFN response. In the present study we sought to investigate the role ofthis novel virus induced apoptotic pathway in the context of arenavirusinfection using the prototypic arenavirus lymphocytic choriomeningitisvirus (LCMV) as a model. Using functional assays and imaging studies,we found that LCMV infection neither results in cytochrome C release,nor in activation of caspases 9, 3 and 7. Interestingly, we found that LCMVwas unable to prevent activation of RIG I/MAVS mediated mitochondrialapoptosis induced by superinfection with sendai virus (SeV) or vesicularstomatitis virus (VSV). The data suggest that LCMV evades activationof mitochondrial apoptosis via RIG I/MAVS, but is unable to block thepathway. Considering the efficient suppression of type I IFN induction byLCMV, our results indicate that arenaviruses evolved to selectively blockIRF3 mediated induction of type I IFN, but not IRF3 mediated regulationof mitochondrial apoptosis.REF 232Inhibition of Crimean Congo hemorrhagic fever virus replication byantiviral compoundsOlivier FERRARIS 1 , Marie MOROSO 2 , Olivier PERNET 3 , MichèleBOULOY 4 , Glaucia PARANHOS BACCALÀ 2 , ChristophePEYREFITTE 11 IRBA, Lyon, FRANCE; 2 Fondation Mérieux, Lyon, FRANCE; 3 UCLA,Los Angeles, USA; 4 Institut Pasteur, Paris, FRANCECrimean Congo hemorrhagic virus (CCHFV) causes viral hemorrhagicfever with high case fatality rates and is endemic in Europe, Africa, andAsia. The causative CCHF virus is an enveloped, segmented negativestranded RNA virus of the family Bunyaviridae, genus nairovirus. Thediscorvery of novel CCHFV inhibitors remains an imortant priority inlight of the lack of currently specific treatment. To enter cells, viruses bindto a variety of host cell receptors, which determine the entry mechanism,host range and pathogenesis of each virus. Following binding, viruses gainaccess to intracellular compartment by fusing with plasma membrane orby internalization in endosomes. Considering this possibility, compoundswere tested for antiviral activity. In this in vitro study, we have evaluatedthe potential antiviral activity of two compounds against Crimean Congohemorrhagic virus. Plaque formation inhibition and virus yield reductionassays of strains of CCHFV in Vero E6 and HuH7 cells were evaluated. The50% inhibitory concentration values for the first tested compound rangedfrom 28 to 43 M, and ranged from 10.8 to 15.7 M for the second. Timeof addition studies demonstrated that compounds might have a direct effecton viral particle infectivity and spread. Our study highlights the interest ofusing compound that target entry pathway to treat CCHF virus infection.This approach has to be considered for the development of future antiviralcompounds targeting CCHF virus.REF 233Development of a cell based assay to image SKI 1/S1P activity for highthoughput screensEmanuela Maria PASI 1 , Joel Ramos DA PALMA 1 , Clara GALAN 1 ,Prudence DONOVAN 2 , Daniel CONSTAM 2 , Antonella PASQUATO 1 ,Stefan KUNZ 11 Institute of Microbiology,University Hospital Center and University ofLausanne, Lausanne, SWITZERLAND; 2 Swiss Federal Institute of TechnologyLausanne, School of Life Sciences, Swiss Institute for ExperimentalCancer Research, Lausanne, SWITZERLANDArenaviruses are lethal human pathogens with no FDA approved vaccinesand drugs. Envelope glycoprotein precursors (GPCs) of arenaviruses arecleaved by the cellular protease Subtilisin Kexin isozyme (SKI 1)/Site 1Protease (S1P), a key regulator of lipid homeostasis, ER stress response,and lysosomal biogenesis. Since this step is fundamental for the formationof infectious virions, SKI 1/S1P is a promising antiviral target. We showedthat SKI 1/S1P inhibitors block arenavirus spread and clear persistentlyinfected cells. However, systemic inhibition of SKI 1/S1P will affect its cellularfunctions with unwanted side effects. Notably, while cellular proteinprocessing takes place in the mid Golgi, the GPCs of Lassa virus (LASV)and lymphocytic choriomeningitis virus (LCMV) undergo maturation inS184 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologythe ER and late Golgi, respectively. We found that the sequence at the cleavagesite (RRLL of LASV GPC and RRLA of LCMV GPC) is crucial forsubcellular cleavage localization. In order to assess SKI 1/S1P processingin specific subcellular compartments in live cells, we have implementeda novel cell based fluorescence sensor assay (CLIP) that consists of twofluorophores divided by a bait sequence (CLIP RRLL or CLIP RRLA)combined with compartment specific targeting signals. This assay allowsthe visualization of the specific location of SKI 1/S1P activity against adefined bait sequence in live cells. This cell based assay is further suitableto screen for inhibitors of SKI 1/S1P with compartment specific activityin a high throughput format.REF 234Niclosamide and curcumin are potent inhibitors of arenavirus infectionAntonella PASQUATO 1 , Florian ZOPPI 1 , Joel RAMOS DA PALMA 1 ,ANGGAKUSUMA 2 , Eike STEINMANN 2 , Stefan KUNZ 11 Institute of Microbiology, Lausanne University Hospital and Universityof Lausanne (CHUV), Lausanne, SWITZERLAND; 2 Institute of ExperimentalVirology, Twincore Centre for Experimental and Clinical InfectionResearch; a joint venture between t, Hannover, GERMANYViral hemorrhagic fevers by arenaviruses are devastating emerging humandiseases. The lymphocytic choriomeningitis virus (LCMV) is the prototypicmember of the arenavirus family. Currently, no FDA approved vaccineor drugs are available against arenaviruses, with the exception of Ribavirin,with limited efficacy. Small molecules with already known mechanismsof action against pathogens are attractive candidates against arenavirusinfections. Curcumin is a small compound isolated from the spice turmeric.It has a variety of beneficial properties, including antiinflammatoryand chemotherapeutic activities. Curcumin is also known as anti viral viadifferent mechanisms of action. Other small organic compounds extractedfrom plants (e.g. green tea catechins) have antiviral properties. Niclosamideis a FDA approved drug against helminthes with antiviral activity dueto its ability to deplete endosomal proton gradients. Here, we show that curcuminand niclosamide are potent inhibitors of LCMV. Curcumin showsvirucidal activity against LCMV with micromolar range IC50. It blocksLCMV primary infections and reduces virus cell to cell propagation. Similarly,niclosamide potently blocks LCMV infection by interference withthe entry step of the virus life cycle. The mechanism of action relies on theability to negatively tune the pH of late endosomes where LCMV fusiontakes place. Low cost drugs such as curcumin and niclosamide that can bedelivered orally and stored at room temperature in tropical climates mayrepresent promising antiarenavirus candidates.REF 235Post translational modification of CCHF non structural glycoproteinsOlivier REYNARD 1 , Ilona NOVAKOVA 1 , Christophe PEYREFITTE 2 ,Viktor VOLCHKOV 11 INSERM U1111 CIRI, Lyon, FRANCE; 2 IRBA, Lyon, FRANCECrimean Congo Haemorrhagic (CCHF) virus belongs to the bunyavirdae,a family of negative strand segmented RNA viruses responsible of severehaemorrhagic fever in man. Pre or post exposure treatments against thedisease are not yet available for human use. The Hyaloma ticks are thenatural reservoir of the virus and most infections occur after tick’s biteand after spread as a nosocomial disease. The M segment of the genomicRNA encodes a polyprotein precursor that is processed by cellularproteases into several structural and non structural proteins. Role of thenon structural proteins of CCHF as well as their posttranslational processingare not yet clearly defined. In this study we investigate processingof non structural protein precursor GP85 which in the virus infected cellsis believed to give rise upon a cleavage by furin to the so called mucinprotein and GP38. We demonstrate that expression of a GP85 constructin HEK293T cells results in appearance of several forms as detected byanti GP38 antibodies and they are characterized by different mobility onSDS PAGE gels (160kDA, 100kDa and 38kDa). We showed that glycosylationof residues nearby the furin cleavage site are crucial for efficiencyof cleavage and this cleavage is cell type dependent. We demonstrated thatthe 160kDa form cannot be explained neither by a glycosylation patternnor by formation of a conventional dimers since its resistance to reducingconditions. Interestingly, We found that mucin protein was able to conferto any non related fused protein a dimer pattern in reducing conditions.REF 236Ebola virus: transcriptional RNA editing versus premature poly adenylationValentina VOLCHKOVA, Jaroslav VORAC, Viktor VOLCHKOVMolecular Basis of Viral Pathogenicity, CIRI, INSERM U1111, UniversitéLyon 1, Lyon, FRANCEEbola virus (EBOV), a member of the Filovirus family causes lethal hemorrhagicfever in man. The EBOV glycoprotein (GP) gene encodes severaltranscripts due to RNA editing at a conserved site consisting of 7 consecutiveuridines. The majority of GP gene transcripts are unedited and encodefor secreted sGP, whereas surface GP is synthesized from edited mRNAscontaining an extra adenosine inserted at the editing site by viral polymerase.Here we demonstrate for the first time that the editing site can serve asa cryptic transcriptional stop signal causing synthesis of truncated mRNA.Northern blot analysis revealed that nearly 50% of GP mRNAs in EBOVinfected cells represent full length GP transcripts whereas others are productsof premature transcription termination. Sequence analysis of shortmRNAs showed a presence of poly(A) tail consisting of 27 44 adenosinesand an absence of translation termination codon at the end of readingframe. Expression of this nonstop mRNA was assayed using a truncatedpoly adenylated mRNA (tr mRNA) synthesized in vitro from a plasmid byT7 polymerase. No specific protein was detected when capped tr mRNAwas used for transfection of 293T cells whereas its in vitro translationresulted in synthesis of a 36kDa GP specific polypeptide. Of note, introductionof stop codon upstream to the poly(A) sequence restored proteinsynthesis indicating that mammalian cells possess a mechanism preventingexpression of aberrant mRNAs. Role and significance of the editingsite as well as function of novel GP gene product in replication of EBOVwill be discussed.REF 237Hepatocyte pathway alterations in response to in vitro Crimean CongoHemorrhagic Fever virus infectionChristophe FRAISIER 1,2 , Raquel RODRIGUES 3 , Vinh VU HAI 1,2 , LucCAMOIN 4,5 , Maya BELGHAZI 6 , Stéphanie BOURDON 1 , PatrickFOURQUET 4,5 , Glaucia PARANHOS BACCALA 3 , LionelALMERAS 1,2 , Christophe Nicolas PEYREFITTE 3,71 Unité de Parasitologie, Institut de Recherche Biomédicale des Armées(IRBA) antenne Marseille, Parc du Pharo, Marseille, FRANCE; 2 Unitéde Recherche des Maladies Infectieuses et Tropicales Emergente(URMITE), Marseille, FRANCE; 3 Emerging Pathogens Laboratory, FondationMérieux, Lyon, FRANCE; 4 Inserm, U1068, CNRS, UMR7258,CRCM, Aix Marseille Univ, UM 105, Marseille, FRANCE; 5 Institut PaoliCalmettes, Marseille Protéomique, Marseille, FRANCE; 6 Aix MarseilleUniversité, CNRS, UMR 7286, Marseille, FRANCE; 7 Unité de virologie,Institut de Recherche Biomédicale des Armées (IRBA) antenne de Lyon,tour CERVI, Lyon, FRANCECrimean Congo hemorrhagic fever virus (CCHFV) is tick borne virus responsiblefor hemorrhagic manifestations and multiple organ failure, witha high mortality rate. In infected humans, damage to endothelial cells andVirologie, Vol 17, supplément 2, septembre 2013S185

5 th European Congress of Virologyvascular leakage may be a direct result of virus infection or an immuneresponse mediated indirect effect. The main target cells are mononuclearphagocytes, endothelial cells and hepatocytes; the liver being a key targetfor the virus which was described as susceptible to IFN host response andto induce apoptosis. However, to better understand the first liver cell alterationsafter virus infection, the protein profile of in vitro CCHFV infectedHepG2 cells was analysed using two complementary quantitative proteomicapproaches, 2D DIGE and iTRAQ. A set of 243 differentially expressedproteins was identified. Bioinformatics analysis (Ingenuity PathwaysAnalysis) revealed multiple host cell pathways and functions altered afterCCHFV infection, with notably 106 proteins related to cell death, including79 associated with apoptosis. Four protein networks emerged withassociated pathways involved in virus entry and exit, protein synthesis,acute phase response, oxidative stress, ubiquitination, or lipid metabolism.These data suggest that CCHFV seems to hijack host proteins topromote its replication and spread. This work gives informations on virushost interactions leading to CCHV pathogenesis, and offers an unparalleledopportunity of the description of possible targets for antiviral research.REF 238Dating the origin of variola virus and other orthopoxvirusesIrina BABKINA, Igor BABKINInstitute of Chemical Biology and Fundamental Medicine, Novosibirsk,RUSSIAThe data on the structure of conserved genes of the New World orthopoxvirusesand unclassified Yoka poxvirus were used for a Bayesian dating oftheir independent evolution. This reconstruction estimates the time whenan orthopoxvirus ancestor was transferred to the North American continentas approximately 50 thousand years ago (TYA) and allows for relation ofthis time interval with the global climate changes (with one of the shorttern warmings during the Last Ice Age). The onset of the Yoka poxvirusevolution was assessed as approximately 90 TYA. Availability of a largenumber of genome sequences of various cowpox virus strains provided fora comprehensive analysis of the orthopoxvirus evolutionary history. Sucha study is especially topical in view of the postulated role of this virusin the evolution of various orthopoxviruses, namely, as an ancestor virus.The computations have demonstrated that the orthopoxviruses divergedfrom the ancestor virus to form the current species about 10 TYA, whilethe ancestor of horsepox virus separated about 3 TYA. An independentevolution of taterapox, camelpox, and variola viruses commenced approximately3.5 TYA. Study of the geographic distribution areas of the hosts ofthese three orthopoxviruses suggests the hypothesis on the region of theirorigin. It is likely that these viruses first emerged in Africa, in the regionof the Horn of Africa, and that the introduction of camels to East Africainduced their divergent evolution.REF 239New transgenic mouse model revealed crucial role of type I interferonsignaling during early stages of Henipavirus infectionKévin P. DHONDT, Cyrille MATTHIEU, Marie CHALONS, JoséphineM. REYNAUD, Branka HORVATCIRI INSERM U1111, CNRS UMR5308, UCBL1, ENS Lyon, Lyon,FRANCEThe Nipah virus (NiV) is highly lethal zoonotic paramyxovirus that emergedin Malaysia in 1998 from a wild reservoir of Pteropus fruit bats. NiV reemerged in Bangladesh few years later causing human outbreaks annually.The study of NiV immunopathogenesis required the development of anadequate small animal model. Therefore, we recently characterized a newanimal model of NiV infection based on mice which have a deletion inInterferon Type I Receptor (IFNAR KO). We showed that in contrast towild type (wt) mice, these mice are highly susceptible to NiV infection bydifferent routes of infection and develop fatal encephalitis with pathologyand immunohistochemical features similar to what was found in humans.We further analyzed cellular and molecular mechanism of resistance of wtmice to lethal NiV infection. Surprisingly, the resistance to NiV was completelypreserved in mice deleted for sensors of the innate immune systemsuggesting the importance of constitutive, rather than virus induced IFNI signaling in the control of NiV infection. Furthermore, while infectionseems to be stopped at very early stages in intraperitoneally injected wtmice, the intracranial NiV infection leads in these mice to systemic spreadof the virus with lethal outcome. These data suggested that some interferontype I (IFN I) producing cells may play the important role in the earlycontrol of NiV infection. On going experiments should elucidate unknownaspects of Nipah virus immunopathogenesis and help us to develop newstrategies to control this highly lethal infection.REF 240Surface proteins of the recently identified African Henipavirus areable to promote viral entry, cell fusion and cytopathic effects in arange of cell lines from Human, Simian and Bat hostsPhilip LAWRENCE 1 , Jan Felix DREXLER 2 , Victor Max CORMAN 2 ,Beatriz ESCUDERO PEREZ 1 , Valentina VOLCHKOVA 1 , MarcelMÜLLER 2 , Christian DROSTEN 2 , Viktor VOLCHKOV 11 CIRI, INSERM U1111, Lyon, FRANCE; 2 Institute of Virology, Universityof Bonn Medical Centre, Bonn, GERMANYThroughout history the emergence of new infectious diseases of animalorigin has caused significant problems for human health. Bats have beenshown to serve as reservoirs for a multitude of infectious agents, includingHenipa, Filo, Corona and Lyssaviruses. Although Henipaviruses aretraditionally restricted to Pteropus bats of South Asia, the recent identificationof distinct viral clades in phylogenetic relation to Henipavirusesin different bat species in five African countries raises the possibility thatthese highly pathogenic viruses may not be as geographically restricted aspreviously imagined. In the absence of isolated infectious African Henipavirus,we have performed experiments with the viral entry proteins Fand G from a representative African strain (M74a) aimed at understandingif this virus presents the same broad cross species tropism as its Henipacounterparts. When the M74a surface proteins are cross expressed withthose of Nipah virus, the M74a G protein is able to replace the NiV Gprotein but with delayed fusion kinetics for a range of cell lines from differentspecies. Co expression of M74a G and F proteins in simian andhost bat cell lines results in rapid cytopathic effects. Pseudotyped MLVreporter infection assays were used to measure the ability of these viralsurface proteins to mediate entry into target cells. Entry mechanisms ofthe African Henipavirus will be discussed further. Results suggest that thisvirus could be expected to display a similar cell tropism to that of the otherHenipa viruses and therefore present a risk for zoonosis.REF 241Crimean Congo hemorrhagic fever virus regulate apoptotic pathwaysin situAli MIRAZIMI 1,2,3 , Yee Joo TAN 4 , Helen KARLBERG 1,51 Swedish Institute for communicable Disease Control, Stockholm,SWEDEN; 2 University of Linköping, Linköping, SWEDEN; 3 NationalVeterinary Institute, Uppsala, SWEDEN; 4 National University of Singapore,Singapore, SINGAPORE; 5 Karolinska Institute, Stockholm,SWEDENCrimean Congo hemorrhagic fever virus (CCHFV) is a member of the Nairovirusgenus of the family Bunyaviridae and it causes a severe disease withhigh mortality rates (∼30%) in human. We have previously demonstratethat CCHFV NP has a conserved cleavage site for caspase 3. Furthermore,we found that this motif, DEVD, is subjected to caspase cleavage duringS186 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyan apoptosis event. We also displayed that caspase activity and apoptosisare induced at late during CCHFV infection. Apoptosis is an importantmechanism by which virus infected cells are eliminated from the host.Many viruses have evolved strategies to delay or suppress apoptosis inorder to secure efficient virus replication, assembly and egress of infectiousviral particles. In this study, we have been interested to investigate ifCCHFV infection can regulate the apoptosis at early post infection. Ourdata displayed that caspase activation are suppressed/delayed early postinfection both upstream and on executor caspase level, insitu. In addition,we suggest that the multifunctional virus structural protein, NP, is involvedin regulation of apoptotic pathways in infected cells. In this study, we alsosuggest that both mitochondrial pathway and death receptors apoptoticpathways are regulated during infection at early and late post infection butin different manner.many cell models such as Vero cells and primary human lung epithelialcells. The activation of host innate immune responses by hCoV EMCand SARS CoV have been shown to be relatively modest. In the presentstudy we assessed the capability of hCoV EMC to infect and replicate inhuman monocyte derived macrophages and dendritic cells and comparedthese responses with SARS CoV. Preliminary data shows that like SARSCoV, hCoV EMC can infect macrophages but both viruses show impairedability to replicate in these cells. A weak induction of interferon(IFN) and IFN 1 gene expression can be seen at 6 h post infection in hCoV EMCinfected macrophages, but at later timepoints this induction is returned tobasal levels. Our data suggests that both of these viruses show relativelypoor ability to replicate in primary human antigen presenting cells whichmay be the reason for a weak ability of these viruses to induce host cellcytokine gene expression.REF 242Phylogenetic analysis of highly pathogenic avian influenza virus H5N1from migratory birds in BangladeshRokshana PARVIN 1,2 , Emdadul H. CHOWDHURY 2 ,Abu.H.M. KAMAL 2 , M. GIASUDDIN 3 , M.R. ISLAM 2 , ThomasW. VAHLENKAMP 11 Institute of Virology, Faculty of Veterinary Medicine, University ofLeipzig, Leipzig, GERMANY; 2 Department of Pathology, Faculty ofVeterinary Science, Bangladesh Agricultural University, Mymensingh,BANGLADESH; 3 Animal Health Research Division, BangladeshLivestock Research Institute, Dhaka, BANGLADESHSince the first outbreak of highly pathogenic avian influenza virus (HPAIV)of subtype H5N1 in Bangladesh in 2007 the virus continues to circulateamong domestic poultry causing severe economic losses. To investigatemigratory birds as possible source of virus introduction genome characterizationof two HPAIV H5N1 isolates from migratory birds (A/migratorybird/Bangladesh/P18/2010 and A/migratory bird/Bangladesh/P29/2010)was performed. After full length amplification and sequence analysis ofthe gene segments encoding HA, NA, M and NS a comprehensive phylogeneticanalysis was performed by comparing the gene segments with otherBangladeshi isolates and representative isolates of all clades described inthe H5 nomenclature. The Bangladeshi H5N1 isolates from different birdsand poultry were broadly grouped into two clades. The majority of isolatesfrom 2007 to 2010 belong to clade 2.2.2 whereas the recent isolates fromchickens and crows of 2011 along with the selected migratory bird isolatesdescribed here are grouped into clade 2.3.2.1. The selected isolates revealedmultiple basic amino acids 338QRERRRKR*G346 at their HA cleavagesite similar to other isolates from clade 2.3.2.1. The receptor bindingsite of HA molecule contained avian like (SAa 2, 3 Gal) receptor specificity.The results verified the presence of HPAIV among migratory birds inBangladesh. Further detailed analyses will reveal whether migratory birdsmay represent the source of the newly introduced clade 2.3.2.1 virus whichwas meanwhile diagnosed in Bangladesh from different avian species.REF 243Lack of efficient replication and induction of host innate immune responsesby the novel betacoronavirus EMC in human monocyte derivedmacrophages and dendritic cellsJanne TYNELL 1 , Esa RÖNKKÖ 1 , Veera ARILAHTI 1 , KristerMELÉN 1 , Bart L. HAAGMANS 2 , Ron A.M. FOUCHIER 2 , PamelaÖSTERLUND 1 , Ilkka JULKUNEN 11 National Institute for Health and Welfare, Helsinki, FINLAND; 2 ErasmusMedical Center, Rotterdam, THE NETHERLANDSThe emergence of a new human coronavirus (hCoV EMC) causing severeacute respiratory infection has alerted the community for a possibility ofanother epidemic similar to the one caused by SARS coronavirus (SARSCoV) in 2003. HCoV EMC has been shown to be able to replicate inVirologie, Vol 17, supplément 2, septembre 2013REF 244Interaction of zoonotic paramyxoviruses with chiropteran cells: Acomparative analysis of Nipah virus and bat derived Henipa like virusglycoproteinsNadine KRÜGER 1 , Markus HOFFMANN 1 , Marcel AlexanderMÜLLER 2 , Jan Felix DREXLER 2 , Michael WEIS 3 , Karl HeinzESSER 4 , Andrea MAISNER 3 , Christian DROSTEN 2 , GeorgHERRLER 11 Institute of Virology/University of Veterinary Medicine Hannover,Hannover, GERMANY; 2 Institute of Virology/Bonn Medical Centre,Bonn, GERMANY; 3 Institute of Virology/Philipps University Marburg,Marburg, GERMANY; 4 Institute of Zoology/University of VeterinaryMedicine Hannover, Hannover, GERMANYBats have been reported to be a natural reservoir for paramyxoviruses withzoonotic potential, e.g. the Nipah virus (NiV), a member of the genus Henipavirus.This virus can lead to severe and often fatal infections in humanswhereas bats generally do not show any clinical symptoms. The detectionof henipa like virus RNA in fecal samples of African bats or organ samplesfrom Indonesian bats and the successful isolation of a bat borne henipavirusin Australia (Cedar virus) raise the question: how likely is such atransmission from a bat virus to the human population? We investigatedthe entry process of an African henipa like virus into immortalized chiropteranand non chiropteran cells and compared it to that of NiV. We focusedon the interaction of the fusion (F) and glycoprotein (G) of NiV and henipalike viruses with cultured cell. To analyze their functional activity we performedfusion assays and screened for syncytia formation following coexpression of NiV or henipa like F and G proteins in chiropteran and nonchiropteran cell lines. Syncytia formation was reduced or abolished by thetreatment of co transfected cells with different cathepsin inhibitors. Forinvestigation of the binding ability of henipa like G proteins to the ephrinB2 (Eph B2) receptor we performed a co immunoprecipitation analysis.The fusion assay revealed the functional activity of henipa like F and G inchiropteran cells in a cathepsin dependent fashion. Furthermore, co immunoprecipitationanalysis indicated that – similar to NiV G henipa like Ginteracts with the Eph B2 receptor.REF 245A Genetic Survey of Crimean Congo Hemorrhagic Fever Virus inTicks from Spain in 2011Ana NEGREDO 1 , Fátima LASALA 1 , Eva RAMÍREZ DEARELLANO 1 , María Dolores FERNÁNDEZ 2,1 , Juan Manuel LUQUE 1 ,Miguel Ángel HABELA 3 , Agustín ESTRADA PEÑA 4 , AntonioTENORIO 11 National Centre for Microbiology, Instituto de Salud Carlos III, Madrid,SPAIN; 2 European Public Health Microbiology Training Programme(EUPHEM), European Centre for Disease Prevention and Control(ECDC), Solna, SWEDEN; 3 University of Extremadura, Cáceres, SPAIN;4 University of Zaragoza, Zaragoza, SPAINS187

5 th European Congress of VirologyCrimean Congo hemorrhagic Fever virus (CCHFV) is a zoonotic virusthat causes outbreaks of hemorrhagic fever in human. The viral cycleis maintained by ticks of Hyalomma spp., serving as vectors and reservoirsof the virus. Vertebral animals suffered an asymptomatic infection.Humans are infected through tick bites or contact with the viremic bloodof patients or livestock. CCHFV belongs to the Nairovirus genus of thefamily Bunyaviridae. It is an enveloped RNA virus with a tri segmentedgenome: S (small), M (medium) and L (Large) segments. The analysis ofthe S, M and L phylogenetic trees topologies document seven lineages,usually coherent between the three segments, although segment re assortmentand recombination events also occur. In Spain, CCHFV genome wasdetected in ticks collected from red deer in a hunting area in Cáceres in2010 and the analysis of the amplified fragment suggested the presence ofan African related strain included in Genotype III (Estrada Peña et al 2011).In order to check the temporal persistence and geographical distributionwe collected ticks from different geographic areas of Spain. Eleven from211 ticks collected in 2011 rendered positive results using an unpublishednested RT PCR with degenerated primers designed on S segment. All thepositive results were obtained in ticks collected in Cáceres, demonstratingthe persistence of virus circulation in the area and no evidence of geographicaldispersion. Nucleotide sequence analysis of the amplified fragmentrevealed the same viral genotype detected in 2010.REF 246Nairoviruses chronically infected Hyalomma derived cell linesCristiano SALATA 1,2 , Helen KARLBERG 2 , Lesley BELL SAKYI 3 ,Giorgio PALÙ 1 , Ali MIRAZIMI 2,4,51 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Swedish Institute for Communicable Disease Control, Solna,SWEDEN; 3 The Tick Cell Biobank The Pirbright Institute, Pirbright,Woking, Surrey, UNITED KINGDOM; 4 Department of Clinical and ExperimentalMedicine, Linköping University, Linköping, SWEDEN; 5 NationalVeterinary Institute, Uppsala, SWEDENCrimean Congo hemorrhagic fever (CCHF) is a zoonotic viral diseasethat is asymptomatic in infected animals, but a serious threat to humans.Human infections begin with nonspecific febrile symptoms that progress toa serious hemorrhagic syndrome with a high case fatality rate. CCHF virus(CCHFV) is classified as a biosafety level 4 pathogen and is the secondmost widespread of all the medically important arboviruses after denguevirus. CCHFV has been found in at least 31 species of ticks, in particular,members of the genus Hyalomma seem to be the principal vectors asthey play an important role as natural reservoir. In the present work, theinfection susceptibility of two Hyalomma derived cell lines to CCHFV andHazara virus (HAZV), an apathogenic virus closely related to the CCHFV,has been investigated in order to compare CCHFV and HAZV virus/hostinteractions in the context of arthropod cells. Preliminary results showedthat the tick cell lines tested were susceptible to infection without anycytopathic effect. It was also discovered that infected cells release progenyvirus to supernatant for at least 28 days post infection. The real time PCRdata analysis suggest that these cells are persistently infected. Currently,a transcriptomics approach to study the cellular response to CCHFV orHAZV infection is under investigation. The results of the comparativestudies on replication of CCHFV and HAZAV in tick cells will be hereindiscussed.REF 247Genetic analysis of Crimean Congo hemorrhagic fever virus strainsin Northern TurkeyHarun ALBAYRAK 1 , Emre OZAN 2 , Cafer EROGLU 3 , AbdullahCAVUNT 21 Ondokuz Mayis University, Faculty of Veterinary Medicine, Departmentof Virology, Samsun, TURKEY; 2 Veterinary Control Institute, Departmentof Virology, Samsun, TURKEY; 3 Ondokuz Mayis University, Faculty ofMedicine, Department of Microbiology, Samsun, TURKEYCrimean Congo hemorrhagic fever virus (CCHFV) is causative agent of atick borne disease with high mortality rates in human. The distribution ofCCHFV includes over 30 countries in Asia, the Middle East, southeasternEurope, and Africa. Aim of the present study was to investigate molecularepidemiology of CCHFV in northern Turkey. The study was performed ona total of 23 confirmed CCHFV in ticks from 2008 to 2009. The hard tickswere collected from variety of mammalian species (cattle, sheep and goat)in northern Turkey (Amasya, Tokat, Sivas, Samsun, Sinop, Ordu and Giresunprovinces). CCHF viral genoms were obtained from seven tick species,and the most abundant were Rhipicephalus turanicus (10/23), Hyalommamarginatum marginatum (5/23), Rhipicephalus bursa (3/23), Hyalommadetritum (2/23), Ixodes ricinus (1/23), H. anatolicum excavatum (1/23), H.anatolicum anatolicum (1/23). All of the CCHF viral strains in northernTurkey were found to belong to the European lineage. Local CCHF viralstrains are grouped into two cluster under European lineage. The majorityof the CCHF viral strains (21/23) were found in the cluster 1, two of themwhich are obtained from Hyalomma detritum and H. anatolicum anatolicumtick species in Alucra location belong to the cluster 2. Pylogeneticanalysis suggest that replication of CCHFV in the tick vectors, whetherHyalomma spp., or other species of ticks has no effect on the viral genomicstructure.REF 248Epidemiological, behavioral and environmental risk factors associatedwith CCHFV seropositivity in humans in GreeceAnna PAPA, Persefoni SIDIRA, Andreas TSATSARIS, AnastasiaKONTANA, Katerina TSIOKA, Elpida GAVANADepartment of Microbiology, Medical School, Aristotle University of Thessaloniki,GREECEBackground: crimean Congo hemorrhagic fever (CCHF) is a viral diseasewith fatality 5 30%. Humans are infected through tick bite or by directcontact with blood or tissues of viremic patients or animals. Aim of thepresent study was to analyze the epidemiological, behavioral and environmentalrisk factors associated with CCHFV seropositivity in humans inGreece.Materials and Methods: The study was based on serological results on3,152 sera, collected from residents from all peripheries of Greece testedfor CCHFV specific IgG antibodies. Age of the patients was 1-97 years(median 58 y); 43.3% were male. The association between seropositivityand categorical variables was estimated by the chi squared test orFisher’s exact test. Univariate logistic regression analysis was performedto calculate the odds ratio and the 95% confidence intervals, and to identifypossible risk factors for inclusion into the final multivariate model.S188 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyA map based on GIS and landcover data of the areas with seropositivitywas constructed. Results: a seroprevalence of 3.77% was observed.Age, gender, periphery, altitude, landcover, history of tick bite, agriculturalactivities and animal contact were shown to be risk factors for CCHFVseropositivity. Conclusions: several environmental, social and geomorphologicpredisposing factors are associated with CCHFV seropositivity.Given that no CCHF cases are reported in Greece, surveillance is neededduring the season when ticks are active in order to gain a better insightinto CCHF epidemiology in Greece.REF 249Human cell tropism and innate immune system interactions of humanrespiratory coronavirus EMC compared to SARS coronavirusFlorian ZIELECKI 1 , Michaela WEBER 1 , Larissa SPIEGELBERG 1 ,Markus EICKMANN 1,2 , Ali Moh ZAKI 3 , Mikhail MATROSOVICH 1,2 ,Stephan BECKER 1,2 , Friedemann WEBER 1,21 Philipps University Marburg, Marburg, GERMANY; 2 German CentreforInfectionResearch, Philipps University Marburg, Marburg, GERMANY;3 Virology Laboratory, Dr. Soliman Fakeeh Hospital, Jeddah, SAUDI ARA-BIAHCoV EMC is a novel human coronavirus associated with severe pneumoniaand kidney failure. Its similarity to SARS CoV raised fears of anepidemic. We demonstrate that HCoV EMC has the ability to replicatein primary cells of the tracheo bronchial epithelium and in continuouscell lines of the target organs lung and kidney. Moreover, HCoV EMCresembled SARS CoV in preventing the upregulation of the antiviralIFN a/ response and the nuclear import of the IFN transcription factorIRF 3. However, HCoV EMC was markedly more sensitive thanSARS CoV to the antiviral state established by ectopic IFN. Thus, HCoVEMC is able to utilize a broad range of human cell substrates, suppressesthe onset of antiviral IFN responses, but does not reach theIFN resistance of SARS CoV. Our ongoing experiments are aimed atthe identification of the antiviral IFN stimulated gene products whichare responsible for the elevated IFN sensitivity of the novel humancoronavirus.REF 250The German G8 Gobal Partnership Program Contribution for theAdvancement of Biorisk ManagementChristine UHLENHAUT 1,3 , Joachimi VON BONIN 21 Robert Koch Institute, Berlin, GERMANY; 2 BioS Programm OfficeG8GPP, Berlin, GERMANY; 3 Gesellschaft für internationale Zusammenarbeit(GIZ) GmbH, Berlin, GERMANYBiosecurity and public health security are often not adequately consideredin the conception and execution of life science research projects.Recent examples and subsequent discussion however shows that theseissues need to be addressed on multiple levels in order to ensure goodscientific practices while at the same time avoiding risks of misuse, e.g.bioterrorism or proliferation of weapons of mass destruction. In a multinationalsetting, a new program that addresses biorisk management in abroader context, was established under the auspices of the German FederalOffice within the ‘Global Partnership Against the Spread of Weapons andMaterials of Mass Destruction’ of the G8 countries. This program combinesdifferent projects in the fields of awareness raising, capacity building,detection and diagnostics, surveillance, biosafety and biosecurity. It alsofurthers the networking among different communities such as the publichealth and the security sector. The Program will achieve its aims throughpari passu partnerships with countries and institutions offering educationand trainings for laboratory staff, decision makers and practitioners ofrelevant institutions as well as by providing equipment to a reasonableextent where needed. Key aspect is the dialogue regarding biorisk managementand non proliferation issues. At the same time the offered supportwill not only strengthen biosecurity but also public health managementand will therefore be of tangible and sustainable benefit for the partnercountries.REF 251Crimean-Congo Hemorrhagic Fever Infection prevalence in FieldCollected Ticks in Endemic Areas of TurkeyMunir AKTAS 1 , Zati VATANSEVER 2 , Nurettin CANAKOGLU 3 , EnginBERBER 3 , Sezayi OZUBEK 1 , Ahmet KALKAN 4 , AykutOZDARENDELI 51 Fırat University Veterinary Faculty, Department of Parasitology, Elazıg,TURKEY; 2 Kafkas University, Kars, TURKEY; 3 Fırat University VeterinaryFaculty, Department of Virology, Elazig, TURKEY; 4 KaradenizTechnical University, Medical Faculty, Department of Infectious Diseases,Trabzon, TURKEY; 5 Erciyess University, Medical Faculty, Department ofMicrobiology, Kayseri, TURKEYTicks are important pests of domestic animals and vectors of the healththreatening agents for humans. Turkey has been experiencing a still increasingoutbreak of Crimean-Congo Hemorrhagic Fever (CCHF), mainly inCentral Anatolia. Host-seeking adult ticks were collected at several villagesduring June-July 2011 and 2012 in CCHF highly endemic area ofTurkey, and specimens were tested for the presence of the disease. Collectionwas made by using human volunteers, where one collector spending15 minutes sitting or standing by the bushy border of the field in order toattract hiding ticks. Also, the ticks were collected by dragging a Whiteflannel cloth (approx 80 × 160 cm) over the suspected area. Sampling wasdone between 10 and 12 in the morning and 14 and 16 hours in the evening.Ticks were collected into vials, kept alive in a cool place at approx.85% relative humidity and later were transferred to the laboratory. Tickswere identified at species level, and divided into 43 pools comprising 2–10tick specimens according to the source species and localities. The pooledticks were stored in tubes and frozen at −80 ◦C for later RNA extraction.The frozen tick pools were crushed using sterile metal rods in liquidnitrogen in 1.5 ml Eppendorf tubes and tested by antigen capture ELISAdeveloped by Firat University, Turkey. Infection rates in tick pools werecalculated using maximum likelihood estimation (MLE) methods with95% confidence intervals (CI) for unequal pool sizes and expressed asMLE of infection rate per 100 ticks. A total of 214 ticks were collected. Ofthe collected ticks, 114 (53.27%) were Hyalomma marginatum, followed89 (41.58%) Rhipicephalus turanicus, 8 (3.73%) Haemaphysalis inermis,2 (0.93%) Hyalomma aegyptium and 1 (0.46%) Rhipicephalus bursa. Ofthe 43 tick pools tested, 6 pools (13.95%) from Hyalomma marginatumwere infected with the virus and MLE of the infection rate was calculatedas 7.45% (CI 3.10-15.01) per 100 ticks. This study was supported byTUB.ITAK (KAMAG 108G126)Virologie, Vol 17, supplément 2, septembre 2013S189

5 th European Congress of Virology16. ZOONOTIC VIRUSES (ANTIGONE,PREDEMICS)Posters: REF 252 to REF 271Among commensal rodents living in urban areas, the wild rat includingthe brown rat, Rattus norvegicus, is a reservoir of known and probablyunknown pathogens. Among the known pathogens, hantaviruses, especiallythe Seoul virus (SEOV) is a growing concern for public health inEurope. The SEOV is responsible for hemorrhagic fever with renal syndrome(HFRS) in humans. Human Life threatening infections by SEOVhave been reported in Asia and are emerging in Europe. Using conventionalapproaches (RT PCR, ELISA), we detected the presence of SEOV virusesin rats trapped in two different locations in the city of Lyon. We confirmedthe circulation of SEOV and the genome of these SEOV strains are underinvestigation, using standard molecular biology methods (cloning of RTPCR fragments and RACE). The second objective of the capture of liverats was to define the virome of the commensal rats using a complementaryand more comprehensive approache. In absence of specific knownsequences of rat viruses, a pathogen discovery method based on the viralenrichment process, in combination with the next generation sequencing(NGS) has been developed to improve the number of viral reads. Lungsamples are currently being analyzed using NGS sequencing coupled tovirus genome enrichment.REF 252Role of the PB2 subunit in the adaptation of avian influenza virus ofsubtype H9N2 to mammalsHanna SEDIRI, Folker SCHWALM, Hans Dieter KLENKInstitut for Virology, Philipps University, Marburg, GERMANYAvian influenza A viruses of the H9N2 subtype are frequently observedin poultry, in Asia and the Middle East and were reported to beoccasionally transmitted to pigs and humans. Moreover, some recentcirculating H9N2 strains have acquired human like receptor specificity.H9N2 viruses have therefore pandemic potential. Some viral proteins,such as the PB2 subunit of the polymerase, are important for host adaptation.It has been described for H7N7, H3N2 and H5N1 viruses that,among others, mutations E627K, D701N, S714I or S714R on the PB2subunit promote adaptation to mammals. In the present study we haveintroduced these mutations in the PB2 subunits of two avian H9N2 isolates,A/Quail/Shantou/2061/2000 and A/Quail/Shantou/782/2000. Wehave analysed the effect of single/double/triple mutations on polymeraseactivity and viral replication. We show that adaptive mutations increasepolymerase activity in minireplicon assays, as well as viral replication ingrowth kinetic studies. We also compared the impact of these mutations inH7N7 and pandemic H1N1 backbones, and observed that the increase inpolymerase activity was most distinct in H9N2 backbones. Furthermorewe investigated the impact of the different mutations on transcription andreplication activity of the polymerase, and have shown that all mutationshave an enhancing effect on replication and transcription activity. Takentogether, these data confirm that mutations in the polymerase subunits PB2may promote adaptation of avian H9N2 viruses to mammalian hosts.REF 253Hantavirus infection and virome of the commensal rats in a urbanareaTatiana DUPINAY 1 , Maria Halima LAABERKI 1 , Florence AYRAL 1 ,Marc ARTOIS 1 , Philippe MARIANNEAU 2 , Catherine LEGRASLACHUER 3,4 , Michel PÉPIN 11 VetAgro Sup, USC1233, Equipe PERS, Marcy l’Etoile, FRANCE; 2 AnsesLaboratoire de Lyon, Unité Virologie, Lyon, FRANCE; 3 UMR CNDynamiqueMicrobienne et Transmission Virale, UMR CNRS 5557 – UCBL USCINRA 1193 – ENVL, Lyon, FRANCE; 4 Profilexpert, Faculté de Médecineet de Pharmacie, 3453 CNRS US7 Inserm, Lyon, FRANCEREF 254Revisiting the genetic diversity of Hantaviruses using a pan virologicalresequencing microarrayClaudia FILIPPONE 1 , Séverine MURRI 2 , Myriam ERMONVAL 1 , TarjaSIRONEN 3 , Misa KORVA 4 , Olli VAPALAHTI 3 , Tatjana AVSICZUPANG 4 , Jean Baptiste PONS 5 , Frank SAUVAGE 6 , PhilippeMARIANNEAU 2 , Noël TORDO 11 Antiviral Strategies Unit, Institut Pasteur, Paris, FRANCE; 2 VirologyUnit, Laboratoire de Lyon, ANSES, Lyon, FRANCE; 3 Department ofVirology, Haartman Institute, Helsinki, FINLAND; 4 Institute of Microbiologyand Immunology, University of Ljubljana, Ljubljana, SLOVENIA;5 SEISE, Ile Molène, FRANCE; 6 UMR CNRS 5558 Université de Lyon,Villeurbanne, FRANCEHantaviruses are enveloped () stranded RNA viruses belonging to thefamily Bunyaviridae, genus Hantavirus that are distributed worldwide.They are not pathogenic in their reservoir hosts (rodents, insectivores andbats) but become pathogenic when transmitted to humans. They provokeHemorrhagic Fever with Renal Syndrome (HFRS) with different degreeof severity in the Old World (Europe, Asia, Africa) and Hantavirus CardioPulmonary Syndrome (HCPS) in the Americas. Divergent species/variantsof hantaviruses circulate in Europe, particularly in France, Belgium,Germany, Scandinavia and the Balkans: Puumala (PUUV), Dobrava Belgradevirus (DOBV), Saarema virus (SAAV), Tula virus (TULV), Seewisvirus (SWSV) and a recent emerging Seoul virus (SEOV). The purpose ofthis study is to evaluate hantavirus genetic diversity and their phylo geographicaldistribution by using a new generation of resequencing microarrayincluding 51 sequences representative of known hantaviruses. RNA tissuesamples from rodents captured throughout Europe were randomly reversetranscribed, cDNA amplified by the phi29 polymerase then hybridizedon the chip. The microarray not only allowed to detect genetic traces ofhantaviruses, but also to characterize the infecting species/variant by interrogationof databases and phylogenetic analysis directly from raw data.This approach is applicable for the discovery of emerging hantavirusesby screening reservoirs. Moreover, the 900 other viral sequences tiled onthe microarray make it suitable for a pan virological analysis without apriori.REF 255High influenza seroprevalence in Belgian wild boarsMutien GARIGLIANY 1 , Fabien GRÉGOIRE 1 , Frédéric FARNIR 1 ,Alain LICOPPE 2 , Daniel DESMECHT 1 , Annick LINDEN 11 University of Liège Faculty of Veterinary Medicine, Liège, BELGIUM;2 Natural and Agricultural Environmental Studies Department, Public Serviceof Wallonia, Gembloux, BELGIUMInfluenza is a seasonal epidemic disease, further responsible for severalpandemic outbreaks in human population throughout recorded history.Human viruses preferentially recognize 2 6 linked sialic acids (SA), whileavian viruses favor 2 3 linked SA. As swine respiratory tract carries bothlinkages in a more balanced fashion, pigs are thought to be favorable tothe emergence of human/avian reassortant viruses potentially giving birthto new pandemic strains, the 2009 pandemic confirming the role of pigsin the epidemiology of influenza. Indoor intensive breeding conditions ofpigs in Western Europe render contacts between swine and birds highlyunlikely. But wild boars may be in close contacts with aquatic birds, theS190 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologynatural reservoir of influenza A virus, and are likely to play the “mixingvessel” epidemiological role traditionally assigned to domestic pigs. Seroprevalencefor influenza in European wild boar seems to be relatively lowin most published studies, ranging from 0% to 7.8%. In the context ofincreasing populations of wild boars in Europe, the aim of this study wasto determine the apparent seroprevalence of influenza in Belgian wildboars and to identify the potential risk factors associated with seropositivity.We identified foci of high H1 influenza seroprevalence (>45%) inspecific hunting territories. If this high prevalence is the result of recurrenttransmission from domestic pigs or reflects an endemic circulation inlocal wild boar population remains to be determined. Viral isolation andphylogenetic studies would be helpful to answer this question.REF 256Hepatitis e virus (HEV): genetic relationship of the first identifiedhuman strain and swine strains in CroatiaLorena JEMERSIC 1 , Jelena PRPIC 1 , Nenad PANDAK 2 , DraganBRNIC 1 , Oktavija DAKOVIC RODE 3 , Tomislav KEROS 1 , Besi ROIC 1 ,Andreja JUNGIC 1 , Tomislav BEDEKOVIC 1 , Ivana LOJKIC 11 Croatian Veterinary Institute, Zagreb, CROATIA; 2 General HospitalSlavonski Brod, Slavonski Brod, CROATIA; 3 3Universitiy Hospital forInfectious Diseases ”Dr. Fran Mihaljevic”, Zagreb, CROATIAHepatitis E is an important health problem with a possibility of zoonotictransmission through contact with infected animals. Hepatitis E virus(HEV) belongs to the genus Hepevirus, family Hepeviridae. Four HEVgenotypes have been recognized so far. Genetic typing of swine HEVstrains, indicate their close relationship to human HEV strains and provethat swine could be recognised as reservoirs of HEV in nature. The aim ofour study was to investigate the potential genetic relationship of a humanHEV strain identified for the first time in Croatia in a patient showingsigns of acute hepatitis, with strains detected in swine from the samegeographical area. Swine samples (blood, spleen and liver) were collectedfrom different age categories and breeding systems (two large commercialfarms and small farms up to 20 animals). In total, 152 swine were includedin this study. All samples were tested by a nested RT PCR protocol inorder to amplify a fragment within the metil transferase gene. Representativepositive samples including the human HEV strain were sequencedand phylogenetically analysed. HEV RNA was detected in swine samplesshowing a high viral prevalence (31.5%) regardless of the breeding systemor age category. The human and swine RNA sequences clustered into phylogeneticgroup 3 proving their close genetic relationship. However, swinesequences further clustered into subgroup 3a and 3e, whereas the humanHEV strain clustered into subgroup 3f showing the highest similarity witha reference swine strain identified in The Netherlands. Even though ourresults support the fact that swine in Croatia are reservoires of HEV theirtrue role in the chain of viral spread is still unknown. The source of thefirst reported human infection in Croatia still remains unidentified. Weconclude that further epidemiological data is needed, including testing ofswine products.are responsive to West Nile Virus. There have been described the sicknesscases of alligators at crocodile farms, and there have been documentedthe cases of people among the staff of the farms being infected. For thecrocodilians the West Nile Virus is a typical emergent infection. The coldblooded vertebrata may serve as reservoir other dangerous arboviral infections.Mosquitoes of Culicidae family (prevalent in all the continents)that live on the warm blooded animals as well as on the cold bloodedones are a component in the encephalomyelitis viral shedding chain. Newemergent infections pose a serious potential hazard not only to the artificiallycreated reptilian and amphibian populations but also to the naturalones. Reptiles and amphibias turn out to be in the environment of newpathogens after they are taken from their wildlife and trapped. Stress,transportation and inappropriate maintenance conditions result in significantdecrease in immunity which makes animals more vulnerable toinfections. Pathogenic agents can evolve and infect new hosts in whichthey have not been present previously. This may lead to extension of thecausative pathogens of dangerous diseases among responsive animals andpeopleREF 258Screening of Influenza H1N1P antibodies in bats from different geographicalregionsElizanbeth LOZA RUBIO 1 , Edith ROJAS ANAYA 1 , Catalina TUFIÑOLOZA 1 , Oscar RICO 21 INIFAP, CENID Microbiología Animal, Mexico City, MEXICO; 2 FMVZUNAM, Mexico City, MEXICOBats are potentially reservoir of many emergent and re emergent zoonosisall over the world. Mexico has so far 138 species of bats in which antibodieshave been detected against rabies, dengue and swine Rubulavirus.Recently, at least 60 new species of paramyxoviruses have been identifiedin bats. Additionally, it was discovered that some of these paramyxovirusesare genetically similar to those affecting human mankind. Concerning toinfluenza virus, is an Orthomixovirus which affects a wide range of hostsincluding birds and mammals. Following the 2009 outbreak of H1N1, thescientific community has reported the presence of the virus in several species,both domestic and wild. So, it is of interest to conduct studies on thesubject, recently identified as a new hemagglutinin into a bat at two locationsin Guatemala. In our country there are no reports on the presence ofantibodies in bats. The objective of this study was to detect antibodies insera against pandemic in different bats species and geographic region. Wecaught bats in the state of Mexico, Puebla, Hidalgo and Morelos, wheredifferent number of samples was taken (29, 23, 5 and 2, respectively). Serawere analyzed by hemagglutination inhibition test using 4 hemagglutinatingunits (HU). The species tested were Desmodus rotundus, Sturniralilium, Sturnira ludovici, Carollia perspicillata, Pteronotus parneli, Artibeusliteratus, Artibeus intermedius, and Myotis microtis Leptonycterisnivalis. All sera were negative. We conclude that apparently this variantvirus is not circulating in the populations analyzed; however we suggestto sample more individuals.REF 257Research of exotic animals, which are a reservoir of infectious diseases,as a component of biosafetyZinaida KLESTOVA, Irina SAVINOVAThe Institute of Veterinary Medicine NAAS of Ukraine, Kiev, UKRAINEThe question of virus diseases of the cold blooded animals and of theirbeing virus carriers or reservoirs has not been adequately studied yet andis a topic of interest. The representatives of all the reptilian classes mayserve as bridging hosts or reservoirs for different viruses, which mayinfect people, mammals and birds. It has been established that reptilesREF 259Seoul hantavirus in Rattus norvegicus in Lyon, FranceLorraine MCELHINNEY 2,4 , Kieran POUNDER 1 , Denise MARSTON 2 ,Florence AYRAL 3 , Andrew BREED 2 , Michael BEGON 1 , CharlotteFEATHERSTONE 2 , Marc ARTOIS 3 , Anthony FOOKS 2,41 University of Liverpool, Liverpool, UNITED KINGDOM; 2 AnimalHealth and Veterinary Laboratories Agency (AHVLA), Weybridge, UNI-TED KINGDOM; 3 Université de Lyon, Marcy L’Étoile, FRANCE;4 National Consortium for Zoonosis Research, Leahurst, UNITED KING-DOMVirologie, Vol 17, supplément 2, septembre 2013S191

5 th European Congress of VirologyHantaviruses (family: Bunyaviridae, genus Hantavirus) are single strandedRNA viruses, which are transmitted to humans primarily via inhalation ofaerosolised virus in contaminated rodent urine and faeces. Whilst infectedreservoir hosts are asymptomatic, human infections can lead to two clinicalmanifestations, haemorrhagic fever with renal syndrome (HFRS) andhantavirus cardiopulmonary syndrome (HCPS), with varying degrees ofmorbidity and mortality. Surveillance in Europe has detected six rodentborne hantaviruses; Dobrava Belgrade Virus (DOBV), Saaremaa virus(SAAV), Seoul virus (SEOV), Puumala virus (PUUV), Tatenale virus(TATV) and Tula virus (TULV). There were over 1900 HFRS cases reportedin France between 2005 and 2010. However, the prevalence of rodentand human cases associated with SEOV in Europe are considered to below, and speculated to be driven by the sporadic introduction of infectedbrown rats (Rattus norvegicus) via ports. Between October 2010and March 2012, 128 brown rats were caught at sites across the Lyonregion in France. SEOV RNA was detected in the lungs of 18 brown rats(14%, 95% CI 8.6–21.3) using a nested pan hantavirus RT PCR (polymerasegene). We did not detect any evidence of a genetic differencebetween infected and non infected rats (cytochrome b gene). Our findingsand the isolation of Seoul virus in UK brown rats, suggest thatSEOV is more prevalent in European brown rats and may contribute toa greater number of the reported HFRS cases in Europe than previouslybelieved.REF 260Cell culture isolation and molecular analysis of HEV strains fromUruguay: evidence of a recent history of the infectionSantiago MIRAZO 1 , Natalia RAMOS 1 , José RUSSI 2 , GustavoCASTRO 3 , Juan ARBIZA 11 Sección Virología. Facultad de Ciencias. UdelaR, Montevideo,URUGUAY; 2 British Hospital, Montevideo, URUGUAY; 3 Ministerio deGanadería, Agricultura y Pesca, Montevideo, URUGUAYHepatitis E virus (HEV) infection is an important public health concernin many developing countries causing waterborne outbreaks and sporadicautochthonous hepatitis. Aultough it is transmitted primarily by the fecal– oral route, zoonotic trasmission from animal reservoirs has also beensuggested. Data regarding the molecular characterization of HEV isolatesfrom South America lacks and further investigation is needed. We recentlyreported the first cases of autochtonous acute HEV infection in Uruguay.Molecular analysis and phylogenetic reconstruction of the viral hypervariableregion and capsid gene showed that they clustered together withinGenotype 3 and were closely related to a set of European strains but weredissimilar to reported South American isolates. The co circulation of subtypes3i and 3 h was observed. Here, we isolated selected Uruguayan HEVstrains in a cell culture based system by blind serial passages in A549 cellline. In vitro replication was confirmed by immunofluorescence assay andRT PCR. To better understand the molecular epidemiology of the HEVisolates we analyzed a larger region of the capsid gene and a conservedfragment of the methyltransferase gene. Additionally, we investigated thecirculation of HEV in domestic pig population in nationwide herds byRT PCR. We confirmed that the HEV infection in Uruguay has probablya European origin and several sources have existed. According to phylogeneticanalysis and since no swine HEV strain was detected in thisstudy, we also suggest that the history of HEV in our country is veryrecent.REF 261Hantavirus circulating among Rattus rattus in Mayotte Island, IndianOceanSéverine MURRI 1 , Frédérik BEAULIEUX 2 , Philippe MARIANNEAU 1 ,Amélie DESVARS 3 , Lénaïg HALOS 4 , Gwenaël VOURC’H 3 , NoëlTORDO 51 Virology Unit, Anses Laboratoire de Lyon, Lyon, FRANCE; 2 Centrede recherche clinique, Hôpital Croix Rousse, Lyon, FRANCE; 3 UR346Animal Epidemiology, INRA, Saint Genès Champanelle, FRANCE;4 Mérial, Lyon, FRANCE; 5 Unit Antiviral Strategies, Institut Pasteur, Paris,FRANCEFollowing the Chikungunya virus epidemics in the Indian Ocean, morethan 3500 animals (primates, bats, rodents, insectivores) were capturedand sampled (blood, serum, organs). The serum of 539 rodents (Rattusrattus, R. norvegicus, Mus musculus) and insectivores (Suncus murinus,Tenrec ecaudatus) captured in 2006 7 in La Réunion island (379), in 2007in Mayotte island (160) were checked for arenaviruses and hantaviruses.RNA was extracted, pooled by groups of 5, screened by nested RT PCR forhantaviruses (Klempa, 2007, EID 13: 520 2; Kang, 2009, Virology 388: 814) and arenaviruses (Vieth, 2007, Trans R Soc Trop Med 101: 1253 64).For each positive pool, samples were screened individually, then sequenced.None of the samples from La Reunion Island was positive for eitherarenavirus or hantavirus. In Mayotte Island, no sample was positive forarenavirus. However 29/160 (i.e. 18%) serums from Rattus rattus testedpositive for hantavirus. The sequence of 246 nucleotides within the polymeraseL coding region showed a limited genetic diversity and a perfectconservation of the amino acid sequence. The phylogenetic analysis indicatedthat these new hantaviruses clustered closer but clearly distinct fromthe hantaviruses Serang virus and Jurong virus. A phylogeographic analysissuggests that several variants seem to have been largely spread andmixed across the Mayotte Island. This work invites to a better surveillanceof hantaviruses in rodents of Indian Ocean islands and to assess the riskof transmission of Haemorrhagic Fever with Renal Syndrome (HFRS) tothe population.REF 262Bat rabies surveillance in France. First report of Isolation of theBokeloh Bat Lyssavirus in France in Myotis nattereri in 2012Evelyne PICARD MEYER 1 , Alexandre SERVAT 1 , EmmanuelleROBARDET 1 , Marie MOINET 1 , Dorothée JOUAN 2 , ChristopheBOREL 2 , Florence CLIQUET 11 ANSES Nancy Laboratory for Rabies and Wildlife, French agency forfood, environmental and occupational health safety, Malzeville, FRANCE;2 CPEPESC Lorraine, Velaine en Haye, FRANCEPassive surveillance of bat rabies has been improved in France since 2000,thanks to the surveillance network constituted by local veterinary servicesand bat handlers from the Chiroptera group (SFEPM, French nationalbat conservation network). Since its inception in 1989, the network hasreported 61 cases of EBLV 1 infection for 2,457 tested bats. All casesinvolved the same species, Eptesicus serotinus in different areas of thecountry. In July 2012, we reported for the first time the presence of theBokeloh Bat Lyssavirus (BBLV), isolated in Myotis nattereri in a forestin North Eastern of France. The Bokeloh Bat Lyssavirus was previouslyS192 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyisolated in 2010 in Lower Saxony, Germany in the same bat species. Theisolate [KC169985] showed 98.8% nucleotide identity with the GermanBBLV isolate [JF311903]. Several organs of the infected bat were investigatedby reference rabies diagnostic methods: fluorescent antibody test,cell culture inoculation test and qRT PCR. Antigen, infectious virus andhigh RNA levels were found in both the brain and salivary glands. Tracesof genomic RNA were also detected in the bladder, kidney, liver/spleen,heart and lung tissues. The results of Lyssavirus distribution investigationsuggest a possible transmission of the virus. To date, the BBLVstrain has never been found in Europe except in 2010 and in 2012 inGermany and France, in two isolated animals. The results of the passivesurveillance of bat rabies in France and the circumstances of discovery andthe results of Lyssavirus distribution investigations will be presented anddiscussed.REF 263Analysis of complete Puumala hantavirus genomes recovered from afatal case of nephropathia epidemica and bank voles (Myodes glareolus)from the site of infectionAngelina PLYUSNINA, Alexander PLYUSNINHaartman Institute, University of Helsinki, Helsinki, FINLANDPuumala virus (PUUV) is a major European hantavirus transmitted tohumans from bank voles and causing nephropathia epidemica. Herewe present the first complete PUUV genome directly recovered froma fatal case together with the results of a search for related geneticvariants in local bank voles. The findings (i) established genetic linkbetween the case and wild type PUUV strains, (ii) revealed that no mutationsaccumulated in the viral genome of PUUV during transmissionto the victim and the fatal infection that followed, and (iii) demonstratedthat the PUUV strain involved was neither unique nor rare geneticvariant.REF 264Optimization of a Commercial Hantavirus IgG Enzyme Immunoassayfor Human Use to Screen Infection Among Wild Rodents in Kirklareli,TurkeyCeylan POLAT 1 , Mehmet Ali OKTEM 1 , Ahmet KARATAS 2 , MustafaSÖZEN 3 , Ferhat MATUR 3 , Hakan ABACIOGLU 11 Dokuz Eylül University, Faculty of Medicine, Department of MedicalMicrobiology, Izmir, TURKEY; 2 Nigde University, Faculty of Arts andSciences, Department of Biology, Nigde, TURKEY; 3 Bülent Ecevit University,Faculty of Arts and Sciences, Department of Biology, Zonguldak,TURKEYCommercial assays for screening of specific Hantavirus antibodies inrodent sera do not exist. We therefore sought to optimize an enzymeimmunoassay (EIA) and an immunoblot assay (IBA) using commerciallyavailable Hantavirus antigens, Euroimmun Anti Hanta virus PoolELISA plates and Euroline Anti Hanta Profile 1 strips developed for usein human samples. The optimized assays were than used to screen 82rodent sera collected from Kirklareli Igneada region in 2009. The optimalserum dilutions were 1/50 and 1/100 for EIA and IBA, respectively. Theoptimal horseradish peroxidase (HRP) conjugated goat anti mouse IgGconcentration for EIA was 1/10,000, while 1/5,000 dilution of alkalinephosphatase (AP) conjugated goat anti mouse IgG dilutions was foundoptimal for IBA. We followed the manufacturer’s recommendations forsubstrate and incubation parameters. Twenty samples were initially positivefor anti Hantavirus IgG by EIA. Of these 16 were Dobrava virus(DOBV) and one was Puumala virus (PUUV) positive with IBA. Thenumber of DOBV positive samples from Apodemus flavicollis, Apodemusagrarius, Microtus guentheri, Apodemus sylvaticus and Apodemusiconus were 9, 3, 2, 1 and 1, respectively. One PUUV positive sample wasfrom Apodemus flavicollis. When immunoblot test was accepted as goldstandard, optimized EIA’s sensitivity and specificity was 100% and 93%,respectively.REF 265Human coronavirus EMC replication induces severe in vitro cytopathologyand is strongly inhibited by cyclosporin A or interferon alphatreatmentClara POSTHUMA 1 , Adriaan DE WILDE 1 , Stalin RAJ 2 , TheoBESTEBROER 2 , Stefan VAN NIEUWKOOP 2 , Monserrat BÁRCENA 1 ,Bart HAAGMANS 2 , Eric SNIJDER 1 , Bernadette VAN DEN HOOGEN 21 Leiden University Medical Center, Leiden, THE NETHERANDS;2 Erasmus MC, Rotterdam, THE NETHERLANDSCoronavirus (CoV) infections are associated with respiratory and entericdisease in humans and animals. The 2003 outbreak of severe acute respiratorysyndrome (SARS) highlighted the potentially lethal consequencesof CoV induced disease in humans. In 2012, a novel CoV (HCoV EMC)emerged, causing 16 human cases thus far, of which 9 had a fatal outcome.We characterized HCoV EMC replication and cytotoxicity in various celllines. Electron microscopy of infected cells revealed extensive membranerearrangements, including the formation of double membrane vesicles andconvoluted membranes, which were previously implicated in the RNAsynthesis of SARS CoV and other CoVs. Following infection, we observedrapidly increasing viral RNA synthesis and release of high titres ofinfectious progeny, followed by pronounced cytopathology. These characteristicswere used in an assay for antiviral compound screening in 96well format, by which cyclosporin A was found to inhibit HCoV EMCreplication in cell culture. Furthermore, HCoV EMC was found to be50 100 times more sensitive to interferon alpha (IFN a) treatment thanSARS CoV, an observation that may have implications for the treatmentof HCoV EMC infected patients. HCoV EMC infection did not prevent theIFN induced nuclear translocation of phosphorylated STAT1, in contrastto infection with SARS CoV where this block inhibits the expressionof antiviral genes. These findings highlight relevant differences betweenthese distantly related zoonotic CoVs in terms of their interaction with andevasion of the cellular innate immune response.REF 266Identification of viruses affecting Venezuelan batsFlor PUJOL 1 , Luzmir BOYER 2 , Mayra H HIDALGO 2 , DomingoJ. GARZARO 1 , Sara PAPO 31 IVIC, Caracas, VENEZUELA; 2 INIA, Maracay, VENEZUELA; 3 INSAI,Maracay, VENEZUELABat (Chiroptera) are reservoirs for zoonotic diseases with potential riskto human and animal health. The aim of this study was the molecularidentification of viruses affecting bats from various regions of Venezuela.Viral RNA or DNA was detected by PCR, using broad spectrum primerstargeting conserved regions of the viral genomes. A total of 54 bats (12vampires, 29 frugivores and 13 insectivores) were analyzed to identify:Lyssavirus (brain), Herpeviruses and Polyomaviruses (trachea and lung),Flaviviruses (heart and liver), and Astroviruses (intestines). Herpesviruses,subfamily Gammaherpesvirinae, were identified in 8 samples. Phylogeneticanalysis suggested their classification in the genus Rhadinovirus, butnot closely related to isolates from Old World bats. Four polyomaviruseswere identified, not closely related to the only bat isolate reported to date.One Astrovirus was identified, related to one clade comprising Old Worldbat and mouse viruses. Neither Flaviviruses nor Lyssaviruses were detectedin this sample group. However Rabies viruses isolated from differentVenezuelan animal species confirmed the circulation of Rabies genotypeVirologie, Vol 17, supplément 2, septembre 2013S193

5 th European Congress of Virology1 viruses in the country, as expected. Viral agents were more frequentlydetected in insectivores bats (viruses detected in 38% of them), althoughthe difference in frequency between different group of bats was not significant.This study allowed the identification of new viral agents in batsfrom New World, where information is still scarce.HEV transmission. The HEV sequences obtained from wild boars wereall of G3f like most of the human HEV sequences. Therefore, these dataare in agreement with the situation observed in other European countriesand the links between HEV infection in pigs, wild boars and humans needto be further analysed to support the hypothesis of a zoonotic transmissionin BelgiumREF 267Sudden increase in seroprevalence of Dobrava Belgrade virus (DOBV)in the province of Trento, ItalyAna Paola RIZZOLI 1 , A. RIZZOLI 1 , V. TAGLIAPIETRA 1 , R. ROSÀ 1 ,H.C. HAUFFE 1 , L. VOUTILAINEN 2 , T. SIRONEN 2 ,H. HENTTONEN 31 Fondazione Edmund Mach – Research and Innovation Centre, SanMichele all’Adige, ITALY; 2 University of Helsinki – Dept. of Virology, Helsinki,FINLAND; 3 Finnish Forest Research Institute, Vantaa, FINLANDDobrava Belgrade virus (DOBV) is considered the most pathogenic ofhantaviruses in Europe, causing HFSR with case fatality rate up to 12%.The first DOBV strain was isolated from lungs of A. flavicollis mice capturedin Slovenia, southeast Europe, and now represents the prototypeDOBV strain (named Slovenia, or Slo/Af) from A. flavicollis. The spatialand temporal distribution of hantaviruses has been monitored in Trentinoregion (northern Italy) since the year 2000 by performing seroprevalencestudies in rodents and humans with IFAT. An intense long term serologicalmonitoring of a population of Apodemus flavicollis has been carriedout. During the period 2000–2008 the antibody seroprevalence of DOBVvaried from 0% to 1.4%. From the year 2010 onwards a sudden increasehas been registered: 3,8% in 2010, 3% in 2011 and 7,7% in 2012. Possibledrivers affecting changes in DOBV seroprevalence in this rodentpopulation will be discussed.REF 268Hepatitis E virus infection in Wild Boars and humans in BelgiumDamien THIRY 1 ,AxelMAUROY 1 , Claude SAEGERMAN 2 ,Bernard BROCHIER 3 , Isabelle THOMAS 3 , Annick LINDEN 4 ,Etienne THIRY 11 Veterinary Virology and Animal Viral Diseases, department of Infectiousand Parasitic Diseases, faculty of veterinary medicine, Liège, BELGIUM;2 Research Unit of Epidemiology and Risk Analysis applied to veterinaryscience (UREAR ULg), Faculty of veterinary medicine, Liège, BELGIUM;3 Virology, Operational Directorate Communicable and InfectiousDiseases, Institute of Public Health, Brussels, BELGIUM; 4 Wildlife healthand pathology, department of Infectious and Parasitic Diseases, Facultyof Veterinary Medicine, Liège, BELGIUMHepatitis E virus (HEV) possesses four genotypes. In Europe, genotype(G) 3 mainly circulates and its route of transmission is highly suspected tobe zoonotic. The aims of this study were to obtain data on apparent viroprevalenceand seroprevalence in wild boar and to compare the differentstrains identified in wild boar and human in Belgium. For the detection ofthe viral infection, a nested RT PCR, an ELISA and a Western blot wereused. A sample of 383 wild boar sera and 69 sera and 61 livers from youngwild boars was obtained during the hunting season in 2010. The humansamples were obtained by the Belgium Scientific Institute of Public Healthand concerned all the sera samples sent by physicians for HEV diagnosis inBelgium. An apparent seroprevalence of 33%(±4.6;125/383) was obtainedin wild boars. Five out of 61 livers and 4/69 sera of young wild boarswere detected viropositive. The sequences obtained belonged to G3f. Inhumans, 25/340 sera in 2010 and 32/437 in 2011 were IgM positive and,from these 25 and 32 sera, 10 and 24, respectively, were viropositive. Fromthese, 4 belonged to G1, 7 to G3 and 1 to G4. In conclusion, the high HEVseroprevalence in wild boars in Belgium raises zoonotic concern aboutREF 269Identification and characterization of virus like particle of Denguevirus types 14inDrosophila expression systemEunbyeol WANG 1 , Wooyoung CHOI 1 , Sunwhan PARK 1 , Seok MinYUN 1 , Kiju CHOI 2 , Myungguk HAN 1 , Chan PARK 11 Division of Arboviruses, Korea CDC, Chungcheongbuk do, SOUTHKOREA; 2 Division of Respiratory Viruses, Korea CDC, Chungcheongbukdo, SOUTH KOREADengue viruses (DENV) are the most prevalent arthropod borne virusesin the world and a public health threat to approximately 40% of the globalpopulation. However, neither licensed vaccines nor effective therapiesexist. Here, we report the production of virus like particle (VLP) of Denguevirus types 1 4 (DENV 1 4) in Drosophila expression system as a vaccinecandidate. We have constructed DNA plasmid containing DENV 2 premembrane(PrM) and envelope (E) proteins (pMT/BiP/V5/His DENV2)and then incorporated respective epitopes on domain III of E proteins ofDENV type 1, 3, and 4 into pMT/BiP/V5/His DENV2 vector. Constructcontaining the full length DENV 2 PrM and E genes and specific epitopesof DENV 1,3, and 4 E genes (pMT/BiP/V5/His DENV1 4) hasbeen shown to express E protein and secrete VLP into cell culture supernatantwhen transfected into Drosophila S2 cells. The VLP particle ofDENV1 4 was detected in electron microscopy. We have determined theoptimal conditions for the expression plasmid by performing a differentcondition of transfection, dose response and time course experiments. Wealso analyzed the glycosylation status of E proteins and found subtledifferences in endoglycosylase sensitivity patterns in cell lysate. Basedon these data, further in vivo studies of the VLP of DENV1 4 is necessaryto determine the immune responses of the dengue virus as a vaccinecandidate.REF 270Comparative phylogenetic analysis of DOBV L and S segments isolatedfrom animal reservoirs in SerbiaValentina NIKOLIC 1 , Novica STAJKOVIC 2 , GoranaSTAMENKOVIC 3 , Mladen VUJOSEVIC 3 , Radovan CEKANAC 2 ,Predrag MARUSIC 4 , Marina SILJIC 1 , Ana GLIGIC 5 , MajaSTANOJEVIC 11 University of Belgrade Faculty of Medicine, Belgrade, SERBIA; 2 MilitaryMedical Academy, Belgrade, SERBIA; 3 Institute for Biological Research“Sinisa Stankovic”, Belgrade, SERBIA; 4 Institute of Public Health,Zajecar, SERBIA; 5 Institute of Virology, Vaccines and Sera Torlak,Belgrade, SERBIAIntroduction: Dobrava Belgrade (DOBV) virus is rodent borne agent withnegative sense three segmented RNA genome. This old world hantavirus isknown to be one of the leading causative agents of hemorrhagic fever withrenal syndrome (HFRS) with mortality rate up to 12%. This study describesphylogenetic analysis of DOBV isolates from Serbia, found in differentrodent hosts. Materials and methods: Rodents were trapped on differentsites in Serbia from 2007 to 2011. Viral RNA was detected by nested RTPCR method, using different pair of primers which afforded amplificationof partial L segment and S segment genes. Evolutionary relationshipsamong all examined sequences were estimated using PAUP and MEGAsoftware packages. Results: Hantaviral sequences identified as DOBVS194 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologywere detected in samples from A. flavicollis, A.agrarius and G. glis. Meannucleotide distance among all examined L segment sequences was 12.6%(0.6 20.1%). S segment sequences included in the study have shown meannucleotide divergence of 10.6% (0 16.5%). Phylogenetic tree, based onL segment, revealed that all isolates from Serbia clustered together withsequences from Greece and Slovenia. All isolates from Serbia, includingthose retrieved from Genbank, were placed in the same cluster on thephylogenetic tree reconstructed using S segment sequences. Conclusion:We detected the presence of DOBV in different animal reservoirs found atdifferent trapping sites in Serbia. Obtained molecular data of both L andS segment sequences is suggestive of geographically related clusteringregardless of virus host.REF 271Detection of Hepatitis E virus in swine liver sausage, in ItalyIlaria DI BARTOLO 1 , Giorgia ANGELONI 1 , Fabio OSTANELLO 2 ,Franco Maria RUGGERI 11 Department of Veterinary Public Health & Food Safety, Istituto Superioredi Sanità, Rome, ITALY; 2 Department of Veterinary Medical Sciences,Università di Bologna Bologna, Italy, Bologna, ITALYHepatitis E is a viral disease usually self limiting with low mortality, butit can become chronic in transplanted patients and be highly lethal duringpregnancy. Hepatitis E virus (HEV) is a small RNA virus, which infectshumans and several animal species. Four mammalian HEV genotypesare known, among which g3 and g4 are considered zoonotic. In Italy, g3strains have been detected in swine farms and slaughterhouses, and wereassociated to human cases. Recently, swine HEV was detected in the porkfood chain in four European countries. We have investigated the presenceof HEV in liver sausage, which is often consumed uncooked in Italy,France, and other countries. In 2012, four packs (300 gr) of pork liversausage were bought at a butcher bench, at 2 different times. Forty fiveportions (200 mg) were contaminated artificially with murine norovirus(MNV). After RNA extraction, samples were analyzed for detection ofHEV, MNV (process control), and porcine adenovirus (pAdV, indexvirus of fecal contamination). HEV was evaluated by RT PCR, targetingseveral genomic regions. Altogether, 2 samples (4.4%) resulted positivefor HEV in at least one PCR. Sequence analysis confirmed swine g3HEV in both samples. PAdV was detected only once, in a HEV positivesample. Attempts to infect A549 cells with a HEV positive liver extractwere repeatedly unsuccessful. This study confirms that pig liver sausagemay contain HEV, possibly resulting from fecal contamination duringimproper slaughtering. Further studies to investigate residual HEVinfectivity in pork, and consumer’s risks are needed.Virologie, Vol 17, supplément 2, septembre 2013S195

5 th European Congress of Virology17. VECTOR BORNE VIRAL INFECTIONS(EUROWN, VECTORIE, WINGS)Posters: REF 272 TO REF 293REF 272Altered protein networks and cellular pathways in severe cases of WestNile virus infectionLionel ALMERAS 1,6 , Christophe FRAISIER 1 , Luc CAMOIN 2,3 ,Stéphanie LIM 4 , Mahfoud BAKLI 1 , Maya BELGHAZI 5 , PatrickFOURQUET 2,3 , Samuel GRANJEAUD 2,3 , Ab OSTERHAUS 4 ,Penelope KORAKA 4 , Byron MARTINA 41 Armed Forces Biomedical Research Institute, IRBA antenne Marseille,Unité de Parasitologie., Marseille Cedex 02, FRANCE; 2 Inserm,U1068, CNRS, UMR7258, CRCM, Aix Marseille Univ, UM 105, Marseille,FRANCE; 3 Institut Paoli Calmettes, Marseille Protéomique.,Marseille, FRANCE; 4 Department of Virology, Erasmus MC, Rotterdam,THE NETHERLANDS; 5 Aix Marseille Université, CNRS, UMR7286, Marseille, FRANCE; 6 Unité de Recherche en Maladies Infectieuseset Tropicales Emergentes (URMITE), WHO Collaborative Center forRickettsioses and Ot, Marseille cedex 5, FRANCEBackground: The recent West Nile virus (WNV) outbreaks in developedcountries have been associated with significantly higher neuropathologyincidence and mortality rate than previously documented. The changingepidemiology and the lack of effective human antiviral therapy or vaccinesmake understanding the pathogenesis of severe disease a priority. Thus, togain insight into the pathophysiological processes in severe WNV infection,a kinetic analysis of protein expression profiles in the brain of WNVinfected mice was conducted using samples prior to and after the onsetof clinical symptoms. Methodology/Principal findings: Using quantitativeproteomic approaches, a set of 148 proteins with modified abundancewas identified. The bioinformatics analysis (Ingenuity Pathway Analysis)of each protein dataset originating from the different time point comparisonsrevealed that four major functions were altered during the courseof WNV infection in mouse brain tissue: i) modification of cytoskeletonmaintenance associated with virus circulation; ii) deregulation of the proteinubiquitination pathway; iii) modulation of the inflammatory response;and iv) alteration of neurological development and neuronal cell death.Conclusion: This study provides novel insights into the in vivo kinetichost reactions against WNV infection and the pathophysiologic processesinvolved, according to clinical symptoms. This work offers useful cluesfor anti viral research and further evaluation of early biomarkers for thediagnosis and prevention of severe neurological disease caused by WNV.REF 273Expressions of virus like particles of Japanese encephalitis virus andWest nile virus in transfected Drosophila cell linesWooyoung CHOI 1 , Eunbyeol WANG 1 , Sunwhan PARK 1 , Seok MinYUN 1 , Kiju CHOI 2 , Myungguk HAN 1 , Chan PARK 11 Division of Arboviruses, Korea CDC, Chungcheongbuk do, SOUTHKOREA; 2 Division of Respiratory Viruses, Korea CDC, Chungcheongbukdo, SOUTH KOREAJapanese encephalitis virus (JEV) and West nile virus (WNV) belong tofamily Flaviviridae and are important mosquito borne human pathogens.Members of the JEV serocomplex including JE and WN viruses, exhibithighly shared antigenicity and cross react well in many serodiagnoses.Virus like particles (VLPs) of flaviviruses generated from the premembrane(PrM) and envelope (E) genes are a promising vaccine candidate.Here we have established expression of VLPs of JEV and WNV in Drosophilacell lines. JEV K05 GS isolate from Korea in 2005 and WNVprototype B956 strain were used in this study. The coding sequences ofPrM and E proteins of JEV and WNV were cloned into the Drosophilaexpression vector (pMT/BiP/V5/His JEV & pMT/BiP/V5/His WNV).Drosophila cell line S2 was transfected with these plasmids. The cell linesexpressing the JEV PrM & E proteins and WNV PrM & E proteins wereconfirmed by western blot analysis using monoclonal antibodies directedagainst flavivirus E protein. The results showed that the recombinantJEV and WNV E proteins had the expected molecular weights of approximate50 kilodaltons, were immnoreactive with monoclonal antibodies, andfound in both the cell lysate and culture supernatant. The VLP particlesof JEV and WNV were detected in electron microscopy, respectively. TheDrosophila expression system is a more convenient and safer approach tothe production of vaccine candidates for JEV and WNV.REF 274Current Status of West Nile Virus (WNV) Infection in TurkeyTaner KARAOGLU 1 , Koray ERGUNAY 2 , Filiz GUNAY 3 , SepandarGARGARI 1 , Seda TEZCAN 4 , Bulent ALTEN 3 , Aykut OZKUL 11 Ankara University, Faculty of Veterinary Medicine, Department of Virology,Ankara, TURKEY; 2 Hacettepe University, Faculty of Medicine,Department of Medical Microbiology, Ankara, TURKEY; 3 Hacettepe University,Faculty of Science, Department of Biology, Ankara, TURKEY;4 Mersin University, Faculty of Medicine, Department of Medical Microbiology,Mersin, TURKEYDespite increased global importance of arboviral infections, there is insufficientinformation in Turkey. Without having troubles on human health, itis hard to establish real awareness on such virus infections even though presenceof serological data indicating virus circulation in animals for years.After occurrence of WNV suggesting clinical human cases in Manisa provincein August 2010, local authorities have focused intensively aimingto obtain information on WNV strains possibly spilling over Turkey. Thispresentation includes virological, entomological and serological data fromfield surveys of regions showing potential risk for WNV circulation and/orinfection. Briefly;WN viruses detected in mosquitoes and horses were shown to be belongedto Lineage 1,No Lineage 2 viruses was able to detect in study areas including TurkishGreek border,No WNV virus was detected in donated human blood samples,Mosquito species carrying WNV were identified by DNA barcoding,Overall seroprevalence of the virus was found approximately 12% and%13 in healthy human beings and various animal species, respectively,using PRNT.Data obtained clearly showed that WNV are detectable countrywide inhuman and equine cases and various mosquito species in Turkey and thusmay cause serious responsible public health problems in near future.REF 275Differentiation of Flaviviruses circulating in Europe using a panel ofmonoclonal antibodies and recombinant proteinsBelen REBOLLO POLO 1 , Ana MORENO 2 , Davide LELLI 2 , EmilianaBROCCHI 2 , Javier SARRASECA 1 ,M a jose RODRIGUEZ 1 , AngelVENTEO 1 , Miguel Angel JIMENEZ 3 , Gamou FALL 4 , MireilleMONDO 4 , Arame BA 4 , Amadou ALPHA SALL 4 , Paolo CORDIOLI 21 INGENASA, Madrid, SPAIN; 2 Istituto Zooprofilattico Sperimantale dellLombardia ed Emilia Romagna, Brescia, ITALY; 3 INIA CISA, Madrid,SPAIN; 4 Istitut Pasteur de Dakar, Dakar, SENEGALFlaviviruses currently circulating in Europe are West Nile virus (WNV),Usutu virus (USUV),Tick borne encephalitis virus (TBEV) and Bagazavirus (BAGV). Diagnosis of flaviviruses is complicated by the presence ofcross reactions especially in regions where more than one virus is endemic.S196 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyThis study describes the preparation of recombinant proteins E and NS1of WNV and USUV and the characterization of Monoclonal antibodies(MAbs) against WNV, USUV and TBEV in order to evaluate their applicabilityin virological identification and serological diagnosis. Viruses usedas immunogen in mice for MAb production were WNV reference and fieldstrains and USUV and TBEV field strain. MAb reactivity was evaluatedby different methods:1 indirect immunofluorescence (IFI) using referenceand field flaviviruses isolated from Europe and Africa,2 Virus neutralizationtest,3 Indirect ELISAs using recombinant E and NS1 proteins ofWNV lineages 1 2 and USUV in Baculovirus expression system,4 CompetitiveELISAs performed to analyze the competition between selectedMAbs and experimental sera obtained from different species. Flaviviruscross reactive MAbs proved to be useful for viral detection in pathologicalspecimens or in infected cell cultures. MAbs specific to immunogenicepitopes have been used for the development of competitive ELISAs forWNV, USUV and TBEV antibody detection. In addition, a double antibodysandwich ELISA based on NS1 specific Mabs able to differentiateWNV L1,L2 and USUV has been developed.This research has receivedfunding from the EU 7th Framework Programm EUROWESTNILE.REF 276Virulence determinants of West Nile Virus Strains circulating inEuropeKhaled ALSALEH 1 , Céline BAHUON 2 , Ana VAZQUEZGONZALEZ 3 , Miguel Angel JIMÉNEZ CLAVERO 4 , AntonioTENORIO 3 , Maha DRIDI 5 , Bénédicte LAMBRECHT 5 , SylvieLECOLLINET 2 , Philippe DESPRÈS 1 , Nathalie PARDIGON 11 Institut Pasteur, Unité des Interactions Moléculaires Flavivirus Hôtes,Paris, FRANCE; 2 Agence Nationale de Sécurité Sanitaire (ANSES), Laboratoirede Santé Animale, Maisons Alfort, FRANCE; 3 Laboratory ofArboviruses and Viral Imported Diseases, National Center of Microbiology,Institute of Health “Carlos III”, Majada, Madrid, SPAIN; 4 Centrode Investigación en Sanidad Animal (CISA) INIA, Ctra, Algete El Casars/n, 28130 Valdeolmos, Madrid, SPAIN; 5 Operational Direction of ViralDiseases, Veterinary and Agrochemical Research Center (CODA CERVAVAR), 99 Groeselenberg, 1180 Br, Brussels, BELGIUMWest Nile virus (WNV) is a neurotropic, mosquito borne enveloped RNAflavivirus. Zoonotic transmission of WNV occurs between avian hosts andornithophilic mosquito vectors, but can cause disease in horse and human,that are incidental dead end hosts. Although Mediterranean WNV strainswere considered as less virulent, recently, several outbreaks and epizooticshave been reported in Eastern and Southern Europe. To investigatethe viral factors involved in the virulence of European strains, we comparedthe highly pathogenic isolate Israel 98 (IS98) and an Italy strainisolated from a magpie in 2008 (IT08). Fifteen amino acid changes and25 nucleotide differences in the 3 ′ UTR were found between the 2 isolates.When introduced separately or combined in an IS98 infectious clone, thesemutations and differences had no effect on viral replication and virus productionin vitro. Moreover chimeras of the 2 isolates did not show anysignificant difference in virus virulence in newborn mice. However, IT08was less pathogenic than IS98 in one day old SPF chickens. Interestingly,IT08 strain but not IS98 was able to induce the fusion of C6/36 cellssuggesting that the isolates are not disseminated in the same manner inmosquitoes. In order to identify viral determinants responsible for thesedifferences, we are currently studying chimeras between the 2 isolates inone day old SPF chickens and in Culex pipiens mosquitoes. In summary,our results indicate that mice might not be discriminant enough to evaluateWNV virulence and the vector might be a virulence limiting factor.REF 277Excretion of West Nile virus in urine during acute infectionLuisa BARZON 1,2 , Monia PACENTI 2 , Elisa FRANCHIN 1,2 , SilvanaPAGNI 1,2 , Thomas MARTELLO 2 , Margherita CATTAI 2 , RiccardoCUSINATO 2 , Giorgio PALÙ 1,21 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Clinical Microbiology and Virology, Padova University Hospital,Padova, ITALYDetection of West Nile virus (WNV) RNA in urine has been anecdotallydescribed and proposed for the diagnosis of WNV infection. This studyreports the application of real time RT PCR for the detection of WNVRNA in urine as a routine procedure in support to the diagnosis of WNVinfection during the large outbreak that occurred in North Eastern Italy in2012. Fourteen out of 32 (43.8%) patients with symptomatic WNV infection,i.e., neuroinvasive disease (WNND) and fever (WNF), had detectableWNV RNA in urine at the time of diagnosis, at a higher rate and load andfor longer time than detection of WNV RNA in blood. At variance, detectionof WNV RNA in urine was less frequent (2 out of 14, 14.2%) inblood donors in whom WNV infection was identified by WNV NAATscreening. Different patterns of WNV viraemia and viruria were observedin patients with WNND, WNF, and in blood donors with subclinicalinfection, with typically both viraemia and viruria observed in patientswith WNND, viruria but no viraemia in patients with WNF, and viraemiabut no viruria in asymptomatic blood donors. Infectious virus wasisolated from urine of a patient with WNND and high WNV RNA loadin urine. In conclusion, this study demonstrated the diagnostic utility ofWNV RNA detection in urine by real time RT PCR for case confirmationin patients with WNND and WNF. The virus was isolated from the urineof a patient with WNND, indicating that WNV excreted in urine may beinfectious.REF 278The Tick Cell Biobank facilitates tick borne arbovirus researchLesley BELL SAKYIThe Pirbright Institute, Pirbright, UNITED KINGDOMThe Tick Cell Biobank is the world’s largest collection of ixodid and argasidtick cell lines. Many tick species of medical and veterinary importanceare represented, including Ixodes ricinus, Hyalomma anatolicum and Ornithodorusmoubata, and attempts to establish new cell lines from EuropeanDermacentor species are ongoing. By supplying tick cell lines and trainingin their maintenance to scientists all over the world, the Tick Cell Biobankunderpins global research into ticks and the many diseases they transmit.Tick cell lines are particularly useful for propagation and study of arbovirusescausing zoonoses including tick borne encephalitis and CrimeanCongo haemorrhagic fever, and livestock diseases such as African swinefever and Nairobi sheep disease, providing a practical alternative to useof live ticks particularly for viruses requiring high level containment. Forexample, material for large scale transcriptomics and proteomics can beproduced in vitro under controlled conditions, and silencing of host cell andvirus genes by RNAi facilitates functional genomics studies. Most existingtick cell lines harbour apparently endogenous viruses, about whichalmost nothing is yet known. It is possible that these putative tick virusesmay play a role in modulation of the tick cell innate immune response,allowing the development of a low level persistent arbovirus infection inthe absence of obvious cytopathic effect that is characteristic of tick celllines.Virologie, Vol 17, supplément 2, septembre 2013S197

5 th European Congress of VirologyREF 279Novel Insights in Anopheles gambiae midgut antiviral responseGuillaume CARISSIMO 1 , Isabelle DIETRICH 3 , EmmanuelBISCHOFF 1 , Christian MITRI 1 , Catherine BOURGOUIN 1 , Anna BellaFAILLOUX 2 , Alain KOHL 3 , Kenneth VERNICK 11 Genetics and Genomics of Insect Vectors, Institut Pasteur, Paris,FRANCE; 2 Laboratory of Insect Vectors, Institut Pasteur, Paris, FRANCE;3 MRC University of Glasgow Center for Virus Research, Glasgow, SCOT-LAND, UNITED KINGDOMMany mosquito species are vectors of human pathogens, such as virusesresponsible for Dengue or Chikungunya, or malaria parasites. The mosquitogenus Aedes is largely responsible for transmission of viruses whilethe transmission of human malaria is exclusively performed by the genusAnopheles. The basis for these pathogen vector specificities is currentlyunknown. However, one alphavirus, the O’Nyong Nyong virus (ONN), isbelieved to be the only human arbovirus transmitted by Anopheles. UsingONN, we have functionally characterized the antiviral immune responseat the midgut infection barrier of A. gambiae. We show that midgut andhemocoel immune compartments have distinct antiviral responses, hintingat different immune pathways for the basis of arbovirus vector specificity.REF 280Continued circulation of West Nile virus lineage 2 in Romania in 20112012Cornelia Svetlana CEIANU, Sorin DINU, Raluca Ioana GATEJ, AniIoana COTAR, Elena FALCUTA, Liviu Florian PRIOTEASA, GabrielaOPRISAN, Daniela BADESCUNational Institute for Research and Development in Microbiology andImmunology CANTACUZINO, Bucharest, ROMANIAWest Nile virus (WNV) is endemic in Romania. Starting with the unprecedentedoutbreak in urban settings in 1996, human neuroinvasive WNVcases have been recorded year after year, in Southeast of the country. The1996 epidemic strain was closely related to isolates from Senegal (1993),Kenya (1998), Volgograd (1999), and belonged to the genetic lineage 1of WNV. In 2010 a significant outbreak, with 57 WNV human cases,occurred between July and October 2010. A change in the epidemiologicalpattern of WNV infection was recorded, with an extension of theaffected area towards North east, in Moldova, and to West, beyond theCarpathians range, in Transylvania. The 2010 WNV outbreak in Romaniawas triggered by the emergence of a neuroinvasive strain from the lineage2 of WNV, highly similar to the strain which caused the 2007 outbreak inVolgograd, in the Volga Delta region. We bring evidence for the continuedcirculation of WNV lineage 2 in Romania in the years which followedthe 2010 outbreak. Eleven and, respectively, fourteen human WNV neurologicalcases were recorded in South and East of Romania in 2011 and2012. A total number of 14108 mosquitoes were collected in Danube Deltaand other areas in South of Romania, distributed in 521 mosquito pools,and screened for the presence of WNV genome using a commercial Taqmanassay. During 2011 transmission season three positive mosquito poolswere found (2 pools of Culex pipiens and 1 pool of Cx modestus), correspondingto a minimum infection rate of 2.09/1000). NS5 region partialsequence and sequence of the 3 ′ UTR region were respectively 99.5% and97% similar to Volgograd 2007 strain. A very active transmission seasonoccurred in 2012: end of August month, which represents the peakof WNV transmission in Romania, the minimum infection rate in mosquitoesreached 16.31/1000 in Danube Delta, and 1.64/1000 in Bucharestarea. A pool of Cx pipiens males was found positive for WNV genome.Mechanisms for WNV maintenance in the area via mosquito vectors arediscussed.REF 281Routine diagnostic data of sentinel travelers as a source for monitoringand surveillance of emerging arboviral diseasesNatalie CLETON 1 , Chantal REUSKEN 1,2 , Jean Luc MURK 2 , MennoDE JONG 3 , Annemiek VAN DER EIJK 2 , Marion KOOPMANS 1,21 National Institute for Public Health and the Environment, Bilthoven,THE NETHERLANDS; 2 Erasmus Medical Center, Rotterdam, THENETHERLANDS; 3 Academic Medical Center Amsterdam, Amsterdam,THE NETHERLANDSBackground: In a large part of the developing world, limited surveillanceis performed. In laboratory information management systems a largeamount of data on diagnostic requests for travelers is available that maybe amenable to trend analyses. Objective: We investigate the usability ofinformation provided by routine diagnostic laboratories to monitor trendsin disease and symptoms in returning travelers. Its potential use for surveillanceof arboviral threats to Dutch travelers and public health in generalis examined using DENV diagnostic requests as a model. Method: Testresults and anonymous information provided by clinicians was receivedfor 10540 diagnostic requests for 8942 patients from diagnostic centers inthe Netherlands from January 2000 to May 2011. The data was evaluatedfor completeness of a predefined minimal dataset and trends in DENVpositive results by travel destination. Population travel data were obtainedfrom a commercial registry, and dengue case notification data by countryfrom WHO DengueNet. Results: Vaccination history was rarely reported(0.4%), despite its importance for interpretation of serology. Traveldestination was completed for 42% of requests and trends in IgM positivetests for this subset correlated to the WHO DENV notifications, withsome discrepancies due to underreporting or underdiagnosis. Hemorrhagicsymptoms showed the highest specificity for patients being DENVIgM positive. We conclude that diagnostic requests can be used to monitortrends, but clinicians would have to provide more detailed vaccination andtravel history.REF 282Investigation for West Nile virus and Toscana virus vectors in Ankaraprovince, Central Anatolia, TurkeyKoray ERGUNAY 1 , Serra ORSTEN 1 , Ozge ERISOZ KASAP 2 ,Filiz GUNAY 2 , Murat OCAL 1 , Bulent ALTEN 2 , Aykut OZKUL 3 ,Durdal US 11 Hacettepe University, Faculty of Medicine, Department of MedicalMicrobiology, Virology Unit, Ankara, TURKEY; 2 Hacettepe University,Faculty of Sciences, Department of Biology, Ecology Division, Ankara,TURKEY; 3 Ankara University Faculty of Veterinary Medicine, Departmentof Virology, Ankara, TURKEYWest Nile virus (WNV) Toscana virus (TOSV) infections have been identifiedin Ankara province since 2009. However, mosquito and sandfly speciesthat participate in virus transmission have not been characterized. In thisstudy, supported by Hacettepe University Research Fund (012 D11 101006), preliminary findings of a field survey, performed in Ankara provinceare presented. Mosquito and sandfly sampling was performed in 7locations during July September, 2012. All mosquitoes, pooled as 2 40individuals and female sandflies were homogenized via established procedures.Mosquito specimens were investigated for WNV via PolymeraseChain Reaction (PCR) and a commercial immunochromatographic antigenassay. Sandfly specimens were evaluated via a pan phlebovirus PCR.A total of 641 sandflies were analysed. Morphological identification ofmales (n:264) revealed Phlebotomus papatasi as the most abundant species(48.5%) followed by Phlebotomus halepensis (39.4%), Phlebotomusmajor sensu lato (5.6%), Phlebotomus perfiliewi perfiliewi (5.3%), Phlebotomustobbi (0.4%), Phlebotomus simici (0.4%) and Phlebotomus sergentiS198 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology(0.4%). Female specimens revealed negative results for phlebovirus RNA.A total of 222 mosquito specimens were investigated which include Culexpipiens sensu lato (75.2%), Anopheles maculipennis sensu lato (22.5%)and Anopheles claviger (2.3%). All pools or individual specimens werenegative WNV assays. No evidence for WNV or phlebovirus infections insandflies and mosquitoes could be identified in this study. Nevertheless,activity of potential vectors was revealed.REF 283Experimental studies of vector competence for West Nile virus (Flaviviridae:Flavivirus) in Italian populations of Culex pipiensClaudia FORTUNA, Maria Elena REMOLI, Eleonora BENEDETTI,Paola BUCCI, Loredana NICOLETTI, Marco DI LUCA, LucianoTOMA, Francesco SEVERINI, Daniela BOCCOLINI, Roberto ROMI,Maria Grazia CIUFOLINIIstituto Superiore di Sanita‘, Rome, ITALYWest Nile virus (WNV) is a mosquito borne flavivirus responsible of severalcases of infection in many European countries. However little is knownabout the potential mosquito vector species. In Italy, the presence and abundanceof Culex pipiens and the increase of WNV cases represent a realemerging health problem. Aim of this study is to evaluate the susceptibilityof Italian populations of Cx. pipiens to WNV and to assess a possible verticaltransmission to the offspring. For this purpose Cx. pipiens laboratorycolonies from field populations collected in Central Italy have been testedfor susceptibility to WNV using “membrane feeding” technique. Experimentalinfections were performed using mosquito females 8 12 days oldand the WN Ma V3 seed, lineage 1 strain, isolated in Vero cells from CSFof a patient from Sardinia outbreak (2011). To evaluate the infection rate,after the WNV oral infection, collections of mosquitoes at different times(0-32 day) were carried out. The virus titre was calculated both by PlaqueForming Units (PFU/ml) and by Real time PCR. The dissemination ratewas also determined analyzing legs and wings of infected mosquitoes. Preliminaryresults indicate that the infection rate (IR) on day 0 was 100%.After 3rd day post infection the IR reached a mean value of 62%. Themean viral titre of infected mosquitoes on day 0 was 3.73 log10 PFU/ml.It decreased at 3rd day, due to the digestion of the blood meal, and thenit increased from the 6th (4.41 log10 PFU/ml) up to 28th day (6.33 log10PFU/ml). These results indicate that Italian colonies of Culex pipiens usedfor experimental infections are able to permit the replication of WNV. Novertical transmission of the virus on the F1 progeny was detected. Furtherexperimental infections will be performed to evaluate the transmissionrate through the saliva analysis of infected mosquitoes. This research wasfunded by EU grant HEALTH.2010.2.3.3 3 Project 261391 EuroWestNile.REF 284Production, purification and characterization of Venezuelan equineencephalitis (VEEV) and Chikungunya virus (CHIKV) nsP1Jaime GUILLÉN 1 , Changqing LI 1 , Irina ALBULESCU 2 , Martijn J.VAN HEMERT 2 , Julie LICHIÈRE 1 , Bruno CANARD 1 , EtienneDECROLY 1 , Bruno COUTARD 11 CNRS and Aix Marseille Université, UMR7257, Architecture et Fonctiondes Macromolécules Biologiques, Marseille, FRANCE; 2 MolecularVirology Laboratory, Department of Medical Microbiology, Center ofInfectious Diseases, Leiden University Medical Cent, Leiden, THENETHERLANDSAlphaviruses are enveloped viruses possessing a single positive strandedgenomic RNA causing emerging infectious diseases. Many of these arthropodborne alphaviruses are considered to be of significant importance interms of public health. Venezuelan equine encephalitis virus (VEEV) isa New World virus and Chikungunya virus (CHIKV) is an Old Worldvirus. nsP1 is implicated in RNA synthesis as the RNA capping enzyme.For alphaviruses, the cap synthesis requires a multi step process involvingmethylation at position N7 of GTP molecule, followed by the formation ofcovalent complex between nsP1 and the N7 methylated GMP and releaseof PPi. It is then suggested that this N7 Methyl GMP is transferred onthe viral mRNA. The understanding of the complex capping process isa key step for the development of inhibitors against nsP1 functions. Inthe present study, we have expressed and purified nsP1 of VEEV andCHIKV. We showed that in presence of [a 32P] GTP and the methyl donormolecule S adenosylmethionine (AdoMet), nsP1 catalyses the N7 methylationof GTP, the hydrolysis of N7GTP into N7GMP, and the formationof a phosphoramide to covalently bind N7GMP on nsP1. The componentsrequired for the reactions were characterized by mean of biochemical andbiophysical methods. Altogether, the results confirmed that VEEV nsP1carries the capping activities and further to decipher the full mechanismare in progress.REF 285Effects of dengue virus infection on the secretion and post translationalmodifications of alpha enolase in HepG2 cellsLuiza HIGA 1,2 , Bruno CURI 1 , Russolina ZINGALI 1,2 , Andrea DAPOIAN 11 Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro,Rio de Janeiro, BRAZIL; 2 Unidade de Espectrometria de Massas e Proteômica,Universidade Federal do Rio de Janeiro, Rio de Janeiro, BRAZILHaemostatic dysfunction is a common feature in severe forms of denguedisease. Previously, our group used a proteomic approach to study theeffects of dengue virus (DENV) infection on protein secretion from HepG2hepatic cells. We showed that infection alters the secretion of several proteinsincluding alpha enolase (ENO1). Recently, ENO1 was described asa plasminogen receptor that modulates its activation. In this work, westudy the effects of DENV infection on the modulation of alpha enolase inHepG2 cells. Our data show that ENO1 secretion correlates with viral loadin a dose dependent manner. However, DENV infection does not affectENO1 intracellular content. Cell viability was not affected by DENV upto 24 h post infection. Two dimensional western blots indicate that ENO1presents 5 isoforms with different isoeletric points. Comparative analysisshowed that the distribution of these isoforms differs between control andinfected cells. The infection of HepG2 cells by DENV leads to an increasein alpha enolase secretion which is dose dependent but it has no effect onENO1 intracellular content. Our data suggest that DENV infection modulatesENO1 post translational modifications. In our future investigations,we will study the effects of ENO1 secreted by infected and control cells onplasminogen activation. The increase of ENO1 secretion by infected cellsmight be associated with fibrinolysis impairment through plasminogenactivation promoting haemostatic dysfunction and playing an importantrole in dengue pathogenesis.Financial Support: CNPq and FAPERJREF 286Experimental West Nile virus infections in different European wildbirdsMiguel Angel JIMENEZ CLAVERO 1 , Elisa PÉREZ RAMÍREZ 1 ,Francisco LLORENTE 1 , Javier DEL AMO 1 , Norbert NOWOTNY 2 ,Paolo CORDIOLI 3 , Ana MORENO 3 , Ramón C. SORIGUER 4 , JordiFIGUEROLA 41 CISA (INIA), Valdeolmos, SPAIN; 2 University of Veterinary Medicine,Vienna, AUSTRIA; 3 Istituto Zooprofilattico Sperimentale della Lobardiae dell’Emilia Romagna (IZSLER), Brescia, ITALY; 4 Estación Biológciade Doñana (CSIC), Sevilla, SPAINVirologie, Vol 17, supplément 2, septembre 2013S199

5 th European Congress of VirologyWest Nile virus (WNV) is an emerging vector borne flavivirus causingencephalitis and other pathologies in humans and animals. It is transmittedby bites of infected mosquitoes, which acquire the infection from wildbirds acting as reservoir hosts. Humans and other mammals like horsesare dead end hosts, that is, although might develop clinical disease theydo not transmit the virus. In North America WNV was first observed in1999, while in Europe/Mediterranean region it re emerged in 1996. Sinceits first description in North America, WNV was associated with wild birdmortality events, a feature that was not evident in Europe/Mediterraneanregion, rising the question as to whether “Old World” bird species areless susceptible to die from WNV infection than “New World” ones. Inan attempt to answer this question we carried out a series of experimentsin which several Old world wild bird species (house sparrows, red leggedpartridges and common coots), were inoculated with different WNVstrains. As a result, the red legged partridge showed a high susceptibilityto WNV disease, in particular with certain Euro Mediterranean strains,while the house sparrow was found less susceptible. Virulence of differentWNV strains varied depending on bird species. Regarding host capacity,both red legged partridges and house sparrows reached viremias capableof transmitting the virus to mosquitoes. By opposite, though susceptibleto the infection, the common coot was unable to sustain a mosquito birdvirus cycle.(Funding: EU Grant HEALTH 2010.2.3.3 3 Project 261391 EuroWest-Nile).REF 287Mosquito, bird and human surveillance of Usutu virus in Germany in2011 and 2012Hanna JOEST 1,2 , Norbert BECKER 1,3 , Martin EIDEN 4 , UteZIEGLER 4 , Ludger ALLERING 2 , Marcus GRAEBERGERBERDING 2 , Stefan BOSCH 5 , Stephan GÜNTHER 2 , MartinGROSCHUP 4 , Jonas SCHMIDT CHANASIT 21 German Mosquito Control Association, Waldsee, GERMANY;2 Bernhard Nocht Institute for Tropical Medicine, Hamburg, GERMANY;3 Department of Zoology, Heidelberg, GERMANY; 4 Friedrich LoefflerInstitut, Riems, GERMANY; 5 Nature and Biodiversity ConservationUnion, Stuttgart, GERMANYUsutu virus (USUV) is a mosquito borne, single stranded RNA virus andbelongs to the Japanese encephalitis virus group within the family Flaviviridae.After the initial detection of USUV in German mosquitoes in August2010, the virus spread in 2011 and caused epizootics among wild and captivebirds in southwest Germany. Since June 2011, 407 dead birds werecollected and tested for the presence of USUV with real time RT PCRs,immunohistochemistry and isolation methods. USUV RNA was detectedby real time RT PCR in 157 birds representing 6 species. The virus wasisolated in cell culture from the heart of 14 Blackbirds. USUV specificantigen was demonstrated by immunohistochemistry in brain, heart, liverand lung of infected Blackbirds. In February 2012 USUV was detectedin hibernating Culex mosquitoes, demonstrating transovarial transmissionof USUV. As expected USUV was re detected in dead birds in June 2012USUV showing that the virus is still circulating in southwest Germany. Toinvestigate the medical importance of USUV, 4200 serum samples fromhealthy blood donors from south west Germany were collected in January2012 and analysed for the presence of USUV specific IgG antibodies. Oneserum samples originating from a healthy blood donor was tested positivein IFA and ELISA. Public health authorities and clinicians in Germanyshould be aware of the risk of USUV infection in humans and considerthis virus in cases of meningoencephalitis.REF 288Matrix M adjuvanted West Nile virus envelope protein vaccine leadsto protection against experimental West Nile virus infectionSofia E MAGNUSSON 1 , Karin H. KARLSSON 1 , Jenny M REIMER 1 ,Silke CORBACH SÖHLE 2 , Justin M RICHNER 3 , Sebastian ULBERT 4 ,Karin LÖVGREN BENGTSSON 1 , Michael S. DIAMOND 3 , LindaSTERTMAN 11 Isconova AB, Uppsala, SWEDEN; 2 University of Zürich, Institute ofLaboratory Animal Science, Zürich, SWITZERLAND; 3 Washington UniversitySchool of Medicine, St Louis, USA; 4 Fraunhofer Institute for CellTherapy and Immunology, Leipzig, GERMANYWest Nile virus (WNV) is an arthropod borne flavivirus and emergingpathogen. In elderly and immunosuppressed individuals infection can leadto neuroinvasive disease. No human WNV vaccine is available thoughveterinary vaccines exist. Recently there have been several WNV outbreaksin the US and Europe hence an increasing need of a human WNVvaccine exists. We have formulated recombinant WNV envelope (E) proteinwith particulate saponin based adjuvant Matrix M and studied antigenspecific immune responses in mice. Mice immunized with Matrix M formulatedWNV E protein developed higher IgG1 and IgG2a serum titers atantigen doses ranging from 0.5 to 10 g compared to mice immunized with3or10g of only antigen. Adjuvanted vaccine also significantly increasedWNV neutralizing antibody titers. This was accompanied by strongcellular recall responses as splenocytes from mice immunized with MatrixM formulated vaccine produced high levels of Th1 and Th2 cytokines andhad antigen specific CD4+ and CD8+ T cell proliferation. Addition ofMatrix M prolonged the immune response duration, as elevated humoraland cellular responses were maintained for 214 days. Importantly, micewere protected from WNV infection if vaccinated with Matrix M formulatedvaccine. In conclusion, addition of Matrix M to WNV E protein leadsto dose sparing and potent humoral and cellular immune responses thatare protective against WNV infection. This suggests that Matrix M adjuvantedWNV E protein is a promising human WNV vaccine candidate.This research was funded by EU FP7 project WiNgS grant no. 261426.REF 289Molecular characterisation of the African orthobunyavirus IleshavirusKarin PACHLER 1 , Daniel RUŽEK 2,3 , Norbert NOWOTNY 1,41 Institute of Virology, University of Veterinary Medicine, Vienna, AUS-TRIA; 2 Department of Virology, Veterinary Research Institute, Brno,CZECH REPUBLIC; 3 Institute of Parasitology, Biology Centre of theAcademy of Sciences of the Czech Republic, Ceské Budejovice, CZECHREPUBLIC; 4 Department of Microbiology and Immunology, College ofMedicine and Health Sciences, Sultan Qaboos University, Muscat, OMANIlesha virus (ILEV) is an arthropod borne virus belonging to the genusOrthobunyavirus, family Bunyaviridae. Orthobunyaviruses are lipid envelopedviruses with a genome of 3 single stranded RNA segments ofnegative polarity designated S (small), M (medium), and L (large). ILEVhas been isolated from humans in several African countries, mostly inrelation with febrile illness and erythema. However, ILEV has also beenreported in association with fatal meningo encephalitis and haemorrhagicfever (Morvan et al., 1994). Despite its known pathogenicity to man,only partial genetic information had been available. In the present study,the complete ILEV genome was determined including the viral noncodingends. Viral RNA was amplified by RT PCR with primers designedon basis of published sequences and degenerate orthobunyavirus primers,S200 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyand PCR fragments were sequenced directly. The viral non coding endswere determined with the SMART RACE method (Matz 2003). Phylogeneticanalyses confirmed the relationship of ILEV to the Bunyamweraserogroup of orthobunyaviruses. ILEV segments S and L exhibited highestnucleotide sequence identities with Bunyamwera virus (86% for S, 80%for L) and Ngari virus (87% for S, 81% for L). The M segment of ILEVhowever shared only a maximum of 70% sequence identity with severalmembers of the Bunyamwera serogroup (e.g. Ngari virus, Northway virus,Cache Valley virus). Notably, all three ILEV segments displayed nucleotideidentities of 99% with the partially available sequences of Mbokevirus, which would suggest that Mboke virus is an ILEV isolate.REF 290Monitoring of Phlebotomus trasmitted viruses activity in Marcheregion, ItalyMaria Elena REMOLI, Claudia FORTUNA, Antonella MARCHI, PaolaBUCCI, Gioia BONGIORNO, Luigi GRADONI, Michele MAROLI,Marina GRAMICCIA, Maria Grazia CIUFOLINIIstituto Superiore di Sanita, Rome, ITALYPhlebotomine sand flies are known to transmit leishmaniases, bacteriaand viruses that affect humans and animals in many countries worldwide.Sand flies are widely distributed in all countries around the Mediterranean,and thus human populations in this area are exposed to sand flytransmitted diseases. Sand fly borne viruses are mainly belonging to thegenus Phlebovirus, the Vesiculovirus and the Orbivirus. The phlebovirusestransmitted by sand flies are a major cause of meningitis, encephalitis andfebrile illnesses. To monitor the stability of the Phlebotomus transmittedviruses “foci” and to investigate about the possible circulation of differentviral variants, surveillance activity was carried out at the end of summer2012 in a known Italian Phlebotomus trasmetted viruses historical focus inFermo commune (Marche region) (Ciufolini. et al., 1992, Parassitologia,34, Suppl. 1: 7 8) monospecific for Phlebotomus perfiliewi. Nine hundredsand fly specimens (480 females (F) and 420 males (M)) were collected,pooled and processed both by nested PCR, using generic primers for thegenus Phlebovirus, and by inoculation in VERO cells for virus isolation.Seven pools resulted positive to RT PCR analysis (5 F and 2 M). Fourof them showed also a CPE in VERO cells. Preliminary study about thepolimerase gene (L segment) sequence analysis showed the confirmationof the presence of Toscana virus and the possible circulation of a newPhlebovirus. Analysis is in progress to characterize the complete genomeof the new isolates. However our data confirm the stability of Phlebovirusfocus in Fermo province. A comparison between historical data onvirus genotypic stability have been planned. This work was supported byEDENext, a collaborative project of the 7th FP (2011 2014) funded by theEuropean Commission under the DG Health; Contract Number: 261504REF 291West Nile Virus interactions with viruses usually infecting the mostprobable West Nile Virus vectorsMaria Paz SANCHEZ SECO 1 , Ana VAZQUEZ 1 , Laureano CUEVAS 1 ,Laura HERRERO 1 , Jason LADNER 2 , Esperanza PEREZ PASTRANA 1 ,Annapaola RIZZOLI 3 , Gioia CAPELLI 4 , Santiago RUIZ 5 , DavidROIZ 3,6 , Jordi FIGUEROLA 6 , Gustavo PALACIOS 3 , AntonioTENORIO 1 , Maria Paz SANCHEZ SECO 11 Instituto de Salud Carlos III, Madrid, SPAIN; 2 National Center forBiodefense and Infectious Diseases, Washington, USA; 3 Research andInnovation Centre Fondazione Edmund Mach, San Michele all’Adige (TN),ITALY; 4 Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD),ITALY; 5 Servicio Control de Mosquitos. Diputación de Huelva, Huelva,SPAIN; 6 Estacion Biologica de Doñana, Sevilla, SPAINPathogenic arboviruses have been the focus of many investigations.However, in the vectors that carry them, other viruses are also present.Interactions between them have been less studied. One such exampleis that of the flaviviruses. This genus contains important public healthchallenges such as West Nile. In recent years, many new flaviviruseshave been described, most of them belonging to the so called “mosquitoonly flaviviruses” group. Other new mosquito only viruses such as birna,alpha, mesoni or negeviruses have also been recently described. With theaim of initiating an investigation about the interactions between virusescirculating in Spain and Italy, we have conducted systematic virus isolationin C636 cells from mosquito pools. When cythopatic effect wasdetected, isolates were analyzed by electron microscopy, consensus PCRand/or sequencing (by generic primers and high throughput sequencingsystems). Sixty six pools of mosquitoes of the species Culex pipiens,Cx. modestus, Cx perexiguus, Ochlerotatus caspius and Aedes albopictuswere collected in Spain. Eighteen viruses were identified (11 flavi,2 reo, 3 parvo (1 denso), 1 ortorthobunya and 1 negevirus). Fifty twopools of the species Cx pipiens, Cx modestus and Ae albopictus were collectedin Italy. Fourteen viruses were identified (6 reo, 4 rhabdo 5 flavi,1 birna, 2 parvo and 2 negeviruses). Most of the positive pools containedtwo or three viruses. Thus, it is possible that a mosquito could beinfected with more than one agent. Possible implication of these will bediscussed.REF 292Wild bird surveillance of West Nile virus in an endemic areaKarin SEKULIN 1 , Jolanta KOLODZIEJEK 1 , Tamas BAKONYI 2 , HelgaLUSSY 1 , Mihai MARINOV 3 , Zdenek HUBALEK 4 , Ivo RUDOLF 4 ,Paul REITER 5 , Norbert NOWOTNY 1,61 1 Zoonoses and Emerging Infections Group, Virology, University of VeterinaryMedicine, Vienna, AUSTRIA; 2 Department of Microbiology andInfectious Diseases, Faculty of Veterinary Science, Szent István, Budapest,HUNGARY; 3 Danube Delta National Institute for Research and Development,Tulcea, ROMANIA; 4 Institute of Vertebrate Biology, Academyof Sciences Kvetna, Brno, CZECH REPUBLIC; 5 Institut Pasteur, Insectsand Infectious Disease Unit, Paris, FRANCE; 6 Department of Microbiologyand Immunology, College of Medicine and Health Sciences, Sultan,Muscat, OMANWest Nile virus (WNV) is maintained in nature in a cycle between Culexmosquitoes and wild birds. To better understand the complex mechanismof WNV maintenance and transmission in an endemic area, a serosurveyand RT qPCR were carried out. Wild birds of 25 different species weresampled from October 2011 to January 2012 and from May to July 2012near Tulcea, in the Danube Delta. Four hundred forty five sera sampleswere screened for WNV Ab with the INGEZIM ELISA and positive serawere conducted to a plaque reduction neutralization test (PRNT). Oral andcloacal swabs of 350 birds (pooled per 4 birds) and different organ samplesand swabs of 83 birds (pooled per bird) were tested for presence of WNVnucleic acid by use of RT qPCR. Sixty out of 445 sera were tested positivein the INGEZIM ELISA whereas only 19 sera were verified with the PRNT.Antibodies specific to WNV were detected in birds of the family Passeridae(4/178), Corvidae (6/76), Sylvidae (2/28), Lanidae (1/28), Orolidae (3/3),Upupidae (2/2), and Ardeidae (1/1). No WNV nucleic acid was detectedin any of the wild birds sampled. Our serological and virological datawere not indicative of any acute/persistent infections or WNV sheddingin the investigated wild birds. However, considering the great biodiversityand large amounts of wild birds in this endemic area our sample size wasrestricted. Moreover, only a limited number of birds may be involved in theWNV transmission cycle. On the basis of our results, we assume that therewas limited WNV circulation in wild birds in Tulcea in winter 2011/12and summer 2012.Virologie, Vol 17, supplément 2, septembre 2013S201

5 th European Congress of VirologyREF 293Detection of antibodies against West Nile virus in spider monkeys insoutheastern MexicoElizabeth LOZA RUBIO 1 , Edith ROJAS ANAYA 1 , FernandoCORTES 2 , Estela ESCRIBANO ROMERO 3 , Ana BLAZQUEZ 3 , JoseMARTINEZ ESCRIBANO 3 , Juan Carlos SAIZ 31 CENID Microbiología, INIFAP, Mexico City, MEXICO; 2 SEMARNAT,Mexico City, MEXICO; 3 INIA, MADRID, SPAINWest Nile virus (WNV), a Flavivirus distributed most widely, is presentinglately variable epidemiological and ecological patterns, including anincreasing virulence that has already caused several deaths in differentcountries. Currently, diagnosis of WNV is achieved mainly by plaquereduction neutralization tests (PRNTs) and lately by an enzyme linkedimmunoassays (ELISAs) developed by our staff. In this test the WNVenvelope recombinant E (rE) protein expressed by Trichoplusia ni insectlarvae with a recombinant baculovirus was evaluated. Concordance withthe WNV based ELISA used routinely was good (95%), as it was withthe reference PRNT (90%), with specificity of 94.4% and sensitivity of88.1%.Since several aspects of the presence of WNV in wildlife is notwell known, we had as goal to standardize this same ELISA in sera of22 spider monkeys of Bacalar, Quintana Roo, Mexico. Once optimized,the rE based ELISA was validated with a battery of human and equinesera characterized previously. Two of 22 sera (9%) resulted sero positive.Production of rE protein in insect larvae allows for an easy, low cost andquite large scale yield of partially purified antigen which is suitable forserological diagnosis of WNV, without the need for manipulation of largequantities of infective virus; however in this case further studies will benecessary, since these monkeys could be reservoir.S202 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology18. EMERGING DISEASES INVETERINARY VIROLOGY (ESVV)Posters: REF 294 to REF 344cDNA clone of FMDV. We managed to get cultured MDBK cells (bovineepithelial cells) persistently infected by FMDV type O (the most commonserotype in the world) O/FRA/1/2001 strain. The construction of the fulllength infectious cDNA clone of this strain is underway. The data fromthis study could be used to improve control strategies to prevent the spreadof the disease worldwide.REF 294Early Step Of JSRV Mediated Cell Transformation May InvolveRALBP1Fabienne ARCHER, Margaux MONOT, Sophie DESLOIRE, BarbaraGINEYS, Alexandra ERNY, Caroline LEROUXUMR754 INRA Univ Lyon1, Lyon, FRANCEJSRV (Jaagsiekte Sheep RetroVirus) is a betaretrovirus that causes a lungadenocarcinoma in sheep. The main targets of JSRV infection are pulmonaryepithelial cells, type II pneumocytes and club cells (Clara). These cellsare at the origin of the tumors developing in the distal lung. It is now clearlyestablished that JSRV induces tumors via the oncogenic properties of itsenvelope (Env), and more precisely the intracellular cytoplasmic tail (CT)of the transmembrane domain (TM). Different pathways such as MAPKand Akt are involved in this transformation. Using a large scale doublehybrid screening in yeast, we identified cellular partners of JSRV and subdomains. We focused on RALBP1 (ralA Binding Protein 1), a GTPaseimplicated in the control of cellular proliferation and in Ras mediated transformation.Plasmids carrying the full length or part of Env and RALBP1were generated. Following transfection, we demonstrated the interactionof Env with RALBP1 in eukaryotic cells by co immunoprecipitation and colocalization of both proteins in the same cellular compartments. RALBP1was overexpressed in tumoral cells compared to normal ones. This interactionmay be a key determinant in the first step of oncogenesis. We arecurrently investigating the role of this interaction using siRNA targetingRALBP1 by measuring the effect on cell transformation and activationof the Akt/MAPK pathways. This experimental model allows studies ontransformation mechanisms and lung cancer tumorigenesis in response toretrovirus infection.REF 295Molecular determinants of foot and mouth disease virus persistenceLela KOPLIKU, Kamila GORNA, Anthony RELMY, Aurore ROMEY,Aude ALLEMANDOU, Stéphan ZIENTARA, Sandra BLAISEBOISSEAU, Labib BAKKALI KASSIMIAnses, Laboratoire de Santé Animale de Maisons Alfort, UMR Virologie1161, Maisons Alfort, FRANCEFoot and mouth disease is a highly contagious and economically importantviral disease of cloven hoofed animals. The causative agent, Footand mouth disease virus (FMDV), is able to cause persistent infectionin ruminants besides acute infection and disease. This asymptomatic carrierstate can occur in initially naïve or vaccinated animals, and can bemaintained for several months (up to 2 years in cattle). Such “carrier”animals represent a potential risk for FMDV transmission to susceptibleanimals. The mechanisms of FMDV persistence and the determining factorsare still unknown. The main objective of this study is to identify theviral and cellular factors involved in the establishment and maintenanceof FMDV persistence. This shall be achieved by identification of changesin host cell gene expression and virus genome associated with virus persistencein cellular models. We expect that our investigations will lead toin vitro modelling of FMDV persistence. Two major tools are needed forthis study, a cell culture model of persistence of FMDV, and an infectiousREF 296Seroprevalence Of Schmallenberg And Akabane Virus Infections InDomestic Ruminants In Northern TurkeyHarun ALBAYRAK, Emre OZAN, Abdullah CAVUNT, Hamza KADIVeterinary Control Institute, Department of Virology, Samsun, TurkeyAt the end of 2011, a new orthobunyavirus, tentatively named Schmallenbergvirus (SBV), was discovered in Germany. This virus has sincebeen associated with clinical signs of decreased milk production, waterydiarrhoea and fever in dairy cows, and subsequently also with congenitalmalformations in calves, lambs and goat kids. In this study, the serumsamples collected from domestic ruminants (cattle, sheep and goat) inmiddle Blacksea region of Turkey where abortion, stillbirth and congenitalmalformations cases were observed in the past years were surveyed forthe presence of specific antibodies from Schmallenberg virus (SBV) andAkabane virus (AKAV). The material consisted of 724 domestic ruminantsincluding 308 cattle, 307 sheep and 109 goats from Sinop and Samsun provinces,northern part of Turkey. Seropositivity rates in cattle, sheep andgoats were detected as 36.03%, 1.63% and 2.75% for SBV and 42.21%,2.28% and 2.75% for AKAV, respectively. Out of 724 serum sa! mplesexamined, 140 (19.34%) were positive for AKAV and 119 (16.44%) werepositive for SBV by enzyme-linked immunosorbent assay (ELISA). Ourstudy demonstrates for the first time the presence of antibodies to SBV inTurkey. These results suggest that AKAV and SBV diseases are widespreadin northern Turkey.REF 297Drug development As Preventive Measures Of Animal Viral InfectionsZinaida KLESTOVA 2 , Olga ZOZ 1 , Viktor TASHUTA 11 Society of Microbiology, Kiev, UKRAINE; 2 EUROSCIENCE, Strasbourg,FRANCEThe viruses that cause diseases in humans and animals, are capable ofmutation and worldwide spread. More dangerous viruses attack humansand animals, causing economic losses. The aim of every country is creationbiosafety systems to prevent viral infections. Obtaining of new preventativeand antiviral drugs one of the ways to achieve this goal. A numberof laboratories carry out screening of new chemicals to detect antiviralactivity. There is a range of antiviral agents, the use of which leads to adecrease in viral activity, but, nevertheless, universal antiviral agent is unknown.The main focus of our work is to determine the antiviral activity ofa number of chemicals against a variety of animal viruses. The study wasconducted in the culture of animal cells: SKEV, BHK 21, Vero, SK 6, RK13. As the virus model we used some strains of the family Herpesviridae.In the course of the studies to determine cytotoxicity and antiviral activityof newly synthesized compounds and well known chemical substancesused for other purposes, we obtained positive results. Among them dataon antiviral activity 5,54 ± 0,04 lg TCID 50/ml. The results confirm thepromise of test substances as anitviral drugs. They can be used in clinicalresearch and clinical practice in veterinary medicine, as well as models ofhuman antiviral drugs.Virologie, Vol 17, supplément 2, septembre 2013S203

5 th European Congress of VirologyREF 298Antiviral activity of extracts of bacteria isolated from soils moundsagainst Feline herpesvirus (FeHV 1) and Feline calicivirus (FCV)Marina PADILLA, Luciana K. KHON, Claudia Beatriz MENEZES,Fabiana FANTINATTI GARBOGGINI, Matheus MARTINI, AnaCaroline BARNABÉ, Ricardo DURÃES CARVALHO, LeonardoCASERTA, Juliana SANTIAGO, Clarice ARNSUNICAMP, Laboratory of Virology, Campinas, BRAZILThis study demonstrated the antiviral properties of extracts from bacteriaisolated from soils mounds in Brazilian northeast against feline herpesvirus(FeHV 1) and feline calicivirus (FCV). Microorganisms isolatedfrom land of termite were incubated in culture medium for four weekat 30 ◦ C and these inoculums were extracted by liquid liquid extractionwith ethyl acetate. Antiviral activity was measured by virus titration techniqueand calculated the percentage of viral inhibition (PI) for with virustreat with samples. The 50% effective concentration (EC50), 50% cytotoxicconcentration (CC50), selectivity index (SI) were determined usingthe MTT method. The objective of this study was to evaluate in vitro 16microorganism extracts for antiviral activity against FeHV 1 and FCV.The extracts were considered active when PI is higher than 90%. Threeextracts were active against FeHV 1: LC04 Ac and MC25 met with 97%of inhibition and LC22 CM with 99% inhibition and against FCV werealso three active extracts: LC16 Ac and LC22 CM with 99% of inhibitionand LC16 met with 98% inhibition. This active extracts were evaluated inMTT assay to calculate the selectivity index. The LC04 Ac and MC25 metshow SI considered promising equal to 16.58 and 6.23 respectively againstFeHV 1. For the FCV the promising value of SI were found to LC16 Acand LC22 CM extracts with SI values equal to 12.59 and 11.00, respectively.These results shows that bacteria isolated from soils mounds have apotential source for discovery of new antiviral drugs.REF 299The Serological, Virological and Pathological Investigations of RiftValley Fever Virus Infection in Aborted Cattle, Sheep, Goats and TheirFoetusesKamil ATLI 5 , Mehmet KALE 1 , Sibel HASIRCIOGLU 1 , OzlemOZMEN 2 , Nuri MAMAK 3 , Sibel GUR 4 , Orhan YAPICI 6 , SibelYAVRU 5 , Mehmet HALIGUR 2 , Oya BULUT 51 University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine, Departmentof Virology, Burdur, TURKEY; 2 University of Mehmet Akif Ersoy,Faculty of Veterinary Medicine, Department of Pathology, Burdur, TUR-KEY; 3 University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine,Department of Internal Medicine, Burdur, TURKEY; 4 University of AfyonKocatepe, Faculty of Veterinary Medicine, Department of Virology, Afyon,TURKEY; 5 University of Selcuk, Faculty of Veterinary Medicine, Departmentof Virology, Konya, TURKEY; 6 University of Kyrgyzstan TurkeyManas, Faculty of Veterinary Medicine, Department of Virology, Bishkek,KYRGYZSTANThis study was investigated the serological, virological and pathologicalof Rift Valley Fever Virus (RVFV) infection in aborted cattle (Holstein;178 pcs), sheep (Domestic of breed; 160 pcs), goat (Hair goat; 66pcs,Honamli goat; 98pcs, Saanen goat; 16pcs) aborted cattle foetuses (8pcs), sheep foetuses (24 pcs), goat foetuses (5pcs). Samples werecollected between July 2009 to September 2010. Blood serum sampleswere collected from aborted cattle, sheep and goats were detected asseropositive for 7 cattle (3.93%), 4 sheep (2.50%) and 18 goats (10.0%)by indirect ELISA. Distribution of according to breeds in determined of18 seropositive goats, 13 Hair goat (19.70%), 2 Honamli goat (2.04%)and 3 Saanen goat (18.75%) was observed. Blood serum samples werecollected from aborted cattle foetuses, sheep foetuses and goat foetuseswere detected as seropositive for 1 cattle (12.50%), 4 sheep (16.67%),1 goat (20.0%). While cytopathic effect (CPE) was not observed in anyof them when leukocyte samples that were collected from aborted cattleand sheep, were inoculated in Vero cell cultures. CPE observed positiveonly 2 (1.11%) goat leukocyte samples. Also, CPE observed as positive3 sheep (12.50%), 1 goat (20.0%) when leukocyte samples that werecollected from aborted sheep&goat foetuses. Liver, spleen and brainsamples collected from aborted cattle, sheep and goat foetuses were notdetermined for RVFV antigen by IP and IF tests. As a result, RVFVinfection was not detected in the Western Mediterranean, Turkey.Key words: Abortion, Pathology, RVFV, Serology, VirologyREF 300Experimental infection of European breed sheeps with 2 differentvirulent strains of Rift Valley fever virusSandra LACÔTE 1 , Marie MOROSO 1 , Pierre SARRADIN 2 , MichelPEPIN 3 , Isabelle SCHWARTZ 4 , Philippe MARIANNEAU 11 Unité Virologie, ANSES Laboratoire de Lyon, Lyon, FRANCE; 2 UE1277Plate forme d’Infectiologie expérimentale, INRA, Nouzilly, FRANCE;3 USC INRA/VAS 1233, équipe pathogènes émergents et rongeurs sauvages,Lyon, FRANCE; 4 VIM, INRA, Jouy en Josas, FRANCERift Valley Fever (RVF) is a mosquito borne disease occurring in humanand animals, mostly in livestock. Outbreaks of severe disease occurredthroughout Africa and more recently in the Arabia peninsula. This disease,resulting in « abortion storms » and with 20% of mortality in ruminants,is caused by a virus called RVF virus (RVFv), belonging to the Bunyaviridaefamily and to the Phlebovirus genus. Human infection results infebrile disease with 1 2% of more severe disease. Improving knowledgeon the susceptibility and pathology induced in European sheep breeds byRVFv and developing effective and safe diagnosis tools and vaccines areimportant in the hypothesis of a future emergence of the disease in Europe.We have characterized the clinical symptoms, the viral replication and theimmune and inflammatory responses developed by 5 month old lambs(Ile de France breed) infected by two different virulent strains of RVFv.Few days after inoculation, lambs developed a febrile disease with hyperthermiacorrelated with viral replication. Humoral immune response wasstudied by following IgM and IgG antibodies production and high titers ofneutralizing antibodies were detected. This successful infection of Ile deFrance sheep by RVFv illustrates the potential risk for disease importationand shows that this model is useful to study transmission, viral cycle ornew vaccine candidates.REF 301Detection and genetic analysis of group C rotaviruses in pigs in theCzech RepublicRomana MOUTELIKOVA, Jana PRODELALOVA, Lucie DUFKOVAVeterinary Research Institute, Brno, CZECH REPUBLICThe objective of our work was to examine epizootiological situation in theCzech pig farms with regard to group C rotaviruses (RV C). We examined aset of total 293 porcine faecal samples or gut contents gathered from sevendifferent farms. The RT PCR detection of double stranded (ds) RNA of RVC was carried out with the use of primers specific for the gene coding proteinVP6. In the set of 220 stool samples from different age groups of pigsa total of 47 (21.4%) was positive for RV C dsRNA. The biggest portionof RV C positive samples was found among specimens from weaning andpost weaning piglets (35%). In the set of 73 specimens from gut content offinisher pigs collected at the abattoir 28 samples (38.4%) were positive forRV C. There are only limited numbers of investigations on porcine RV Cin Central Europe; many questions remain unanswered in respect of theirgenetic heterogeneity as well as their possible genetic relatedness withhuman strains. In the present study, we determined the near full lengthnucleotide sequences of VP4, VP6 and VP7 genes of selected porcine RVC strains. These sequences were aligned with each other and also withS204 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyother VP4, VP6 and VP7 sequences of porcine, bovine and human RV Cstrains available in GenBank. Subsequently, the phylogenetic and molecularevolutionary analysis was conducted using MEGA version 4. Thestudy was supported by the Ministry of Agriculture of the Czech Republic(Grant No. QH81061) and the Ministry of Education, Youth and Sportsof the Czech Republic (AdmireVet; Grant No. CZ.1.05/2.1.00/01.0006,ED0006/01/01).REF 302A Serological Investigation of Rift Valley Fever (RVF) Infection inCamel (Camelus dromedarius), Goitred gazella (Gazella subgutturosasubgutturosa) and Domestic Anatolian water buffaloes (Bubalusbubalis Linneaus)Sibel YAVRU 3 , Sibel GUR 1 , Mehmet KALE 2 , Orhan YAPICI 6 , NuriMAMAK 4 , Nural EROL 51 University of Afyon Kocatepe, Faculty of Veterinary Medicine, Departmentof Virology, Afyon, TURKEY; 2 University of Mehmet Akif Ersoy,Faculty of Veterinary Medicine, Department of Virology, Burdur, TUR-KEY; 3 University of Selcuk, Faculty of Veterinary Medicine, Departmentof Virology, Konya, TURKEY; 4 University of Mehmet Akif Ersoy, Faculty ofVeterinary Medicine, Department of Internal Medicine, Burdur, TURKEY;5 University of Adnan Menderes, Faculty of Veterinary Medicine, Departmentof Virology, Aydin, TURKEY; 6 University of Kyrgyzstan TurkeyManas, Faculty of Veterinary Medicine, Department of Virology, Bishkek,KYRGYZSTANRift Valley Fever (RVF) which is an acute Arboviral infection characterizedzoonotic is a member of the genus Phlebovirus, in the familyBunyaviridae. Since first determination of the disease in 1915, many studieshave been done. But there is little data about the infection in ourcountry. This study was investigated the serological of RVFV infectionwhich collected blood serum samples in camel, goitred gazella, domesticAnatolian water buffaloes by the Indirect RVFV ELISA (IDVet, France).Camel samples (72 pcs) in the different farms province of Aydin, gazellasamples (82 pcs) at Ceylanpinar State Farm were collected in 2005. Anatolianwater buffaloes samples in majority of small farms between 1999 and2001 were obtained from various provinces. These provinces are Afyon(168 pcs),Amasya (80 pcs), Samsun (69 pcs), Ankara (35 pcs), Sivas (21pcs), Tokat (19 pcs), Konya (10 pcs) and Elazig (8 pcs). As a result of thetest sample of 82 goitred gazella found to be seronegative for RVF, whilethe one of camel samples (1/71, 1.3%), was positive. While DomesticAnatolian water buffaloes negative in all the samples obtained from Sivas,Tokat, Konya and Elazig, Amasya, 12 (12/80, 15%), Ankara, 5 (5/35,14.2%), Samsun, 8 (8/69, 11.5%) and Afyon, 10 (10/168, 5.9%) sampleswere detected seroposiitive. A total of 35 water buffaloes samples weredetermined seropositive for RVFV.Key words: Buffalo, Camel, Gazella, Rift Valley Fever, SerologyREF 303First detection of Schmallenberg Virus Infections in SloveniaJoze GROM, Ivan TOPLAK, Vasilij COCIANCICH, DanijelaRIHTARIC, Tomislav PALLERToplak Ivan, Grom Jože, Cociancich Vasilij, Rihtaric Danijela, TomislavPaller University of Ljubljana, Veterinary faculty, NVI, Gerbiceva 60, 1000Ljubljana, SLOVENIAIn November 2011, Germany reported the first occurrence of novel Orthobunyavirus,named Schmallenberg virus (SBV), later the same virusemerged in number of European countries. The first clinical case of SBVinfections, confirmed by laboratory diagnosis of SBV in Slovenia, wasidentified in January 9th 2013 from a herd of sheep, but to the end ofMarch 2013 additional 12 bovine herds were identified SBV positive byreal time RT PCR. To identify the time of first introduction of new virusinfection on territory of Slovenia retrospective analysis of 42 cattle samplescollected from June to October 2012 were screening for SBV antibodiesusing ELISA test. First SBV antibody positive blood samples were identifiedfrom sample collected in August 29th 2012. High prevalence (82.8%)of SBV antibodies in cattle was detected from 87 randomly selected cattlesamples collected between January and February 2013. The detectionof virus and high seroprevalence on our territory means, that a new viraldisease has already spread to majority of our farms and that it may resultin increased losses due to abortions in pregnant animals and losses in neonatallambs, calves and kids. Slovenia is the first country of Balkan areawhere disease was confirmed with high prevalence and this new virus willbe probably detected also in other Balkan countries soon.REF 304Epidemiology of Bluetongue Infection in Northeast and SoutheastAnatoliaTaner KARAOGLU 1 , Irfan OZGUNLUK 2 , Yakup YILDIRIM 3 , CigdemOGUZOGLU 1 , Seval Bilge DAGALP 1 , Aykut OZKUL 1 , FerayALKAN 1 , Yilmaz AKCA 1 , Ibrahim BURGU 11 Ankara University, Faculty of Veterinary Medicine, Ankara, TURKEY;2 Harran University, Faculty of Veterinary Medicine, S.Urfa, TURKEY;3 Kafkas University, Faculty of Veterinary Medicine, Kars, TURKEYBluetongue is an important disease of ruminants and is caused by an arthropodborne Orbivirus, Reoviridae. The aim of this study was to establish awarning system to enable the early detection of BT virus serotype invasionsin northeast and southeast Anatolia through the deployment of ‘sentinelherds’ formed of seronegative individuals. According to serological monitoringdata, two sentinel herds were constituted: one from 114 seronegativecattle from the herd I in the northeast Anatolian region; and the other form101 seronegative cattle from the herd II in the southeast Anatolian region.All the cattle from the two sentinel herds were sampled 11 times. None ofthe 114 individuals from herd I showed seroconversion during any samplingperiod. However, seroconversion was observed in 32 cattle from theherd II at various sampling dates. The cross virus neutralization test performedagainst BT 4, BT 9, BT 16 on the blood sera showed seroconversionin 32 individuals, of which 28 had specific antibody against BT 4, BT 9and BT 16, 3 had them against BT 9 and BT 16, and 1 had them againstonly BT 9. The results showed that the antibody titers against BT 9 weremuch higher, so that these antibodies had low cross reaction with BT 4and BT 16. While antibody titers against BT 4 and BT 16 varied betweenundiluted samples to 1/80, antibody titers against BT 9 were detected between1/40 and 1/1280. The data had been obtained showed that BT 9 wasdetected as the dominant circulating virus type in the area.REF 305Seroprevalence of five arboviruses in sentinel cattle as part of nationalsurveillance in Korea, 2009 2012Yeon Hee KIM 1,2 , Jae Ku OEM 1 , Koung Ki LEE 1 , Seong Hee KIM 1 ,Myoung Heon LEE 1 , Se Chang PARK 21 Animal and Plant Quarantine Agency, Anyang, REPUBLIC OF KOREA;2 College of Veterinary Medicine and Research Institute for VeterinaryScience, Seoul, REPUBLIC OF KOREAFrom 2009 2012, national arbovirus surveillance was conducted on sentinelcattle, fewer than 1 year to investigate the possible circulationarboviruses in South Korea. We investigated the presence of antibodies toAkabane virus, Aino virus, Chuzan virus, Bovine ephemeral fever (BEF)virus, Ibaraki virus using serum neutralization test in 1,000 blood samplescollected every year. In 2009, seropositive rates for each virus were lessthan 13.3%, whereas the individual seropositive rates for each virus were40.2% for Akabane virus, 33.2% for Aino virus, 29.1% for Chuzan virus,7.5% for Ibaraki virus, 2.9% for BEF virus in 2010. The seropositiveVirologie, Vol 17, supplément 2, septembre 2013S205

5 th European Congress of Virologyrates identified in 2011 and 2012 were 8.3%, 11.9% for Akabane virus,6.9%, 6.8% for Aino virus, 4%, 3.7% for Chuzan virus, 6.3%, 4% for BEFvirus, 14.1%, 3.3% for Ibaraki virus, respectively. The results indicatedthat among the 5 viruses, Akabane, Aino, Chuzan virus infection wereprevalent in the natural transmission cycle in 2010. In addition, it wasrelated with a large scale outbreak of Akabane viral encephalomyelitis incattle reported in the southern part of Korea in 2010. Continued seroprevalencesurvey will help ensure that it monitors to be useful tool for naturalarboviral disease surveillance.REF 307First detection of Deformed wing virus (DWV) in honey bee Apismellifera intermissa in AlgeriaNoureddine ADJLANE, Haddad NIZARUniversity of Boumerdes, Department of Biology, ALGERIAThe honey bee is threatened by many pathogens; deformed wing virus(DWV) is the virus most prevalent and most dangerous in the world today.In Algeria, no study has been done to determine the presence of this virusin Algeria. The objective of this study was to evaluate the influence of thevirus on mortality of bees, and its relationship with the parasitic mite Varroadestrutor. The study was conducted in an apiary located in the centralregion of Algeria. Used PCR showed the presence of deformed wing virusin honey bee colonies Apis mellifera intermissa in Algeria. This is the firstdetection of this virus in Algeria, 42% of the apiary samples are contaminatedwith the virus. This study highlighted the role of Varroa and itsassociation with the virus DWV in mortalities recorded at the same apiary.REF 308A Serological survey of selected pathogens in wild boar (Sus scrofa)in northern TurkeyHarun ALBAYRAK 1 , Emre OZAN 2 , Abdullah CAVUNT 21 Ondokuz Mayis University, Faculty of Veterinary Medicine, Departmentof Virology, Samsun, TURKEY; 2 Veterinary Control Institute, Departmentof Virology, Samsun, TURKEYDuring the hunting season in March 2012, a total of 93 blood sampleswere collected from wild boars (Sus scrofa) shot in the area of northernTurkey (Samsun and Gumushane provinces). These blood samples wereexamined by enzyme immunoassay (ELISA) for the presence of antibodiesto classical swine fever virus (CSFV), Aujeszky’s disease virus(ADV), porcine reproductive and respiratory syndrome virus (PRRSV),porcine respiratory coronavirus (PRCV), swine influenza virus (SIV), porcineparvovirus (PPV), swine vesicular disease virus (SVDV), hepatitis Evirus (HEV), African swine fever virus (ASFV), porcine rotavirus (PRV),transmissible gastroenteritis virus (TGEV) and bovine viral diarrhoe virus(BVDV). Out of 93 serum samples examined, 65 (69.9%) were positive forPRV, 22 (23.7%) were positive for ADV, 5 (5.4%) were positive for BVDV,4 (4.3%) were positive for PPV and 2 (2.2%) were positive for PRRSV. Allsera were negative for ASFV, SVDV, HEV, SIV, PRCV, TGEV and CSFV.The results, recorded for the first time in Turkey, supported the hypothesisthat wild boar act as a potential reservoir of selected viruses, and thus havea role in the epidemiology of these diseases.REF 309A serologic investigation for Peste des petits ruminants infection insheep, cattle and camels (Camelus dromedaries) in Aydin province,West AnatoliaHarun ALBAYRAK 1 , Sibel GUR 21 Ondokuz Mayis University, Faculty of Veterinary Medicine, Departmentof Virology, Samsun, TURKEY; 2 Afyon Kocatepe University, Faculty ofVeterinary Medicine, Department of Virology, Afyonkarahisar, TURKEYPeste des petits ruminants (PPR) virus infection is a contagious diseaseof ruminants, mainly sheep and goats. Presence of PPR has been knownsince 1996 in Turkey. In this study, the infection was investigated in sheep,cattle and camels (Camelus dromediarius) in Aydin province where oneof the limited locations for camel rearing having been in Turkey. Bloodserum samples were obtained from a dairy cattle herd, PPR related clinicaldisorders observed a sheep flock and a slaughterhouse for camels. As aresult of C ELISA test, PPRV specific antibody presence was found to be88% (44/50) and 18% (22/122) in sheep and cattle, respectively. Positivitywas not detected in any of the camel samples (n=18) probably due to closedmanagement conditions.REF 310Molecular Surveillance for Tick Borne and Mosquito Borne Flavivirusesin Blacksea Region of TurkeyHarun ALBAYRAK 1 , Emre OZAN 2 , Abdullah CAVUNT 2 , HamzaKADI 2 , Mitat KURT 3 , Cenk BOLUKBASI 4 , Zafer PEKMEZCI 4 , SelmaKAYA 31 Ondokuz Mayis University, Faculty of Veterinary Medicine, Departmentof Virology, Samsun, TURKEY; 2 Veterinary Control Institute, Departmentof Virology, Samsun, TURKEY; 3 Veterinary Control Institute, Departmentof Parasitology, Samsun, TURKEY; 4 Ondokuz Mayis University, Facultyof Veterinary Medicine, Department of Parasitology, Samsun, TURKEYSpecies within the Flavivirus genus pose public health problems aroundthe world. Increasing cases of Dengue and Japanese encephalistis virus inAsia, frequent outbreaks of Yellow fever virus in Africa and South America,and the ongoing spread of West Nile virus throughout the Americas,show the geographical burden of flavivirus diseases. Climatic changestrigger geographical distribution of vector borne flavivirus. In this study,a total of 2340 adult ticks (675 hard tick pools) collected from varietyof mammalian species (cattle, sheep, goat and buffalo) and 3226 mosquitoes(142 mosquito pools) including Culex spp., Anopheles spp., Aedesspp., and Culicoides spp., trapped in Blacksea region of northern Turkeywere surveyed for the presence of RNA from mosquito borne flaviviruses(MBFV) and tick borne flaviviruses (TBFV) by reverse transcriptase polymerasechain reaction (RT PCR) assay. No flavivirus genomic RNA wasdetected in any sample. This is the first study about both TBFV and MBFVinfections in Turkey.REF 311Detection of antibodies against Equine Herpesvirus 1 and EquineHerpesvirus 4 By Indirect ELISAOguzhan AVCI 1 , Sibel YAVRU 1 , Selim TOKGOZ 2 , Mehmet KALE 31 University of Selcuk, Faculty of Veterinary Medicine, Department ofVirology, KONYA, TURKEY; 2 Veterinary Control Institute, ADANA, TUR-KEY; 3 University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine,Department of Virology, BURDUR, TURKEYEquine herpesviruses are significant causes of serious illness and mortalityin domestic horse population worldwide. Most mammalian speciesare susceptible to at least one type of herpesvirus. Herpesviruses establishlatency, meaning that the virus persists in the horse for the longterm, possibly for life, without causing clinical disease. Equine herpesvirusis a common DNA virus that occurs in horse populations worldwide.Equine herpesviruses are in the family Alphaherpesviridae and are envelopeddouble stranded DNA viruses. Equine herpesvirus 1 (EHV 1) andEquine herpesvirus 4 (EHV 4) are genetically and antigenically relatedviruses. The two most common strains are EHV 1, which causes abortion,respiratory disease and neurologic disease; and EHV 4, which usuallycauses respiratory disease only but can occasionally cause abortion. Theaim of the present study was to estimate the existence of EHV 1 andEHV 4 antibodies in domestic horses in five different province (Adiyaman,Diyarbakir, Gaziantep, Kilis, Sanliurfa) of South East Anatolia, Turkey.S206 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyIn this study, 150 blood sera samples of unvaccinated domestic horseswere tested for equine herpesvirus (EHV 1, EHV 4) specific antibodies bycommercially available indirect Enzyme Linked Immunosorbent Assay(ELISA, Svanovir, EHV 1/EHV 4 Ab kit, Svanova Biotech AB, Sweden).EHV 1 and EHV 4 specific antibodies were detected in 52 (34.66%) and101 (67.33%) of the 150 tested sera, respectively. The results indicatedthat EHV 1 and EHV 4 infections appear to be widespread in domestichorses in South East Anatolia.Key words: EHV 1, EHV 4, ELISA, horseREF 312Detection of antibodies against Blue Tongue Virus in Yaks (BOSGRUNNIENS) in Issyk KulOguzhan AVCI 1 , Orhan YAPICI 2 , Oya BULUT 1 , Mehmet KALE 3 ,Sibel YAVRU 1 , Atilla SIMSEK 11 University of Selcuk, Faculty of Veterinary Medicine, Department of Virology,KONYA, TURKEY; 2 University of Kyrgyzstan Turkey Manas, Facultyof Veterinary Medicine, Department of Virology, BISHKEK, KYRGYZS-TAN; 3 University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine,Department of Virology, BURDUR, TURKEYBluetongue virus (BTV) is the type species of the genus Orbivirus withinthe family Reoviridae. There are 26 BTV serotypes that are determinedby the specificity of interactions between the virus outer capsid with neutralizingantibodies generated during infection of the ruminant host. Theaim of this study was to describe the seroprevalence rate of bluetonguevirus (BTV) in unvaccinated yaks in Issyk Kul. A total of 168 serumsamples were collected from yaks that were randomly selected betweenJuly August 2011. Antibodies to BTV in sera were detected using a commerciallyavailable competitive enzyme linked immunosorbent assay (cELISA, VMRD, USA). The test was performed as per the manufacturer’sinstructions. The results showed that 4 (2.38%) of the samples werepositive for BTV antibodies. This study indicates that antibodies againstBTV are uncommon among the yaks in Issyk Kul. In spite of the significanteconomic and animal health impacts of BTV infection in sheep, todate there has been no assessment of the Culicoides spp. involved. Despitethe low results large scale research is necessary for the prevention ofBTV infection. In conclusion, the seroprevalence of BTV in yaks has beeninvestigated for the first time in Issyk Kul.Key words: BTV, yak, ELISAREF 313New subtypes of small ruminant lentiviruses (SRLVs) detected inSlovenian goat and sheep flocksDarja BARLIC MAGANJA 3 , Urska KUHAR 1 , Joze GROM 21 University of Ljubljana, Veterinary Faculty, Ljubljana, SLOVENIA;2 University of Ljubljana, Veterinary Faculty, Ljubljana, SLOVENIA;3 University of Primorska, Facutly for Health Sciences, Izola, SLOVENIASmall ruminant lentiviruses (SRLVs) of the Retroviridae family infectgoats and sheep worldwide. Phylogenetic analysis divides them into fiveprincipal genotypes (A E). The Slovenian SRLV strains characterized byphylogenetic analysis of two genomic regions (1.8 kb gag pol fragmentand 1.2 kb pol fragment) were clustered into A and B genotypes. Theyare highly heterogeneous, with ovine strains belonging to genotype A andcaprine strains to genotypes A and B. A cluster composed of four sheepvirus sequences was clearly divergent from all other subtypes in group Aand could not be assigned to any of them and two goat virus sequenceswere found to belong to genotype A and could not be assigned to existingsubtypes. The results of phylogenetic analysis performed in our studyrevealed two new subtypes within genotype A, subtype A14 and subtypeA15.REF 314Characterization of pandemic influenza A(H1N1) 2009 viruses isolatedfrom Pig and Cat in France since 2009Emilie BONIN 1,2 , Séverine HERVÉ 1,2 , Aure SAULNIER 1,2 , StéphaneQUÉGUINER 1,2 , Nicolas BARBIER 1,2 , Stéphane GORIN 1,2 , GaëlleSIMON 1,21 Anses, Laboratoire de Ploufragan Plouzané, Unité Virologie ImmunologiePorcines, Ploufragan, FRANCE; 2 Université Européenne de Bretagne,FRANCEIn April 2009, a novel influenza A(H1N1) virus (H1N1pdm) was detectedin people in Mexico and the United States. The virus spread rapidly andcaused a flu pandemic in humans within a few months. The new viruscontains a combination of genomic segments from swine origin that hadnever been identified previously. Soon after the virus started to spread globallyin humans, its introduction into pig holdings was noticed in manycountries, including European member states. Transmission of H1N1pdmto French pigs was evidenced to have occurred in Brittany as early asJanuary 2010. However, the virus does not seem to have been maintainedwithin the pig population in this region of high pig density where almost50% of the herds are infected by European enzootic swine influenza viruses(SIVs) for many years. By contrast, H1N1pdm was regularly isolated frompig herds located in central part of France since October 2010, demonstratingthat the virus spread in the French pig population, especially inareas with most naive animals. Not only restricted to humans and pigs,H1N1pdm virus was also detected in turkeys, cheetahs, minks, seals, ferrets,dogs and cats. In November 2009, it was isolated from a domesticcat in France, after several members of the family contracted the infection.Whole genomes of H1N1pdm viruses isolated from pigs and cat inFrance were sequenced. Phylogenetic and polymorphism analyses showedthat all genomic segments were very closely related to respective counterpartsfrom other contemporary pandemic strains isolated in Europe andto reference strain A/California/04/2009. To date, no reassortant virusesbetween H1N1pdm and enzootic SIVs were detected in France unlike inother European countries, but may appear. The spread of H1N1pdm intopig population is an additional risk for the emergence of new influenzaviruses with zoonotic potential.REF 315Serological and virological investigation of Canine Adenovirus Infectionin dogsOya BULUT 1 , Orhan YAPICI 2 , Oguzhan AVCI 1 , Atilla SIMSEK 1 ,Kamil ATLI 1 , Irmak DIK 1 , Sibel YAVRU 1 , Sibel HASIRCIOGLU 3 ,Mehmet KALE 3 , Nuri MAMAK 41 University of Selcuk, Faculty of Veterinary Medicine, Department of Virology,Konya, TURKEY; 2 University of Kyrgyzstan Turkey Manas, Facultyof Veterinary Medicine, Department of Virology, Bishkek, KYRGYZSTAN;3 University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine, Departmentof Virology, Burdur, TURKEY; 4 University of Mehmet Akif Ersoy,Faculty of Veterinary Medicine, Department of Internal Medicine, Burdur,TURKEYAdenoviruses are linear, double stranded DNA viruses which infect a widevariety of mammals and birds. Two have been identified in the dog: CanineAdenovirus type 1 (CAV 1) which infects most of the major organs causing,amongst other diseases, canine infectious hepatitis, and CAV 2 whichcauses canine infectious laryngotracheitis and enteric diseases. The aimof this study is to define the serological and virological status of CAVinfection in dogs. In this study, blood serum samples were taken from111 dogs that show clinical signs from the clinics of Veterinary MedicineFaculty, Selcuk University. Also 77 dogs were sampled from Ispartaand Burdur dog shelters by random sampling, regardless of the clinicalfindings. Clinical findings included a systemic disease, characterized byfever, vomiting, mucopurulent oculonasal discharge and conjunctivitis,Virologie, Vol 17, supplément 2, septembre 2013S207

5 th European Congress of Virologysevere moist cough, signs of pulmonary disease and dehydration. In serologicalstudies, serum samples were investigated for the presence of CAVantibodies by indirect ELISA. Antibodies to CAV were detected in 103(54.7%) of the examined sera samples. Leukocyte samples were processedand inoculated onto confluent monolayers of MDCK cells using standardvirological techniques. Cells were incubated at 37 ◦ C after inoculation andobserved daily for the appearance of cytopathogenic effect. After thirdpassage, cells were examined by direct Immunofluorescence test (IFT) forCAV antigens. We have not seen any morphological changes in MDCKcells and CAV antigens were not detected in any of leukocytes.Key words: CAV, ELISA, IFTREF 316Experimental infection in mice with avian coronavirus (Gammacoronavirus)isolated from chickensMatheus CAVALHEIRO MARTINI 1 , Márcia Mercês AparecidaBIANCHI DOS SANTOS 1 , Leonardo CARDIA CASERTA 1 , RicardoDURÃES CARCAVLHO 1 , Marina AIELLO PADILLA 1 , Helena LAGEFERREIRA 2 , Clarice WEISS ARNS 11 Laboratory of Animal Virology, Institute of Biology, University of CampinasUNICAMP, Campinas, BRAZIL; 2 Laboratory of Avian Disease,University of São Paulo College of Animal Science and Food Engineeringof USP, Pirassununga, BRAZILCoronaviruses, which are single stranded, positive sense RNA viruses, isthe causative agent of a wide variety of existing and emerging diseasesin humans and other animals. Infectious bronchitis virus (IBV) belongsto the genus Gammacoronavirus of the Coronaviridae family and is associatedto a highly contagious upper respiratory tract disease in chickens.IBV has been mainly detected in epithelial cells causing lesions in thenasal turbinates, trachea, kidney, gonads, oviduct, lungs and airsacs. Thisstudy was conducted to investigate if an IBV sample, obtained from anavian host (Gallus gallus), is able to replicate in a murine model. TenBalb/C mice 4 wks old were inoculated with 50 L fluid containgning104.0TCID50/mL of IBV/BRAZIL/2007/Unicamp801. Also, a controlgroup with four Balb/C mice was sham inoculated with 50 L of tissueculture fluid. Five infected and two control animals were euthanatized at3th and 10th days post inoculation; samples from sinus, tracheas, lung andkidney were collected at necropsy. Viral RNA was detected by real timePCR, based on the UTR gene, with copy numbers varying from 3,97 × 106to 1,21 × 108 (Ct values:35.98 to 38.58) at 3th d.p.i and 4,13 × 103 to1,74 × 108 (Ct values:35,94 to 38.51) at 10th d.p.i; no RNA was detectedin the control group. These results showed that an IBV sample was able toreplicate in a mammal host. To date there is no report of avian coronavirusreplication in mammals. A mammal species could constitute a host rangein which the virus can evolve, as well as a space for recombination betweenthe avian and mammal coronavirus. As a consequence, new variantsmight be produced.REF 317Susceptibility of Chicken embryonic Stem Cell Derived Keratinocytesto Marek’s Disease Virus infectionMathilde COUTEAUDIER 1 , Katia COURVOISIER GUYADER 1 ,Bertrand PAIN 2 , Caroline DENESVRE 1 , Jean François VAUTHEROT 11 INRA UMR 1282 ISP, Biova, Centre INRA de Tours, Nouzilly, FRANCE;2 INRA UMR 1288 IGFL, (ENSL/CNRS/UCBL1), Centre INRA de ClermontFerrand Theix, Villeurbanne, FRANCEMarek’s Disease Virus (MDV) is a very potent oncogenic alphaherpesviruswhich replicates to high titres in feather follicle epithelium (FFE)cells, from which it is efficiently shed as free virus. Investigations on thereplication of MDV in FFE are of major importance for a better knowledgeon viral dissemination. No in vitro cell system can afford an environmentthat may reproduce the one found in the cells of the differentiated FFE. Toenlarge the array of our investigations we explored the possibilities offeredby the differentiation of pluripotent stem cells from chicken. We thereforeexamined whether chicken embryonic stem cells (cES) could be inducedto differentiate toward the keratinocyte lineage, and whether those keratinocytesor their progenitors at intermediate stages of differentiation couldsupport MDV replication. From cES BP25, we derived several homogenouscell populations which showed typical morphological characteristicsof chicken keratinocytes. In addition, we showed a down regulation of pluripotencymarkers over the time, meanwhile the expression of late markersgenes of epidermal differentiation was upregulated. MDV replication wasexamined in cell populations showing either a typical keratinocyte morphologyor an “epithelial cell” phenotype, by using cell adapted or virulentvirus strains. The permissiveness of these differentiated cells was comparedto the one of the parental pluripotent cES. The ongoing experimentsare focussed on the characteristics of virus replication and morphogenesisin those cell populations.REF 318Antibody prevalence against Caprine Arthritis Encephalitis in SaanenGoats in Adana ProvinceIrmak DIK 2 , Orhan YAPICI 1 , Oguzhan AVCI 2 , Kamil ATLI 2 ,Sibel YAVRU 21 University of Kyrgyzstan Turkey Manas, Faculty of Veterinary Medicine,Department of Virology, Bishkek, KYRGYZSTAN; 2 University of Selcuk,Faculty of Veterinary Medicine, Department of Virology, Konya, TURKEYCaprine arthritis encephalitis is a viral disease of small ruminant that iscaused by the caprine arthritis encephalitis virus (CAEV). CAEV infectionoccurs worldwide and like other lentiviruses, they cause lifelong persistentinfection, which may be essentially symptomless or results in a variety ofinflammatory, degenerative, or immunosuppressive diseases in a variableproportion of infected hosts. This virus causes chronic disease of the joints,encephalitis, slowly progressive pneumonia, and mastitis in goat. The aimof this study is to define seroprevalence of CAEV infection in Saanengoats in Adana in the Mediterranean region of Turkey. In the present study,blood serum samples were collected from 150 Saanen goats unvaccinatedagainst CAEV in Adana. Sera samples were analyzed for presence ofantibodies against CAEV by commercially available enzyme linked immunosorbentassays (ELISA; ID Screen, France). The test was performed asper the manufacturer’s instructions. Antibodies to CAEV were detectedin 4 (2.66%) of the examined sera samples. Results of this study carryout that CAEV has a low prevalence in Saanen goats in Adana. As a vaccinationprogramme for CAEV infections is not implemented in Turkey.Further studies should focus on explaining the risk factors associated withCAEV prevalence in Saanen goat populations in an attempt to control newinfections.Key words: Caprine arthritis encephalitis virus, ELISA, Saanen goat,SerumS208 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF 319Role of the Seg 10 and NS3 proteins in modulating Bluetongue virushosts interactionsNajate FTAICH 1 , Claire CIANCIA 1 , Gerald BARRY 2 ,EvaVERONESI 3 , Maxime RATINIER 2 , Marc BAILLY BECHET 4 , SimonCARPENTER 3 , Damien VITOUR 5 , Massimo PALMARINI 2 ,Christophe TERZIAN 1 , Frederick ARNAUD 11 UMR754, INRA, Université Claude Bernard, Ecole Pratique des HautesEtudes, Université de Lyon, SFR BioSciences Gerland Lyon Sud, Lyon,FRANCE; 2 MRC University of Glasgow Centre for Virus Research,Institute of Infection, Immunity and Inflammation, College of Medicaland V, Glasgow, UNITED KINGDOM; 3 Institute for Animal Health,Vector borne Viral Diseases Programme, Pirbright, Surrey, UNITEDKINGDOM; 4 Laboratoire de Biometrie et Biologie Evolutive, CentreNational de la Recherche Scientifique, UMR 5558, Université Lyon,Villeurbanne, FRANCE; 5 ANSES, Maisons Alfort Laboratory for AnimalHealth, ANSES, INRA, ENVA, UMR 1161 Virology, Maisons Alfort,FRANCEBluetongue virus (BTV) is an arbovirus (for arthropod borne virus) andthe etiological agent of bluetongue, a hemorrhagic disease of ruminantsthat can cause high levels of morbidity and mortality. BTV is transmittedbetween mammalian hosts by species of Culicoides biting midges. BTV isa complex non enveloped virus that belongs to the genus Orbivirus (Reoviridaefamily). It possesses a 10 segmented double stranded RNA (dsRNA)genome that encodes 7 structural proteins (VP1 7) and 5 non structuralproteins (NS1, NS2, NS3/NS3A and NS4). Several evidences indicatethat the NS3 proteins, encoded by the segment 10 (Seg 10), play a majorrole in BTV egress, suggesting that it may also influence viral replicationrate and therefore the pathogenicity in mammals and/or the vector competencein insects. To assess whether the Seg 10 nucleic acid sequencesand/or NS3 proteins of various BTV strains or serotypes affect BTV lifecycle, we generated reassortants viruses in a BTV 1 backbone with fivedifferent Seg 10 of various BTV strains or serotypes. Preliminary resultssuggest that indeed, depending on the Seg 10 considered, differences existin terms of viral replication rate and cytopathic effects in sheep choroidplexus cells. Further in vitro and in vivo experiments are ongoing to betterunderstand how different Seg 10/NS3 proteins can modulate BTV hostsinteractions.REF 320Evidence of vertical transmission of circovirus type 2 (PCV2) fromsows infected before or during gestationBéatrice GRASLAND, Roland CARIOLET, Lionel BIGAULT, CéciliaBERNARD, Anne Cécile HERNANDEZ NIGNOL, Lise GOURVES,André KÉRANFLEC’H, André JESTIN, Nicolas ROSEANSES Ploufragan/Plouzané, Ploufragan, FRANCEPorcine circovirus type 2 (PCV2) is associated with several diseases includingreproductive failure. Our aim was to determine if transplacentaltransmission of PCV2 followed by virus transmission to piglet depends onthe time of infection before or during the gestation. Four sows served asnegative controls. Ten sows, free of PCVs, were infected nasally at days65, 40, 35, 0, +35, +62 or +91 relating to the beginning of gestation. Thesows were killed few days before farrowing for hysterectomia. All fetuseswere identified and euthanized. Viral genomic loads in serum and organsand PCV2 specific antibodies levels were assessed. The trial was performedaccording EU regulations on animal experimentation. All infectedsows became viremic and seroconverted. All the piglets born to infectedsows were seronegative. All the piglets from the sows inoculated at +35days, before the time of foetus immuno competency, were negative for thepresence of PCV2. Few piglets from sows infected at days 65, 40, and +91had low genomic loads in organs. Moderate to high viral genomic loadswere detected in the organs of seronegative pigs from sows infected at days35, 0 and +62. In conclusion, placental transmission of the PCV2 from anon immune sow to fetuses occurs when the infection of the dam occursbefore gestation or after the time of the foetus immuno competency. Thelack of antibodies raised the question of the presence of immuno tolerantpersistently infected animals.Acknowledgements: this work was financially supported by an award ofBoerhinger Ingelheim animal health.REF 321Experimental infection of calves with porcine circovirus (PCV) type2Mohammad Yahya HALAMI, Hermann MÜLLER, Thomas W.VAHLENKAMPInstitute of Virology, Faculty of Veterinary Medicine, University of Leipzig,Leipzig, GERMANYCircoviruses are known to infect pigs and birds and cause a broad range ofsevere clinical symptoms. Recently circoviruses were identified in humanand chimpanzee faeces and new circovirus genomes were described fromchickens, goats, cows, and bats. Porcine circovirus type 2 (PCV 2), whichcauses PCV 2 associated disease (PCVAD) with severe economic losses,was detected in rodents, cattle, and calves affected with bovine neonatalpancytopenia (BNP). The aims of this study are to investigate the susceptibilityand immune response of calves to experimental PCV 2 inoculation.Animal groups were inoculated with (i) tissue culture grown PCV 2, (ii)bone marrow from calves with BNP, and (iii) immunized with a commercialinactivated PCV 2 vaccine. The results showed that the animalsinoculated with PCV 2 or with bone marrow from calves with BNP displayedswelling in lymph nodes, reddening of oral and ocular mucosa,abnormal behavior and diarrhoea (7 18 days p.i.). PCV 2 specific antibodieswere detected in the immunized and PCV 2 infected animals fromday 7 and 11 p.i. onwards, respectively, but not in the bone marrow inoculatedanimals. PCV 2 was detected by PCR only in the PCV 2 infectedanimals especially in the lymph tissues with high copy numbers betweendays 4 (5 log10 copies/g) and 46 (5.67 log10 copies/g) p.i. In conclusion,experimental PCV 2 inoculation of calves results in seroconversion andthe detection of PCV 2 in lymph tissues for an extended period of timewhich seems to indicate that host specificity of PCV 2 is not as restrictedas previously thought.REF 322Identification of single nucleotide polymorphisms on feline interferon gene and association with the outcome of feline coronavirus infectionLi En HSIEH, Ling Ling CHUEHGraduate Institute of Veterinary Medicine, School of Veterinary Medicine,National Taiwan University, Taipei, TAIWANFeline infectious peritonitis (FIP) is an immune mediated, highly lethaldisease caused by feline coronavirus (FCoV) infection. Currently no protectivevaccine and effective treatment for the disease is available. Cellularimmunity is thought to be important to against FCoV infection and preventthe occurrence of FIP. Diseased cats showed a significant decrease ofthe level of interferon . As single nucleotide polymorphisms (SNPs) ofhuman interferon gene (hIFNG) at the position +874 has been identifiedto be related with the outcomes of variable viral infection, e.g.Dengue virus and severe acute respiratory syndrome coronavirus. Thisstudy aim to identify hIFNG +874 homolog on the feline genome andinvestigate its association with the outcome of FCoV infection. Throughthe genetic analysis, hIFNG +874 homolog was mapped to the felineIFNG (fIFNG) on nucleotide 886 from the start codon. The region includingfIFNG +886 was amplified and sequenced from the blood samplesVirologie, Vol 17, supplément 2, septembre 2013S209

5 th European Congress of Virologyof 35 and 27 FCoV asymptomatically infected and FIP cats, respectively.However, no polymorphism was identified in all cats at fIFNG+886. Investigation was further expended to identify other SNPs onfIFNG and 15 SNPs were identified on the intron regions. The associationbetween identified SNPs and the outcome of FCoV infection will bediscussed.that this superinfection exclusion was mediated by differential inhibitionof cp and ncp viruses by type I interferon (IFN), as recombinant IFNalpha inhibited both biotypes to a similar extent. However, the underlyingmechanism of this ncp specific superinfection exclusion remains to bedetermined.REF 323Arboviruses pathogenic for domestic and wild animalsZdenek HUBÁLEK 1 , Ivo RUDOLF 1 , Norbert NOWOTNY 21 Institute of Vertebrate Biology ASCR, v.v.i., Brno, CZECH REPUBLIC;2 Institute of Virology, University of Veterinary Medicine, Vienna, AUSTRIAThis review contains background data on taxonomy, history, arthropodvectors, vertebrate hosts, animal disease, and geographic distribution ofall arboviruses known to date causing disease in endotherm vertebrates(except those viruses affecting exclusively man). Nearly 50 arbovirusespathogenic for animals have been documented worldwide, belongingto seven families: Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae,Rhabdoviridae, Orthomyxoviridae and Asfarviridae. They are transmittedto animals by five groups of haematophagous arthropods (ticks Ixodidaeand Argasidae; mosquitoes Culicidae; biting midges Ceratopogonidae;sandflies Phlebotominae; cimicid bugs Cimicidae. Important arboviraldiseases in endotherm animals are tick borne louping ill, Nairobi sheepdisease (NSD), African swine fever (ASF); mosquito borne Eastern, Westernand Venezuelan equine encephalomyelitides (EEE, WEE, VEE), WestNile encephalitis (WNE), Israel turkey meningoencephalitis, Cache Valleydisease, Rift Valley fever (RVF); sandfly borne (vesicular stomatitis(VS); midge borne Akabane disease, Schmallenberg disease, African horsesickness (AHS), bluetongue (BT), epizootic haemorrhagic disease of deer.Diseases due to some of these viruses occasionally cause very significanteconomic losses in domestic animals – e.g., EEE, WEE and VEE, WNE,NSD, RVF, Akabane fever, Schmallenberg disease (emerging recently inEurope), AHS, BT, VS, or ASF; all of these (except for Akabane andSchmallenberg diseases) are notifiable to the World Organisation for AnimalHealth (OIE).REF 324Biotype Specific Superinfection Exclusion in Bovine Viral DiarrheaVirus InfectionsLinda HÜSSER, Kay Sara SAUTER, Matthias SCHWEIZERInstitute of Veterinary Virology, University of Berne, Berne,SWITZERLANDThe pestivirus bovine viral diarrhea virus (BVDV) exists as two biotypes,cytopathic (cp) and noncytopathic (ncp).Ncp BVDV may infect fetusesin utero leading to the birth of persistently infected (PI) animals that areat risk of developing fatal mucosal disease (MD).From animals sufferingfrom MD both, an ncp and the antigenically homologous cp biotype canbe isolated (called a “virus pair”).We aimed to clarify the interactions ofsuch virus pairs in PI animals. We superinfected PBMCs isolated from PIor control animals with either ncp or cp BVDV of a virus pair that is nothomologous to the persistent BVDV strain and assessed the efficiency ofviral replication.The cp biotype grew to similar titers in control and in PIPBMCs whereas the growth of superinfecting ncp BVDV was severelysuppressed only in PI PCMCs.This selective viral growth was confirmedusing strain specific RT PCR that permits the differentiation of thesuperinfecting biotypes from the persisting ncp virus.These ex vivo experimentscould be confirmed in an in vitro model.Furthermore FACS analysisusing a strain specific antibody revealed that only the cp biotype wasable to superinfect persistently BVDV infected turbinate cells, whereasno cells harboring superinfecting ncp virus were detectable. It is unlikelyREF 325Derivation of a complete louping ill virus genome sequence from thespinal cord of an infected lambNicholas JOHNSON 1 , Denise MARSTON 1 , Karen MANSFIELD 1 ,Rebecca MEARNS 2 , Richard ELLIS 1 , Anthony FOOKS 1,31 Animal Health and Veterinary Laboratories Agency, Addlestone, UNI-TED KINGDOM; 2 Animal Health and Veterinary Laboratories Agency,Penrith, UNITED KINGDOM; 3 National Consortium for ZoonosisResearch, Leahurst, UNITED KINGDOMLouping ill virus (LIV) causes a febrile illness primarily in sheep andgrouse that can lead to fatal encephalitis. There is also evidence that thisvirus is zoonotic. LIV is found predominantly within the upland areas of theUK and is transmitted by Ixodes ricinus ticks. The only published sequenceof the LIV genome was submitted to GenBank in 1996 from a virus thathad been serially passaged following its isolation from a tick caught inScotland in 1963 (isolate 369/T2/GenBank NC 001809). In order to assessthe ability to derive full length genomes from a clinical sample we haveextracted total RNA from a spinal cord removed from a young sheep thatdied suddenly near Penrith, England. LIV was confirmed in the spinal cordby immunohistochemistry and detection of flavivirus by RT PCR. NextGeneration Sequencing recovered a partial genome sequence with only77 reads (0.1% of total) being of LIV origin reflecting the relatively smallproportion of virus genome in relation to host nucleic acid. The remainingsequence was obtained using directed PCR. The genome is 10865 basepairs in length and shares 95.6% sequence identity with the published LIVgenome (NC 001809). This study represents the first attempt to recovercomplete LIV genome from a clinical sample.REF 326Phylogenetic analysis of two novel Mengo virus strains isolated fromMarmosets in The United Arab EmiratesJolanta KOLODZIEJEK 1 , Tom BAILEY 2 , Ulrich WERNERY 3 , NicolaHEIDLER 1 , Helga LUSSY 1 , Norbert NOWOTNY 1,41 Institute of Virology, University of Veterinary Medicine, Vienna, AUS-TRIA; 2 Dubai Falcon Hospital, Dubai, UNITED ARAB EMIRATES;3 Central Veterinary Research Laboratory, Dubai, UNITED ARAB EMI-RATES; 4 College of Medicine and Health Sciences, Sultan QaboosUniversity, Muscat, OMANCytopathic viruses were isolated from brain tissues of several marmosets(Callithrix jacchus), which succumbed due to neurological diseasebetween 2005 and 2006, and were submitted to our institute for furtherinvestigations. Two virus isolates were selected for detailed genetic characterizationand phylogenetic analysis. For the initial RT PCR investigations,encephalomyocarditis virus (EMCV) specific primers were used. Basedon the nucleotide sequences of the obtained amplification products and ofMengo virus reference strain sequence L22089, further 56 primer pairswere designed in order to amplify the complete viral genomes. All RTPCR products were sequenced. The obtained sequences were aligned andverified by BLAST search. The phylogenetic analysis was performed byemploying the MEGA4 program. The two isolates exhibited 99% identityto each other at both nucleotide and amino acid levels. Complete aminoacid sequences of their polyproteins revealed 95% identity to Mengo virusstrain “M” and 89 94% identities to the porcine, murine and simian EMCviruses. The identities to rat Theilo and human Cardio and Saffold virusesS210 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologywere only 52 54%. The phylogenetic analysis of the entire polyproteinperformed on altogether 37 amino acid sequences confirmed the classificationof cardioviruses into the two species Encephalomyocarditis virusand Theilovirus, and revealed that the two virus isolates from marmosetscan be considered novel EMCV strains with the closest genetic relationshipto the “old” Mengo virus strain “M”, which was already isolated 50years before.REF 327Prevalence of hepatitis E virus in population of free living and gameenclosure wild boarsMonika KUBANKOVA 1,4 , Jiri LAMKA 2 , Vladimir ZAKOVCIK 3 , PetraVASICKOVA 11 Veterinary Research Institute, Brno, CZECH REPUBLIC; 2 Charles Universityin Prague, Faculty of Pharmacy in Hradec Kralove, HradecKralove, CZECH REPUBLIC; 3 Central Military Veterinary Institute atHlucin, Hlucin, CZECH REPUBLIC; 4 University of Veterinary and PharmaceuticalSciences Brno, Faculty of Veterinary Medicine, Brno, CZECHREPUBLICHepatitis E virus (HEV) is a small, non enveloped RNA virus, which isclassified in the genus Hepevirus, family Hepeviridae. Isolates of HEVare divided into four major genotypes and several subtypes accordingto sequence comparisons and phylogenetic analysis. Mammalian HEVstrains have been obtained from domestic pigs, wild boars, rabbits, rats,deer and mongoose and possibly cattle and sheep. The existence of HEVantibodies have been identified in other animal species as well. HepatitisE is considered to be as a zoonotic disease and domestic pigs, wild boarsand perhaps other animal species are natural reservoirs of the virus. In theCzech Republic, HEV RNA has been detected by triplex real time reversetranscription polymerase chain reaction (qRT PCR) in free living wildboars (17.3%) and wild boars originated from game enclosures (28.6%).In one game enclosure was detected prevalence up to 62.5% in wild boars.The HEV prevalence in game enclosures is significantly higher than infree living wild boars, since the breeding conditions are different from thenatural environment. Subsequently, sequence and phylogenetic analysesof obtained HEV isolates suggested a potential cross species transmissionand circulation of HEV in the Czech Republic. The discoveries of pathogenesis,clinical manifestation epizootology and epidemiology of HEV inwild animals are still missing.This work was supported by grants: NT13884 4/2012 and AdmireVetCZ1.05/2.1.00/01.0006 ED0006/01/01.software, phylogenic trees were constructed by neighbor joining accordingto Kimura 2 parameter model in MEGA 5.1 software.Analysis of phylogenic trees (P4b and fpv140) showed a wide diversity ofstrains. Most of isolates cluster in Canarypox virus clade (84%), mainlyin subclades B1 (Canarypox) and B2 (Starlingpox). The others clusterin Fowlpox virus clade, mainly in subclade 2 (Turkeypox). This studyshows that a wide diversity of avipoxviruses can affect a unique hostspecies, even in a restricted geographic area (most of the observed diversityrefers to birds from Morocco) and suggests that vaccination alone canhardly protect Houbara bustard against avipoxvirus infections. Finally itquestions the taxonomy of avipoxviruses, which classically attributes thehost name to the virus species and provides clues for a better understandingof avipoxviruses epidemiology.REF 329Monoclonal antibodies against accessory protein 7b of feline coronavirusTanja LEMMERMEYER 1 , Benjamin LAMP 1,2 , Heinz Jürgen THIEL 11 Institute of Virology, Department for Veterinary Medicine, Justus LiebigUniversität, Giessen, GERMANY; 2 present adress: Institute of Virology,Department for Pathobiology, University of Veterinary Medicine, Vienna,AUSTRIAFeline infectious peritonitis virus (FIPV), the virulent biotype of felinecoronavirus (FCoV), causes feline infectious peritonitis (FIP), a lethaldisease. FIPV is thought to arise by mutation(s) during persistent infectionwith feline enteric coronavirus (FECV). Nucleotide sequence analysesindicated that mutations within the genes S, 3abc and 7ab are involved inthe transition from FECV to FIPV. This study aims at the generation ofmonoclonal antibodies (mAbs) against the 7b protein of FCoV in order tocharacterize this viral antigen and to develop novel diagnostic reagents.First the 7b protein was expressed as GST fusion protein and purified byaffinity chromatography. After immunization of mice with the 7b proteinmAb producing hybridomas were generated. Two reactive mAbs termed5B6 and 14D8 were identified by ELISA. Both mAbs detected bacteriallyexpressed 7b by immunoblot. The specific binding region was mappedwithin the amino acids 50 65 of 7b (total length: 206 aa). The isotypesof 5B6 and 14D8 were IgG2a and IgG1, respectively. Both mAbs detectedthe authentic viral protein in FIPV (strain 79 1146) infected cells byimmunoblot and immunofluorescent assay (IFA). The mAbs also showedcross reactivity with bacterially expressed 7b of FCoV strain Black. Itwill be interesting to study the biosynthesis of this protein in FCoV infectedcells which is expected to be glycosylated and secreted. Moreover itsintracellular localization will be determined.REF 328Avipoxvirus diversity in Houbara bustard in captivityGuillaume LE LOC’H 1,2,3 , Christelle CAMUS BOUCLAINVILLE 1,2 ,Mariette DUCATEZ 1,2 , Stéphane BERTAGNOLI 1,21 INRA, UMR 1225, Toulouse, FRANCE; 2 Université de Toulouse, INPENVT, Toulouse, FRANCE; 3 Emirates Center for Wildlife Propagation,Missour, MOROCCOAvipoxvirus sp. is a serious threat for the success of reinforcementprograms of some endangered birds species such as Houbara bustard(Chlamydotis sp.). Avipoxvirus infections can lead to high morbidity andcompromise the survival of wild release birds. In order to better understandits epidemiology and adapt the prophylaxis strategy, the diversityof Avipoxvirus sp. strains was studied in Houbara bustard. From 2008to 2012, 70 pox lesions from houbara bustards in 3 breeding projects(Morocco, UAE and Uzbekistan) were sampled and two regions of theviral genome (P4b: 578pb and fpv140: 2 3 kb) were amplified and sequenced.After alignment of obtained and published sequences with ClustalXREF 330Infection of cats with a new recombinant FCoVI/CCoVI strain harboringspecific forms of non structural protein gp3Anne Laure PHAM HUNG D’ALEXANDRY D’ORENGIANI 1 , LidiaDUARTE 1 , Cristina HORHOGEA 2 , Carine PINHAS 1 , AstridVABRET 3 , Nicole PAVIO 1 , Sophie LE PODER 11 UMR 1161 Virologie, INRA, ENVA, ANSES, Maisons Alfort, FRANCE;2 Université des Sciences Agricoles et Médecine Vétérinaire Ion Ionescude la Brad Iasi, Iasi, ROMANIA; 3 Laboratoire de Virologie, EA 2128,CHU de Caen, Caen, FRANCEOne of the main characteristics of Coronaviruses is their rapid genetic evolution,allowing cross species transmission, sometimes leading to humanepidemic such as SARS in 2002 and the new hCoV EMC strain in theMiddle East. Despite these new threats originating from animal reservoirs,mechanisms of these inter specific host transmissions remain unclear. Bothcanine (CCoV) and feline (FCoV) Coronaviruses are subdivided into twoVirologie, Vol 17, supplément 2, septembre 2013S211

5 th European Congress of Virologyserotypes and the close genetic relationship between them leads to potentialcross species infections. Serotype I canine strain (CCoVI) differs by thepresence of a unique open reading frame ORF3, coding for an accessoryglycoprotein gp3, with unknown function. In a phylogenetic study performedto characterize Coronavirus infected cats, structural genes S, M and Nwere sequenced and allowed to detect a new recombinant FCoVI/CCoVIstrain on 5 cats out of 26 samples. In these strains, the S gene clusters withFCoVI type, whereas downstream genes ORF3, M and N are grouped withCCoVI genotype. Moreover, ORF3 found in cats samples, displayed oneor two identical deletions, never described in canine samples. In a possiblecross species virus transmission context, our aim is to understand the roleof gp3 and determine if the deletions observed play a role during the adaptationof the recombinant canine strain to the feline species. More globally,this study will give an overall view of molecular mechanisms implied inthe inter specific host transmission of Coronaviruses.Acute gastroenteritis is one of the most common diseases in humans aswell as farm animals. Small, non enveloped RNA viruses are recognizedas important causes of this disease. The objective of the study was to determinethe prevalence and genetic diversity of members of genera Sapovirus,Kobuvirus, and Mamastrovirus in asymptomatic pigs in the Czech Republic.A total of 196 fecal samples were tested by reverse transcriptionPCR (RT PCR) assay using gene specific primers. Phylogenetic analysiswas carried out using the neighbor joining method with a bootstrap testof 1000 replicates using MEGA software package v4.0. The most prevalentvirus was kobuvirus (87.3%), followed by astrovirus (34.2%) andsapovirus (10.2%). Statistically significantly highest number of positivesamples was detected in the age group of post weaning pigs. Consideringthe possible zoonotic potential of monitored viruses, their geneticdiversity and degree of similarity to human strains was also determined.In conclusion, the circulation of porcine sapoviruses, kobuviruses, andastroviruses in the Czech Republic was shown thus extending their Europeandistribution. The high prevalence rate of gastroenteritis producingviruses in clinically healthy pigs confirmed the role of pigs as a reservoirof a heterogeneous viral population representing a constant threat tohuman health. The study was supported by the Ministry of Agricultureof the Czech Republic (Grant No. QH81061) and the Ministry of Education,Youth and Sports of the Czech Republic (Admirevet; Grant No.CZ.1.05/2.1.00/01.0006, ED0006/01/01).REF 331Prevalence and genetic diversity of porcine bocavirus 1 in apparentlyhealthy pigs in the Czech RepublicJana PRODELALOVAVeterinary Research Institute, Brno, CZECH REPUBLICBocaviruses (family Parvoviridae) are pathogens causing various diseasesin both humans and animals. Porcine bocavirus 1 (PBoV1) was firstlyidentified in Sweden in 2009 and it is potentially associated with swinedisease. The virus was detected in pigs suffering from porcine circovirusassociated disease and respiratory disease but also from clinically healthypigs. The aim of the present study was to detect and measure the prevalenceof PBoV1 and analyse partial PBoV1 sequences. Faecal samples were collectedfrom different farms located in the South Moravia region, tested byPCR specific for VP1/VP2 gene of PBoV1 and sequenced. Among 179samples obtained from apparently healthy pigs of different age groups, theprevalence was 22.4%. Higher infection rate was found in post weaningpigs (75.5%, 34/45) compared with finisher pigs (8.2%, 5/61) and saws(3%, 1/33). No PBoV1 positive samples were detected in nursing piglets(0/40). PBoV1 positive samples were obtained from three out of eightfarms. By comparative sequence analysis of 496 bp fragments of VP1/VP2gene from domestic pigs and sequences available in GenBank we detectedonly minor differences between PBoV1 s. This result indicates thatPBoV1 s circulating within the apparently healthy domestic pig populationin South Moravia region were highly similar. This work was supportedby the Ministry of Agriculture of the Czech Republic (Grant No. NAZVQH81061) and the Ministry of Education, Youth, and Sports of the CzechRepublic (Grant No. AdmireVet CZ.1.05/2.1.00/01.006, ED006/01/01).REF 333Serological surveilance for selected viral agents in ROE DEER(Capreolus Capreolus) and RED DERR (Cervus Elaphus) in CroatiaBesi ROIC, Lorena JEMERSIC, Svjetlana TERZIC, Andreja JUNGIC,Dragan BRNIC, Tomislav KEROS, Jelena PRPICCroatian Veterinary Institute, Zagreb, CROATIADuring the hunting season of 2009, blood samples from 32 roe deer(Capreolus capreolus) and 55 red deer (Cervus elaphus) were collectedin four Croatian regions. Sera were tested by enzyme linked immunosorbentassay (ELISA) for the presence of antibodies against bovine herpesvirus 1 (BHV 1), parainflurnza 3 virus (PI 3), bovine respiratory syncytialvirus (BRSV), bovine viral diarrhea virus (BVDV) and bluetonguevirus (BTV). Specific antibodies were detected for bovine herpes virus1 (BHV 1) in 12.50% (4 positive/32 tested) roe deer and in 25.45% (14positive/55 tested) red deer. Additionally, positive results were obtainedagainst parainfluenza 3 virus (PI 3) in 68.75% roe deer (22 positive/32 tested)and 83.63% red deer (46 positive/55 tested). Most of the seropositivesera were recorded among red deer in the eastern part of Croatia which isknown as a district with high density populations of wildlife species. Noantibodies were detected against any of the other viral agents assayed inthis study. The presence of BHV 1 and PI 3 virus antibodies indicate thatboth deer species have been exposed and have responded serologically toviral agents which are related or identical to the domestic livestock pathogens.This study provides the first survey for the presence of antibodies toviral diseases in cervids from Croatia.Key words: roe deer (Capreolus capreolus), red deer (Cervus elaphus),viral disease, serology, CroatiaREF 332Prevalence and genetic diversity of porcine sapoviruses, kobuviruses,and astroviruses in asymptomatic pigs in the Czech RepublicLucie DUFKOVA 1 , Ivana SCIGALKOVA 2 , Romana MOUTELIKOVA 1 ,Hana MALENOVSKA 1 , Jana PRODELALOVA 11 Veterinary Research Institute, Brno, CZECH REPUBLIC; 2 Faculty ofScience/Masaryk University, Brno, CZECH REPUBLICREF 334Molecular characterization of VP2 Gene of infectious pancreaticnecrosis virus from Mexican isolatesEdith ROJAS ANAYA 1 , Elizabeth LOZA RUBIO 1 , Celene SALGADOMIRANDA 1,2 , Gary GARCIA ESPINOSA 21 INIFAP, CENID Microbiología Animal, México City, MÉXICO; 2 FMVZ,UNAM, México City, MÉXICOInfectious pancreatic necrosis virus (IPNV), is considered one of the mostimportant viral pathogen in salmonids and remains a serious problem inthe aquaculture industry. To better understand the genetic and biologicaldiversity of these viruses in Mexico it is necessary to further study thefeatures which include genetic characterization and virulence that mayimpact on the production of biologicals. The aim of this study was toperform the molecular characterization of the VP2 gene Mexican IPNVisolates from different geographical regions. Seven isolates were obtainedfrom rainbow trout hatchery, during 2006 and 2007 of the major farmsproducing trout: Michoacan, Puebla and State of Mexico. We designedS212 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologytwo pairs of primers and RT PCR was standardized to amplify 1359 bp ofthe VP2 gene. Then, the nucleotide sequence and amino acid deductionwere made in order to perform a phylogenetic analysis. Sequences wereassembled using multiple alignment ClustalW2. For the construction ofdendrograms, we used the Neighbor Joining method from 1000 bootstrapreplicates. The phylogenetic tree shows seven genogroups IPNV isolatesthat have been previously reported. IPNV Mexican isolates are includedin the genogroup I and showing a close relationship with the Buhl strainisolated from the United States of America. This is the first report of themolecular characterization of the whole VP2 gene in several isolates.extraction to PCR result). The molecular tools being developed alloweda rapid and quantitative detection of both gammaherpesviruses in horses.The study was performed on nasal swabs collected from 172 horses agedfrom 6months to 23 years which all exhibiting clinical signs of respiratorydisease. The data revealed a high rate of EHV 2/EHV 5 co infection, aswell as a significant influence of age on both viral detection and viralloads. Further studies are warranted in order to precise the mechanismsbeing involved during gammaherpesvirus infections in horses.Fortier, et al. Vet J 2010; 182: 346-8.Nordengrahm, et al. Vet Research 2002; 33: 251-9.REF 335Evaluation of Bovine Viral Diarrhoea Virus Infections in Repeat BreederDairy Cows in Burdur Region (2010 2012 years)Hasbi Sait SALTIK, Mehmet KALE, Sibel HASIRCIOGLU,Kamil ATLIMehmet Akif Ersoy University, Burdur, TURKEYBetween 2010 and 2012 years, was made a study about the presence ofBovine viral diarrhoea virus (BVDV) infection in blood samples and vaginaldischarges from dairy cattle which applied artificial insemination morethan 3 times and having repeat breeder syndrome. The presence of BVDVantigen (Ag) is examined in blood samples by ELISA and in vaginaldischarges by Fluorescent Antibody Test (FAT). In 2010, twelve of 16dairy cattle’s blood samples were found to be positive for the presence ofBVDV Ag and we did not come up with BVDV Ag in vaginal discharge ofsame animals. In 2011, thirteen of 144 dairy cattle’s blood samples werefound to be positive for the presence of BVDV Ag as well as only one vaginaldischarged were found positive of the same animals for BVDV Ag. In2012, seventy eight of 146 blood samples were found to be positive for thepresence of BVDV Ag plus 6 vaginal discharges were positive of the sameanimals. Consequently, BVDV ELISA (Ag) was much better than BVDVFAT (Ag) to detect infected animals as well as increased number of dairycattle with repeat breeder syndrome and seroprevalance of those animalswere detected between 2010 and 2012 years in territory of Burdur.Key words: BVDV, repeat breeder syndrome, ELISA, FATREF 336Characterisation of Equid Herpesvirus 2 and 5 quantitative PCRaccording to the norm AFNOR XP U47 600 2: determination of theviral loads in nasal swabs according to ageErika HUE 1,2,3 , Stephane PRONOST 1,2,3 , Albertine LEON 1,2,3 , EricRICHARD 1,3 , Loic LEGRAND 1,2,3 , Kerstin BORCHERS 4 , GuillaumeFORTIER 1,2,31 Frank Duncombe Laboratory, Caen, FRANCE; 2 Normandie Univ, SF4206 ICORE, EA 4655 U2RM, Caen, FRANCE; 3 Hippolia Foundation,Caen, FRANCE; 4 Freie Universität Berlin, Institut für Virologie, Berlin,GERMANYInfections by gammaherpesviruses are common among horses worldwide(Nordengrahm et al., 2002). EHV 2 and EHV 5 are involved, in respiratorytroubles with clinical manifestations such as nasal discharge, pharyngitisand swollen lymph nodes (Fortier et al., 2010). These viruses may also beresponsible for poor performance syndrome, causing significant economicimpact for horse industry. The aim of this study was 1/to develop andvalidate two distinct quantitative PCR to quantify EHV 2 and EHV 5 and2/to investigate viral loads of EHV 2 and EHV 5 in the airways according toage among equine population. Each quantitative PCR (EHV 2 and 5) wascharacterized according to the norm AFNOR XP U47 600 2. This approachdefined the specificity, linearity, quantifiable sensibility and accurancyof the qPCR and of the complete analytical PCR method (from DNAREF 337Comparison of manual and automated nucleic acid extractionmethods for detection of peste des petits ruminants virus RNAMurat SEVIK 1 , Oguzhan AVCI 2 , Baris INCE 31 Molecular Microbiology Laboratory, Veterinary Control Institute, Konya,TURKEY; 2 Department of Virology, Faculty of Veterinary Medicine,Konya, TURKEY; 3 Agriculture and rural development support Institution,Afyonkarahisar, TURKEYPeste des petits ruminants (PPR) is an economically important contagiousdisease of sheep and goats. PCR based techniques have been successfullyused for rapid diagnosis of PPR. The method used for isolation of RNAfrom tissue samples is an important concern when using reverse transcriptionPCR (RT PCR) methods for the detection of PPR virus (PPRV).In this study, a commercial kit for manual preparation (Qiagen) and anautomated processing technique (Roche Diagnostics) for RNA extractionwere compared in terms of performance. During May October 2012, 16small ruminants (14 sheep and 2 goats), each from different flocks, withPPR suspect submitted to laboratory were chosen to compare manual andautomated extraction methods for the detection of PPRV. One step RTPCR was used for the detection of PPRV RNA. Amplification was carriedout using primers based on based on fusion (F) protein gene of virus.Lyophilized freeze dried live PPR vaccine (Nigeria 75/1 vaccine strain)was used as positive control for the extraction and RT PCR. PPRV nucleicacid was detected in 6 of 16 tissue samples that were manually extracted,while viral RNA was detected in 4 of 16 extracts prepared by the robot.Two samples that were negative with automated extraction were weaklypositive in manual extraction. It can be concluded from the present studythat the manual extraction performed slightly better than the automatedextraction. Therefore, it is suitable to use manual extraction method whensmall numbers of samples needed to be examined.Key words: Peste des petits ruminants virus, RNA extractionREF 338Comparative evaluation of F gene based conventional RT PCR andN gene based real time RT PCR for the detection of peste des petitsruminants virus in clinical samplesMurat SEVIK 1 , Oguzhan AVCI 21 Molecular Microbiolgy, Veterinary Control Institute, Konya, TURKEY;2 Department of Virology, Faculty of Veterinary Medicine, Konya, TURKEYPeste des petits ruminants (PPR) is an acute and highly contagious viraldisease causing high morbidity and mortality in small ruminants. Severalreverse transcription PCR (RT PCR) methods have been developed for therapid and specific detection of peste des petits ruminants virus (PPRV) infield samples. However, these are being replaced by more sensitive androbust real time RT PCR assays. Two different genes have been mostly usedas targets for amplification by RT PCR, namely fusion (F) and nucleocapsid(N) protein genes. In this study, a conventional RT PCR assay targeting Fgene and a real time RT PCR assay targeting the N gene for detection ofVirologie, Vol 17, supplément 2, septembre 2013S213

5 th European Congress of VirologyPPR viral RNA were compared in terms of performance. During JanuaryOctober 2012, 32 sheep, each from different flocks, submitted to KonyaVeterinary Control Institute with PPR suspect. One step RT PCR, whichamplified a 448 bp fragment in the F gene, detected PPRV in 9 samples.Twelve of these 32 samples were found to be positive by real time RT PCR,and most infected animals (9/12) showed a high viral load with individualcycle threshold (Ct) values are less than 30. The present study suggeststhat N gene based one step real time RT PCR was more suitable for therapid and specific detection of PPRV in clinical samples.Key words: Peste des petits ruminants virus, RT PCR, Real time RT PCR,F gene, N genecoated with VP7 monoclonal antibody was added to form immuno complex.Signal DNA annealed to DNA strands covalently bound to the GNPwere released by heating and characterized by PCR and real time PCR. Adetection limit of 0.1fg/ml was measured for VP7. Further, a simple GNPAmodified from BCA was developed. GNP was prepared through coatedwith designed single stranded signal DNA and VP7 monoclonal antibody.After immuno complex was formed (GNP VP7 MMP) and purified, thesignal DNA present in the immuno complex was detected by PCR and realtime PCR. The GNPA has a VP7 detection limit of 0.01fg/ml which is 1order of magnitude more sensitive than that of BCA. These approachescan reliably detect BTV, and it is important to prevent bluetongue.REF 339Comparative study of liquid phase blocking ELISA (LPBE) and solidphase competition ELISA (SPCE) methods for the detection of antibodiesto the structural proteins of foot and mouth disease types Oand A virusesMurat SEVIK 1 , Feridun ÖZTÜRK 21 Veterinary Control Institute/Molecular Microbiology, Konya, TURKEY;2 Department of Virology/Faculty of Veterinary Medicine/Selcuk University,Konya, TURKEYTwo foot and mouth disease virus (FMDV) structural protein antibodydetection methods, liquid phase blocking ELISA (LPBE) and solid phasecompetition ELISA (SPCE), were compared in the study. These methodswere compared using sera collected from cattle with no history of exposureto FMDV (n=30), cattle (n=180) vaccinated with oil adjuvanted bivalentvaccine (containing O1 Manisa, A22 Iraq FMDV strains) and internationalreference sera (positive, weak positive and negative) for FMDV serotypesO and A. The results showed that the SPCE had a better specificity (96.6%for serotype O and 100% for serotype A) than the LPBE (90% for serotypeO and 93.3% for serotype A). Sensitivity of LPBE (97.2% for serotypeO and 98.3% for serotype A) was almost equivalent to that of the SPCE(98.3% for serotype O and 98.8% for serotype A). It can be concludedthat SPCE is more suitable than LPBE for use as a screening test for thedetection of antibodies against structural proteins of FMDV.This study was summarized from a PhD thesis and supported by ScientificResearch Projects Coordination Center of Selcuk University, Konya,Turkey (Project No: 09202025).Key words: Cattle, foot and mouth disease, structural protein, LPBE,SPCEREF 341Baculovirus expressed type specific spike proteins in the differentiationof type 1 and 2 feline coronavirus infectionYing Ting WANG 1 , Cho Hua WAN 2 , Ling Ling CHUEH 11 Graduate Institute of Veterinary Medicine, School of Veterinary Medicine,National Taiwan University, Taipei, TAIWAN; 2 Graduate Instituteof Molecular and Comparative Pathology, School of Veterinary Medicine,National Taiwan University, Taipei, TAIWANFeline infectious peritonitis (FIP), is a fatal disease caused by feline coronavirus(FCoV). FCoVs are divided into serotypes I and II. Despite thehigher prevalence of type I FCoV infection in epidemiological survey usingboth molecular or serological assay, infection of type II FCoV appears tobe highly significantly correlated to FIP. This study aim to develop a novelserological assay to differentiate two types of FCoV infection throughthe baculovirus expressed type specific spike proteins from type I and IIFCoV. Two type specific regions in spike gene corresponding to the putativereceptor binding domain of spike protein from type II virus and therelated region from type I virus were selected, amplified and cloned intoa baculovirus expression system to produce two type specific recombinantviruses. A novel indirect immunofluorescence assay (IFA) using S.frugiperda (Sf 9) insect cells infected with the recombinant baculovirusexpressing two FCoV type specific spike proteins was developed. TheIFA test was first characterized using two type confirmed FCoV positivesera and two negative sera from SPF cats. Clinical sera from 42 FIP suspectedanimals were subjected for further test, twenty four (24/42, 57%)and five (5/42, 11%) sera were type I and II seropositive respectively.Thirteen (13/42, 30%) sera were positive to both types of recombinantproteins. These type specific IFA test is capable to distinguish two typesof virus infection. The relationship between serotypes and the diseasemanifestation will be evaluated further.REF 340Assays for detection of bluetongue virusHuiqiong YIN, Minxian JIA, Shu YANG, Rui WANG, Jianguo WANG,Jingang ZHANGViral Safety Laboratory of the National Center of Biomedical Analysis,Institute of Transfusion Medicine, the Academy of Militar, Beijing, CHINABluetongue caused by bluetongue virus (BTV) has been included in theWorld Organization for Animal Health (OIE) list of notifiable diseases.Reliable assays of BTV detection are essential for fighting against bluetongue.A series of techniques were developed for BTV detection includingRT PCR, real time RT PCR, ELISA, bio barcode assay (BCA) and goldnanoparticle probe based assay (GNPA). BTV RT PCR and real time RTPCR diagnostic kits detect BTV NS1 gene through a pair of primers anda TaqMan probe respectively. The kits had been approved by Ministry ofAgriculture of the People‘s Republic of China. The ELISA kit detects BTVVP7 through anti VP7 monoclonal antibody. After preclinical study, clinicaltrial is being applied. In BCA, VP7 was captured by gold nanoparticle(GNP) coated with polyclonal antibody. Magnetic microparticle (MMP)REF 342Comparative investigation of border disease virus infection in sheepflocks in Konya Province, TurkeyOguzhan AVCI, Sibel YAVRUUniversity of Selcuk, Faculty of Veterinary Medicine, Department of Virology,Konya, TURKEYThe aim of the present study was to determine the serological and virologicalstatus of animals persistently infected (PI) with border disease virus(BDV), with focus on the prevalence of pestiviruses in sheep and lambs.This study is the first to our knowledge, to investigate the role of viralinfections in abortions in the sheep population of Konya Province, Turkey.The prevalence of antibodies against BDV was as follows: sheep,79% (n=1000); lambs, 43.4% (n=500); and rams, 6% (n=50). Of 1000leukocyte samples examined with a direct enzyme linked immunosorbentassay (ELISA), 11 (1.1%) (first sampling) were antibody positive. Thirtysheep were determined to be PI and were sacrificed. Of 1011 sheep leukocytesamples, 14 were examined using a direct immunoperoxidase (IP)S214 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyassay for BDV antigen, and 11 of 327 (3.36%) tissue samples were positive.The BDV genome was detected in 14 of 63 (22.2%) sheep leukocytesamples and 11 of 327 (3.36%) tissue samples by one step reverse transcriptionpolymerase chain reaction (RT PCR). The BDV genome couldnot be detected in any vaginal swab samples. The sensitivity and specificityrates between direct ELISA and direct IP, were respectively 100%and 98.8% and the values were, respectively, 78.57% and 98.6% betweendirect ELISA and one step RT PCR and 100% and 97.5% between onestep RT PCR and direct IP.This study was supported financially by the Scientific Research Councilof Selcuk University (08102001) and summarized from PhD thesis ofOguzhan Avci.Key words: BDV, direct ELISA, immunoperoxidase, one step RT PCR,sheepREF 343Serologic and virologic investigation of BHV 1, BVDV and BHV 4 incattle with metritisSibel YAVRU 1 , Oguzhan AVCI 1 , Mehmet KALE 21 University of Selcuk, Faculty of Veterinary Medicine, Department ofVirology, Konya, TURKEY; 2 University of Mehmet Akif Ersoy, Facultyof Veterinary Medicine, Department of Virology, Burdur, TURKEYBovine herpesvirus type 1 (BHV 1), Bovine viral diarrhoea virus (BVDV),and Bovine herpesvirus type 4 (BHV 4), are well known, importantpathogens of cattle that give rise to substantial economic losses due toreproductive failures and increased calf mortality, as well as enteric andrespiratory disease. The purpose of the present study was to evaluate thepossible effects of BHV 1, BVDV and BHV 4 involving metritis in theselected unvaccinated dairy cattle herds in Afyon province of Turkey byserologically and virologically methods. A total of 63 dairy cattle withclinical signs of metritis were sampled in order to investigate the presenceof BHV 1, BVDV and BHV 4 infections. The sera samples weretested for presence of antibodies to BHV 1, BVDV and BHV 4 using ancommercially available indirect Enzyme Linked Immunosorbent Assay(ELISA, Biox, Belgium). Leukocyte samples were tested for presenceof BVDV viral genome using Reverse Transcription Polymerase ChainReaction (RT PCR) and PCR for BHV 1 and BHV 4 viral genome. Antibodieswere detected in 6 (9.52%) of 63 against BVDV and 51 (80.95%)of 63 against BHV 4. All sera samples were negative for BHV 1 antibodies.BHV 4 genome were detected in 8 (12.69%) of 63 leukocytesamples while BVDV and BHV 1 genomes could not be found. Presenceof BVDV and BHV 4 antibodies in unvaccinated animals indicates thatthese cattle had contracted infection. The serologic and virologic resultsof the study strongly suggest that BHV 4 infections may play a director indirect role in causing bovine metritis; therefore their importancein the aetiology of metritis and their economical impact needs furtherattention.Key words: BHV 1, BVDV, BHV 4, metritisREF 344Immunomodulatory effects of EHV 4 on PBMC and a new equinetracheal epithelial cell lineErika HUE 1,2,3 , Christine FORTIER 1,3 , Kerstin BORCHERS 4 ,Guillaume FORTIER 1,2,3 , Stephane PRONOST 1,2,31 Frank Duncombe Laboratory, Caen, FRANCE; 2 Normandie Univ, SF4206 ICORE, EA 4655 U2RM, Caen, FRANCE; 3 Hippolia Foundation,Caen, FRANCE; 4 Freie Universität Berlin, Institut für Virologie, Berlin,GERMANYEquine alphaherpesvirus 4 (EHV 4) was a major cause of respiratorydisease in horses throughout the world. It causes significant economicimpact to the equine industry because of respiratory problems and losttime for training and performance. Currently, no specific treatment wasavailable in the field to cure these equine infections and the efficacy of thedifferent vaccine was limited. Moreover, only few data were available onthe immune response induced by EHV 4.. To improve new vaccine andtreatments, it’s necessary to better understand the immune responses ofnatural target cells during infection of EHV 4. The aim of this study wasto 1/determine the immunological profile induced by EHV 4 infection onperipheral blood mononuclear cells (PBMC) and 2/develop and characterizea new epithelial cells line to characterize the immunological profile..Modulation of the expression of major cytokines involved during viralinfection (IFN alpha, IFN beta, IFN gamma, TNF alpha, IL 1 beta, IL 6and IL 18) was measured by real time quantitative PCR (RT PCR). Preliminaryresults indicate that EHV 4 stimulated IFN gamma production inPMBC. The equine tracheal epithelial cell model was characterised withspecific antibodies. This new equine tracheal epithelial cell line will helpto determine the immunological profile during EHV 4 infection in airwayepithelial cells.Virologie, Vol 17, supplément 2, septembre 2013S215

5 th European Congress of Virology19. VIRAL EVOLUTION ANDQUASISPECIESPosters: REF 346 to REF 359REF 346Use of deep sequencing to evaluate the intrinsic heterogeneity ofhuman influenza type A viruses directly in nasal swabsCyril BARBEZANGE 1,2 , Hervé BLANC 1 , Ofer ISAKOV 4 , VincentENOUF 2,3 , Noam SHOMRON 4 , Sylvie VAN DER WERF 2,3 , MarcoVIGNUZZI 11 Institut Pasteur, Viral Populations and Pathogenesis, Paris, FRANCE;2 Institut Pasteur, Molecular Genetics of RNA Viruses, Paris, FRANCE;3 Institut Pasteur, National Influenza Centre, Paris, FRANCE; 4 Universityof Tel Aviv, Sackler Faculty of Medicine, Department of Cell and DevelopmentalBiology, Tel Aviv, ISRAELIn 2009, a new influenza type A virus of H1N1 subtype (H1N1pdm09)entered the human population and caused the first pandemic of the 21stcentury. After this pandemic wave, it replaced the previously circulatingH1N1 virus and, along with the H3N2 subtype, is now responsible forthe seasonal influenza epidemics. The genome of influenza type A virusesis composed of eight single stranded RNA segments of negative polarity.So far, the evolutionary potential of influenza viruses has been mainlydocumented by consensus sequencing data. However, RNA virus polymerasesare considered to have low fidelity and a virus thus exists asa cloud of closely related sequences (referred to as a quasispecies) thatcould influence its fitness and its adaptability. Interest in the quasispeciesnature of influenza viruses has only recently increased with the developmentof next generation sequencing (NGS) technologies that allow awider study of the genetic variability. NGS deep sequencing methodologieswere developed to determine the whole genome genetic heterogeneityof the three subtypes of influenza viruses that circulated in humans between2007 and 2012 in France. For each subtype, between 20 and 30specimens, collected from mild and severe human cases of influenza,were selected to cover the different epidemic seasons. We are presentinghere the results of the comparison performed to identify subtype/severitysignatures focusing on mutation frequencies and specific nucleotidepolymorphisms.REF 347Isolation and characterization of Coxsackie virus B3 RdRp variantsin vitroStéphanie BEAUCOURT, Nina GNÄDIG, Olve PEERSON, MarcoVIGNUZZIInstitut Pasteur, Paris, FRANCEThe crystal structure of the CVB3 RdRp reveals a similar H bond networkto the poliovirus involving amino acids 1, 64, 239 and 241 and thought toparticipate in fidelity. In order to generate fidelity variants of CVB3, eachamino acid was substituted into these positions by site directed mutagenesis.As expected, many substitutions were non viable (1 and 241) orless stable (64, unlike poliovirus). G64A, G64S and G64Q were stablebut presented severe replication defects. A large number of position 64and 239 substitutions resulted reversion to wild type at these positions.However, each reversion was accompanied by a new mutation (P48K,S164P, A239G, A239S, or S299T) that also affected fidelity. By structuralmodeling, we generated another 50 mutations across the polymerase, targetingactive site conformational changes occurring during catalysis. Fromthese, we isolated six different low fidelity polymerase mutants (I176 V,I230F, F2323 V, F232Y, Y268H, Y268W). We also isolated a high fidelityvariant (A372 V) by passaging the wild type virus in mutagens, and 8 moremutations were generated in motif D, that are structurally very close toA372, in order to try to find similar effects on fidelity. Together, our datashow that structural modeling can identify both unique and shared determinantsof RdRp fidelity in picornaviruses and that viral RdRp fidelity ismore flexible than originally thought, thereby providing new tools to studyvirus evolution, virulence and attenuation.REF 348Evolution of Anelloviridae: analysis of serial sequences within 17 yearsSandra BÉDARIDA 1 , Bertrand DUSSOL 2 , Yvon BERLAND 2 , PhilippeDE MICCO 1 , Philippe BIAGINI 11 Equipe “Emergence et co évolution virale”, UMR 7268 EFS CNRS AixMarseille University, Marseille, FRANCE; 2 Centre de Néphrologie etTransplantation Rénale, CHU Conception, Marseille, FRANCEThe Anelloviridae family is composed of multiple viral genera and speciesinfecting humans and animals; currently, more than 200 variants havebeen described, including prototype genus Torque Teno Virus (TTV). Despitesuch advances, many aspects related to the biology, natural historyand implication for host health of these circular single stranded DNAviruses are still a matter to debate. In order to tentatively gain insightsabout the evolution rate of these viruses, we characterized and analyzedserial sequences belonging to genus TTV, based on the study of severalblood samples obtained from a hemodialysis patient (follow up ∼17years). Viral DNA was extracted from plasma samples, and a sequenceindependent molecular approach described previously in our laboratory,i.e. RCA SISPA (Biagini et al., J Gen Virol 2007), was performed. SENvand DXL2 related sequences identified in these serial samples servedas templates for three specific PCRs located on the ORF1 of the viralgenomes. Following cloning and sequencing, sequences analysis (∼2kbeach) demonstrated the presence of highly conserved regions and alsoregions with noticeable variability; an evolution rate in the range 10 3 104 mutations per site per year was estimated when analyzing central partof DXL2 ORF1. Details of the protocol and precise analysis of moleculardata obtained are exposed.REF 349Evolution of viral RNA dependent polymerasesJiri CERNY 1,2 , Barbora BOFIKOVA 3,4 , Libor GRUBHOFFER 1,2 ,Daniel RUZEK 2,51 Faculty of Science, University of South Bohemia in Ceske Budejovice,Ceske Budejovice, CZECH REPUBLIC; 2 Institute of Parasitology, BiologyCentre of the Academy of Sciences of the Czech Republic, CeskeBudejovice, CZECH REPUBLIC; 3 Faculty of Science, Charles Universityin Prague, Prague, CZECH REPUBLIC; 4 Faculty of Tropical AgriSciences,Czech University of Life Sciences, Prague, CZECH REPUBLIC;5 Veterinary Research Institute, Brno, CZECH REPUBLICRNA viruses evolve rapidly. Fast accumulation of many mutations leadsto high diversity of viral proteins. Many viral proteins originating fromthe same ancestor share only low sequence homology. Despite that, theirtertiary structure remains conserved. RNA dependent polymerases displaythe highest degree of conservation. They contain many highly or evenabsolutely conserved amino acids residues and share remarkable structuralhomology. As they were found in all RNA virus families as well as in allviruses reproducing via DNA intermediate, they are ideal candidates forpossible reconstruction of evolutionary history of all RNA viruses. Toreconstruct evolutionary history of viral RNA dependent polymerases, weused both sequence and structural data. We unified sequence data from theS216 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologypolymerase structure based sequence alignment and structural data gainedfrom “morphological” comparison of individual polymerase structures.The result of our work is the first highly accurate phylogenetic tree ofviral RNA dependent polymerases having significant statistical support forall branches. It shows evolutionary relations among all RNA dependentpolymerases with known tertiary structure and sheds some light in earlyevolution of RNA viruses.REF 350Structural Allocation of Extrachromosomal DNA containing parts ofthe 5 ′ Non coding region of Hepatitis C Virus in PBMCs of HCVnegative human individualsReinhard H.D.R. DENNIN 1 , Jianer D.R. WO 21 Institute of Medical Microbiology and Hygiene, University of Luebeck,Lübeck, GERMANY; 2 State Key Laboratory for Diagnosis & Treatmentof Infectious Diseases, Institute of Infectious Diseases, Hangzhou, P RCHINABackground: The HCV is a positive single stranded non retroviral RNAvirus. It is both hepatotropic with a high rate of hepatocellular carcinoma,and it is lymphotropic too, associated with auto immune disorders. Experimentalfindings: (i) Previous studies performed with regular PCR haveshown DNA sequence sections of HCV’s 5’ non coding region (5’ NCR)up to 272 bp in the total DNA extract from peripheral white blood cells(PBMC) of all healthy, HCV negative tested individuals. The homologywith a HCV GT 1 reference strain varies form 92% to 99%. BLAST alignmentsshow no assignment to any known HCV geno/subtype. (ii) Inorder to discriminate against any contamination pre PCR digestion protocolswere performed with restriction enzymes being sensitive (no digest)against methylation(s) within their cutting codes. The results show up inindividual patterns of methylation, i.e., methylomes. (iii) The total DNAextract from PBMCs was subjected to isolation for episomal circular (ec)DNA structures by making use of the Plasmid Safe ATP dependent DNasedigest protocol TM . This results in about 25 kbp ec DNA fractions. By bothregular PCR and the Rolling Circle Amplification (RCA) DNA sequencescould be generated from it with high homology of the range shown aboveto the 5 ′ NCR of HCV. Contiguous DNA sequences of this length are notpresent in the human genomic chromosomal DNA, the longest found thereinbeing up to 41 bp. Conclusion: The DNA sections of the 5 ′ NCR ofHCV are part of extrachromosomal ec DNA fraction.REF 351Global circulation of slowly evolving trichodysplasia spinulosa associatedpolyomavirus and its adaptation to the human populationthrough Middle T antigenMariet FELTKAMP 1 , Siamaque KAZEM 1 , Chris LAUBER 1 , Els VANDER MEIJDEN 1 , Sander KOOIJMAN 1 , Seweryn BIALASIEWICZ 2 ,Richard WANG 3 , Alexander GORBALENYA 11 Leiden University Medical Center, Leiden, THE NETHERLANDS;2 Queensland Children’s Medical Research Institute & Sir Albert SakzewskiVirus Research Centre, Herston, AUSTRALIA; 3 University of Texas,Southwestern Medical Cente, Dallas, USAThe trichodysplasia spinulosa associated polyomavirus (TSPyV) is oneof the new human polyomaviruses associated with disease. Based onseroepidemiological data, up to 70% of the population is infected withTSPyV. So far nothing is known about its genomic heterogeneity. Genomicsequences of TSPyV from lesional skin of 11 TS patients fromdifferent continents collected over 16 years were amplified and sequenced.Comparative analysis of these and two previously reported TSPyVgenomes revealed 30 single nucleotide polymorphisms (SNPs) over agenome of 5232 nts (0.6% diversity), as well as two multiple nucleotidedeletions in two isolates. The majority of SNPs and both deletionsclustered in the NCCR. Most other SNPs appeared silently in the VP capsidproteins and Large T antigen, while the putative Small and Middle Tantigens shared one non synonymous SNP (NSSNP). In addition, MiddleT antigen had three unique NSSNPs. Phylogenetic analysis revealed thepresence of three distinct TSPyV lineages that may have diverged fromBornean orangutan polyomavirus some 4000 years before present. Thephylogenetic pattern of NSSNPs in the putative Middle T antigen wasindicative of purifying selection. In summary, the TSPyV isolates fromclinical samples show little genetic heterogeneity. However, the observedpattern of predicted amino acid variations in the putative Middle T antigenindicates selective pressure on this protein, likely due to its role inTSPyV adaptation to the human population, and suggests its expressionin vivo.REF 353Long term reassortment and co evolution pattern of influenza A virusin TaiwanHsin Fu LIU 1,2 , Jih Hui LIN 3 , Shu Chun CHIU 3 , Yung Cheng LIN 2 ,JuChien CHENG 4 , Ho Sheng WU 3 , Marco SALEMI 51 Department of Medical Research, Mackay Memorial Hospital, New TaipeiCity, TAIWAN; 2 Institute of Bioscience and Biotechnology, NationalTaiwan Ocean University, Keelung, TAIWAN; 3 Research and DiagnosticsCenter, Centers for Disease Control, Taipei, TAIWAN; 4 Departmentof Medical Laboratory Science and Biotechnology, China Medical University,Taichung, TAIWAN; 5 Department of Pathology, Immunology, andLaboratory Medicine, and Emerging Pathogens Institute, University ofFlorida, Gainesville, Florida, USAOur recent study of influenza A evolutionary history suggested that Taiwanis a microcosm of flu pandemics. We investigated their reassortmentand co evolution pattern of the virus in detail, which may be importantto predict the potential emergence of pandemic strains. 343 full genomesequences of human influenza A isolated in Taiwan between 1979 and2009 were studied. Co evolving sites and significant associations amongsites were detected with Bayesian graphical models method in Spidermonkeyof the Datamonkey website. Recombination was tested for each dataset to avoid misinterpretations. H1N1 and H3N2 have different reassortmentpatterns. The internal genes of H1N1 moved as a unit, separatelyfrom the co evolving HA and NA genes; but the HA and NA of H3N2tended to segregate with different internal gene segments. In particular,H3HA always segregated as a group with the PB1, PA and M genes,while N2NA consistently segregated with PB2 and NP. Nineteen pairs ofinteracting sites were identified in H1N1 and 18 pairs in H3N2, mostlylocated in or close to Sa or Sb antigenic sites for H1N1, and sites B orD for H3N2. These sites are located toward the top of globular head ofHA molecule and believed to be antibody binding sites. The co evolutionof amino acids was not limited to intra segment changes but also atinter segment level. Overall, these results shed new light on the influenceof epistatic interactions within and between influenza A genes and mayhelp in the early recognition of potential new epidemic variants in thefuture.Virologie, Vol 17, supplément 2, septembre 2013S217

5 th European Congress of VirologyREF 354From the 1980s to our days: the evolutionary history of Europeanbrown hare syndrome virus (EBHSV) in SwedenAna M LOPES 1,2,3 , Dolores GAVIER WIDÉN 4,5 , Ghislaine LE GALLRECULÉ 6,7 , Jacques LE PENDU 3 , Pedro J ESTEVES 1,8 , JoanaABRANTES 1,31 CIBIO/UP, Centro de Investigação em Biodiversidade e Recursos Genéticos/Universidadedo Porto, InBio, Laboratório Associado, Ca, Vairão,PORTUGAL; 2 Departamento de Zoologia e Antropologia, Faculdadede Ciências da Universidade do Porto, Porto, PORTUGAL; 3 INSERM,U892, Université de Nantes, Nantes, France; 4 Department of Pathologyand Wildlife Disease, National Veterinary Institute (SVA), Uppsala, SWE-DEN; 5 Department of Biomedical Sciences and Veterinary Public Health,Swedish University of Agricultural Sciences (SLU), Uppsala, SWEDEN;6 Anses, Ploufragan Plouzané Laboratory, Avian and Rabbit, Virology,Immunology, and Parasitology Unit, Ploufragan, FRANCE; 7 EuropeanUniversity of Brittany, FRANCE; 8 CITS, Centro de Investigação em Tecnologiasda Saúde, IPSN, CESPU, Gandra, PORTUGALEuropean hares (Lepus europaeus) and mountain hares (L. timidus) arehighly decimated by European brown hare syndrome (EBHS), a viraldisease causing severe acute hepatitis. The aetiological agent of EBHS,European brown hare syndrome virus (EBHSV), is a single stranded, positivesense RNA virus ∼7.4 kb in length. EBHSV and the closely relatedrabbit hemorrhagic disease virus (RHDV) belong to the genus Lagovirus,family Caliciviridae. The first outbreak of EBHS was diagnosed in Swedenin 1980. Livers from 104 hares found dead between 1982 and 2008in Sweden were tested for the presence of EBHSV. Complete sequencesof the capsid protein VP60 gene were obtained for 54 EBHSV positivesamples. Phylogenetic and selection analyses were conducted to assess theevolution of the virus. The inferred phylogenetic tree is divided into twomain branches: one grouping the most recent samples and the other groupingthe older samples. The tree shows that evolution of EBHSV follows achronological pattern that, although described for RHDV, had not yet beenobserved for EBHSV. Analyses of selection revealed 4 sites under positiveselection located within hypervariable regions E (3) and F (1), according toRHDV structure. Signatures of positive selection at these exposed regionsof the capsid have also been detected for RHDV and suggest an immunemediated mechanism for EBHSV evolution. This study represents the firstdescription of EBHSV evolution in the area of original diagnosis of thevirus and highlights the importance of using complete sequences of theVP60 gene to infer EBHSV evolution.REF 355Phylogeny of introduced Dengue virus type 1 strains in EuropeLeticia FRANCO 1 , Lieselotte CNOPS 2 , Ivan KUROLT 3 , DavidPOLUDA 4 , Marjan VAN ESBROECK 2 , Andreas NEUMAYR 5 ,Francisca MOLERO 1 , Lourdes HERNANDEZ 1 , Christoph HATZ 5 ,Antonio TENORIO 11 National Center of Microbiology, Instituto carlos III, Madrid, SPAIN;2 Department Clinical Sciences, Institute of Tropical Medicine, Antwerp,BELGIUM; 3 University Hospital for Infectious Diseases, Zagreb, CROA-TIA; 4 Division of Infectious Diseases and Tropical Medicine, MedicalCenter of the University of Munich, Munich, GERMANY; 5 Swiss Tropicaland Public Health Institute, Basel, SWITZERLANDDengue is caused by 4 different but antigenically related viruses, DENV 1to DENV 4, transmitted to humans through the bites of Aedes mosquitoes.The disease is endemic in 100 countries from Asia, America, Africa andOceania. In Europe, in the past century, Dengue epidemics occurred inGreece and other Mediterranean countries were the main Ae. Aegypti waspresent. After that, it disappeared from Europe, but another competentvector, Ae. albopictus, was introduced in late 70 s. In recent years, Ae.aegypti was reintroduced in south Russia and in Madeira island (Portugal).In 2010, dengue re emerged in the French Riviera and Croatia, withsmall outbreaks. Two years later, in October 2012, a sustained and explosiveepidemic appeared in the Madeira. Both, 2010 and 2012 outbreakswere caused by DENV 1. The aim of this study was to describe the phylogenyand spatio temporal dynamics of the DENV 1 recently introduced inEurope, taking in advantage the ongoing collaborative European initiativeon imported dengue and chikungunya infections. We analyzed the completeE sequences from imported and autochthonous cases of DENV 1infections related to the recent outbreaks in Europe. Phylogenetic analysisrevealed that all DENV 1 strains belong to genotype V. The strains introducedin France and Madeira clustered within different South Americansublineages, whereas the Croatian strain appeared to be introduced fromIndia. In conclusion, our data suggest that three different introductionsof DENV 1 genotype V were the responsible for the three independentoutbreaks in Europe.REF 356Next generation sequencing of a longitudinal tick borne encephalitisvirus study in a micro focus in Central EuropeStefan FREY 1 , Dirk HÖPER 2 , Manfred BEER 2 , Gerhard DOBLER 1 ,Sandra ESSBAUER 11 Bundeswehr Institute of Microbiology, Munich, GERMANY; 2 FriedrichLoeffler Institut, Greifswald Riems, GERMANYTick borne encephalitis virus (TBEV) is a member of the genus Flavivirusin the family Flaviviridae and is transmitted by ticks. The temporaldynamics of the TBEV genome structure within a single natural focus ispoorly understood. In our study we analysed for the first time TBEV fullgenomes from a single TBEV focus in Central Europe over a time periodof 4 years. From 2009 to 2012 a total of 5787 Ixodes ricinus ticks werecollected monthly by flagging. In real time RT PCR 24 ticks were testedpositive for TBEV RNA. For detailed analysis of the genomes cDNAlibraries of 9 selected TBEV isolates were made and sequenced using 454pyrosequencing. For these isolates complete genomes were assembledfrom roughly 3100–35,000 reads (representing 5.8–35.0% of the reads).Comparison of the open reading frames revealed a strong conservationat nucleotide (nt) and amino acid (aa) level. A single aa exchange wasdeduced in 7 out of the 9 strains in the NS5 region encoding the RNAdependent RNA polymerase and in one strain an additional aa substitutionwas deduced encoding the helicase. Metagenome analyses of thecomplete genomes give new insights into TBEV genome variations in tickhosts and in cell culture. Herein we show first data which imply that TBEVwithin one micro focus has a highly conserved genome for at least 4 years.Changes in nt sequences which could be acquired by spontaneous mutationsmay have implications for the replication and are subject of furtheranalyses.REF 357Genetic variability and molecular evolution of the human RespiratorySyncytial Virus (RSV) circulating in Milan between 2006 and 2012Marianna MARTINELLI, Alessandra ZAPPA, Silvia BIANCHI, DanielaCOLZANI, Elena Rosanna FRATI, Antonella AMENDOLA, ElisabettaTANZIDepartment of Biomedical Sciences for Health, Università degli Studi diMilano, Milan, ITALYRespiratory Syncytial Virus (RSV) is a major cause of lower respiratorytract infections in children. It is classified into two groups, RSV A andB, and several genotypes of each group have been reported. This studyaimed to assess the genetic diversity of both RSV groups by sequencingS218 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologya part of G gene in samples collected between 2006 and 2012 from childrenaged under 3 years in Milan, Italy. The molecular characterizationof sequences was conducted using BioEdit software. Phylogenetic treeswere constructed by Neighbor Joining method. To estimate the selectionpressures, codon specific dN and dS values were inferred using Datamonkeyserver. Potential N and O glycosylation sites were predicted usingNetNGlyc 1.0 and NetOGlyc 3.1 servers. All RSV A sequences clusteredinto two genotypes, GA2 and NA1 (similarity range: 97 99%), whereasRSV B sequences grouped into BA4 genotype (similarity range: 95 99%).Compared to the reference strains, 31 RSV A and 8 RSV B amino acidsubstitutions were identified among the sequences studied. Even if 5 positivelyselected codons were found in RSV A and one in RSV B sequences,no evidence of positive selection in both sequencing alignements emerged.Different patterns of O and N glycosilation sites were idenfided inboth groups of sequences. In conclusion, the results showed that there wasnot a variability in RSV A and B genotypes circulation in Milan duringthe study period. Although G protein showed a high genetic variability, itaccumulated almost exclusively neutral substitutions.REF 358Growth of Porcine Influenza Viruses in Differentiated RespiratoryEpithelial CellsFandan MENG 1 , Darsaniya PUNYADARSANIYA 2 , Wei YANG 1 ,Xiaofeng REN 3 , Georg HERRLER 11 Institute for Virology, University of Veterinary Medicine, Hannover, GER-MANY; 2 Institute of Virology, Mahanakorn University of Technology,Bangkok, THAILAND; 3 College of Veterinary Medicine, Northeast AgriculturalUniversity, Harbin, CHINASwine are an important host for the epidemiology and interspecies transmissionof influenza A viruses. The differentiated epithelial cells of therespiratory tract are the primary target cells for influenza virus infection.We have recently reported precision cut lung slices (PCLS) as a model systemto analyze the infection of porcine influenza viruses in their naturaltarget cells (Punaydarsaniya et al., PlosOne 6:e28429, 2011). Comparisonof a porcine virus of the H3N2 subtype with an avian virus of the H9N2subtype revealed differences in the virulence as indicated by various parameters:(i) duration of the growth cycle, (ii) amount of infectious virusreleased into the supernatant, and (iii) extent of the ciliostatic effect. Herewe compared five porcine viruses of the three subtypes currently prevalentin the swine populations (H3N2, H1N1, H1N2). The data collected werecompared with the pathogenicity of the viruses determined in pigs. In thisway, we show which of the above parameters is the best indicator of thepathogenicity of porcine influenza viruses.REF 359The adaptation of avian influenza viruses to the respiratory epitheliumof pigsWei YANG 1 , Fandan MENG 1 , Darsaniya PUNYADARSANIY 2 ,Markus HOFFMANN 1 , Juergen STECH 3 , Dirk HOEPER 3 , MartinBEER 3 , Christel SCHWEGMANN WESSELS 1 , Xiaofeng REN 4 , GeorgHERRLER 11 Institute of Virology, University of Veterinary Medicine, Hannover, GER-MANY; 2 Institute of Virology, Mahanakorn University of Technology,Bangkok, THAILAND; 3 Friedrich Loeffler Institut, Bundesforschungsinstitutfür Tiergesundheit, Greifswald, GERMANY; 4 College of VeterinaryMedicine, Northeast Agricultural University, Harbin, CHINAPigs are an important host for influenza A viruses and may play a crucialrole in the interspecies transmission. To analyze the infection byinfluenza viruses, we have established precision cut lung slices (PCLS)from the porcine lung as a culture system for differentiated respiratoryepithelial cells. In PCLS, the differentiated epithelial cells are maintainedin their original setting. As differentiated repiratory epithelial cells are theprimary target cells for influenza virus infections, PCLS provide an interestingsystem to analyze the adaptation of avian influenza viruses to therespiratory epithelium of pigs. Avian influenza viruses of the H9N2 subtypewere subjected to several passages in PCLS. Adaptation of the avianviruses to growth in porcine cells was evident in a shortening of the growthcycle. Sequence analysis revealed that few amino acid changes occurredduring the different virus passages. The importance of the individual mutationsis currently analyzed by generating recombinant viruses that containthe respective mutated proteins. Our study will help to understand theprocesses involved in the adaptation of H9N2 influenza viruses to newhosts.Virologie, Vol 17, supplément 2, septembre 2013S219

5 th European Congress of Virology20. VIRUS EPIDEMIOLOGYPosters: REF 360 to REF 396REF 360Three year follow up of HPV positive and negative urine in womenfrom general population in finistere, West Brittany, FranceChristopher PAYAN 1 , Sylvain ROSEC 2 , Patricia AMOUROUX 3 , AdissaTRAN 1 , Yvon FOLL 4 , Morgane THELOHAN 4 , FrancoiseBOMMELAERT 4 , Francoise CHARLES 5 , Edith POSTEC 3 , MichelCOLLET 31 Virology Lubem Chru, Brest, FRANCE; 2 CIC INSERM 0502, Brest,FRANCE; 3 GYNECOLOGY CHRU, Brest, FRANCE; 4 ADEC29, Brest,FRANCE; 5 CYTOLOGY CHRU, Brest, FRANCEObjectives: We have conducted the PapU29 study for detection of HPVDNA in urine for cervical cancer screening in general population (n=3115between 2008 2010). Since, a large proportion of women with HPV infection(24%) will clear their virus (about 1/3), we expect little appearanceof lesions. The aim of our study was to follow their cytology histologyafter 3 years from inclusion. Methods: HPV DNA detection and quantificationwas assessed by real time PCR as previously described (Payan, JClin Microb 2007) and typing using the LipA HPV test (Innogenetics). Weobtained 5 groups: 1) negative PCR (n=2343), 2) positive PCR with normalcytology (n=664), 3) positive PCR with abnormal cytology but normal colposcopy(n=26), 4) positive PCR with abnormal cytology and CIN1 (n=12)and 5) positive PCR with abnormal cytology and CIN2+ (n=14). Cytologyand colposcopy was obtained during 3 years after. Results: In group 1,no abnormal cytology was observed among 362 cases, out of 2 cases withASCUS and CIN1 lesions but with negative HPV control, in groups 2 to 4no lesions was observed on control cytology, half of the CIN1 women had atreatment, and in group 5, all cases were treated (11 conisations, 2 hysterectomies)with normal cytology after 1 year. Complete analysis is ongoing.Conclusion At present, there is no appearance of new lesions in womenwith urine HPV analysis. This strategy allows the detection of CIN2+lesions in women who do not participate to cytology screening and earlytreatment with no relapse. Grants from the French Ligue contre le Cancer.REF 361Presence of Parvovirus B19 Infection in Patients with Thyroid GlandDisease with and without Autoimmune ComponentZaiga NORA KRUKLE 1 , Svetlana CHAPENKO 1 , SabineGRAVELSINA 1 , Alina SULTANOVA 1 , Egils CUNSKIS 2 , ModraMUROVSKA 11 August Kirchenstein Institute of Microbiology and Virology, Riga StradinšUniversity, Riga, LATVIA; 2 Riga Eastern Hospital, Clinic, Riga, LATVIAIntroduction: Viral infections including parvovirus B19 are frequentlycited as a major enviremental factor implicated in autoimmune thyroiddiseases and subacute thyroiditis. Objective: To investigate the presenceand activity of B19 infection in patients with thyroid gland diseases withand without autoimmune component. Design/Methods: 82 patients wereenrolled in the investigation. TPOAb, TRAb, TGAb and virus specific IgGand IgM were detected using ELISA; B19 infection using nPCR and RTPCR. Results: Virus specific IgG had 29/37 (78.4%) patients with highthyroid specific autoantibody titre. Parvoviral DNA was detected in 11/29patients: in 4/11 patients B19 genomic sequence was found in thyroid specimensonly, in 1/11 – blood sample only, in 2/11 – plasma samples only.Only 1/11 patient was parvoviral DNA positive in blood and plasma specimens,1/11 – in blood and tissue and 2/11 – in plasma and tissue. 30/45(66.7%) patients without elevated thyroid specific autoantibody titre hadvirus specific IgG. B19 DNA was detected in 17/30 patients. 9/17 patientshad viral DNA in tissue, 2/17 – in blood, 3/17 in blood and tissue and3/17 – in plasma and tissue. None of the patients had B19 specific IgM.mRNA transcription was not detected in any of tissue or blood samples.Conclusion: Results allow suggest that B19 is involved in pathogenesisnot only in autoimmune thyroid disease that is related with lymphocyticinfiltration but also in subacute thyroiditis that indicate on possiblecell surface primary receptor Gb4Cer (globoside) expression in thyroidtissue.REF 362Hepatitis E prevalence in patients with acute alcoholic hepatitis: asingle center experienceStephanie HAIM BOUKOBZA 1,2,3 , Philippe ICHAI 2,3,4 , AudreyCOILLY 2,3,4 , Olga YORDANOVA 1 , Mouna BOUAMOUD 4 , JeannineSAVARY 1 , Faouzi SALIBA 2,3,4 , Didier SAMUEL 2,3,4 , Anne MarieROQUE AFONSO 1,2,31 AP HP, Hôpital Paul Brousse, Virologie, Villejuif, FRANCE; 2 Univ ParisSud, UMR S 785, Villejuif, FRANCE; 3 INSERM U785, Villejuif, FRANCE;4 AP HP, Hôpital Paul Brousse, Centre Hépato Biliaire, Villejuif, FRANCEObjectives: To assess hepatitis E (HEV) prevalence in patients with acutealcoholic hepatitis (AAH), and the possible role of HEV as a cofactor ofAAH severity. To update guidelines for HEV prevention in this population.Methods: Patients admitted for HAA in intensive care unit from 2007 to2011 were included. The following characteristics were collected: age,gender, number of previous AAH episodes, treatment by corticoids, liverbiopsies, prothrombin rate, bilirubin, lymphocytes, platelets, MELD score,Child Pug score, C reactive protein, Factor V, aminotransferase level. Onpatients with available sera at admission, HEV serology (Beijing WantaiBio Pharm, China) and HEV RNA (Ceeram ® , France) were retrospectivelytested.Results: Of 131 patients, mean age was 48,6 years, 67,2% were men, 13%have experienced a previous HAA episode, 64,9% received corticoids,90,1% were cirrhotic. Mean prothrombin rate was 35,3, bilirubin was277, lymphocytes were 1300, platelets were 126708 G/L, MELD score29, Child Pug score was C (90,1%), B (8,4%) and A (1,5%), C reactiveprotein was 43,1%, factor V was 42,5%, aminotransferase level were 212for AST and 139 for ALT. Markers of HEV infection were assessed in92/131 patients: IgG were present in 28,3%. None presented a positiveHEV RNA at time of AAH, but one patient had a positive IgM indicativeof recent infection. Conclusion: HEV prevalence in patients presentingwith AAH from Northern France assessed with the Wantai assay is high.However, concomitant HEV, found in 1%, does not appear as a significantrisk factor of AAH severity.REF 363First case report in Italy of genotype 3 human hepatitis E virus acquiredin FranceMaria Rosaria CAPOBIANCHI 1 , Anna Rosa GARBUGLIA 1 , NicolePAVIO 2 , Daniele LAPA 1 , Alessandrini ANNA IDA 31 Laboratory of Virology, National Institute for Infectious Diseases “LazzaroSpallanzani, Rome, ITALY; 2 AnsesLaboratoire de Santé Animale,maison Alfort, Paris, FRANCE; 3 Clinica Malattie infettive IRCCS AziendaOspedaliera San Martino, Genova, ITALYGenerally in Europe several cases of hepatitis E related to genotype3 infectionhad been described as “authoctnous”. Furthemore no HEV acutehepatitis cases have been reported as imported case from an Europeancountry. In this report we described a case of acute hepatitis E in a menS220 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology(PEGE) from Genova (Italy), who had his vacation in Corsica and in SouthernFrance at the end of August begining September 2011. He boughtfigatelli in a supermarket of Corsica and he stored them at +4 ◦ C untiluse (21 September 2011). During the last week of october he presenteda progressive outset of fever, fatigue, loss of appetite and he wasadmitted to the Infectious Disease Service with a provisional diagnosisof nonA nonC acute hepatitis. Anti hepatitis E virus IgM and IgGwere positive in ELISA assay (Radim, Italy). Molecular analysis showedthat the HEV strain belonged to genotype3.Comparison of the PEGEORF2 region against 98 GT3 sequences isolated in pig, wild boar orhuman revealed that PEGE sequence was closest related to GQ426992sequence belonged to a patient from Marseille (France), with an identityof 97.7%. PEGE was 98.3% similar to an second human strain (Fr 08V22)isolated in an other locality of Southern France. In contrast PEGE wasonly 78.6 91.7% similar to the human and swine HEV strains isolated inItaly. In this contest the possibility that PEGE was infected with a frenchHEV strain by ingesting contaminated food seems the most plausiblehypothesis.Conclusion:Although hepatitis E is mainly an authochthounousdisease in Europe, such as epidemiological feature is significant toPublic Health, imported infection should be considered also in patientswith acute hepatitis symptoms,which traveled in developed countries.Thiswork was partially supported by the European Union Seventh FrameworkProgramme [FP7/2007 2013] under Grant Agreement n o 278433PREDEMICS.REF 364Dengue Diagnosis and Epidemiology by a Regional Reference Laboratoryin 25 years, Rio de Janeiro, Brazil: an overviewFlavia DOS SANTOS, Ana Maria DE FILIPPIS, Eliane ARAUJO,Monique LIMA, Fernanda NOGUEIRA, Nieli FARIA, JaquelineSIMOES, Simone SAMPAIO, Priscila NUNES, Bianca GONÇALVES,Manoela HERINGER DA SILVA, Clarice RODRIGUES, Carla SOUSA,Dinair LIMA, Rita Maria NOGUEIRAOswaldo Cruz Institute, Fiocruz, Rio De Janeiro, BRAZILDengue activity in Brazil has been evidenced by a large number of cases.DENV 1, DENV 2 and DENV 3 were introduced in Rio de Janeiro, in1986, 1990 and 2000, respectively. In 2010, DENV 4 reemerged after28 years. DENV 1 caused an explosive epidemic in 1986 1987, DENV2 introduction in 1990 caused the first DHF/DSS cases. DENV 3 causedan epidemic in 2002 with the largest number cases. In 2008, thecountry experienced the most severe epidemic in terms of morbidity andmortality in children. Since 1986, the laboratorial diagnosis has playedan important role in the surveillance and epidemiology. Virus isolationand IgM ELISA were first used. After DENV 2 introduction in 1990,the immune response characterization performed by the heamaglutinationinhibition test (HI), was replaced by an IgG ELISA. In the 90 ′ s,the RT PCR and sequencing were used for nucleic acid detection andcharacterization. NS1 capture tests were used for the early diagnosis ofdengue after 2007. A total of 47,346 suspected cases were received bythe Laboratory of Flavivirus, IOC/FIOCRUZ, RJ, Regional ReferenceLaboratory for the Brazilian Ministry of Health, from 1986 to 2011. Atotal of 32,374 cases were tested by MAC ELISA, 25,037 were submittedto virus isolation, and 829 cases to IgG ELISA. The RT PCR was performedin 7,441 cases and NS1 ELISA in 1,124.Virus isolation and RTPCR identified the serotype in 4,990 cases. The implementation of newtechniques over the years constituted an important and reliable tool forthe disease surveillance in Brazil. Support by CNPq FAPERJ PAPES VIFIOCRUZ MSREF 365Molecular and epidemiological profiles of Hepatitis C virus genotype4 in Regione Lazio, ItalyAnna Rosa GARBUGLIA 1 , Daniele LAPA 1 , Catia SIAS 1 , AngelaTESTA 2 , Maria Rosaria CAPOBIANCHI 11 Laboratory of Virology, National Institute for Infectious Diseases “LazzaroSpallanzani, Rome, ITALY; 2 Clinical Departement, National Institutefor Infectious Diseases “Lazzaro Spallanzani”, Rome, Italy, Rome, ITALYIn Europe HCVgenotype 4 (GT4)shows an increasing prevalence. Thisprevalence is particularly high in Italy,and reaches values of 8,4% inspecific areas. Among patients(Pt) attending INMI L Spallanzani clinicalsetting we observed a prevalence of 13%.The goal of this study isto provide a picture of GT4 strains circulating in Regione Lazio. GT4subtypes were determined by direct sequencing of a PCR amplified productsfrom NS5B and core regions.The phylogenetic tree was inferred bythe Neighbor Joining(NJ) method with bootstrap values based on 1,000replicates.Sixty seven Pt were included in the analysis;46 were italian and21 were linving in Rome,but they were born abroad.The mean age was45years(range 18 71). Among italian Pt we found 42 GT4d, 3 GT4a and1 GT4r, never described in Italy. Among foreign Pts 11 people harbouredGT4a, GT4d was observed in 8 Pt(38%), most of which were drug addicts.GT4c and o were identified into 2 foreign Pts.The average pairwise nucleotideidentity within the entire group of GT4d patients strains was 95.8%in NS5B region. Three italian Pts showed the same sequences. The pairwisenucleotide identity within the entire group GT4a was 90.6%. A 100%concordance was found between the the subtype assignement in the NS5Band core region, implying no evidence of recombination. The phylogenetictree indicated 2 distinct monophyletic clades for GT4a and GT4d.The 4d strains isolated in foreign Pt were grouped in the same clusters ofitalian 4d strains.New variants had been found.Conclusion:The significativeprevalence of GT4d among immigrated Pt drug users suggests needleexchange as main route of transmission in this population. The study indicatesthe introduction of new subtypes and existence of GT4variants inItaly. Clinical research and drug development program must be stimulatedto further deter the spread of GT4.REF 366Seroepidemiology and molecular epidemiology of enterovirus 71 inRussiaLudmila AKHMADISHINA 1 , Tatiana EREMEEVA 1 , OlgaTROTSENKO 2 , Olga IVANOVA 1 , Mikhail MIKHAILOV 1 , AlexanderLUKASHEV 11 Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow,RUSSIA; 2 Khabarovsk Institute of Epidemiology and Microbiology, Khabarovsk,RUSSIAEnterovirus 71 (EV71) is an emerging human pathogen causing massiveepidemics of hand foot and mouth disease (HFMD) with occasionalsevere neurological complications in Asia. EV71 also circulates in Europe,however it does not cause large outbreaks. The reason for distinct epidemiologicalpatterns of EV71 infection in Europe and Asia remainunknown. In six regions of Russian Federation, seroprevalence of EV71ranged from 5% to 20% in children aged 1 2 years and from 19% to 83% inchildren aged 3 5 years. There was no correlation between EV71 seroprevalenceand income level and population density. In the European regionsof Russia seroprevalence among children aged 3 5 years was 21 27%.Significantly higher rates were observed in Eastern regions of Russia withVirologie, Vol 17, supplément 2, septembre 2013S221

5 th European Congress of Virologyhigh prevalence of Asian ethnic background (41% and 83%) and commoncross border contacts with China (45%). Only 24 out of over 4000 nonpolio enteroviruses isolated in Russia and neighbouring countries in 20012012 were identified as EV71, genotype C. Only two isolates belongedto GtC1. Subgenotype C2 dominated from 2001 until 2012. Subtype C4emerged in Russia in 2009 and became common in 2011 2012. Phylogeneticevidence suggests that 18 non identical EV71 isolates resulted from14 independent introductions, presumably from Europe (GtC1 and GtC2)and China (GtC4). Despite high seroprevalence and detectable virus circulation,there has been no outbreaks of HFMD and neuroinfection causedby EV71 in Russia. It is therefore likely that host factors play an importantrole in EV71 epidemiology and pathogenesis.REF 367Seroprevalence of hepatitis B in the Mohamed V Military Hospital inRabatNada BENJELLOUN, Mohamed Rida TAGAJDID, BouchraBELFQUIH, Hicham ELANNAZ, Saâd MRANILaboratory of Virology, HMIM V, Faculty of Medicine and Pharmacy,University Mohamed V Souissi, Rabat, MOROCCOIntroduction: this study was conducted to determine the seroprevalenceof hepatitis B virus in the Mohammed V military hospital (HMIMV) andto study clinical and biological characteristics of the patients included inthe study.Patients and Methods: this is a retrospective study conducted in thelaboratory of Virology over a period of 13 months between 01/01/2012and 18/02/2013. Epidemiological (age, sex, service) and biological data(HBsAg, anti HBc Ab, Ac antiHBs, Ac antiHBe, HBeAg, viral load, lookingfor a co infection) of patients with HBsAg positive were collected onthe package “Laboserver ”. Serological tests were performed by ELISA.Determination of the HBV viral load was performed by real time PCRCobasTaqman instruments. Results: 9723 samples were tested for HBsAgduring the study period. The HBsAg was positive in 261 cases (2.68%).Among patients with HBsAg positive, 162/169 had positive anti HBc Ab,90/98 had positive anti HBe, 2/105 had anti HBs positive and 41/134 had anundetectable viral load. Co infection HBV HIV was detected in 3 patientsof 162 tested, and no co infection HBV HCV was encountered. Discussion:in Morocco, there is little information available on the prevalenceof hepatitis B, but it was listed in the area of low endemicity, with a prevalenceof HBsAg between 2 and 7%, this data is approved by our study(2.68%). This decline in the incidence and prevalence of HBV infectionis particularly due to the vaccination program. Median seroprevalence ofanti HBc Ab and anti HBe Ab followed a trend similar to that of HBsAg,as shown in our study. In the literature, the average age of infected patientswas 41 ± 12.4 years, with a male predominance, these data are consistentwith those obtained HMIMV. The study of viral load can’t be used on firstline because of the high cost and technical complex.REF 368Monitoring and comparison of the burden of different respiratoryviruses in primary healthcare units and in hospitals at NationalInfluenza Centre in SloveniaNatasa BERGINC, Katarina PROSENC TRILARLaboratory for Virology, National Institute of Public Health, Ljubljana,SLOVENIAFrom week 38/2011 to 11/2013 3147 swabs of patients with influenzalike illness and acute respiratory tract infection, their personal and clinicaldata were collected from 45 primary healthcare(PHC) doctors and 2hospitals(H). 6 age groups(AG) were formed: 0 3, 4 7, 8 14, 15 19, 20 64,>65 years of age(YA). Influenza(Flu), respiratory syncytial virus(RSV),adenovirus(AV), enterovirus(EV), rhinovirus(RV), parainfluenza, metapneumovirus,bocavirus were detected in multiplex RT RT PCR. Resultsare analysed to monitor the burden of this viruses in PHC and in H. In PHCin AG of 0 3YA Flu, RSV, AV are most often detected (37%, 25%, 20%respectively) while in other AGs Flu is most often detected (70% 83%). InH in young chilrden of 0 3YA AV, RV, RSV, EV, Flu are most often detected(27%, 26%, 20%, 10%, 9% respectively). In children and teenagersAV, Flu, RV are most often detected (18%, 16%, 7%, AG 4 7YA; 25%,25%, 25%, AG 8 14YA; 36%, 19%, 16%, AG 15 19YA respectively). Inadults and elderly Flu is mostly detected (73%, AG 20 64YA; 70%, AG>65YA respectively), followed by RSV, AV, RV (6%, 12%, 6%, AG 2064YA; 14%, 5%, 5%, AG >65YA respectively). Young children are moreoften diagnosed with bronciolitis than pneumonia (30%, 7% respectively)but adults and elderly with pneumonia than bronciolitis (22%, 6%, AG 2064YA; 30%, 15%, AG >65YA respectively). Children and teenagers areequally diagnosed with both. In PHC Flu represents the highest burden inall AGs. In H AV, RV, RSV, Flu represent the highest burden in childrenand Flu represents the highest burden in adults and elderly.REF 369Characteristisc of influenza season 2012/2013 and differences in comparisonto previous Influenza Seasons in SloveniaNatasa BERGINC, Katarina PROSENC TRILARLaboratory for Virology, National Institute of Public Health, Ljubljana,SLOVENIAAt National Influenza Centre specimens from patients with influenza likeillness and acute respiratory tract infections with personal, clinical andepidemiological data are collected from a sentinel of 45 primary healthcaredoctors and 2 hospitals. Detection and subtyping of influenza (Flu)is performed with RT RT PCR: FluA subtypes AH1, AH1pdm, AH3,AH5 and FluB lineages Victoria (Vic), Yamagata (Yam). Usually in theinfluenza season (IS) one subtype predominates (IS 2004/05 FluB, IS2006/07 AH3, IS 2007/08 AH1, IS 2009/10 AH1pdm). It is commonthat in the end of an IS another subtype overcomes the predominant one(most often FluB overcomes FluA: IS 2008/09 FluB overcame AH3). InIS 2010/11 AH1pdm and FluB had equal shares trough the whole IS.During the pandemic in 2009 an unusual IS was observed with 2 peaks: insummer and in autumn with predomination of AH1pdm. An average peakof an IS lasts 6 weeks (W). IS 2012/13 differs from others in 2 aspects:constant simultaneous circulation of 4 different Flu and a duration. Atthe start of IS there were 70% FluA, 30% FluB. That changed graduallyduring the IS to 30% FluA, 70% FluB. Amongst FluA H1pdm and H3were present in relatively stable ratio (70%, 30% respectively). AmongstFluB Yam and Vic were constantly present with a changing ratio: at thebeginning Yam predominated over Vic, in the middle they were presentin equal ratios and towards the end Yam overcame Vic again. The firstFlu (AH1pdm) was detected in W49/2012, the peak lasted from W4/2013to at least W12/2013 (abstract submission), so it was at least 8 weekslong.REF 370Hepatitis E virus epidemiology: is serology sufficient?Sylvia BRUISTEN 1 , Kelly VAN DIJK 1 , Martijn VAN ROOIJEN 1 ,David KWA 2 , Boris HOGEMA 3 , Gini VAN RIJCKEVORSEL 11 Public health service of Amsterdam, Amsterdam, THE NETHERLANDS;2 OLVG General Hospital, Amsterdam, THE NETHERLANDS; 3 SanquinBlood bank, Amsterdam, THE NETHERLANDSNon travel related hepatitis E virus (HEV) infections are reported toincrease in many industrialized countries. A high prevalence of HEVin the general population may be indicative for the threat for HEVS222 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologytransmission to immune compromised patients who may experienceserious clinical consequences. We aimed to establish the added valueof detecting HEV by PCR compared to IgM and IgG serology in a lowrisk general population and a selected hospital patient group with suspicionof viral hepatitis in Amsterdam and surroundings, the Netherlands.At the Public Health Laboratory, Amsterdam a real time Taqman PCRtargeting the Orf3A region of HEV was validated. A panel with knownHEV genotypes was used to establish the sensitivity and specificity. Storedplasma samples (n=90), collected in 2004 among the general population inAmsterdam were used. Serum samples (n=30) collected in 2012 from differentoutpatient clinics and hospital wards from patients with complaintsrelated to viral hepatitis were tested by PCR and for IgM and IgG HEVantibodies (Wantai kit). Nucleic acids were isolated from plasma using aTriPure kit (Roche diagnostics). PhHV was used as internal control in thereal time PCR.The HEV PCR targeting the orf3A region had a sensitivity of 83% and100% specificity relative to the validation panel and could detect at leastgenotypes 1 and 3. When applied to the 90 plasma samples none werepositive in the HEV PCR. However, 44 samples were HEV IgG positive,and one was also HEV IgM positive. A HEV IgG prevalence of around 50%was thus noted and even when considering that we used a biased sampleselection, the HEV prevalence was much higher than among healthy blooddonors in the Netherlands (27%). Testing the sera of the 30 non ABChepatitis patients showed one positive HEV PCR result. This sample wasfrom a 65 year old male who was seen with hepatitis like complaints. Hewas also IgM and IgG positive, indicating a recent infection. An additional5 sera (from 3 males and 2 females) were both IgG and IgM positive. Theadded value of performing HEV PCR would thus have been very low forthese hospital patients. We conclude that in non travel related hepatitispatients the serology for HEV suffices to establish HEV as a cause. HEVPCR may remain of added value in suspected cases of acute hepatitis andin immune compromised persons.REF 371The results of the implementation epidemiological surveillance systemand control and response measures to influenza, acute viral respiratoryinfections (ARI) and severe acute respiratory infection (SARI)Spinu CONSTANTIN, Scoferta PETRU, Cojocaru RADU, EderVERONICA, Spinu IGOR, Gostev IGOR, Gheorghita STELANational Center for Public Health, Chisinau, REPUBLIC OF MOLDOVAThe health system of the Republic of Moldova has a nominated infectionssurveillance system, developed with the support of the World Bankand adjusted to the requirements of WHO, ECDC and CDC, which isconnected to the European network EuroFlu and global FluNet WHOinfluenza surveillance, ARI and SARI. This system includes the NationalInfluenza Center accredited by WHO, a biosafety level BSL 2 and BSL 2+,with modern equipment, supplies and qualified personnel able to performthe techniques of classical virology and molecular biology. Exploring theepidemiological surveillance system for influenza during 2012 (week 40)2013 (week 12) made it possible to highlight: extensive spreading, mediumintensity 12,770/0000, apogee at week 08/2013, the trend of increas anddiscreas respectively 2.8 0/0000 and 2,24 0/0000 Influenza morbidity wasincluded within weeks 04/2013 12/2013, identified viruses: A (H1N1)pdm, A (H3N2) and B dominant strain A (H1N1)pdm (51.44%), the rate ofpositive samples during apogee, increas and discreas, was 48.8%, 58.5%and 47.7% respectively, were mostly affected person aged 15 64 years(63.2%) and children 0 14 years (32.0%). Categories of pregnant womenand patients with immunocompromised status with confirmed influenzawere 22, 1% and 22.5%, respectively. ARI morbidity in apogee periodwas 468,60/0000 and reached epidemic threshold 189,70/0000 and bySARI – 76,6 0/0000 increased compared to the same period of last season– 48,30/0000. Control and response measures: making epidemiologicalsurveillance based on specific and nonspecific indicators allowed to vaccinateabout 100 000 at risk population, predict in real time the evolutionof influenza morbidity, health system impact assessment, then appreciatedas moderate, confirm clinical biodiagnostical influenza, arguing the needfor initiation of therapy avoid severe postinfluenza complications, appreciatingthat Oseltamivir and Zanamivir sensitive influenza virus strainsidentified, demonstrating isolated strains belonging and place global phylogenetictree and argue the need to extend the at risk population forimmunization with vaccine recommended by WHO for the season 20132014.REF 372Active cytomegalovirus infection among patients in the clinics of theUniversity of Szeged between 2008 and 2012Judit DEÁK 1 , Laura Elizabeth BROWN 1,2 , Judit NÉMETH 1,2 , RozáliaPUSZTAI 21 Institute of Clinical Microbiology, Faculty of Medicine, University ofSzeged, Szeged, HUNGARY; 2 Department of Medical Microbiology andImmunobiology, Faculty of Medicine, University of Szeged, Szeged, HUN-GARYThe incidence of active cytomegalovirus (CMV) infection was determinedamong patients in the clinics of the University of Szeged, Hungaryfrom January 2008 to December 2012. This study included all consecutivepatients who were examined for active CMV infection. Duringthe 5 year period 2151 samples (EDTA/blood, urine or liquor) from 994patients (485 male, 509 female) were investigated. CMV load was monitoredby real time quantitative PCR (Roche). IgM and IgG antibodiesspecific for CMV were tested in sera by ELISA (Sorin). CMV activeinfection occurred in 135 (13.6%) patients ranging from 0 days to 75years old (mean age 34 years). Twenty four, 15, 39, and 22% of themwere in age groups 60 years, respectively. Theactive CMV infections were linked to malignant disease (36%), congenital/perinatalinfection (20%), renal disease (14%), and other diseases(30%), such as neurological disorder (7 cases), autoimmune disease (5cases), etc. Antiviral treatment (ganciclovir, valganciclovir or acyclovir)was initiated in 75 (55.6%) patients and CMV load was monitored. Theantiviral treatment was effective in 50 patients (66.6%). In conclusion, theincidence of active CMV infection is substantial among patients with malignantdisease, congenital infection, and renal disease and may affect theoutcome of disease. More extensive studies of statistical impact investigatingactive CMV infection, especially in patients with malignant disease,are necessary. The impact of antiviral agents on clinically meaningfuloutcomes in these patients should be determined.REF 373Viral hepatitis C in hemodialysisMohamed EL AMRANI 1 , Mohamed Amine HAMZI 1 , WafaeARRACHE 1 , Driss KABBAJ 1 , Taoufik DOBLALI 2 , BouchraBELFQUIH 2 , Mohammed Reda TAGAJDID 2 , Hicham EL ANAZ 2 ,Nadia TOUIL 2 , Saad MRANI 2 , Mohammed BENYAHIA 11 Department of nephrology, dialysis and renal transplantation, Rabat,MOROCCO; 2 Department of human and molecular virology, Rabat,MOROCCOBackground: Hepatitis C virus (HCV) infection is the first hepatitis etiologyin chronic hemodialysis. Our objective is to define the anti HCVantibody prevalence, the seroconversion rate and the main HCV infectionrisk factors in hemodialysis patients in the military teaching hospitalMohammed V. Patients and methods: ambispective study concerning141 hemodialysis patients, between April 2010 and September 2012.HCV serology and PCR were both performed. Results: the mean age was52.6 years with a male predominance. The median hemodialysis durationwas 43.0 months. Predominant initial nephropathy was diabetes (25.5%).Virologie, Vol 17, supplément 2, septembre 2013S223

5 th European Congress of VirologyBlood transfusion was noted in 46.2% of cases. HCV prevalence was20.5%. Two HCV seroconversion cases (1.4%) were diagnosed. Conclusion:beside longer hemodialysis duration, blood transfusion remains amajor risk factor of HCV infection in hemodialysis patients despite thesystematic anti HCV screening in blood donors. The nosocomial transmissionis certain in hemodialysis departments. More respect of Hygienerules and adapted strategy are needed to prevent HCV transmission.REF 374Preliminary results on Human T cell lymphotropic virus HTLV ½identification among the blood donorsSpinu IGOR, Guriev VLADIMIR, Spinu CONSTANTINNational Center for Public Health, Chisinau, REPUBLIC OF MOLDOVACurrently, habilitate requirements submitted by the structures of the ECto the service of blood include testing of mandatory donated blood, bloodcomponents, including stem cells to the presence of markers of viral hemotransmisibileinfections: infection with viral hepatitis B, C, HIV infection,cytomegalovirus and infection with HTLV 1 and HTLV 2 (Human Tcell lymphotropic virus HTLV ½). Laboratory investigations algorithmin national blood service achieved by the insurance for the biosafety riskexclusion from the hemotransmisibile transmitting of viral etiology infectionsincludes testing of the donors for human imunodificiency virus andviral hepatitis B and C. In this context a special scientific practically interestpresents data to presence of this Human T cell lymphotropic virusamong on blood donors. The results show that during investigation ofblood samples collected from donors in number of 258 people aged 19 to59 years, five persons were HTLV positive. Preliminary obtained resultsallowing us to following: to organize together with donor service additionalresearches on markers HTLV for donors of blood; studying the possibilityof supplementing the donors blood investigation on infections by viruseshepatitis B, C and HIV, with additional HTLV virus, in the National Centersfor blood transfusion, reducing the risk of post transfusional disease,caused by the named virus; extending these studies and the other contingentsof population in particular with increased risks of infection withhepatitis viruses B, C and HIV, taking into account the fact that associateways of transmission.REF 375Distribution of Torque Teno Viruses (TTV) in healthy Bulgarian populationZlatko KALVATCHEV 1 , Iliya TSEKOV 1 , Kalina SHISHKOVA 2 ,Rumen POPOV 3 , Stoyan SHISHKOV 21 Military Medical Academy, Center for Virology Diagnosis, Sofia, BUL-GARIA; 2 University of Sofia “St. Kl. Ohridski ”, Faculty of Biology,Laboratory of Virology, Sofia, BULGARIA; 3 Military Medical Academy,Center of Transfusional Haematology, Sofia, BULGARIABackground: Torque teno viruses (TTV) are novel circular, single strandedDNA viruses first identified in patients with post transfusion hepatitisof non A G type. The prevalence and genetic heterogeneity of TTV isshown in patients with various diseases, but TTV are not linked to a particularcondition. TTV are widespread and relatively little is known abouttheir pathogenic potential, especially among healthy humans. Also datais not present for the prevalence of TTV in the Bulgarian population.The present study analyzed TTV loads in two groups of clinically healthypeople that donated blood or performed prophylactic examinations.Methods: Groups of 40 healthy people and 40 blood donors, which werenegative for common pathogens such as HIV, HBV, HCV, CMV, EBVwere included. DNA extraction from blood samples was performed withthe QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Germany) and TTVsequences were amplified in nested PCR with primers for the 5 ′ UTR ofthe viruses. The amplification product was visualized on an ethidium bromidestained 2% agarose gel. Results and Discussion: This is the firstreport to demonstrate TTV among Bulgarians with a prevalence of 46/80(57.5%). These results indicate the widespread of TTV among healthyindividuals and correspond to the data published by other authors, whichreport the viruses in up to 90% of the human population. The adaptedPCR system is effective and can be used for large scale screening studiesconcerning the prevalence of TTV in Bulgaria, as well as to clarify therole and significance of these viruses in human pathology.REF 376Human parechovirus 4 Caused Neonatal Cases with Suspected Sepsisin FinlandPekka KOLEHMAINEN 1,2 , Anne JÄÄSKELÄINEN 1,3 , HannimariKALLIO KOKKO 1,3 , Tea NIEMINEN 4 , Marjaleena KOSKINIEMI 1 ,Sisko TAURIAINEN 2 , Maija LAPPALAINEN 31 Haartman Institute/University of Helsinki, Helsinki, FINLAND;2 University of Turku, Turku, FINLAND; 3 HUSLAB/Helsinki UniversityCentral Hospital, Helsinki, FINLAND; 4 HUS/Helsinki University CentralHospital, Helsinki, FINLANDHuman parechoviruses (HPeVs), close relatives of enteroviruses, havebeen associated with neonatal sepsis like syndrome, central nervous systeminfections, respiratory symptoms and gastrointestinal infection. Whilemost HPeV types are primarily associated with infections of a mildoutcome, HPeV3 has been observed to be responsible for more severeinfections. HPeV4, originally extracted from a child with fever, has notbeen linked to more severe infections. In October, 2012 two neonates aged1 and 2 months were hospitalized with suspected sepsis in Finland. Bothof the patients had high fever and one of them had marbleizing skin, whichdeveloped in three days into an enterovirus like macular rash. No causativeagent was detected with routine bacterial and viral diagnostics from blood,urine, spinal fluid and stool samples. Samples were then directed to HPeVreal time PCR. Both patients were positive for HPeV in stool and one alsoin serum. The causative agent was typed to be HPeV4 in both cases. Inthis study we show that HPeV4 may cause more serious infections withdermatological manifestations and sepsis like symptoms. Additionally, weconfirm that more severe HPeV infections occur also in Finland. Theseare first reported HPeV4 cases in Finland. Patients with neonatal sepsis orsuspected sepsis require often intensive care and thus clarifying its causativeagent is essential. These cases underline the importance of HPeVinfection diagnosis as equally vital as that of enteroviral infection.REF 377Extensive genetic variation of seasonal A/H3N2 influenza viruses inGreece during the winter period 2011 2012Athanasios KOSSYVAKIS 1 , Angeliki MELIDOU 2 , Maria EXINDARI 2 ,Georgia GIOULA 2 , Antonios KALLIAROPOULOS 1 , VasilikiPOGKA 1 , Afroditi MOUTOUSI 1 , Mary EMMANOUIL 1 , AndreasMENTIS 1 , Sotirios TSIODRAS 3 , Petros KARAKITSOS 4 , NikolaosMALISIOVAS 21 National Influenza Reference Laboratory for S. Greece, Hellenic PasteurInstitute, Athens, GREECE; 2 Laboratory Department, MedicalSchool, Aristotle University of Thessaloniki, Thessaloniki, GREECE;3 Hellenic Centre for Disease Control and Prevention, Athens, GREECE;4 Department of Cytopathology, University General Hospital “Attikon”,School of Medicine, National and Kapodistrian University of, Athens,GREECEObjectives: this study aimed to identify emergence of genetic and antigenicvariation of A (H3N2) strains circulated in Greece during 2011-2012winter period. Methods: respiratory specimens from influenza like ill-S224 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyness cases were sent weekly by sentinel GPs to the two Greek NationalInfluenza Centres. Specimens were also received from outpatients and hospitalisedcases. RNA was extracted from 36 influenza A(H3N2) strains. RTPCR and sequencing was performed and nucleotide sequences were usedfor the molecular and phylogenetic analysis using the neighbour joining(NJ) method implemented in MEGA 5.0. Results: phylogenetic analysisof Greek H3N2 strains indicated clustering within the Victoria/208 cladei.e. groups 3A, 3B, 3C and 6. Interestingly no isolates fell into the geneticPerth/16 clade characterized by the H3N2 component of the 2011 12influenza vaccine. Variations on antigenic sites accumulated on the A, E,C and D, while B antigenic sites were conserved. Amino acid analysis ofA/Athens/16/2012 strain revealed the emergence of 4 amino acid substitutionsin 2 different antigenic sites indicating potential antigenic drift. All ofthe Greek A(H3N2) examined strains possessed the L183H substitutionthat affected the HA receptor binding site. Variations were also observedon the N linked glycosylation sites of HA. All 2011 12 isolates hadan altered potential N linked glycosylation site at amino acids 144–146.Four Southern greek stains that belonged to the 3C phylogenetic group,also possessed an additional glycosylation site, due to substitution S45N.These alterations may have influenced the function of the glycoprotein ofthose viruses.Conclusion: Our findings confirmed the genetic instability of influenzatype A (H3N2) viruses and the importance of continuous molecular surveillancefor the effective management of viral epidemics.REF 378Molecular characterisation of Human Hepatitis E virus from ItalyGiuseppina LA ROSA 1 , Marta FRATINI 1 , Marcello IACONELLI 1 ,Michele MUSCILLO 1 , Simonetta DELLA LIBERA 1 , MicheleEQUESTRE 2 , Angela CANDIDO 2 , Stefania TAFFON 2 , PaolaCHIONNE 2 , Elisabetta MADONNA 2 , Stefano DETTORI 2 , RobertoGIUSEPPETTI 2 , Roberto BRUNI 2 , Anna Rita CICCAGLIONE 21 Department of Environment and Primary Prevention, Istituto Superioredi Sanità, Rome, ITALY; 2 Department of Infectious, Parasitic and ImmuneMediated Diseases, Istituto Superiore di Sanità, Rome, ITALYHuman hepatitis E virus (HEV) is an emerging pathogen in industrialisedcountries. We re analysed a collection of serum samples previouslyconfirmed as HEV positive by anti HEV IgM and IgG assays as well as byReal Time PCR, with the aim of shedding light on the molecular epidemiologyof HEV in Italy. Samples were analyzed by published and newlydesigned PCR assays, using four sets of primers targeting the ORF1 andORF2, followed by phylogenetic analysis. Despite the use of four differentassays, not all samples tested positive by nested PCR. Moreover,no single method was able to detect all positive samples. Most sequencesgrouped with genotype 1, and originated from patients who had travelledto endemic areas, while the minority belonged to genotype 3, and originatedfrom Italian patients with no travel history. Phylogenetic analysisshowed a match between sequences derived from patients with travel relatedHEV and sequences from the geographical regions in which infectionwas acquired (mostly Bangladesh and India). Sequences from patientswith no travel history clustered on the same branch with published swineHEV isolates. Although autochthonous cases do occur, this study confirmsthat HEV in Italy is predominantly travel related. Broad range methodsfor molecular diagnosis of HEV are still in need of improvement. Thestudy was co funded by the Italian National Centre for the Prevention andControl of Diseases (CCM) project Hepatitis E Surveillance in Italy: anemerging disease in industrialised countries, and by the EU 7th Frameworkprogramme PREDEMICS project.REF 379Human Hepatitis E virus in urban wastewaters in ItalyGiuseppina LA ROSA, Marta FRATINI, Marcello IACONELLI,Simonetta DELLA LIBERA, Michele MUSCILLODepartment of Environment and Primary Prevention, Istituto Superiore diSanità, Rome, ITALYIn industrialised countries, Hepatitis E infection (HEV) is usuallyassociated with travel to endemic areas, but a growing number of sporadiccases are also seen in patients with no travel history. The objective ofthe present study was to investigate the occurrence of HEV in Italythrough the molecular screening of raw sewage samples collected fromurban wastewater treatment plants (WTPs). Three sets of broad rangePCR primers targeting the ORF1 (MTase and RdRp) and ORF2 (capsid)were used to examine 200 sewage samples collected throughout Italy(38 WTPs). PCR products were then subjected to sequencing andphylogenetic analysis. Despite the use of different assays, HEV RNAwas detected only in two samples, both belonging to genotype 3. On thephylogenetic tree, sequences clustered on the same branch with HEVisolates identified in serum samples of Italian patients with autochthonousHEV (no travel history). Although different studies indicate that HEVin Italy is predominantly travel related, in this study no genotype 1was detected. The study was co funded by the Italian National Centrefor the Prevention and Control of Diseases (CCM) project Hepatitis ESurveillance in Italy: an emerging disease in industrialised countries, andby the EU 7th Framework programme PREDEMICS project.REF 380High seroprevalence of Hepatits E virus among Egyptian BloodDonorsLobna METWALLY 2 , Endale TADESSE 1 , Alaa El Din SAAD ABD ELHAMID 31 Department of Medical Laboratory Science, College of Medicineand Health Sciences, Hawassa University, Hawassa, ETHIOPIA;2 Department of Microbiology, Faculty of Medicine, Suez Canal University,Ismailia, EGYPT; 3 Department of Clinical Pathology, Faculty of Medicine,Suez Canal University, Ismailia, EGYPTAim: the aim of this study was to assess seroprevalence of hepatitis E virus(HEV) among blood donors attending blood transfusion center of SuezCanal University Hospital during the study period. Method: 488 subjectsconsisted of 137 anti HCV positive donors, 35 HBsAg positive donors, and316 blood donors who were negative HBsAg, anti HCV and HIV attendingSuez Canal University Hospital blood transfusion center were included inthis study. All the study subjects were tested for anti HEV by ELISA andthe positive samples were further tested for HEV RNA by reveres transcriptasepolymerase chain reaction (RT PCR) method. Results: in a total of488 subjects, 102 (20.9%) were positive for anti HEV by ELISA. Anti HEVwas detected in (56/316) 17.7%, (10/35) 28.57%, and (36/137)26.28%, ofblood donors negative for both HBsAg & Anti HCV, HBsAg positive andAnti HCV positive donors, respectively. No significant (P>0.05) associationwas found between anti HEV positivity and HBsAg positivity andanti HCV positivity subjects. Anti HEV among blood donors with normalALT level (40) who were negative for HCV Ab, HBs Ag, and HIVwas 19.6%(43/219) and 13.4%(13/97) respectively. There was no statisticalsignificance (P>0.05) between the increase ALT level and Anti HEV.Conclusion: Seroprevalence of HEV antibody among blood donors in ourstudy in Ismailia, Egypt is high, but we cannot recommend screening of allblood donors for HEV until more data becomes available and until moreis known about the parenteral rout of transmission of HEV.Virologie, Vol 17, supplément 2, septembre 2013S225

5 th European Congress of VirologyREF 381Phylogenetic analysis in an outbreak of Echovirus 30 aseptic meningitisin ItalyMaria Grazia MILIA 1 , Francesco CERUTTI 1 , Gabriella GREGORI 1 ,Elisa BURDINO 1 , Tina RUGGIERO 1 , Tiziano ALLICE 1 , AlexPROIETTI 1 , Barbara SIMONCELLI 1 , Filippo LIPANI 2 , Gianni DIPERRI 2 , Valeria GHISETTI 11 Laboratory of Microbiology and Virology, Infectious Diseases Department,Amedeo di Savoia Hospital, Torino, ITALY; 2 Department ofInfectious Diseases, Amedeo di Savoia Hospital and University of Torino,Torino, ITALYBackground/Aim: Enteroviruses (EVs) are RNA viruses causing a varietyof diseases, including aseptic meningitis. Recent EVs aseptic meningitisoutbreaks have been reported in European countries. We report an outbreakof aseptic meningitis from October 18th to November 2nd, 2012 involving10 adult patients in the area of Turin, North Western Italy.Methods: All patients were parents or close relatives of children aged

5 th European Congress of Virologyisolation of viruses in cell cultures has proved to be an interesting tool toinvestigate the circulation of eventual PV. Also, the surveillance of NPEVmight allow us to associate any outbreaks of HEV infection in the populationwith the constant presence of certain viral serotypes in the raw sewagesamples.REF 385Epidemiological Surveillance of Measles in the North Central Regionof Emilia Romagna, ItalyGiulia PICCIRILLI 1 , Maria Grazia PASCUCCI 2 , Liliana GABRIELLI 1 ,Bianca Maria BORRINI 2 , Laura MOSCHELLA 2 , Gabriella FRASCA 2 ,Alba Carola FINARELLI 2 , Angela CHIEREGHIN 1 , EvangeliaPETRISLI 1 , Maria Paola LANDINI 1 , Tiziana LAZZAROTTO 11 Operative Unit of Clinical Microbiology, Laboratory of Virology, StOrsola Malpighi General Hospital, University of Bologna, Bologna,ITALY; 2 Public Health Service, Emilia Romagna Region, Bologna, ITALYIntroduction: Measles is the most frequent cause of vaccine preventablechildhood deaths. The WHO’s European Regional Committee has set 2015as the target year for the elimination of Measles Virus (MV) from allmember states. As in other European countries, various measles outbreakshave recently occurred in Italy. The purpose of this study was to confirmsuspected cases of measles and identify the MV genotypes circulating inEmilia Romagna Region from April 2010 to June 2012. Material andMethods: The samples tested (156 urine, 5 saliva) were related to 161index cases of the various outbreaks recorded in Emilia Romagna. Allsamples were subjected to F gene amplification by single round PCR.Only positive samples were subjected to subsequent amplification of Ngene a region (450 nucleotides of the COOH terminal) by nested PCR,for molecular characterization of the virus isolated. Results: Out of 161samples, 100 (62.1%) showed positive results for MV genome detection.To date, 90 out of 100 positive samples were subjected to genotypinganalysis. Four different genotypes were identified, D4 (47/90; 52.2%), D8(36/90; 40%), D9 (1/90; 1.1%) and B3 (6/90; 6.7%). Conclusion: Ourdata show the circulation of a limited number of viral genotypes exactlyas described for all countries with endemic transmission of MV. On thecontrary, reports from countries in MV elimination phase show that thereis the co circulation of more strains. The predominant genotype circulatingin Emilia Romagna is the D4 genotype which is prevalent throughout Italyas well as in other European countries.the ESU showed that the percentage of affected children aged 0 9 yearsis significantly decreasing while the number of affected individuals aged10 39 years is increasing with time. Furthermore the seroprevalence dataon the healthy Lebanese adults did not exceed 72% and this was remarkablylower when compared to earlier studies in Lebanese adults. Bothobservations confirm HAV epidemiological shift in Lebanon and hencean increased risk of HAV outbreaks among adults. Conclusion: In mostMENA countries including Lebanon a campaign for universal childhoodHAV vaccine should be implemented. A catch up vaccination approachdirected at 10-50 years of age groups should also be considered.REF 387Risk factors for swine Influenza in different States In MexicoEdith ROJAS ANAYA 1 , Catalina TUFIÑO LOZA 1 , Elizabeth LOZARUBIO 1 , Jose Juan MARTINEZ MAYA 2 , Fernando DIOSDADO 1 ,Atalo MARTINEZ LARA 1 , Luis GÓMEZ NÚÑEZ 1 , Maria EugeniaMANJARREZ 3 , Carlos CABELLO 3 , Dionicio CORDOVA 1 , ArturoGARCIA 11 CENID Microbiología, INIFAP, México city, MEXICO; 2 FMVZ UNAM,México city, MÉXICO; 3 INER, México city, MÉXICOIn Mexico, there are few studies about risk factors involved in the spreadof influenza virus in swine farms. The aim of this study was to identify riskfactors for swine flu virus in the states of Guanajuato, San Luis Potosi,Queretaro, Michoacán and Jalisco. 2048 samples were taken from 256pig farms in order to determine serological frequencies of endemic H1N1,H3N2 and pandemic H1N1using inhibition hemagglutination test. Identificationof possible risk factors associated with exposure of each subtype,was made by Chi square, Odds ratio of 0.05 with a significance and alogistic regression analysis for factors with a value of P 1, P

5 th European Congress of Virologyanti HCV were part of the technical staff, which practically excludes theirlikelihood of infection in hemodialysis units. In the south of the country thestudy was conducted in one hemodialysis unit. For anti HCV marker wereinvestigated 34 blood samples from patients, of which 11 (32,3%) werepositive. Samples from health workers, numbering 9, were negative for antiHCV marker. Overall, the level of infected hemodialysis patients is 39%,and among medical staff of hemodialysis units 5,7%, higher, compared tothe estimated index in the general population 3,5%.REF 389Serodiagnostic studies of human parvovirus 4Kestutis SASNAUSKAS 1 , Paulius TAMOSIUNAS 1 , KarolisSIMUTIS 1 , Indre KODZE 1 , Regina FIRANTIENE 2 , ReginaEMUZYTE 2 , Rasa BURNEIKIENE 1 , Aurelija ZVIRBLIENE 11 Institute of Biotechnology of Vilnius university, Vilnius, LITHUANIA;2 Faculty of Medicine of Vilnius university, Vilnius, LITHUANIAHuman parvovirus 4 (PARV4) is a recently discovered new member ofthe Parvoviridea family not closely related to any of the known humanparvoviruses. PARV4 has been isolated from plasma of individuals withsymptoms of acute viral infection, however, till now PARV4 has not beenassociated with any disease and its prevalence in human population is notyet clearly established. In the current study, the major capsid protein VP2of PARV4 was generated in yeast Sacharomyces cerevisiae and used forserological detection of virus specific IgG and IgM in the sera of lowrisk individuals. One hundred seventy serum specimens obtained frompatients with acute respiratory diseases were tested for PARV4 specificIgG and IgM antibodies. Sixteen (9.4%) seropositive individuals werediagnosed, including 10 IgG positive, 12 IgM positive and 6 both IgGand IgM positive. Seven of 16 seropositive individuals were 3 11 yearsold children. None of them had an evidence of parenteral exposure toPARV4 infection. Our data demonstrate that recombinant yeast derivedVP2 protein self assembled to virus like particles represent a useful tool forstudying the seroprevalence of PARV4 infection. The presence of PARV4specific antibodies in a low risk group may indicate the possibility ofalternative routes of virus transmission.REF 390Anti HTLV I/II Seroprevalance in healthy blood donors in Izmir,Turkey. The first diagnosed two cases in TurkeyRuchan SERTOZ 1,2 , Ajda TURHAN 2 , Aysu DEGIRMENCI 2 , ServetBIÇEROGLU 2 , Bahar BASKIR 3 , Selda ERENSOY 1 , YesimAYDINOK 21 Ege University Medical Faculty Clinical Microbiology Department,Izmir, TURKEY; 2 Ege University Medical Faculty Blood Bank, Izmir,TURKEY; 3 Ankara University Statistics Department, Ankara, TURKEYHuman T cell lymphotropic virus type I (HTLV I) is the first humanretrovirus to be associated with malignant disease namely, adult T cell leukemia/lymphoma.HTLV I has also been associated with several diseases.HTLV I has a worldwide distribution with major endemic foci in the Caribbeanand Southern Japan. HTLV II is a closely related retrovirus that sharesconsiderable genomic homology with HTLV I but has not been proven tobe a pathogen. Major routes of transmission are blood transfusion, breastmilk and sexual activity. In this study, we examined the seroprevalance ofHTLV I/II among healthy blood donors attended to Ege University Hospitalin Izmir. 50.000 healthy blood donors were examined for the presenceof anti HTLV I/II antibody in their sera. Serum specimens were testedwith an enzyme immunoassay (ARCHITECT rHTLV I/II Reagent kit,Germany). Borderline and positive reactivity was investigated repeatedly.HTLV/II confirmatory test (INNO LIA HTLV I/II, Innogenetics, Ghent,Belgium) was used for confirmation of repeatedly reactive samples. Pearson’scorrelation analysis, hierarchical clustering methods (HierarchicalCluster Analysis Methods) were used for statistical analysis. 54 sampleswere repeatedly reactive and two were confirmed positive. Two donors arethe first HTLV confirmed donors in Turkey. Screening of blood donors forHTLV I/II, risk and cost effective analysis and management of positivedonors must be discussed.REF 391Does the distribution of HBV strains in blood donors reflect that ofHBV strains circulating in the general population?Tatjana TALLO 1,2 , Diana MOOR 1 , Irina RESHETNJAK 1 , TatianaKUZNETSOVA 1 , Tatjana PLAHHOVA 3 , Valentina TEFANOVA 1 ,Helene NORDER 2,41 National Institute for Health Development, Tallinn, ESTONIA; 2 SwedishInstitute for Communicable Disease Control, Solna, SWEDEN; 3 NorthEstonain Medical Centre Blood Centre, Tallinn, ESTONIA; 4 GöteborgsUniversitet, Göteborg, SWEDENAn unremunerated blood donation system was introduced in Estonia in2004. We compared demographic characteristics, HBsAg prevalence andrelative HBV genotype/subgenotype distribution in donors before (1998-2003) and after (2004-2009) the introduction of this system. We alsostudied whether distribution of HBV strains in donors reflected that ofHBV strains circulating in the population.121 out of a total of 208 (58.2%)HBsAg positive sera were selected for the present study. From these, 82and 39 sera were collected during 1998-2003 and 2004-2007, respectively.After introduction of the unremunerated blood donation system in Estonia,the total number of blood donors, donations as well as the number ofrepeated donors increased by factors of 1.2, 1.1 and 1.4 times, respectively.HBsAg prevalence decreased by 2.4 and 3.5 times, respectively. For 1998-2003, HBV strains were classified into genotype D (75%) – subgenotypesD1 16%, D2 66%, D3 18%; genotype A (22%) subgenotype A2 100%, andgenotype C (3%). For 2004-2007, the distribution was: genotype D (91%)– subgenotypes D1 26%, D2 – 44%, D3 – 29%, and genotype A (9%) –subgenotype A2 100%. Introduction of the unremunerated blood donationsystem in Estonia was effective in improving the quality of donors. Basedon the analysis of previously published data, epidemiological and phylogeneticdata we can conclude that distribution of HBV strains in blooddonors independently on blood donation system may reflect strains naturallycirculating in general population as well as an ongoing indigenousepidemiological processes.REF 392Mumps in vaccinated adolescents – Portugal 2012/2013Elsa VINAGRE 1 , Paula PALMINHA 1 , Eugénio CORDEIRO 2 , CarlosRIBEIRO 1 , Carla ROQUE 11 National Institute of Health, Dr. Ricardo Jorge IP, Lisbon, PORTU-GAL; 2 Department of Public Health.Regional Directors of Health Centre,Coimbra, PORTUGALMumps (epidemic parotitis) is a contagious disease caused by a RNA virus.The mumps virus has only one serotype but 12 different genotypes (A, B,C, D, F, G, H, I, J, K, L, N). The MMR vaccine containing Rubini strain wasintroduced in Portugal in 1987. Despite its high immunization coveragea large outbreak of mumps with notification of 19415 cases occurred in1997. Thereafter, the MMR composition was changed and the Rubini strainwas replaced by the Jeryl Lynn, which has been administered since 1998.Between November 2012 and January 2013 there were 48 reported casesof mumps in two high schools of Aveiro district in the centre of Portugal;98% of cases occurred in vaccinated adolescents. All adolescents had twodoses of MMR one containing the Rubini strain and the other the JerylLynn strain. The first 6 cases were laboratory confirmed. All cases showedpositive IgM and IgG. The mumps virus was detected in 2 cases. The viruswas identified as belonging to genotype G. The phylogenetically distanceS228 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyof this virus and the vaccine strains used (which belongs to genotype A) aswell as the vaccine effectiveness (during outbreaks) of 61.6% to 70% forJeryl Lynn strain and 0% to 12.4% for Rubini may explain the high numberof cases in this outbreak involving vaccinated teenagers. Additional studiesare needed to clarify this vaccine failure.Acknowledgements: The authors gratefully acknowledge to the patientsand to the public health staff which contributed, respectively, with samplesand information about each mumps cases.REF 393Results of the retrospective analysis of HPV genotypes at YeditepeUniversity Hospital from 2008 to 2012Gulden CELIK(YILMAZ) 1 , Iskender KARALTI 2 , Yesim GUROL 1 ,Cagatay ACUNER 1 , Burcu OKSUZ 1 , Pinar OZCAN 3 , YaseminOZTURK 1 , Baki EKCI 4 , Sahap AKSACLI 1 , N. Cem FICICIOGLU 3 ,Ozcan GOKCE 41 Yeditepe University Faculty of Medicine, Medical Microbiology, Istanbul,TURKEY; 2 Yeditepe University Faculty of Medicine, Faculty of HealthSciences, Istanbul, TURKEY; 3 Yeditepe University Faculty of Medicine,Obstetrics and Gynecology, Istanbul, TURKEY; 4 Yeditepe UniversityFaculty of Medicine, General Surgery, Istanbul, TURKEYObjective: to evaluate the results of retrospective analysis of HPV genotypesdetermined by multiplex PCR and reverse hibridization techniquesin the Microbiology Laboratory Yeditepe University Hospital. Methods:149 clinical samples (125 of them female and 23 of them male) werecollected from patients of different ages and sex who applied to differentclinics of Yeditepe University Hospital tested for HPV from May 2008to April 2012. Nucleic acid isolation of the genital samples were madeby recommended isolation kit (Roche Magna Pure Compact Nucleic AcidKit) by the company. For HPV DNA detection and typing, multiplex PCRand reverse hybridization techniques were used by Hybribio HPV Genoaraykit. Results: HPV DNA was detected in 47.2% of the female and 58%of the male patients. The HPV PCR positivity for the women and men, forhigh risk group was 67.8%, 13%; for low risk group 27.1%, 86.7% andundefined risk group 5.1% and 0.3% respectively. Of the 34 high risk HPVpositive samples which were from departmant of Obstetrics and Gynecoloy;type 16 was detected in 10 patients; type 31 in 7 patients; each oneof type 58 and type 66 in 3 patients; each one of type 18, type 52, type 33and type 56 in 2 patients; and each one of type 59, type 39 and type 45 inone patient. Of the 12 low risk HPV positive samples; type 6 was detectedonly in 5, type 11 only in 3, type 44 only in 2, type 43 and type CP8304only 1 patients. Type 53 was detected in 8 patients. More than one HPVgenotypes (mixed infection) were detected in9 clinical samples.Conclusion: although the study group was small, variability of HPVgenotypes was found to be worth presenting.REF 394Global WHO Measles-Rubella Laboratory Network – molecular surveillanceto support the disease elimination initiativesMick N. MULDERS, David FEATHERSTONE, Paul ROTA, JosephICENOGLE, Kevin BROWN, Richard MYERS, Peter STREBEL1 World Health Organization, Geneva, SWITZERLAND; 2 Independentlaboratory consultant, Hastings, NEW ZEALAND; 3 Centers for DiseasesControl and Prevention, Atlanta, USA; 4 Centers for Diseases Control andPrevention, Atlanta, USA; 5 Public Health England, London, UK; 6 PublicHealth England, London, UK; 7 World Health Organization, Geneva,SWITZERLANDIn the Global Vaccine Action Plan, measles and rubella are targeted forelimination in at least five of the six WHO Regions by 2020. Rapidand accurate diagnosis of measles and rubella is essential for monitoringprogress and detecting outbreaks. WHO coordinates a global MRLaboratory Network (LabNet) that provides valuable global informationabout the circulation of measles and rubella virus. It consists of 690 national,sub-national and regional laboratories, serving almost all memberstates. The laboratories follow standardized testing protocols and reportingmechanisms that are constantly reviewed and improved as technologicalinnovations occur. The LabNet relies on a strong quality assurance programthat monitors the performance of all laboratories through annualproficiency testing and continuous assessment. It also provides vital informationfor the immunization programs as it documents the successes ofvaccination efforts to interrupt measles and rubella chains of transmission,monitor virus transmission patterns and help document successfulelimination strategies. Examples of which will be given including theWHO genotyping databases MeaNS and RubeNS. Both databases havebeen developed by Public Health England together with the WHO GlobalMR LabNet to develop a web-accessible and quality-controlled nucleotidedatabase as tool to track measles and rubella sequence diversity andmonitor elimination of virus strains. At the time of submission, MeaNScontained well over 12000 viral sequences, and RubeNS is still in its pilotphase with close to 1000 sequences. The databases have been developedspecifically for the MR LabNet with only secure access.http://www.who-measles.orgREF 395Molecular Characterization of Barley Yellow Dwarf Virus isolatesfrom Different Regions of TunisiaMaryem BOUALLEGUE 1 , Maha MEZGHANI-KHEMAKHEM 1 ,Hanem MAKNI 1,2 , Mohamed MAKNI 11 RU “Genomics of devastating insects of agricultural interest crops”GIRC, Faculty of Sciences of Tunis, Tunis El-Manar University, Tunis,TUNISIA; 2 ISAJC Bir El Bey, Tunis University, Tunis, TUNISIAThe Barley Yellow Dwarf Virus assigned to the Luteoviridae, is transmittedin a persistent manner and circulated by several aphid species mainlyRhopalosiphum padi, Sitobion avenae and Schizaphis graminum. Thisvirus infects many hosts of Poaceae and causes reducing yield in grainwhich leading to serious agronomic and economic damage. In the presentstudy, serological and molecular tests were done in order to analyze theORF3 coding for the coat protein (CP) and characterize the BYDV isolatesoccurring in Tunisia. For this purpose, barley samples, collected followinga North-South trend, were subjected to a DAS-ELISA using antibodiesagainst three variants of BYDV PAV, MAV and RPV. Results revealed thatthe South area is the most affected area and showed a prevalence of PAVvariant. Then, for positive samples, CP gene were amplified, cloned andsequenced. A total of six PAV isolates were identified and named PAV-TN1to PAV-TN6. Phylogenetic analysis showed that Tunisian isolates presenta strong similarity with the Moroccan isolate (PAV-MA9517),the Frenchisolate (PAV-FH1) and the German one (PAV-G) suggesting that the virusmay be transmitted by the same migration aphid.REF 396The Rapid Implementation of a Novel Sampling and Molecular TestingAlgorithm to Facilitate the Management of a Large Outbreak ofMeasles in South WalesCatherine MOORE 1 , Simon COTTRELL 2 , Jorg HOFFMANN 3 ,Michael CARR 4 , Rachel JONES 11 Wales Specialist Virology Centre, Public health Wales MicrobiologyCardiff, University Hospital of Wales, CARDIFF, UNITED KINGDOM;2 Public Health Wales Communicable Disease Surveillance Centre, Templeof Peace and Health, CARDIFF, UNITED KINGDOM; 3 Health ProtectionDivision (Mid and West Wales), Public Health Wales, SWANSEA, UNITEDKINGDOM; 4 National Virus Reference Laboratory (NVRL),UniversityCollege Dublin, DUBLIN, IRELANDVirologie, Vol 17, supplément 2, septembre 2013S229

5 th European Congress of VirologyDuring November 2012, an increase in measles virus clinical notificationswas first described in populations with

5 th European Congress of Virology21. VIRUS DISCOVERY ANDMETAGENOMICS (EMPERIE)Posters: REF 397 to REF 416REF 397The pathogenesis and genetic diversity of rodent torque teno virusesShoko NISHIYAMA 1 , Bernadette DUTIA 1 , Peter SIMMONDS 1,2 ,Colin SHARP 11 Department of infection and immunity, The Roslin Institute and R(D)SVS,University of Edinburgh, Edinburgh, UNITED KINGDOM; 2 Centre forImmunity, Infection and Evolution, University of Edinburgh, Edinburgh,UNITED KINGDOMTorque teno virus (TTV) is a single stranded circular DNA virus and,despite its widespread nature in the human population, its pathogenesis isstill unknown. Factors complicating TTV research include its huge geneticdiversity, difficulties identifying an uninfected control population, the lackof a small animal model and lack of a good cell culture system for viralpropagation. Recently we have identified a TTV homologue (RoTTV)in wild rodents. RoTTV was frequently observed in wood mice (Apodemussylvaticus) and field voles (Microtus agrestis). RoTTV infectionswere also found in bank voles (Myodes glareolus) but not in Mus musculuspopulations. Analysis of complete genome sequencing shows thatseveral genetic variants are found in wild rodent population with two distinctspecies containing several diverse genotypes. Furthermore, multiplevariants were present in single individuals, consistent with infection patternsseen in humans. RoTTV transcripts in infected wild wood mice havealso been detected and fully sequenced. Predicted protein coding regionsfrom these transcripts have been expressed in cell culture and show thedifferent expression patterns. Using cloned genomic DNA, it has also beenpossible to observe the transcription from the virus in vitro and develop anin vivo model system in naïve laboratory wood mice. This research representsthe first detailed characterisation of anellovirus diversity in the wildUK rodent population and the establishment of a tractable small animalmodel for the study of viral pathogenesis.REF 398Isolation and characterization of novel orthobunyavirus (Californiaencephalitis virus) in Finnish mosquitoesNiina PUTKURI 1 , Satu KURKELA 1,2 , Lev LEVANOV 1 , EiliHUHTAMO 1 , Antti VAHERI 1 , Tarja SIRONEN 1 , Olli VAPALAHTI 1,2,31 Haartman Institute, Department of Virology, Faculty of Medicine, Universityof Helsinki, Helsinki, FINLAND; 2 HUSLAB, Helsinki UniversityCentral Hospital, Helsinki, FINLAND; 3 Department of Veterinary Biosciences,Faculty of Veterinary Medicine, University of Helsinki, Helsinki,FINLANDWe have recovered a new California encephalitis virus isolate of the genusorthobunyavirus in Finland. Genus orthobunyavirus (family bunyaviridae)includes a large group of mosquito borne viruses found all over the worldwhich many are important human and veterinary pathogens. Inkoo virus(INKV) has been the only representative of the genus in Finland sinceits isolation 1964. However, ongoing research and laboratory diagnosticshave been neglected for decades. We recovered 5 orthobunyavirus isolatesfrom altogether 1940 mosquitoes collected from eastern Finland duringat early autumn in 2007 and 2008 by inoculating Vero cells. We sequencedcomplete S and M segments and partial L segments and analyzedphylogenetic and serological relationships between the new isolates andother California encephalitis group viruses. Genetic and serological findingssuggest that the new virus isolates,called Möhkö virus (MÖHKV),are most closely related to clusters of Russian orthobunyavirus isolatesnamed Chatanga virus and further to Snowshoe hare virus and La Crossevirus. Some Chatanga virus isolates appear very similar to MÖHKV isolates(polyprotein similarity 98%) but majority are considerably divergent(polyprotein similarity 89-92%). S segment N protein identity to SSHVand LACV is 92-93% and 90-91%, M segment polyprotein identity 87%and 82%, respectively. MÖHKV is not closely related to INKV, whichwas not found in this study. Many of the California encephalitis groupviruses are well known human pathogens but the association of MÖHKVto human infection requires more investigation.REF 399Two Novel Parvoviruses in Frugivorous New and Old World BatsMarta CANUTI 1 , Anna Maria EIS HUEBINGER 2 , Martin DEIJS 1 ,Michel DE VRIES 1 , Jan Felix DREXLER 2 , Samuel K. OPPONG 3 ,Marcel A. MÜLLER 2 , Stefan M. KLOSE 2,4 , NeleWELLINGHAUSEN 5 , Veronika M. COTTONTAIL 4,6 , Elisabeth K. V.KALKO 4,7 , Christian DROSTEN 2 , Lia VAN DER HOEK 11 Department of Medical Microbiology, Academic Medical Centre (AMC),Amsterdam, THE NETHERLANDS; 2 Institute of Virology, University ofBonn Medical Centre, Bonn, GERMANY; 3 Department of Wildlife andRange Management, Kwame Nkrumah University of Science and Technology,Kumasi, GHANA; 4 Institute of Experimental Ecology, University ofUlm, Ulm, GERMANY; 5 Gaertner & Collegues Laboratory, Ravensburg,GERMANY; 6 Institute of Medical Microbiology and Hygiene, Universityof Ulm, Ulm, GERMANY; 7 Smithsonian Tropical Research Institute,Balboa, PANAMABats, a globally distributed group of mammals with high ecologicalimportance, are recognized as natural reservoir hosts for viral agents ofsignificance to human and animal health. We evaluated pools of bloodsamples obtained from two phylogenetically distant bat families: flyingfoxes (Pteropodidae), Eidolon helvum in West Africa, and two speciesof New World leaf nosed fruit bats (Phyllostomidae), Artibeus jamaicensisand Artibeus lituratus in Central America. A sequence independentvirus discovery technique (VIDISCA) was used in combination with highthroughput sequencing to detect two novel parvoviruses: a PARV4 likevirus (Eh BtPV 1) in E. helvum from Ghana and the first member of aputative new genus in A. jamaicensis from Panama (Aj BtPV 1). Thoseviruses were circulating in the corresponding bat colony at rates of 7–8%.Aj BtPV 1 was also found in Artibeus lituratus (5.5%). Both viruses weredetected in the blood of infected animals at high concentrations: up to10E8 and to 10E10 copies/ml for Aj BtPV 1 and Eh BtPV 1 respectively.Eh BtPV 1 was also detected in all organs collected from bats (brain, lungs,liver, spleen, kidneys and intestine) and spleen and kidneys were identifiedas the most likely sites where viral replication takes place. Our studyshows that bat parvoviruses share common ancestors with known parvovirusesof humans and livestock. We also provide evidence that a varietyof Parvovirinae are able to cause active infection in bats and that theyare widely distributed in these animals with different geographic origin,ecologies and climatic ranges.REF 402Identification of a novel herpes simplex virus type 2 (HSV 2) variantSonia BURREL 2 , Nathalie DESIRE 1 , Laurent DACHEUX 3 , LaureDIANCOURT 4 , Marie Edith LAFON 5 , Emiliana ABRAO 1 , SophieSEANG 6 , Eric CAUMES 6 , Valérie CARO 4 , Hervé BOURHY 3 , HenriAGUT 1,2 , David BOUTOLLEAU 1,21 UPMC Univ Paris 06, ER1 DETIV, Paris, FRANCE; 2 Service de Virologie,Hôpitaux Universitaires La Pitié Salpêtrière, Charles Foix, AP HP,Paris, FRANCE; 3 Unité de Dynamique des Lyssavirus et Adaptation àVirologie, Vol 17, supplément 2, septembre 2013S231

5 th European Congress of Virologyl’Hôte, Centre National de Référence de la Rage, Institut Pasteur, Paris,FRANCE; 4 Plateforme de Génotypage des Pathogènes et Santé Publique,Institut Pasteur, Paris, FRANCE; 5 Service de Virologie, Hôpital Pellegrin,Bordeaux, FRANCE; 6 Service des Maladies infectieuses et Tropicales,Hôpitaux universitaires La Pitié Salpêtrière – Charles Foix, AP HP, Paris,FRANCEHerpes simplex virus type 2 (HSV 2) is one of the most common sexuallytransmitted viruses. The viral genome variability reported so far is verylow, and few studies have reported the existence of distinct HSV 2 genogroups.Here we report the description of a new genetic variant of HSV2. The genotypic resistance testing of one HSV 2 isolate from a HIVinfected man revealed an unexpected high 2.4% divergence within UL30(DNA polymerase) gene in comparison with the reference strain HG52.The retrospective screening of numerous HSV 2 clinical isolates usinga specific real time PCR assay designed within UL30 gene permitted theidentification of 11 additional HSV 2 variant (HSV 2 v) isolates, leading toan overall prevalence of 2.2%. Phylogenetic analysis based on UL30 geneand US4 (glycoprotein G) gene, previously studied for HSV classification,evidenced that HSV 2 v isolates clustered in a genetic group distinct fromthe one of HSV 2 classical isolates. HSV 2 v isolates exhibited a specificmolecular signature in the DNA polymerase C terminus. They were genotypicallyand phenotypically susceptible to acyclovir and foscarnet. AllHSV 2 v were isolated from patients originating from West and CentralAfrica, and 8 patients were co infected by HIV. These results raise thequestion of the origin of this new virus and further studies are warrantedto assess its relationships to other human and primate simplexviruses. Preliminaryresults indicate that HSV 2 v might represent a zoonosis or resultfrom a recombination mechanism with a chimpanzee alphaherpesvirus.REF 404Molecular studies and genetic diversity among the begomovirusesinfecting Jatropha species in IndiaSUNIL KUMAR SNEHI, A. SRIVASTAVA, S.K. RAJCSIR National Botanical Research Institue, Lucknow, INDIAThe genus Jatropha family Euphorbiaceae has 476 species and distributedthroughout the world. Many excellent characteristics, including high yield,resistance to drought and biofuel oil have generated the interest of manyresearchers to J. curcas, while other species are of ornamental value or traditionallyused for their medicinal values. The virus diseases have a majorproblem throughout the India and other tropical and subtropical countriesresulting in great economic loss to Jatropha growers. The full length viralgenome was amplified from DNA isolated from infected J. curcas by RCAusing Ø29 DNA polymerase. Two begomovirus species Jatropha mosaicIndia virus (HM230683) and Jatropha curcas mosaic virus (JN692494)were identified from J. curcas and both the begomoviruses were consideredas monopartite begomovirus because of absence of DNA B moleculein their genome. The DNA A genome of CYVMV (EU727086) and DNA (EU604296) molecule associated with yellow vein mosaic disease of J.gossypifolia were identified for the first time in India. A new member of thegenus begomovirus, Jatropha yellow mosaic India virus (FJ177030) hasbeen identified first time in J. gossypifolia from India. Three begomovirusspecies associated with mosaic disease on ornamental Jatropha species viz.Jatropha mosaic India (HQ848382) on J. podagrica, ToLCPV (HQ848381)on J. multifida and PLCV (JQ043440) on J. integerrima were identified onthe basis of ∼1.2 kb. These results indicated that genetic diversity existsamong the begomoviruses infecting various species of Jatropha grown inIndia.REF 403Identification and molecular characterization of Potato spindle tuberviroid infecting ornamentals in CroatiaJasna MILANOVIC 1 , Jasna MILANOVIC 1 , Vesna KAJIC 1 , SnjezanaMIHALJEVIC 21 Institute for Plant Protection, Zagreb, CROATIA; 2 Ruder Boškovic Institut,Zagreb, CROATIASince the initial report of Potato spindle tuber viroid (PSTVd) presence inornamental solanaceous plants in the Netherlands, similar findings havebeen reported from various European countries. In order to assess theviroid presence in Croatia and to enable enforcement of European Unionlegislation, an extensive survey was conducted in 2009. During a four yearsof monitoring, 149 samples were tested: 62 Solanum jasminodes, 20 Solanumrantonnetii, 19 Brugmansia spp., 21 Petunia spp. and 27 Surfinia spp.Samples were collected from nurseries and consisted of eight randomlyupper leaves taken from individual plants. Total RNA was extracted fromsymptomless leaf tissue with a commercial kit (Qiagen, Germany) andused as template for one step RT PCR. The full length genome, correspondingto the viroid size (359 bp) was obtained using specific primers PSTVd32/33. Eight samples (6 S. jasminodes and 2 S. rantonnetii) were foundpositive in this assay. The amplicons were purified, cloned in pGEM T easyplasmid vector (Promega, USA) and sequenced (Macrogen Europe, theNetherlands). BLAST nucleotide sequence analysis confirmed their respectivePSTVd identity and phylogentic relationships were evaluated usingneighbour joining (NJ) implemented through MEGA 4.0. Although infectedplants did not show any symptoms, they might be sources of inoculafor crops like potato and tomato. Therefore, it is extremely important touse certified seed, apply sanitary measures and conduct regular inspectionmonitoring to prevent the spread this quarantine pathogen in the future.REF 405A novel putative tick orbivirus detected in a cell line derived from thetick Amblyomma americanumHoussam ATTOUI 1 , Mourad BELHOUCHET 1 , Fauziah MOHDJAAFAR 1 , Pilar ALBERDI 2 , Colin SHARP 2 , Ulrike MUNDERLOH 3 ,Timothy KURTTI 3 , Lesley BELL SAKYI 11 The Pirbright Institute, Pirbright, UNITED KINGDOM; 2 The RoslinInstitute and R(D)SVS, University of Edinburgh, Edinburgh, UNITEDKINGDOM; 3 University of Minnesota, Saint Paul, UNITED STATESIn addition to supporting replication of a wide range of arbovirusestransmitted by ticks, mosquitoes, midges and sandflies, many tick celllines harbour apparently endogenous viruses. Until recently, only oneof these “tick viruses”, the orbivirus St Croix River virus (SCRV) thatchronically infects cell lines derived from Ixodes scapularis and Rhipicephalusappendiculatus, had been identified and sequenced. Here wereport identification and sequence analysis of a second putative tick orbivirusdetected in a cell line, AAE12, derived from embryos of the lonestar tick Amblyomma americanum. The virus was initially detected bya combination of electron microscopy, RT PCR with pan orbivirus primersand deep sequencing. The single primer amplification technique wasused to further characterise genomic segments. This virus represents thefirst strain of a novel orbivirus species that, along with SCRV, we considerto be an ancestral orbivirus. Results will be presented of attempts toinfect cell lines derived from other tick species, mosquitoes and mammalswith this virus, and from screening of additional tick cell lines for itspresence.S232 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF 406Characterization of an infectious insect endogenous retrovirus froma Drosophila cell lineFranck TOURET, François GUIGUEN, Christophe TERZIANUMR 754, INRA,UCBL,EPHE, Université de Lyon, SFR Biosciences GerlandLyon Sud, Lyon, FRANCEInsect endogenous retroviruses (IERVs) are present in the genome ofseveral insect species. It was previously shown that gypsy is an active endogenousretrovirus in Drosophila melanogaster, which could be transmittedto individuals fed with gypsy containing extracts. Here we demonstrate theproduction of gypsy virions from the Drosophila cell line S1 (Schneider1) The gypsy virions are able to infect the permissive drosophila cell lineSS (Stable Strain), as shown by the production of gypsy envelope proteinin SS infected cells. We also demonstrated that this infection process canbe inactivated by heat and U V treatments. Finally, using electron microscopyand immunogold staining experiments, we have shown that virionspresent in the supernatant of S1 cells share morphological properties withvertebrate infectious retroviruses.REF 407Viral metagenomics in drug naïve, recent onset schizophrenia patientswith prominent negative symptomsMarta CANUTI 1 , Nico Jm VAN BEVEREN 2 , Jitschak G STOROSUM 3 ,Seyed Mohammad JAZAERI FARSANI 1 , Michel DE VRIES 1 , MartinDEIJS 1 , Maarten F JEBBINK 1 , Barbera Dc VAN SCHAIK 4 , AntoineHc VAN KAMPEN 4 , Antoinette C VAN DER KUYL 1 , Lieuwe DEHAAN 3 , Lia VAN DER HOEK 11 Department of Medical Microbiology, Academic Medical Center (AMC),Amsterdam, THE NETHERLANDS; 2 Delta Center for Mental HealthCare, Rotterdam, THE NETHERLANDS; 3 Department of Psychiatry, AcademicMedical Center, Amsterdam, THE NETHERLANDS; 4 Departmentof Clinical Epidemiology, Biostatistics and Bioinformatics, AcademicMedical Center, Amsterdam, THE NETHERLANDSSchizophrenia is a severe and disabling mental disorder of largely unknownaetiology. Although several lines of evidence suggest a virus or(endogenous) retrovirus involvement at the time of onset of (a subpopulationof) schizophrenia, the unequivocal identification of one or moreinfectious agents, by means of an undirected catch all technique, hasnever been conducted. In this study VIDISCA, a state of the art virusdiscovery method, was used in combination with Roche 454 high throughputsequencing as a tool to determine the possible presence of viruses(known or unknown) in blood of 23 recent onset drugs naïve schizophrenicpatients with prominent negative symptoms. Two viruses (Torqueteno virus and GB virus C) were detected. Both viruses are commonlyfound in healthy individuals and no clear link with disease was everestablished. When the hypothesis of endogenous retroviral involvementin schizophrenia was tested with specific molecular analysis and deepsequencing one sample was positive for human endogenous retrovirusestype K (HML 2) RNA. No specific predominant strain was detected, instead119 different variants were found. The higher expression/replicationlevel of HERVs in schizophrenic patients detected in previous studiesmight be a result of associated factors, like therapy, rather than a cause.In conclusion, these findings indicate no evidence for viral, retroviral orendogenous retroviral involvement in sera at the time of onset of schizophrenia.It is a priority to focus future research on the prenatal and perinatalperiod.REF 408Viral metagenomics: A systematic and representative analysis of thefall 2012 gastroenteritis outbreak in GermanyNicole FISCHER 1 , Malik ALAWI 2 , Daniela INDENBIRKEN 3 , NicoleWALZ 3 , Marina HÖHNE 4 , Claus Thomas BOCK 4 , Martin MIELKE 4 ,Martin AEPFELBACHER 1 , Adam GRUNDHOFF 11 University Medical Center Hamburg Eppendorf/Institute for MedicalMicrobiology and Virology, Hamburg, GERMANY; 2 University MedicalCenter Hamburg Eppendorf/HEXT Bioinformatic Support, Hamburg,GERMANY; 3 Heinrich Pette Institute, Leibniz Institute for experimentalVirology, Hamburg, GERMANY; 4 Robert Koch Institute, Berlin, GER-MANYIn fall 2012, the largest foodborne gastroenteritis outbreak in Germanyaffected approximately 11.000 individuals. Here, we have analyzed a subsetof outbreak samples using an unbiased metagenomic approach forthe detection and characterization of viral (and bacterial) pathogens. Wecompare the results to conventional diagnostic PCR in order to evaluatethe benefit of NGS approaches for the management of future outbreaks.RNA and DNA libraries prepared from 18 fecal samples were subjectedin a blinded fashion to next generation sequencing (NGS) on IlluminaHiSeq and MiSeq sequencers. Data were analyzed with a bioinformaticpathogen detection and analysis pipeline we developed. We detectednorovirus sequences in 8 of the 18 samples. High read coverage allowedus to assemble complete viral genomes from most samples. GI as wellas GII sequences were detected in 3 samples while 4 samples containedeither GI or GII. Our data are in good agreement with conventional diagnosticPCR results: ct values correlated well with the number of readsdetected by NGS. Low intergenetic diversity between the identified norovirussequences highly suggest a common source of infection which was,based on multiple norovirus genotypes per sample, contaminated withsewage. Conclusions: These results illustrate the potential of metagenomicapproaches in the detection of viral and/or bacterial sequences: NGSoffer unbiased, open approaches which are supportive of PCR based strategies.Both techniques together exhibit the best possible molecular analysisof future epidemics.REF 409Identification of a new genotype of Torque Teno Mini Virus (TTMV)Seyed Mohammad JAZAERI FARSANI 1 , Maarten F. JEBBINK 1 ,Martin DEIJS 1 , Marta CANUTI 1 , Karel A. VAN DORT 2 , NeeltjeKOOTSTRA 2 , Lia VAN DER HOEK 11 Laboratory of Experimental Virology, Department of Medical Microbiology,Academic Medical Center, University of Amsterdam, Amsterdam,THE NETHERLANDS; 2 Laboratory of Viral Immune Pathogenesis,Department of Experimental Immunology, Academic Medical Center, Universityof Amsterdam, Amsterdam, THE NETHERLANDSAlthough Human torque teno viruses (TTVs) was first discovered in 1997,still many aspects of this virus are not clear. Several studies have mentionedthe possible association of TTVs with a broad range of different diseasesin humans. TTVs reveal a remarkable heterogeneity of genotypes and itcould be that some genotypes are more pathogenic than others. Thereforeidentification of all genotypes is essential. In this study, using VIDISCA454 (virus discovery cDNA AFLP, Amplified Fragment – Length Polymorphismcombined with Roche 454 high throughput sequencing) we describea new genotype of TTMV in two HIV 1 positive patients. The patients hadtwo unusual characteristics; both patients have suffered a pneumococcalVirologie, Vol 17, supplément 2, septembre 2013S233

5 th European Congress of Virologypneumonia during follow up and both patients had extremely low B cellscounts. The low B cell count might be caused by the infection with TTMV,or it could be that an infection with the TTMV is poorly cleared due to thelow B cell immunity.REF 410Powerful sequence similarity searches identify homologs in manyapparently “orphan” viral proteins: a guide to genome annotatorsDavid KARLIN 1,2 , Durga KUCHIBHATLA 3 , Westley SHERMAN 3 ,Betty CHUNG 4 , Shelley COOK 5 , Georg SCHNEIDER 3 , BirgitEISENHABER 31 Department of Zoology, Oxford, UNITED KINGDOM; 2 Departmentof Structural Biology (STRUBI), Oxford, UNITED KINGDOM;3 Bioinformatics Institute (A*Biomedical Sciences Institutes), Singapore,SINGAPORE; 4 Department of Plant Sciences, Cambridge, UNITEDKINGDOM; 5 Life Sciences Parasite and Vectors Division, London, UNI-TED KINGDOMGenome sequences of new viruses often report many “orphans” or “taxonspecific” proteins apparently lacking homologs. However, we show thatcommonly used sequence similarity detection methods such as Blast orPsi Blast overlook homologs (predictably, since viral proteins evolve veryfast). We analysed a dataset of proteins characterized as “genus specific” byBlast, using powerful methods developed recently, such as profile profilecomparison (HHblits, HHpred). Homologs were detected in other generafor a quarter of these proteins. In depth manual analyses on a subset of theremaining sequences, guided by contextual information such as taxonomy,gene order or domain order, identified distant homologs for a third ofthem. Extrapolating these results, a combination of powerful automatedmethods and manual analyses may uncover distant homologs for overhalf of proteins found to be “orphans” by Blast. As a case in point, were analyzed the genome of a bee pathogen, chronic bee paralysis virus(CBPV), and identified homologs, and probable functions, for almost allits “orphan” proteins. CBPV encodes a previously unrecognized enzyme,a new type of virion membrane protein homologous to that of insect andplant viruses with a different morphology, and a virion glycoprotein alsofound in these viruses. We used only software available through web based,user friendly interfaces, and will present recommendations for virologistson how to use these methods to annotate (or re annotate) genomes.REF 411Unbiased detection of infectious agents in respiratory syndromes ofpoultry using a metagenomic approachEtienne LIAIS 1,2 , Guillaume CROVILLE 1,2 , Jérome MARIETTE 3 ,Christopher KLOPP 3 , Cécile DONNADIEU 4 , Jérome LLUCH 4 ,Mariette DUCATEZ 1,2 , Jean Luc GUÉRIN 1,21 Université de Toulouse, INP, ENVT, Toulouse, FRANCE; 2 INRA, UMR1225, Toulouse, FRANCE; 3 Plateforme bioinformatique Toulouse MidiPyrénées, UBIA, INRA, Castanet Tolosan, FRANCE; 4 GeT PlaGe, Genotoul,INRA, Castanet Tolosan, FRANCELittle is known so far on respiratory viruses in poultry. In order to identifyall the viral pathogens in duck respiratory tract, tracheal swabs were collectedon birds showing respiratory clinical signs and/or egg drop syndromesand were submitted to a metagenomic analysis. Using a high throughputsequencing approach (Miseq, Illumina), we aimed at describing therespiratory viral and bacterial flora of ducks showing respiratory clinicalsigns and/or egg drop syndromes. Viral nucleic acids were concentratedby centrifugation and DNase/RNase treatment. DNA and RNA wereextracted and PCR amplified using random primers (Victoria & al., 2009).Around 2.8 million reads (of about 150 nucleotides) were generated.The assembled contigs and singlet sequences were compared to Genbankvirus database using GAAS software. Sequences of bacteriophages(Siphoviridae, Myoviridae, Podoviridae, Microviridae) and animal viruses(Retroviridae, Reoviridae, Adenoviridae„Picornaviridae, and Paramyxoviridae)were mainly identified. These sequences were submitted to aphylogenetic analysis and their putative association with respiratory pathologywas also subsequently assessed. Altogether, we described an avianrespiratory virome. Deep sequencing is a powerful approach to detectmost infectious agents in a clinical case. It also allows for the detectionof the opportunistic pathogen flora, unrelated to clinical signs. This studyincreases our understanding of the viral diversity in ducks and highlightsthe complexity of co infections in poultry respiratory tract.REF 412A novel virus discovery approach to identify unrecognizable virusesBas OUDE MUNNINK 1 , Seyed Mohammad JAZAERI FARSANI 1 ,Martin DEIJS 1 , Marta CANUTI 1 , Jiri JONKERS 1 , JoostVERHOEVEN 1 , Greet IEVEN 2 , Herman GOOSSENS 2 , MatthewCOTTEN 3 , Lia VAN DER HOEK 11 Amsterdam Medical Center, Amsterdam, THE NETHERLANDS;2 University of Antwerp, Antwerpen, BELGIUM; 3 Sanger Institute, Cambridge,UNITED KINGDOMViral infections remain a major cause of human diseases. Nevertheless,it is assumed that there are pathogenic viruses that have escaped identification,while the threat of new viruses adapting to human hosts remainsunabated. Discovery of new viruses has in the last decade been boostedby viral metagenomics: second generation virus discovery whereby tenthousands of sequences from a clinical sample are generated. With thedevelopment of the VIDISCA 454 technology, we were one of the firstto exploit these new possibilities, and identified several novel viruses.However, this experience also showed the limitations of next generationsequencing based approaches. In clinical samples positive identificationis entirely based on similarity with known virus families. Unknown virusfamilies still escape detection. We have developed a novel VIDISCA basedapproach which will allow identification of viruses that lack similarity toknown viruses. Key thereby is that infected patients show an adaptiveimmune response resulting in virus specific antibodies in blood. The IgGin convalescent serum can be used to capture and enrich viruses. Whenthe enriched fraction is used as input in VIDISCA 454, unrecognizablenew viruses can be detected. This new method was evaluated with severalvirus infections: 13 respiratory and 7 intestinal infections. Identificationvia Enrichment VIDISCA 454 allowed identification of all 13 respiratoryviruses, and 6 of 7 intestinal viral infections.REF 413Bufavirus, a Novel Human Virus in the Family ParvoviridaeElina VÄISÄNEN 1 , Tung G. PHAN 2,3 , Eric DELWART 2,3 , EveliinaTARKKA 4 , Klaus HEDMAN 1,4 , Maria SÖDERLUND VENERMO 11 Department of Virology, Haartman institute, University of Helsinki, Helsinki,FINLAND; 2 Blood Systems Research Institute, San Francisco, USA;3 University of California, San Francisco, USA; 4 Helsinki University CentralHospital Laboratory Division, Helsinki, FINLANDParvoviruses are small, non enveloped viruses with 4 6 kb ssDNAgenomes. They infect diverse animals from insects to humans, and causediseases with mild to severe symptoms. Since 2005, nine new humanparvoviruses have been found and last year, metagenomic analysis offeces of children with acute diarrhea in Burkina Faso revealed a novelhighly divergent parvovirus provisionally named Bufavirus. The initialbufavirus sequence showed in the NS1 region

5 th European Congress of Virology72% similarity in the VP1 area. A highly sensitive quantitative PCR wasdesigned on the conserved NS1 region. We analyzed fecal samples fromgastroenteritis patients (n=124) in Finland, and found bufavirus DNAto be present in 2.4% of the samples. Furthermore, we amplified fromthe original fecal supernatants the VP2 genes of both bufaviruses andcreated recombinant baculoviruses for protein expression. EM analysisof the purified protein showed parvovirus like capsids of 20 25 nm indiameter. We have found the first bufaviruses outside Africa, in stoolcollected in Finland, thus indicating a wider circulation of the virus. Inaddition, we have produced for bufavirus antibody detection VP2 VLPs,and serological assay development is ongoing. We will elucidate thepathogenicity and epidemiology of this virus.REF 414Complete Genome Sequence of a Newfound Hantavirus Harbored bythe Doucet’s Musk Shrew in GuineaRichard YANAGIHARA 1 , Se Hun GU 1 , Violaine NICOLAS 2 , AudeLALIS 2 , Nuankanya SATHIRAPONGSASUTI 11 University of Hawaii at Manoa, Honolulu, USA; 2 Muséum Nationald’Histoire Naturelle, Paris, FRANCEElucidation of the molecular phylogeny of Tanganya virus (TGNV) andAzagny virus (AZGV), two crocidurine shrew borne hantaviruses recentlydetected in the Therese’s shrew (Crocidura theresae) in Guinea and WestAfrican pygmy shrew (Crocidura obscurior) in Côte d’Ivoire, respectively,has been severely hampered because their genomes have not been fullysequenced. To address this issue, ethanol fixed intercostal muscle specimensfrom 22 Therese’s shrews and 39 West African pygmy shrews, aswell as 11 Doucet’s musk shrews (Crocidura douceti), captured in southwesternGuinea during May 2011 to February 2012, were analyzed forhantavirus RNA by RT PCR. Although TGNV and AZGV were not detected,a genetically distinct hantavirus, designated Bowé virus (BOWV), wasidentified in a Doucet’s musk shrew. This report represents the first completegenome sequence of a genetically distinct crocidurine shrew bornehantavirus from sub Saharan Africa. Sequence comparison of the 431,1,145 and 2,158 amino acid gene products of the S, M and L segments,respectively, revealed that BOWV differed by 24.1 53.4%, 17.0 59.9%and 14.6 39.7%, respectively, from all other representative rodent, shrewand mole borne hantaviruses. Phylogenetic analysis, using maximum likelihoodand Bayesian methods, under the GTR+I+G model of evolution,showed that BOWV shared a common ancestry with TGNV and AZGV.Whole genome analysis of many more hantaviruses from sub SaharanAfrica will better clarify how the radiation of African shrews might havecontributed to the phylogeography of hantaviruses.REF 415Taxonomy of the new bunyaviruses: Khurdun virus (KHURV;Orthobunyavirus), Issyk kul virus (IKV; Nairovirus), Khasan virus(KHASV; Phlebovirus) and Razdan virus (RAZV; Phlebovirus), isolatedin Northern EurasiaDmitry LVOV, Sergey ALKHOVSKY, Michael SHCHELKANOV,Alexey SHCHETININ, Petr DERYABIN, Eugene SAMOKHVALOV,Asya GITELMAN, Andrey BOTIKOVThe D.I. Ivanovsky Institute of Virology, Moscow, RUSSIAIssyk Kul virus (IKV) is the etiologic agent of Issyk Kul fever. IKV wasisolated from bats and their argasid ticks, mosquitoes and birds (Passeriformes)in Kirghizia, Tajikistan, Kazakhstan and in Transcaucasia.Genome of IKV was sequenced and IKV has been classified to Nairovirus.IKV proteins are most similar (40%) to that of Crimean Congo hemorrhagicfever virus and Dugbe virus. Razdan virus (RAZV) was isolated fromticks (Dermatocentor marginatus) in Transcaucasia. RAZV has 90% identitywith Bhanja virus (BHAV) and 85% with Forecariah virus, thus RAZVbelongs to BHAV group in genus Phlebovirus. Khasan virus (KHASV)was isolated in Far East from Haemophysalis longicornis ticks. Based ongenomic data KHASV was classified to Phlebovirus genus. KHASV has32-45% identity with Uukuniemi group viruses and 25% with Toscanavirus. Phylogenetic analysis places KHAS between tick borne and mosquitoesborn phleboviruses. Novel Khurdun virus (KHURV) is associatedwith coots (Fulica atra) in Volga river estuary and has 25-30% identity withviruses of Orthobunyavirus genus. KHURV L and S segments are typicalfor orthobunyaviruses, including canonical terminal nucleotide sequences.KHURV S segment doesn’t have ORF for NSs. KHURV M segment isshorter (3,200 nt.) and apparently doesn’t encode NSm protein. KHURVGn protein is truncated and comprises 679 aa. According to obtained datawe proposed KHURV to be classified as a member of genus Orthobunyavirus.However, based on its distinctive M segment features KHURVmay belong to the novel genera “Khurduvirus” in the Bunyaviridaefamily.REF 416Enrichment of viral proteins annotation with Argot2Enrico LAVEZZO 1 , Luisa BARZON 1 , Marco FALDA 1 , PaoloFONTANA 2 , Giorgio PALÙ 1 , Stefano TOPPO 11 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Research and Innovation Centre, Fondazione Edmund Mach, San Micheleall’Adige (TN), ITALYThe vast majority of viral genes and related proteins are still functionallyunknown. In order to address this issue we developed a tool, calledArgot2, to predict protein function exploiting the Gene Ontology (GO)controlled vocabulary. Argot2 has been recently assessed in an internationalchallenge [1] and ranked second overall. As a proof of principleof the capability to enrich viral proteins annotation, we tested Argot2 onthe 168 coding sequences belonging to the clinical isolate NC 006273,strain Merlin, of the human cytomegalovirus. Predictions were comparedwith annotations already available in GOA database for the same set ofproteins. Our tool was able to produce at least one annotation for 158proteins, while only 141 were already annotated in GOA, and the totalnumber of predicted GO terms was higher with Argot2 (905 vs. 705). Theintersection between the two sets was 595, confirming the reliability ofthe tool, and the majority of terms missed by Argot2 were generic andpoorly informative. As an example, we found annotations for 2 proteinspreviously unannotated: US26, predicted to be involved in modulating hostimmune response, and UL30, predicted to possess hydrolase and helicaseactivity. We propose Argot2 as a tool to enrich viral proteins functionalannotation and update GOA. Certainly, the predictions are provisional andneed to be experimentally validated, but they can serve as a starting point todrive experimental design. References: 1. Radivojac, P., et al. A large scaleevaluation of computational protein function prediction. Nature Methods,2013.Virologie, Vol 17, supplément 2, septembre 2013S235

5 th European Congress of Virology22. INNOVATIVE METHODS INVIRAL DIAGNOSISPosters: REF 417 to REF 460In Brazil, dengue became a public health problem after the introduction ofDENV 1 in 1986. In July of 2010, DENV 4 was isolated in Roraima. Thedetection of DENV NS1 as an alternative method useful for the early diagnosisof dengue has been shown. However, in secondary infections NS1 isless likely to be available for capture in an immunoassay due to immunecomplex formation. We aimed to analyze the NS1 ELISA sensitivity forearly diagnosis of DENV 4 cases recently reported and aiming to improvethe sensitivity, results were compared to those by dissociating immunecomplexes from primary and secondary cases. DENV 4 sera (n=471)confirmed by virus isolation and/or were analyzed. The IgG—ELISAwas performed immune response characterization. The Platelia TM DengueNS1 Ag ELISA (BioRad Laboratories, France) was used for NS1 capture.To improve the test sensitivity, two dissociation protocols were used: acid(AD) and heat meadiated (HD). Positive NS1 was observed in 54.38% ofprimary and 39.07% of secondary cases. The overall NS1 assay sensitivityincreased to 70.48% and 77.49% (p=0,017), after the AD and HDprocedures, respectively. After the HD procedure, a significant NS1 sensitivityincrease was observed in primary (82.01%) and secondary cases(73.10%, p=0,002).The NS1 assay results should be interpreted with cautionwhen used alone due to the false negative results and the addition ofa HD step prior to the assay to improve the sensitivity on endemic areaswhere secondary infections are more frequently reported is suggested.Support by: CNPq FAPERJ PAPES VI FIOCRUZ MSREF 417Immunoglobulin M, nonstructural 1 antigen and virus detection todengue virus for developing diagnostic algorithmMyung Guk HAN, Jooyoun BAE, Young Eui JEONG, Jung Eun CHO,Chan PARKNational Institute of Health, Cheongwon gun, KOREADengue fever caused by dengue virus (DENV), a member of Flaviviridae,leads to large global disease burden. Detection of immunoglobulinM (IgM) and nucleic acid to DENV, and virus isolation have been usedfor laboratory diagnostic assays for dengue fever. Nonstructural 1 (NS1)antigen which releases from DENV infected cells provides a useful diagnostictool. The measure of duration and intensity of immune response andthe first detection time of antibody and antigen to DENV are influencedby sensitivity and specificity of assays and specimen collection time. Todiagnose a current DENV infection, the demonstration of seroconversionin paired sera is required. Specimens are collected at wide time range ofinfection episode and the paired sera are usually not available in mostof cases. Therefore, it is strongly required to develop dengue diagnosticalgorithm to support the likelihood of a correct laboratory diagnosis. In thestudy, sera were collected from 325 dengue suspected patients who traveleddengue endemic country and were tested by IgM ELISA, NS1 antigenELISA and nested RT PCR for DENV. IgM ELISA, NS1 antigen ELISAand RT PCR showed 43.4%, 49.5% and 15.1% of positive rate, respectively.The positive rate was increased by combination of assay, 70.5% inIgM ELISA and NS1 antigen ELISA, 44.3% in IgM ELISA and RT PCR,50.2% in NS1 antigen ELISA and RT PCR, 70.8% in three assays. Theseresults suggest that combined result of NS1 antigen and IgM ELISAs givesmore accurate diagnostic result for dengue fever.REF 418Antigen Antibody Dissociation Significantly Improves the NS1 captureELISA Sensitivity for Dengue Virus Type 4 Diagnosis in BrazilMonique LIMA, Rita NOGUEIRA, Ana Maria DE FILIPPIS, PriscilaNUNES, Carla SOUSA, Manoela HERINGER DA SILVA, Flavia DOSSANTOSFlavivirus Laboratory, Oswaldo Cruz Institute/FIOCRUZ, Rio de Janeiro,BRAZILREF 419The results of Hepatitis E antibody prevalence study vary seriouslywhen using different serological testsVratislav NEMECEK 1 , Petr DITE 2,3 , Marek MALY 11 National Institute of Public Health, Prague, CZECH REPUBLIC;2 University of Defense, Hradec Kralove, CZECH REPUBLIC; 3 CentralMilitary Health Institute, Prague, CZECH REPUBLICThe incidence of reported hepatitis E cases in the Czech Republic increasedin the past decade from 0,1 cases/100 000 in the year 2001 to 1,6/100 000in the year 2012. It is important to determine the seroprevalence data onanti HEV antibodies in the general population in the Czech Republic. Serafrom the last available serological survey from the year 2001 were tested.1719 sera from common healthy population of the age 15 64 were testedin duplicate for anti HEV IgG antibodies using Dia.Pro test (DiagnosticBioProbes s.r.l., Italy). Overall seroprevalence of anti HEV IgG value was2,33% (40 positive/1719 sera). Sera with S/CO value >0,5 (n=118) wereexamined also with two additional anti HEV IgG EIA tests. MP HEVELISA IgG (MP Diagnostics, Singapore) and RecomWell IgG (MikrogenDiagnostik, SRN) revealed 10 and 49 positive sera resp. Among those40 Dia.Pro positive sera only 10 (25%) were positive with the MP testand 22 (57,5%) were positive with the RecomWell test. Only 7 sera wereidentically positive in all three tests. Immunoblot Recomline test (MikrogenDiagnostik, SRN) was used for confirmatory testing of sera whichhave been found positive at least in one test. Immunoblot test confirmed43 from 49 RecomWell positive samples, 7 from 10 MP HEV ELISApositive samples and 18 from 40 DiaPro positive samples. We estimatedprevalence of HEV IgG antibodies in the Czech Republic in the year 2001in range 0,6 2,5% depending on used EIA test. Our results indicate thatthe used test can complicate the comparison of prevalence data.Supported by IGA MZ grant NT/13884/2012.REF 420Influenza virus infection induced the rising of cellular temperatureAyae HONDA, Minetaka MARUYAMA, Kosuke ISHII, NanakoKAWAGUCHI, Fumito ARAIDepartment of Frontier Bioscience, Hosei University, Tokyo, JAPAN;2 Department of Mechanical Science & Engineering, Nagoya University,Nagoya, JAPANInfluenza virus infection influences the gene expression of the cell, andafter virus infection a big amount of viral genes express in the cell. Wesupposed that these changing in the cell may induce physical and chemicalchanging of cell, and these changing may induce the activation of cellularmetabolism and consumption of energy. Whereas a big amount of energywill produce. Under this idea, the tool for measuring the temperature wasdeveloped and measured the temperature of influenza virus infected anduninfected cells. Interestingly the temperature of influenza virus infectedcell was risen about 4 K compared, however we could not detected anytemperature rising on influenza virus uninfected cell. We would like todiscuss this result.S236 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF 421Development of a new diagnostic tool for the detection of HumanParechovirus in RT PCRCome BARRANGER, Jérôme BES, Manon DUBE, Lionel GARNIER,Stéphane MAGROBioMérieux, site de Verniolle, Verniolle, FRANCEObjectives: parechoviruses are responsible for infections that, althoughmostly asymptomatic, may still lead to severe diseases (eg encephalitis,meningitis, pneumonia...) The bioMérieux Parechovirus R gene ® significantlyimproves the diagnosis of HPeV allowing early detection andimproved sensitivity with main serotypes of human Parechovirus. Theaddition of the internal control (IC) allows to check extraction step, thuspreventing a false negative result due to presence of inhibitor or extractionissue. Methods: Nucleic acids were extracted from 200 L ofCSFon NucliSENS ® easyMAG ® and eluted in 50 L. 0.15 L of reversetranscriptase was added to 15 L of Parechovirus r gene ® amplificationpremix. Then 10 L of eluted samples were added. Parechovirusesand IC were detected at 530 nm & 560 nm respectively on Bio Rad DxReal Time System or Applied Biosystems 7500Fast. Results: On theParechovirus QCMD 2012 Panel, 9 of 10 samples were correctly identified.Analytical Sensitivity study on the HPeV 1 and HPeV2 specieswas performed in CSF. This study shows a high level of sensitivity foreach parameter. Intra/inter assay variability studies were carried out onHPeV1 and HPeV2 cultured cells diluted in CSF. Coefficients of variationwere under 1.5% (intra assay) and 3.0% (inter assay). Specificitystudy showed no cross reaction among 22 viruses that could be present inCSF. Conclusion: The high quality associated with its compatibility withthe major extraction and real time PCR platforms allows an immediateintegration of Parechovirus R gene ® 71 020 in most routine diagnosticlabs.REF 422Evaluation of the clinical and analytical performances of viral serologicalassays on the Liaison XL platformMathias F.C. BEERSMA, Janienne KLAASSE, Sandra M.J.SCHERBEIJN, Martin SCHUTTENErasmus Medical Centre, Virology, Rotterdam, THE NETHERLANDSThe Liaison ® XL platform (DiaSorin) is an automated chemiluminescenceanalyzer offering a virtually complete panel of virologic IgG andIgM assays. Objectives: To evaluate the performances of the serologicalrepertoire of the Liaison XL in comparison with either Architect(Abbott) or VIDAS (Biomerieux); To validate the current Liaison assaysfor EBV, HSV, and parvo B19 on the XL platform. Methods: A total of30 assays (5 total Ig, 12 IgG, 9 IgM, 3 antigen, one IgG avidity) weretested using selective panels of clinical samples and parallel testing indaily routine. Results: Comparable performances (100% concordance)were found for the HIV Ab, HBcore IgM, HAV IgM, VZV IgG/M, andmumps IgG/M XL assays. Better performances (specificities) were foundfor the anti HBcore, anti HCV (genotypes 1 4), HAV Ig, and measlesIgM. Balanced discrepant results within the cut off range occurred forHIV p24 Ag, HBe Ag, anti HBe, CMV IgG/M, CMV IgG avidity, andmeasles IgG. Levels of HBsAg (genotypes A) and anti HBsAg testedhigher than the international standards (NIBSC, WHO), whereas rubellaIgG levels tested lower. Liaison XL showed optimal sensitivity for HBsAgsubtypes A G and HBsAg mutation panels. Validation of the existingEBV, HSV, and B19 assays on the Liaison XL platform showed 100%concordance, although s/co ratios were variable. Conclusions: The LiaisonXL platform offers a complete, sensitive and highly specific panelof viral serologic assays. Anti HBsAg and rubella IgG titers on the XLplatform may require attention during their implementation in clinicalpractice.REF 423Comparative evaluation of the Real Time PCR based LiPA Assay forthe detection and the genotyping of Human Papillomavirus in cervicalspecimens versus Clart ® HPV 2 micro array and conventional INNOLiPA extra genotyping testsThomas BOURLET 1 , Isabel MICALESSI 2 , Sylvie PILLET 1 , MichelHUSS 3 , Julie JACQUET 1 , Jean Philippe KLEIN 4 , Michèle COTTIER 4 ,Céline CHAULEUR 3 , Bruno POZZETTO 11 Virology Unit, GIMAP EA 3064, University Hospital of Saint Etienne,PRES of Lyon, Saint Etienne, FRANCE; 2 Applied Molecular BiologyResearch (AMBIOR) group, University of Antwerp, Universiteitsplein 1,B2610, Antwerp, BELGIUM; 3 Gynaecology Obstetrics Department, UniversityHospital of Saint Etienne, PRES of Lyon, Saint Etienne, FRANCE;4 Cytology Unit, LINA EA 4624, University Hospital of Saint Etienne, PRESof Lyon, Saint Etienne, FRANCEThe detection and genotyping of Human Papillomavirus (HPV) by moleculartechniques is of high interest for the screening of cervical cancer. Theanalysis of these HPV related markers is also valuable for epidemiologicalstudies, vaccine trials and fundamental research purposes. A new two stepapproach combining a real time PCR based on SPF10 primers for broadspectrum HPV detection and a reverse line probe assay (LiPA) used forthe typing of 28 different HPV genotypes has been developed (Real TimeLiPA Assay (Innogenetics)). A prospective study was conducted on 110cervical specimens with cytopathological findings of ASCUS or LSIL, inorder to compare this new assay to the Clart ® HPV2 test (Genomica) andthe conventional INNO LiPA HPV genotyping extra assay (Innogenetics).A positive result was asserted when a sample was detected positive forHPV by at least 2 out of 3assays. The sensitivity of SPF10 real time PCR,INNO LiPA extra and Clart ® HPV2 was 95.2, 100 and 93.6%, respectively.Complete or partial agreement’s rates of 86.4, 87.5 and 84.6% were foundbetween SPF10 Real Time and Clart ® HPV2 assays, SPF10 Real Time andINNO LiPA extra assays, and between INNO LiPA extra and Clart ® HPV2 tests, respectively. The 8 most frequent HPV types attributed to cervicalcancer (HPV 16, 18, 31, 33, 35, 45, 52 and 58) were equally detected bythe 3 assays. Since only positive samples obtained by the SPF10 real timePCR are further genotyped by the LiPA assay, this new approach offersreduced hands on time, workload and cost for HPV diagnosis.REF 424Antibody responses in humans infected with West Nile Virus in Europeand their implications for diagnostics developmentStefan CHABIERSKI 1 , Luisa BARZON 2 , Petra FIEBIG 3 , Uwe G.LIEBERT 3 , Anna PAPA 4 , Michael S. DIAMOND 5 , Giorgio PALÙ 2 ,Sebastian ULBERT 11 Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, GER-MANY; 2 Padova University, Department of Histology, Microbiology andMedical Biotechnologies, Padova, ITALY; 3 Institute of Virology, LeipzigUniversity, Leipzig, GERMANY; 4 Department of Microbiology, MedicalSchool, Aristotle University of Thessaloniki, Thessaloniki, GREECE;5 Departments of Medicine, Molecular Microbiology, Pathology & Immunology,Washington University School of Medicine, St.Louis, USAThe zoonotic West Nile Virus (WNV) belongs to the family Flaviviridae,similar to Yellow Fever Virus or Japanase Encephalitis Virus. Most WNVinfections result in no or flu like symptoms. In some cases, especially inolder or immunocompromised persons, severe neurological disease (suchas encephalitis or meningitis) can develop. The serologic detection ofWNV infections is complicated by the cross reactivity of antibodies againstrelated flaviviruses. In the context of the EU funded WINGS project, westudied the human antibody response to European WNV strains responsiblefor outbreaks in Italy and Greece in 2010, caused by lineage 1 and2 strains, respectively. The WNV structural proteins were expressed as aseries of overlapping fragments fused to a carrier protein, and binding ofVirologie, Vol 17, supplément 2, septembre 2013S237

5 th European Congress of VirologyIgG in sera from infected persons was analyzed. The results demonstratethat, although the humoral immune response to WNV in humans is heterogeneous,several dominant peptides are recognized. In addition, the dataindicate that some peptide sequences can be used for the development ofa specific serologic WNV test as they show only marginal cross reactivitywith antibodies against related flaviviruses.REF 425Comparison of workflow and data analysis for the quantificationof Cytomegalovirus (CMV) using an in house method versusQIAsymphony ® RGQ systemTim CONIBEAR, Jennifer MERRIT, Ana KUBLIK, Aysha KHANOM,Claire ATKINSONDepartment of Virology, Royal Free Hospital, London, UNITED KING-DOMObjectives: quantification of Cytomegalovirus (CMV) viral load is animportant tool in the management of CMV infections in immunocompromisedpatients. The new artus CMV QS RGQ integrated assay protocoloffers an automated solution to viral load determination. The workflowand data analysis generated with the new system was compared to an inhouse quantitative PCR method. Methods: following routine diagnostictesting, whole citrated bloods were batched into groups of 19 to facilitateoptimal testing. Eight integrated assays were performed on the QIAsymphonyaccording to the manufacturer’s instructions, utilising full barcodescanning and sample tracking. The time taken for each protocol step wasrecorded by the operator according to a designated protocol. Whole citratedblood aliquots (n=116) were tested prospectively using routine diagnosticsamples tested with the in house assay. In addition, a serial dilutionof the 1st WHO CMV international standard (NIBSC) in triplicate wastested in two separate assays. Data from all RGQ runs was analysed usingthe Rotor Gene AssayManager and compared to the laboratory in housedata. Results: the total time taken to generate a viral load result was notsignificantly reduced (average 9 minutes) however the average user handsoff time increased significantly (by 62 ± 3 minutes) using the QIAsymphonyRGQ system. There was a high level of concordance between inhouse laboratory generated data with those from the QIAsymphony RGQ.Furthermore the data generated by the new system provided results incopies/ml that were equivalent to the international standard in IU/ml (averagedeviation from the mean log10 -0.04). Conclusion: the artus CMVQS-RGQ integrated assay protocol offers an efficient and accurate alternativeto current in-house protocols for quantitation of CMV in wholeblood. The automated extraction and assay setup platform affords the usera significant increase of hands-off time which will effectively reduce thecost per test and therefore be considered during any cost benefit analysis.This study also showed good agreement and correlation of data using bothcurrent in-house protocols and the automated system. In conclusion, theQIAsymphony ® RGQ system is a versatile platform that is able to providesignificant improvements in sample handling efficiency and accuracywithin a routine virology laboratory setting.REF 426Detection of infectious viral particules of the Hepatitis A Virus (HAV)By using a pre treatment in combination with quantitative real time(RT qPCR)Coralie COUDRAY 1 , Audrey FRAISSE 1 , Sandra MARTIN LATIL 1 ,Laurent GUILLIER 2 , Sylvie PERELLE 11 Maisons Alfort Laboratory for food Safety, Food and Water Virology Unit,Maisons Alfort, FRANCE; 2 Maisons Alfort Laboratory for food Safety,Modelling of Bacterial Behaviour Unit, Maisons Alfort, FRANCEHuman enteric viruses are important agents of foodborne diseases. HepatitisA virus (HAV) is a single strand RNA virus belonging to thePicornaviridae family. HAV is responsible for hepatitis around the worldand is transmitted mainly via the faecal oral route, either by personto person contact or by ingestion of contaminated water and food.Because of the absence of a reliable cell culture method, RT qPCRis now widely used for the detection of HAV in food samples. Howeverthis approach detects virus nucleic acids of both infectious and noninfectious viruses, which limits conclusions in terms of public healthconcern. The use of photoinductible molecules like propidium monoazide(PMA) or ethidium monoazide (EMA), combined to PCR basedassays has been yet described in bacteria, parasites and fungi to distinguishviable from non viable particles. Recently, this approach has alsobeen tested in some RNA viruses. The aims of this study were to show1) EMA and PMA binding to HAV RNA under light photoactivation, 2)to test the efficacy of an associating treatment PMA/EMA and surfactantsto distinguish between infectious and non infectious HAV, 3) to usethe most promising treatment based on PMA/EMA/surfactant RT qPCRfor HAV to establish kinetic thermal inactivation curves and 4) finallyattempt to show the influence of the molecular RT qPCR models in theseassays. To conclude, we developed a molecular detection method for theHAV genome by EMA IgepalCA630 RT qPCR which provides a bettercorrelation between infectious titers and molecular titers with thermalinactivation.REF 427Viral agents in the pathogenesis of cardiomyopathyJudit DEAK 1 , Marta HÖGYE 2 , Miklos CSANÁDY 3 , GabriellaTERHES 4 , Beatrix KELE 5 , Robert SEPP 6 , Bela IVÁNYI 71 Department of Clinical Microbiology, University of Szeged, Szeged,HUNGARY; 2 2nd Department of Medicine and Cardiology Center,University of Szeged, Szeged, HUNGARY; 3 2nd Department of Medicineand Cardiology Center, University of Szeged, Szeged, HUNGARY;4 Department of Clinical Microbiology, University of Szeged, Szeged,HUNGARY; 5 Department of Clinical Microbiology, University of Szeged,Szeged, HUNGARY; 6 2nd Department of Medicine and Cardiology Center,University of Szeged, Szeged, HUNGARY; 7 Department of Pathology,University of Szeged, Szeged, HUNGARYParvoiruses are ubiquitous pathogens of humans and animals. QuantitativePCR methods are suggested for the determination of viral nucleic acids indilatative cardiomyopathies (DCMs), and some other cardiac diseases.Certain authors, have detected viral nucleic acids in autopsy samples,without cardiac diseases, at similar rates as in DCM patient samples.In our study in the past 9 years (2004-2012), 93 adult endomyocardialbiopsy samples were received from the 2nd Department of Medicine andCardiology Centre. Samples were prepared on the day of collection. Afternucleic acid isolation from the clinical samples by the Qiagen method,these were frozen or amplified with adenovirus, cytomegalovirus, EpsteinBarr virus, herpes simplex virus 1, herpes simplex virus 2, parvovirus B 19and enterovirus primers. Qualitative RT PCRs, were used; 1 5 PCRs wereperformed on the biopsy samples/patients. 406 PCRs were performed.The clinical samples contained 1 viral nucleic acid each in 20 patients; 2viral nucleic acids were positive in 2 patients, parvovirus nucleic acid in 11patients, CMV in 4 patients, and EBV, enterovirus and HSV 1 in 3 patientseach. Adenovirus and HSV 2 were not detected. Many research articleshave been published on this topic, but there is not yet a validated molecularmicrobiological method. Not only classical cardiotropic enteroviruseshave been detected in DCM, but also a high percentage of members ofthe Herpesviridae family. The possibility of antiviral therapy should betaken, into consideration in the event of DCM of HSV, EBV or CMVorigin.S238 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyREF 428New approaches for standardization and validation of quantitativeqRT PCR assays for quantitation of yellow fever on clinical sampleswith high quality parametersAlice FERNANDES 1,2 , Gisela TRINDADE 1 , Anna YAMAMURA 1 ,Constança BRITTO 2 , Vanessa DE PAULA 3 , Ana DUARTE 1,2 , KellyLÚCIO 1 , Renan VIEIRA 1 , Sheila LIMA 11 LATEV/Bio Manguinhos/Fiocruz, Rio de Janeiro, BRAZIL;2 LABIMDOE/Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro,BRAZIL; 3 LADTV/Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro,BRAZILThe development and production of viral vaccines involves several stepsthat need the monitoring of viral load throughout the process (antigen production,purification, inactivation). Currently, these steps are monitored byplaque lysis titration assay, whose results take 7 10 days to come out. Withthe advent of real time RT PCR, we have a faster approach available to thisissue. In this context, the development, standardization and validation of atechnique to quickly and efficiently quantify the yellow fever (YF) virus inthe aforementioned stages is extremely important. To accomplish that, weconstructed a plasmidial standard curve and validation parameters wereevaluated. Furthermore, we defined the limits of detection and quantificationof the test. To ensure high quality, internal controls were establishedin order to avoid false negative results. The statistical analysis revealedan excellent correlation between the results obtained in RNA copies/mLquantified by qRT PCR and the viral titer calculated by lysis plaques tests(R=0.96). In addition, a correlation factor for conversion of the real timePCR data to plaque assays was generated. The results analysis showed thatthe validation experiments sufficed all parameters defined by the qualitycontrol sector. The technique herein standardized proved to be effective fordetermining YF viral load both in vivo and in vitro, thus becoming a veryimportant tool in all projects developed in LATEV, and may eventually beadopted as the gold standard laboratory analysis and quality control forvaccine production.or AdV R gene. With a throughput of up to 14 samples per run in 1 hour,EZ1 has the same capacity than MP and is easier to use but it is also moreexpensive.REF 430Development of A Sensitive and Specific Epitope Blocking ELISA forUniversal Detection of Antibodies to Human Enterovirus 71 StrainsFang HE, Jimmy KWANGTemasek Life Sciences Laboratory, Singapore, SINGAPOREHuman Enterovirus 71 (EV71) is a common cause of hand, foot and mouthdisease (HFMD) in young children. It is often associated with severe neurologicaldiseases and mortalities in recent outbreaks across the Asia Pacificregion. Currently, there is no efficient universal antibody test available todetect EV71 infections.Methodology/Principal Finding: In the present study, an epitope blockingELISA was developed to detect specific antibodies to human EV71viruses in human or animal sera. The assay relies on a novel monoclonalantibody (Mab 1C6) that specifically binds to capsid proteins in wholeEV71 viruses without any cross reaction to any EV71 capsid proteinexpressed alone. The sensitivity and specificity of the epitope blockingELISA for EV71 was evaluated and compared to microneutralization usingimmunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses.Further, 200 serum sample from human individuals who werepotentially infected with EV71 viruses were tested in both the blockingELISA and microneutralization. Results indicated that antibodies to EV71were readily detected in immunized animals or human sera by the epitopeblocking ELISA whereas specimens with antibodies to other enterovirusesyielded negative results. This assay is not only simpler to perform but alsoshows higher sensitivity and specificity as compared to microneutralization.Conclusion: the epitope blocking ELISA based on a unique Mab1C6 provided highly sensitive and 100% specific detection of antibodiesto human EV71 viruses in human sera.REF 429Comparison of the EZ1 XL advanced and the Magna Pure instrumentsfor the extraction of whole blood before DNA quantification ofCMV, EBV, HHV 6 and AdenovirusMarie GUEUDIN, Alexandre LOUVEL, Jean Christophe PLANTIERCHU de Rouen, Laboratoire de virologie, Rouen, FRANCEBackground: the extraction is a key step for real time PCR and can befacilited by ease to use instrument like EZ1 XL advanced (Qiagen). Herewe have compared the EZ1 and the Magna Pure LC (MP) (Roche) beforean amplification with CMV, EBV, HHV 6 and Adenovirus (AdV) R genekits (Argène Biomerieux). Methods: whole blood samples (200 l) wereextracted (CMV n=156, EBV n=75, HHV 6 n=49, AdV n=32) with MPand EZ1 before amplification. Viral loads (VL) below the threshold wereconsidered as “detected”. The concordance of the results was verified.The agreement was evaluated by the intra class correlation coefficient(ICC) (acceptable if ICC>90%). Results: no known negative sampleswas positive above the threshold. CMV: 45 samples positive in the 2assays, 1 positive EZ1 (784 copies/ml) detected with MP, 1 positive MP(519 copies/ml) detected with EZ1. EZ1 VL were on average 0.03 Loglower than MP VL, ICC=95%. EBV: 46 samples positive in the 2 assays,2 positive MP (520 and 731 copies/ml) detected with EZ1. EZ1 VL wereon average 0.19 Log lower than MP VL, ICC=96%. HHV 6: 25 samplespositive in the 2 assays, 1 positive MP (1315 copies/ml) detected withEZ1. EZ1 VL were on average 0.12 Log lower than MP VL, ICC=97%.AdV: 9 samples positive in the 2 assays. EZ1 VL were on average 0.13 Loglower than MP VL, ICC=97%. Conclusion: The extracts obtained withEZ1 or MP give similar results when amplified with CMV, EBV, HHV 6REF 431Evaluation of a new rapid test for the detection of norovirus antigenin comparison with real time RT PCRPascale HUYNEN 1 ,AxelMAUROY 2 , Catherine GÉRARD 3 , RaphaëlBOREUX 1 , Marie Rose BRUCCULERI 1 , Cécile MEEX 1 , Marie PierreHAYETTE 1 , Julie DESCY 1 , Etienne THIRY 2 , Patrick DE MOL 1 ,Pierrette MELIN 11 Department of Medical Microbiology, University Hospital of Liège,Liège, BELGIUM; 2 Department of Infectious and Parasitic Diseases,University of Liège, Liège, BELGIUM; 3 Department of Biomedical andPreclinical Sciences, University of Liège, Liège, BELGIUMObjectives: noroviruses (NoV) are recognized as the leading cause ofgastroenteritis worldwide. Diagnosis of NoV infection mainly relies onmolecular methods. A detection of viral antigens can also be performed byimmunochromatographic assays. In outbreak settings, these rapid detectiontests (RDT) may be useful. The aim of this study was to compare theperformances of the new RDT ImmunoCardSTAT! ® Norovirus (MeridianBioscience ® , Europe) with a real time RT PCR. Methods: on the basis ofthe symptoms, 205 samples from patients were selected. Their status wasdetermined by real time RT PCR (Stals et al, J Virol Methods, 2009): 68positive, and 137 negative of whose 16 samples were positive for otherenteropathogens in order to evaluate the specificity of the RDT. Fifty of the205 samples originated from nosocomial NoV outbreaks during which agenotyping of the strains was performed. Results: we observed an overallagreement between the RDT and the RT PCR of 81%. The specificity andthe sensitivity of the RDT were respectively 96.3% and 50%. Regarding toVirologie, Vol 17, supplément 2, septembre 2013S239

5 th European Congress of Virologythe 50 samples from the nosocomial NoV outbreaks (genotypes GII.4 andGII.16), the RDT showed a specificity of 100% and a sensitivity of 68.8%(95%CI). None interference was observed with enteropathogens tested.Conclusion: according to our evaluation study, this RDT is very specificbut exhibits inadequate sensitivity to be used for the diagnosis of sporadiccases. However, in front of gastroenteritis outbreaks, this TDR is an effectivemethod for the early detection of NoV and the fast implementation ofprevention measures.REF 432No additional value of throat swabs in the diagnosis of enterovirusinfectionsMischa JAGER, Saara VAINIO, Wim ANGVU University Medical center, Amsterdam, THE NETHERLANDSIntroduction: it is currently unknown what the additional value is ofsampling other sites than cerebrospinal fluid, such as throat or feces, for thediagnosis enterovirus (EV) meningitis. The presence of EV RNA in fecesor throat is considered to be a strong indication of EV sepsis or meningitis.This study aims defining the additive value of testing throat and/or fecalsamples for diagnosis of EV infections. Methods: we analyzed samplesthat were tested with an EV PCR between 1 January 2007 and 1 October2012. In total, 566 patients had one or more samples tested for EV, 322of them were below the age of one year. We used a composite referencestandard with the following definition: patients that were positive in any ofthe PCR’s for CSF, throat or feces were regarded as EV positive. Results:in the group of patients with both CSF and throat swab testes, a smallgroup had a negative CSF PCR but a positive throat PCR. However, inall patients that had also their feces tested, the feces PCR was positive,indicating that a throat PCR does not provide extra information when afeces PCR is performed. In the group of patients with both CSF and fecesPCR, we observed that a substantial number of patients had a positivefeces PCR and negative CSF PCR. EV meningitis would not be missedwhen a PCR on feces and CSF is done.Conclusions: our data show that including a throat swab in the diagnosisof EV infection has only limited additional value and can be omitted. Thiswill lead to a reduction in costs without compromising sensitivity, whichis important in the current economic situation.REF 433Fast and accurate RSV A/B detection by PCR on a BDmax platformRuud JANSEN, Chau NGUYEN, Wil VAN DER REIJDENRegional Laboratory for Public Health, Haarlem, THE NETHERLANDSPatients on a pediatric ward are screened for RSV infection to preventspread of this highly contagious respiratory virus. Patients that are testedpositive for RSV are nursed in isolation to prevent the spread of thevirus. The decision whether a patient should be isolated upon admission ismade on the outcome of fast diagnostic tests, such as antigen or moleculartests. Antigen tests are fast, but have a low positive predictive value (PPV),risking the spread of RSV by false negative results. The molecular teststhat are based on reverse transcriptase PCR (rtPCR) however, are superiorto the antigen tests with respect to sensitivity and specificity. In thisreport we describe an in house developed rtPCR for RSV A and B on aBDmax platform. The BDmax is a fully automated platform that combinesRNA/DNA isolation with real time PCR. The test detects both RSV A andB in a single run and the time to result is only 2 hours. We compared for 60samples the RSV BDmax test with the BinaxNow antigen test and founda PPV of 65% for the antigen test that missed 7 of the 20 BDmax positivesamples. When comparing the BDmax test to a routine multiplex moleculartest (Respifinder, Pathofinder, NL) we found a good concordance of bothtests. Finally, we tested the QCMD 2012 proficiency panel for the in housetest on the BDmax. All samples were correctly analyzed on the BDmax,again demonstrating the good performance of the test. We conclude thatthe BDmax is a flexible and reliable platform for the implementation of inhouse developed tests in a clinical molecular laboratory setting.REF 434Development of biplex real time RT PCR for detection and differentiationof human parainfluenza virusesSaoussen KACEM 2 , Bénédicte MOUREZ 1 , Astrid VABRET 1 ,Abdelhalim TRABELSI 2 , François FREYMUTH 11 Laboratory of human and molecular virology, Caen, FRANCE;2 Laboratory of microbiology of Sahloul hospital, Sousse, TUNISIAObjectives: Development of biplex real time PCR for detection anddifferentiation of human parainfluenza viruses (HPIV) 1 to 4. Clinicalevaluation and comparison with conventional methods: direct fluorescenceassay (DFA) and viral isolation techniques (VIT). Material and methods:primers and probes used for real time PCR have been described and evaluatedin original publications (Templeton et al., 2005, Garbino et al.,2009) and tested in silico. Each PCR was primarily set up as a monospecificassay and then combined in two biplex reactions and optimizedfurther. Both assays have the same PCR protocol. Amplification, detectionand data analysis were performed with SMARTCycler, Cepheid ® . Retrospectivestudy used 304 nasopharyngeal aspirates to evaluate the biplexPCR and compare obtained results with those of conventional techniques.Results: monospecific PCR parameters were maintained in biplex assays.Optimization focused on concentration of MgCl2, probes, and dNTP. PCRspecificity and repeatability testing showed no non specific reaction andno variation exceeding ±1Ct. The biplex PCR positive specimens included92 samples that were positive by DFA/VIT and 9 additional ones.Discrepant results were tested by a conventional PCR. Obtained resultswere supporting our findings except for one sample. Conclusion: biplexreal time PCR was found to be a sensitive and specific alternative for classicalDFA and VIT. Conventional methods remain the gold standard fordetection of respiratory viruses. However, rapid laboratory diagnosis canbe critical for clinical management of patients.REF 435Evaluation of the new Abbot Architect assays for the detection of EBVantibodies in routine diagnosticsTorbjörn KJERSTADIUS 1 , Evfa JANSSON 1 , Jan ALBERT 1,21 Karolinska University Hospital, Solna, Stockholm, SWEDEN; 2 MCT,Karolinska Institute, Stockholm, SWEDENObjective: The aim of the study was to evaluate the performance of 3 newautomated Abbot Architect EBV diagnostic test of IgG antibodies againstEpstein Barr virus Nuclear Antigen 1 (EBNA 1) and Viral Capsid Antigen(VCA) and IgM antibodies against VCA. Material and methods:204consecutive samples, and 59 selected samples were analyzed by Biotest(EIA) for IgG EBNA, VCA IgG and VCA IgM and Abbott Architect(CMIA) for EBNA IgG, VCA IgG and VCA IgM. Dissenting sampleswere also analyzed by bioMerieux Vidas (ELFA) EBNA IgG, VCA/EAIgG or VCA IgM. A cross reactivity panel of 61 samples were analyzedby Architect VCA IgM. Results: If Biotest results were used as goldstandard the sensitivity for EBNA 1 IgG, VCA IgG and VCA IgM were96,5%, 100% and 94,3% respectively and the specificity 97,8%, 84,6%and 92,8% respectively.If a combination of Biotest, Architect and Vidasresults (2 out of 3) were used as gold stand the sensitivity for EBNA 1 IgG,VCA IgG and VCA IgM were 98,8%, 100% and 100% respectively andthe specificity 98,9%, 95,6% and 98,4% respectively. The concordancewas 89,1%. Crossreactity was shown for CMV, VZV, Mycoplasma andhepatitis A. Conclusion: The Architect assays show good performanceS240 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyand could be used as routine diagnostics of samples from persons withsuspected EBV infection and virus screening before transplantation andimmunomodulatory treatment. The relatively high prevalence of suspectednon specific IgM reactivity is noteworthy.REF 436Biotin IGM antibodies in human blood: a previously unknown factoreliciting false results in Biotinylation Based ImmunoassaysXumeng LI 1 , Tingting CHEN 1 , Carola WEIGEL 2 , Lea HEDMAN 1 ,Maria SÖDERLUND VENERMO 1 , Anna Maria EIS HÜBINGER 3 ,Klaus HEDMAN 1,41 Department of Virology, Haartman Institute University of Helsinki,Helsinki, FINLAND; 2 Department of Clinical Chemistry and ClinicalPharmacology, University of Bonn Medical Centre, Bonn, GERMANY;3 Department of Medical Microbiology, Immunology and Parasitology,University of Bonn Medical Centre, Bonn, GERMANY; 4 Department ofVirology and Immunology, Helsinki University Central Hospital LaboratoryDivision, Helsinki, FINLANDBiotin is an essential vitamin that binds streptavidin or avidin with highaffinity and specificity. As biotin is a small molecule that can be linked toproteins without affecting their biological activity, biotinylation is appliedwidely in biochemical assays.We recently discovered the occurrence of biotin reactive IgM antibodiesin a small proportion of human sera. Among >1000 human sera, we foundseveral samples to contain high affinity biotin IgM antibodies. In competitiveinhibition assays, their affinities ranged from 2,1 × 103to6,1× 105 mol/L. Using four inhouse and one commercial virus IgM EIAs, we nextshowed that the biotin IgM can cause false positivities in these serodiagnosticassays. We have purifyed from the sera the biotin IgM antibodiesto examine whether they influence biotinylation based assays widely usedin clinical chemistry or clinical immunology. Our findings provide newinformation on biotinylation based diagnostics and a new perspective fortheir validation, and open a new research venue in immunobiology ofvitamins.REF 437Evaluation of six new assays for the detection of Parvovirus B19 IgMand IgG antibodiesThoai Duong LY, Catherine COIGNARDLaboratoire Biomnis, Ivry sur Seine, FRANCEObjectives: In this study, the performance (specificity and sensitivity) of6 new serological Parvovirus B19 assays: LIAISON ® Biotrin ParvovirusB19 IgG, IgM (DiaSorin), Parvovirus B19 ELISA PKS IgG, IgM (MedacGmbH) and Parvovirus ELISA IgG and IgM (Vircell) was compared tothat of Biotrin EIA IgG and IgM. Alls assays use recombinant proteinsobtained from baculovirus. Methods: 214 unselected serum samples, submittedto the laboratory for IgG and IgM Parvovirus testing, were examinedby these 8 assays. Frozen serum specimens (N=38) from 19 patients wereused for sensitivity of primary infection study. Samples that were discordantbetween methods were tested by Immunoblot Parvovirus B19ViraStripe ® IgG, IgM (Viramed Biotech AG) for confirmation. RelativeIgM specificity was calculated based on the consensus result of 3 out of4 IgM assays. Results: The Vircell, LIAISON ® and Medac had an overallagreement of 98%, 97.1% and 96.1% respectively versus Biotrin EIAwith IgG. The LIAISON ® , Medac and Vircell had an overall agreement of97.2%, 96.7% and 95.8% respectively versus Biotrin EIA with IgM. Therelative specificity of IgM was 98.1%, 98.1%, 96.7% and 94.9% for BiotrinEIA, LIAISON ® , Medac and Vircell respectively. With Immunoblotthe specificity of IgM was 97.2%, 96.7%, 96.7% and 95.3% for Medac,Biotrin EIA, LIAISON ® and Vircell. Conclusions: LIAISON ® , Medacand Vircell showed a good overall agreement (superior to 96%) versusBiotrin EIA with IgG. LIAISON ® , Biotrin EIA and Medac presented ahigh IgM specificity (> 96%). Vircell IgM was less sensitive in primaryinfection.REF 438Performance of the liaison Xl automated immunoassay platform forblood donor screening on viral and treponema markersKerstin MALM, Elisabeth KRAGSBJERG, Sören ANDERSSONDepartment of Laboratory Medicine, Örebro University Hospital, Örebro,SWEDENBackground: Sensitivity and specificity is of utmost importance whenscreening blood donors for viral and bacteriological blood borne infections.Assay platforms designed for this purpose need to have highthroughput, easy management and appropriate analysis menu. Recently anew such platform has been introduced: Liaison XL from DiaSorin (Dia-Sorin S.p.A, Saluggia, Italy). This platform offers all the markers neededfor infection screening of blood donors. We have carried out a performanceevaluation of this platform, including Hepatitis B surface antigen (HBsAg),Hepatitis B core antibodies (anti HBc), Hepatitis C antibodies (anti HCV),HIV p24 antigen, HIV antibodies, Human T lymphotropic virus types 1and 2 (HTLV 1/2) and Treponema pallidum antibodies. Methods: For sensitivity,selected panels of samples previously analysed on the Architectfrom Abbott (Abbott Laboratories, Abbott Park, Illinois, USA; gold standardfor this evaluation) immunoanalyser were used. These samples wereconfirmed positive for HBsAg, anti HBc, anti HCV, HIV ag/ab, anti HTLV1/2 and antibodies to Treponema pallidum respectively. Specificity analysiswas assessed by analysis of samples from blood donors, previously runon the Architect platform and found non reactive for each marker. Up to1000 donor samples (both new and regular donors) will be tested in the evaluation.Previously non specific reactive samples were also run for everytested marker, as well as samples with autoimmune antibodies and antibodiesfor other infections. Results: To date, 312 samples positive for thetested markers (HBsAg n=50, anti HBc n=50, anti HCV n=75, Treponemaab n=56, anti HIV 1 n=32, anti HIV 2 n=21, anti HTLV 1 n=24 anti HTLV2 n=4) have been tested, and found positive, suggesting a high sensitivity.86 negative blood donors have been tested, where to this date, 1 sample hasshown a low false positivity in the HBsAg assay. All these samples werefrom first time donors. Conclusions: The Liaison XL platform for bloodborne infection screening provides a rational and easy way of testing atlarge scale. So far, the sensitivity of the tested markers seems to be veryhigh, and the specificity fulfills the requirements of blood donor screening.REF 439Preparation of armored RNA virus like particles as a control for realtime reverse transcription PCR detection of RNA virusesPavel MIKEL, Petra VAŠÍCKOVÁ, Radek TESARÍK, Pavel KULICH,Petr KRÁLÍKVeterinary Research Institute, v.v.i., Brno, CZECH REPUBLICRNA viruses are pathogenic agents of many serious infectious diseasesaffecting humans and animals, e.g. the flu, viral hepatitis, HIV/AIDS,rotavirus enteritis, tick borne encephalitis. Modern molecular biologicalmethods are used to detect pathogenic RNA viruses. Nowadays the mostused method is real time reverse transcription (RT) PCR (qRT PCR) thatenables both the detection and quantification of RNA viruses. Compared toDNA analysis, detection of RNA viruses is more sensitive and complicatedprocess, particularly due to the need of RT prior to PCR. Obstacle to theroutine analysis of RNA viruses by qRT PCR represents mainly the lack ofreliable RNA positive controls and standards that allow (through isolation,reverse transcription and PCR) control of all analytical steps. Thereforearmored RNA virus like particles (aRNA) were prepared using MS2 phagepackaging system. These particles carry selected unique sequence, whichVirologie, Vol 17, supplément 2, septembre 2013S241

5 th European Congress of Virologyserve as a positive control within all steps of the analysis. The key featureof aRNA is protection of specific RNA fragment against ubiquitous RNasedegradation due to its enclosing in phage proteins. Prepared particles arenot infectious and are not able to replicate, thus risk of laboratory contaminationis decreased. In comparison to RNA, stability of aRNA is higher; itcan be stored at different conditions for a long time without degradation.Moreover the aRNA mimics target viral pathogens, which increases thespecificity of the analysis. This work was supported by grants: NT138844/2012 and AdmireVet CZ1.05/2.1.00/01.0006 ED0006/01/01.REF 440Diagnostic performance of eight commercial Hepatitis E virus specificIgM and IgG ELISA assaysSuzan PAS 1 , Roel STREEFKERK 1 , Mark PRONK 1 , Robert DE MAN 2 ,Matthijs BEERSMA 1 , Albert OSTERHAUS 1 , Annemiek VAN DEREIJK 11 Department of Viroscience, ErasmusMC, Rotterdam, THE NETHER-LANDS; 2 Department of Hepatogastroenterology, ErasmusMC, Rotterdam,THE NETHERLANDSHepatitis E virus (HEV) genotype 3 is recognized as an emerging pathogenin industrialized countries. Following introduction of a new HEV IgMassay in November 2012 and reports suggesting commercial HEV ELI-SAs may not sensitively detect HEV genotype 3 or 4, we evaluated theperformances of eight commercially available HEV serum antibody IgMand IgG specific ELISAs. We used a sensitivity panel consisting of 88well defined samples from patients with PCR confirmed HEV infectionon basis of time after infection, immune status and genotype. A specificitypanel was constituted of 10 sera of acute infections of hepatitis A, B and Cvirus, human cytomegalovirus, Epstein Barr virus, B19 virus and 22 seraof healthy blood donors. The analytical performance of all eight assayswas assessed by dilution series of sera with known HEV genotype 1 or3 antibodies and the WHO HEV antibody standard. Based on the analyticalperformance six assays were selected for further clinical validation.Mikrogen new IgG assay was considerably more sensitive than its comparatorsfor genotype 3. Receiver operator curve analysis resulted in highestarea under the curve of 0.974, 0.971 and 0.994 for the Wantai, Mikrogennew and DiaPro HEV IgM assays, respectively. Cohen’s Kappa coefficientshowed highest concordance (>0.9) for Wantai and DiaPro for IgM, 0.893for Wantai and Mikrogen nov2012 for IgG. Highest specificity was foundfor the IgM Wantai assay. Our study shows that current commercial HEVELISAs can be used to diagnose HEV genotype 3 infection in a clinicalsetting without confirmational testing.REF 441Development of a panel of urine samples for evaluation of HPV testsin general populationChristopher PAYAN 1 , Sylvain ROSEC 2 , Marie Christine LEGRAND 1 ,Adissa TRAN 1 , Alexandra DUCANCELLE 3 , Marianne COSTE 4 ,YvonFOLL 5 , Francoise BOMMELAERT 5 , Francoise CHARLES 6 , EdithPOSTEC 7 , Michel COLLET 71 Virology Lubem Chru, Brest, FRANCE; 2 Cic Inserm 0502, Brest,FRANCE; 3 Virology Chu, Angers, FRANCE; 4 Virology Chu, Nantes,FRANCE; 5 Adec 29, Brest, FRANCE; 6 Cytology Chru, BREST, FRANCE;7 Gynecology Chru, BREST, FRANCEObjectives: we developed a HPV DNA test in urine for cervical cancer andprecancer lesions (CIN2+) screening in general population (PapU29 study;Payan 2007). The aim of our study is to propose a panel of urine samples toevaluate commercial HPV tests. Methods: Urine samples were obtainedfrom 25 65 years old women (n=3115). A pool of negative samples allowedthe production of a panel of SiHa cells containing 1.5 HPV16 DNA per cellin urine (from 1 to 7 log copies per mL). We produce 4 groups of urinesamples: 1) HPV positive with CIN2+ lesions (n=14), 2) HPV positivewith CIN1 lesions (n=8), 3) HPV positive with normal cytology (n=14), 4)HPV negative with normal cytology (n=10). Using this panel, we evaluatethe HPV mRNA EasyQ test (bioMérieux) and the HPV Real time PCRkit (Innogenetics). Results: SiHa panel limit of detection was at 2 log.Using the Innogenetics HPV DNA test, urine samples were all positive ingroups 1 and 2, in 93% in group 3 and in 20% in group 4, whereas, usingBioMerieux mRNA HPV test, positivity was observed respectively in 29%,25%, 7% and 0%. Among the 46 tested urine samples, 23 had undetectablecell control (B globin), but all samples were validated with an internalcontrol (DICO, Argene/BioMerieux). Evaluation of other HPV DNA tests(M2000RT,Abbott; Cobas4600,Roche) is on progress. Conclusion: wedemonstrate that other commercial Rt PCR methods could be appropriatefor HPV DNA testing in urine and that HPV mRNA could be detected inurine.Grants from the Ligue contre le Cancer and kit supplies from BioMerieux,Innogenetics, Abbott and Roche.REF 442Validation of a new easyMAG protocol for extraction of viral DNAfrom whole blood: application to cytomegalovirus DNA load monitoringSylvie PILLET, Thomas BOURLET, Bruno POZZETTOUniversity Hospital of Saint Etienne, Saint Etienne, FRANCEA new on board version (Vnew) of the whole blood (WB) extraction protocolon NucliSENS EasyMAG (bioMérieux) was evaluated in comparisonwith the off board protocol (V1) previously validated (Pillet et al. 2009).The sample input volume of WB was 200 L and elution volumes of 50 Land 70 L were tested with the Vnew protocol. After amplification with theCMV R gene ® kit (bioMerieux), results of CMV DNA load were expressedin IU/ml. All the 8 samples from QCMD Proficiency panel WB CMV 2012were correctly identified and the viral loads obtained with the Vnew protocolwere more closely related to the consensus values, whatever the elutionvolume used, compared to V1 protocol. Analysis of 120 clinical WB specimensshowed a concordance between V1 and Vnew protocols of 88.33%(kappa=0.744, 95% CI [0.620; 0.869]). Fourteen samples exhibited discrepantresults and corresponded to viral loads

5 th European Congress of Virologycells, and virus like particles of YFV was expressed in HEK 293T cells.Furthermore, we developed a panel of 31 hybridomas against the YFV,among which two monoclonal antibodies were well characterized. Withthese reagents it was possible to standardize two serologic immunoassays(sandwich ELISA) for the detection of IgG or IgM directed against YFVproteins in simian and human samples. Recombinant NS1 and VLPs showedto be good antigens for the detection of anti YFV immunoglobulins.And the ELISAs with these antigens showed high concordance in reactivityprofiles of samples when compared to a similar test using the nativeviral particle as antigen. Besides proving the usefulness of the developedtools for diagnosis of yellow fever, it was also possible to evidence the presenceof antibodies against NS1 protein in samples of primary vaccinationwith 17DD strain, in contrast with previous literature data.REF 444Real time label free measurement of virus infectivityAure SAULNIER 1 , Damien SOULET 1 , Cédric CHARRETIER 2 ,Nolwenn NOUGAREDE 11 Immunological and Microbiological Characterization Platform, AnalyticalR&D EU department, Sanofi Pasteur, Marcy l’Etoile, FRANCE;2 Virology and Immunology Platform, Analytical R&D EU department,Sanofi Pasteur, Marcy l’Etoile, FRANCEDifferent basics methods are commonly used to titer viral infectivity. Theseendpoint assays are time consuming and long lasting. Most of them arebased on direct examination of virus cythopathic effects and a specificvirus staining is often required to discriminate viral spreading. Real timecell analyzer (RTCA) system was developed to monitor dynamic label freeand non invasive cellular events using microelectronic biosensor technology.In this study, feasibility to use cell sensor technologies to substituteclassical limiting dilution method was evaluated. Large panel of virus families,known to be cytopathogenic, lytic or not, was investigated to have arepresentative view of the device performance. We compared in two celllines (Vero, MRC5) in parallel classical titration method to the real timeimpedance signal acquired with the xCELLigence RTCA (ACEA Biosciences).A comprehensive representation of the cell culture behavior wasobtained: the real time monitoring of the biological status of cells (cellularindex) showed a virus induced dose dependent impedance drop or changesfollowing infection. The time corresponding to 50% decrease/modulationin cell impedance was inversely proportional to virus infectious dose. Anextrapolation of final titers can be made sooner, few days post infection,based on the timing of the first impedance drop/modulation, for all virusfamilies tested on at least one cell substrate. We conclude that RTCA systemprovide a broad new tool, quantitative and high throughput, to realtime characterizing viral growth in cell culture.REF 445Utility of the QIAGEN artus PCR assays for the detection of humanherpes virus genomes in formalin fixed paraffin embedded clinicalsamplesOliver SCHILDGEN, Verena SCHILDGEN, Ramona Liza TILLMANN,Michael BROCKMANNKliniken der Stadt Köln gGmbH, Cologne, GERMANYObjectives: Qualitative and quantitative pathogen detection from formalinfixed paraffin embedded (FFPE) material is a major challenge in moleculardiagnostics, especially if no further unfixed clinical samples are availablefor microbiological or virological diagnostics. Thus the aim of the presentstudy was to analyse the utility of the artus real time PCR assays for thedetection of human herpes viruses (HSV1, HSV2, VZV, CMV, and EBV)in FFPE samples. Methods: Between April 2010 and September 2012we received requests for a total number of 91 clinical samples putativelypositive for human herpes viruses. FFPE material was subject to DNAextraction using the QIAGEN FFPE kit (QIAGEN GmbH, Hilden, Germany).PCRs for human herpesviruses were carried out using the artusassays for the detection of HSV1/2, EBV, CMV, and VZV according tothe manufacturer’s protocols. Results: In total, 27 samples were tested forHSV1 and HSV2, of those 3/27 (11.1%) were positive for HSV1 and 1/27samples was positive for HSV2. Of 50 samples tested for CMV, 6 werepositive (12%). 1/12 samples tested for VZV was positive for VZV and0/2 samples tested for EBV were positive. Conclusions: The PCR resultsshow that human herpes viruses can be detected from FFPE material withcommercial real time assays in an off label manner. These assays have theadvantage to detect small DNA fragments, which are a typical feature ofDNA extracted from FFPE material.REF 446Evaluation of a parechovirus PCR on cerebrospinal fluid samplespreviously considered as PCR negativeEvelyne SCHVOERER 1,2 , Aurélie VELAY 1,2 , Hélène JEULIN 1,2 , SibelBERGER 1 , Chantal FINANCE 1 , Véronique VENARD 11 Centre Hospitalier Universitaire Nancy Brabois, Laboratoire de Virologie,Vandoeuvre lès Nancy, FRANCE; 2“ Stress, IMmunité, PAthogènes”(SIMPA), EA 7300, Vandoeuvre lès Nancy, FRANCEBackground: It was admitted recently that parechoviruses can beresponsible for meningitis in young children, justifying the research ofparechoviruses in cerebrospinal fluid (CSF) to explore central nervoussystem syndromes. Aim: We are retrospectively evaluating a PARECHO-VIRUS R Gene PCR (bioMérieux, France), on CSF samples from childrenunder 5 year old, previously considered as negative for enteroviruses.Methods: The study consists of analyses performed on 50 CSF samplescollected from January 2012 August 2013. Until now, 36 CSF have beentested for parechovirus. After nucleic acid extraction on easyMAGTM(bioMérieux), PCR amplification (ABI 7500) has been realized with:ENTEROVIRUS R Gene kit, PARECHOVIRUS R Gene Kit, and theSuperscript III reverse transcriptase (RT) supplied with the PARE-CHOVIRUS R Gene Kit combined with the ENTEROVIRUS premix(PARECHOVIRUS R Gene template). Results: Two CSF samples out of36 were parechovirus positive. These parechoviruses have been detectedin children of less than 1 month old showing fever and typical signs ofmeningitis. Moreover, the combination of the ‘PARECHOVIRUS RT’ andthe ENTEROVIRUS premix has permitted the detection of enterovirusesin three out of 36 CSF samples. Conclusion: The RT supplied with thisPARECHOVIRUS kit allows us to save time when simultaneously usedwith enteroviruses and parechoviruses PCR detection kits. These preliminaryresults have highlighted that enteroviruses could be detected in threepreviously negative CSF samples, and parechoviruses in two additionalsamples.Virologie, Vol 17, supplément 2, septembre 2013S243

5 th European Congress of VirologyREF 447Evaluation of EBV VCA IgM, VCA IgG and EBNA 1 IgG tests usingnovel Abbott Architect CMIA AssayVlasta STEPANOVA 1 , Lenka PLISKOVA 2 , Miroslav FAJFR 1 ,EvaVEJRAZKOVA 3 , Jan TRBUSEK 41 Inst. Clin. Microbiology,University Hospital and Faculty of Medicine,Charles University, Hradec Kralove, CZECH REPUBLIC; 2 Inst. Clin.Biochemistry and Diagnostics,University Hospital and Faculty of Medicine,Charles University, Hradec Kralove, CZECH REPUBLIC; 3 IVthClinic of Internal Diseases, University Hospital and Faculty of Medicine,Charles University, Hradec Kralove, CZECH REPUBLIC; 4 AbbottLaboratories s.r.o., Prague, CZECH REPUBLICThe serologic diagnosis of Epstein Barr virus (EBV) infection wasextended by a novel, rapid fully automated chemiluminiscent imunoassayon microparticles, CMIA, Architect, Abbott. The aim of our study was todetermine VCA IgM, VCA IgG and EBNA 1 IgG antibodies by Abbotttest and compare the results with those obtained by microplate ELISAtests. Materials,methods: VCA IgM, VCA IgG and EBNA 1 IgG weredetected by CMIA, Architect, Abbott and by microplate ELISA tests, ETIVCA IgM, VCA IgG, DiaSorin, Italy, EBNA 1 IgG, Test Line, CZ in totalof 405 serum samples (185 males, 220 females, age 5–90, different Dg.).Results: seronegativity accordance of Abbott tests with ELISA was in 16to 17 samples, primoinfection in 20 to 21 samples, CMIA VCA IgG andEBNA 1 IgG pozitivity (past infection) accordance in 311 to 317 samples,transient EBV infection in 9 samples to 6 in ELISA, isolated CMIA VCAIgG pozitivity in 19 samples to 8, isolated CMIA EBNA IgG pozitivity in2 samples to 6 in ELISA assays. Cross reactivity of VCA IgM with CMVIgM was found in 4 samples in Abbott assay and in 5 samples in ELISA,VCA IgM positivity due to EBV reactivation in 14 samples in Abbottassay to 11 in ELISA, other reactivity was documented in 10 and 14cases in both types of tests. The precision of Architect CMIA assays wasperfect, the maximum SD was 0,16, maximum CV% 4,67. Conclusion:the discrepancy in results should be confirmed by WB, other tests andsubsequent sample due to patient clinical symptoms. Architect assayoffers the automated, rapid, sensitive and specific complete EBV serologytest.with high quality in house EIAs, these new Luminex based antibody assaysappear to be highly sensitive and specific. We next wish to design Luminexbased assays of novel types for antimicrobial IgM antibodies, as well asIgG avidity and IgG conformation dependence (“ETS”).REF 449Performance evaluation of Rubella and CMV IgG and IgM assaysfrom BioPlex ® 2200 ToRC multiplexed panels (Bio Rad)Marie Josee WENDLING, Hicham BENYELLES, Francoise STOLLKELLERInstitut de Virologie, Strasbourg, FRANCEObjectives: BioPlex2200 multiplexed ToRC assays offer the ability toreport Toxoplasma, Rubella and CMV results in a single test. The aim ofthis study is to evaluate the performances of the BioPlex2200 Rubella andCMV IgG and IgM immunoassays by comparing with our routine method(Liaison ® XL, DiaSorin). Methods: 200 routine samples sent to the laboratoryfor Rubella and CMV testing, 5 Rubella IgM positive samples and 4CMV seroconversion panels were submitted to BioPlex2200 ToRC panels.Results were compared to Liaison ® XL. In case of discrepancy, Liaison ®for Rubella IgG and Vidas ® (bioMérieux) for Rubella IgM, CMV IgG andIgM were used to arbitrate. Results: Rubella: The concordance for IgGand IgM is respectively 92.5% and 97.0%.The BioPlex2200 IgG assaymay be more sensitive than the Liaison ® XL assay. Regarding the abilityto detect IgM, BioPlex2200 properly detected 4 samples out of 5 (thediscrepant sample result was close to the equivocal range). CMV: Theconcordance for IgG and IgM is respectively 99.0% and 97%. Regardingthe ability to detect seroconversion, BioPlex2200 IgM assay was moresensitive than Liaison ® XL for 2 panels out of 4 and less sensitive forone. The BioPlex2200 IgG assay is as performant as Liaison XL. Conclusion:The formulation of this kit combining several assays is original andinnovative. The system is easy to use. BioPlex2200 CMV assays performancesare very good on IgG, good on IgM. Both are very good in case ofseroconversion. BioPlex2200 Rubella IgM assay showed a concordance(97%) equal to the data claimed in the package insert.REF 448Microsphere based multiplex antibody assay for Human ParvovirusB19 and Bocaviruses 1 3Yilin WANG 1 , Lea HEDMAN 1,2 , Arun KUMAR 1 , JonasBLOMBERG 3 , Maija LAPPALAINEN 2 , Maria SÖDERLUNDVENERMO 1 , Klaus HEDMAN 1,21 Department of Virology, Haartman Institute, University of Helsinki,Helsinki, FINLAND; 2 Helsinki University Central Hospital LaboratoryDivision, Helsinki, FINLAND; 3 Section of Virology, Department ofMedical Sciences, Uppsala University and Uppsala University Hospital,Uppsala, SWEDENDetection of antibodies is widely used in diagnosis of autoimmune andinfectious diseases. Conventional antibody detection procedures focuson one or a few microbes at a time. The recently introduced multiplexassay performed in suspension (Luminexcorp, Austin, Texas) may enablethe detection of antibodies against 50 100 species of microbes simultaneously.The system comprises dyed polystyrene beads and two specificfluorochromes. Up to 100 different beads can carry up to 100 differentanalytes. The beads are detected upon bypassing two distinct barcode readerlasers. Semiquantitative results are obtained as Median FluorescentIntensity (MFI) values. This approach has the potential of saving time,labour and cost.We set up IgG assays for human parvovirus B19 and the emerging humanbocaviruses (HBoV) 1 3, using as antigen recombinant virus like particles(VLPs) made up of the corresponding capsid proteins (VP2). ComparedREF 450Analytical Performance Characteristics of Automated Abbott Real-Time CMV for CMV Quantitation in Plasma and Whole BloodHong “Judy” YU 1 , Shiaolan HO 1 , Sara JONES 1 , Catherine BARRY 1 ,Won CHOI 1 , Erika WEBB 1 , John STEPHENS 1 , Klara ABRAVAYA 1 ,Karin PFEIFER 21 Abbott Molecular Inc., Des Plaines, USA; 2 Abbott GmbH & Co., Wiesbaden,GERMANYObjectives: Abbott RealTime CMV is an integrated real time PCR assayincluding automated sample preparation and real time PCR for the quantitationof cytomegalovirus (CMV) in human plasma and whole blood.The assay is intended to monitor CMV viral load in patients undergoingtransplantation. This study established the analytical performance characteristicsof Abbott RealTime CMV. Methods: Abbott RealTime CMVwas performed on the automated m2000 real time PCR system. Limit ofdetection (LOD), lower limit of quantitation (LOQ), linearity, and precisionwere determined using serial dilution panels of CMV strain AD169in pooled CMV negative human plasma and whole blood. Assay reproducibilitywas assessed using QCMD external quality control panel and/orAcrometrix OptiQuant ® CMVtc Calibration Panel across 12 labs. Results:Abbott RealTime CMV LOD and LOQ were determined to be 31.20 IU/mlusing the plasma protocol and 62.40 IU/ml using the whole blood protocol.The linearity range was from 31.20 IU/ml to 156 million IU/ml, and 62.40IU/ml to 156 million IU/ml respectively. Assay precision was found to be=0.320 log IU/ml regardless of sample prep protocol. Evaluation of internalS244 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyand external quality control panels in 12 different laboratories confirmedexcellent precision and reproducibility of the assay. Conclusion:Abbott RealTime CMV demonstrated excellent analytical performance.A large study including 12 labs corroborated internally established precisionand reproducibility. The assay contributes to the consistency in CMVquantitation in different labs across the world.REF 451Evaluation of a new Hepatitis E Virus (HEV) ELISA based on recombinantORF2 genotype 1 and 3 proteinsThorsten ZACHER, Sevgi UZUN, Friedhelm STRUCK, OliverBÖCHER, Bernd KRÄMER, Erwin SOUTSCHEKMIKROGEN GmbH, Neuried, GERMANYThe new ELISA, recomWell HEV IgG, IgM (Mikrogen GmbH, Germany),is based on state of the art purified ORF2 fragments, genotype 1 andgenotype 3, which are immobilized on polystyrene plates. The ELISA isdesigned for the sensitive and specific screening for anti HEV antibodiesin sera from patients with a clinical picture of acute non A, B, C hepatitisas well as in intensive care hospitals with immunosuppressed patients. Thediagnostic efficiency of the ELISA is beneficial in the routine laboratory.The evaluation includes: i) 63 HEV IgM and IgG double positive sampleswith acute hepatitis signs and excluded A, B, C hepatitis, ii) 200 blooddonor samples, iii) 135 clinical samples of patients with potentially similarclinical pictures (acute HAV, EBV, CMV, Parvovirus B19 IgM and IgGdouble positives with serological signs of acute infections, e.g. low IgGavidity findings in CMV and EBV samples), iv) 125 samples from patientswith specific immunological engagements, that are pregnant women, SLEpatients, IgM rheumatoid factor positives, and v) 50 potentially interferingsamples, i.e. lipemic, hemolytic and icteric samples. The new recomWellHEV assays display highly specific (IgM 99.1%, IgG) and sensitive (IgG96.3%, IgM 96.8%) detection of anti HEV antibodies.REF 452Development and evaluation of a high sensitive and specific real timeRT PCR for detection of hepatitis E virus in human plasma and serumThorsten ZACHER, Michaela SANDER, Monika BERNATZ, KerstinKUNTH LEHMANN, Bernd KRÄMER, Oliver BÖCHER, ErwinSOUTSCHEKMIKROGEN GmbH, Neuried, GERMANYThe hepatitis E virus (HEV) is one of the most common causes of fecalorally acquired hepatitis infection. HEV is endemic in such regions asIndia, Pakistan, and Southeast Asia;usually infections result from contaminateddrinking water. In the industrialised countries of the northernhemisphere HEV infections are sporadic, but with increasing incidence.This HEV infections are often autochthon, and assumed to be caused byzoonotic transmission from swine or other animals to humans. The newampliCube HEV assay (Mikrogen GmbH, Germany) has been developedas a real time RT PCR test which detects all four known genotypes (1–4)of HEV in humans. The test includes an inprocess control to monitor theextraction of nucleic acid and the presence of potential inhibitors of theRT PCR reaction.The assay has been validated for the detection of HEVinfections in plasma and serum of human specimens. Sensitivity, specificityas well as intra and inter assay variation has been evaluated to verifythe ampliCube HEV assay specifications. Dilution series of samples withdefined numbers of the WHO International Standard für HEV were analysedto determine the limit of detection (LOD). The new ampliCube HEVassay is highly specific (specifcity 100%) and sensitive (10 IU/RT PCR)for the detection of HEV RNA in humans.REF 453Development of a method for HEV detection in pig liver products:Application in a HEV French surveillance planSandra MARTIN LATIL 1 , Catherine HENNECHART COLLETTE 1 ,Laurent GUILLIER 2 , Corinne DANAN 3 , Julien SANTOLINI 3 , LaurineBOUTEILLER 31 ANSES, Maisons Alfort Laboratory for Food Safety, Food and Water VirologyUnit, Maisons Alfort, FRANCE; 2 ANSES, Maisons Alfort Laboratoryfor Food Safety, Modelling of Bacterial Behaviour Unit, Maisons Alfort,FRANCE; 3 DGAL, Paris, FRANCEHEV infection is a growing public health concern in industrialized countrieswhere the disease is mainly autochthonous caused by zoonotic HEVeither genotype 3 or 4. Besides zoonotic transmission via direct contactwith infected animals, foodborne transmission of HEV is associated witheating uncooked or undercooked meat of viscera of deer, boars, and pigs.In France, foodstuffs with pig’s liver were suspected to be at the originof autochthonous cases of HEV infection between 2007 and 2009. Thedevelopment of sensitive and reliable techniques for the detection of HEVis needed to ensure the safety of pig liver products. Due to low concentrationsof virus recovered and the presence of inhibitory substances in thesamples, RT qPCR has been one of the most promising detection methods.The general strategy for the detection of food borne viruses in food samplesconsists of 3 steps: 1) elution/concentration of virus, 2) purification of theviral RNA and 3) quantitative molecular detection of the purified RNA.One of the general requirements for viral diagnosis concerns the use ofa process control to monitor the efficiency of the entire sample process.The aims of this study were (i) to develop a detection method for HEV inpig liver products by using MNV 1 as process control; (ii) to validate themethod from artificially contaminated samples and (iii) to analyse for thepresence of HEV in 70 pig liver samples collected during a french surveillanceplan. To conclude, we have developed and validated a methodallowing an accurate quantification of HEV in pig liver products.REF 454A Liquid Handling System (L.H.S) to automate ARGENE ® assays setup from extracted samplesJerome BES 1 , Erwan LEVILLAIN 1 , Marina BERTRAND 1 , RaphaëlVEYRET 2 , Michel GUY 2 , Lucia BORDICCHIA 3 , Pascal MARGRY 1 ,Christelle RATINEAU 4 , Stéphane MAGRO 1 , Côme BARRANGER 11 BioMérieux, Site de Verniolle, Verniolle, FRANCE; 2 BioMérieux, CentreChristophe Mérieux, Grenoble, FRANCE; 3 BioMérieux Italia, Via di Campigigliano,Bagnio a Ripoli, ITALIE; 4 BioMérieux, Chemin de l’Orme,Marcy l’Etoile, FRANCEObjectives: Interest in automation of Molecular workflow is increasingin clinical diagnostic laboratories, as it saves time in routine procedures,reduces tedious manual pipetting steps, and improves throughputand traceability. We are introducing a flexible liquid handling system(L.H.S) dedicated to PCR set up that allows automatic handling of 48extracted samples with up to 3 quantitative or 6 qualitative PCR assayswithin 30 minutes. Methods: Nucleic acids are extracted from clinicalsamples using NucliSENS ® easyMAG ® and eluted in 50 L or 100 L.easyMAG ® vessels, containing eluted nucleic acids, and all PCR reagentsare loaded on dedicated holders and transported to the deck of the L.H.S.Assay setup is performed using an user friendly software. Eluates andreagents can be dispensed in various reaction supports for use with commonqPCR instruments. Results: LHS Turn Around Time for PCR setup was compared to manual dispensing according to several routine laboratoryworkflows. For this purpose, samples were tested with differentcombinations of Argene ® real time qPCR kits. Further technical validationincluding absence of carry over, intra assay reproducibility as wellas short clinical validation comparing LHS and manual distribution willbe presented to demonstrate the reliability of the LHS. Conclusion: ThisVirologie, Vol 17, supplément 2, septembre 2013S245

5 th European Congress of Virologyversatile and compact LHS allows automation of PCR set up, improvesthroughput and reliability. It can easily be integrated in any laboratoryworkflow to link extraction and amplification steps.REF 455Transmission electron microscopic quantification of viruses grown incell culturesHana MALENOVSKÁVeterinary Research Institute, Brno, CZECH REPUBLICInfectious titration of viruses is laborious and alternative protocols forquantification of viruses are needed. In this study, suspensions of Canineadenovirus 1 (CAdV 1), Feline calicivirus (FCV) and Bovine herpesvirus1 (BoHV 1) were quantified comparatively by transmission electronmicroscopy (TEM) and TCID50 to investigate their correlation. Each ofthe viruses was grown in five replicates until complete cytopathology wasrecorded (time 0), then frozen. In order to evaluate the influence of timingon virus harvest, they were left for additional periods of 16, 32 and 48 hoursat 37 ◦ C. At each time point, the infectious ability was characterised byTCID50 and the number of virions quantified by TEM. The virus particlecount determined by TEM did not change for any of the viruses throughoutthe experiment. The relationship between virus particle counts withTCID50 at time 0 showed good linearity response; their ratio was almostconstant. The proportion of non infectious particles did not change throughoutthe experiment for either CAdV 1 or BoHV 1. However, a decreasein virus infectious ability disclosed by TCID50 indicated that the fractionof non infectious particles in FCV increased 300 000 times when time0 and 48 hours were compared. In TEM quantification account must betaken of harvesting time as virus counts need not necessarily correlate withvirus infectious ability.Supported by the Ministry of Agriculture and the Ministry of Education,Youth and Sports of the Czech Republic (Grant no. MZe 0002716202,AdmireVet; Grant No. CZ.1.05/2.1.00/01.0006 ED 0006/01/01).REF 456A portable point-of-entrance system for the detection of lumpy skindisease virus using recombinase polymerase amplification assayAyman EL DEEB 3 , Ahmed ABD EL WAHED 1 , Mohamed ELTHOLOTH 2 , Mohamed SHALABY 3 , Frank HUFERT 1 , ManfredWEIDMANN 11 Institute of Virology, University Medical Center, Goettingen, GER-MANY; 2 Virology Department, Mansoura University, Mansoura, EGYPT;3 Virology Department, Cairo University, Giza, EGYPTLumpy skin disease is viral disease of cattle characterized by fever, followedshortly by the development of nodular lesions in the skin thatsubsequently undergo necrosis. Early diagnosis of its infectious agentshelps to diminish their impact by adequate outbreak management. Samplescollected from animals in the field or at quarantine stations are sent longdistances to the laboratory for PCR analysis because portable PCR isneither available nor suitable for on-site screening. The recombinase polymeraseamplifications (RPA) assay is an isothermal DNA amplification anddetection technology. In contrast to PCR, RPA is performed at a single temperature(42 ◦ C) and yield a result after only 5-15 minutes. In this study,we describe the development of a real-time RPA assay for the detection oflumpy skin virus (LSDV). Molecular DNA standards representing a part ofthe GPCR gene of LSDV were prepared. The assay sensitivity was determinedby probit analysis of eight assay runs of serial dilutions of the molecularstandard. The assay specificity was evaluated against a panel of virusesconsidered for differential diagnosis with LSDV. The assays were validatedusing clinical samples from 22 LSDV-infected cattle. The LSDV wasrapid (15 minutes) and showed an analytical sensitivity of 179 moleculesdetected. No cross reactivity with other viruses causing similar clinicalpictures were observed. In comparison to real-time PCR, LSDV RPAssensitivity was 100%. In conclusion, LSDV RPA yielded similar resultsas the corresponding PCR assays, but RPA were quicker and much easierto handle in the field. Thus RPA could be easily implemented to performdiagnostics at quarantine stations or farms for rapid on-site viral detection.REF 457Using EQA results to better understand the observed variation withinquantitative molecular testing and support standardisationCaterina DI LORENZO 1 , Paul S. WALLACE 1 , Hubert G.M.NIESTERS 2 , Anton M. VAN LOON 31 QCMD, Glasgow, UNITED KINGDOM; 2 University Medical Centre,Groningen, THE NETHERLANDS; 3 University Medical Centre, Utrecht,THE NETHERLANDSMolecular technologies have been the mainstay of infectious disease diagnosticsfor over twenty years. In addition, over the last decade, quantitationand the determination of pathogen load has become a core diagnosticparameter. For some pathogens such as HIV 1 and HCV, there is a clearrelationship between disease status and pathogen load. In addition, thedevelopment of international standards and the improved 2nd and 3rdgeneration commercial molecular assays available in this area have helpedconsiderably in improving test reproducibility within the laboratory,which in turn helps facilitate comparison of results across laboratories. Incontrast, for many other pathogens now routinely tested, the relationshipbetween pathogen load and disease status still remains unclear. For thesepathogens there are often not many commercial assays available. There area wide range of in house developed assays utilising different extraction andamplification methods and there is also a lack of suitable reference materialor international standards. This makes it difficult to compare quantitativeresults from laboratory to laboratory. Using the EQA data obtained over thelast decade, analysis of the overall variation observed within the reportingof quantitative EQA data from a range of EQA programmes will be made.A comparison between the variation observed within programmes wherean International Standard in known to be available, where a Standard hasonly just been introduced and also where no International Standards arepresent will be performed.REF 458Using proficiency testing programmes as a tool to assess and improvediagnostic methods for infectious diseasesCaterina DI LORENZO 1 , Paul S. WALLACE 1 , HubertG.M. NIESTERS 2 , Anton M. VAN LOON 31 QCMD, Glasgow, UNITED KINGDOM; 2 University Medical Centre,Groningen, THE NETHERLANDS; 3 University Medical Centre, Utrecht,THE NETHERLANDSProficiency testing is an essential tool used by laboratories to evaluate theperformance of existing and evolving molecular diagnostic technologies.The rapid evolution of molecular based diagnostic techniques and theever increasing number of clinically relevant pathogens, pose significantchallenges for proficiency testing providers as testing programmes mustcontinually evolve to meet the requirements of today’s laboratories.Quality Control for Molecular Diagnostics (QCMD) is responsible forthe coordination of international proficiency testing (PT) programmesand has kept at the forefront by adding new PT programmes eachyear covering a wide range of emerging and re emerging pathogens.Data from over 1500 laboratories in more than 100 countries using awide range of molecular diagnostics methods were collected and theresults were analysed to determine trends in the use and performanceof molecular diagnostics for a wide range of human pathogens and toevaluate laboratory performance over time. Results on QCMD PT panelsshowed that laboratories have improved precision of their (quantitative)assays in recent years. Furthermore the percentage of false positive resultsS246 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyhas been decreasing, although it remained an issue in specific areas, i.e.detection of fungal pathogens. Commercial assays were the favouredmethod of choice, however in house tests still remained an important partof the diagnostics, particularly where commercial assays are not readilyavailable. A move towards the use of real time assays was also evidentwhilst the use of non PCR methods was uncommon.REF 459Quality assurance: Monitoring assay performance with external runcontrols from QnosticsAlastair RICKETTS 1 , Joanna PHILLIPS 1 , Sadhia MOHAMMED 1 , PaulWALLACE 11 Qnostics, Glasgow, UNITED KINGDOM; 2 QCMD, Glasgow, UNITEDKINGOMThe application of nucleic acid based detection technologies has becomeroutine within the modern clinical microbiology laboratory with molecularassays available for the causative agents for a wide range of humaninfectious diseases. Each of these assays brings a requirement to monitorperformance and laboratories are finding themselves under increasingpressure to become fully accountable for the quality of the services theyprovide. The challenge for laboratory medicine is to be able to demonstratethat the results from a patient’s sample will be consistent and independentof which laboratory generated the results. The performance of an assayis monitored through the use of internal and external controls. Internalcontrols monitor the intra sample fitness and external controls monitorthe inter run performance. By monitoring both internal and externalcontrols a laboratory can identify trends and shifts in assay performance,allowing them to more readily demonstrate control and consistency ofthe assay during the QC review process. In effect monitoring the assaycontrols allows laboratories to balance the requisite high error detectionrate while maintaining a low false rejection rate. In order to achieve this,the use of well characterised, consistent, external controls are of greatbenefit as laboratories require controls that are traceable and consistentbetween technologies and institutions. The data presented shows how theuse of well characterised controls can be used to monitor performance anddemonstrates control in a clinical setting.REF 460Impact of External Quality Assurance on Improvement of Virus Diagnosticsand Virus Safety Testing of BloodHeinz ZEICHHARDT 1,3 , Vanessa LINDIG 1 , Carlos TELLEZCASTILLO 1,4 , Oliver DONOSO MANTKE 2 , Hans PeterGRUNERT 1,2,31 Charité University Medicine Berlin, Campus Benjamin Franklin, Instituteof Virology, Berlin, GERMANY; 2 Gesellschaft fuer BiotechnologischeDiagnostik (GBD) mbH, Berlin, GERMANY; 3 INSTAND e.V. Gesellschaftzur Foerderung der Qualitaetssicherung in medizinischen Laboratoriene.V., Duesseldorf, GERMANY; 4 Researcher Excellence Grant HLT08INFECT MET in the European Metrology Research Project (EMRP) ofEURAMETCorrect laboratory diagnosis of virus diseases is the basis for reliabletherapy of patients and virus safety testing of blood. Preventive measurementsagainst virus diseases as well as solid epidemiological datarely on good virus diagnostic results. In Europe accreditation of laboratoriesaccording to international norms (e.g. ISO EN DIN 15189)and certification of manufacturers of diagnostic test kits, i.e. in vitrodiagnostic (IVD) medical devices give the administrative fundament fordiagnostic laboratories on the one hand and for manufacturers of diagnostictest kits on the other hand. Specified test materials applied inexternal quality assessment (EQA) schemes are efficient tools to measurethe competence of diagnostic laboratories and the reliability ofdiagnostic test kits. The presentation will concentrate on an internationalEQA scheme network for quality assurance and standardizationwhich has resulted in improvement of virus diagnostics for the benefitof patients and blood services. INSTAND EQA schemes in virusdiagnostics have been implemented in Germany in 1988 (>40 EQAschemes with >1100 participating laboratories from >40 countries). Theseschemes are performed on behalf of the German Medical Association(Bundesaerztekammer) under the scientific auspices of the German Associationagainst Virus Diseases (DVV) and Society of Virology (GfV).Examples for improved virus diagnostics as outcome of EQA schemeswill focus on diagnostics of HIV/AIDS and hepatitis B and C with emphasison training aspects and surveillance of performance of IVD medicaldevices.Virologie, Vol 17, supplément 2, septembre 2013S247

5 th European Congress of Virology23. VIRAL DIAGNOSIS IN CHRONICINFECTION AND DURING PREGNANCYPosters: REF 461 to REF 491REF 461Mother to infant transmission of Hepatitis E virusMaysaa EL SAYED ZAKI 1,2 , Amena ABD EL AAL 1 , AhmedBADAWY 2,1 , Douaa RAAFAT EL DEEB, 1 , Nermin YOUSSEF ABOEL KHEIR 11 Clinical Pathology Mansoura Faculty of medicine, Mnasoura, EGYPT;2 Obsteric and Gynaecology Mansoura Faculty of Medicne, Mnasoura,EGYPTThe Aim is to study (1) The seroprevalence of HCV, HBV and HEV inpregnant women complaining of acute hepatitis in Mansoura UniversityHospital Egypt (2) The rate of mother to infant transmission of thosemarkers to their infants. Method sera of 124 affected pregnant womenpresented with acute hepatitis were tested for IgG for hepatitis C, hepatitisB s antigen (HBsAg) and/or core IgM and specific IgM and IgG for HEVby ELISA and nested PCR for HEV. Moreover, 54 of their neonates weretested similarly for those virological markers Among pregnant women thehighest prevalence for hepatitis serological markers was for specific IgGfor HEV 24(19.4%), followed by HCV IgG (17.7%) and HBsAg (9.7%),while IgM for HEV was positive only in one patient. By nested PCRfor HEV, viremia was found in 10 patients (8.1%). All affected womenhad mild symptomatic disease with complete recovery regarding theiracute hepatitis conditions, however, affected pregnant women had significantlypremature rupture of membranes compared to non infected patients(P=0.0001). In their neonates the highest prevalence for serological markerswas for HEV IgG (11.1%), HEV IgM and HCV IgG (1.8% for each).HEV viremia was detected by nested PCR in 5 neonates (9.3%). Amongthe affected neonates by presence of hepatitis E viremia, prematurity wascommon (41.1%) with signs of congenital infections as respiratory distresssyndrome (62.2%) and sepsis (41.4%). The mother transmission of hepatitisE virus was 50.6% .Conclusion: The study predicts that seroprevalenceof hepatitis E in our community is commonREF 462Epidemiological surveillance of influenza virus in N. Greece during2012 1013 influenza seasonGeorgia GIOULA, Maria EXINDARI, Angeliki MELIDOU, NikolaosMALISIOVASLaboratory Department, Medical School, Aristotle University of Thessaloniki,Thessaloniki, GREECEInformation on annual activity of influenza is essential. The aim of thisstudy is the report of the epidemiological data of influenza activity, during2012-2013 influenza season in northern Greece. During 2012-2013 winterseason (weeks 46 20), 224 respiratory specimens from representativeinfluenza like illness patients were sent to the National Influenza Centre ofN. Greece. 52 of the above specimens were received from Sentinel GPs,while the remaining 171 samples belonged to outpatients or hospitalizedcases. RNA was extracted and Real Time RT PCR was performed usingthe recommended CDC protocol. Influenza virus was detected in 60 outof the 224 specimens (26.78%). The majority of them belonged to type A(96.66%), while the predominant subtype was A(H1N1)pdm09 (72.41%).Influenza morbidity was higher in adults aged 51 60 years old. 15 peopledied, 11 of whom were infected by A(H1N1)pdm09 influenza strain. Sexdistribution showed similar infection rates at both sexes. Co infection wasobserved in one case, with A(H3N2) and B influenza viruses. It is worthmentioning that the pandemic strain, which was not present during lastyear’s influenza season (2011-2012), was mostly detected in specimensderived from ICU hospitalized patients (81.3%), while A(H3N2) influenzavirus was detected in the majority of sentinel samples (p= 0.00014). Inconclusion, the continuous monitoring of the characteristics of circulatingviruses is an essential tool for understanding the epidemiological and virologicalfeatures of influenza viruses, for monitoring their matching withseasonal vaccine strains, and for tuning vaccination strategies.REF 463GB virus C Infection among Lithuanian population with Hepatitis C(HCV) or Human immunodeficiency virus (HIV) infectionAgne VALINCIUTE 1 , Silvija KIVERYTE 2 , Mykolas MAURICAS 11 Immunology Department, State Research Institute Centre for InnovativeMedicine, Vilnius, LITHUANIA; 2 Laboratory of Molecular Diagnostics,Center of Laboratory Diagnostics, Vilnius University Hospital SantariskiuClinics, Vilnius, LITHUANIAGB virus C (GBV C), also known as hepatitis G virus (HGV), is an envelopedvirus with about 9,4Kb genome length single strand RNA. Basedon similarity in genome structure with HCV, the GBV C virus is classifiedas the Flaviviridae family virus. The GBV C virus has a worldwide distributionand virus infections are common among healthy blood donors aswell as among immunocompromised individuals. Previous studies showedthe high GBV C co infection rate in hepatitis C virus (HCV) and Humanimmunodeficiency virus (HIV) positive individuals. Based on genetic differencesbetween the 5 ′ untranslated region (5’UTR) sequences of GBVC isolates, virus is classified into seven major genotypes and in severalsubtypes. The distribution of GBV C genotypes varies geographically andinformation is still incomplete. The aim of this study is determined thefrequency of GBV C and the GBV C genotypes among the HCV and HIVpositive individuals in Lithuania and compare with the results obtainedfrom other countries. This is the first study that characterizes the presenceof GBV C in co infection with HCV and HIV in Lithuania. In thisstudy, GBV C RNA was isolated from serum samples of patients withknown HCV or HIV infection. The nested reverse transcriptase reactionwas used to synthesize the complementary DNA (cDNA) and the fragmentof 211 bp from 5’UTR region was amplified by nested RT PCR.The PCR products were purified and sequenced. The sequences obtainedfrom GenBank were used to compare the sequences of the isolates in thestudy.REF 464HHV 6 associated leukocytoclastic vasculitis in an immunocompetentadult patient and the possible role for HHV 6 chromosomal integration(CIHHV 6)Calvario AGATA 1 , Baldanti FAUSTO 2 , Foti CATERINA 3 , CitarellaFRANCESCA 1 , Miragliotta GIUSEPPE 1 , Scarasciulli MARIA 11 Microbiology And Virology Dept. Policlinico, Bari, ITALY; 2 MolecularVirology Unit, Virology And Microbiology Dept. Fondazione Irccs PoliclinicoSan Matteo, Pavia, ITALY; 3 Internal Medicine And Clinical OncologyDept. Dermatology Section, Bari, ITALYA 43-year-old woman was admitted to our Hospital because of a 10 dayhistory of scattered papulo vesicular lesions localized on the back andlimbs. Complete physical examination did not disclose other pathologicalsigns. The patient was otherwise healthy and denied pharmacologicaltreatment to the onset of the cutaneous lesions. Laboratory routine testswere negative. Skin biopsy, cutaneous swab, serum,whole blood were analyzed.Real time PCR (RT PCR) was carried out in order to detect HHVS248 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology6,HSV 1,HSV 2,VZV,EBV and CMV DNA. HHV 6 chromosomal integration(CIHHV 6) was determined by virus DNA amplification in hairfollicles. Cutaneous swab and skin biopsy as well as serum and wholeblood resulted positive for HHV 6 DNA (3.2 × 102,2.7 × 103,1.5 × 102and 4.5 × 106copies/ml,respectively) whereas were negative for the otherviruses. Since the presence of high viral copy number in whole blood togetherwith lower level in serum is suggestive of CIHHV 6, hair follicles wereinvestigated for HHV 6 DNA and CIHHV 6 was established. Histologywas consistent with a small vessel neutrophilic/leukocytoclastic vasculitis.The skin rash spontaneously disappeared within 4 weeks. Six monthslater the persistence of HHV 6 DNA was confirmed by RT PCR in bothserum and whole blood. No clinical recurrence was observed in the following12 months.According to the most recent data, our finding supportsthat the expression of HHV 6 genes after viral chromosomal integrationmight contribute to explain, at least in part, the development of immunemediated diseases, such as leukocytoclastic vasculitis.REF 465Comparison of Viral Load Assays for the Quantification of HCVGenotypes 14intheSetting of Low Viremic SamplesPatrick BRAUN 1 , Frank WIESMANN 1 , Annemarie BERGER 3 , RolfKAISER 2 , Gudrun NAETH 1 , Robert EHRET 4 , Heribert KNECHTEN 11 PZB Aachen, Aachen, GERMANY; 2 Institute of Virology, Cologne,GERMANY; 3 Institute of Frankfurt, Frankfurt, GERMANY; 4 MedicalLaboratory Berg, Berlin, GERMANYBackground: The aim of this study was to assess the variation between5 quantitative HCV RNA assays in the range of clinical decision points,recently established i.e. for treatment with protease inhibitors.Methods: Serial dilutions from the 3rd WHO and PEI standards [nominal:1000, 500, 200, 100, 25, 10 & 5 IU/mL] were tested with 5 HCV viral load[VL] assays (artus HCV QS RGQ [Qiagen], COBAS Ampliprep/COBASTaqMan (CTM) HCV v1 and v2 [Roche], RealTime HCV [Abbott] andVersant HCV RNA 1.0 [Siemens]) in triplicate in a single run. Interassaycoefficients of variation (CV) were determined for serial dilutions of 1000,100 & 25 IU/ml [gt1:N=6/gt2,3,4: N=1 each). 20 clinical gt1 samples weremeasured in triplicates to determine the differences in VL between theassays.Results: From 100 1000 IU/ml, the max. difference from nominal valuesranged from CTM v2 results of 0.51 log [PEI] and 0.91 log [WHO] toartus results of +0.25 log [PEI] and 0.07 [WHO]. Within the dilution of 525 IU/mL for both standards, artus “detected” 9/18, CTM v1 14/18, CTMv2 7/18, RealTime 16/18 and Versant 10/18, respectively. The mean CV%for clinical gt1 samples at 1000/100/25 IU/mL was 27%/35%/81% (artus),21%/30%/54% (CTM v1), 30%/32%/65% (CTM v2), 15%/18%/26%(RealTime) and 28%/31%/46% (Versant), respectively. The widest discrepancybetween two assays was observed between artus and RealTime[0.68 0.75 log].Conclusion: High sensitivity and accuracy of HCV quantification assaysare crucial for reliability of clinical decisions concerning treatment continuation.REF 466Performance Evaluation of Toxoplasma IgG and IgM assays fromBioPlex ® 2200 ToRC Multiplexed Panels (Bio Rad)Ermanno CANDOLFI, Elodie HAAR, Denis FILISETTI, OdileVILLARDInstitut de Parasitologie et de Pathologie Tropicale, Centre National deRéférence pour la Toxoplasmose, laboratoire associé, Un, Strasbourg,FRANCEObjectives: BioPlex ® 2200 multiplexed ToRC assays offer the ability toreport Toxoplasma, Rubella and CMV results in a single test. The aimof this study is to evaluate the performances of the BioPlex ® 2200 ToxoplasmaIgG and IgM immunoassays by comparing with our routine method(Architect, Abbott). Moreover very specific retrospective samples werealso assessed using BioPlex ® 2200 kits. Methods: 200 routine sampleswere submitted to BioPlex ® 2200 assays and compared to Architect’sresults (142 IgG/IgM, 3 IgG/IgM+, 33 IgG+/IgM, 22 IgG+/IgM+). Moreover41 sera with low titers of IgG (and IgM negative) and 27 seroconversionpanels were also assessed. Finally 44 samples from chronic infection(IgG positive) with persistent IgM, and 53 possibly interfering sampleswere also tested. Discrepant results were arbitrated on Western Blot IgGII(LDBIO) and ISAGA IgM test (bioMérieux). Results: Relative sensitivityand specificity of BioPlex ® 2200 Toxoplasma assays compared to Architectare respectively of 96.4% and 99.3% for IgG, and 86.4% and 92.3%for IgM. All 41 samples with IgG low titer were confirmed positive onBioPlex ® 2200. However, Toxoplasma IgG titers were overestimated inlatent toxoplasmosis. In acute toxoplasmosis, for 5 seroconversion casesout of 27, a delay is observed for the detection of IgG but the concordancefor IgM is perfect. Finally the IgM assay is fortunately less sensitive toresidual IgM.Conclusion: BioPlex ® 2200 Toxoplasmosis assays demonstrate satisfactoryperformances with a good overall concordance with our routinemethod.REF 468Anti HCV Signal to Cut off ratios for prediction of HCV infectionRoselyne FALCOU BRIATTE 4 , Guillaume DAUSSANGE 1,2,3 , PascaleTRIMOULET 1,2,3 , Christine MASSON 4 , Christophe HILLAIRET 5 ,Hervé FLEURY 1,2,31 Laboratoire de Virologie, CHU de Bordeaux, Bordeaux, France; 2 CNRS,Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux,France; 3 Univ. Bordeaux Segalen, Microbiologie Fondamentale et Pathogénicité,UMR 5234, Bordeaux, France; 4 Bio Rad, Marnes la Coquette,France; 5 Beckman Coulter Inc., Villepinte, FRANCEObjectives: the screening and diagnosis of hepatitis C virus (HCV) infectioninvolve the detection of specific antibodies (anti HCV) and of HCVRNA. Positive result of anti HCV must be confirmed by another immunoassayand/or by HCV RNA quantification to detect viremia. The aim ofthis study is to determine specific Signal to Cut off (S/Co) ratios with thecommercial kit that is used routinely in our laboratory for the prediction ofanti HCV or HCV RNA positivity. Methods: A chemiluminescence immunoassay(CIA) system with random access (UniCel ® DxI 800, BeckmanCoulter) was used to detect anti HCV status (Access ® HCV Ab PLUS,Bio Rad) between January and September 2012. Positive results of antiHCV were followed by an enzyme immunoassay (EIA) (MONOLISA ®Anti HCV PLUS version2, Bio Rad) used on the ETI MAX 3000 system(DiaSorin) to confirm anti HCV positivity and/or HCV RNA quantification.The Statistical Analyse it ® 2.30 software was used for data analysis.ROC curves were analyzed to ascertain the optimal S/Co ratios to predictHCV infection. Results: With the CIA method, the S/Co ratios weredetermined on 15,552 patient samples. 4.7% of patients had positive testresults. A S/Co ratio

5 th European Congress of VirologyREF 469Novel neutralizing antibody types and ELISA antibodies togH/gL/pUL128 131, gH/gL and gB complexes allow to define retrospectivelythe onset of primary human cytomegalovirus infectionGiuseppe GERNA 1 , Daniele LILLERI 1,2 , Anna KABANOVA 2 , ElenaPERCIVALLE 1 , Chiara FORNARA 1 , Maria Grazia REVELLO 1 ,Antonio LANZAVECCHIA 21 Fondazione IRCCS Policlinico San Matteo, Pavia, ITALY; 2 Institute forResearch in Biomedicine, Bellinzona, SWITZERLANDDuring primary HCMV infection of pregnant women, it is critical to knowthe onset of infection. We took advantage of novel neutralizing and ELISAIgG antibodies directed to HCMV glycoprotein complexes to address thisissue. Neutralizing antibodies were determined against VR1814 at passageHUVEC/454 and/117, and AD169m131 at passage HELF/36. Results showedthat the neutralizing antibody geometric mean titer (GMT) vs VR1814HUVEC/454 on ARPE 19 was 51 in the first 15 days and 511 at days 16 30p.i. At the same times, GMT to VR1814 HUVEC/117 and AD169m131 onHELF were

5 th European Congress of VirologyConclusion: Our method is very specific and allows detection of co infectionswhile commercial methods are more sensitive but failed to detect coinfections. The accuracy of our method was confirmed by the identificationof expected HBV genotypes (A,D: most frequent genotypes in Europe) inpatient and in QCMD samples.REF 473An exercise in instrument consolidation and redundancy in atertiary teaching hospital: Comparative evaluation of Diasorin Liaison/LiaisonXL Hepatitis B Serology AssaysCraig LEEMAN, Elizabeth MARLAND, Elizabeth GALVIN, SpirosREPOUSIS, Jane CORNWALLSydPath St Vincent’s Hospital, Darlinghurst AUSTRALIASt Vincent’s Hospital is one of Australia’s leading tertiary teaching hospitalsand recognised as a centre for excellence in clinical care, research andtraining with specialty areas including transplantation and HIV/AIDs care.The need to streamline pathology services by centralisation or consolidationaffects laboratories of all sizes. From 2011 to 2013 a project has beenundertaken within SydPath to reduce test platforms and subsequent costswhile remaining compliant with ISO and cGMP requirements for redundancyfor provision of donor services. The solution sought was to increasethroughput and capacity while eliminating restrictive batch testing of traditionalELISA technology without compromising result quality and patientcare. Stage one realized automation and consolidation of EBV, HSV, Parvo,MMV, Toxo and Hep A testing to LIAISON/LIAISON XL. Stage twoprovides consolidation and redundancy for Hepatitis B testing. Aim: Toevaluate LIAISON/LIAISON XL murex Hepatitis B CLIA assays vs currentAbbott Architect CMIA and BioMerieux VIDAS ELFA. Method: 150characterised retrospective and prospective samples tested for HBsAg, sAgneut, HBeAg/Ab, HBc IgM to determine concordance/serostatus; PelispyT38 & T17, Bio Rad Virotrol III & Viroclear assessed limit of detection,linearity and precision; HBsAg mutant panel challenged mutant detectioncapability. Results: Excellent concordance. Superior HBsAg mutantdetection capability demonstrated by LIAISON XL murex HBsAg Quantassay. Throughput, capacity and consolidation were achieved improvingpatient care and clinical outcomes.REF 474Reconsidering primary HPV testing in cervical cancer screeningCarlo LIVERANI 1,21 Fondazione IRCCS, Ospedale Maggiore Policlinico, Milano, ITALY;2 University of Milan, Milano, ITALYInappropriate testing for HPV types on healthy subjects increases costswithout benefit and potentially results in overtreatment (1). HPV testingalso has a negative psychosocial impact on women, increasing anxiety,stress, and concerns on sexual relationships (2,3). Giving the fact thatrecently HPV testing has been shown to have similar sensitivity but moreoverdiagnosis than cytology (4), and also giving the fact that false negativeresults may be higher than previously suspected (5), primary screeningwith HPV tests in European countries should be reconsidered. Resourcessaved in molecular testing may well be addressed in implementing vaccinationstrategies which are still underused, and may possibly includemales as well as women (6,7).References1. Solomon D, et al. Statement on HPV DNA Test Utilization. Acta Cytol2009; 53 (3): 247-8.2. Rosen NO, et al. The impact of intolerance of uncertainty on anxietyafter receiving an informational intervention about HPV: a randomisedcontrolled study. Psychol Health 2010; 25 (6): 651-68.3. Kwan TT, et al. Psychological burden of testing positive for highrisk human papillomavirus on women with atypical cervical cytology:a prospective study. Acta Obstet Gynecol Scand 2011; 90 (5):445-51.4. Malila N, et al. The HPV test has similar sensitivity but more overdiagnosisthan the Pap test A randomised health services study on cervicalcancer screening in Finland. Int J Cancer 2012 Sep 18. doi: 10.1002/ijc.2785.5. Liverani CA, et al. High risk HPV DNA subtypes and E6/E7 mRNAexpression in a cohort of colposcopy patients from Northern Italy withhigh grade histologically verified cervical lesions. Am J Transl Res 2012;4 (4): 452-7.6. Oscarsson MG, et al. Young women’s decision making process for HPVvaccination. Sex Reprod Healthc 2012; 3 (4): 141-6.7. Jin XW, et al. Human papillomavirus vaccine: Safe, effective, underused.Cleve Clin J Med 2013; 80 (1): 49-60.REF 475Development of a new diagnostic tool for the detection and quantificationof Parvovirus B19 by Real Time PCRPatricia MARECHAL, Gwendoline FOUCAUD, Stéphane MAGRO,Come BARRANGERBioMérieux, Verniolle, FRANCEObjectives: Parvovirus B19 is an important human pathogen causing avariety of diseases with outcomes ranging from asymptomatic to severe,such as chronic anemia immunocompromised patients or fetal hydrops anddeath after maternal infection. The bioMérieux Parvovirus B19 R gene ®real time PCR assays allows to quantify the three B19 genotypes on themajor extraction and real time PCR platforms. The results expressed incopies/mL can be converted to international units by using a conversionfactor determined on the basis of the 2nd WHO International Standard forParvovirus B19.Methods: Viral DNA was extracted from 200 L of whole blood orplasma on NucliSENS ® easyMAG TM and eluted in 50 L. 10 L ofpurified nucleic acids were added to 15 L of ready to use amplificationpremix. An Internal control added before extraction step was usedas extraction/inhibition control. Parvovirus B19 and internal control wererespectively detected at 530 nm and 560 nm on ABI 7500 Fast.Results: On the Parvovirus B19 QCMD Panel 2012, 100% (8/8) of thesamples were correctly identified and quantified. Analytical sensitivitystudies performed in whole blood and plasma specimens showed a highlevel of sensitivity.Coefficient of variations below 3% were observed for the intra/inter assayreproducibility studies carried out on the 2nd WHO IS Parvovirus B19diluted in whole blood negative samples.Conclusion: The high quality associated with its compatibility with themajor extraction and real time PCR platforms allows immediate integrationof this tool into most routine diagnostic laboratories.REF 476Does the HCV INNO LIPA 2.0 assay correctly assigns genotype 4?Camelia MOKHTARI 1 , Arielle ROSENBERG 2 , Stephanie HAIMBOUKOBZA 1 , Eric MARCHADIER 1 , Anne Marie ROQUE AFONSO 11 Virologie,AP HP Hôpital Paul Brousse, Villejuif, 94800, France;2 Virologie,AP HP Hôpital Cochin, Paris, 75006, FRANCEThe strategy used to treat HCV infection depends on the viral genotype particularlywith the upcoming direct antiviral agents. For therapeutic trials,European medicine authorities recommend the use of NS5B sequencingor the reverse hybridization assay Inno Lipa 2.0, based on the simultaneousdetection of 5’UTR and Core regions. However, one of our patients,Virologie, Vol 17, supplément 2, septembre 2013S251

5 th European Congress of Virologyinfected by HCV genotype 4r, as determined by NS5B sequencing, wasdenied inclusion in a genotype 4 dedicated trial because the centralizedscreening method (InnoLipa) gave an undetermined result. To study theability of the Inno Lipa assay to correctly assign genotype 4 for this particularsubtype, we tested retrospectively all genotype 4r samples identifiedat our center. From 2008 to 2012, 20 patients (2%) infected by genotype 4rwere identified. InnoLipa assigned genotype 4e in 10 samples, as determinedby the band pattern 1,2,5,16,23. An undetermined result was obtainedfor the remaining 10 samples: a pattern showing an additional band on line3 was observed in 8 cases, an additional reactivity on line 9 was observedin one case and the missing of line 16 was observed in one case. In conclusion,although Versant HCV genotype 2.0 assay is easiest to implement inroutine genotyping, NS5B sequencing must remain the reference method,particularly for therapeutic trials.REF 477Evaluation of a rapid test device for the detection of hepatitis C virusinfection at the point of first contactSandeep RAMALINGAM 1 , Mina OHARA 2 , Jacky SHAW 2 , PeterMCCULLOCH 1 , Clifford LEEN 31 Department of Microbiology, Royal Infirmary of Edinburgh, Edinburgh,SCOTLAND; 2 Substance Misuse Directorate, NHS Lothian, Edijnburgh,SCOTLAND; 3 Department of Infectious Diseases, Western General Hospital,Edinburgh, SCOTLANDIntroduction: Diagnosis of hepatitis C virus (HCV) infection is primarilylaboratory based. In high risk groups, a proportion would not return forresults, thereby reducing the effectiveness of intervention projects.Objectives: To determine if a rapid test device for HCV could be used in theclinic and if its performance characteristics allowed its safe introductioninto clinical practice.Methods: Individuals attending the Substance Misuse Directorate forHCV screening were consented. Samples were tested by ImmunoFlowHCV (IFHCV) prior to being sent to the laboratory for routine testing.Difficulty in processing specimen or reading results were noted alongwith whether participants wished to know the result immediately if thetest was introduced.Results: All 135 individuals tested expressed their desire for knowingresults immediately. The sensitivity, specificity, positive and negative predictivevalues for diagnosing antibody status and active HCV infectionswere 75%,99%,96%,93%; and 100%,96%,86%, 100% respectively. Therewere three acute HCV infections, all of whom were detected by IFHCV.Conclusions: IFHCV was cheap and easy to use in the clinic.ThoughIFHCV was less sensitive in detecting HCV antibody, the assay had goodperformance characteristics in identifying active HCV infections. IFHCVcould hence be used to give a preliminary result and organize furtherappointments at the first clinic visit whilst waiting for confirmation ofresults from the laboratory.REF 478Towards standardization of HDV RNA quantificationRoland SAHLI 1 , Christopher DOERIG 2 , Christine ESTRADE 1 , AmalioTELENTI 1 , Darius MORADPOUR 21 Institute of Microbiology, Centre Hospitalier Universitaire Vaudois andUniversity of Lausanne, Lausanne, SWITZERLAND; 2 Division of Gastroenterologyand Hepatology, Centre Hospitalier Universitaire Vaudoisand University of Lausanne, Lausanne, SWITZERLANDChronic infection by hepatitis D virus (HDV) is a severe complication inpatients with hepatitis B. HDV RNA quantification in plasma is importantto confirm the diagnosis of chronic hepatitis D and to monitor the responseto treatment. HDV RNA quantification has been difficult to standardize.We explored the contribution of RNA extraction, cDNA synthesis andPCR assays to viral load determination. Two RNA extraction methods, 3RNA melting temperatures prior to cDNA synthesis, and 3 PCR assays(HDVL, Le Gal et al., 2005; HDVR, in house multiplex assay; HDVG,Ferns et al., 2012) were assessed in clinical samples and international qualitycontrol samples. HDV RNA quantification was influenced mainly byviral sequences and PCR assay. HDVL underestimated loads by 5 foldcompared to HDVR. However, HDVR was susceptible to sequence polymorphismsin a low percentage of genotype 1 strains downstream the 3’ endof the forward primers binding site, resulting in 100 fold lower viral loadestimates relative to HDVL or HDVG. HDVG offered the best compromisebetween viral load estimates and independence to viral sequence variation.In addition, fast thermocycling diminished the PCR assay dependent readoutand an endogenous extraction control could correct for inhibitors.Selecting PCR assays with fast thermocycling and with primers havingtheir 3’ end near loop structures of the cDNA target may allow for optimalstandardization of HDV RNA quantification. Definition of an internationalstandard would be required to allow for comparison of HDV RNA resultsobtained in different laboratories.REF 479Comparison of two immunoblot assays for the confirmation of hepatitisC antibodiesHelvi Holm SAMDAL 1 , Vethanayaki SRIRANGANATHAN 1 , RagnhildVassbø EID 1 , Bente HAGH 2 , Svein Arne NORDBØ 21 Department of Microbiology, Oslo University Hospital, Ullevål, Oslo,NORWAY; 2 Department of Medical Microbiology, Trondheim UniversityHospital, Trondheim, NORWAYObjective: to compare recomLine HCV IgG (Mikrogen) and Chiron ®RIBA ® HCV 3.0 SIA (Novartis) in verifying repeatedly reactive resultsobtained in primary HCV antibody testing. Methods: Sera from 13 blooddonors and 78 patients, reactive in Abbott Architect Anti HCV test, wereanalyzed by Chiron ® RIBA ® SIA HCV 3.0 containing NS5 and c33crecombinant proteins and c100p, 511pandc22p synthetic peptides, andrecomLine HCV IgG containing Core1, Core2, Helicase, NS3, NS4 andNS5 recombinant proteins. Interpretation was done according to manufacturersguidelines. Samples from all blood donors and 51 patients were alsoanalyzed by PCR (In house and CAP/CTM (Roche). Results: the resultswere in accordance for 59 sera. Seven sera were RIBA positive, but indeterminateor negative in recomLine. Follow up gave no indication of HCVinfection, including negative PCR results. Three sera were RIBA indeterminateand recomLine positive. PCR analyses were positive in two of thesera, which reacted strongly against RIBA c22p. The difference is due todifferent antigen representation and interpretation criteria. Seventeen sera,indeterminate in RIBA (mainly reactive against c33c), were negative inrecomLine and PCR analyses. Five sera, negative in RIBA and PCR, wereindeterminate in recomLine. Conclusion: in our study, recomLine reducedthe number of indeterminate results compared to RIBA. For three patientsrecomLine was more sensitive in detecting HCV infection. RecomLineseems well suited for confirmation testing for HCV antibodies.REF 480Evaluation of a novel real time PCR assay for Parvovirus B19 (B19 V)genome detection and quantificationAurélie SCHNURIGER 1,2 , Kenda SALOUM 1 , Yanne MICHEL 1 ,Valérie MARINHO 1 , Isabelle ANSART 1 , Antoine GARBARGCHENON 1,21 APHP Trousseau hospital, Virology Department, Paris, FRANCE; 2 ERLINSERM U1057/UMR7203, UPMC University Paris 6, Paris, FRANCES252 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyMolecular diagnosis of B19 V requires accurate detection and quantificationof viral load ranging from values very high in acute infection, tovery low in chronic infection. We evaluated the Parvovirus B19 R gene ®assay (bioMérieux), in comparison with an “in house” quantitative realtime PCR, both targeting the NS1 gene. We analysed 320 serum samples,selected prospectively (n=100) or retrospectively (n=220). qPCR valuesin copies/ml were converted in IU/ml, using the WHO B19 standard.The 2 assays were 100% concordant for prospective samples. Quantification(8 positive sera) was similar (R2=0.95). The R gene ® PCR gavelower copies/ml values (approx. 2 Log) than the in house PCR, but IU/mlvalues were similar (mean 0.4 Log). Concordance for the 220 retrospectivesamples was 99.6%. R gene ® PCR specificity was 100%. Viral loadsfor 71 positive sera (including low, medium and high values) were highlycorrelated (R2=0.98), with similar results on the range 10E2 10E10 IU/ml.Acute infection sera (n=5) showed in both cases high viral loads (10 13Log IU/ml) but were under quantified by the in house assay (1.2 LogIU/ml) and slightly over quantified by the Argene assay (+0.3 Log IU/ml).Detection and accurate quantification of B19 V in these sera required amandatory manual reanalysis of raw results for both assays. The ParvovirusB19 R gene ® assay offers a good practicability and gives verysatisfying results. This assay is suitable for molecular diagnosis of B19 Vinfection, allowing accurate quantification over a large range of viral loadvalues.REF 481Calibration of microneutralisation assays of proven CMV primaryinfections in early pregnancyKatrin SCHWEINZER 1 , Karl Oliver KAGAN 2 , Harald ABELE 2 ,Markus HOOPMANN 2 , Ingo RETTIG 3 , Gerhard JAHN 1 , KlausHAMPRECHT 11 Institute of Medical Virology, University of Tuebingen, Tuebingen, GER-MANY; 2 Department of Obstetrics and Gynaecology, University Hospitalof Tuebingen, Tuebingen, GERMANY; 3 Department of Internal Medicine,University Hospital of Tuebingen, Tuebingen, GERMANYBackground: In the absence of a general CMV seroscreening in pregnancy,the detection of CMV primary infection in Germany is still adiagnosis by accident. Additional parameters for viral monitoring besideCMV IgG/IgM levels, avidity and gB2 absence are needed for better definitionof the infection onset. Actually, there exists no defined CMV IgGWHO standard. Patients and Objective: Patient sera were derived fromproven CMV primary infections during pregnancy. We wanted to correlateundefined CMV IgG levels (AU or IU/ml) with the correspondingmicroneutralisation (NT) titers using PEI units (Planitzer et al., 2011).Methods: For calibration of our NT assays we created reference pools ofseronegative and seropositive mothers from our prospective cCMV studywith immunoblot proven high IgG avidity. The reference sera included aCMV specific HIG (Cytotect ® ), CMV IgG reference (12/1996), Paul EhrlichInstitut, and anti CMV QC1 (NIBSC). Results: Calibration of bothHFF and RPE dependent NT assays showed distinct differences with respectto IgG reference pools. The dynamic range of NT50 values showedthe advantage of RPE versus HFF target cells. The NT capacity was foundto be drastically increased in RPE cells compared to HFF cells. NT antibodiesin RPE cells were detectable very early in pregnancy. Conclusion:We succeeded to establish a new standardized NT assay for RPE cells.Further investigations have to verify the prognostic value of our CMVNT assay in prenatal diagnosis and its suitability in monitoring of HIGtreatment.REF 482Performance of Qiagen HCV real time assay to monitor Hepatitis CVirus viral loadVincent THIBAULT 1 , Syria LAPERCHE 2 , Monique THEVENIN 1 ,Jacques MASSON 1 , Sepideh AKHAVAN 11 GH Pitie Salpetriere/AP HP/Virology, Paris, FRANCE; 2 Institut Nationalde Transfusion Sanguine/Virology, Paris, FRANCEHepatitis C virus (HCV) infection treatment monitoring relies on accurateand sensitive assessment of HCV viral load (VL). This study was designedto assess the performance of Qiagen HCV real time RT PCR assayon a large variety of genotypes. Samples (n=106) corresponding to clinicalspecimens covering all genotypes (Gt) and several subtypes from HCVinfected patients were analyzed in parallel with Abbott M2000 rt/sp HCVRealtime and Qiagen Qiasymphony/rotorgene (artus HCV QS RGQ Kit).Additional samples (n=20, Gt 1 4) and serial dilutions of the WHO standardwere also tested with Roche Cobas Taqman v1.0 and v2.0 in parallelwith previously indicated techniques. Although there was a strong correlation(r2=0.95) between Abbott and Qiagen techniques, a mean bias of0.446 log IU/mL in favor of Qiagen was observed. This bias was detectedacross all genotypes (1 6) but was statistically less important (0.228) for Gt2 samples (p=0.005). Despite a good correlation between all techniques,the mean biases over Abbott for Roche v1 and v2 techniques were 0.530and 0.114 logIU/mL, respectively. Serial dilutions of the WHO standardindicated a good linearity of all techniques but also important discrepanciesbetween each other according to HCV VL. Qiagen HCV VL assayrepresents a good alternative to Abbott and Roche techniques. Identificationof biases and discrepancies between techniques, particularly in thelower quantification range, should be considered when monitoring treatments.Recommendations to monitor a patient along time with a singletechnique should still be the rule.REF 483A New Quantitative PCR for Human Parvovirus B19Mari TOPPINEN 1 , Päivi NORJA 1 , Maria SÖDERLUND VENERMO 1 ,Klaus HEDMAN 1,21 Department of Virology, Haartman Institute, University of Helsinki, Helsinki,FINLAND; 2 Hospital District of Helsinki and Uusimaa, LaboratoryServices, HUSLAB, Helsinki, FINLANDParvovirus B19 is a small, ssDNA virus associated with a wide rangeof diseases from mild childhood erythema to fetal death. After primaryinfection, the viral genomes persist lifelong in solid tissues of most types.Quantification of the viral DNA is important both in timing of primaryinfection, and the assessment of tissue persistence. In this study, we presenta new PCR assay to detect and quantify all three B19 genotypes. A qPCRwas designed based on the hydrolysis probe format. Via comprehensiveanalysis of B19 sequences, a primer pair and a probe were designed toamplify and quantify a 154 bp amplicon of the NS1 area. Optimization ofthe assay was done by adjusting the PCR parameters, by using as templategenotype 1 3 plasmids, and clinical material. In addition, the 1st WHOInternational Reference Panel for Parvovirus B19 Genotypes served asa control. Our new qPCR amplifies and quantifies all three B19 genotypesequally. The assay sensitivity is 10 copies/reaction of genotype 13 plasmid templates and =50 copies/reaction of ex vivo viral genomes.Most B19 infections occur asymptomatically or with mild symptoms.However, during primary infection, the B19 levels in blood can be upto 1013 copies/ml. While traditional diagnostics of B19 infection relies onVirologie, Vol 17, supplément 2, septembre 2013S253

5 th European Congress of Virologyserological methods, quantitative PCR aside of serology is increasinglyrecommended. Due to its small size and non enveloped structure B19 isresistant to most viral inactivation methods. For safety of blood productsurveillance, quantitative PCR amplifying all B19 genotypes is mandatory.REF 484Age dependent differences in levels of rubella antibodies of pregnantwomen in NorwayRegine BARLINN 1 , Kirsti VAINIO 1 , Helvi Holm SAMDAL 2 , SveinArne NORDBØ 3 , Hanne NØKLEBY 4 , Susanne G. DUDMAN 11 Department of Virology, Norwegian Institute of Public Health, Oslo,NORWAY; 2 Department of Medical Microbiology, Drammen Hospital,Vestre Viken Hospital Trust, present institution; Department of Microbiol,Drammen, NORWAY; 3 Department of Medical Microbiology St. OlavHospital, Trondheim University Hospital, Norway, Trondheim, NORWAY;4 Division of Infectious Disease Control, Norwegian Institute of PublicHealth, Oslo, NORWAYAim: Rubella infection in pregnancy may lead to abortion, fetal deathor congenital rubella syndrome. Rubella vaccination was introduced as amonovalent vaccine in the national vaccination programme in 1978 witha single dose at the age of 15 for girls. MMR was introduced in 1983 asa two dose schedule at the age of 15 months and 13 years. The last majorrubella outbreak in Norway was registered in 1985. While natural infectionsusually give lifelong protection against rubella, there is uncertainty oflong term immunity following two doses of MMR because vaccine inducedimmunity may wane over time. The aim of this study was to determinethe seroprevalence of rubella antibodies in pregnant women in differentage groups in two different counties in Norway. Methods: Sera were collectedfrom pregnant women in age groups; =19, 20 24, 25 29, 30 34, 3539 and=40 years old. Anti rubella IgG were determined by using a chemiluminescence immunoassay Results: Of the 2000 pregnant women testedanti rubella IgG was positive in 94.4%. When differentiated by age, serumlevels of rubella IgG was lowest in age group 25 29 years with a positivityrate of 91, 0%. Women born before vaccination with two doses of MMRstarted, had both a higher positivity rate and significantly higher levels ofrubella antibody titre compared to those born after; 96,1% and 82,2 IU/mlversus 92,9% and 41,7 IU/ml. Conclusion: It is likely that most of thewomen in age group 4 to 6 have been naturally immunized or boostedduring childhood resulting in a stronger immune response compared tothe younger women who probably have obtained their antibodies purelyfrom vaccination. The significantly lower anti rubella IgG titres found inthe 3 youngest age groups in our study highlights the need for continuedantenatal screening in order to ensure postpartum vaccination for womenwith deficient rubella immunity.REF 485Does Early Acquisition of Human Cytomegalovirus (CMV) Precludesthe Need for Primary CMV Infection Screening during Pregnancy?Lobna METWALLY 2 , Noha KAMEL 1 , Nahed GOMAA 2 , WaleedSAYED AHMED 3 , Soha YOUNIS 11 Department of clinical pathology, Faculty of Medicine, Suez CanalUniversity, Ismailia, EGYPT; 2 Department of Microbiology, Faculty ofMedicine Suez Canal University, Ismailia, EYGPT; 3 Department of Obstetricsand Gynecology, Faculty of Medicine, Suez Canal University,Ismailia, EYGPTIntroduction: Primary CMV infection during pregnancy carries a greaterrisk for congenital fetal infection compared to secondary infection.CMV specific IgG avidity is a powerful tool for distinguishing primaryfrom non primary CMV infection. Objective: To determine the prevalenceof primary CMV infection among Egyptian pregnant women usingthe CMV IgG avidity testing. Methodology: A cross sectional descriptivestudy conducted at Suez Canal University Hospital, Ismailia, Egypt. 546pregnant women were screened for CMV IgM and IgG using a commerciallyavailable Enzyme Linked Immunosorbant Assay (ELISA). CMVIgM positive women were tested by an IgG avidity assay using Enzymelinked fluorescent assay by VIDAS. Results: All 546 pregnant women(100%) were seropositive for anti CMV IgG. Forty women (7.3%) werepositive for IgM antibodies. All the 40 samples showed high or intermediateCMV IgG avidity index, and those women were considered to be at alow risk for CMV infection transmission. Eleven of the forty women withCMV IgG avidity results were followed up till the end of pregnancy andtheir babies did not show any evidence of congenital CMV infection.Conclusion: To our knowledge this is the first study in Egypt utilizingthe IgG avidity testing to differentiate between primary and non primaryinfection in CMV IgM positive patients. All women included in the studywere determined to be at a low risk for primary CMV infection, hence theuse of universal screening tests for primary CMV infection in pregnancyor public health interventions in Egypt are not justified.REF 486HCMV DNA load in maternal blood, amniotic fluid and newbornblood in pregnancies complicated with primary HCMV infectionFurione MILENA, Dossena LUCA, Zavattoni MAURIZIO, BaldantiFAUSTOMolecular Virology Unit, Virology and Microbiology Department, FondazioneIRCCS Policlinico S. Matteo, Pavia, ITALYHCMV DNA was determined in 194 whole blood (WB), and 67 amnioticfluid (AF) samples from 67 pregnancies complicated with primaryHCMV infection referring to our Institution in the period 2008 2012, andin 19 WB samples from as many corresponding newborns using the Qia-Symphony and CMV QS RGQ assay (Qiagen,Hilden, DE). WB sampleswere also analyzed from 10 HCMV seronegative and 30 pregnant womenwith remote HCMV infection. Results were expressed as IU/ml followingidentification of WB and AF conversion factor using WHO InternationalStandard (0.90 and 1.15, respectively). HCMV DNA was negative in allWB from controls. In contrast, HCMV DNA was positive in WB of 11/13(84.61%) pts in the first month of infection and positivity decreased overtime (19/26, 73.08% 2nd mos; 22/33, 66.67% 3rd mos; 18/34, 52.94%4th mos; 7/23, 30.43% 5th mos; 7/26, 26.92% after the 5th mos). MedianHCMV DNA levels decreased in parallel (299 IU/ml at 1st mos; 35 at 2ndmos; 33 at 3rd mos; 8 at 4th mos) reaching negativity whitin the 5th mosof infection. Of analyzed AF, 36 (53.73%) were HCMV DNA positive(median 267,502 IU/ml, range 8 13,522,505) whereas 31 (46.27%) werenegative. WB samples were available from 11 newborns from 36 pregnancieswith positive AF, and all were HCMV DNA positive (median 2379IU/ml, range 57 7182). WB samples were available from 8 newborns from31 pregnancies with negative AF, and 6 were HCMV DNA negative, while2 were HCMV DNA positive (578 and 23 IU/ml, respectively).REF 487Vertical transmission of dengue infection: a case reportPriscila NUNES, Priscila NUNES, Rita NOGUEIRA, Ana SOARES,Flavia DOS SANTOS, Marcelo PELAJO, Barbara OLIVEIRA, Ana DEFILIPPISFiocruz, Rio de Janeiro, BRAZILAlthough dengue is endemic in several regions of Brazil, there are fewreports of dengue infection in pregnant and the consequences for thefetus. This study reports dengue infection resulting in maternal and fetaldeath. In November of 2010 a pregnant woman aged 23 years old, wasadmitted to the maternity of a municipal Hospital, in Rio de Janeiro, Brazil,complaining of abdominal pain, vomiting and diarrhea, fainting andS254 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyinaudible fetal heartbeat. The ultrasound revealed a single fetus with noactive movements, little or absente amniotic fluid and placenta with reduceddimensions. The fetus was 32/33 weeks presenting placental abruptionwith apparently early detachment. During the caesarean there was largeextravasation of blood in cavity, deterioration of clinical picture, hypothermia,anuria, hemodynamic instability, mydriasis, absent reflexes anddeath. Tissues of spleen, placenta and umbilical cord were paraffin embeddedto investigate dengue virus. The RNA was extracted from sections of3 m and RNA extraction was performed using the kit Purelink FFPERNA Isolation Invitrogen. Real time PCR and RT PCR was performedand DENV 2 detected by both methods in the umbilical cord.Considering that dengue infection during pregnancy may represent a risk tomother and concept, measures to strengthen epidemiological surveillanceand virological diagnosis should be implemented to this specific groupespecially in dengue endemic areas.REF 488Prognostic significance of intra-uterine detection of infectious agentsOlga OSTROVSKAYA, Nataliya IVAKHNISHINA, ElenaNAGOVITSYNA, Marina VLASOVAResearch Institute of Mother and Child Health Care, Khabarovsk, RUSSIADNA of infectious agents was analyzed by a PCR method in placentalsamples of 66 pregnant women who had placental insufficiency and ahigh risk of intrauterine infection development. Infectious agents werefound in 72.7% cases of placental analysis. Ur.urealyticum (51.5%) andM. hominis (24.2%) were most frequent foiund, followed by type 4, 5 and6 herpesviruses. Enteroviruses, C. trachomatis, S. pneumoniae were foundin single cases. No other pathogens were detected.Infected placentas were significantly more frequently associated withabnormal peri-natal features such as untimely discharge of amnioticfluid, decompensation of chronic placental insufficiency, delivery ofpremature newborns with very to extremely low body weight, withdevelopmental delay and clinical signs of neonatal infection (pneumonia,hepatosplenomegaly,necrotizing enterocolitis, retinopathy, etc.)Detection of intrauterine infectious agents in placenta may be used forprediction of the newborn’s condition and early administration of adequatetherapy.REF 489Case report of a congenital Echovirus 11 infection acquired early inpregnancyChristelle VAULOUP FELLOUS 5 , Mikael TASSIN 1 , JelenaMARTINOVIC 2 , Audrey MIRAND 3 , Hélène PEIGUE LAFEUILLE 3 ,Olivier PICONE 4 , Alexandra BENACHI 11 AP HP, Department of Gynecology and Obstetrics, Antoine BéclèreHospital, Clamart, FRANCE; 2 AP HP, Unit of Fetal Pathology, AntoineBéclère Hospital, Clamart, FRANCE; 3 Laboratory of Virology, EnterovirusNational Reference Centre, Clermont Ferrand Hospital, ClermontFerrand, FRANCE; 4 Department of Gynecology and Obstetrics, FochHospital, Suresnes, FRANCE; 5 AP HP, Laboratory of Virology, RubellaMaterno foetal infection National Reference Center, Paul Brousse Hospital,Villejuif, FRANCEEnterovirus (EV) maternal infection during pregnancy and its relation tofetal pathology are not well described. When reported, the main manifestationsof EV congenital infections are intra uterine fetal death ormyocarditis. No information on intrauterine Echovirus 11 infection or theeffect of transplacental Echovirus 11 infection on development of the fetushas been described in literature up to date (excluding late pregnancy infections).We report here a case of an extreme form of pulmonary hypoplasiain a neonate, characterized by total failure of development of terminal respiratoryunits. This bichorial biamniotic twin pregnancy, was marked byspontaneous demise of the co twin at 14 weeks of gestation (WG), as wellas by positive PCR for EV (Echovirus 11 serotype) in the amniotic fluid,performed for moderate pericardial effusion at 22 WG. No sign of cardiacdisease were further observed, but at 32 WG an ultrasound examinationrevealed bilateral abnormal lung development, and pulmonary hypoplasiawas confirmed by MRI. After spontaneous delivery at 38 WG, and despitepediatric reanimation, the child died one hour after birth. The coexistenceof a congenital EV infection and a severe primary pulmonary hypoplasia,in this case, challenges our understanding of the pathogenesis of thissevere pulmonary growth arrest. The study of more cases of congenitalEV infections acquired early in pregnancy is needed to better understandthis puzzling lethal condition.REF 490Infants born to HBsAg(+) Mothers Are they fully protected afteractive passive vaccination?Radka KOMITOVA 1 , Maria NESHEROVA 2 , Maria ATANASOVA 31 Infectious Diseases Dept, University Hospital, Plovdiv, BULGARIA;2 Neonatology Unit, Selena Hospital, Plovdiv, BULGARIA; 3 Microbiologyand Immunology Laboratory, Medical University, Plovdiv, BULGARIAIntroduction: Infants born to HBsAg(+) mothers are at ongoing risk ofhepatitis B virus (HBV) infection. Despite the timely active-passive vaccination,newborns may still become HBV infected because of nonresponseto vaccination.Aims: to confirm that the child born to an HBsAg(+) mother is fullyprotected by active-passive immunization.Case presentation: A girl was born on 07.04.2012 by cesarean sectionto a mother with chronic HBV infection [(HBsAg(+), antiBHctotal(+),HBeAg(-), antiHBe(+)]. The infant received the 1-st shot hepatitis B vaccineas well as hepatitis B immunoglobulin (HBIG) within 6 hours afterbirth. The test conducted on 07.01.2013 (3 months after administration ofthe third vaccine shot) revealed an HBsAg(-) result and antiHBs level 1225IU/ml, i. e. she is thoroughly protected and does not need further management.A second study is underway with a girl born to an HBsAg(+)mother on 30.11.2012. She also received the 1-st shot hepatitis B vaccineas well as HIBG within 6 hours after birth. At the age of 9 months shewill be tested for HBsAg and antiHBs level to make sure protection ispresent. In case of HbsAg(-) and antiHBs level

5 th European Congress of Virologyinjecting drug users and individuals from countries with HIV epidemics.In accordance with the ‘2008 European Guideline on HIV testing’ fourthgeneration screening assays are recommended for simultaneously detectionof anti-HIV-1 antibodies and HIV-1 p24 antigen as well as HIV-2antibodies. The guideline requests confirmation of any reactive or indeterminatescreening test result, using a line-immunoassay or western blotthat distinguish between the different individual HIV-1 and HIV-2 antibodies.This study evaluated two immunoblot line assays to be used as HIVconfirmation assays.REF 590Studies of human cytomegalovirus (HCMV) maternal fetal transmissionand pathogenesis in ex vivo infected human decidual organculturesDana WOLF 1 , Yiska WEISBLUM 1,2 , Zichria ZAKAY RONES 2 , RonitHAIMOV KOCHMAN 3 , Debra GOLDMAN WOHL 3 , SimchaYAGEL 3 , Amos PANET 21 Clinical Virology Unit, Hadassah Hebrew University Medical Center,Jerusalem, ISRAEL; 2 Department of Biochemistry and the Chanock centerfor Virology, IMRIC, Jerusalem, ISRAEL; 3 Department of Obstetrics andGynecology, Hadassah Hebrew University Medical Center, Jerusalem,ISRAELHuman cytomegalovirus (HCMV) is the leading cause of congenitalinfection, associated with severe birth defects, placental damage, andintrauterine growth retardation. The mechanism of transmission via thematernal fetal interface is largely unknown, and there are no animal modelsfor congenital HCMV infection. The initial stages of infection are believedto occur in the maternal decidua representing the maternal aspectof the chimeric human placenta. To gain insight into these critical earlyevents of transmission and pathogenesis, we have recently establishedan ex vivo model of HCMV infection in first trimester decidual organcultures. Using both laboratory derived and clinical HCMV strains, wedemonstrated the broad viral target cell range, with consistent cell to cellmode of spread in the decidual tissue. Antiviral drugs as well as neutralizingHCMV antibodies exhibited inhibitory activity on viral spread inthe decidua. We have further employed the decidual infection model tostudy the tissue innate immune response to HCMV infection, revealing aprofound effect of the virus on decidual innate immune and angiogeniccytokine/chemokine expression profile. Significantly, a unique virus inducedstimulation of a decidual innate immune response, with formationof proinflammatory environment was demonstrated. The ex vivo infecteddecidual cultures can serve as a surrogate human model that couldpotentially address viral and tissue determinants of HCMV maternal fetaltransmission, damage, and immunopathogenesis, and could facilitate evaluationof the effects of new antiviral interventions in the maternal fetalinterface.S256 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology24. TYPING OF VIRUSES (MOLECULAREPIDEMIOLOGY, TAXONOMY)Posters: REF 492 to REF 507REF 492HPV 16 variants isolated from general population in finistere, WestBrittany, FranceChristopher PAYAN 1 , Aureliane MICHAUD 1 , Stephanie GOURIOU 1 ,Sylvain ROSEC 2 , Sophie VALLET 1 , Adissa TRAN 1 , Yvon FOLL 3 ,Francoise BOMMELAERT 3 , Francoise CHARLES 4 , Edith POSTEC 5 ,Michel COLLET 51 Virology Lubem Chru Ufr Medecine, Brest, FRANCE; 2 Cic Inserm0502, Brest, FRANCE; 3 Adec29, Brest, FRANCE; 4 Cytologie Chru, Brest,FRANCE; 5 Gynecology Chru, Brest, FRANCEObjectives: From a HPV DNA test in urine (PapU29 study; Payan, 2007),we typed all HPV 16 positive samples to determine variants (European,African 1 and 2, Asian, North American and Asian American). In ourstudy, 50% of CIN2+ and 24% of CIN2 cases had HPV 16. Our aim wasto find out if some variants were more present in our region and if some ofthem were more virulent than others. Methods: Urine samples were obtainedfrom 25 65 years old women (n=3115) and HPV 16 positive (n=88),we performed PCR with SiHa HPV 16+ cells as control. Since the concentrationrange was fluctuent (from 1 to 7 log copies per mL), several PCRin the E6 gene and adjustments were necessary. We established that threePCR were needed including two nested. They were followed by electrophoresisand, when positive, DNA samples were sequenced and analyzedusing bioinformatic tools (Staaden Package and BioEdit). Results: Twogroups have been characterized. First, samples with more than 3 log DNAcopies per mL (n=42), 83% of them have been sequenced successfully,and second, samples below 3 log, only 15.2% were sequenced (n=46).Most of the samples sequenced presented an European variant, 57% werethe mutant 350G and with some minor differencies; only two were an Af2 variant located in the same city.Conclusion: It does not appear that there is a link between variant andCIN2+ cases, we demonstrated that the European variant is more presentthan any other in West Brittany. Nevertheless, more HPV 16 patients shouldbe done to confirm those results. Grants from the French Ligue contre leCancer.REF 493Genotypic prediction of HIV 1 isolates tropism in MoroccoHicham EL ANNAZ 1 , Patricia RECORDON PINSON 2 , RidaTAGAJDID 1 , Bouchra BELEFQUIH 1 , Safae EL KOCHRI 1 , RachidABBI 1 , Siham OUMAKHIR 1 , Herve FLEURY 2 , Saad MRANI 11 Université Mohamed V Souissi, Faculté de Médecine et de Pharmaciede Rabat. Laboratoire de virologie, Hôpital Militaire d’instru, Rabat,MOROCCO; 2 Laboratoire de Virologie, CNRS UMR5234, WHO Accredited(HIV Resistance), Universite Victor Segalen, Bordeaux, FRANCEDetermination of human immunodeficiency virus tropism plays a crucialrole in AIDS progression and is necessary prior to the use of CCR5 antagonists.The aim of this study is to investigate predicted HIV 1 co receptorusage in HIV 1isolates in Morocco. Methods: A total of 45 MoroccanHIV 1 infected patients followed up at the Mohammed V Military TeachingHospital in Rabat were included in this study. All patients were drugNaive to treatment. The viral RNA was used for reverse transcription polymerasechain reaction (RT PCR) followed by a nested PCR of V3 region ofgp120 env gene. The obtained fragments were sequenced on both strands.HIV 1 subtype analysis was determined by phylogenetic analysis of RTand gp120 sequences. The genotypic interpretation of HIV 1 tropism wasperformed using the Geno2phenocoreceptor approach. The Geno2phenointerpretation was made with the false positive rates (FPRs) for predictionof X4 variants FPRs of 10% and with the clinical parameters data (the viralload and the CD4 value). Results: Among the 45 HIV 1 infected treatmentnaive patients, 38 (84%) are males. Their mean age is 41 [35 48] years.The WHO clinical classification indicated that 13% of these patients are atstage 1, 25% at stage 2, 30% at stage 3 and 32% at stage 4. The median HIV1 viral load and CD4 cell count HIV 1 infected patients is 45700 [10140151000] copies/ml and 228 [107 419] cells/mm3 respectively. The viralsubtypes are subtype B (71%), CRF02 AG (20%) and C (9%). All subtypeC appear to use CCR5 for cell entry (R5 strains), while 87% of subtypeB and 90% of CRF02 AG are also R5, indicating a higher prevalence ofR5 (89%) than X4 (2%) or R5X4 (9%). The co receptor usage of HIV 1seems to be not associated with the different subtypes. Conclusion: Thehigher prevalence of R5 suggest that CCR5 antagonists will be promisingdrugs for future AIDS treatment in Morocco.REF 494Recent Emergence and Spread of a Phylogenetic Lineage of RabiesVirus in NepalLaurent DACHEUX 2 , Ganesh R. PANT 1 , Rachel LAVENIR 2 , Frank Y.K. WONG 3 , Andrea CERTOMA 3 , Florence LARROUS 2 , Dwij R.BHATTA 4 , Hervé BOURHY 2 , Vittoria STEVENS 31 Rabies Vaccine Production Laboratory, Kathmandu, NEPAL 2 InstitutPasteur, Unit Lyssavirus Dynamics and Host Adaptation, National ReferenceCentre for Rabies, Paris, FRANCE; 3 Australian Animal HealthLaboratory, CSIRO Animal Food and Health Sciences, Geelong, AUS-TRALIA; 4 Central Department of Microbiology, Tribhuvan University,Kathmandu, NEPALRabies is a zoonotic disease that is endemic in many parts of the developingworld, especially in Africa and in Asia. However its epidemiologyremains largely unappreciated in much of these regions, such as in Nepal,where limited information is available about the spatiotemporal dynamicsof the main etiological agent, the rabies virus (RABV). In this study,we describe for the first time the phylogenetic diversity and evolution ofRABV circulating in Nepal, as well as their geographical relationshipswithin the broader region. A total of 24 new isolates obtained from Nepaland collected from 2003 to 2011 were full length sequenced for both thenucleoprotein and the glycoprotein genes, and analysed using neighborjoining and maximum likelihood phylogenetic methods with representativeviruses from all over the world. Despite Nepal’s limited land surfaceand its particular geographical position within the Indian subcontinent,our study revealed the presence of a surprising wide genetic diversity ofRABV, with the co existence of three different phylogenetic groups: anIndian subcontinent clade and two different Arctic like sub clades withinthe Arctic related clade. This observation suggests at least two independentepisodes of rabies introduction from neighbouring countries. In addition,specific phylogenetic and temporal evolution analysis of viruses within theArctic related clade has identified a new recently emerged RABV lineagewe named as the Arctic like 3 (AL 3) sub clade that is already widelyspread in Nepal.Virologie, Vol 17, supplément 2, septembre 2013S257

5 th European Congress of VirologyREF 495Cytomegalovirus gB, gpUL144 and pUS28 genotypes distributionamong Polish childrenMiroslawa STUDZINSKA 1 , Edyta PARADOWSKA 1 , MiroslawaSTUDZINSKA 1 , Patrycja SUSKI 1 , Agnieszka JABLONSKA 1 ,Katarzyna DZIERZANOWSKA FANGRAT 2 , BeataKASZTELEWICZ 2 , Malgorzata WISNIEWSKA LIGIER 3 , TeresaWOZNIAKOWSKA GESICKA 3 , Barbara ZAWILINSKA 4 , Zbigniew J.LESNIKOWSKI 11 Laboratory of Molecular Virology and Biological Chemistry, Instituteof Medical Biology Polish Academy of Sciences, Lodz, POLAND;2 Department of Microbiology and Clinical Immunology, Children MemorialHealth Institute, Warsaw, POLAND; 3 III Department of Peadiatrics,Research Institute Polish Mother’s Memorial Hospital, Lodz, POLAND;4 Department of Virology, Jagiellonian University Medical College, Cracow,POLANDHuman cytomegalovirus (HCMV) is the most common cause of viralintrauterine infection, affecting 0.5 2.0% of all live births. The associationof genetic polymorphisms in some particular genes with the occurrence ofcongenital infection is debated. The aim of our studies was to determinethe distribution of the HCMV genotypes isolated from Polish children andto recognize the relationship between genotype, viral load, and clinicalsequelaes. We analyzed the gB, gpUL144 and pUS28 genetic variations incongenitally HCMV infected newborns (n=34) and infants with postnatalor unproven congenital HCMV infection (n=90). Genotyping was performedby sequence analysis of PCR amplified fragments, and viral load wasmeasured by quantitative real time PCR. Total genomic DNA was extractedfrom blood, plasma, peripheral blood mononuclear cells (PBMC) and urinesamples. Our results demonstrated that HCMV genotypes gB1and A1 wereprevalent in congenitally infected newborns (p

5 th European Congress of Virologyfollowed by EV A (25%). The distribution of types in VIRO Typenedwas comparable to regular surveillance. VIRO typened also registered thedetection of 3 oral poliovirus (OPV) vaccine strains (2 OPV2 and 1 OPV3).Clinical data were only available in 20% 30% of patients. Meningitis wassignificantly more found among EV B strains, while Hand Foot Mouthdisease is frequently registered for EV A strains. Analysis of the sequencedata showed the circulation of specific strains.Conclusions: The VIRO Typened concept generates information on circulatingEVs comparable to regular surveillance. With Typened clinicaldata could be linked to specific EV species. Complete submission of clinicaldata in the future should provide more in depth analysis of typespecific illnesses. The sequence data allowed the analysis of strain specificsurveillance rather than the current type specific surveillance enablinganalysis of immune divergent strains.REF 499Molecular epidemiology and clinical association of enterovirus andparechovirus types in neonates and young infants in SpainMaria CABRERIZO 1 , Carmen MUÑOZ ALMAGRO 2 , Diana ROULA 2 ,Esther PEREZ 2 , Maria Pilar ROMERO 3 , Ines MARTÍNEZ RIENDA 4 ,Antonio MORENO DOCON 5 , Almudena OTERO 1 , GloriaTRALLERO 11 Instituto de Salud Carlos III, National Centre for Microbiology, Madrid,SPAIN; 2 Hospital Sant Joan de Déu, Barcelona, SPAIN; 3 Hospital dela Paz, Madrid, SPAIN; 4 Hospital de Cruces, Bilbao, SPAIN; 5 HospitalVirgen de la Arrixaca, Murcia, SPAINEnteroviruses (EV) and parechoviruses (HPeV) are the main viral causesof neonatal sepsis and meningitis. The relative frequencies of specific EVand HPeV types in infections affecting children under 1y were determinedovera3ysurveillance period in Spain, and compared with those observedin patients over 1y. Positive samples included were 461 EV and 24HPeV from children under 1y, and 587 EV from patients over 1y. Thus,79 and 88% of the EV and HPeV, respectively, could be directly typed bysequencing of VP1. All HPeV were type 3, exclusively in infants up to7m, whereas 27 different EV types were identified in children under 1yr.CV B3, CV B4, E 11, E 18 and E 25 were statistically more frequent ininfants than in patients over 1y, while E 6, E 30 and E 13 were prevalentin patients over 1y (p=0.02). Incidence of CV B and EV A in infants werealso statistically higher (p=0.006). Regarding clinical outcome in childrenunder 1y, frequency detection of predominant EV types as E 11 and CVB4, was similar in meningo encephalitis and febrile syndromes. Only E 6and HPeV 3 caused more meningo encephalitis and neonatal sepsis thanfever (p=0.001). All myocarditis cases (7) were caused by CV B, whileCV A are associated with HFMD (p=0.001). In neonatal sepsis (11 cases),different viruses were detected, including HPeV and CV B. In conclusion,HPeV 3, E 11 and CV B types were prevalent in neonatal and young infantinfections in Spain during 2010-2012, while not others that frequently circulatedin the same period. Overall, those types presented with greaterdisease severity.Methods: A multiplex of Real Time PCRs (RT PCR) was developed andvalidated on the VP1 gene to differentiate the 4 genotypes of BKV. 150BKV positive samples (17 plasma, 133 urine) were tested with these specificassays. Of these 150 samples, 50 were additionally confirmed bysequencing the 1630 1956 nucleotide fragment. Results: For every genotype,a 100% specific and internally controlled assay was developed. Theprecision of the 4 RT PCRs remained within 1SD and the limit of detectionwas log 3 copies/ml. Of the 150 BKV positive samples, 105 (70%)were genotype I, 8 (5.3%) genotype II, 19 (12.7%) genotype IV and 3(2%) genotype I+IV. In 15 (10%) samples genotyping was not successfuldue to a low viral load. By sequence analysis the genotypes of 46 of the50 and 2 of the 3 samples with the double infection could be confirmed.Conclusions: This study describes a new multiplex RT PCR for detectionof the genotypes of BKV. It proved to be a rapid, cheap and sensitive genotypingtool compared to sequencing, and can detect double infections withdifferent BK genotypes. This method will be of value to obtain insight inthe relation of BKV genotype with nephropathy and hemorrhagic cystitis.REF 501New method targeting VP2 capsid gene for direct enterovirus genotypingin clinical specimensWafa IBRAHIM 1,2,3 , Nabila BOUKHADRA 1 , PhilippeBERTHELOT 1,2 , Dorsaf NASRI ZOGHLAMI 3 , Shabir OMAR 1 ,Thomas BOURLET 1,2 , Bruno POZZETTO 1,2 , Sylvie PILLET 1,21 University Hospital of Saint Etienne, Saint Etienne, FRANCE; 2 Facultyof Medicine of Saint Etienne, University of Lyon, Saint Etienne, FRANCE;3 Faculty of Pharmacy of Monastir, Monastir, TUNISIATyping of human enterovirus (HEV) by sequencing the viral capsid (VP) 1coding region is the gold standard for HEV typing, even in the absence ofcell culture of the specimen thanks to the CODEHOP strategy. We describean alternative method based on the same strategy but in the VP2 codingregion. By testing 10 fold dilutions of reference and clinical strains, thenew test was shown to be at least as sensitive as the VP1 method, except forpoliovirus 1. A total of 98 specimens [48 cerebrospinal fluids (CSF) and50 peripheral specimens] taken from patients of the University Hospital ofSaint Etienne and found positive for HEV RNA by routine techniques wereanalyzed by the VP1 and VP2 methods. The concordance between bothtyping techniques was of 70.41% (P

5 th European Congress of Virologywith particular ethnic group and frequently used as a genetic marker forhuman migration in both prehistoric and modern times. The aims of thisstudy were to determine the frequency of JCV urinary shedding and genotypedistribution. Material and methods: Urine samples collected from107 healthy individuals were tested for presence of JCV DNA by PCR.A semi nested PCR was performed for amplification of 495 bp fragmentwitin VP1coding region and amplified fragments were directly sequenced.The genotypes of JCV isolates were determined by comparison to prototypesequences of the known genotypes in BioEdit software. Results: JCVDNA was detected in 31.7% of healthy individuals. Males had a higherexcretion rate than did the females (62% vs. 38%) and difference wasstatistically significant. In Serbian population genotype 1 was most prevalent41.2%, followed by genotype 4 in 31.4% and genotype 2 in 26.4%.A new variant of subtype 1A with a nucleotide substitution (C>G) at aposition 1940 was found in two samples. Conclusion:Considering geographicalposition and knowing that distribution of JCV genotypes mayreflect migration patterns, it is not surprising that the most prevalent genotypesin Serbia are 1 and 4 (European types) followed by genotype 2B, 2Cand 2D (Eurasian types).REF 503HIV 1 epidemic in Portuguese injecting drug users may be evolvinginto a unique molecular epidemiological patternJoão PIEDADE 1,2 , Carina SOUSA 1 , Sandra VIDEIRA E CASTRO 1 ,Elizabeth PÁDUA 3 , Ricardo PARREIRA 1,2 , Aida ESTEVES 1,21 Grupo de Virologia, Unidade de Microbiologia Médica, Instituto deHigiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, POR-TUGAL; 2 Unidade de Parasitologia e Microbiologia Médicas (UPMM),Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,Lisbon, PORTUGAL; 3 Lab. Nac. Referência IST VIH/SIDA e Hepatites Be C, Dep. de Doenças Infecciosas, Instituto Nacional de Saúde Dr. RicardoJorge, Lisbon, PORTUGALHIV 1 is characterised by a high degree of genetic diversity. HIV 1 geneticvariants are classified in 4 phylogenetic groups (M P) and group M issubdivided into 9 subtypes (A–D, F–H, J, K). Fast genetic recombinationgives rise to mosaic viruses, some of which gain epidemic proportions (circulatingrecombinant forms, CRF). According to their coreceptor (CCR5or CXCR4), HIV 1 variants can also be classified as R5, X4 or dual/mixedtropic. The aims of this study were to assess the genetic diversity of protease(PR), reverse transcriptase (RT), integrase (IN) and C2V3C3 codingsequences and to estimate the frequency of coreceptor usage in HIV 1strains circulating among 61 intravenous drug users from the Greater Lisbon.Viral RNA was amplified by RT nested PCR to originate PR, RT, INand C2V3C3 amplicons of 460, 650, 906 and 565 bp, respectively. 158DNA sequences were analysed, from 49 samples successfully amplifiedfor, at least, one of the regions. Subtype classification was achieved byphylogenetic analysis with MEGA4 and recombinant analysis was carriedout by bootscanning. A significant degree of HIV 1 diversity was shown.This epidemic is dominated by B and G subtypes, and their recombinantforms, but other genetic forms (F1, CRF02 AG) were also found. On thewhole, non B subtypes were identified in 58.9% (93/158) of the sequences.It is also significant that 45.2% (19/42) of the concatenated sequences studiedwere derived from inter genotype recombinants. Finally, coreceptorusage prediction classified 30 V3 amino acid sequences as R5 and 5 as X4or dual/mixed tropic.REF 504The value of real time sequence based information in surveillance ofhealthcare associated viral infectionsJ.C. RAHAMAT LANGENDOEN 1 , D.S. LUIJT 2 , A.D. PRENGER 3 ,M.WAGELAAR 3 ,A.OTT 2 , H.G.M. NIESTERS 11 Department of Medical Microbiology, Division of Clinical Virology, Universityof Groningen, University Medical Center Groningen, Groningen,THE NETHERLANDS; 2 Laboratory for Infectious Diseases, Groningen,THE NETHERLANDS; 3 Municipal Health Service, Groningen, THENETHERLANDSObjectives: sequence based information can serve as a tool to definetransmission routes. As most laboratories have not incorporated sequenceanalysis in their daily routine, information is mostly available retrospectively.Reducing the time to obtain sequence based information shouldbenefit the understanding of transmission and guide the implementationof appropriate infection control measures. Methods: in August 2012, realtime sequencing is introduced at the UMCG, a large tertiary referral hospital.A set of viruses, particularly noro, rhino, parecho and enterovirus, ischaracterized immediately after detection. To gain insight in viral diversityoutside the UMCG, a regional network is set up in which a regionallaboratory and the municipal health service submit samples for typing. Incase of an outbreak of gastroenteritis or respiratory illness in healthcareassociated institutions a limited set of clinical and epidemiological data iscollected.Results: sequence analysis results were available less than a week afterdetection. Several clusters of identical viruses were identified, especiallywith norovirus, confirming clonal transmission. Real time sequencing alsoenabled us to rapidly detect pseudo outbreaks where several norovirusgenotypes were found, providing evidence for multiple introductions ofdifferent strains rather than an ongoing transmission. Conclusion: realtime sequence analysis contributes to the understanding of transmissionof healthcare associated viral infections and enables us to focus infectioncontrol interventions adequately and timely.REF 505Frequency of distribution and variation of anal HPV genotypes andcorrelation with lifestyle and sexual behaviors among HIV infectedand non infected MSMs, compared to cervix samplesEszter UJHELYI, Csaba KOSA, Eszter SZABO, Edit BABARCZI, JanosSZLAVIK, Denes BANHEGYI, Istvan VALYI NAGYUnited Saint Istvan and Saint Laslo Hospital, Budapest, HUNGARYBackground: Anal cancer is one of the leading causes of death in nonAIDS defining cancers. Most of these cancers are associated with highrisk HPV (HR HPV) infection. No survey was made on anal HPV infectionin Hungarian MSM population before. We evaluated incidencesof cytological abnormalities and different genotypes, known and suspectedrisk factors, compared cervical and anal pattern. Materials andMethods: After obtaining informed concern, cervical and anal cytobrushwere taken and HPV genotyping with PCR (Roche Linear Array HPVgenotype).Every patient were inquired and tested about their sexual behavior,socioeconomic factors, drug use, and other known and suspected riskfactors. Risk assessments on this cross sectional cohort study were madeby Chi squared and odds ratio were calculated.S260 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyResults: HPV was detectable from 258 cervix 39,9%, and from 92 analexamination 39,9%. 51% of cervix sample had only 1, genotype. Incontrast 15% of anal samples had only one 1, HPV genotype. 88,8% ofanal samples HR, and 75,0% of anal samples had LR HPV genotype. FromHIV infected group 44% had other known sexually transmitted diseases(syphilis, gonorrhea, hepatitis B or C). Anal bleeding during sex (OR:1,66)fisting (OR1,18) passive role (OR:1,14) were independent risk factors forHR HPV after analysis. 57% of patients had more than 100 sexual partners.Conclusion: Hungarian MSM population is severely co infected with HPVand HR HPV. Smoking and high risk sexual behaviors are strong predictorfor acquiring HR HPV co infections. Early male vaccination programmight give the chance in preventing this potentially carcinogen infectionswab specimens. Epidemiologically, the typing results gave a comprehensivepicture of prevalent adenovirus genotypes during the four year periodin Finland.References1. Ylihärsilä M, Valta T, Hattara L, Karp M, Harju E, Hölsä J, Saviranta P,Waris M, Soukka T. Oligonucleotide array in well platform for detectionand genotyping human adenoviruses by utilizing upconverting phosphorlabel technology. Anal Chem 2011; 83: 1456-61.2. Ylihärsilä M, Harju E, Arppe R, Hattara L, Hölsä J, Saviranta P,Soukka T, Waris M. Genotyping of clinically relevant human adenovirusesby array in well hybridization assay. Clin Microbiol Infect 2012.doi: 10.1111/j.1469 0691.2012.03926.x.REF 506Epidemiological study of Human Adenovirus Genotypes in Finland,2009 2012Minna YLIHÄRSILÄ 1,2 , Tiina YLINEN 1 , Tero SOUKKA 2 , PetriSAVIRANTA 3 , Matti WARIS 11 Department of Virology, University of Turku, Turku, FINLAND;2 Department of Biotechnology, University of Turku, Turku, FINLAND;3 Medical Biotechnology Centre, VTT Technical Research Centre of Finland,Turku, FINLANDThere is scarce data on the epidemiology of human adenoviruses in Finland.In this study, an array in well hybridization assay [1,2] was used togenotype adenovirus types of cases detected in Finland between January2009 and December 2012. A total of 499 adenovirus positive respiratory,ocular swab, stool, and other types of specimens were included in thestudy. The specimens were first amplified with real time PCR based onthe adenovirus hexon gene, and the PCR products were typed by the arrayin well assay. The E04 (n=110, 22%), C02 (87, 17%) and C01 (46, 9%)genotypes were detected in every season. Overall, epidemic activity oftype B03 (160, 32%) was observed in the autumn of 2010, after that onlysporadic cases were found. Genotypes D08, D19 and D37 (42, 8%), whichare known to cause epidemic keratoconjunctivitis, were detected in ocularREF 507ViralZone: recent updates to the virus knowledge resource.Patrick MASSON, Chantal HULO, Edouard DE CASTRO, LydieBOUGUELERET, Ioannis XENARIOS, Philippe LE MERCIERSuisse Institute of Bioinformatics, 1 rue Michel Servet, 1211 Genève 4,SuisseViralZone (viralzone.expasy.org) is a web resource that links sequencedata with virus biology knowledge, including virion structure, replicationcycle and host-virus interactions. The information is divided into viral factsheets for each viral genus, that describe virion shape, molecular biologyand epidemiology, with links to the corresponding sequences in GenBankand UniProt. Each viral genus page contains detailed illustrations, text,and PubMed references. This new update provides detailed view of viralmolecular biology through 133 viral ontology pages that describe commonsteps of viral replication cycles shared by several viral genera. Thisviral ontology is also represented in in the form of Gene ontology (GO)terms, and UniProtKB keywords. In this way, users can navigate from thedescription of a replication-cycle event to any viral genus concerned, andcan access to the sequences of genes related to this event.Virologie, Vol 17, supplément 2, septembre 2013S261

5 th European Congress of Virology25. RESPIRATORY VIRUS INFECTIONSPosters: REF 508 to REF 538REF 508Candidate plant derived universal Influenza A nanovaccines conferprotection against lethal homologous and heterologous virus challengePeter IVANOV, Tatiana GASANOVA, Natalia PETUKHOVALomonosov Moscow State University, Moscow, RUSSIAConservative Influenza M2e antigen (23 aa) has been overexpressed onthe surface of tobacco mosaic virus (TMV U1) particles. Three versionsof M2e were inserted into the TMV coat protein (CP) gene. In additionto the consensus human M2e sequence, we introduced mutations by thesubstitution of cysteines with either serine or alanine residues. Agrobacteriummediated infections of Nicotiana benthamiana produced as much as4 g of TMV M2e ala per 1 kg of systemic leaves. Genetic stability of viralvectors was demonstrated. Chimeric virions consisted of two proteins andcontained up to 90% of CP M2e. Immunized mice did not lose weightcomparing with the control animals. The amount of antibodies specificto M2e exceeded those specific to TMV particle itself (maximum ratio5/1). Immunogold microscopy proved that the foreign antigens were uniformlydistributed and tightly packed on the surface of chimeric particles.The majority of TMV CP epitopes are hidden from the immune systemby the M2e antigens exposed on the particle surface. IgG1/IgG2a ratiovaries from 0.7 (Ser) to 3.2 (Ala). The anti M2e IgG response declinedinsignificantly during the 7 month period. Vaccinated mice were resistantto 5 LD50 of A/PR/8/34 (H1N1) and TMV M2e ala conferred partial protection(70% of survival rate) against heterologous strain (4 aa changes inM2e) influenza A/California/07/2009 (H1N1). Virus titer in lungs was twoorders lower than in the non immune animals. Therefore, a new generationcandidate universal Influenza A nanovaccines with specific rod shapedepitope geometry has been obtained.REF 509R.I.P. Rest in Peace: Human rhinoviruses target the death kinaseRIPK1 to interfere with caspase dependent cell deathMark LÖTZERICH, Urs F. GREBERInstitute of Molecular Life Sciences, University of Zurich, Zurich, SWIT-ZERLANDVirus infections can trigger apoptosis by activation of death receptorsor pattern recognition receptors, such as tumor necrosis factor receptor(TNFR), double strand RNA sensor toll like receptor 3 (TLR3) or RNAhelicase melanoma differentiation associated gene 5 (MDA5). Besides initiatorcaspase 8 and executor caspases this can involve regulatory kinases,such as receptor interacting protein kinase 1 (RIPK1). RIPK1 transmitsapoptotic signals via its death domain to caspase 8. We addressed howhuman rhinoviruses (HRV1a, 2, 14, 16, 37) interfere with cell death processesin Hela OHIO and primary human nasal epithelial cells. HRVinfection induced low levels of activation of caspases 8 and 9. Host chromatinrelocalized to the nuclear periphery and lamins were partially cleavedindependent of caspase activity. There was no sign of DNA fragmentation,poly ADP ribose polymerase cleavage, or activation cleavage of caspases3 and 7. Instead, the death domain of RIPK1 was cleaved to 60, 47 and37 kDa fragments. Ruptintrivir, an inhibitor of HRV 3C protease blockedthe formation of the 60 kDa RIPK1 fragment and abrogated cytopathiceffects. RIPK1 was found in a complex with 3C protease in infected cellsand recombinant 3C protease cleaved RIPK1. Collectively, the data suggestthat HRV induces upstream activation signals for apoptosis but blockexecution of apoptosis by 3C protease mediated cleavage of RIPK1. Thisleads to a non classical death pathway, which is controlled by viral genesrather than the host apoptosis machinery.REF 510Analyzing the receptor binding of influenza A viruses by using solublechimeric proteinsAnne Kathrin SAUER 1 , Chi Hui LIANG 2 , Maren BOHM 1 , ChristelSCHWEGMANN WESSELS 1 , Chung Yi WU 2 , Chi Huey WONG 2 ,Georg HERRLER 11 Institute of Virology, University of Veterinary Medicine, Hannover, GER-MANY; 2 Genomics Research Center, Academia Sinica, Taipei, TAIWANInfluenza A viruses can be found in many avian and mammalian speciesand are known for their ability to cross species barriers and adapt to newhosts. One major aspect for switching the host is the binding specificityof the surface glycoprotein hemagglutinin. It binds to cell surface sialicacids and viruses from different host species preferentially recognize sialicacids in certain linkage types. Avian influenza strains generally showa preference for 2,3 linked sialic acids whereas human strains show ahigher affinity to sialic acids linked in 2,6 conformation to galactose.Many studies use two plant lectins to stain for 2,3 and 2,6 linked sialicacids in cells and tissues: SNA (Sambucus nigra agglutinin) and MAA(Maackia amurensis agglutinin). But these two lectins have of course theirown individual binding properties. Using only these lectins is not sufficientto analyze the binding properties of HAs considering the high amount ofdifferent oligosaccharides even on a single cell and the variety of differentHA subtypes. To circumvent this problem we utilize soluble forms of severalHA subtypes to investigate the binding properties of the influenza HA,exploiting the receptor specificity of each subtype. Soluble hemagglutininsof the avian subtypes H5, H7 and H9 as well as human and porcineH1 subtypes have been tested on different cell lines and tissue sectionsfrom avian trachea and the porcine respiratory tract. Glycan array analysisof solHAs and lectins was performed to get a detailed insight into receptorbinding properties of influenza HAs.REF 511Efficient uncoating of influenza A virus requires exposure to mildlyacidic pH and a high concentration of potassium prior to fusion inlate endosomesSarah STAUFFER 1 , Yuehan FENG 1 , Firat NEBIOGLU 1 , LassiLILJEROOS 2 , Butcher SARAH J. 2 , Paola PICOTTI 1 , Ari HELENIUS 11 Institute of Biochemistry, ETH Zurich, Zurich, SWITZERLAND;2 Institute of Biotechnology, University of Helsinki, Helsinki, FINLANDFollowing endocytic uptake, Influenza A virus (IAV) is transported tolate endosomes where virus fusion is induced by the viral hemagglutinin,with an optimum of pH 5.0 for the strain X31 (A/Aichi/2/1968 (H3N2)).Acidification of the viral core in endosomes prior to fusion promotes efficientviral ribonucleoprotein (vRNP) uncoating. At mildly acidic pH theM2 channel mediates proton translocation into the viral core, resulting indisassembly of matrix protein 1 (M1) oligomers and release of vRNPsinto the cytoplasm following fusion. To date, core specific viral proteinprotein interactions and their dissociation upon acid exposure have mainlybeen analyzed in vitro. Here, we aimed to assess the pre fusion requirementsof IAV uncoating in the context of infection, using pH 5.0 mediatedfusion at the plasma membrane. Mild acidification (pH 5.8) as well astreatment of IAV at high K+ concentrations mimicking the endosomalenvironment prior to fusion increased viral uncoating and infection significantly.In particular, core disassembly in vitro and in vivo, as monitoredby M1 dissociation and vRNP exposure, was facilitated when virions werepre acidified. We applied limited proteolysis combined with targeted massspectrometry in order to probe conformational changes in the virion. M1S262 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologywas found to undergo an irreversible molecular change following pre acidification.Our findings suggest that the gradual decrease of pH and thehigh K+ conditions in endosomes is required to prime the IAV virion forefficient uncoating following fusion at pH 5.0 in the late endosomes.REF 513Complete genome sequences of elephant endotheliotropic herpesviruses1A and 1B determined directly from fatal casesGavin WILKIE 1 , Andrew DAVISON 1 , Mick WATSON 2 , Karen KERR 1 ,Stephanie SANDERSON 3 , Tim BOUTS 4 , Falko STEINBACH 5 , AkbarDASTJERDI 51 MRC–University of Glasgow Centre for Virus Research, Glasgow, UNI-TED KINGDOM; 2 ARK Genomics, The Roslin Institute and Royal (Dick)School of Veterinary Studies, University of Edinburgh, Edinburgh, UNI-TED KINGDOM; 3 Chester Zoo, Chester, UNITED KINGDOM; 4 ZSLWhipsnade Zoo, Dunstable, UNITED KINGDOM; 5 Animal Health andVeterinary Laboratories Agency Weybridge, Addlestone, UNITED KING-DOMA highly lethal hemorrhagic disease associated with infection by elephantendotheliotropic herpesvirus (EEHV) poses a severe threat to Asian elephanthusbandry. We have used high throughput methods to sequencethe genomes of the two genotypes that are involved in most fatalities,namely EEHV1A and EEHV1B (species Elephantid herpesvirus 1, genusProboscivirus, subfamily Betaherpesvirinae, family Herpesviridae). Thesequences were determined from postmortem tissue samples, despite thedata containing tiny proportions of viral reads among reads from a host forwhich the genome sequence was not available. The EEHV1A genome is180,421 bp in size and consists of a unique sequence (174,601 bp) flankedby a terminal direct repeat (2,910 bp). The genome contains 116 predictedprotein coding genes, of which six are fragmented, and seven paralogousgene families are present. The EEHV1B genome is very similar to that ofEEHV1A in structure, size, and gene layout. Half of the EEHV1A geneslack orthologs in other members of subfamily Betaherpesvirinae, such ashuman cytomegalovirus (genus Cytomegalovirus) and human herpesvirus6A (genus Roseolovirus). Notable among these are 23 genes encodingtype 3 membrane proteins containing seven transmembrane domains (the7TM family) and seven genes encoding related type 2 membrane proteins(the EE50 family). The availability of the genome sequences will facilitatefuture research on the epidemiology, pathogenesis, diagnosis, andtreatment of EEHV associated disease.REF 514Investigaton of BDV, PPR and BTV infections in sheep in KyrgyzstanOrhan YAPICI 1 , Orhan YAPICI 1 , Oya BULUT 2 , Oguzhan AVCI 2 ,Mehmet KALE 3 , Mambetali TURSUMBETOV 4 , Sibel YAVRU 2 , AtillaSIMSEK 2 , Kudaybergen ABDIKERIMOV 41 University of Kyrgzstan Turkey Manas, Faculty of Veterinary Medicine,Department of Virology, Bishkek, KYRGZSTAN; 2 University of Selcuk,Faculty of Veterinary Medicine, Department of Virology, Konya, TURKEY;3 University of Mehmet Akif Ersoy, Faculty of Veterinary Medicine, Departmentof Virology, Burdur, TURKEY; 4 Institute of Veterinary Research,Bishkek, KYRGZSTANThe aim of this study is to define seroprevalence of Border Disease Virus(BDV), Peste De Petits Ruminants (PPR) and Blue tongue Virus (BTV)infections in sheep. Blood serum samples were collected from Issyk Kul(144), Naryn (208), Talas (189) and Çuy (114) in Kyrgzstan. Serumsamples (655) were analyzed for presence of antibodies against BDV, PPR,and BTV by commercially available enzyme linked immunosorbent assays(ELISA). The tests were performed as per the manufacturer’s instructions.Seropositivity to BDV, PPR, and BTV were found to be 7.32%, 35.11%,and 36.94%, respectively. Abortions of sheep are the cause of considerableeconomic losses for the farmers. These infectious viral agents are easilyspreading among animals. Abortions among small ruminants in Kyrgzstanshould be attributed to mixed infections. Furthermore these infectionspathogenes are should be further investigated. Etiological agents of viralabortion in sheep needs further investigation. Preventing diseases is themost efficient and cost effective way of managing disease. After the determinationof the situation of causative viral agents, eradication control andmonitoring programs should be prepared.Keywords: BDV, BTV, ELISA, PPR, sheepREF 515The Respiratory Virus Network an initiative to collect and providedata on respiratory virus diseases via internetOrtwin ADAMS 1 , Rolf KAISER 2 , Barbara GÄRTNER 3 , BenediktWEISSBRICH 41 Institute for Virology, Düsseldorf, GERMANY; 2 Institute for Virology,Cologne, GERMANY; 3 Institute for Medical Microbiology and HospitalHygiene, Homburg, GERMANY; 4 Institute for Virology, Würzburg,GERMANYThe Respiratory Virus Network (RespiVir) started 2009 from an initiativeof the section “Clinical Virology” of the Gesellschaft für Virologie(GN). Meanwhile the network consists of more than 30 laborato¬riesfrom Austria, Switzerland and Germany. The online accessible databasecollects data from positive and negative results from different respiratoryviruses, detection methods and age and gender of hospitalized patients.Frequencies and localization of the different virus species are vi¬siblefor the participants and in the near future also for the public. In additionthe results are reported with comments to the participants in monthlyreports. Specialized centers perform (sub)typing or sequence analysis forcertain viruses. The database contains more than 250000 test results from27000 patients. This allows to monitor more precisely than in the pastthe yearly RSV activity, which allows a better guided start and stop ofthe RSV prophylaxis with Palavizumab (Synagis) in premature babies. Asecond example was the detection of high activity with HMPV during theperiod of the H1N1 new variant in 2009/2010. Rhinoviruses have peaksin spring and autumn, but can be detected throughout the year. We seeRespiVir as a tool that allows to collect the data from meanwhile routinelyperformed diagnostics and provide an added value from the sum of thedata by addressing the question, which viruses are cir¬culating currently,are there regional differences, etc. The structure of this network allowsa virtual biobank for the participants. For the future it is also planned toserve as a platform for bacterial respiratory infections like B. pertussis,M. pneumoniae, C. pneumoniae as well as for collecting data of viral andbacterial gastroenteritis.REF 516Respiratory viruses in hospitalised adults in Bern, Switzerland duringthe 2012/2013 seasonAlexander LUETHI, Maria Teresa BARBANI, Samuel ZUERCHER,Jacqueline STEINLIN, Sarah WASMER, Meri GORGIEVSKIVirology, Institute for Infectious Diseases, University of Bern, Bern, SWIT-ZERLANDBackground: Respiratory viruses are a significant cause of morbidityand mortality especially in immunocompromised patients. A sensitive andspecific assay is required to provide optimal clinical care and reduce therisk of nosocomial infections. We evaluated the novel Argene Multi Well(MWS) r gene real time PCR assay to detect respiratory viruses fromadult patient samples. Methods: Respiratory specimens (431 upper/119lower respiratory tract) from adult patients with respiratory infection wereprospectively collected and tested from October 2012 to April 2013 byVirologie, Vol 17, supplément 2, septembre 2013S263

5 th European Congress of Virologythe Argene PCR assay for different respiratory viruses. 68 QCMD 2012respiratory samples were analysed as controls.Results and Conclusions: Patient samples (median age 61 years, range:16 90yrs, 61% male, 31.7% Immunocompromised) were analyzed asrequested by the clinic: 550 for Influenza A/B, 335 for RSV/hMPV,AdV/hBoV and Rhino and 281 for hCoV/hPIV. From Immunocompetent/Immunocompromisedpatients, 33.3/39.1% were positive for onerespiratory virus, 2.1/2.8% showed dual viral infections. Influenza A wasthe most common detected virus (15.8%/10.9%), followed by Influenza B(11.0%/5.1%), Rhinovirus (6.7%/11.2%), RSV (3.8%/13.4%), Coronavirus(7.7%/7.2%), Adenovirus (0%/4.4%), Parainfluenzavirus (0.6%/2.7%)and Metapneumovirus (2.0%/0%). Bocavirus was not identified in anypatient sample. 98.5% of the QCMD results were correctly identified. TheArgene MWS r gene real time PCR assay is a highly sensitive and specificassay showing good clinical correlation and was easily implementedwithin routine diagnostics.REF 517Comparison of two automated PCR platforms for Influenza virusTina Vasehus MADSEN, Derya CARKACI, Jørgen ENGBERG, XiaohuiChen NIELSENSlagelse Hospital, Slagelse, DENMARKBackground: A precise and quick Influenza (Flu) test is important duringthe Flu season. Since February 2012 we have applied the automatedGeneXpert (GX) from Cepheid routinely for Flu testing. Beckton Dickinsonrecently released a Flu kit for the automated BD MAX (MAX). Thepurpose of this study was to evaluate the Flu kit on MAX in a routinesetting and compare the performance with GX.Results: 120 consecutive samples sent for routine testing for Flu were collectedfrom January to March 2013 and tested on GX and MAX. The reportpossibilities from GX are Flu A, Flu A (H1N1)2009 v, Flu B, and negative.The report possibilities from MAX are Flu A, Flu B, and negative.Concordant results were found in 112 (93.3%) samples, among which 17(15%) were Flu A, 15 (13%) Flu B and 80 (71%) samples were negative.Results from eight (6.7%) samples were discordant by the two systems.Two samples were reported as Flu A (H1N1)2009 v positive by GX, whilenegative on MAX. Four samples were negative by GX, while MAX reportedthree samples positive for Flu B and one sample inconclusive. Twosamples were inconclusive by GX, while both were negative by MAX.Turn around time on GX was 70 minutes, and on MAX 2 3 hours.Conclusion: A high concordance was found between the Flu kits fromGX and MAX. We suggest that both platforms can be used in a routinesetting. Reasons for discordant results for a few samples could either be asensitivity issue of the two assays, or be related to the specific sample. Inan outbreak situation GX might be more convenient because of the shortturn around time.REF 518Clinical Validation of Novel Biochip Array Technology (BAT) forRapid Respiratory Pathogen DetectionJames Patrick MCKENNA 1,2 , Martin CROCKARD 3 , Jason O’NEILL 3 ,Catherine POLLOCK 3 , Rachana THAPLIYAL 3 , Peter V. COYLE 21 Queens University Belfast, Belfast, N. IRELAND; 2 Regional Virus Laboratory,Belfast Health and Social Care Trust, Belfast, N. IRELAND;3 Randox Laboratories, Crumlin, N. IRELANDRespiratory tract infections (RTI) are caused by infection with a heterogeneousrange of viral and bacterial pathogens which frequently producesimilar clinical presentations. Specific diagnosis of RTI relies almost entirelyon laboratory investigations which at present remain laborious andcomplicated due time required to culture bacterial pathogens, the geneticdiversity of many of the viral pathogens involved and the limited capacityof current molecular based technology to adequately differentiate betweenmultiple targets. Randox laboratories have recently developed a novel respiratorypathogen detection assay based on innovative multiplex BiochipArray Technology (BAT) capable of simultaneous same day detection of22 common and emerging viral and bacterial respiratory pathogens froma single sample. Clinical validation of the Respiratory BAT using a widerange of respiratory clinical specimens is essential to determine assay specificityand sensitivity and to give insights into overall assay performance.This study outlines initial validation work carried out with a prototype RespiratoryBAT using 512 clinical specimens collected from patients withconfirmed respiratory infection from Jan. 2010 to Nov. 2012. Details ofclinical validation of BAT and performance in relation to specific RealTime PCR are presented. BAT technology represents a viable alternativeto Real Time PCR has the potential to revolutionise current laboratorydiagnosis of RTI.REF 519Recombinant immunofluorescence assay for differential diagnosis ofnovel human coronavirus EMC/2012 infectionsBenjamin MEYER 1 , Doreen MUTH 1 , Tabea BINGER 1 , UdoBUCHHOLZ 2 , Andreas NITSCHE 2 , Andreas SANEWSKI 3 , UlfDITTMER 4 , Norbert WEVERING 5 , Thomas BAUER BALCI 6 ,Christian DROSTEN 1 , Marcel A. MÜLLER 11 Institute of Virology, University of Bonn Medical Centre, Bonn, GER-MANY; 2 Robert Koch Institute, Berlin, GERMANY; 3 Country HealthDepartment of Essen, Essen, GERMANY; 4 Institute for Virology, UniversityHospital Essen, Essen, GERMANY; 5 Ruhrland hospital, Essen,GERMANY; 6 Country Health Department Oberbergischer Kreis, Gummersbach,GERMANYIn 2012 a novel human coronavirus (hCoV EMC) emerged on the Arabianpeninsula, necessitating the development of rapidly available laboratorydiagnostics. Serological assays are a prerequisite to evaluate transmissionevents of past infections. Conventional CoV serological immunofluorescenceassays (IFA) use virus infected cells to detect IgG and IgMantibodies. Due to known cross reactivity between CoVs within the samegenus and the high seroprevalence of circulating hCoVs like OC43 andNL63, conventional IFA can lead to false positive results. We developed arecombinant IFA (rIFA) using heterologously expressed spike and nucleocapsidproteins of different prototype hCoVs (SARS CoV, hCoV EMC,OC43, NL63). The assay was evaluated during a case contact study followingthe exposure of health care workers to an hCoV EMC infectedpatient in a hospital in Essen, Germany. Patient and contact sera were firstscreened for IgM and IgG antibodies by conventional IFA. Subsequentlyall hCoV EMC positive sera were tested using the established rIFA andvirus neutralization assay. We could confirm the presence of IgM and IgGantibodies in the patient serum as well as neutralizing activity. In addition,we could show that two contacts had hCoV EMC cross reactive IgMantibodies most likely due to recent infections with hCoV NL63 or hCoVOC43. Cross reactivity was minor using the spike proteins making themthe preferred substrates for differential CoV serology. The presented rIFAprovides a biosafe and reliable confirmatory assay to rapidly differentiatebetween infections of different hCoV.REF 520Sensitivity of influenza A/B molecular detection on BD MAXFlorence MORFIN 1 , Emilie FROBERT 1 , Vanesssa ESCURET 1 , VincentFRANCART 2 , Pauline SILVESTRE 3 , Donat DE GROOTE 3 , RenaudCLOSE 3 , Bruno LINA 11 Laboratory of Virology, French National Influenza Centre (NIC), EMR,Université Lyon 1, Hospices Civils de Lyon, Lyon, FRANCE; 2 BectonDickinson France, Le Pont De Claix, FRANCE; 3 Diagenode, Liege, BEL-GIUMS264 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyMolecular biology technics for the detection of influenza have proventheir utility on the occasion of 2009 pandemic episode. The objective ofthis study was to evaluate the sensitivity of a fully automated molecularassay for the detection of influenza A and B: the Diagenode FluA/B assayoptimized for use on the automated BD MAX. The evaluation has beenperformed on 3 reference strains (2012/2013 vaccine strains), diluted from10 1 to 10 8 . 132 clinical samples positive for influenza A or influenza Bfrom 2011 2012 winter season were also retrospectively tested. Thesesamples were previously tested positive using our NIC reference technicsfor the detection of influenza A and influenza B and were stored at 20 ◦ C.All samples were tested on BD MAX and results were compared with thoseof our NIC reference technics. On reference strains, there was a perfectconcordance of BD MAX and NIC technics for the detection of A/H1N1strain. For A/H3N2 and B strains, there was a slight decrease in sensitivityon the last or two last dilutions, corresponding to a detection limit (Ctvalues >38). All types of samples tested (naso pharyngeal aspirates, nasalswabs and bronchoalveolar lavages) had valid results. The internal controlwas highly reproducible. 120 influenza A positive samples were tested.6 were not detected by BD MAX including 4 with high Ct values (35 to37). After a second test performed on these 6 samples, 3 were detectedpositive by BD MAX. 12 influenza B positive samples were tested; 4were not detected (all with high Ct values from 36 to 39) and 1 wasdetected after a second test. The limit of detection of the FluA/B molecularassay on BD MAX is satisfactory and proves its potential value for routinedetection of influenza viruses. The FluA/B assay on BD MAX appears tobe an interesting, sensitive, fully automated test for the routine moleculardetection of influenza viruses.REF 521Strategies to overcome invalid RespiFinder ® SMART 22 results causedby amplification inhibitionBart PEETERS, Isabel MICALESSI, Annick SMISMANS, Britt VANMEENSEL, Johan FRANSLaboratory of Clinical Microbiology, Imelda hospital, Bonheiden, BEL-GIUMThe RespiFinder ® SMART 22 assay (PathoFinder) is a multiplex PCR,detecting and differentiating 18 respiratory viruses and 4 bacteria. Aninternal control is included in the assay to assess amplification inhibition.A result is reported as invalid when the internal control is not amplified anda negative result is obtained for the 22 respiratory pathogens. This assay,which is performed in our daily routine, exhibits an inhibition rate of 5.9%.To circumvent false negative results due to inhibition, the manufacturerrecommends sample pretreatment with dithiothreitol or dilution of thesample extract. The aim of our study was to evaluate an alternative samplepretreatment procedure to avoid repeat testing of samples with invalidresults. First, 10 nasopharyngeal swab samples were retested after a onetime freeze thaw cycle with freezing for at least 15 h and all invalid resultswere resolved. After that, the minimum freezing time was determined on4 samples and a 30 min step was already sufficient to resolve the inhibitionproblem. Finally, a one time freeze thaw cycle was performed on 3 positivesamples and this pretreatment did not have any effect on the outcome of theassay. In conclusion, invalid results for the RespiFinder SMART 22 assaymay be avoided by subjecting all samples to a one time freeze thaw cyclewith a freezing step of at least 30 min. This sample pretreatment provides afast, low cost and easy to apply solution to prevent amplification inhibitionand to obtain higher turnaround times.REF 522Development of a new diagnostic tool for the detection of HumanCoronaviruses & Human Parainfluenzaviruses in a duplex RT PCRCécile RESA, Jérôme BES, Manon DUBE, Lionel GARNIER, MatthieuVIGNOLES, Stéphane MAGRO, Côme BARRANGERbioMérieux Site de Verniolle, Verniolle, FRANCEObjectives: Human Coronavirus (HCoV) and Parainfluenzaviruses(HPIV) are frequently involved in respiratory infections in young children,the elderly, and the immunodepressed. The bioMérieux HCoV/HPIVr gene ® real time PCR assay allows the detection of the 4 HCoV species(NL63, OC43, HKU1 & 229E) and the 4 HPIV species (HPIV 1; 2; 3 &4) with a high sensitivity.Methods: Nucleic acids were extracted from 200 L of nasopharyngealspecimens, after a Proteinase K pre treatment, on NucliSENS ®easyMAG TM and eluted in 50 L. 0.15 L of reverse transcriptase wasadded to 15 L of HCoV/HPIV r gene ® amplification premix. Then 10 Lof eluted samples were added. HCoV and HPIV were detected at 530 nm& 560 nm respectively on Bio Rad Dx Real Time System.Results: On the Coronavirus QCMD 2012 Panel, all samples (10) werecorrectly identified. On the Parainfluenzavirus QCMD 2012 Panel, 9 of10 samples were correctly identified. Analytical Sensitivity study on the4 HCoV and the 4 HPIV species was performed in respiratory specimens.This study shows a high level of sensitivity for each parameter. Intra/interassay variability studies were carried out on HCoV and HPIV cultured cellsdiluted in nasopharyngeal negative samples. Coefficients of variation wereunder 2.5% (intra assay) and 4.0% (inter assay). Specificity study showedno cross reaction among 56 other respiratory viruses/bacteria.Conclusion: The high quality associated with its compatibility with themajor extraction and real time PCR platforms allows an immediate integrationof HCoV/HPIV r gene ® 71 045 in most routine diagnostic labs.REF 523Equivalent performance results in Sofia fluorescent immunoassayanalyzer for Respiratory Syncytial Virus (SOFIA FIA RSV ® )toPCRtests in infantsAstrid VABRET 1,2,3 , Cecile RESA 3 , Julia DINA 1,2,3 , Amélie HEBERT 2 ,Celine TOURNUS 1 , Jean Baptiste DAVY 1,3 , Fabien MISZCZAK 1,3 ,Stephanie GOUARIN 1 , Joëlle PETITJEAN 3 , Jacques BROUARD 3,41 University Hospital of Caen, Laboratory of Virology, Caen, FRANCE;2 Reference Natioanl Center for Paramyxoviridae and Measles, Caen,FRANCE; 3 EA4655, University of Caen Basse Normandie, Caen,FRANCE; 4 University Hospital of Caen, Pediatric Department, Caen,FRANCERespiratory Syncytial Virus (RSV) is a common ubiquitous pathogen responsiblefor cold like symptoms in most adults and children. In infants,RSV is more likely to move into the lower respiratory tract and has beendetected in the majority of infants hospitalized for bronchiolitis during theRSV season. Sofia FIA RSV (Quidel, San Diego, CA) is an automatedrapid diagnostic test (RDT) that may improve the performance of classicalmanual RDT. The objective was to assess its performances with aprotocol in “real life laboratory” conditions. Four hundred and one (401)nasal swabs were sampled from patients of different groups of age duringthe RSV season. All these fresh samples were tested for RSV using directimmunofluorescence assay (DFA), Sofia FIA RSV, and PCR test. In allVirologie, Vol 17, supplément 2, septembre 2013S265

5 th European Congress of VirologyPCR positive respiratory samples, a standardized viral load (expressed asRSV RNA copies per 100 cells) was calculated using the kit Cell controlr gene ® Biomerieux. The Sofia FIA RSV displayed sensitivities of 78.8%(from 47.3% to 95%, in patients >2 years old and in infants, respectively)as opposed to PCR. The negative predictive value was 91.3% (from 89%to 96.6% in >2 years old and in infants, respectively). The cell loads in the123 respiratory specimens were between 3 and 5 Log10 cells per PCR. Nosignificant difference is observed in the cell loads in respiratory specimensfrom patients of different age groups. Nevertheless, the respiratory viralloads dramatically decreased in patients above 2 years of age. Sofia FIARSV ® is a sensitive and specific method with a high negative predictivevalue for patients of different age groups. Its performances prove equivalentto molecular tests in infants throughout the RSV circulation perioddue to high respiratory viral loads in this group. This fact can be explainedby the quasi systematic RSV primo infection before the age of 2 years.Methods: Nasopharyngeal swabs were prelevated from patients with acuterespiratory illness admitted from November 2012 to March 2013. Thesamples were tested with xTAG Respiratory Virus Panel Fast (Luminex).Results: A total of 227 samples were received, most of them (77.3%) frompediatric (0 14 years old) patients. More than half (56.3%) of the sampleswere positive for at least one virus, the predominant viruses being influenzaA and influenza B. Adenovirus, RSV and Rhinovirus/Enterovirus (RV/EV)were detected most frequently in children less than 5 years old. Elevensamples (8.5%) were positive for two viruses and one sample was positivefor three viruses. Co infection was significantly associated (p=0.02)with age under 5 years. Adenovirus and RV/EV were more frequentlydetected with other viruses, and all the viruses of the panel (influenza,parainfluenza, RSV, adenovirus, RV/EV, Coronavirus, Metapneumovirus,Bocavirus) were identified in co infections.Conclusion: Co infections with two or more respiratory viruses are oftendetected in pediatric patients with acute respiratory illness.REF 524The role of newly discovered viruses in the development of lowerrespiratory tract infections in KuwaitSahar ESSA 1 , Haya ALTAWALAH 1 , Abdulla OWAYED 2 , MossaKHADADHA 3 , Nasser BEHBEHANI 3 , Widad AL NAKIB 11 Department of Microbiology/Faculty of Medicine/Kuwait University,Kuwait, KUWAIT; 2 Department of Pediatrics/Faculty of Medicine/Kuwaituniversity, Kuwait, KUWAIT; 3 Department of Medicine/Faculty of Medicine/Kuwaituniversity, Kuwait, KUWAITIntroduction: More than 80% of the cases of respiratory tract infectionsare viral in origin. Although mostly causing self limited upper respiratorytract infections (URTI), they can be associated with serious lower respiratoryinfections (LRTI) with a high level of morbidity, particularly inchildren. Little is known about the role of newly discovered viruses in thedevelopment of URTI and LRTI in Kuwait and the Gulf region. Methods:The aim of this study is to investigate the role of seven newly discoveredviruses which include coronaviruses (HCoV) NL63, OC43, and 229E,human metapneumovirus (hMPV), bocavirus, human polyomaviruses WU(WUV) and KI (KIV) by using sensitive molecular techniques.Results: Seven hundred thirty five hospitalized patients were screenedduring three and a half year period, from September, 2010 to April, 2013.Of the 285 patients with viral respiratory infection HCoV OC43 was detectedin twenty five patients (8.8%), both HCoV 229E and bocavirus weredetected in fourteen patients (4.9%), hMPV in fifteen patients (5.3%),WUV in ten patients (3.8%), KIV in four patients (1.4%), and HCoVNL63, was not detected in any of our patients’ samples.Conclusion: We conclude that newly discovered viruses do play a rolein the development of both URTI and serious LRTI in Kuwait. Rapididentification of viral infections can help control nosocomial transmission,reduce overall antibiotic use and improve the treatment and managementsof these infections.REF 525Multiple respiratory viral co infections in patients with acute respiratoryinfectionDragos FLOREA 1 , Angelica VISAN 1,2 , Anca DRAGANESCU 1 ,George JUGULETE 1,2 , Monica LUMINOS 1,2 , Dan OTELEA 11 National Institute for Infectious Diseases “Matei Bals”, Bucharest,ROMANIA; 2 University of Medicine and Pharmacy “Carol Davila”,Bucharest, ROMANIAObjective: the aim of the study was to investigate the prevalence of respiratoryviruses and the rate of viral co infection in patients hospitalizedwith acute respiratory infection in “Matei Bals” Institute.REF 526High proportion of multiple infections with respiratory viruses inyoung children with acute respiratory tract infectionsAtsushi KAIDA 1 , Hideyuki KUBO 1 , Nobuhiro IRITANI 1 , Koh IchiTAKAKURA 1 , Jun Ichiro SEKIGUCHI 1 , Minori OHYAMA 1 , UraraKOHDERA 2 , Masao TOGAWA 3,4 , Kiyoko AMO 3 , Masashi SHIOMI 3 ,Seiji P YAMAMOTO 1 , Kaoru GOTO 1 , Atsushi HASE 1 , TsutomuKAGEYAMA 51 Osaka City Institute of Public Health and Environmental Sciences,Osaka, JAPAN; 2 Nakano Children’s Hospital, Osaka, JAPAN; 3 OsakaCity General Hospital, Osaka, JAPAN; 4 Osaka City Sumiyoshi Hospital,Osaka, JAPAN; 5 Influenza Virus Research Center, National Institute ofInfectious Diseases, Tokyo, JAPANObjectives: Respiratory virus is a major etiologic agent of acute respiratorytract infections (ARTIs) in young children. This study was conductedto detect multiple respiratory viruses and to analyze their co infection andmutual association using statistical analysis.Methods: During January 2010 – December 2011, 1,044 respiratory clinicalspecimens were obtained from children (< 6 years of age) with ARTI.To detect viral genes, multiplex real time PCR was conducted for respiratoryviruses (human metapneumovirus [hMPV], respiratory syncytial virus[RSV A, B], human parainfluenza virus types 1–4 [HPIV 1–4], humanbocavirus 1 [HBoV 1], human coronavirus [229E, OC43, HKU1, NL63],influenza virus [A, A (H1N1)2009, B, C], human adenovirus [HAdV],human enterovirus, and human rhinovirus [HRV]).Results: The 1,414 viruses were detected from 891 (85.3%) specimens,of which 388 (43.5%) specimens were multiple virus positive (2 viruses,279;=3 viruses, 109). The most detected was HRV (n=390). The proportionof multiple virus positives was higher in the 0–35 month age group. Thecombinations of HPIV 1 and HPIV 3, HPIV 1 and RSV A, HPIV 3 andhMPV, hMPV and RSV A, hMPV and FLUA (H1N1) 2009, RSV A andRSV B, and HRV and FLUA (H1N1) 2009 were negatively correlated. Incontrast, HAdV and HBoV 1 were positively correlated.Conclusions: Young children are highly susceptible to respiratory viruses.Viral co infections are common.REF 527Respiratory viruses in patient with acute respiratory tract infectionsin AYDIN, TURKEYSevin KIRDAR 1 , Neriman AYDIN 1 , Ayse YENIGUN 2 , EmelCEYLAN 31 Adnan Menderes University, Department of Medical Microbiology,Aydin, TURKEY; 2 Adnan Menderes University, Department of PediatricDiseases, Aydin, TURKEY; 3 Adnan Menderes University, Department ofPulmonary Medicine, Aydin, TURKEYS266 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyIntroduction and aim: Acute respiratory tract infections (ARTIs) aresome of the most common diseases that occur in both children and adults,Their rapid and accurate diagnosis is essential to patient management. Theaim of this study to evaluate with respect to types, ratio, and co infectiontrends of infected respiratory viruses.Material and methods: Between January 2010 and December 2012, 807clinical specimens were collected from patient submitted to Adnan MenderesUniversity Hospital in Aydin. The screening of respiratory viruseswas performed with a commercial multiplex PCR based method. Results:The mean and median ages of all patients were 9,45 and 4,60 years (range,1 to 91 years), respectively. Of the 807 submitted clinical samples 364(45,1%) specimens were positive for at least one respiratory virus. The percentageof the viruses detected were 39,3% for human rhinovirus, 13,7%for human respiratory syncytial virus A, 2,5% for human respiratory syncytialvirus B, 7,1% for human adenovirus, 14,0% for influenza A virus,1,1% for influenza B virus, 4,9% for human metapneumovirus,2,5% forhuman coronavirus OC43, 0,3% for human coronavirus 229E/NL63, 2,2%for human parainfluenza virus (HPIV) 1, 0,5% for HPIV 2, and 3,0%for HPIV 3. The co infection analysis showed 9,34% of double or tripleinfections.Conclusion: Human rhinovirus virus was the most common virus in allpatients. Early detection of a viral respiratory infection is crucial for theimplementation of timely and appropriate treatment and restriction ofantibiotic therapy.REF 528Unexpected high prevalence of HMPV acute infection among children

5 th European Congress of VirologyREF 531Comparison of Idaho Tecnology FilmArray ® Respiratory Virus paneland Clart ® Pneumovir Microarray Genomica for multiplexed detectionof respiratory viruses in hospitalised patientsAla NASSEREDDIN, Nikolai KIRKBY, Claus BOHN CHRISTIANSEN1 Department of Clinical Microbiology, Rigshospitalet, Copenhagen,DENMARK; 2 Department of microbiology, Rigshospitalet, Copenhagen,DENMARK; 3 Department of microbiology, Rigshospitalet, Copenhagen,DENMARKObjective: To evaluate the clinical performance of two microarray systemsfor detection of respiratory viruses in hospitalised patients. Study design:In this prospectively study (February 4 to March 27, 2013) routine sampleswere included. Samples with inconclusive result in one of the assays wereexcluded. Only the first sample from each patient was included. Methods:CLART PneumoVir: RNA/DNA were extracted with EasyMac. Fragmentsof the viral genomes were amplificated by RT PCR and analysed. The FilmArrayRV integrates extraction, amplification, detection, and analysis in apouch. Viral targets: Influenza A/B, RSV, HMPV, Adenovirus, Rhinovirus,Enterovirus, PIV, Bocavirus and Coronavirus.Results: 121 samples were evaluated. Five samples gave inconclusiveresults in one of the assays. In 82/116 samples (70,6%) at least one viruswere detected in one or both assays. In 51 samples (43,9%) identical resultswere found (43 one virus, 7 two viruses and 1 three viruses). In 34 samples(29,3%) a negative result were found in both assays.In 12 samples (10,3%) FilmArray RP detected additional viruses. In 2samples (1,7%) PneumoVir detected additional viruses.In 8 samples (6,9%) FilmArray RP detected a virus while Pneumovir werenegative. In 8 samples (6,9%) Pneumovir detected a virus while FilmArrayRP were negative. One sample gave discrepant results.Conclusion: FilmArray Respiratory virus panel detected a co infectionin additional 12 samples compared to 2 for Pneumovir. Morestudies are needed to clarify the clinical implications of the viral coinfections.REF 532Enterovirus 68 circulation in hospitalized patients with respiratorysyndrome during 2010 2012 in Northern ItalyAntonio PIRALLA 1 , Alessia GIRELLO 1 , Michela GRIGNANI 2 ,Antonietta MARCHI 2 , Gianluigi MARSEGLIA 2 , Fausto BALDANTI 11 Molecular Virology Unit, Virology and Microbiology Department, FondazioneIRCCS Policlinico San Matteo, Pavia, ITALY; 2 Department ofPediatrics, Fondazione IRCCS Policlinico San Matteo, University of Pavia,Pavia, ITALYEnterovirus 68 (EV D68) was associated with mild to severe respiratoryinfections. In the last four years, circulation of different EV D68 strainshas been documented worldwide. Here, we describe the phylogenetic characterizationof nine EV D68 strains identified in hospitalized patients inthe 2010 2012 period and 12 additional EV D68 Italian strains previouslyidentified in 2008 in Northern Italy. From January 2010 to December 2012,a total of 889 respiratory from 588 patients hospitalized at the FondazioneIRCCS Policlinico San Matteo were positive for HRV or HEV. Extractednucleic acids were amplified by one step RT PCR with primer specific forVP1 region of EV D68 and purified positive PCR products were directlysequenced. Overall, 9/588 (1.53%) patients were EV D68 positive. Ofthese, 5/9 (77.7%) were pediatric and two (22.2%) were adults. Five outof seven (74.4%) pediatric patients had lower respiratory tract infectionwith oxygen saturation 90% of specimens, compared to 71% for GXF v2.0. False negativeswere observed in the low copy number range but not in core samples. Alsoin patients, overall concordance was good (>90%), with >75% of all discre-S268 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologypancies occurring in low titer specimens (Ct>34). Finally, reproducibilitywas high with CVs

5 th European Congress of Virology26. VIRAL INFECTIONS INTRANSPLANT ANDIMMUNOCOMPROMISED PATIENTSPosters: REF 539 to REF 562REF 539Determination of anti HCMV T Cell response of potential recipientsand donors in cellular immunotherapy of leukemic patients aftertransplantation of hematopoetic stem cells (SCT)Sarka NEMECKOVA 1 , Katarina BABIAROVA 1 , Jitka KRYSTOFOVA 1 ,Petr HAINZ 1 , Kamila ZURKOVA 1 , Marketa STASTNA MARKOVA 21 Institute of Hematology and Blood Transfusion, Department of experimentalvirology, Prague, CZECH REPUBLIC; 2 Institute of Hematologyand Blood Transfusion, Transplantation Ward, Prague, CZECH REPU-BLICCytomegalovirus (CMV) reactivation in immunosuppressed patients isassociated with morbidity and mortality. The testing CMV specific T cellresponse can help to find the patients which are in a high risk of CMVdisease. Such patients would profit from adoptive transfer of CMV specificT cells. Cellular immune response against CMV of 14 patients after allogeneicSCT was monitored for 180 days by ELISPOT interferon gammawith the aim to find patients with insufficient control of CMV reactivation.The second aim was to compare various forms of CMV antigen forin vitro stimulation. The patients after SCT were classified according tothe anti CMV T cell response, the presence of CMV DNA in the blood andthe response of T cells after stimulation with anti CD3 antibody into fourgroups: 1) CMV DNA (neg.), anti CMV T cells + (pos.); 2) CMV DNA,anti CMV T cells; 3) CMV DNA +, anti CMV T cells, CD3+; 4) CMVDNA +, anti HCMV T cells, CD3. Patients of groups 3,4 would benefitfrom adoptive transfer. By testing the recall response of T cells of donorsagainst pp65, IE 1, US3, UL36, US29, UL55 peptide pools, pp65, IE 1epitopes or CMV lysate we found that inclusion of US3, UL36, US29,UL55 as testing antigens can increase significantly the overall responseof anti CMV T cells. We observed that stimulation with the long 30merpeptides from pp65 or IE 1 sequence induced activation of CD8 and CD4antiviral T cells. The long peptides could be used for T cell activationof multiple epitopes if the MHCI binding minimal CTL epitopes are notknown.Supported by NT/13898/2012 grant.donor/recipient status (D R). Virus load (VL) was measured by RochePCR. In all 10 D+R patients primary HCMV infection led to a developmentof total and IgG3 HCMV ABs, but 2 patients showed no IgG1 response.Total HCMV IgG, and IgG3 AB RLUs remained stable post TX within 1log during follow up in all 9DR+andD+R+ patients without detectableVL and in 86% and 96% of 28 patients with detectable VL, respectively.In contrast, IgG1 subclass ABs were variable in 46% of the patients (RLUvariation >1log) who developed HCMV VL. In 7 cases IgG1 declinedprior to viremia and in 6 patients IgG1 increased during virus replication.HCMV total IgG and IgG3 AB responses are stable over time, while IgG1ABs exhibit substantial variability in relation to virus replication. Theclinical implications of these data will be further elucidated.REF 541Detection of TS polyomavirus from tonsillar tissues of children andadultsMohammadreza SADEGHI 1 , Lea HEDMAN 1,2 , Leena MaijaAALTONEN 3 , Maria SÖDERLUND VENERMO 1 , Klaus HEDMAN 1,21 Department of Virology, Haartman Institute, University of Helsinki, Helsinki,FINLAND; 2 Department of Virology and Immunology, HelsinkiUniversity Central Hospital Laboratory Division, Helsinki, FINLAND;3 Department of Otorhinolaryngology Head and Neck Surgery, HelsinkiUniversity Central Hospital, Helsinki, FINLANDBackground: in the past few years several new polyomaviruses of humanshave been discovered. Trichodysplasia spinulosa associated polyomavirus(TSPyV) was identified in a patient with trichodysplasia spinulosa (TS), arare follicular skin disease of immunocompromised patients. Seroepidemiologicalstudies indicate that TSPyV is ubiquitous and infects ∼ 70% ofhealthy individuals. We have shown that the tonsil may be a site of WUPyVinitial infection and of MCPyV persistence. Whether TSPyV persistenceoccurs in the lymphoid system is unknown.Methods: to determine whether tonsils harbor TSPyV, we tested a total of229 matched pairs of tonsillar tissues and sera from asymptomatic childrenor adults (overall mean age 21.0 years). The samples were also screenedfor the single copy human RNase P gene by real time quantitative PCR(qPCR). TSPyV was detected by qPCRs with Taq Man probes and primerpairs targeting the VP1 or LT genes. Results: TSPyV DNA was detectedin 8 of 229 (3.5%) tonsillar tissues, from both cohorts, and in none ofthe corresponding sera. Sequence analysis of the 8 virus positive samplesconfirmed the identity of TSPyV.Conclusions: these data provide the first evidence of TSPyV in tonsillartissue and suggest that (i) tonsils may serve as an initial site of TS polyomavirusinfection and that (ii) lymphoid tissue may be a persistence sitefor this virus.REF 540Human Cytomegalovirus specific IgG subclass antibody kinetics inthe follow up after lung transplantationBenedikt SIMON, Irene GÖRZER, Elisabeth PUCHHAMMERSTÖCKLDepartment of Virology, Med Uni Wien, Vienna, AUSTRIAHuman cytomegalovirus (HCMV) may cause severe infections in lungtransplant recipients (LTRs). HCMV specific AB response limits virusreplication, but the kinetics of subclass AB response after transplantation(TX) are not clear.We analyzed the HCMV specific IgG AB kinetics of total IgG and of thedominant IgG AB subclasses 1 and 3 after TX and assessed whether theseare associated with HCMV replication in the follow up. HCMV AB titers of53 LTRs were measured repeatedly post TX using the MEDAC CMV IgGELISA, modified for subclass 1 and 3 ABs. ODs were expressed in relativelight units (RLUs). Cut offs were established in patients with HCMVREF 542The Reactivation of Immunomodulating Beta Herpesviruses Infectionin Patients Undergoing Microvascular Free Flap SurgeriesArnis VILKS 1,2 , Romans DZALBS 4 , Santa RASA 3 , Zaiga NoraKRUKLE 3 , Viesturs BOKA 1 , Biruta MAMAJA 2,1 , ModraMUROVSKA 31 Riga Eastern Clinical University Hospital Gailezers, Riga, LATVIA;2 Riga Stradins University, Department of Anaesthesiology and Reanimatology,Riga, LATVIA; 3 Riga Stradins University, A. Kirchenstein Instituteof Microbiology and Virology, Riga, LATVIA; 4 Riga Stradins University,Faculty of Medicine, Riga, LATVIABeta herpesviruses HHV 6 and HHV 7 are immunomodulating viruses.They have the ability to impair host defence system seriously and maycontribute to other infectious pathogens due to immune suppression. Wehave investigated the effect of long lasting microvascular free flap surgeryS270 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyupon general or regional anaesthesia on reactivation of HHV 6 and HHV7.58 patients undergoing long lasting (average 5.7 h) microvascular free flapsurgery were enrolled in this study. Anaesthesia methods that were appliedgeneral anaesthesia (GA) (n=35) or regional anaesthesia (RA) (n=23)depending of tissues defect and surgery planned donor place. Peripheralblood samples were collected before and 10 days after surgery, DNAisolated from whole blood and cell free blood plasma and viral genomicsequences detected by nPCR using corresponding primer pairs. Before thesurgery latent HHV 6 infection was detected in 13 and 6 patients with GAand RA, respectively but latent HHV 7 infection in 6 and 17 patients withRA and GA, respectively. Two patients in each group had active HHV 6infection, 3 patients with GA and 4 with RA – active HHV 7 infection.After surgery reactivation in GA group was observed in 7 patients (in onepatient HHV 6 and in 6 patients HHV 7, respectively). In the RA grouponly in one patient reactivation of HHV 7 was detected. After surgery reactivationof HHV 6 and HHV 7 occurs more frequent in patients to whomGA is applied (p

5 th European Congress of VirologyHuman cytomegalovirus (HCMV) may account for severe morbidityand mortality in transplant patients, although monitoring strategies havegreatly improved outcome. Beside this, evaluation of cellular immune responsemay be used to optimize clinical management. Herein, the relationbetween HCMV specific immune status and antiviral strategies has beenstudied in renal transplant patients. Cellular immune response to HCMVwas studied by Elispot assay in 328 renal patients (M/F 218/110; meanage 55.7 years; 201 in the first year posttransplantation). HCMV DNAemiawas evaluated concomitantly on whole blood specimens. Anti HCMV prophylaxiswas administered in high risk patients (D+/R), while pre emptivestrategy was adopted in the others. HCMV specific immune status wasrelated to clinical features, including anti viral strategies. Overall, 218patients (66.5%) evidenced recovery of immune response within 3 monthsposttransplantation. HCMV infection at follow up was significantly morefrequent in patients who did not recover immune response (p10ˆ5 copies/ml (p

5 th European Congress of VirologyObjectives: a study of the impact of viral co infections, in vitro and in vivoduring the follow up of 36 umbilical cord blood stem cell recipients inBordeaux (2011 2012).1) Experimental study: to analyse the impact of AdV HCMV co infectionin vitro on the replication of each virus (infections, co infections andsuper infections of MRC5 cells, with laboratory strains HCMV AD169and AdV5) and cell gene expression (interferon beta mRNAs).2) Clinical study: to describe the natural history of viral co infections incord blood recipients, with HCMV, EBV, HHV6, BKV and AdV monitoringand analyse the clinical outcome according to the existence of singleviral infections or co infections.Results: HCMV AdV co infection enhanced HCMV replication in MRC5cells; this tendency observed during in vitro simultaneous co infection reacheda difference of 2 log10 for HCMV viral load in culture supernatantswhen AdV superinfection was applied 48 hours after HCMV infection(quantitative real time PCR assays for HCMV UL83 and AdV Hexon genesin culture supernatants). Confocal microscopic examination of infectedcells demonstrated simultaneous co infection in certain cells (immune fluorescencein infected cells with antibodies directed against HCMV pp65,HCMV IE1 and pan AdV, Argène Biomérieux, France). Interferon betamRNAs relative quantification indicated a two to three fold increase in coinfected cultures compared to single infections. Clinically, only 4 patientsout of 36 presented no viral opportunistic infection. Single infections werediagnosed in 7 patients (19%), dual infections in 9 (25%) and in 16 patients(44%) three viruses or more were detected during the first term followingtransplantation. The analysis of patient’s outcome, taking into accountmajor clinical endpoints (viral disease, Graft versus Host Disease anddeath) and virological data (viral loads and kinetics) is presently beingperformed.Conclusions: Viral co habitation in cord blood recipients is frequent andcould have a clinical impact. Viral co infection experiments confirm thepossible existence of co activation mechanisms in dually infected cells.REF 551Comparison of two assays for CMV viral load expressed in InternationalUnits in plasma and whole bloodPaolo RAVANINI, Tiziana DI FATTA, Maura BANDI, Anna MariaNICOSIA, Maria Grazia CROBUAOU Maggiore della Carità, Novara, ITALYRecently an international standard for CMV has been established by WHO.This improvement can lead to a better comparison among the results obtainedwith different diagnostic assays and biological materials (plasma andwhole blood).In this study, the results of two CMV viral load assays were compared toverify the concordance of the results expressed in International Units (IU),the sensitivity of the assays and the different biological materials, and to seta possible conversion factor between results from plasma and whole blood.Two Real Time PCR assays were used (RealTime CMV, Abbott; AlertCMV Q PCR, Nanogen). All results were expressed in IU/ml. Plasma andwhole blood samples, collected at the same time for both materials from47 kidney transplant recipients, were tested with both assays. The resultssuggest that both plasma and whole blood can be effectively used with bothassays for the monitoring of CMV viral loads in transplant recipients. Bothassays show good sensitivity in plasma (92.3 and 94.9% respectively),while in whole blood the Abbott assay shows better sensitivity (84.6 vs71.8%). In both cases, the sensitivity in plasma appears higher than inwhole blood. The concordance of the results in IU/ml is good between thetwo assays (100% of the results under 0.5 Log variation for both materials).The mean viral load difference between whole blood and plasma resultsis 1.63 fold. This conversion factor should be taken into account in theassessment of different threshold values for preemptive therapy when theviral load is determined in plasma or whole blood.REF 550Low level DNAemia of Parvovirus B19 (genotypes 1–3) in Adult TransplantRecipients is not Associated with AnaemiaSusanne MODROW, Annelie PLENTZ, Michael WÜRDINGER,Matthias KUDLICHUniversity of Regensburg, Institute for Medical Microbiology, Regensburg,GERMANYAfter acute parvovirus B19 (B19 V) infection of immunocompetentindividuals, viral genomes persist lifelong in various tissues. In immunocompromisedpatients, acute B19 V infection may be associated withsevere anaemia. It is unclear whether reactivation of latent B19 V genomesmay contribute to persistent viraemia and anaemia in transplant recipients.We retrospectively analyzed the impact of B19 V infection in 371 adulttransplant recipients (kidney, liver, heart, bone marrow). The patients’ pretransplantation serostatus was determined. 1431 serum or plasma samplesobtained in monthly intervals during six months following transplantationwere analyzed for the presence of B19 V DNA by qPCR which allowsdiscrimination between B19 V genotypes 1 3.Overall, 82% of the patients were seropositive indicating past B19 V infection.B19 V DNA was detected in of 4.0% of all patients and classified asgenotype 1 in 12, genotype 2 in one and genotype 3 in two patients, theDNA load ranging from

5 th European Congress of VirologyREF 553The prevalence of DNA HSV 1 and HSV 2 in Polish patients subjectedto allogeneic hematopoietic stem cell transplantationSylwia RYNANS 1 , Agnieszka TOMASZEWSKA 2 , TomaszDZIECIATKOWSKI 1,3 , Katarzyna MOCKO 4 , Anna MAJEWSKA 1 ,Maciej PRZYBYLSKI 1,3 , Kazimierz HALABURDA 5 , GrazynaMLYNARCZYK 11 3Medical University of Warsaw, Chair and Department of Medical Microbiology,Warsaw, POLAND; 2 Institute of Hematology and TransfusionMedicine, Department of Haemopoetic Stem Cell Transplantation, Warsaw,POLAND; 3 2Central Clinical Hospital in Warsaw, Department ofMicrobiology, Warsaw, POLAND; 4 Warsaw University of Life Sciences,SGGW, Interfaculty Department of Biotechnology, Warsaw, POLAND;5 Medical University of Warsaw, Department of Hematology, Oncologyand Internal Medicine, Warsaw, POLANDHerpes simplex viruses type 1 (HSV 1) and 2 (HSV 2) belong to widespreadcontagious factors. A situation might becomes dangerous whenpatient is subjected to hematopoietic stem cells transplantations (HSCT)with the profound immunosuppression. A retrospective review of serasamples coming from collection of Department of Microbiology fromgroup of 54 adult recipients of allogeneic HSCT was made. All ofthem received anti viral prophylaxis, using oral form of acyclovir. Serumsamples taken before transplant were examined for presence of specificanti HSV 1 and anti HSV 2 antibodies in IgM and IgG classes. After transplantationquantitative real time PCR was used to determine viral load inplasma samples in range 0 100 days. Achieved results showed that antibodiesdirected against the HSV 1 in IgG class were appearing in the 87%examined persons and anti HSV 2 IgM antibodies in 3% patients. ViralDNA was detected in plasma samples in 15 (27,8%) of the 54 recipients,mostly between day 21st and day 45th of transplantation. All of themdeveloped fever of unknown origin, and over 50% had GvHD features.All of positive patients have HSV 1 viremia; none of HHV 2 DNA wasdetected. There is a high frequency of detectable HSV 1/2 viral load inalloHSCT recipients and it may lead to an increased risk for fatal symptomaticdisease. The availability of quantitative real time PCR method meansthat results are available in a clinically helpful time frame, which shouldassist with implementing timely therapeutic intervention and assessingresponse to treatment.REF 554Use of multiplex real time PCR assay for detection and differentiationviral haemorrhagic cystis in patients undergoing allogeneichaematopoietic stem cell transplantationSylwia RYNANS 1 , Tomasz DZECIATKOWSKI 1,2 , MaciejPRZYBYLSKI 1,2 , Agnieszka TOMASZEWSKA 3 , KazmierzHALABURDA 4 , Grazyna MLYNARCZYK 11 Medical University of Warsaw, Chair and Department of MedicalMicrobiology, Warsaw, POLAND; 2 Central Clinical Hospital in Warsaw,Department of Microbiology, Warsaw, POLAND; 3 Institute of Hematologyand Transfusion Medicine, Department of Haemopoetic Stem CellTransplantation, Warsaw, POLAND; 4 Medical University of Warsaw,Department of Hematology, Oncology and Internal Medicine, Warsaw,POLANDHaemorrhagic cystitis (HC) is characterized by painful haematuria dueto haemorrhagic inflammation of the urinary bladder mucosa. This complicationin group of allogeneic haematopoietic stem cell transplantation(HSCT) is associated with the reactivation of urotropic viruses, such ashuman adenoviruses (HAdV) and polyomavirus BK (BKV). The majoraim of this study was to develop a duplex real time PCR assay for thesimultaneous detection of HAdV and BKV using TaqMan hydrolyzingprobes. Second aim was a retrospective review of group of 64 adult recipientsof allogeneic HSCT with history of haemorrhagic cystitis. 92 serumand 141 urine samples were examined for presence of HAdV/BKV DNA,using quantitative real time PCR to determine viral load in range 40 80days after HSCT. Viral DNA was detected in the sera samples of 31 persons(48%): BKV in 9 (14%) and HAdV in 22 (34%). Viruria was detected in:BKV in samples taken from 24 (37,5%) and HAdV from 55 (86%) individuals,showing symptoms of late haemorrhagic cystitis. None of describedindividuals died during detectable viremia period. Obtained quantitativeresults usually were at low to medium level, placed between 100-1500copies/ml for sera and 400-18000 copies/ml for urine samples, respectively.There is a high frequency of detectable HAdV viremia in HSCTrecipients with signs of haemorrhagic cystitis. Furthermore, establishmentof appropriate procedures for monitoring active HAdV/BKV infection isimportant to clarify the virus infection in transplant recipients.REF 555Cytomegalovirus and human herpesvirus 7 co infections in Polishadults subjected to allogeneic haemopoietic stem cell transplantationsSylwia RYNANS 1 , Agnieszka TOMASZEWSKA 2 , TomaszDZIECIATKOWSKI 2,3 , Anna KRYSKO 4 , Maciej PRZYBYLSKI 2,3 ,Karolina PIEKARSKA 5 , Kazimierz HALABURDA 6 , GrazynaMLYNARCZYK 11 Medical University of Warsaw, Chair and Department of Medical Microbiology,Warsaw, POLAND; 2 Institute of Hematology and TransfusionMedicine, Department of Haemopoetic Stem Cell Transplantation, Warsaw,POLAND; 3 Central Clinical Hospital in Warsaw, Department ofMicrobiology, Warsaw, POLAND; 4 Warsaw University of Life Sciences– SGGW, Interfaculty Department of Biotechnology, Warsaw, POLAND;5 Medical University of Warsaw, 2nd Faculty of Medicine, Warsaw,POLAND; 6 Medical University of Warsaw, Department of Hematology,Oncology and Internal Medicine, Warsaw, POLANDHuman herpesvirus 7 (HHV 7) is widespread around the world and mayalso be a possible cofactor for cytomegalovirus (CMV) infection in haematopoieticstem cell transplant recipients (HSCT). In case of viral diseaseswhere specific treatment is available, real time PCR assays constitutereliable diagnostic tools enabling timely initiation of appropriate therapy.The presence of CMV and HHV 7 was confirmed by the detection of DNAisolated from 1027 plasma samples. A group of 69 HSCT recipients wasexamined in early post transplant period using quantitative real time PCR.Within the study period, 62% of patients had at least once CMV DNAemia, while HHV 7 DNA was found in 43% of subjects. Co infection wasdetected in the plasma samples collected from 18 patients (26%). Patientswith concomitant HHV 7 DNA emia had significantly higher number ofCMV DNA copies compared with those without HHV 7 infection (1986vs. 432 copies/ml) but there was no difference in duration of CMV DNAemia between these groups. On the other hand, while the load of HHV 7DNA was comparable between patients with CMV DNA emia and withoutCMV DNA emia, the duration of HHV 7 DNA emia was significantlylonger in the first group (38.5 vs. 14 days). HHV 7 DNA emia is veryfrequently detected in Polish HSCT recipients. In those, who have subsequentCMV reactivation, the coexistence of the viruses may negativelyaffect the kinetics of infection with either of them. Therefore the investigationof concomitant HHV 7 DNA emia could affect the prognosis ofpost transplant patients suffering from CMV reactivation.REF 556Effect of a TNF alpha antagonist based therapy on the VZV specificT cell immunity in a cohort of psoriatic patientsAlda SALDAN 1 , Elena SANDINI 2 , Daniel TINTO 1 , StefanoPIASERICO 2 , Andrea PESERICO 2 , Giorgio PALÙ 1 , Davide ABATE 11 Department of Molecular Medicine, University of Padova, Padova,ITALY; 2 Dermatology Unit, Padova General Hospital, Padova, ITALYIntroduction: biological antagonists of TNF (etanercept, adalimumab)are currently used for the treatment of severe psoriasis. One of theS274 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyside effects of TNF therapy is an increased rate of VZV infections.In the present study we examined the VZV specific T cell immunityin TNF treated patients versus standard therapy (CsA, methotrexate,steroids).Methods: we enrolled 64 psoriatic patients including 41 subjects receivinganti TNF therapy and 23 subjects as control group. For these patientswe determined VZV specific T cell response with an ELISPOT assay atbaseline, +3 and +6 months after starting treatment. Clinical evaluation ofpatient’s status was done in double blind fashion.Results: in both cohorts, we observed a consistent reduction in diseaseseverity, with an 86% average decrease of the Psoriasis Area SeverityIndex (PASI), suggesting an equal efficacy of both treatments. Duringthe follow up study, we observed 2 cases of symptomatic VZV infectionwhen etanercept (Enbrel) was used. A statistically significant (p< 0.0037)difference of VZV specific T cell response was observed in TNF versusstandard therapy group, with TNF group developing consistentlyhigher levels of VZV T cell immunity. Patients receiving etanercept showedhigher levels of VZV immunity compared to adalimumab (Humira)group.Conclusion: the results were unexpected based on the clinical observations.We speculate that the higher levels of immunity observed in theTNF group derives from subclinical VZV reactivations that may increasethe levels of VZV specific immunity.REF 557Association between BK polyomavirus serostatus and post transplantationviremia in a kidney transplant cohort of living related donorrecipient pairsHerman WUNDERINK 1 , Caroline VAN DER BLIJ DE BROUWER 1 ,Els VAN DER MEIJDEN 1 , Hans DE FIJTER 2 , Louis KROES 1 , AnnVOSSEN 1 , Joris ROTMANS 2 , Mariet FELTKAMP 11 Leiden University Medical Center/Department of Medical Microbiology,Leiden, NETHERLANDS; 2 Leiden University Medical Center/Departmentof Nephrology, Leiden, NETHERLANDSIntroduction: In kidney transplant patients, BK virus can cause polyomavirusassociated nephropathy (PVAN). PVAN can cause irreversiblekidney damage or loss of the graft. Here we investigated if BKV serostatusof donors (D) and recipients (R) influenced the development of BKVviremia post transplantation (TX).Methods: 198 living related D R pairs established between 2007 2010were included. 396 pretransplant sera were tested for the presence of BKVVP1 directed antibodies using in house Luminex based serology. BKV loadwas measured with RT PCR in plasma samples taken 1.5, 3 and 6 monthsafter TX.Results: 95% of D and 94% of R were BKV seropositive at time of TX.Viremia any time after TX was observed in 30% of all R, 90% of whichwere seropositive and 10% seronegative. All viremic R had a seropositiveD. 50% of seronegative R developed viremia, and 30% of seropositive R.Seropositive R from seronegative D did not show viremia. D BKV serostatuswas significantly correlated with development of viremia (p=0,047),whereas R BKV serostatus was not (p=0,127).Conclusion: Overall BKV seroprevalence was high and viremia frequentlyobserved. Seronegative R were more often viremic thanseropositive R, and therefore seem more at risk of PVAN. In R of seronegativeD, no viremia was observed, indicating that BKV reactivationoriginates from the D organ. Taken together, our data suggest that the riskof PVAN in R from BKV seronegative D is extremely low.REF 558No influence of HIV viraemia status on HCV RNA value in immunocompetentHIV HCV co infected patients: preliminary results of alongitudinal retrospective studyMonica BASSO 1 , Saverio Giuseppe PARISI 1 , Mario CRUCIANI 2 ,Marta FISCON 1 , Teresa DALLA ZUANNA 1 , Renzo SCAGGIANTE 1 ,Marzia Maria FRANZETTI 1 , Andrea SATTIN 1 , Carlo MENGOLI 1 ,Giorgio PALÙ 11 Department of Molecular Medicine,University of Padova, Padova,ITALY; 2 Centre of Community & Medicine and HIV Outpatient Clinic,Verona, ITALYBackground: the aim of this study was to evaluate if HCV RNA load canbe influenced by HCV related clinical factors and by HIV viraemia duringan observation period of 36 months.Patients and Methods: HIV HCV co infected patients followed at theInfectious Disease Unit of the Padova Hospital were included if they regularlyattend to the Unit,had no urgent indication to antiviral therapy orrefused.Studied variables:age,gender,HCV genotype,diagnosis of advancedliver fibrosis,ongoing HAART or not in 2012,HCV RNA in 2012,CD4count in 2009 and 2012,successful HIV viraemia suppression or not in2011 (interval A) and in 2010 2009 (interval B).Linear regression analysis,ANOVA,thePearson’s chi square or the Fisher’s exact test wereused.Results: 98 patients were studied (M/F 70/28,median age 48 years,genotype 1 51%).Median HCV RNA was 1723701 (425378-5313631)IU/ml,26.5% of patients had advanced liver fibrosis.Most were on HAART(86.7%):included untreated patients,HIV viraemia was undetectable in66/98 (67.3%) of patients in interval A and in 46/85 (54.1%) in intervalB (patients with discordant virological suppression in the 2 years wereexcluded from analysis).Median CD4 count was 525(340 710) in 2009and 550 (350 770) in 2012.No studied variable showed significant correlationwith HCV RNA load,included HIV viraemia control in interval Aand B.Discussion: in immunocompetent co infected patients HCV RNA load hasno correlation with age,gender,CD4 count,advanced liver fibrosis,HCVgenotype,ongoing HAART at determination,undetectable or detectableHIV viremia in a 36 months periodREF 559Dramatic outcome of HIV related complication in childhoodNajwa GUEROUAZ, Ahlam ABDOU, Fatima Zahra LAMCHAHEB,Mohamed AIT OUGHAROUI, Badr HASSAMDepartment of dermatology, Rabat, MOROCCOIntroduction: the molluscum contagiosum (MC) is a common and oftenbenign viral infection in children. It is caused by a poxvirus and is manifestedby smooth umbilicated papules in the center.Observation: child aged 10 years born from an unmarried mother. Hisvaccinations were up to date. He showed a cervical vegetating lesion discoveredincidentally while hospitalized for epilepsy. HIV viral serologywas positive in both children and his mother. The evolution was spectacularby the rapid spread of the initial injury to the rest of the neck, axillaand external genitalia. Lesions were nodular with a wide implantationbase and umbilicate center, pink, soft consistency, measuring 1 4 cm. Skinbiopsy demonstrates molluscum contagiosum. Treatment was based on10% potassium hydroxide. The evolution was fatal in a few months dueto HIV complications.Virologie, Vol 17, supplément 2, septembre 2013S275

5 th European Congress of VirologyDiscussion: the MC is frequently observed in children between 3 and16 years, especially in atopic and immunocompromised patients. It isfrequently found on the cephalic region, trunk and limbs. In patients seropositivefor HIV, genital regions are most commonly affected and theinfection occurs in stage C of AIDS. In the immunocompromised, evolutioncan be dramatic with a major expansion of tumors, deep penetrationinto the dermis and subcutis to the fascia muscle sometimes requiringsurgical and local treatments.Conclusion: our case illustrates the severity of MC in immunocompromisedchildren. Therapy is often difficult and recurrences are common, sothe combination of several techniques is recommended.REF 560Monitoring of the efficacy of Hepatitis B vaccination and infection inrenal disease and transplantation patientsSanaa AL ATTAS 1 , Hamed ADETUNJI 21 Biology department, Faculty of Science, King Abdulaziz University,Jeddah, SAUDI ARABIA; 2 Oxford Brookes University, Oxford, UNI-TED KINGDOM/Associate Professor, College of Public Health & HealthInformatics Qassim University, Bukairiyah, SAUDI ARABIA; 3 AssociateProfessor, College of Public Health and Health Informatics, Qassim University,Bukairiyah, SAUDI ARABIABackground: Reports show that patients with end stage renal failure andtransplants present with reduced immunity to infections especially withHepatitis B Virus. This may make the patients respond poorly to hepatitisB vaccine with resultant poor seroconversion rate. This study examinesthe efficacy of hepatitis B vaccine, in chronic renal failure (CRF), Post-Kidney Transplantation (PKT) and renal patients as compared to controlshealthy. Methods: A total of 303 subjects participated: One hundred andthirty three immunocompetent and immunocomprised individuals and 170controls were enrolled in this study. They were categorized into fourgroups. Group I: Comprised 54 Chronic Renal Failure, Group II: Comprised21 End Stage Renal Disease patients, Group III: Comprised 38Post Kidney Transplant patients, Group IV: Control group comprised 170vaccinated blood donors and hospital staff. Detection of serological markersHBsAg, Anti HBs, HBeAg, Anti HBe, HBcIgG, and HBcIgM wereperformed by enzyme immune assays (EIA). The vaccination schedulecomprised recombinant hepatitis B vaccine (Engerix B) 40 g intramuscularlyat 0, 1, and 6 months. Results: Our results have documentedsuccessful vaccination in only group I, and III, with primary seroconversion20.4% and 15.8% respectively while none in group II, seroconvertand 100% of group lV demonstrated successful vaccination. The remainingpatients were HBV infected.Conclusions: Controlling HBV infection in end stage of renal disease stillpose great deal of challenges. We highly recommend early vaccination inchronic renal cases.REF 561Longitudinal testing for HHV 6 post Stem Cell Transplantationreveals different patterns of HHV 6 DNAemiaCoretta VAN LEER BUTER 1 , Lilly GARD 1 , Bart SPAN 2 , AnneliesRIEZEBOS BRILMAN 1 , Bert NIESTERS 11 Division of Clinical Virology, Department of Medical Microbiology, Universityof Groningen Medical Center, Groningen, THE NETHERLANDS;2 Department of Hematology, University of Groningen Medical Center,Groningen, THE NETHERLANDSBackground: several recent studies have shown that HHV 6 DNAemiais frequently found in hematological stem cell transplantation (SCT)recipients, especially those who had myelo ablative transplantations andthose receiving cord blood transplantations. Reactivations of HHV 6 areassociated with the development of acute Graft versus Host Disease(GvHD), CMV reactivations, poor engraftment and a higher overallmortality.Methods: frozen whole blood samples from 30 consecutive SCT patientswere tested for HHV 6 by quantitative PCR. All samples taken withinthe first year of transplantation were analyzed retrospectively (total 607samples). Patient data about the underlying hematological disease, type oftransplantation (myelo ablative, reduced intensity, cord blood, sibling, ormatched unrelated donations) were recorded from existing medical files,as well as the occurrence of acute or chronic GvHD and overall outcome.Results: twenty nine of 30 patients had at least one blood sample whichtested positive for HHV 6 DNA. 18 patients had occasional samples withHHV 6 DNAemia and 11 patients had consistent HHV 6 DNAemia Inthe group with consistent HHV 6 DNAemia, the mortality was significantlyhigher (p

5 th European Congress of Virology27. NEUROTROPIC VIRUSESPosters: REF 563 to REF 576REF 563Sylvatic rabies in the North East of Italy:monitoring and evaluationof the effectiveness of prophylaxis in workers at risk andtravelersNadia INGLESE 1 , Cristiano SALATA 1 , Paola DE BENEDICTIS 2 ,Graziella Carpene’ ROSANNA MEL 3 , Monica Bevilacqua SIMONESCHMORAK 4 , Patrizia Rossi ELISABETTA PAGANI 5 , PaoloMULATTI 2 , Giorgio PALU’ 1 , Lebana BONFANTI 2 , FrancoMUTINELLI 2 , Pierlanfranco D’Agaro DANIELA SANTON 6 , TolindaGALLO 7 , Stefano MARANGON 2 , Paola TOMAO 8 , NicolettaVONESCH 81 University of Padua, Padua, ITALY; 2 Istituto Zooprofilattico Sperimentaledelle Venezie (IZSVe), Legnaro, ITALY; 3 Unita’ Locale Socio Sanitarian.1 (ULSS1) di Belluno, Belluno, ITALY; 4 Azienda Sanitaria dell’AltoAdige, Comprensorio Sanitario di Merano, Merano, ITALY; 5 AziendaSanitaria dell’Alto Adige, Comprensorio Sanitario di Bolzano, Bolzano,ITALY; 6 University of Trieste; Istituto di Ricovero e Cura a CarattereScientifico (I.R.C.C.S.), Trieste, ITALY; 7 Azienda per Servizi Sanitari n.4(ASS4), Udine, ITALY; 8 Istituto Nazionale per l’Assicurazione contro gliInfortuni sul Lavoro (INAIL) Settore Ricerca, Monteporzio Catone, Rome,ITALYRabies is a zoonotic viral encephalitis almost invariably fatal in bothhumans and animals. Rabies virus infects mammals through infected salivavia bites or scratches, although atypical exposures have been documented.In late 2008, wildlife rabies re emerged in Northeastern Italy. Since then,287 animal cases have been documented with the last one diagnosed in ared fox in February 2011. No human cases have been reported linked to therecent epidemic and Italy has been declared as free from rabies in February2013.Several oral fox vaccination campaigns and extensive monitoring ofterritories subject to the epidemic have been implemented together witheducation and preventive vaccination of workers at risk of viral exposureas forestry and wildlife workers, veterinarians, shelters ‘operatorsand laboratory personnel. We have evaluated the level and persistence ofserological response in workers at risk and travelers vaccinated followingdifferent immunization schedules. In 113 cases post exposure prophylaxishas been administered, five or four doses, with Rabipur (Novartis), Verorab(Sanofi Pasteur) vaccine. Serum samples from about 300 vaccinatedvolunteers at risk and travelers were collected from the Northeastern territoriesrecently subject to the epidemic. All sera have been tested using anELISA (Enzyme Linked Immunosorbent Assay) and a neutralising test,namely FAVN (Fluorescent Antibody Virus Neutralisation). The resultsof the ongoing analysis will be reported.REF 564Specific immune response induced by rabies G recombinant proteinin baculovirus and maizeEdith ROJAS ANAYA 1 , Elizabeth LOZA RUBIO 1 , FernandoESQUIVEL GUADARRAMA 2 , Estela ESCRIBANO ROMERO 3 , AnaBELEN BLAZQUEZ 3 , Juan Carlos SAIZ 3 , Graciela TAPIA PEREZ 41 Inifap, Cenid Microbiologia, Mexico City, MEXICO; 2 Faculty of medicine,Uaem, Cuernavaca, MEXICO; 3 INIA, Madrid, SPAINThe objective of this study was to analyse the protective capability of thespecific immune response induced by rabies G recombinant protein in twodifferent eukaryotic expression systems: baculovirus and maize. For thispurpose, groups of mice were inoculated as follows: group 1 received acommercial inactivated rabies vaccine dose intramuscularly (i.m.); group2, 100 g of plant derived recombinant G protein orally; group 3, 100 gof baculovirus expressed G protein i.m.; group 4, 100 g of baculovirusexpressed G protein orally; and group 5, 100 g of untransformed maizeorally. The levels of specific antibodies were evaluated every 15 days bya rapid fluorescent focus inhibition test, and the levels of IFN and IL 4were measured every 5 days after immunisation. The mice were challengedwith a lethal dose (10 6 LD50) of the virus at 60 days post vaccination.Antibodies were detected in all groups except negative control. The resultsshowed the presence of antibodies against the G protein in the serum fromanimals immunised with the G protein expressed in baculovirus and maize.In addition, there were significant differences in the splenocyte expressionof IFN and IL 4 from the different immunisation protocols. Most of themice (83%) immunised orally with maize or i.m. with baculovirus extractssurvived viral infection, whereas those immunised orally with baculovirusexpressed protein died. These results showed that the G protein expressedboth in maize and baculovirus can not only induce neutralising antibodiesbut also can mount a Th1/Th2-type response that is able to protect animalsfrom viral challenge.REF 565The AMPK/Sirtuin1 axis modulation during neuronal infection withHerpes Simplex Virus type 1Carola OTTH 1 , Carolina MARTIN 1 , Elizabeth DIMTER 1 , MelissaHOTT 1 , Alexei CUEVAS 1 , Margarita CONCHA 21 Instituto de microbiologia clinica, facultad de medicina, UniversidadAustral de Chile, Valdivia, CHILE; 2 Instituto de bioquimica y microbiologia,facultad de Ciencias, Universidad Austral de Chile, Valdivia,CHILEThe pathogenic mechanisms of Herpes Simplex Virus type 1 (HSV 1) arenot well known. However, data suggest that it is able to establish latencyand in the CNS, and that this condition would not be harmless. Currently, itis unclear whether a neuron, that undergoes viral reactivation, survives andresumes latency or is killed. Taking in consideration that the stress sensorAMP dependent kinase (AMPK) and Sirtuin 1 (Sirt1) are involved in neuroprotectionwe hypothesized that HSV 1 could modulate AMPK/Sirt1pathways as a strategy to establish latency in neurons through inhibitionof apoptosis and stimulation of mitochondrial biogenesis. Therefore weevaluated AMPK, Sirt1, and its downstream targets PGC 1a and p53, inneuronal cultures infected with HSV 1 through western blot, RT qPCRand immunofluorescence analyses. During early infection, the levels ofactive AMPK (p AMPK) decreased significantly as well as its phosphorylationtarget PGC 1a. However at 18 hpi, coinciding with the expressionof the early viral protein ICP8, p AMPK recovered its baseline levelsand simultaneously, PGC1a and TFAM transcript reached their maximallevels. Similarly, at early infection times (2hpi) acetylated p53 peaked,decreasing thereafter in parallel with an increase of Sirt1. However a newrise in acetylated p53 was observed at 18 hpi. These results suggest thatat different times, HSV 1 could interfere with the pro apoptotic response,modulating the AMPK/Sirt1 axis in neurons.Funding FONDECYT 1120464.REF 566Prevention of Fatal Measles Encephalitis by Brain penetrant FusionInhibitorsJeremy WELSCH 1 , Jeremy WELSCH 1 , Aparna TALKER 2 , CyrilleMATHIEU 2 , Antonello PESSI 3 , Anne MOSCONA 2 , MatteoPOROTTO 2 , Branka HORVAT 11 CIRI, INSERM U1111, Lyon, FRANCE; 2 Department of Pediatrics andof Microbiology and Immunology, Weill Medical College of Cornell University,New York, USA; 3 3JV Bio and CEINGE, Via Gaetano Salvatore486, Napoli, ITALYVirologie, Vol 17, supplément 2, septembre 2013S277

5 th European Congress of VirologyMeasles virus (MV) infection causes an acute childhood disease, whichmay be associated with infection of the central nervous system andsevere neurological disease for which specific treatment is currentlynot available. We generated potent inhibitors of MV entry, spreadingand MV induced cell fusion by conjugation to cholesterol and dimerizationof peptides derived from the C terminal heptad repeat (HR)region of the MV fusion protein. The peptides efficiently inhibited MVinfection in SLAM transgenic brain explants, where neurons are primarytargets of measles infection and express the MV fusion protein.After a single intraperitoneal injection, the dimeric cholesterol conjugatedpeptide was found in the brain after 24 h, thus demonstrating itscapacity to penetrate blood brain barrier. When administered to eithersuckling or adult transgenic mice expressing human MV receptor CD150,which are highly susceptible to MV infection, the dimeric cholesterolconjugated peptide protected both from developing fatal encephalitis followingintranasal MV infection. Altogether, these results highlight thepotential of fusion inhibitors for the prophylaxis of MV infection inthe non vaccinated and immunocompromised population and open newpossibilities for the treatment of lethal neurological complications ofmeasles.REF 567Expression and activation by EBV and EBVgp350 of HERV W humanendogenous retroviruses in blood cells and astrocytes: inference formultiple sclerosisAntonina DOLEI 1 , Giuseppe MAMELI 1 , Luciana PODDIGHE 1 ,Alessandra MEI 1 , Elena ULERI 1 , Caterina SERRA 1 , RobertoMANETTI 21 Department of Biomedical Sciences, University of Sassari, Sassari,ITALY; 2 Department of Clinical and Experimental Medicine, Universityof Sassari, Sassari, ITALYProposed co factors triggering multiple sclerosis (MS) are the Epstein Barrvirus (EBV), and the potentially neuropathogenic MSRV (MS associatedretrovirus) and syncytin 1, of the W family of human endogenous retroviruses(HERV W). There is epidemiological evidence that late EBV primaryinfection/seroconversion is a risk factor for MS development. During MS,no MS specific EBV expression occurs, but a continuous HERV W expression.In search of links, the expression of HERV W/MSRV/syncytin 1,with/without exposure to EBV or to EBVgp350, was studied on PBMCfrom healthy donors and MS patients, and on astrocytes, by discriminatoryenv specific RT PCR assays and by flow cytometry. Basal expressionof HERV W/MSRV/syncytin 1 occurs in astrocytes, monocytes, NK,and B, but not in T cells. This uneven expression is amplified in naïveMS patients, and reduced during therapy. EBVgp350 activates MSR-Venv and syncytin 1 in monocytes and B, but not in T cells, nor inthe highly expressing NK cells. In conclusion, in vitro EBV activatesHERV Ws in cells from blood and brain. In vivo, pathogenic outcomeswould occur in late, symptomatic, EBV primary infection, or in predisposinghost genetic backgrounds. In the blood, HERV Wenv activationmight induce immunopathogenic phenomena linked to its superantigenicproperties. In the brain, toxic mechanisms against oligodendrocytescould inducing inflammation, demyelination and axonal damage. A possiblemodel could include EBV as initial trigger of future MS, yearslater, and HERV W/MSRV/syncytin 1 as actual contributor to MSpathogenicity.REF 568The treatment of multiple sclerosis patients with natalizumab inhibitsthe expression of human endogenous retroviruses of the HERV Wfamily, in a longitudinal cohort studyCaterina SERRA 2 , Giannina ARRU 1 , Giuseppe MAMELI 2 , StefaniaLEONI 1 , Alessandra MEI 2 , Caterina SERRA 2 , Roberto MANETTI 1 ,Maura PUGLIATTI 1 , Stefano SOTGIU 1 , Antonina DOLEI 21 Department of Clinical and Experimental Medicine, University of Sassari,Sassari, ITALY; 2 Department of Biomedical Sciences, University ofSassari, Sassari, ITALYMultiple Sclerosis (MS) is a complex immune mediated neurodegenerativedisease. The endogenous retroviruses of the HERV W family (MSRV, MSassociated retrovirus, and syncytin 1, only an env protein) were proposedas MS co factors, and as biomarkers of MS progression and therapy outcome,in patients under treatment with interferon beta. To clarify if HERVW/MSRV/syncytin 1 evaluation could be used as biomarker in patientsexposed to the immunosuppressive natalizumab monoclonal antibody, alongitudinal evaluation of twenty two relapsing remitting MS patientsduring one year of therapy was carried out. Peripheral blood mononuclearcells were collected at study entry and after 3, 6 and 12 monthsof treatment. Cell subpopulations and expression of MSRVenv/syncytin1/HERV Wenv were analysed by flow cytometry and by discriminatoryenv specific RT PCR assays. The therapy was effective, as none of thepatients relapsed during therapy, at variance with the pre entry year. Arelative increase of B cells was observed at 3 6 months (p=0.033). B cells,NK and monocytes but not by T cells expressed the HERV Wenv protein,as expected (Mameli et al., PLOS One 2012;7:e44991). The MSRVenv andsyncitin 1 RNA levels were reduced at 6 12 months of therapy (p=0.0001).Accordingly, at month 12, the plasma membrane levels of the HERVWenv protein were reduced (p=0.0001). The study shows for the firsttime that effective therapy with natalizumab determines a strong decreaseof HERV W/MSRV/syncytin 1 expression as compared with pretreatmentvalues.REF 569Tracing of neuronal connections and neuronal function with pseudotypedrabies virus vectorsMaximilian EIZINGER, Alexander GHANEM, Karl KlausCONZELMANNLMU, Munich, GERMANYAn understanding of how the brain processes information requires knowledgeof the architecture of its underlying neuronal circuits, as wellas insights into the relationship between architecture and physiologicalfunction. Novel genetically modified rabies virus (RABV) variants haveenabled the investigation of specific monosynaptic connections. The applicationof G gene deleted rabies viruses (G RABV) leads to a singleround infection in neurons. Transsynaptic transmission to the presynapticneurons, known as “monosynaptic tracing”, is achieved by providingthe RABV G in trans. Specificity in regard to target cells is achievedby pseudotyping of the RABV vectors. This technology, in combinationwith other genetic, physiological, optical and computational tools,has enormous potential for the visualization of neuronal circuits, andfor monitoring and manipulating their activity. RABV vectors expressingS278 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virologyfluorescent proteins allow specific labeling of cells and cellular compartments.Furthermore, functional proteins as calcium indicators, channelrhodopsinsor e.g. ferritin are expressed from novel vectors. Here we areproviding an update on the rabies virus vector toolbox, allowing bothretrograde and anterograde labeling of neurons, mono synaptic tracing ofneuronal connections, and studying the function and activity of individualneurons.REF 570CMV encephalitis in AIDS patientsSven GRÜTZMEIER 1 , Johan BERGSTRÖM 1 , Eric SANDSTRÖM 1 ,Inger NENNESMO 21 South hospital, Stockholm, SWEDEN; 2 Department of Pathology, KarolinskaUniversity Hospital, huddinge, Stockholm, SWEDENA cohort of 243 patients with HIV infection was followed 1989 1996 atVenhälsan from diagnosis of the infection to death. All were investigatedfor reactivation of CMV infection by measuring CMV antigen and/orCMV PCR in the blood and signs of CMV encephalitis and CMV retinitis,as well as other opportunistic infections. 221 patients died with CD4count 100 × 106/L. Postmortem brain examinations were performed on 78/221 and 6/22 respectively.Of the 78 patients 70 (90%) had signs of reactivated CMV infectionmeasured by blood tests and on morphological examination 43 (55%) hadCMV encephalitis, 16 (21%) toxoplasmosis, 15 (19%) CNS lymphoma,6 (8%) progressive multifocal leukoencephalopathy (PML), 3 (4%) HIVencephalitis and 3 (4%) fungal infection. CMV lesions in the brain wereprimarily found along the walls of the ventricles. 15 patients (19%) hadmore than one opportunistic infection. Of the patients with CMV encephalitis90% also had CMV retinitis. Of the six autopsied patients with CD4counts >100 × 106/L the brain was normal in 2 patients, 2 had infarction,1 HIV encephalitis and 1 glioblastoma. CMV encephalitis is the predominantCNS opportunistic infection in this material and also appears to bethe cause of death either alone or together with other opportunistic infections.Opportunistic CNS infections are not common if the patients diewith a CD4 count >100 × 106/L. HIV encephalitis is not common in anypatient group, one reason could be that all patients had been treated withzidovudin for their HIV infection.REF 571From rabies virus to new drugs candidatesMonique LAFON, Mireille LAFAGE, Christophe PREHAUDInsitut Pasteur NeuroImmunologie Virale, Paris, FRANCERabies virus is a strictly neurotropic virus which multiplies in the centralnervous system of mammals. During the propagation of the virus into thenervous system from one neuron to the next order neuron, the infectedneurons rarely encounter apoptosis. Avoiding premature apoptosis can beseen as an adaptive mechanism to facilitate rabies virus propagation fromthe site of inoculation by bite, up to the salivary glands where it is excreted.We showed that virulent strains of rabies virus, not only avoid prematureapoptosis but also enhance the survival of neurons in culture by activatingsurvival and neurogenesis signaling pathways. Rabies virus infectionenhances the regrowth of axons of adult mouse dorsal root sensory neuronsand promotes neurite outgrowth of human neuroblastoma cells (SH SY5Ycells lines). Among the five proteins of the virus, we determined that theenvelope glycoprotein, the G protein, drives the neuro survival phenotype.We identified the active polypeptide part of the protein and the major roleplayed by its cellular partner, the microtubule associated serine threoninekinase 2, MAST 2. When delivered in cells by a lentivector, the activepolypeptide was observed to favour the healing of axons of human postmitotic dopaminergic neurons (NT2 N cells) which have been woundedby needle scratches. This new neuro protective compound might foundpromising application in neuro degenerative medicine, demonstrating thatviruses can be a source of new drug candidates.REF 572Characterization of the Mouse Neuroinvasiveness of Selected EuropeanStrains of West Nile VirusStephanie LIM, Penelope KORAKA, Sander VAN BOHEEMEN, JoukeROOSE, Dick JAARSMA, David VAN DE VIJVER, AlbertOSTERHAUS, Byron MARTINAErasmus Medical Centre, Rotterdam, THE NETHERLANDSWest Nile virus (WNV) has caused outbreaks and sporadic infections inCentral, Eastern and Mediterranean Europe for over 45 years. Most strainsresponsible for the European and Mediterranean basin outbreaks are classifiedas lineage 1. In recent years, WNV strains belonging to lineage 1and 2 have been causing outbreaks of neuroinvasive disease in humans incountries such as Italy, Hungary and Greece, while mass mortality amongbirds was not reported. This study characterizes three European strainsof WNV isolated in Italy (FIN and Ita09) and Hungary (578/10) in termsof in vitro replication kinetics on neuroblastoma cells, LD50 values inC57BL/6 mice, median day mortality, cumulative mortality, concentrationof virus in the brain and spinal cord, and the response to infectionin the brain. Overall, the results indicate that strains circulating in Europebelonging to both lineage 1 and 2 are highly virulent and that Ita09 and578/10 are more neurovirulent compared to the FIN strain.REF 573Association between grey matter volumes and Herpes Simplex VirusType 1 specific humoral immunity in Alzheimer’s diseaseRoberta MANCUSO 1 , Francesca BAGLIO 2 , Monia CABINIO 2,4 , ElenaCALABRESE 3 , Ambra HERNIS 1 , Milena ZANZOTTERA 1 , FrancaGUERINI 1 , Raffaello NEMNI 3,4 , Mario CLERICI 1,41 Laboratory of Molecular Medicine and Biotechnology, Don GnocchiFoundation IRCCS, Milan, ITALY; 2 Magnetic Resonance Laboratory, DonC. Gnocchi Foundation IRCCS, Milan, ITALY; 3 Dept. Neurorehabilitation,Don C. Gnocchi Foundation IRCCS, Milan, ITALY; 4 Department ofPhysiopathology and Transplantation, University of Milan, Milan, ITALYBackground and aims: HSV1 is suspected to be a risk factor for Alzheimerdisease (AD): it can infect the regions of the central nervous systemwhich are affected by AD associated pathological changes (hippocampal,temporal region). Therefore we evaluated the possible correlations betweenHSV1 humoral immunity and cortical grey matter (GM) volumein AD patients. Materials and methods: 134 subjects were enrolled inthe study: 83 AD (mean age 77+7 yrs; 50 M) (NINCDSADRDA criteria/DSMIV R) and 51 healthy controls (HC) age and sex matched. Patientsunderwent a Mini Mental State Evaluation (MMSE) and HSV1 IgG antibodies(Ab index) were tested in serum by ELISA test. 44 AD HSV1seropositive patients underwent a Magnetic Resonance Imaging (MRI)examination to investigate cortical gray matter (GM) volume (Voxel BasedMorphometry study). ApoE genotyping was also obtained. To highlightthe correlation between GM and HSV1 values, a regression analysis wasperformed on MRI data including for each subject GM maps and HSVantibody values. Statistical analyses were performed on SPM8 software.Results: Seroprevalence and Ab titers were comparable in two groups.High HSV1 titers (Ab index >75 ◦ percentile) were present in 35.8% of ADvs. 16% of HC (p=0.024). Notably, HSV1 Ab titers were positively correlatedwith cortical volume bilaterally in temporal and orbitofrontal cortex(clusters corrected FWE

5 th European Congress of VirologyREF 574Possible Involvement of Beta herpesviruses HHV 6 and HHV 7 andparvovirus B19 Infection in the Development of EncephalopathyModra MUROVSKA 1 , Svetlana CHAPENKO 1 , Santa RASA 1 , SilvijaROGA 2,3 , Zaiga NORA KRUKLE 11 A.Kirchenstein Institute of Microbiology and Virology, Riga Stradins University,Riga, LATVIA; 2 Chair of Pathology, Riga Stradins University,Riga, LATVIA; 3 Riga 1st Hospital, Riga, LATVIAEncephalopathy is a syndrome of global brain dysfunction with unclearaetiology. HHV 6 and HHV 7 are neurotropic viruses associated with awide variety of neurologic disorders. Involvement of B19 in neurologicpathologies is rarely reported.Our study aims to investigate the frequency of B19 and HHV 6, HHV7 infection markers in meningeal and brain tissues of individuals withunspecified encephalopathy. 34 individuals with and 34 without signs ofencephalopathy were enrolled in the study. nPCR was used to detect B19,HHV 6 and HHV 7 DNA in autopsy specimens (dura and pia mater, braintissue) and to define HHV 6 variant. Significantly higher frequency of viralDNAs detection was found in specimens from individuals with encephalopathycomparing to the control group (32/34 vs. 25/34, p=0.044). Nodifference was detected regarding the presence of B19 (17/34 vs. 9/34,p=0.079) and HHV 7 DNA (21/34 vs. 21/34). Frequency of single HHV6 infection (8/34 vs. 1/34, p=0.027) and co infections (29/34 vs. 14/34,p=0.0003) was higher in encephalopathy specimens, single HHV 7 infectionin control group’s samples (2/34 vs. 9/34, p=0.044). Frequency of B19infection/co infection was higher in pia mater, HHV 6 in brain tissue andpia mater, HHV 7 in brain tissue specimens of individuals with encephalopathy.In all HHV 6 positive samples HHV 6B variant was recognized.Meningeal and brain tissues are sites of B19, HHV 6, HHV 7 persistency.Simultaneous study of these viral infections and their activity stages arerequired to identify the relation with the development of encephalopathy.REF 575Identification of viral determinants involved in Borna Disease Virusinterference with human neurogenesisChloé SCORDEL 1 , Marielle COCHET 1 , Marion SZELECHOWSKI 2 ,Daniel GONZALEZ DUNIA 2 , Marc ELOIT 1 , Muriel COULPIER 11 UMR1161 de virologie, ENVA INRA ANSES, Maisons Alfort, FRANCE;2 INSERM, U1043, Toulouse, FRANCEBorna disease virus (BDV) is a neurotropic virus causing neurologicaldisorders and encephalitis in horse and sheep. BDV antigens can be foundin human and the infection could be associated with the development ofpsychiatric disorders. By using human Neural Progenitor Cells (hNPCs)in culture, we previously demonstrated that BDV interferes with humanneurogenesis by inducing the death of newly formed neurons. Our researchnow focuses on the identification of viral determinants responsible for thisinterference. We used lentiviral vectors containing genes encoding 3 viralproteins, the X protein, the nucleoprotein (N), and the phosphoprotein (P)and demonstrated that these 3 proteins neither interfere with hNPCs proliferationnor with atroglial differentiation. Likewise, the X protein does notimpair neuronal differentiation. On the contrary, N and P strongly impairneuronal differentiation, resulting in a 25 and 50% decrease in the numberof neurons, respectively. No death was observed in differentiated cellsexpressing N and P, indicating that the two proteins inhibit neuronal differentiationrather than trigger neuronal apoptosis. We next focused on thecharacterization of the molecular pathways impaired by P. We found analteration of two proteins known to be involved in neurogenesis: the transcriptionalrepressor REST was up regulated and one of its targets, SCG10,was down regulated. Our results demonstrate that the phosphoprotein ofBDV specifically impairs neurogenesis. We are currently pursuing thecharacterization of the cellular and molecular mechanisms altered.REF 576Human endogenous retroviruses of the HERV W family are upregulatedby the JC polyomavirus in human astrocytesElena ULERI 1 , Elena ULERI 1 , Serra CATERINA 1 , GiuseppeMAMELI 1 , Ilker K SARIYER 2 , Kamel KHALILI 2 , Antonina DOLEI 11 Department of Biomedical Sciences, University of Sassari, Sassari,ITALY; 2 Department of Neuroscience, Center for Neurovirology, TempleUniversity School of Medicine, Philadelphia, USAThe ubiquitous JCV polyomavirus causes progressive multifocal leukoencephalopathyin patients severely immunosuppressed or with autoimmunediseases treated with immunosuppressive antibodies. In most of thesediseases, upregulation of HERV W/MSRV/syncytin 1 endogenous retroviruseswas reported. Since the HERV W/MSRV/syncytin 1 are potentiallyneuropathogenic, we wondered about their interactions with JCV, thatcould contribute to neurodegeneration. Thus, human primary fetal astrocytesand the U87MG cell line were infected by JCV or transfected withexpression plasmids carrying early or late JCV genes, to monitor HERVW/MSRV/syncytin 1 expression, or that of constructs carrying the syncytin1 promoter. Infection by JCV upregulated both MSRV like and syncytinenv transcripts. Transient transfection showed that both JCV early and lateproteins stimulate the transcription of HERV W/MSRV/syncytin 1 envgenes. As for the HERV W promoter activity in presence of JCV proteins,gene reporter experiments with constructs carrying the full length syncytin1 promoter, or promoter deletion mutants, co transfected with JCVearly or late plasmids were carried out. Data indicate that the full lengthpromoter is upregulated by both JCV proteins. Deletion of the upstream(cellular) regulatory region indicate that the stimulatory effect lies on the5 ′ LTR (viral) moiety. Due to the identities between the 5 ′ LTR of MSRVand syncytin 1, it is likely that up regulation by JCV antigens of bothMSRV and syncytin 1 requires mainly the viral moiety of their promoters.S280 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of Virology28. GASTROINTESTINAL VIRALINFECTIONSPosters: REF 578 to REF 589REF 578Human Enteric Adenoviruses 40 and 41 in 293 and Caco 2 cellsCasandra MANGROO 1 , Dr. Martha BROWN 1,21 Department of Laboratory Medicine & Pathobiology, University ofToronto, Toronto, CANADA; 2 Department of Molecular Genetics, Universityof Toronto, Toronto, CANADAFastidious human enteric adenoviruses, serotypes 40 and 41 (HAdV 40/41;species F), are important agents of paediatric enteritis. Unlike other adenoviruses,they grow to high titre in the small intestine but typically donot cause disease elsewhere in the body. In cell culture, infectivity is poor,even in 293 cells, whose endogenous HAdV 5 E1 sequences complementthe low level E1 expression of HAdV 40/41. Recent work in this laboratoryidentified a major, though incomplete, block in virion uptake in 293and A549 (lung epithelial) cells. A comparable block in intestinal epithelial(Caco 2) cells, grown under conditions optimized for susceptibility toHAdV 40/41, was unexpected. Caco 2 cells are less susceptible to infectionas cells become differentiated with time, and are most susceptible insub confluent undifferentiated cultures, in particular, when the edges areexposed. Recent experiments using HAdV 41 EGFP and 293 cells haveaddressed the hypothesis that virions are stable during passage through thestomach, but require modification by intestinal conditions for successfulentry/genome delivery. Consistent with the hypothesis, virion infectivitywas increased by exposure to synthetic gastric fluid followed by syntheticintestinal fluid, as determined by flow cytometric analysis of infectedcultures. The unexpected finding was that exposure to gastric fluid alonewas sufficient for the increased infectivity. Subsequent experiments willaddress structural modifications induced by gastric conditions and stepsin the entry pathway facilitated by these modifications.REF 579Validation and performance of an internally controlled multiplexedreal time PCR run on the BDMAX for the detection of Norovirus(NoV) in 7 day working systemKate TEMPLETON, Juliet KENICER, Julie WHITE, PeterMCCULLOCHNHS Lothian, Edinburgh, UNITED KINGDOMThe study aim was to assess performance of a multiplexed NoV GI/GIIPCR with EAV internal control on the BDMAX. A secondary objectivewas to assess BDMAX as a suitable platform for routine testing by nonprofessionally registered staff.The initial validation phase collected 337 samples in January and February2012. 312 samples were faecal specimens and 25 were vomit; all patientshad diarrhoea or vomiting or both. 1 ml suspensions were made by adding25 mg of solid sample or two drops of liquid sample to H2O containingEAV, spun down and extracted both on the easyMAG (Biomerieux) andthe BDMAX (Becton Dickinson). PCR multiplex was performed and analysedon the ABi 7500 or the BDMAX. Following the validation phase theassay was run on the BDMAX for routine diagnosis and then switched backto the easyMAG and ABi to compare performance. The BDMAX NoVmultiplex was 94% sensitive (96/102) and 99% specific (233/235.) 8.6%of positives were GI and the rest were GII. The assay on the BDMAX had2% inhibition and 10.6% inhibition on the EasyMAG/ABi. 1414 sampleswere tested routinely: 361 on the EasyMAG/ABi 7500 and 1053 on theBDMAX. Mean turnaround time was 3 Hrs 47mins on EasyMAG/ABiand 3 Hrs 55mins on BDMAX. During 2011/12, only 29% of NoV testsamples were achieving 15:15 cut off time for resulting. Over the sameperiod during 2012/13 this improved to 75%. The BDMAX could be runmore frequently with minimal hands on time and uncomplicated ease ofuse. The validated NoV Multiplex PCR performed well on the BDMAXand brought significant practical advantages including walk away functionality,minimal hands on time and low level of technical ability requiredto operate the machine. This allowed routine NoV diagnosis to be carriedout 7 days by non professionally registered staff and helped minimise theimpact of NoV sample numbers on other molecular tests.REF 580New Sydney 2012 strain of norovirus detected in patients with acutegastroenteritis in PortugalInês COSTA 1 , João MESQUITA 3 , Elisabete VEIGA 1 , MónicaOLEASTRO 1 , Maria SÃO JOSÉ NASCIMENTO 21 Laboratório Nacional de Referência das Infeções Gastrintestinais,Departamento de Doenças Infeciosas, do Instituto Nacional de S, Lisboa,PORTUGAL; 2 Laboratório de Microbiologia, Departamento de CiênciasBiológicas, Faculdade de Farmácia da Universidade do Porto, Porto,PORTUGAL; 3 Escola Superior Agrária, Instituto Politécnico de Viseu,Viseu, PORTUGALNorovirus (NoV) is the leading cause of diarrheal disease across all agegroups as well as half of all gastroenteritis outbreaks worldwide. Surveillancesystems showed an increase in NoV activity in late 2012 relatedto the emergence of a new NoV genotype II.4 variant, termed Sydney 2012,with the first report coming from Australia in March 2012. Until date, Sydneyvariant circulation in Portugal has not been reported. Aim: To studythe genetic variation of NoVs strains circulating in Portugal in patientswith acute gastroenteritis from May 2011 to December 2012. Methods:A total of 36 NoVs positive samples were studied. Viral RNA was extractedusing NucliSens ® easyMAG TM (bioMérieux) and tested with primerstargeting the POL and the VP1 major capsid genes with Qiagen One StepRT PCR Kit. PCR products were sequenced with Big Dye Terminator v1.1in the Sequencer Analyser ABI Prism 3130 xl (PE Applied Biosystems).NoVs sequences were analysed using the Food borne Viruses in Europe(FBVE) network NoV genotyping tool and NCBI Blast. Results: Overall,14 (39%) samples were positive for the Sydney GII.4. variant. Theremaining 22 (61%) were also identified as GII.4 with a predominance ofthe New Orleans 2009 variant (16/22, 72.7%). Sydney variant was onlydetected from samples dating from June 2012.Conclusion: This is the first report of the circulation of NoV GII.4 Sydneyvariant in Portugal.REF 581Molecular dating in the evolution of primate bocavirusesIgor BABKIN 1 , Irina BABKINA 1 , Aleksandr TUMENTSEV 2 , ArtemTIKUNOV 1,2 , Aleksander KURILSHIKOV 1 , Nina TIKUNOVA 11 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk,RUSSIA; 2 Novosibirsk State University, Novosibirsk, RUSSIAHuman bocavirus (HBoV) is associated with acute gastroenteritis inhumans, occurring mostly in young children and elderly people. Fourbocavirus genotypes have been found so far. Since there were no data onthe contribution of HBoV to gastroenteritis in Russia, 1781 fecal samplescollected from infants hospitalized with acute gastroenteritis in Novosibirsk,Russia during one year were tested for the presence of nucleicacids from HBoV and three major gastrointestinal viruses (rotavirus A,norovirus II, and astrovirus). HBoV was detected only in 1.9% of thesamples. Complete genome sequencing of three Novosibirsk isolates wasperformed. An evolutionary analysis of these sequences and the availableVirologie, Vol 17, supplément 2, septembre 2013S281

5 th European Congress of Virologysequences of human and great apes bocaviruses demonstrated that thecurrent HBoV genotypes diverged comparatively recently, about 60–300years ago. The independent evolution of bocaviruses from chimpanzeesand gorillas commenced at the same time period. This suggests that theseisolates of great apes bocaviruses belong to separate genotypes within thespecies of human bocavirus, which is actually the primate bocavirus. Therate of mutation accumulation in the genome of primate bocaviruses hasbeen estimated. It has been demonstrated that HBoV1 diverged from theancestor common with chimpanzee bocavirus approximately 60–80 yearsago, while HBoV4 separated from great apes bocaviruses about 200–300years ago. The hypothesis postulating independent evolution of HBoV1and HBoV4 genotypes from primate bocaviruses has been proposed.REF 582Frequency of Rotavirus and Adenovirus in Children Between05yearsold in AnkaraGülendam BOZDAYI 8 , Kayhan CAGLAR 1 , Melda MERAL 1 , Ali AdilFOUAD 1 , Aylin ALTAY 1 , Yildiz DALLAR BILGE 2 , AyseKALKANCI 1 , Kamruddin AHMED 31 Gazi University, Faculty of Medicine, Department of Medical Microbiology,Ankara, TURKEY; 2 Ministry of Health, Ankara Training andEducation Hospital, Department of Pediatrics, Ankara,TURKEY; 3 OitaUniversity, Research Promotion Project, Yufu, Oita, JAPANObjectives: Acute gastroenteritis that observed in children are composedby mostly rotaviruses, human caliciviruses, enteric adenoviruses andastroviruses. Adenoviruses were isolated from 1.4% 10% of gastroenteritiscases which are observed in developing countries. In this study, determinationof rotavirus group A and adenovirus serotype A F incidence wasaimed in children patients that applied to hospital because of acute gastroenteritis.Material and Methods: Stool samples were collected from286 patients attended to hospital with diarrhea and vomiting complaints.Rotavirus antigen in stool was detected by ELISA (Rotaclone, MeridianDiagnostics, Inc., Cincinnati, Ohio, USA). Adenovirus DNAs were acquiredfrom stool samples using ZR Fecal DNA KitTM (Zymo Research Corp.Irvine, USA). Acquired DNAs amplificated in LightCycler ® 2.0 (Roche,Germany) using WAYGENE ® Adenovirus nucleic acid detection kit (Berlin,Germany). Results: Rotavirus ELISA positivity was found in 77 (27%)of 286 patients involved in the study. Adenovirus PCR was found positivein 20 (7%) of the same patients’. While both rotavirus and adenovirus positivitywas detected 6 (2.2%) of patients, 6 (30%) of adenovirus positive 20patients, were rotavirus positive. Ten (50%) of adenovirus positive patientswere type F, 5 (25%) of were type B, 2 (13%) of were type E and 1 (7%)of was found type A, however 2 (13%) of them could not be determined.Conclusion: Our results were evaluated as similar results with previousstudies in our country. In proportion to similar studies,we indicated a lowerresult as 2.2% about association of two viruses in our study. This situationmakes us think that both two viruses could be observed at the same time,at the same host and could have been taken by the same way.REF 583Rotavirus genotypes G9 and G2 are co dominantly circlating inAnkara, Turkey: a shift of genotype distribution by emerging G9strainsGulendam BOZDAYI 1 , Aylin ALTAY 1 ,Ayça YENIARAS 2 , YildizDALLAR BILGE 2 , Kamruddin AHMED 31 Gazi University, Faculty of Medicine, Department of Medical Microbiology,Ankara, TURKEY; 2 Ministry of Health, Ankara Training andEducation Hospital, Department of Pediatrics, Ankara, TURKEY; 3 OitaUniversity, Research Promotion Project, Yufu, Oita, JAPANObjectives: Globally, rotavirus is the leading cause of diarrhea in childrenunder 5 years of age. The purpose of this study was to detect the frequencyof rotavirus infection and genotype distribution in children under 5 yearsold attending with acute diarrhea to a hospital in Ankara, Turkey. Materialsand Methods: Stool specimens were collected from 269 childrenattended to Ankara Training and Education Hospital with acute diarrheafrom January 2010 to July 2011. Rotavirus antigen in stool was detectedby commercially available ELISA kit androtavirus genomic dsRNA wasextracted from the ELISA positive samples using phenol chloroform isoamylalcohol method. Amplification of genotype specific geneswas carriedout by nested RT PCR. Results: Among 269 samples, 29.7% were positivefor rotavirus. Rotavirus mainly affected (45.7%) children of 12-23 monthsage group. In spring and winter roatavirus infection was more frequent,36.7% and 36.4%, respectively. G and P types were detected in 88.7% and91.2% of rotavirus positive samples, respectively. The frequency of differentG type detection was as follow: G2 (35.2%) and G9 (45.1%) werethe most prevalent types.G1, G3, G4, G1+G9 and G2+G9 were detectedin 8.4%,5.6%, 2.8%, 1.4% and 1.4% of samples, respectively.WithinP genotypes, P8 was detected in 93.1%, P4 in5.5% and P4+P8 in 1.4%of samples. The dominant G and P genotype combinations were, G2P[8](33.8%) and G9P[8] (39.4%). Conclusion: The frequency of P genotypedistribution in this study was similar to that of detected in previous years.Compared with previous study there was a significant increase of genotypeG9 and G2 in 2010-2011. Almost half of G genotypes were G9 indicatingthat this genotype is emerging in Ankara, Turkey. It is necessary to continuesurveillance in the coming yearsto understand the epidemiology andevolution of genotype G9 in Ankara and other parts of Turkey.REF 584Norovirus infections and prevalence of IgG antibodies to genotypeGII.4 in a Spanish populationJavier BUESA 1,2 , Noelia CARMONA VICENTE 1 , ManuelFERNANDEZ JIMENEZ 1 , Juan M. RIBES FERNANDEZ 1 , Carlos J.TELLEZ CASTILLO 11 School of Medicine, University of Valencia, Microbiology, Valencia,SPAIN; 2 Hospital Clinico Universitario, Valencia, SPAINNoroviruses (NoVs) are responsible for most of the outbreaks of acutegastroenteritis worldwide, as well as a common cause of sporadic cases.The prevalence of different NoVs genotypes as the cause of acute gastroenteritisin the region of Valencia (Spain) during a three year period(2008 10) was investigated. In addition, the prevalence of IgG antibodiesto NoV genotype GII.4 in sera from a random sample of individualsduring the same period of time was determined. Baculovirus expressedvirus like particles (VLPs) of NoV GII.4 2006b and a recombinant P2polypeptide of NoV GII.4 2008 produced in Escherichia coli were usedas coating antigens in enzyme immunoassays to study NoV GII.4 specificantibodies. Serum samples from 434 individuals of different ages collectedbetween 2009 and 2010 were assayed. NoVs were detected in 42 (77.3%)outbreaks and in 7.8% sporadic cases of acute gastroenteritis. GenogroupGII strains were predominant causing both outbreaks and sporadic cases.Different genotype GII.4 viral variants were found throughout the studyperiod (GII.4 2006a, 2006b, 2008 and 2010), being the GII.4 2010 variantthe most frequently detected (40%). Antibodies to the P2 polypeptide andto GII.4 VLP were detected in practically all (99%) of the serum samplesanalysed. Titers of antibodies to NoV VLPs and to the P2 domain graduallyincrease with age, reaching their highest concentrations in the forthdecade of life. Our results suggest that exposure to NoV genotype GII.4occurs early in childhood but re infections are common.REF 585Detection and first molecular characterization of Canine parvovirus(CPV) on dogs in TurkeyTaner KARAOGLU, Sepandar GARGARI, Aykut ÖZKULAnkara University, Faculty of Veterinary Medicine, Department of Virology,Ankara, TURKEYS282 Virologie, Vol 17, supplément 2, septembre 2013

5 th European Congress of VirologyAs a worldwide distributed pathogen,Canine Parvovirus type 2 (CPV 2)firsemerged in dogs in 1978,almost simultaneously in Europe and NorthAmerica, and was responsible for hemorrhagic gastroenteritis and myocarditisin puppies.Afterward new antigenic variants designated CPV 2aand CPV 2b,became widespread during 1979 to 1980 and 1984, respectively.RecentlyCPV 2c has been identified initially in Italy and subsequentlyin Vietnam. In Europe, all CPV genotypes(CPV 2a, CPV 2b and CPV2c) have been reported from Italy.In South America,Uruguay has reportedthat CPV 2c is the predominant type.In Asia, India has reportedthe prevalence of CPV 2a and CPV 2c.This is the first report focusingon molecular characterization of CPV 2 on dogs in Turkey. To achievethis aim, fecal samples/rectal swabs from CPV suspected dogs were collectedfrom private veterinary pet clinics in Ankara, Turkey.The fecalsamples/rectal swabs obtained from the suspected dogs were tested forthe presence of CPV vDNA using in house PCR targeting ORF7 ofviral genome.Amplicons obtained from positive fecal samples were submittedto nucleotide sequencing for further phylogenetic analysis.Partialsequences obtained from VP1 coding gene of local CPVs were submitted toGenBank.Phylogenetic analysis (Neighbor Joining) with bootstrap analysis(1,000 replicates) and Kimura 2 parameter correction was conducted byusing the MEGA software package v5.0. Data obtained showed that CPVsdetected in fecal samples were diagnosed as canine originated parvovirusandare closely related each other based on partial ORF7 sequences.REF 586Viral gastroenteritis in hospitalized pediatric patientsFrancesca ROVIDA 1 , Michela GRIGNANI 2 , Giulia CAMPANINI 1 ,Antonio PIRALLA 1 , Kodjo Messan Guy ADZASEHOUN 1 , AntonellaSARASINI 1 , Gian Luigi MARSEGLIA 2 , Fausto BALDANTI 11 Molecular Virology Unit, Virology and Microbiology Department, FondazioneIRCCS Policlinico S. Matteo, Pavia, ITALY; 2 Department ofPediatrics, Fondazione IRCCS Policlinico S. Matteo, University of Pavia,Pavia, ITALYGastrointestinal viral syndromes are one of the most common cause ofmorbidity and mortality in children worldwide. In the period April 2011April 2012, 173 stool samples from as many pediatric patients hospitalizedat our Institution with gastroenteritis were examined for the presence ofrotavirus (RV), norovirus (NoV), astrovirus (HAstV), adenovirus (HAdV),rhinovirus (HRV), enterovirus (EV), parechovirus (HPeV), bocavirus(HBoV), coronavirus (HCoV), sapovirus (SaV), cosavirus (HCoSV) eaichi virus (AiV) with specific molecular assays. Viral gastrointestinalagents were detected in 93/173 (54%) patients. Viruses as a single pathogenwere diagnosed in 60 (65%) patients, as multiple infections in 33(35%). The most common viruses identified were RV (35/93, 38%), HAdV(9/93, 10%) and HRV (5/93, 6%), while NoV (4/93, 4%), EV (4/93, 4%),HAstV (2/93, 2%) and HBoV (1/93, 1%) were less frequently detected.SaV, HPeV and HCoV were detected only in multiple infections. Noneof the patients (0%) was positive for HCoSV and AiV infections. Thehighest number (42/93, 45%) of viral gastroenteritis was detected in childrenof ages 13 36 months, while the lowest number (9/93, 10%) wasidentified in children of age >60 months. RV is the leading cause of viralgastroenteritis in hospitalized pediatric patients. A potential implication ofHRV in gastroenteritis is suggested. The investigation of non conventionalgastrointestinal viruses is fundamental to implement the laboratory diagnosticroutine and to supply more epidemiological data on the circulationof gastrointestinal viruses.REF 587Genetic Diversity and Molecular Evolution of Human AstrovirusSergei SOKOLOV 1 , Igor BABKIN 2 , Artem TIKUNOV 2 , NinaTIKUNOVA 21 State Research Center of Virology and Biotechnology Vector, Koltsovo,RUSSIA; 2 Institute of Chemical Biology and Fundamental Medicine,Novosibirsk, RUSSIAHuman astrovirus (HAstV) is one of etiologic agent of acute gastroenteritisin humans, mostly in young children. HAstVs are classified intoeight genotypes (HAstV 1 – HAstV 8) perfectly correlated with eightserotypes, with HAstV 1 being the most common around the world. Inthe present study the prevalence, time distribution, genotype frequenciesand clinical significance of astrovirus infection from children with acutegastroenteritis were determined during 8 year period in Novosibirsk,Russia. To examine genetic diversity of HAstV, specimens positive forastrovirus were genotyped. Five different genotypes (HAstV 1 – HastV4 and HAstV 6) were detected, with HAstV 1 being the most prevalenttype. Complete genome sequencing of one HAstV 6 and three of HAstV 3isolates were performed. Further analysis of the nucleotide sequences andother complete genomes from GenBank revealed a number of predictedrecombination break points. The rate of HAstV evolution was estimatedbased on genome fragments without the recombination events and itappeared to be typical for RNA viruses.REF 588A case control study of acute gastroenteritis in Slovenian children 0 6years of age: is there something new?Andrej STEYER 1 , Tjaša CERAR 1 , Adela FRATNIK STEYER 1 , ŠtefanGROSEK 2 , Monika JEVŠNIK 1 , Marko KOLENC 1 , Tatjana MRVIC 3 ,Miroslav PETROVEC 1 , Marko POKORN 3 , Mateja POLJŠAKPRIJATELJ 1 , Barbara ŠOBA 1 , Marija TRKOV 4 , Tina URŠIC 1 , FrancSTRLE 31 Institute of Microbiology and Immunology, Faculty of Medicine, Universityof Ljubljana, Ljubljana, SLOVENIA; 2 Department of PediatricSurgery and Intensive Care, University Medical Centre Ljubljana, Ljubljana,SLOVENIA; 3 Department of Infectious Diseases, University MedicalCentre Ljubljana, Ljubljana, SLOVENIA; 4 Institute of Public Health of theRepublic of Slovenia, Ljubljana, SLOVENIAObjectives: The etiology of acute gastroenteritis was studied in a prospectivecase control study to review and optimize the diagnostic procedurein hospitalized children up to 6 years of age.Methods: Stool samples were collected from cases and controls fromOctober 2011 to September 2012. Real time (RT) PCR was used to detecta broad spectrum of enteric pathogens (bacteria, parasites, viruses).Results: Stool samples from 292 cases and 89 controls were assessed. Thedetection rates of viruses were 69.2% and 14.6%, of bacteria 6.5% and4.5% and of parasites 1.7% and 1.1% in cases and controls, respectively. Atleast one pathogen was found in 90.8% of cases and 20.2% of controls. Incases, rotaviruses were the most prevalent pathogen (detected in 55.5%),followed by adenoviruses (19.5%) and genogroup II noroviruses (13.7%).A substantial detection rate was observed also for pathogenic E. coliVirologie, Vol 17, supplément 2, septembre 2013S283

5 th European Congress of Virology(VTEC, EPEC, EIEC) (7.9%), Salmonella sp. (5.8%) and Campylobacterspp. (5.1%). A relatively high percentage of C. difficile in cases (8.6%) (25/292) was found, but it was detected mostly as a co pathogen withrotaviruses, adenoviruses and noroviruses.Conclusions: Rotaviruses, adenoviruses and noroviruses GII should beincluded in the first choice of testing, combined with bacterial pathogens,especially Campylobacter spp., Salmonella sp., and pathogenic E. coli.The significance of C. difficile needs further evaluation as it was detectedmainly as co pathogen with the most prevalent viruses.REF 589Occurrence and genetic diversity of viral causative agents of acutegastroenteritis in Novosibirsk, Russia, 2003 2012Nina TIKUNOVA 1 , Elena ZHIRAKOVSKAYA 1 , Artem TIKUNOV 1 ,Sergei BODNEV 1 , Tatiana YUN 1 , Igor BABKIN 1 , AleksandrKURILSCHIKOV 1 , Sergei SOKOLOV 1 , Sergei NETESOV 21 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk,RUSSIA; 2 Novosibirsk State University, Novosibirsk, RUSSIAThis study assessed the molecular epidemiologic characteristics of acutegastroenteritis in hospitalized children

5 th European Congress of VirologyIndex by Author an Abstract n oAAaltonen LM, REF 541Aaskov J, REF O28Abacioglu H, REF O161, REF 264Abate D, REF 545, REF 556Abbas W, REF 003Abbi R, REF 493Abd El Aal A, REF 461Abd El Rahmann S, REF 162Abd El Wahed A, REF 456Abdelwhab ES, REF O63Abdikerimov K, REF 514Abdou A, REF 559Abdullah H, REF 076Abel T, REF 163Abele H, REF 481Aberle JH, REF 043, REF O51Abitayeva M, REF 037Abrantes J, REF 354Abrao E, REF 402Abravaya K, REF 450Accardi R, REF 004Acuña R, REF 148Acuner C, REF 393Adams D, REF O82Adams O, REF 515Ader Ebert N, REF 149Adetunji H, REF 560Adjlane N, REF 307Adler B, REF O50Adler H, REF O119Admiak B, REF O07Adzasehoun KMG, REF 586Aepfelbacher M, REF 408Aferchichi A, REF 121Affabris E, REF 063Agata C, REF 464Agresti C, REF 063Agteresch E, REF O147Aguilar Setién A, REF 0166Agut H, REF 133, REF 134, REF 135, REF 402Ahlm C, REF 068Ahmadian G, REF 042Ahmed K, REF 582, REF 583Ahmed W, REF 002Ahola T, REF O08Aiello Padilla M, REF 316Ait Arkoub Z, REF 133, REF 134, REF 135Ait Ougharoui M, REF 559Ait Youcef H, REF 472Aitgoughoulte M, REF 036Aitor N, REF O31Ajobiewe OJ, REF 131Akca Y, REF 304Akhavan S, REF O76, REF 482Akhmadishina L, REF 366Akhrymuk I, REF 021Aksacli S, REF 393Aktas M, REF 251Al Attas S, REF 560Al Hello H, REF O144, REF 352Al Nakib W, REF 524Alain S, REF O111Alam M I, REF 107Alawi M, REF 204, REF 408Albayrak H, REF 247, REF 296, REF 308,REF 309, REF 310Alberdi P, REF 405Albert J, REF 435Albertini A A, K10Albina E, REF 075Albonetti C, REF O10, REF 183Albornoz A, REF O32Albrecht J C, REF 001Albulescu I, REF 199, REF 284Albulescu L, REF O117Alcaro S, REF 137Alcina A, REF 226Alexuyk P, REF 037Alkan F, REF 304Alkhovsky S, REF 415Allander T, REF O44Allemandou A, REF 295Allering L, REF 287Allice T, REF 381Almazan F, REF O31Almeras L, REF 237, REF 272Alpha Sall A, REF 275Alsaleh K, REF 276Altawalah H, REF 524Altay A, REF 582, REF 583Alten B, REF 274, REF 282Altmann A, REF 074Altmeyer R, SP5_4Altwegg M, REF 533Alvarez K, REF 126Alves L, REF 149, REF 156Alvisi G, REF O140Amara A, REF 168Amberkar S, REF O140Amendola A, REF 357, REF 529Amirache F, REF 014, REF 015, REF O52Amo K, REF 526Amouroux P, REF 360Anastasiadou A, REF 027Andersson B, REF O44Andersson C, REF O91Andersson S, REF 438Andouard D, REF O111Andraud M, REF O65André G, REF O12André P, REF O101, REF O109, REF O138,REF 173, REF 191, REF 543Andrée P, REF 382Andreo U, REF O136Andrus L, REF O136Ang W, REF 432Angeloni G, REF 271Angius F, REF 100Anna Ida A, REF 363Annunziata C, REF 016Ansart I, REF 480Antoniou K, REF 078Aouni M, REF 181Aquino V, REF 116Arai F, REF 420Arancibia Y, REF 198Araujo E, REF 364Arbiza J, REF 260Arcangeletti M C, REF 070, REF 206Archambaud M, REF 006Archer F, REF 294Archer J, REF O108Archimbaud C, REF O24Ardèvol M, REF 104Arenzana-seisdedos F, SP5_5Ari A, REF 141Arias-Goeta C, K19Arilahti V, REF 024, REF 243Arnaud F, REF 319Arnberg N, REF 166Arns C, REF 298Arrache W, REF 373Arribas M, REF O142Arru G, REF 568Artacho A, REF O108Artarini A, REF 190Artois M, REF 253, REF 259Artusi S, REF O114Aschman N, REF 089Asnical S, REF O38Atanasova M, REF 490Atasheva S, REF O53Atkinson C, REF 425Atli K, REF 049, REF 299, REF 299, REF 315,REF 318, REF 335Attoui H, REF 405Aubard Y, REF O111Auboeuf D, REF 007, REF O154Aubry V, REF O35Aumayr M, REF 195Ausina V, REF 104Avci O, REF 049, REF 311, REF 312, REF 315,REF 318, REF 337, REF 338, REF 342,REF 343, REF 514Avila Sánchez M, REF 149Avsic Zupang T, REF 254Awada H, REF 386Aydin N, REF 527Aydinok Y, REF 390Ayral F, REF 253, REF 259BBa A, REF 275Babarczi E, REF 505Babiarova K, REF 539Babkin I, REF O100, REF 238, REF 581,REF 587, REF 589Babkina I, REF O100, REF 238, REF 581Babudieri S, REF O22Bacharach E, REF 175, REF 213, REF 218Backes S, REF 077Badawy A, REF 461Badescu D, REF 280Badillo A, REF O101Bae J, REF 417Baeshen N, REF 009Baglio F, REF 573Bahuon C, REF 276Baikov I, REF O09Bailer S M, REF 169Bailey T, REF 326Bailly B, SP5_5Bailly Bechet M, REF 319Bailly J L, REF O24Virologie, Vol. 17, supplément 2, septembre 2013S285

5 th European Congress of VirologyBaize S, REF O89Baker S, REF O99Bakkali Kassimi L, REF 295Bakker A, REF 127Bakli M, REF 272Bakonyi T, REF 292Bakthiari K, REF O25Baldanti F, REF O148, REF 532, REF 586Balloco C, REF 546Bally M, REF 219Bamford D, REF O113Bandi M, REF 551Banhegyi D, REF 505Banko A, REF 496Baquero E, K10Barak S, REF O85Barbani M T, REF 516Barbarit C, REF O28Barbezange C, REF 346Barbi M, REF 384Barbier N, REF O65, REF 314Barbier V, REF 091Bárcena M, REF 265Barclay W S, REF O107, REF 190Barclay W, REF 011Bardin P, REF 229Barhoom S, REF 213Barlic Maganja D, REF 313Barlinn R, REF 484Barnabé A C, REF 298Barnaud E, REF O127Barnes A, REF O82Barnett J, REF O168Barranger C, REF 421, REF 454, REF 475,REF 522Barrón B L, REF 064, REF 114, REF 122,REF 497Barron M J, REF 157Barry C, REF 450Barry G, REF 319Bartenschlager R, REF 098, REF O140,REF 194, K7Bartolini G, REF 038Baruchel A, REF O149Barzon L, REF 006, REF O10, REF 012, REF O29,REF O38, REF 046, REF 050, REF O55,REF 077, REF O155, REF 183,REF 277, REF 416, REF 424Bascuñana E, REF 104Basileo M, REF 038Baskir B, REF 390Bassereau P, REF 219Basso M, REF 558Batetta B, REF 100Battistone A, REF 384Bauby H, REF O107Baudin F, REF O33Baudin M, REF O124Bauer Balci T, REF 519Baule A, REF 069Baumert T F, REF 123Bavari S, REF 069Bayoumy A, REF O30Bayram E D, REF 141Bayry J, REF 062Beach N N, REF O143Beaucourt S, REF 347Beaufrère A, REF 056Beaulieux F, REF 261Beaumont T, REF 127Bechter K, REF 074Becker Dreps S, REF O95Becker J, REF 132Becker N, REF 287Becker S, REF 249Bédarida S, REF 348Bedekovic T, REF 256Bedin F, REF 180Beer M, REF 356, REF 359Beersma M F C, REF 422, REF 440Begon M, REF 259Begu D, REF O57Behbehani N, REF 524Bejaoui Olhmann Y, REF O72Bejvl I, REF O150Belalov I, REF O145Belefquih B, REF 493Belen Blazquez A, REF 564Belfquih B, REF 139, REF 367, REF 373Belghazi M, REF 237, REF 272Belhouchet M, REF 405Bell Sakyi L, REF O42, REF 246, REF 278,REF 405Bello F, REF 128Bello Morales R, REF 226Belouzard S, REF 178Ben Khalifa Y, REF 006Ben M’hadheb Gharbi M, REF 181,REF 182Ben Tal N, K19Benachi A, REF 489Benedetti E, REF 283Benfield C T O, K4Bengrine A, REF 178Benjelloun N, REF 367Benkirane M, K6Benschop K S, REF O160Benschop K, REF O15, REF 127, REF 498Bensenane M, REF 144Benussi M, REF 011Benyahia M, REF 373Benyelles H, REF 449Berber E, REF 251Berdance E, REF O57Berenguer M, REF 549, REF O108Berezin V, REF 037, REF 120Berger A, REF 465Berger S, REF 446Bergeron C, REF O46Bergeron E, REF O93Berginc N, REF 368, REF 369Bergström J, REF 570Bergstrom T, REF O07, REF O41Berisio R, REF 153Berland Y, REF 348Bernard C, REF 130, REF 320Bernardin C, REF 088Bernatz M, REF 452Berri F, REF 075Bertagnoli S, REF 328Berthelot P, REF 501Bertoletti A, REF O82Bertrand J B, REF 130Bertrand M, REF 454Bes J, REF 421, REF 454, REF 522Bespalov M, REF O08Bestebroer T, REF 265Bhatia S N, REF O136Bhatta D R, REF 494Biagini P, REF O18, REF 348Bialasiewicz S, REF 351Bianchi Dos Santos M M A, REF 316Bianchi S, REF 357Bianco É M, REF 110Bianco G, REF 137Biasolo M A, REF O114Biçeroglu S, REF 390Bichurina M, REF O162Bieringer M, REF 156Biesinger B, REF 001Bigault L, REF 320Billaud G, REF O71Bily T, REF O20Bin H, REF O37Binda S, REF 384Binger T, REF 519Biolatti M, REF O105Bischoff E, REF 279Bishop K N, REF 218Biswal S, REF 069Bitto D, REF 150Björkström N, REF O81Blackledge M, REF O116Blagrove M, P2_4Blahova M, REF O82Blaise Boisseau S, REF 295Blanc H, REF O141, REF 346Blasi E, REF 147Blazevic J, REF 165Blazquez A, REF 293Blight J, P2_4Bloch W, REF 157Blomberg J, REF 448Blomqvist S, REF O144, REF 352Blondeau J, P1_4Bloyet L M, REF 023, REF 092Boccolini D, REF 283Böcher O, REF 451, REF 452Bock C T, REF 408Bodeus M, REF 472Bodnev S, REF 589Boerma E, REF 537Bofikova B, REF 349Bogdanow B, REF O36, REF 212Bogner E, REF 094, REF 132,REF 142Bogoyavlenskiy A, REF 037, REF 120Bogs J, REF O63Bohm M, REF 510Bohn Christiansen C, REF 531Boka V, REF 542Boland G, REF 491Boldorini R, REF O152Bolfa P, REF 065Boller K, REF 163Bolukbasi C, REF 310Bommelaert F, REF 360, REF 441, REF 492Bondarenko T, REF 215Bonfanti L, REF 563Bongiorno G, REF 290Bonin E, REF 314Bonne I, K19Bonnefoy E, REF 025Bonvicini F, REF 186S286Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyBoorn Konijnenberg D V D, REF 107Boot H, REF O13Borchers K, REF 336, REF 344Borchers K, REF 344Borderia A, K19Bordi L, REF 019Bordicchia L, REF 454Bordignon J, REF 110, REF 443Borel C, REF 262Boreux R, REF 431Borgogna C, REF O152Borkner L, REF O50Borner C, REF 001Borrini B M, REF 385Bosch B J, REF O83, REF 151, REF 158Bosch S, REF 287Bosnjak M, REF 086Botikov A, REF 415Bouallegue M, REF 395Bouamoud M, REF 362Boucher C, K8Boucherit V C, REF 218Bougueleret L, REF 507Boukhadra N, REF 501Bouloy M, REF 025, REF 232Bouquet J, REF O127Bourdon J C, REF O79, REF 087Bourdon S, REF 237Bourgouin C, REF 279Bourhy H, REF 006, REF O122, REF O167,REF 402, REF 494Bourlet T, REF 423, REF 442, REF 501, REF 544Bouscambert M, REF O46Bouteiller L, REF 453Boutel D, REF 027Boutet Robinet E, REF O157Boutolleau D, REF 133, REF 134, REF 135,REF 402Bouton C, REF 197Bouts T, REF 513Boyer L, REF 267Bozdayi G, REF 582, REF 583, REF 547Bradt V, REF 165Braun M, REF O81Braun P, REF 465Breed A, REF 259Breitbart M, REF O18Breithaupt A, REF O63Bressanelli S, REF O62Bressollette Bodin C, REF O151, REF 548Brisbarre N, REF 018Britto C, REF 428Brnic D, REF 256, REF 333Brocchi E, REF 275Brochier B, REF 268Brochot E, REF 178Brockmann M, REF O158, REF 445Bronowicki J P, REF 144Brossard A, REF 181Brouard J, REF 523Brown D M, REF 578Brown K, REF 394Brown L E, REF 372Brucculeri M R, REF 431Bruisten S, REF 370Brulois K, REF 094Brun P, REF O27Brunel J, REF 205Bruni R, REF 378Bruzzone R, REF O59Bryazhikova T, REF 111Bryon M, REF 128Bua G, REF 186Bubba L, REF 384Buc H, REF O23Bucardo F, REF O95Bucci P, REF 283, REF 290Buchholz C, REF 163, REF 0130Buchholz U, REF 519Büchner S, REF 055Buchy P, REF 129Buckland R, REF O52Buesa J, REF 584Buffaz C, REF 543Bulut O, REF 049, REF 299, REF 312, REF 315,REF 514Bumbarov V, REF 175Bumbasirevic V, REF 086Buonaguro F, REF 016Buonaguro L, REF 016Buonocore L, K10Burdino E, REF 381Burgu I, REF 304Burkard C, REF 151Burke D, REF 471Burmistrz M, REF 228Burneikiene R, REF 389Burrel S, REF 133, REF 134, REF 135, REF 402Burri D, REF 170Burton D R, REF 048Burtseva E, REF 530Butcher S J, K14Butcher S, REF 222Butovskaya E, REF O34CC Van Der Kuyl A, REF 407Cabanillas L, REF O142Cabello C, REF 387Cabinio M, REF 573Cabrerizo M, REF 499Cadoré J L, REF 065Caglar K, REF 582Caglioti C, REF 019Caignard G, REF O12, REF 230Caillet C, REF O23Calabrese E, REF 573Calattini S, SP4_3Calderaro A, REF 070, REF 206Calistri A, REF O114, REF 0134, REF 136Calliste C, REF O111Calvo G, SP4_2Camerer E, REF 075Camilloni B, REF 038Camoin L, REF 237, REF 272Campadelli Fiume G, REF 003, REF 0131Campadelli G, REF 0131, REF 586Campbell J, REF O99Camsusou C, REF O65Camus Bouclainville C, REF 328Canakoglu N, REF 251Canard B, REF O115, REF 126, REF 202,REF 284Candido A, REF 378Candolfi E, REF 466Cannas V, REF 137Cannie K, REF 040Cantisani M, REF 153Canuti M, REF O98, REF O165, REF 399,REF 407, REF 409, REF 412, REF 529Capelli G, REF O37, REF 291Capobianchi M R, REF 019, REF 363, REF 365Capone C, REF 063Cappellesso R, REF 006Cappy P, REF 074Caprani A, REF 017, REF 058, REF 059Capua I, REF 011Caputo A, REF 046, REF 050, REF O55Carbajo Lozoya J, REF 115, REF 123Carbone A, REF O87Cardia Caserta L, REF 316Carell T, REF O11Carfi A, REF 159Cariolet R, REF 320Carissimo G, REF 279Carkaci D, REF 517Carli I, REF 136Carmeliet P, REF 075Carmona Vicente N, REF 584Carnec X, REF 168Caro V, REF O167, REF 402Carpenter S, REF 319Carr M, REF O163, REF 396Carron C, REF O79, REF 224Cartet G, REF 224Casalegno J S, REF O46, REF O71Casanovas S, REF 104Caserta L, REF 298Cassany A, REF O57Castagliuolo I, REF O27, REF O29, REF O38Castaño C, REF 004Castelain S, REF 178Castillett C, REF 019Castro G, REF 260Caterina F, REF 464Caterina S, REF 576Catoi C, REF 065Cattai M, REF O38, REF 277Cattaneo R, REF 020, REF 028, REF 0129,REF 161, REF 205Cauldwell A, K13Caumes E, REF 402Cavalheiro Martini M, REF 316Cavalier A, REF O135Cavallo R, REF 546Cavazzana Calvo M, REF 0134Cavunt A, REF 247, REF 296, REF 308,REF 310Cecuda Adamczewska V, REF 057Ceianu C S, REF 280Cekanac R, REF 270Celik (Yilmaz) G, REF 393Cerar T, REF 588Cermelli C, REF 147Cerný J, REF 196, REF 349Certoma A, REF 494Cerutti F, REF 381Cervantes Gonzalez M, REF O167Ceylan E, REF 527Chabas D, REF 472Chabierski S, REF 040, REF 424Chaffey P, REF O127Chalons M, REF O92, REF 239Chambon M, REF O24Virologie, Vol. 17, supplément 2, septembre 2013S287

5 th European Congress of VirologyChamond N, REF 181Chan D P, REF 022Chan M, REF 200Chan P K, REF 022, REF 083Chan P, REF 200Chane Woon Ming B, REF 184Chapenko S, REF 361, REF 574Charles F, REF 360, REF 441, REF 492Charneau P, REF 095Charreau B, REF O151Charretier C, REF 444Chatre E, REF 095Chauleur C, REF 423Chauveau E, REF O68Chen A A, REF O136Chen P, REF 068Chen T, REF 436Cheng J C, REF 353Cheng S, REF 201Cherepanov P, REF O121Chevallier P, REF 548Chevallier Queyron P, REF 543Chevret S, REF O149Chezzi C, REF 070, REF 206Chiantore M V, REF 004Chiaramonte I, REF 384Chichev E, REF 530Chiereghin A, REF 385Chinh N T, REF O99Chionne P, REF 378Chisari F V, SP4_2Chiu S C, REF 353Chmelik V, REF 043, REF O51Cho J E, REF 417Choi K, REF 269, REF 273Choi W, REF 269, REF 273, REF 450Choi Y, REF O26, REF 047Choudhury B, REF O67Chowdhury E H, REF 242Chueh L L, REF 322, REF 341Chung B, REF 410Chung J, SP4_2Ciancia C, REF 319Ciccaglione A R, REF 378Cicin Sain L, REF O50Cifuentes N, REF 148Cintra A, REF 116Ciufolini M G, REF 283, REF 290Claas E, REF 498Claassen Y, REF 127Claus C, REF 071Clerici M, REF 573Cleton N, REF O123, REF O169, REF 281Cliquet F, REF 262Close R, REF 520Cnops L, REF 355Cochet M, REF 575Cociancich V, REF 303Coffey L, K19Cohen G H, REF 159Coignard C, REF 437Coilly A, REF 362Collet A, REF O115, REF 202Collet M, REF 135, REF 360, REF 441,REF 492Colombo A A, REF O148Colzani D, REF 357Combredet C, REF 030Comoli P, REF O148Concha M, REF 565Condamine E, REF 219Conibear T, REF 425Connell J, REF O163, REF 471Constam D, REF 233Constant S, REF O43Constantin S, REF 371, REF 374Contamin B, SP5_3Contrant M, REF 184Convert S, REF 562Conzelmann K K, REF O11, REF 569Cook S, REF 410Cooperman B, REF 213Corbach Soehle S, REF O38Corbach Söhle S, REF 288Cordeiro E, REF 392Cordier A G, REF O72Cordier F, REF O23Cordioli P, REF 275, REF 286Cordova D, REF 387Corman V M, REF 0166, REF 240Cornelissen J, REF O147Cornwall J, REF 473Corona A, REF O110, REF 137Correa Basurto J, REF 114Correia B, REF 217Cortes F, REF 293Corti D, REF O73Cosby S L, REF 076Cosconati S, REF O110Cosset F-L, REF 014, REF 015, REF 036,REF O52, REF O87Costa C, REF 014, REF 015, REF O52,REF 546Costa I, REF 580Costanzi G, REF 012Coste Burel M, REF O151Coste M, REF 441Costi R, REF O110Cotar A I, REF 280Cotin S, REF O111Cotten M, REF 412Cottier M, REF 423Cottiglia F, REF 137Cottontail V M, REF 399Cottrell S, REF 396Couderc T, SP5_4Coudray C, REF 426Coulpier M, REF 575Coupeau D, REF 189Courvoisier Guyader K, REF 317Cousin G, REF O12Coutard B, REF 284Couteaudier M, REF 317Cowley D, REF O99Coyle P V, REF 518Crean M, REF 471Cremer J, REF O13Crespillo A J, REF 226Crobu M G, REF 551Crockard M, REF 518Crosnier R, REF O76Crotty S, REF 048Croville G, REF 411Crowe Jr J E, REF 048Cruciani G, REF 117Cruciani M, REF 558Crussard S, REF 056Csanády M, REF 427Cuevas A, REF 565Cuevas L, REF 291Cunskis E, REF 361Cupic M, REF 103, REF 496, REF 502Curbishley S, REF O82Curi B, REF 285Curtil C, REF 191Curtin F, REF 130Curtoni A, REF 546Cusack S, REF 023Cusinato R, REF O38, REF 277Czosnek H, REF O60DDa Palma J R, REF 233Da Palma J, REF 170Da Poian A, REF 285Dacheux L, REF O167, REF 402, REF 494Daga M, REF 113Dagalp S B, REF 304Dakovic Rode O, REF 256Dal Bello F, REF 136Dal Pozzo F, REF O69Dalla Zuanna T, REF 558Dallar Bilge Y, REF 582, REF 583Dalle J H, REF O149Dallmeier K, REF 128Dalpke A, REF 533Damas M, REF 079Dambrinne G, REF 189Damond F, REF 470Danan C, REF 453Daniela Santon P D, REF 563Danilenko D, REF 111, REF 203Danjoy M, REF 562Dao Thi V L, REF O87Dao-Thi L, SP4_3Dargel C, REF 185Dastjerdi A, REF O67, REF O168, REF 513Daussange G, REF 468Dauzonne D, REF O12Davidkin I, REF O164Davis G D, REF 013Davison A, REF 513Davison N, REF O168Davy J B, REF 523De Andrea M, REF O105De Benedictis P, REF 563De Boer M, REF 158De Borba L, REF 066De Castro E, REF 507De Conto F, REF 070, REF 206De Fijter H, REF 557De Filette M, REF 039, REF 040De Filippis A M, REF 364, REF 418De Filippis A, REF 487De Giovanni C, REF 0131De Groote D, REF 520De Haan C, REF 151De Haan K, REF O15De Haan L, REF 407De Haan X, REF O146De Jong M, REF 281De Jong S J, REF 001De Koening M, REF O152De La Fuente J, REF O42S288Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyDe La Grandière M A, REF O69De Leo A, REF 125De Man R, REF O147, REF 440De Micco P, REF 018, REF 348De Mol P, REF 431De Oliveira F, REF 074De Paula V, REF 428De Saint Vis B, REF 056De Simone A, REF 153De Souza Campos H, REF 101De Swart R L, REF O103De Swart R, REF O54De Vries E, REF O146De Vries M, REF O98, REF O165, REF 399,REF 407De Vries R D, REF O54De Vries R, REF O146De Wilde A, REF 265De Wispelaere M, REF O21Deák J, REF 372, REF 427Dealng L, REF 128Deandrea M, REF O152Deaville R, REF O168Debart F, REF 202Decimo D, REF 171Decoster A, REF O172Decroly E, REF O115, REF 284Decroly E, REF O115Dediego M L, REF 004Degirmenci A, REF 390Deijs M, REF O98, REF O165, REF 399, REF 407,REF 409, REF 412, REF 529Dekhtiarenko I, REF O50Dekkers D, REF O83Del Amo J, REF 286Del Valle A, REF 497Del Vecchio C, REF 0134, REF 136, REF 137Delamballerie X, REF 128Delepierre M, REF O23Delfau Larue M H, REF 005Delic D, REF 103Dell’oste V, REF O105Della Libera S, REF 378, REF 379Delmas O, REF 006Delogu R, REF O97Deloire A, REF 191Delpeyroux F, SP5_4Delwart E, REF O18, REF 413Demant P, REF 096Demmers J, REF O83Denesvre C, REF O157, REF 317Denisova O, REF O47, REF 108Dennin R H D R, REF 350Deryabin P, REF 415Descamps V, REF 178Deschamps T, REF O35Descy J, REF 431Desire N, REF O76, REF 402Desloire S, REF 294Desmecht D, REF 255Desprès P, REF O12, REF O21, REF 030,REF 276Desvars A, REF 261Dettori S, REF 378Deubel V, REF 129Devaux P, REF 020, REF 205Devignot S, REF 001, REF O93Devillier P, REF O72Dhondt K P, REF 230, REF 239Dhondt K, REF O92Di Bartolo I, REF 271Di Camillo B, REF O10, REF O29, REF 077,REF O155Di Caro A, REF 019Di Fatta T, REF 551Di Leva F S, REF O110Di Lorenzo C, REF 457, REF 458Di Luca M, REF 283Di Nauta A, REF 546Di Nunzio F, REF 095Di Perri G, REF 381Di Santo R, REF O110Diamond M S, REF 288, REF 424Diamond M, REF 039Diancourt L, REF 402Diaz J J, REF 224Diaz O, REF O101, REF O138, REF 173Diaz Soria C, REF O106Dib L, REF O87Dietrich I, REF 279Dijkman R, REF O07, REF O45, REF O83,REF O126Dik I, REF 049, REF 315, REF 318Dimaio D, REF O86Dimitrova M, REF 197Dimter E, REF 565Dina J, REF 523Dinu S, REF 280Diosdado F, REF 387Diot C, REF O128Disehler A, REF O57Distinto S, REF 137Dite P, REF 419Dittmer U, REF 519Divocha V, REF 109, REF 118Dixon M J, REF 157Djordjevic J, REF 103DME Osterhaus ADoblali T, REF 373Dobler G, REF 356Doceul V, REF O68Doerig C, REF 478Dolei A, REF O22, REF 567, REF 568, REF 576Døllner H, REF 383Dolmazon C, REF 065Dolnik O, K15Domingo E, REF O143Donato C, REF O99Donnadieu C, REF 411Donoso Mantke O, REF 460Donovan P, REF 233Doorbar J, REF O152Dordor A, K11Dorenlor V, REF O65Dorobantu C, REF O117Dos Santos F, REF 364, REF 418, REF 487Dosnon M, REF 205Douam F, REF O87Doyle T, REF O107Draganescu A, REF 525Dreux F M, SP4_3Drew T, REF O67Drexler J F, REF O125, REF O145, REF 0166,REF 240, REF 244, REF 399Dridi M, REF 276Dron A G, REF 191, REF 543Drosten C, REF O07, REF 035, REF O40,REF O83, REF 115, REF 123,REF O125, REF O126,REF 0166,REF 240, REF 244, REF 399, REF 519Drouet E, REF O33Duarte A, REF 428Duarte Dos Santos C N, REF 066Duarte Dos Santos C, REF 110Duarte L, REF 330Dube M, REF 421, REF 522Dubin G, REF 228Dubos F, REF O172Dubreil L, REF 093Dubuisson J, REF 178Duc Dodon M, REF 008Ducancelle A, REF 441Ducatez M, REF 328, REF 411Dudman S G, REF 484Dufkova L, REF 301, REF 332Dulovic M, REF 086Dumarest M, REF O127Dunford L, REF O163Duong V, SP5_4Dupinay T, REF 253Dupont J B, REF 093Dupressoir T, REF 029Duprex W P, REF O54, REF O103Dupuis Maguiraga L, REF O28Durães Carcavlho R, REF 298, REF 316Durand C, REF 129Durantel D, REF 036Dussol B, REF 348Dutia B, REF O30, REF 397Duval R E, REF 121Duverlie G, REF 178Dzalbs R, REF 542Dzeciatkowski T, REF 005, REF 552, REF 553,REF 554, REF 555Dzierzanowska Fangrat K, REF 495EE Magnusson S, REF 288Ebert Ader N, REF 152Echavarria M, SP1_3Eckerle I, REF O40Effantin G, K11Ehret R, REF 465Ehrlich M, REF 175, REF 213, REF 218Eibach D, REF O71Eickmann M, REF 249Eid R V, REF 479Eiden M, REF 287Eifler M, REF O120, REF 212Eis Hübinger A M, REF 399, REF 436Eisenberg R J, REF 159Eisenhaber B, REF 410Eizinger M, REF 569Ekci B, REF 393El Amrani M, REF 373El Anaz H, REF 373El Annaz H, REF 493El Deeb A, REF 456El Kochri S, REF 493El Sayed Zaki M, REF 461El Tholoth M, REF 456Elannaz H, REF 139, REF 367Elberse K, REF O103Eliopoulos A, REF 034Virologie, Vol. 17, supplément 2, septembre 2013S289

5 th European Congress of VirologyElis E, REF 218Elisabetta Pagani P R, REF 563Ellis R, REF 325Eloit M, REF 575Elroy Stein O, REF 213Else-Kröner-Fresenius Foundation, REF O112Elsterova J, REF O20Emerman M, REF O104Emmanouil M, REF 377Emonet S, REF 018Emuzyte R, REF 389Enache L S, REF O138, REF 173, REF 191Eng K E, REF 221Engberg J, REF 517Engelmann F, REF 094, REF O172Enjuanes L, REF 004, REF O137Enouf V, REF 346Ensser A, REF 080, REF 094, REF 0133Eono F, REF O65Equestre M, REF 378Eremeeva T, REF O145, REF O162,REF 366Erensoy S, REF 390Ergör G, REF O161Ergunay K, REF 274, REF 282Erhart J, REF O42Erisoz Kasap O, REF 282Ermonval M, REF 254Erny A, REF 294Eroglu C, REF 247Erol N, REF 302Eropkin M Y, REF 203Eropkin M, REF 111Eropkina E, REF 111Escribano Romero E, REF 293, REF 564Escriou N, REF O12Escudero Pérez B, REF O90, REF 240Escuret V, REF O46, REF 520Esposito F, REF O110, REF 137Esquivel Guadarrama F, REF 564Essa S, REF 524Essaidi Laziosi M, REF O139Essbauer S, REF 356Esser K H, REF O125, REF 244Essere B, REF O135Esteban J I, REF O143Esteves A, REF O17, REF 143, REF 503Esteves PJ, REF 354Estrada A, REF O40Estrada Peña A, REF 245Estrade C, REF O171, REF 478Etienne D, REF 202Etienne L, REF O104Evander M, REF O124Eveno E, REF O65Everest D, REF O168Exindari M, REF 377, REF 462Eydoux C, REF 126FF Jebbink M, REF 407Fahd M, REF O149Failloux A-B, REF 279Faivre Moskalenko C, REF 171Fajfr M, REF 447Falanga A, REF 153Falcou Briatte R, REF 468Falcuta E, REF 280Falda M, REF 416Fall G, REF 275Famiglietti M, REF 063Fantinatti Garboggini F, REF 298Faria N, REF 364Farinati F, REF O29Farnir F, REF 255Farrar J, REF O99Fassan M, REF 006Fassina A, REF 006Faure Della Corte M, REF 549Faure M, K3Faury N, REF 192Fausto B, REF 464, REF 486Favard C, REF 171Fazakerley J K, REF O42Featherstone C, REF 259, REF 394Fedorov R, REF O119Fedosyuk S, REF 195Fedyakina I, REF 530Feghoul L, REF O149Feichtinger S, REF O80, REF 209Feldmann F, REF 094Felgner P L, REF 048Feltkamp M, REF 185, REF 351, REF 557Fenaux H, REF 133Fender A, REF 184Feng Q, REF 033Feng Y, REF 013, REF 511Ferguson B J, K4Fernandes A, REF 428Fernandez Jimenez M, REF 584Fernández M D, REF 245Fernandez R, REF 004Ferraris O, REF O46, REF 084, REF 232Ferreira De Lima Neto D, REF 041Fester N, REF 082Fichez A, REF O71Ficicioglu N C, REF 393Fiebig P, REF 424Figueiredo L, REF 116Figuerola J, REF O37, REF 286,REF 291Filippone C, REF 254Filisetti D, REF 466Finance C, REF 446Finarelli A C, REF 385Finkelshtein D, REF O85Finnegan C, REF O67, REF O168Fiore L, REF O97, REF 384Fiore S, REF 384Fiorucci G, REF 004, REF 063Fiorucci M, REF 056Firantiene R, REF 389Firouzi R, REF 130Fischer A, P1_4Fischer G, REF O51, REF 123Fischer K, REF 055Fischer L, REF O112Fischer N, REF 204, REF 408Fischl W, REF O140Fiscon M, REF 558Fisher E A, REF O136Flamand M, REF 025Flatley B R, REF O136Fleckenstein B, REF 0133, REF O156,REF 208, REF 220Fletcher N, REF O82Fleury H, REF 468, REF 493Florea D, REF 525Florian F, REF 094Florys K, REF 057Foka P, REF 187Foll Y, REF 360, REF 441, REF 492Fontana P, REF 416Fooks A, REF 259, REF 325Fornara C, REF 469Forstova J, REF 060, REF 061Fortier C, REF 344Fortier G, REF 336, REF 344Fortuna C, REF 283, REF 290Fouad A A, REF 582Foucaud G, REF 475Foucault Grunenwald M L, REF 075Fouchier R A M, REF 035, REF 243Fouchier R REF O83, REF O126,Fournier C, REF 178Fournier E, REF O135Fourquet P, REF 237, REF 272Fragnoud R, REF 180Fraiberk M, REF 060, REF 061Fraile Ramos A, REF 226Fraisier C, REF 237, REF 272Fraisse A, REF 426Francart V, REF 520Francesca C, REF 464Franceschini A, REF O78Franchin E, REF O38, REF 277Francioso A, REF 125Francisco Sampaio Bonafé C, REF 041Franco L, REF 355Francois C, REF 178Frans J, REF 521Franssila R, REF 045Franz S, REF O106Franzetti M M, REF 558Frasca G, REF 385Frasson I, REF O34Frati E R, REF 357Fratini M, REF 378, REF 379Fratnik Steyer A, REF 588Frecha C, REF 014, REF O52Frédéric C, REF O19Frenkiel M P, REF O21Frenzke M, REF 0129Fresquet J, REF O87Frey S, REF 356Freymuth F, REF 434Friberg A, REF 216Friesland M, SP4_2Frippiat J P, REF 144Frobert E, REF 520Froeyen M, REF 128Frolov I, REF O53Frolova E, REF 021, REF O53Früh K, REF O49Ftaich N, REF 319Fulcher A, REF 225, REF 229Full F, REF 0133Furione M, REF O70Fusil F, REF 036Fusil L F, SP4_3Fuzik T, REF 172S290Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyGGabriel membersGabrielli L, REF 046, REF O55, REF 385Gaidot N, REF O72Gaillard C, REF 056Galabov A S, REF 112, REF 138Galan C, REF 233Galdiero M, REF 153Galdiero S, REF 153Gallego I, REF O143Gallinella G, REF 186Gallo T, REF 563Galvin E, REF 473Gambino M, REF 384Gang Z, SP5_4Garbarg Chenon A, REF 480Garbuglia A R, REF 363, REF 365García A, REF 387, REF 497Garcia Espinosa G, REF 334García Sastre A, REF 001, REF O146Gard L, REF 500, REF 561Gargari S, REF 274, REF 585Garigliany M, REF 255Gariglio M, REF O105, REF O152Garnier L, REF 421, REF 522Gärtner B, REF 515Garzaro D J, REF 267Gasanova T, REF 508Gastaminza P, SP4_2Gatej R I, REF 280Gatta V, REF 0131Gatti D, REF O105Gatti R, REF 206Gaucherand P, REF O71Gaudin Y, K10Gaudy Graffin C, REF 547Gavana E, REF 248Gavazzi C, REF O135Gavier Widén D, REF 354Gawanbacht A, REF 001Gazzolo L, REF 008Geffers R, REF O126Geldreich A, REF 197Genini E, REF O73Gennart I, REF 189Georger C, REF 093Georgopoulou U, REF 187Gérard C, REF 431Gérard N, REF O151Gerber M, REF 002Gerlier D, REF 023, REF 092, REF O139,REF 205Germi R, REF 130Germini D, REF 070, REF 206Gerna G, REF O73, REF 469Gessain A, REF 005, REF 007, REF O154Geyer M, REF 063Ghanem A, REF 569Gharbi J, REF 181, REF 182, REF 382Ghildyal R, REF 225, REF 229Ghisetti V, REF 381Gianni T, REF 003Giehler F, REF 001Gijselaar D, REF O13Gil P, REF 075Gillespie T, REF O40Gineys B, REF 294Gioula G, REF 377, REF 462Girard A, REF 014, REF 015Girello A, REF O148, REF 532Giron M C, REF O27Gitelman A, REF 415Giuseppe I, REF 019Giuseppe M, REF 464Giuseppetti R, REF 378Gjata B, REF 093Gligic A, REF 270Gnädig N, REF 347Godeke G J, REF O169Goehringer F, REF 144Goeijenbier M, REF O25Goelz R, REF O159Goerzer I, REF O150Gohrbandt S, REF O63Göhring K, REF O112Gokce O, REF 393Goldman Wohl D, REF 590Gomaa N, REF 485Gómez Núñez L, REF 387Gonçalves A R, REF 154Gonçalves B, REF 364Gonzalez Dunia D, REF 575Goodfellow I, K12Goossens H, REF 412Goracci L, REF 117Göransson A, REF O44Gorbalenya A E, REF O115Gorbalenya A, REF 351Gorgievski M, REF 516Gorin S, REF 314Gorna K, REF 295Gorovits R, REF O60Görzer I, REF 540Gosselin Grenet A S, REF 029Goto A, REF 091Goto K, REF 526Gottardello L, REF 046, REF 050, REF O55Göttlinger H, REF 089Gottner G, REF 491Gouarin S, REF 523Goubau P, REF 472Goudeau A, REF 547Goujon C, REF O107Goulidaki N, REF 176Gouma S, REF O13Gouriou S, REF 492Gourves L, REF 320Gout O, REF 005Gozhenko A, REF 109Gräber M, REF O63Gradoni L, REF 290Graeber Gerberding M, REF 287Graf C, REF 124Graf J, REF 098Graf L, REF 207, REF 209Gramiccia M, REF 290Grangeot Keros L, REF O72Granjeaud S, REF 272Grasland B, REF 320Grassi A, REF 077, REF O155Gravelsina S, REF 361Grazia Revello M, K23Greber U F, REF O58, REF O118, REF 509Greco A, REF 082Grégoire F, REF 255Gregori G, REF 381Grey F, REF O30Gribaudo G, REF 188Grierson S, REF O67Grignani M, REF 532, REF 586Grigorov B, REF 092Grijalva A, REF O108Grimes J, REF 006Grishkovskaya I, REF 119Grom J, REF 303, REF 313Groot Koerkamp M, REF O153Groschup M H, REF 164Groschup M, REF 287Grosek Š, REF 588Gross C, REF O156, REF 220Grubhoffer L, REF O42, REF 096, REF 196,REF 349Gruet A, REF 205Gruffat H, REF O35Gruffaz M, REF 036Grundhoff A, REF 204, REF 408Grunert H P, REF 460Grützmeier S, REF 570Gu S H, REF 414Guérin El Khourouj V, REF O149Guérin J L, REF 411Guerini F, REF 573Guerouaz N, REF 559Guerrero N S, REF 128Guerrier L, REF O75Guettari N, REF 013Gueudin M, REF 074, REF 429, REF 470Gugliesi F, REF O105Guigon G, REF O167Guiguen F, REF 406Guillaume T, REF 548Guillemot J C, REF 126Guillén J, REF 284Guillier L, REF 426, REF 453Guillon P, SP5_5Guivel-benhassine F, REF 168Gunay F, REF 274, REF 282Günaydin G, REF O95Günther S, REF 287Guo J T, REF 105Gupta N, REF 062Gupta S, REF 068, REF O81, REF O121Gur S, REF 299, REF 302, REF 309Gurdasani D, REF O106Guriev V, REF 388Gurol Y, REF 393Gushchina E, REF 085Gut W, REF 528Gützkow T, REF O125Guy B, SP3_1Guy M, REF 454Gyan E, REF 547HH Hidalgo M, REF 267Haagmans B L, REF 243Haagmans B, REF O83, REF 265Haar E, REF 466Haarman E G, REF O160Haas J, REF 123Habela M Á, REF 245Habjan M, REF 001Hacein-Bey-Abina S, P1_4Hacot S, REF 224Virologie, Vol. 17, supplément 2, septembre 2013S291

5 th European Congress of VirologyHadji Khodadad S, REF 042Haffner P, REF 192Haftek Terreau Z, REF 095Hagbom M, REF O96Hagemeier C, REF O36, REF O120, REF 211,REF 212Hagh B, REF 479Haglund M, REF O41Hahn B H, REF O104Hahné S, REF O13Haim Boukobza S, REF 362, REF 476Haimov Kochman R, REF 590Hain J, REF 145Hainz P, REF 539Halaburda K, REF 553, REF 554, REF 555Halami M Y, REF 321Halbach T, REF O11Halbherr S, REF O64Haligur M, REF 299Haliloğlu T, K19Hall M, REF O122Hall W, REF O163Halos L, REF 261Haloschan M, REF O150Hamilton S, REF 207Hammarström L, REF O95Hamming O, REF O126Hamprecht F, REF O140Hamprecht K, REF O112, REF O159, REF 481Hamzi M A, REF 373Han M G, REF 417Han M, REF 269, REF 273Hannoun C, REF O07Hanson P, REF O102Hanss M, REF 075Harder J, REF O11, REF O40Hartmann G, REF 107Hartmann R, REF O126Hase A, REF 526Haselhorst T, SP5_5Hashimoto S, REF O160Hasircioglu S, REF 299, REF 315, REF 335Hassam B, REF 559Hassan J, REF O163, REF 471Hatesuer B, REF 072Hatz C, REF 355Hauber J, REF 099Hauffe R H C, REF 267Haveri A, REF O164Hayette M P, REF 431He F, REF 430He Q, REF 201Heaney L, REF 076Hebert A, REF 523Hedman K, REF 045, REF 413, REF 436,REF 448, REF 483, REF 541Hedman L, REF 045, REF 436, REF 448,REF 541Hegde P, REF 062Heidler N, REF 326Heinz F X, REF 043, REF O51, REF 165Helenius A, REF O78, REF O88, REF 154,REF 222, REF 511Helle F, REF 178Hellert J, REF O119Helynck O, REF O12Hemida M, REF O102Heng S, SP5_4Hennechart Collette C, REF 453Hennig J, REF 216Henquell C, REF O24Hepojoki J, REF 044Herbein G, REF 003Herbreteau C H, REF 129Heringer Da Silva M, REF 364, REF 418Hernandez L, REF 355Hernandez Nignol A C, REF 320Hernis A, REF 573Herold S, REF 005Herpe Y E, REF 178Herrant M, SP5_2Herraud J M, SP5_2Herrero L, REF 291Herrler G, REF O66, REF O125, REF O137REF 155, REF 162, REF 244, REF 358,REF 359, REF 510, REF 538Herrmann C, REF 061Hervé S, REF O65, REF 314Hesse M, REF 155Higa L, REF 285Hillairet C, REF 468Hinz A, REF 089Ho S, REF 450Hobelsberger D, REF O40Hober D, REF O172Hochdorfer D, REF 160Hoegner K, REF 005Hoelen H, REF O49Hoeper D, REF 359Hoffmann J, REF 396Hoffmann M, REF O125, REF 244, REF 359Hoffmann T, REF 178Hofko M, REF 533Hogema B, REF 370Högye M, REF 427Höhne M, REF 408Holmes E C, REF O122Holstege F, REF O153Honda A, REF 420Hoopmann M, REF 481Hooykaas M, REF O153Höper D, REF 356Horhogea C, REF 330Horn G, REF 175Horsington J, REF O56Horst D, REF O49Horvat B, REF 092, REF 230, REF 239,REF 566Horvath G, REF 035Horvath P, REF O78Høsøien Skanke L, REF 383Hossain D, REF O124Hott M, REF 565Hourmant M, REF O151Hovi T, REF O144Hsieh L E, REF 322Huang K J, REF 179Huang S, REF O43Hubalek Z, REF 292, REF 323Hübner D, REF 071Hue E, REF 336, REF 344Hué S, REF O107Hufert F, REF 456Hug M, REF 533Huhtamo E, REF 398Huiskonen J T, K14Huiskonen J, REF 150Huissoud C, REF O71Hukkanen V, REF O113Hulo C, REF 507Huss M, REF 423Hüsser L, REF 324Hutterer C, REF 209Huynen P, REF 431IIaconelli M, REF 378, REF 379Ianiro G, REF O97Ibrahim W, REF 501Icard V, REF O109, REF O138, REF 173,REF 543Icenogle J, REF 394Ichai P, REF 362Ieven G, REF 412Igor G, REF 371Igor S, REF 371, REF 374Illiaquer M, REF 548Imbert I, REF O115Imbert Marcille B M, REF O151, REF 548Imler J L, REF 091Ince B, REF 337Incoronato N, REF 153Indenbirken D, REF 204, REF 408Ingianni A, REF 100Inglese N, REF 563Iorio A M, REF 038Iritani N, REF 526Isac M, REF 388Isakov O, REF 346Isel C, REF O135Ishii K, REF 420Islam MR, REF 242Istrate C, REF O17Iuliano M, REF 004Ivakhnishina N, REF 488Ivanov P, REF 508Ivanova O, REF O145, REF O162,REF 366Iványi B, REF 427Izquierdo L, REF 178JJaarsma D, REF 572Jääskeläinen A, REF 376Jablonska A, REF 067, REF 495Jacob R, REF 001Jacob Y, REF O12, REF 095, REF O128,REF 230Jacquet J, REF 423Jacquot F, REF 129Jaensson T, REF O41Jager M, REF 432Jahn G, REF O112, REF O159,REF 481Jaksch P, REF O150Jamin M, REF O33, REF O116Janes V, REF O15Jans D, REF 225, REF 229Jansen R, REF 433Jansson E, REF 435Janvier G, REF O12Jany S, REF O39, REF 053Jarmer J, REF 043Javanbakht H, REF 036S292Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyJazaeri Farsani S M, REF O165, REF 407,REF 409, REF 412,REF 529Jeanson Leh L, REF 093Jebbink M F, REF O165, REF 409Jebbink M, REF O98Jemersic L, REF 256, REF 333Jensen L, REF 225Jeong Y E, REF 417Jepson P, REF O168Jestin A, REF 320Jeulin H, REF 144, REF 446Jevšnik M, REF 588Jia M, REF 340Jian L, SP5_5Jiménez Clavero M A, REF 276, REF 286Jiménez Guardeño J M, REF 004Jimenez M A, REF 275, K17_1Jo J, REF O82Jochmans D, REF 128Joest H, REF 287Johnson N, REF 325Jolivet M, REF 129Jones R, REF 396Jones S, REF 450Jonkers J, REF 412Jonsdottir H R, REF O45, REF O126Jónsdóttir H R, REF O07Jonsson N, REF O44Jorda P, REF O12Jordana Lluch E, REF 104Jouan D, REF 262Jovanovic T, REF 103, REF 496, REF 502Jubert C, REF 549Jugulete G, REF 525Julkunen I, REF 024, REF 243Jung J U, REF 094Jungic A, REF 256, REF 333Junglen S, REF O40Jungnickl D, REF 094KKabala W, REF 228Kabamba B, REF 472Kabanova A, REF O73, REF 469Kabbaj D, REF 373Kacem S, REF 434Kaderali L, REF O140, REF 177Kadi H, REF 296, REF 310Kagan K O, REF 481Kageyama T, REF 526Kaida A, REF 526Kainov D, REF O47, REF 108Kaiser L, REF O43Kaiser R, REF 465, REF 515Kajic V, REF 403Kakkola L, REF O47, REF 108Kakoulidis I, REF 027Kale M, REF 299, REF 302, REF 311,REF 312, REF 315, REF 335,REF 343, REF 514Kalinke U, REF 053Kalkan A, REF 251Kalkanci A, REF 582Kalko E K V, REF 399Kalliaropoulos A, REF 377Kallies R, REF 0166Kallies S, REF 115Kallio Kokko H, REF 376Kalluvya S, REF 145Kalmer M, REF O156, REF 220Kalvatchev Z, REF 375Kamal A H M, REF 242Kamel N, REF 485Kandoussi N, REF 139Kang Q, REF 013Kann M, REF O57Kann N, REF O07Kantele A, REF 045Karakitsos P, REF 377Karalic D, REF 103, REF 502Karalti I, REF 393Karamichali E, REF 187Karaoglu T, REF 274, REF 304, REF 585Karatas A, REF 264Karbalayi M, REF 042Karlas A, REF 190Karlberg H, REF 241, REF 246Karlin D, REF 410Karlsson Hedestam G B, REF 221Karlsson K H, REF 288Kasang C, REF 145Kaserer M, REF O39Kasloff S, REF 011Kassab S, REF 549Kassab T, REF 121Kasztelewicz B, REF 495Kati S, REF 079Kato S, REF 048Kaufer B B, REF 082Kaukinen P, REF O08Kaveri S, REF 062Kawaguchi N, REF 420Kaya S, REF 310Kayman T, REF O161Kazem S, REF 351Kedracka Krok S, REF 228Keil G, REF O66Kele B, REF 427Kellam P, REF O106Keller M, REF 164, REF 197Kempkes B, REF 216Kenicer J, REF 579Kéranflec’h A, REF 320Keros T, REF 256, REF 333Kerr K, REF 513Kerviel A, REF 171Kesik Brodacka M, REF 057Khadadha M, REF 524Khadr N A A A, REF 146Khadr N, REF 140Khalili K, REF 576Khan G, REF 002Khan M J, REF 116Khanna M, REF 113Khanom A, REF 425Khon L K, REF 298Khosravi M, REF 149, REF 156Kiani N A, REF O140Kieser A, REF 001, REF O156Kilcher S, REF O58, REF O88Kim D Y, REF O53Kim S H, REF 305Kim Y H, REF 305Kindler E, REF O07, REF O126Kirchhoff F, P3_3Kirchhoff J, REF O66Kirchoff N, REF 124Kirdar S, REF 141, REF 527Kirkby N, REF 531Kirkwood C, REF O99Kiselev O, REF 051Kisilev V N, REF 215Kiveryte S, REF 463Kjerstadius T, REF 435Klaasse J, REF 422Klein J P, REF 423Klenk H D, REF 252Klepetko W, REF O150Klestova Z, REF 257, REF 297Klimova R, REF 530Klingl S, REF 097Klingström J, REF 068, REF O81Klinker H, REF 145Klopp C, REF 411Klose S M, REF 399Klumpp K, REF 036Kluver N, REF 491Knapp B, REF O51Knebel Mörsdorf D, REF 157Knechten H, REF 465Knezevic A, REF 502Kochs G, REF 001Kodze I, REF 389Koehler C, REF O16Koen G, REF O15, REF 127Kohdera U, REF 526Kohl A, REF 279Koishi A C, REF 110Kojzarova M, REF 060, REF 061Kokkinaki C, REF 078Kolehmainen P, REF 376Kolenc M, REF 588Kolesnikova L, K15Koller C, REF 124Kolodziejek J, REF 292, REF 326Komarova A, REF 030Komitova R, REF 490Komurian-pradel FKongola G, REF 145König Schuster M, REF 124Kontana A, REF 248Kontaxis G, REF 195Kontio M, REF O164Kooijman S, REF 351Koopmans M, REF O13, REF O123, REF O169,REF 281, REF 498Kootstra N, REF 409Kopecký J, REF 096Kopf M, REF O88Kopliku L, REF 295Kopp A, REF O40Koraka P, REF 272, REF 572Koriazova L, REF 048Korotkov A, REF 051, REF 052Kortekaas J, REF 158Korva M, REF 254Kosa C, REF 505Koskiniemi M, REF 376Kossyvakis A, REF 377Kotlyarov R, REF 051Kotta Loizou I, REF 194Koutsilieri E, REF 145Kovaleva A, REF 051Virologie, Vol. 17, supplément 2, septembre 2013S293

5 th European Congress of VirologyKowalinski E, REF 023Kragsbjerg E, REF 438Králík P, REF 439Krämer B, REF 451, REF 452Kraus B, REF 055Krausze J, REF O119Kravic Stevovic T, REF 086Kress A K, REF O156, REF 208, REF 220Krispenz U, REF 491Kroes L, REF 557Krokstad S, REF 383Kroneman A, REF 498Krüger N, REF O125, REF 244Krukle Z N, REF 542Krummenacher C, REF 159Krysko A, REF 555Krystofova J, REF 539Krzyzaniak M AKrzyzaniak M, REF 222Kubankova M, REF 327Kublik A, REF 425Kubo H, REF 526Kuchibhatla D, REF 410Kudelko M, REF O59Kudlich M, REF 550Kuhar U, REF 313Kuiken T, REF O25Kuivanen S, REF 044Kulemzin S, REF 021Kulibin A, REF 085Kulich P, REF 439Kumar A, REF 003, REF 045, REF 448Kumar B, REF 113Kumar P, REF 113Kumar Singh A, REF 223Kumar Snehi S, REF 404Kummerer B, REF O08Kundi M, REF O48Kunth Lehmann K, REF 452Kunz S, REF 154, REF 170, REF 231, REF 233,REF 234Kupriyanov V, REF 052Kurås Skram M, REF 383Kurilschikov A, REF O100, REF 581, REF 589Kurkela S, REF 398Kurolt I, REF 355Kurt M, REF 310Kurtti T, REF 405Kurz M, REF 123, REF 124Kushch A, REF 085, REF 530Kuslii A, REF 215Kuznetsova T, REF 391Kwa D, REF 370Kwang J, REF 430Kweder H, REF O52Kwok K, REF O59LLa Rocca A, REF O67La Rosa G, REF 378, REF 379Laaberki M H, REF 253Labernardière J L, REF 230Labetoulle M, REF O19LabEx EcofectLabois C, REF O71Labrousse F, REF O111Lachuer J, REF 007, REF O154Lacôte S, REF 300Lacroix Desmazes S, REF 062Ladner J, REF 291Lafage M, REF 571, REF O23Lafon M E, REF O23, REF 402, REF 571Lage Ferreira H, REF 316Lagoda O, REF 118Lagresle C, P1_4Laham Karam N, REF 218Laib Sampaio K, REF O112Lalis A, REF 414Lalle E, REF 019Lam W Y, REF 022, REF 083Lambrecht B, REF 276Lamchaheb F Z, REF 559Lamka J, REF 327Lamp B, REF 329Landini M P, REF 385Landolfo S, REF O105, REF 188Lanfredini S, REF O152Lang A, REF 130Langereis M, REF 033Langlois R, REF 077Lanzavecchia A, REF O73, REF 469Lapa D, REF 019, REF 363, REF 365Laperche S, REF 482Lappalainen M, REF 376, REF 448Lara E, REF O68Larcher T, REF O28Larrous F, REF 494Larson G, REF O95Lasala F, REF 245Lauber C, REF 351Laurent D, SP5_4Laurent S, REF 189Lavenir R, REF 494Lavezzo E, , REF O10, REF O29, REF O38,REF 183, REF 416Lavigne M, REF 095Lavillette D, REF 015, REF O87, REF 088,SP4_3Lawrence P, REF 069, REF O90, REF 240Lay SLazarevic I, REF 103, REF 496, REF 502Lázaro E, REF O142Lazear E, REF 159Lazzarotto T, REF 046, REF O55, REF 385Le Gall Reculé G, REF 354Le Grand R, REF O28Le Houerou C, REF 548Le Loc’h G, REF 328Le Mercier P, REF 507Le Pelletier A, REF O122Le Pendu J, REF 354Le Poder S, REF 330Le VB, REF 075Le Vern Y, REF O157Lebbink R J, REF O49, REF O153Lebreton F, REF O71Lechgar L, REF 472Lecollinet S, REF 276Lecomte E, REF 093Lecuit M, SP5_4Lee K K, REF 305Lee M H, REF 305Leeman C, REF 473Leen C, REF 477Leendertz F, REF O40Léger A, REF 093Legoff J, REF O149Legrand L, REF 336Legrand M C, REF 441Legras Lachuer C, REF 005, REF 253Lelli D, REF 275Lemey P, REF O122Lemke G, REF 168Lemmermeyer T, REF 329Lemon K, REF O103Lempp F, REF O84Leon A, REF 336Leone S, REF 063Leoni S, REF 568Leparc Goffart I, REF 018Lepault J, K10Lepiller Q, REF 003Lépine B, REF 129Lepri E, REF 038Lerma L, REF 010Leroux C, REF 065, REF 294Leroy E M, REF 0166Leruez M, SP1_1Lesnikowski Z J, REF 495Letienne J, REF 126Levanov L, REF 398Levillain E, REF 454Levy C, REF 014, REF 015, REF O52Lew E, REF 168Leyrat C, REF O33, REF O116Leyssen P, REF 128Li C, REF 284Li F, REF 061Li M Y, REF O59Li X, REF 436Liais E, REF 411Liang C H, REF 510Lichière J, REF 284Licoppe A, REF 255Lieber D, REF 160, REF 169Liebert U G, REF 071, REF 424Lijnen R, REF 075Liljeroos L, REF 222, REF 511Lilleri D, REF O73 , REF 469Lim F, REF 010Lim S M, K17_2Lim S, REF O39, REF 272, REF 572Lima A, REF O17Lima D, REF 364Lima M, REF 364, REF 418Lima S, REF 428Lin J H, REF 353Lin Y C, REF 353Lina B, REF O46, REF O71, REF 075,REF 087, REF O135, REF 171,REF 224, REF 520Lindberg A M, REF O44Linden A, REF 255, REF 268Lindig V, REF 460Lines L, REF O46Link B, REF O75Lipani F, REF 381Lipoldová M, REF 096Litwinska B, REF 528Liu H F, REF 353Liverani C, REF 474Ljunggren H G, REF O81Llorente F, REF 286Lluch J, REF 411S294Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyLobert P E, REF O172Lojkic I, REF 256Lollini P L, REF 0131Lomonte P, REF O19Long J, K13Longhi S, REF 205Lopes A M, REF 354Lopes Â, REF O17López Guerrero J A, REF 226López Labrador F X, REF O108López Martínez R, REF 114López N, REF O108Loregian A, REF 117, REF 188Losemann C, REF O125Losito S, REF 016Lotteau V, REF 069, REF O101, REF 129,REF O138, REF 173, REF 191Lötzerich M, REF O118, REF 509Louber J, REF 023Louvel A, REF 429Louwen R, REF O103Lövgren Bengtsson K, REF 288Loza Rubio E, REF 258, REF 293, REF 334,REF 387, REF 564Lozach P Y L, REF 148Lozach P Y, REF O78, REF 154Lu C, REF 538Luangsay S, REF 036Luca D, REF 486Lucas Hourani M, REF O12, REF 230Lúcio K, REF 428Luco S, REF 006Ludlow M, REF O54Luedtke N, REF O58Luehrs T, REF O119Luethi A, REF 516Luganini A, REF 188Luijendijk A, REF O103Luijt DS, REF 504Luis E, REF O31Lukashev A, REF O145, REF 366Lulf S, REF 063Lulla A, REF 221Luminos M, REF 525Lundgren O, REF O96Lundin A, REF O07Lundin S, REF O96Lundqvist Å, REF O41Lupan A, REF O12Luque J M, REF 245Lussy H, REF 292, REF 326Luteijn R, REF O49Lvov D, REF 415Ly T D, REF 437Lynn H, REF O56Lysholm F, REF O44MMa Lauer Y, REF 115, REF 123Ma X, REF O86Maaskant J, REF O147Maccario R, REF O148Maccioni E, REF 137Macé M, REF O14Madec F, REF O65Madeddu G, REF O22Madonna E, REF 378Madsen T V, REF 517Maeder M, SP5_3Maertens G, REF O121Maetzig T, REF O121Magaldi T G, REF O86Maggioni AMagro S, REF 421, REF 454, REF 475, REF 522Maida I, REF O22Maisner A, REF 244Maisonneuve P, REF O23Majewska A, REF 553Majinge C, REF 145Majzoub K, REF 091Mäkelä S, REF 024Makni H, REF 395Makni M, REF 395Makówka A, REF 528Malatesta P, REF 0131Malecki M, REF O158Malenovska H, REF 332, REF 455Maleševic M, REF 123Malim M H, REF O107, REF 190Malisiovas N, REF 377, REF 462Malm K, REF 438Malolina E, REF 085Maluquer DE Motes CMaly M, REF 419Mamaja B, REF 542Mamak N, REF 299, REF 302, REF 315Mameli G, REF O22, REF 567, REF 568,REF 576Manaresi E, REF 186Mancip J, REF 036Mancuso R, REF 573Manet E, REF O35Manetti R, REF 567, REF 568Mangenot S, REF 219Mangino G, REF 004, REF 063Mangroo C, REF 578Manjarrez M E, REF 064, REF 387Mann M C, REF O156, REF 208Mansfield K, REF 325Mansur D S, K4Mansuroglu Z, REF 025Mantelli B, REF 046, REF 050, REF O55Mantovani S, REF 0134, REF 546Manuguerra JMarangon S, REF 563Marcati G, REF 050, REF O55Marcato V, REF 025Marcel V, REF O79Marcello A, REF O32Marchadier E, REF 476Marche P, REF 130Marchi A, REF 290, REF 532Marconi P, REF O27Mardones G, REF 198Marechal P, REF 475Margry P, REF 454Maria S, REF 464Marianneau P, REF 253, REF 254, REF 261,REF 300Marie Edith L, REF 549Mariette J, REF 411Marinho V, REF 480Marinov M, REF 292Marland E, REF 473Maroli M, REF 290Marolleau B, REF 093Marquez Jurado S, REF O31Marschall M, REF O105, REF 207, REF 209Marseglia G L, REF 586Marseglia G, REF 532Marston D, REF 259, REF 325Martel C, REF 095Martello T, REF 277Martín B, REF 010Martin C, REF 198, REF 565Martin Latil S, REF 426, REF 453Martina B E EMartina B, REF O25, REF O39, REF 272,REF 572Martinelli M, REF 357, REF 529Martinello V, REF 012Martinez D, REF 075Martinez De La Puente J, REF O37Martinez Escribano J, REF 293Martinez Lara A, REF 387Martinez Maya J J, REF 387Martínez Rienda I, REF 499Martínez Romero C, REF O146Martini M, REF 298Martinot A, REF O172Martinovic J, REF 489Martró E, REF 104, REF O108Marusic P, REF 270Maruyama M, REF 420Marzi A, REF 094Marzook N B, REF O56Masalova O, REF 530Masi G, REF 006, REF O38, REF 183Masiero S, REF 050, REF O55Mason P W, REF 066Massardier J, REF O71Massari S, REF 117, REF 188Massawe I, REF 145Masson C, REF 468Masson J, REF 482Masson P, REF 507Massoud M, REF O71Mastromarino P, REF 125Mateo M, REF 069, REF 161Mathieu C, REF O92, REF 566Matrosovich M, REF O174, REF 249Matsen F A, REF O104Matthee S, REF 0166Matthieu C, REF 239Mattia E, REF 125Matur F, REF 264Matveev A, REF O09Matveev L, REF O09Mauduit F, REF 192Maufrais C, REF O167Mauricas M, REF 463Maurin G, REF O87Maurizio Z, REF 486Mauroy A, REF O69, REF 268, REF 431Mavilio F, REF 093Mavingui P, REF 088Mavromara P, REF 187, REF 194, REF 215May T, REF 144, REF 160Maylin S, REF O75Maynard Muet M, REF O109Mazaheri E, REF 147Mcbride R, REF O146, REF 229Mcculloch P, REF 477, REF 579Mcdonnell V, REF 471Virologie, Vol. 17, supplément 2, septembre 2013S295

5 th European Congress of VirologyMcelhinney L, REF 259Mcgarvey L, REF 076Mcgowan S, REF O67Mcgregor A, REF O26, REF 047Mcinerney G M, REF 221Mckeating J, REF O82Mckenna J P, REF 518Mckenzie C, REF O56Mcquaid S, REF O54Mcvey C, REF 217Mearns R, REF 325Medici M C, REF 070, REF 206Medits I, REF 165Meertens L, REF 168Meex C, REF 431Mehrle S, REF O84Mei A, REF O22, REF 567, REF 568Mei Z, SP5_5Meier R, REF O78Meignin C, REF 091Meijers J, REF O25Mekki Y, REF O71Meleddu R, REF 137Melén K, REF 243Melenevskaya E, REF 111Melhem N, REF 386Melidou A, REF 377, REF 462Melin P, REF 431Menculini G, REF 038Mendes Pereira Ardisson Araújo D, REF 210Méndez Tenorio A, REF 122Menezes C B, REF 298Meng F, REF 358, REF 359, REF 538Mengoli C, REF 558Menotti L, REF 0131Mentis A, REF 377Meral M, REF 582Mercer J, REF O58, REF O88Mercorelli B, REF 117, REF 188Merits A, REF O08, REF O28, REF 221Merrit J, REF 425Mesquita J, REF 580Messaoudi I, REF 094Messaoudi MMettenleiter T C, REF O63Metwally L, REF 380, REF 485Mey C, SP5_4Meyer B, REF 519Meyer T, REF 190Mezghani-khemakhem M, REF 395Micalessi I, REF 423, REF 521Michallet M, REF 562Michaud A, REF 492Michel Y, REF 480Mielke M, REF 408Miest T, REF 0129Miglio U, REF O152Mignion C, REF 189Miguet N, REF 089Mihaljevic S, REF 403Mikel P, REF 439Mikhailov M, REF 366Milanovic J, REF 403Milena F, REF 486Milenkovic M, REF 086Milhas S, REF 126Milia M G, REF 381Milic N, REF 103Militello V, REF 006, REF 077, REF O155Miller R, REF O40Milpied N, REF 549Mingorance L, SP4_2Minnaar R, REF O15Minner M, REF 013Miorin L, REF O32Mirand A, REF O24, REF 489Miranda M, REF 217Mirandola P, REF 070, REF 206Mirasoli M, REF 186Mirazimi A, REF O91, REF O93, REF 241,REF 246Mirazo S, REF 260Mirey G, REF O157Miszczak F, REF 523Mitreski S, REF 547Mitri C, REF 279Mlewa M, REF 145Mlynarczyk G, REF 552, REF 553, REF 554,REF 555Mocko K, REF 553Modrow S, REF 550Mohammed S, REF 459Mohd Jaafar F, REF 405Mohr C F, REF O156Mohty M, REF 548Moinet M, REF 262Mokhtari C, REF 476Molenkamp R, REF O98Molero F, REF 355Molloy J, P2_4Mompelat D, REF O87Moncorgé O, REF O107, REF 190Mondo M, REF 275Monot M, REF 294Moor D, REF 391Moore C, REF 396Moormann R, REF 158Moradpour D, REF 478Moraes Da Conceição T, REF 101Morais Ribeiro B, REF 210Morand P, REF 130Moravkova A, REF 060, REF 061Moraz M L, REF 154Morchikh M, REF 095Moreau P, REF 192Morelli X, REF 126Moreno A, REF 275, REF 286Moreno Docon A, REF 499Morere L, REF O111Morfin F, REF 520Morgado F D S, REF 210Mork A K, REF 162Mornex J F, REF 065Moroso M, REF 084, REF 232, REF 300Mortreux F, REF 005, REF 007, REF O154Mosca L, REF 125Moschella L, REF 385Moscona A, REF 566Moshe A, REF O60Motamedi N, REF O86Mothes W, REF 061Mou H, REF O83Moules V, REF O135Moullier P, REF 093Mourez B, REF 434Moutelikova R, REF 301, REF 332Moutousi A, REF 377Mrani S, REF 139, REF 367, REF 373,REF 493Mrvic T, REF 588Mukasheva E, REF 530Mulatti P, REF 563Mulders M N, REF 394Muller C P, REF O52Müller H, REF 321Müller M A, REF O07, REF 035, REF 115,REF 123, REF O125, REF 244,REF 399, REF 519Müller M, REF O40, REF O83, REF O126,REF 240Muller V, REF 116Muller Y, REF 097Münch R, REF 163Munderloh U, REF 405Munier Lehmann H, REF O12Munier S, REF O128Muñoz Almagro C, REF 499Muñoz J, REF O37Mura M, REF O22Muratore G, REF 117Muriaux D, REF 171Murk J L, REF 281Murovska M, REF 361, REF 542, REF 574Murri S, REF 254, REF 261Muscillo M, REF 378, REF 379Musetti C, REF O152Mutabagani M, REF 193Muth D, REF O07, REF 035, REF 123,REF O126, REF 519Müther N, REF 0132Mutinelli F, REF 563Muylkens B, REF 189Myers R, REF 394Myoung J, REF 094NNadai M, REF O34Naeth G, REF 465Naffakh N, REF O128Nagel C H, REF 099, REF 0132Nagovitsyna E, REF 488Naing Z, REF 209Nakouné E, REF O122Nakowitsch S, REF 124Nannetti G, REF 117Nanni P, REF 0131Napierkowski I, REF 142Nardis C, REF 125Nasir W, REF O95Nasri Zoghlami D, REF 501Nassereddin A, REF 531Naughtin M, REF 095Naumenko V, REF 085Nazaret N, REF 007, REF O154Nebioglu F, REF 511Nedellec S, REF O72Neef A, REF O58Neefjes J, REF O49Neffati H, REF 382Negre D, REF 014, REF 015Negredo A, REF 245Neipel F, REF 080Nemecek V, REF 419Nemeckova S, REF 539S296Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyNémeth J, REF 372Nemirov K, REF 069Nemni R, REF 573Nennesmo I, REF 570Nesherova M, REF 490Netesov S, REF 589Neumayr A, REF 355Neves E, REF O17Newsome T, REF O56Neyts J, REF 128Ng S, REF O136Ngai K L, REF 083Ngai K, REF 200Nguyen C, REF 433Nguyen D T, REF O103Nguyen Hoang A T, REF 068Ni Y, REF O84Nichol S, REF O93Nicolas V, REF 414Nicoletti L, REF 283Nicosia A M, REF 551Nielsen X C, REF 517Niemann I, REF 227Niemeyer D, REF 035Nieminen T, REF 376Niesters B, REF 498, REF 500, REF 537,REF 561Niesters H G M, REF 457, REF 458, REF 504,REF 534Nieto Torres J L, REF 004Nietsch L, REF 383Nikolic V, REF 086, REF 270Nishiyama S, REF 397Nitiema L, REF O95Nitsche A, REF 519Nizar H, REF 307Njouom R, SP5_2Nogueira F, REF 364Nogueira R M, REF 364Nogueira R, REF 418, REF 487Nøkleby H, REF 484Nolf M, REF 065Nolskog P, REF O41Nora Krukle Z, REF 361, REF 574Norberg P, REF O41Nordbø S A, REF 383, REF 479, REF 484Norder H, REF 391Nordgren J, REF O17, REF O95, REF O96Norja P, REF 483Nougarede N, REF 444Nouh H H, REF 146Nouh H, REF 140Novakova I, REF 235Novellino E, REF O110Novick D, REF O85Nowotny N, REF 286, REF 289, REF 292,REF 323, REF 326Nunes Duarte Dos Santos C, REF 443Nunes P, REF 364, REF 418, REF 487Nygårdas M, REF O113, REF 0132OO’neill J, REF 518Obrepalska Steplowska A, REF 073Ocal M, REF 282Odunze O, REF 131Oem J K, REF 305Ogliastro M, REF 029Oguzoglu C, REF 304Ohara M, REF 477Ohlmann T, REF 171Ohyama M, REF 526Okasha H A S, REF 140 , REF 146Oksuz B, REF 393Oktem M A, REF 264Olagolden S, REF 131Oleastro M, REF 580Oliveira B, REF 487Oliveira Resende R, REF 210Oliveira V C, REF 210Olsson G, REF O41Omar S, REF 076, REF 501Onland G, REF 491Openshaw P, K25Oppong S K, REF 399Oprisan G, REF 280Ora A, K14Orsi C F, REF 147Orsten S, REF 282Ortega E, REF 122Ortiz V, REF O108Osseman Q, REF O57Ostanello F, REF 271Osterhaus A D M E, REF O54 , REF O103Osterhaus A, REF O25, REF O83, REF O147REF 272, REF 440, REF 572,Österlund P, REF 024, REF 243Osterrieder N, REF 082Ostrovskaya O, REF 488Otelea D, REF 525Otero A, REF 499Ott A, REF 504Otth C, REF 198, REF 565Ottmann M, REF O46, REF 069, REF 171Otto V, REF 031, REF 097Ouachée M, REF O149Oude Munnink B B, REF O98, REF O165,REF 412, REF 529Ouermi D, REF O95Ouldali MOumakhir S, REF 493Överby A K, REF 105Owayed A, REF 524Ozan E, REF 247, REF 296, REF 308, REF 310Ozcan P, REF 393Ozdarendeli A, REF 251Ozgunluk I, REF 304Ozkul A, REF 274, REF 282, REF 304, REF 585Ozmen O, REF 299Özsu S, REF 141Öztürk F, REF 339Ozturk Y, REF 393Ozubek S, REF 251PPaavilainen H, REF O113 , REF 0132Pacenti M, REF O38, REF 050, REF O55,REF 277Pachler K, REF 289Pachot A, REF 180Padalko E, REF O170Padilla M, REF 298Pádua E, REF 143, REF 503Paeschke R, REF 132Page A, REF 069Pagni S, REF 277Paillart J C, REF O135Pain B, REF 317Pajkrt D, REF O160Palacios G, REF 291Palero F, REF O108Paller T, REF 303Pallier C, REF O76Palma Ocampo H K, REF 026Palmarini M, REF 319Palminha P, REF 392Palù G, REF 006, REF O10, REF 012, REF O27,REF O29, REF O34, REF O38, REF 046,REF 050, REF O55, REF 077, REF O114,REF 117, REF 0134, REF 136, REF O140,REF O155, REF 183, REF 188, REF 246,REF 277, REF 416, REF 424, EF 545,REF 556, REF 558, REF 563Paludan S, P3_1Palumbo M, REF O34Palus M, REF 096, REF O20Pamplona Mosimann A L, REF 443Panas M, REF 221Pancer K, REF 528Pancher M, REF O76Pandak N, REF 256Panet A, REF 590Paniagua M, REF O95Pannetier D, REF O89Pant G R, REF 494Panthu B, REF 171Papa A, REF 027, REF 248, REF 424Papo S, REF 267Paradowska E, REF 067, REF 495Paramithiotis EParanhos Baccala G, REF 084, REF 180,REF 232, REF 237Pardigon N, REF 276Pariani E, REF 529Parisi S G, REF 0134, REF 136, REF 558Park C, REF 269, REF 273, REF 417Park S C, REF 305Park S, REF 269, REF 273Parker J, REF 076Parolin C, REF 0134, REF 136, REF 137Parreira R, REF 143, REF 503Parvin R, REF 242Pas S, REF O147, REF 440, REF 498Pascucci M G, REF 385Pasi E M, REF 233Pasquato A, REF 154, REF 170, REF 233,REF 234Pastorino B, REF 128Pati D, REF 113Paulson J, REF O146Pavio N, REF O127, REF 330, REF 363Paya C, REF 056Payan C, REF 360, REF 441, REF 492Peake J, REF O67Peerson O, REF 347Peeters B, REF 521Peigue Lafeuille H, REF O24, REF 489Peijun R, SP5_4Pekmezci Z, REF 310Pelajo M, REF 487Pellegrinelli L, REF 384Pepin J F, REF 192Pépin M, REF 253, REF 300Perales C, REF O143Virologie, Vol. 17, supplément 2, septembre 2013S297

5 th European Congress of VirologyPérard J, REF O33Percario Z A, REF 063Percivalle E, REF O73, REF 469Perelle S, REF 426Perera Lecoin M, REF 168Peres E, REF 008Perez E, REF 499Perez Pastrana E, REF 291Pérez Ramírez E, REF 286Pernet O, REF 232Perrin Cocon L, REF O138, REF 173Perron H, REF 074, REF 130Perrone R, REF O34Persichini T, REF 063Pescatori L, REF O110Peserico A, REF 556Pessi A, REF 566Peta E, REF 006, REF 077, REF O155Petermann P, REF 157Peters B, REF 048Petit M A, REF 036Petit T, REF 230Petitjean J, REF 523Petrisli E, REF 385Petrovec M, REF 536, REF 588Petru S, REF 371Pettersson J, REF O41Petukhova N, REF 508Peyrefitte C N, REF 084, REF 232, REF 235,REF 237Pfaller C, REF 028Pfeffer S, REF 184Pfeifer K, REF 450Pfeiffer J, P2_1Pham Hung D’alexandry D’orengiani A L,REF 330Phan T G, REF 413Philip P, REF 002Phillips J, REF 459Phuc H L, REF O99Piasentin Alessio E, REF 546Piaserico S, REF 556Picard Meyer E, REF 262Piccirilli G, REF 385Piceghello A, REF 546Pichalova R, REF 172Picone O, REF O72, REF 489Picot V, SP5_1Picotti P, REF 511Piedade J, REF O17, REF 143, REF 503Piekarska K, REF 555Pietrek M, REF O119Pietschmann T, REF 177Pillet S, REF O46, REF 423, REF 442, REF 501,REF 544Pilorge L, REF 134Pinatel C, REF 005, REF 007, REF O154Piñero C, REF 010Pinhas C, REF 330Pinkas Kramarski R, REF 175, REF 213Pinto S, P2_4Piralla A, REF O148, REF 532, REF 586Piras E, REF 100Pizzuto M S, REF 011Plahhova T, REF 391Planas R, REF 104Planche L, REF 548Plantier J C, REF 074, REF 429, REF 470Platonov A, REF O37Plattet P, REF 149, REF 152, REF 156Plentz A, REF 550Pleschka S, REF 005Pliskova L, REF 447Ploy M C, REF O111Plucienniczak A, REF 057Plucienniczak G, REF 057Plumet S, REF 018Plyusnin A, REF 263Plyusnina A, REF 263Poddighe L, REF O22, REF 567Podsiadlowski L, REF O40Poenisch M, REF O140Poets C F, REF O159Pogka V, REF 377Pohlmann A, REF 099Pokorn M, REF 588Polat C, REF O161, REF 264Poljšak Prijatelj M, REF 588Pollock C, REF 518Poluda D, REF 355Pompei R, REF 100Pons J B, REF 254Popara M, REF O42Popov R, REF 375Popow Kraupp T, REF O48Poranen M, REF O113Porcherot M, REF O138, REF 173Porotto M, REF 566Portais J C, REF O101Portoukalian J, REF 130Pospieszny H, REF 073Postec E, REF 360, REF 441, REF 492Posthuma C C, REF O115, REF 265Potapchuk M, REF 051, REF 052Potempa J, REF 228Potier P, REF 088Pounder K, REF 259Power U, REF 076Pozzetto B, REF O46, REF 423, REF 442,REF 501, REF 544Prehaud C, REF O23, REF 571Preiser W, REF 145Prenger AD, REF 504Primache V, REF 384Priniski L, REF 020Prioteasa L F, REF 280Prizan Ravid A, REF 218Procter D, REF O56Prodelalova J, REF 301, REF 331, REF 332Proietti A, REF 381Pronk M, REF 440Pronost S, REF 336, REF 344Prosenc Trilar K, REF 368, REF 369Protzer U, REF 098Prpic J, REF 256, REF 333Przybylski M, REF 552, REF 553, REF 554,REF 555Puchhammer Stöckl E, REF O150, REF 540Pugliatti M, REF 568Pumpens P, REF O57Punyadarsaniy D, REF 358, REF 359Pusztai R, REF 372Putkuri N, REF 398Pyrc K, REF 228Python S, REF 002Pythoud C, REF 231QQesari M, REF O27Qihan L, SP5_5Qiu Y, REF O102Quartu S, REF 019Quéguiner S, REF 314Quer J, REF O143Querat G, REF 126, REF 128, REF 202Quideau S, REF 138Quint K, REF O152RRaafat El Deeb, D, REF 461Rabaa M, REF O99Rabe B, REF O57Rabezanahary H, SP5_3Rabilloud J, REF 092Radeke M J, REF 028Radreau P, REF 191Radu C, REF 371Radzimanowski J, REF 171, REF 219Rafii El Idrissi Benhnia M, REF 048Rahamat Langendoen JC, REF 498, REF 504Rahn E, REF 157Raj S K, REF O83, REF 265, REF 404Raj V S, K21_bRajatonirina S, SP5_2Rajput R, REF 113Ramalingam S, REF 477Ramanan V, REF O136Rambaut A, REF O122,Ramdasi R, REF 168Ramia S, REF 386Ramière C, REF O101, REF O109, REF O138,REF 173, REF 191, REF 543Ramírez De Arellano E, REF 245Ramírez Salinas G, REF 114Ramos Da Palma J, REF 234Ramos N, REF 260Randriamarotia M, SP5_3Rang A, REF 001Raoul H, REF 129Raquin V, REF 088Rasa S, REF 542, REF 574Rasbach A, REF 0130, REF 163Raschbichler V, REF 169Rasschaert D, REF 189Ratineau C, REF 454Ratinier M, REF 319Ravanini P, REF 551Ravin N, REF 051, REF 052Rawlinson W D, REF 207, REF 209Rawlinson W, REF O111Rayne F, REF 549Rebers S, REF O98Rebollo Polo B, REF 275Recordon Pinson P, REF 493Redlberger Fritz M, REF O48Regimbeau J M, REF 178Regla Navas J A, REF 004Reichel A, REF 227Reid S P, REF 069Reimer J M, REF 288Reimerink J, REF O169Reiter P, REF 292Relmy A, REF 295Remoli M E, REF 283, REF 290Remy S, REF O157S298Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyRen HRen P, SP5_4, SP5_5Ren X, REF 358, REF 359Renault T, REF 192Renesto P, REF 089Rennick L J, REF O54Repousis S, REF 473Resa C, REF 522, REF 523Reshetnjak I, REF 391Ressing M, REF O49, REF O153Rethwilm A, REF 145Rettig I, REF 481Reusken C, REF O123, REF 0166, REF O169,REF 281Reuter N, REF 031, REF 094Reutterer B, REF 124Revello M G, REF O73, REF 469Reyes J, REF 056Reyes Leyva J R, REF 026Reynard O, REF 069, REF 235Reynard S, REF O89Reynaud J M, REF 239Reznik V, REF O145Ribeiro C, REF 392Ribes Fernandez J M, REF 584Ricciardi Jorge T, REF 443Rice C M, REF O136, REF O143Richard E, REF 336Richner B, SP5_4Richner J M, REF 288Richter E, REF 212Richter S N, REF O34, REF O114Richter U, REF O119Ricketts A, REF 459Ricklin M, REF O64Rico O, REF 258Riezebos Brilman A, REF 500, REF 537,REF 561Rihtaric D, REF 303Rimbert M, REF 548Rimmelzwaan G F, REF 075Ringkjøbing Jensen M, REF O116Riordan B O, REF O163Ristic B, REF 086Riteau B, REF 075Rith S, REF 129Rittà M, REF 546Ritter C, REF O119Ritter J, REF 543Rizzoli A P, REF 267Rizzoli A, REF O37, REF 291Robardet E, REF 262Roche D, REF 191Roche S, REF O62Roda A, REF 186Rodighiero I, REF 070, REF 206Rodrigues C, REF 364Rodrigues R, REF 084, REF 237Rodrigues Zanello P, REF 110Rodríguez A, REF 064Rodriguez J, REF O101Rodriguez M, REF 275Rodriguez R, REF O45, REF O126Roelofs J, REF O25Roga S, REF 574Rogee S, REF O127Roic B, REF 256, REF 333Roivainen M, REF O144, REF 352Roiz D, REF O37, REF 291Rojas Anaya E, REF 258, REF 293,REF 334, REF 387,REF 564Roland Marquet R, REF O135Rolland J P, REF O135Rolland T, REF O128Romanenkova N, REF O162Romanik A, REF 057Romanovskaya A, REF O113Romeo G, REF 004, REF 063Romero M P, REF 499Romey A, REF 295Romi R, REF 283Rönkkö E, REF 243Roose J, REF 572Roossinck M, P2_2Root M, REF O26 , REF 047Roque Afonso A M, REF O14, REF 362,REF 476Roque C, REF 392Roques P, REF O28Rosa Calatrava M, REF O79, REF 087,REF O135, REF 224Rosa R, REF O37Rosanna Mel G C, REF 563Rosas Murrieta N H, REF 026Rose J K, K10Rose N, REF O65, REF 320Rosec S, REF 360, REF 441, REF 492Rosenberg A, REF 476Rota P, REF 394Rotanet Italy Study Group , REF O97Roth A, REF O41Rotmans J, REF 557Rottier P, REF O83, REF O146, REF 151,REF 158Roula D, REF 499Roulin P S, REF O118Rousseau A, REF O19Roux L, REF O139Rovida F, REF 586Röyttä M, REF 0132Rozaeva N, REF O162Rozen Gagnon K, REF O141Rubinstein M, REF O85Rückert J, REF O119Rudiguoz R C, REF O71Rudnicka K, REF 067Rudolf I, REF 292, REF 323Rueckner A, REF O16Ruediger A T, REF 174Ruggeri F M, REF O97, REF 271Ruggiero T, REF 381Ruggli N, REF 002Ruigrok R, REF O116Ruiz S, REF O37, REF 291Ruml T, REF 172Russi J, REF 260Russier M, REF O89Russo L, REF 153Ružek D, REF O20, REF O42, REF 096,REF 289, REF 349Ryabova L A, REF 197Rynans S, REF 552, REF 553, REF 554,REF 555SSaad Abd El Hamid A E D, REF 380Saade F, REF O111Saba E, REF 562Saczynska V, REF 057Sadeghi M, REF 541Sadoul R, K11Saegerman C, REF 268Saelens X, REF 108Sahli R, REF O171, REF 478Saiz J C, REF 293, REF 564Sajen O, REF 388Sakellariou P, REF 194Sakuntabhai ASala A, REF 147Salame Jaffa M, REF 386Salasc F, REF 029Salát J, REF 096Salata C, REF 246, REF 563Saldan A, REF 545, REF 556Saleh M, P3_2Salemi M, REF 353Salgado Miranda C, REF 334Saliba F, REF 362Sallusto F, REF O73Salmona M, REF O75Saloum K, REF 480Saltik H S, REF 335Saludes V, REF 104Saluzzo J F, REF 129Salvatierra K, REF O108Samdal H H, REF 479, REF 484Samokhvalov E, REF 415Sampaio S, REF 116, REF 364Samuel C E, REF 028Samuel D, REF 362Sanavia T, REF O10, REF O29Sanchez David R Y, REF 030Sanchez Seco M P, REF 291Sanchez Seco M, REF O37Sander M, REF 452Sanders N N, REF 039, REF 040Sanderson S, REF 513Sandhu M, REF O106Sandini E, REF 556Sandrin VSandström E, REF 570Sane J, REF O13Sanewski A, REF 519Santiago J, REF 298Santolini J, REF 453Santos Lopez G, REF 026São José Nascimento M, REF 580Sarah J B, REF 511Sarasini A, REF 586Sardet A, REF O172Sargueil B, REF 181Sariyer I K, REF 576Sarradin P, REF 300Sarraseca J, REF 275Sasnauskas K, REF 389Sathirapongsasuti N, REF 414Sattin A, REF 558Sattler M, REF 216Sauer A K, REF 510Saulnier A, REF 314, REF 444Sauter K S, REF 324Virologie, Vol. 17, supplément 2, septembre 2013S299

5 th European Congress of VirologySauvage F, REF 254Savary J, REF 362Savinova I, REF 257Saviranta P, REF 506Savolainen Kopra C, REF O144, REF 352Sawtell N, REF O19Saxena L, REF 113Sayed Ahmed W, REF 485Scaggiante R, REF 558Scarpa M, REF O29Schablowsky C, REF 211Schambach A, REF O121Scheinost O, REF O51Scheller C, REF 145Scherbeijn S M J, REF 422Scherer M, REF 031, REF 097Schierhorn K L, REF 005Schildgen O, REF O158, REF 445Schildgen V, REF O158, REF 445Schilling E M, REF 031Schipper M, REF O147Schlegel M, REF 0166Schmidt A M, REF 001Schmidt Chanasit J, REF 0166, REF 287Schmidt F I, REF O88Schmidt K, REF 164Schmidt M S, REF 080Schmidtke M, REF 132Schmitt C, K19Schmukler E, REF 175, REF 213Schneider C, REF O88Schneider G, REF 410Schneider M, REF 074Schneider Schaulies J, REF 156, REF 163Schnierle B, REF 055Schnitzler P, REF 533Schnuriger A, REF 480Schoehn G, K11Scholtès C, REF O109, REF O138, REF 173,REF 191, REF 382, REF 543Scholz B, REF 094Schönefeld K, REF 071Schreiner W, REF O51Schudt G, K15Schuette T, REF O159Schughart K, REF 072Schultz R, REF O107Schulz T, REF 079, REF O119,REF O121, K1Schutten M, REF 422Schuurman A C, REF O160Schuurman T, REF 534Schvoerer E, REF 144, REF 446Schwaiger J, REF O51Schwaller L, REF O87Schwalm F, REF 252Schwartz I, REF 300Schwartz O, REF 168Schwegmann Wessels C, REF O137, REF 359,REF 510Schwegmann Weβels C, REF 123, REF 174Schweinzer K, REF 481Schweizer M, REF 324Schwendener R, REF O27Scigalkova I, REF 332Scordel C, REF 575Scott G M, REF 209Scrima N, REF O62Scull M A, REF O136Seang S, REF 402Seaux L, REF O170Seddighzadeh S, REF 042Sediri H, REF 252Segalés J, REF O18Segarra A, REF 192Seidah N G, REF 170Sekiguchi J I, REF 526Sekulin K, REF 292Selisko B, REF 126Semary W M, REF 146Semary W, REF 140Sepp R, REF 427Serra C, REF O22, REF 567, REF 568Serra V, REF 063Sertoz R, REF 390Servant Delmas A, REF O71Servat A, REF 262Severini F, REF 283Sevik M, REF 337, REF 338, REF 339Sevvana M, REF 097Sewald X, REF 061Sgarabotto D, REF 545Shahwan K, REF O137Shai B, REF 175, REF 213Shalaby M, REF 456Shami A, REF 193Shams Ara M, REF 042Shanshan X, SP5_5Sharma S, REF O17, REF O95Sharp C, REF 397, REF 405Shaw J, REF 477Shchelkanov M, REF 415, REF 530Shchetinin A, REF 415Shedden D, REF 535Sheldon J, REF O143Sherman W, REF 410Shevtsova A, REF O139Shields M, REF 076Shin D L, REF 072Shiomi M, REF 526Shishkov S, REF 375Shishkova K, REF 375Shomron N, REF 346Shpacovitch V, REF O174Shumilina E, REF O145Shurtleff A, REF 069Sias C, REF 365Sidira P, REF 248Sidoti F, REF 546Siewert S, REF O137Siljic M, REF 086, REF 270Silva Da Costa L, REF 101Silvestre P, REF 520Simeone S, REF 546Simmonds P, REF 397Simoes J, REF 364Simon B, REF 540Simon F, REF O75, REF O149Simon G, REF O65, REF 314Simoncelli B, REF 381Simoncello I, REF 046Simone Schmorak M B, REF 563Simonovic J, REF 103Simpore J, REF O95Simsek A, REF 049, REF 312, REF 315, REF 514Simutis K, REF 389Sinesi F, REF 546Sinigaglia A, REF 006, REF O10, REF O29,REF O38, REF 077, REF O155,REF 183Sinzger C, REF 160Sironen T, REF 254, REF 267, REF 398Siu L, REF O59Six E, P1_4Skern T, REF 119, REF 195Sliva K, REF 055Smilansky Z, REF 213Smirnova T D, REF 203Smismans A, REF 521Smith G L, K4Smits S L, K21_BSmits S, REF O83Smorodinsky N I, REF 175Smura T, REF O144, REF 352Snijder E J, REF O115, REF 128Snijder E, REF 199, REF 265Snijders F, REF O98Snyder R O, REF 093Soares A, REF 487Šoba B, REF 588Sobh M, REF 562Sobo K, REF O43Sodeik B, REF 099, REF 0132Söderlund Venermo M, REF 413, REF 436,REF 448, REF 483,REF 541Soehle S, REF 039Sokolov S, REF 587, REF 589Sokolova N, REF 120Sol C, REF O98Soler E, REF 104Sommer C, REF O140Soria M E, REF O143Soriguer R C, REF 286Soriguer R, REF O37Sotgiu S, REF 568Souii A, REF 181, REF 182Soukka T, REF 506Soulet D, REF 444Sourvinos G, REF 034, REF 078, REF 176Sousa C, REF 143, REF 364, REF 418,REF 503Soutschek E, REF 451, REF 452, REF 491Sova T, REF 118Sowinskeya I, REF O57Sözen M, REF 264Spagnol L, REF O27Span B, REF 561Spandidos D, REF 078, REF 176Spanevello F, REF 0134Sparrer K, REF O11Spel L, REF 158Spiegelberg L, REF 249Spinu C, REF 388Sprikkelman A B, REF O160Squarzon L, REF O38, REF 046, REF 050,REF O55Sriranganathan V, REF 479Stainton K, P2_4Stajkovic N, REF 270Stamenkovic G, REF 270Stamminger T, REF 031, REF O80, REF 094,REF 097, REF 209, REF 227Stanojevic M, REF 086, REF 270S300Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyStanway G, REF 009, REF 193Stapleford K A, K19Stapleford K, REF O141Stastna Markova M, REF 539Stauffer S, REF 511Stavrakaki E, REF 034Stech J, REF O63, REF 359Stech O, REF O63Steinbach F, REF O67, REF O168,REF 513Steinberger J, REF 119Steinlin J, REF 516Steinmann A E, REF 234Stela G, REF 371Stepanova L, REF 051, REF 052Stepanova V, REF 447Stephens J, REF 450Sterk P J, REF O160Sterkers G, REF O149Stertman L, REF 288Stevanovic G, REF 496Stevens V, REF 494Steyer A, REF 588Stiasny K, REF 043, REF O51, REF 165Stich A, REF 145Sticht H, REF 001, REF 031, REF 097,REF 207, REF 227Stittelaar K, REF O25Stoeck I K, REF O140Stokke A, REF 383Stoll Keller F, REF 449Stoltz M, REF O81Storm R, REF 166Storosum J G, REF 407Strassl R, REF O150Strating J, REF O117Stratta P, REF O152Strebel P, REF 394Streefkerk R, REF 440Stricker R, REF 072Strle F, REF 588Strobel S, REF 208Stronin O, REF O09Strottmann D, REF 066Struck F, REF 451Studzinska M, REF 067, REF 495Stump J, REF 031, REF 097Stürzl M, REF 094Su M, REF 123Subissi L, REF O115Suhrbier A, REF O28Suliman T, REF 035Sultanova A, REF 361Sültmann H, REF O84Summerfield A, REF 002Sumner R P, K4Sundquist W, K11Sundstrom K, REF 068, REF O81Suomalainen M, REF O58Susi P, K14Suski P, REF 067, REF 495Sutaria R, REF O82Suter R, REF 002Sutter G, REF O39, REF 053Svensson L, REF O95, REF O96Svensson M, REF 068Svirtlih N, REF 103Svorcan P, REF 103Swanson C M, REF 190Szabo E, REF 505Szelechowski M, REF 575Szewczyk B, REF 057Szlavik J, REF 505TTabarés E, REF 010, REF 226Tabarrini O, REF 117, REF 188Tabrizi R, REF 549Tadesse E, REF 380Taffon S, REF 378Tagajdid M R, REF 139, REF 367,REF 373Tagajdid R, REF 493Tagliapietra A V, REF 267Takakura K I, REF 526Talbi C, REF O122Talker A, REF 566Tallo T, REF 391Tamosiunas P, REF 389Tan Y J, REF 241Tangy F, REF O12, REF 030, REF 230Tanner L B, REF O118Tanzi E, REF 357Tanzi E, REF 529Tapia Perez G, REF 564Tapparel C, REF O43Tarantola A, SP5_2Tarbouriech N, REF O116Tardy V, REF O71Tarkka E, REF 413Tas A, REF 128Tashuta V, REF 297Tassin M, REF 489Tauriainen S, REF 376Tchendjou P, SP5_2Tefanova V, REF 391Teifke J P, REF O63Telatin V, REF 046Telenti A, REF 478Telles J, SP5_1Tellez Castillo C J, REF 584Tellez Castillo C, REF 460Temchura V, REF O174Templeton K, REF 579Tennert K, REF 0133Tenoever B, REF 077Tenorio A, REF O37, REF 245, REF 276,REF 291, REF 355Terhes G, REF 427Terrien E, REF O23Terrier O, REF O79, REF 087, REF 224Terzian C, REF 319, REF 406Terzic S, REF 333Tesarík R, REF 439Testa A, REF 365Testoni B, REF 036Tetard M, REF O78Textoris J, REF O79Tezcan S, REF 274Thabti I, REF 121Thapliyal R, REF 518Theiss J, REF 204Thelohan M, REF 360Thenoz M, REF 007, REF O154Thevenin M, REF 482Thibault V, REF O76, REF 482Thiberge J M, REF O167Thiel H J, REF 123, REF 329Thiel V, REF O07, REF O45, REF O83, REF 115,REF O126Thier K, REF 157Thimmulappa R, REF 069Thiry D, REF 268Thiry E, REF O69, REF 268, REF 431Thomas B, REF 229Thomas D, REF O135Thomas I, REF 268Thomas M, REF 209Thompson C, REF O99Thompson Da Poian A, REF 101Thumann S, REF 216Thuong T C, REF O99Tikunov A, REF O100, REF 581, REF 587,REF 589Tikunova N, REF O09, REF O100, REF 581,REF 587, REF 589Tillmann R L, REF O158, REF 445Tinto D, REF 545, REF 556Tischler N, REF O81, REF 148Todd D, REF O18Togawa M, REF 526Tokgoz S, REF 311Toma L, REF 283Tomao P, REF 563Tomaszewska A, REF 553, REF 554, REF 555Tommasino M, REF 004Tonnerre P, REF O151Toplak I, REF 303Toppinen M, REF 483Toppo S, REF O38, REF 416Tordo N, REF 254, REF 261Tornesello M L, REF 016Torres M, REF 010Torta F T, REF O118Touch S, SP5_4Touil N, REF 373Touraine J L, REF 130Tourbiez D, REF 192Touret F, REF 406Tournaire B, REF 093Tournamille J F, REF 547Tournus C, REF 523Touzot F, P1_4Tovilovic G, REF 086Tozzi P, REF 038Trabaud M A, REF O109Trabelsi A, REF 434Trajkovic V, REF 086Trallero G, REF 499Tramontano E, REF 137, REF O110Tran A, REF 360, REF 441, REF 492Tran F, REF 088Tran G M K, REF 017, REF 058, REF 059Tran Van V, REF 088Trancard S, REF 192Trapp Fragnet L, REF O157Travers A, REF 192Traversier A, REF 224Trbusek J, REF 447Trevisan M, REF O10, REF 012, REF 077,REF O155, REF 183Trimoulet P, REF 468Trindade G, REF 428Trkola A, K9Virologie, Vol. 17, supplément 2, septembre 2013S301

5 th European Congress of VirologyTrkov M, REF 588Trotsenko O, REF O145, REF 366Trybala E, REF O07Tsatsaris A, REF 248Tsekov I, REF 375Tseng Y T, REF 179Tsergouli K, REF 027Tsiodras S, REF 377Tsioka K, REF 248Tsitoura E, REF 078, REF 187Tsouchnikas G, REF 043Tsui S K, REF 022, REF 083Tsybalova L, REF 051, REF 052Tuan H M, REF O99Tufiño Loza C, REF 258, REF 387Tumentsev A, REF O100, REF 581Turhan A, REF 390Turmagambetova A, REF 037, REF 120Tursumbetov M, REF 514Tuyet P T N, REF O99Tykalová H, REF O42, REF 196Tynell J, REF 243Tyulenev Y, REF 085UÜberla K, REF O174Uda S, REF 100Uecker R, REF O120, REF 212Ugurlu SE, REF 534Uhlenbruck S, REF O66Uhlenhaut C, REF 250Ujhelyi E, REF 505Ulbert S, REF O38, REF 039, REF 040, REF 288,REF 424Ulbrich P, REF 172Uleri E, REF O22, REF 567, REF 576Ulmer A, REF 145Ulrich R G, REF 0166Upadhyay A, REF 105Urban S, REF O84Uršic T, REF 536, REF 588Us D, REF 282Usami Y, REF 089Uzun S, REF 451VVabret A, REF 330, REF 434, REF 523Vaccari G, REF 004Vacher L, REF 129Vaheri A, REF 044, REF 398Vahlenkamp T W, REF O16, REF 242, REF 321Vaillant C, REF 095Vainio K, REF 484Vainio S, REF 432Väisänen E, REF 413Valentina P, SP5_1Valette M, REF O46Valiente Moro C, REF 088Valinciute A, REF 463Vallejo Ruiz V, REF 026Vallet S, REF 492Vallino M, REF O105Valyi Nagy I, REF 505Van Aalderen W M, REF O160Van Amerongen G, REF O54, REF O103Van Belle S, REF O43Van Beveren N J, REF 407Van Binnendijk R, REF O13Van Boheemen S, REF 572Van De Vijver D, REF 572Van Den Brand J, REF O25Van Den Hoogen B, REF 265Van Der Avoort H, REF 498Van Der Blij De Brouwer C, REF 557Van Der Eijk A, REF O147, REF 281, REF 440Van Der Hoek L, REF O98, REF O165, REF 399,REF 407, REF 409, REF 412,REF 529Van Der Linden L, REF O117Van Der Meijden E, REF 185, REF 351,REF 557Van Der Poel W H MVan Der Reijden W, REF 433Van Der Schaaf S, REF 537Van Der Schee M G, REF O160Van Der Werf S, REF 346Van Dijk K, REF 370Van Doorn R, SP5_4Van Dort K A, REF 409Van Eijk H, REF O15Van Esbroeck M, REF 355Van Gorp E, REF O25Van Haagen C, REF O25Van Hemert F, REF O98Van Hemert M J, REF 128, REF 284Van Hemert M, REF 199Van Hooff S, REF O153Van Kampen A H, REF 407Van Kuppeveld F J, REF O118Van Kuppeveld F, REF 033, REF O117Van Leer Buter C, REF 537, REF 561Van Leeuwen W, REF O49Van Loon A M, REF 457, REF 458Van Meensel B, REF 521Van Nieuwkoop S, REF 265Van Raaij M, REF 223Van Rijckevorsel G, REF 370Van Rooijen M, REF 370Van Schaik B D, REF 407Van Tongeren H, REF O117Van Wittenberghe L, REF 093Vancova M, REF O20Vandenbogaert M, REF O167Vanhems P, REF 562Vanhorssen J, REF 130Vapalahti O, REF 044, REF 398Varbanov M, REF 121Varghese F, REF O08Varjak M, REF 221Varsani A, REF O18Vasickova P, REF 327, REF 439Vassena A, REF 205Vassilaki N, REF 194Vassileva Pencheva R, REF 112Vatansever Z, REF 251Vauloup Fellous C, REF 489Vauloup Fellous C, REF O72Vautherot D, REF O157Vautherot J F, REF O157, REF 317Vavrušková Z, REF 196Vazquez A, REF O37, REF 291Vazquez Gonzalez A, REF 276Veiga E, REF 580Veiga J, REF O17Veijo H, REF 0132Veitinger S, REF 001Veits J, REF O63Vejrazkova E, REF 447Velay A, REF 144, REF 446Venard V, REF 446Vene S, REF O41Venteo A, REF 275Verburgh R J, REF O54Verheije H, REF 151Verhoeyen E, K2Verhoeven J, REF 412Verhoeyen E, REF 014, REF 015, REF O52Vernick K, REF 279Vernin C, REF 005, REF 007,REF O154Veronesi E, REF 319Veronica E, REF 371Versluis J, REF O147Vetter B, REF 212Veyret R, REF 454Vidalain P O, REF O12, REF 230Videira E Castro S, REF O17, REF 143,REF 503Vieira R, REF 428Vieyres G, REF 177Vignoles M, REF 522Vignuzzi M, REF O141, REF 346, REF 347Vigouroux S, REF 549Vilhelmova N, REF 138Vilks A, REF 542Villar M, REF O42Villard O, REF 466Villaudy J, REF 008Villegas P, REF 122Vinagre E, REF 392Vinh H, REF O99Vinje J, K28Visan A, REF 525Vitoriano Freitas D, REF 210Vitour D, REF O68, REF 319Vivès R, REF O92Vladimir G, REF 374Vlahava V M, REF 034Vlasova M, REF 488Vogel B, REF 0133Vogelzang Van Trierum S E, REF 075Voigt S, REF O36Vojtíšková J, REF 096Volchkov V, REF 069, REF O90, REF 235,REF 236, REF 240,Volchkova V A, REF 069Volchkova V, REF 236, REF 240Volle R, REF O24Volz A, REF O39, REF 053Von Bonin J, REF 250Von Brunn A, REF 115, REF 123Von Brunn B, REF 123Von Einem J, REF O36Von Itzstein M, SP5_5Von Mering C, REF O78Vonderstein K, REF 105Vonesch N, REF 563Vorac J, REF 236Vossen A, REF 557Vourc’h G, REF 261Vrati S, REF 062Vu Hai V, REF 237Vu Tra My P, REF O99Vujosevic M, REF 270S302Virologie, Vol. 17, supplément 2, septembre 2013

5 th European Congress of VirologyWWagelaar M, REF 504Wagner S, REF 209Wågø A G, REF 383Walker E, REF 225, REF 229Wallace P S, REF 457, REF 458Wallace P, REF 459Walz N, REF 408Wan C H, REF 341Wang C T, REF 179Wang E, REF O53, REF 269, REF 273Wang I H, REF O58Wang J, REF 340, REF 538Wang P G, REF O59Wang R, REF 340, REF 351Wang S M, REF 179Wang Y T, REF 341Wang Y, REF 448Ward C, REF O107Waris M, REF 506Wash R, REF O106Wasmer S, REF 516Waters A, REF O163Watier L, REF O122Watson M, REF O42, REF 513Wattel E, REF 005, REF 007, REF O154Weaver S, REF O53Webb E, REF 450Webel R, REF 207, REF 209Weber F, REF 001, REF 035, REF O93, REF 105,REF O124, REF 249Weber M, REF 001, REF O93Weber S, REF O63Weidmann M, REF 456Weidner Glunde M, REF 079, REF O119Weigel C, REF 436Weis Arns C, REF 041Weis M, REF 244Weisbach H, REF O36, REF 211Weisblum Y, REF 590Weisheit S, REF O42Weiss Arns C, REF 316Weissbrich B, REF 145, REF 515Weissenhorn W, REF 089, REF 171, REF 219Wellinghausen N, REF 399Welsch J, REF 566Welsch K, REF 177Welsh J, REF 092Wendling M J, REF 449Wenk M R, REF O118Wennerberg K, REF O08Wernery U, REF 326Wessels U, REF O63Westerhuis B, REF 127Wevering N, REF 519Whitbeck J, REF 159White J, REF 579Wicht O, REF 151Wiebusch L, REF O36, REF O120, REF 211,REF 212Wieczorek P, REF 073Wiertz E, REF O49, REF O153Wiesmann F, REF 465Wight D J, REF 218Wigzell H, REF O96Wijma JJ, REF 534Wildenbeest J G, REF O160Wilkie G, REF 513Willems L, REF O153Willenbring R, REF 161Williams Ç, REF 009Winkler M, REF O36Winter C, REF 155, REF 162Wirth D, REF 160Wisniewska Ligier M, REF 495Wo J DR, REF 350Wodrich H, REF O57, REF 549Wolf D, REF 590Wolff N, REF O23Wolff T, REF 005Wolthers K, REF O15, REF 127, REF O160,REF 498Wong C H, REF 510Wong F Y K, REF 494Wood C, REF 535Woskobojnik I, REF 132Wozniakowska Gesicka T, REF 495Wrzesinska B, REF 073Wu C Y, REF 510Wu H S, REF 353Wu N H, REF 538Wu Z, REF 538Wunderink H, REF 557Würdinger M, REF 550Wychowski C, REF 178XXavier J, REF 095Xenarios I, REF 507Xiang Y, REF 048Xinsheng C, SP5_5YYabukarski F, REF O116Yadin H, REF 175Yagel S, REF 590Yakovenko M, REF O162Yamamoto S P, REF 526Yamamura A, REF 428Yanagihara R, REF 414Yang D, REF O102Yang S, REF 340Yang W, REF 358, REF 359, REF 538Yaniv R, REF 175Yapici O, REF 049, REF 299, REF 302, REF 312,REF 315, REF 318, REF 514Yavru S, REF 049, REF 299, REF 302, REF 311,REF 312, REF 315, REF 318, REF 342,REF 343, REF 514Ye X, REF O102Yeniaras A, REF 583Yenigun A, REF 527Yeung A C, REF 083Yi G, SP5_4Yildirim Y, REF 304Yin H, REF 340Ylihärsilä M, REF 506Ylinen T, REF 506Yordanova O, REF 362Younis S, REF 485Youssef Abo El Kheir N, REF 461Yu H J, REF 450Yüksel S, REF O54, REF 269, REF 273Yun T, REF 589Yver M, REF O135ZZaatari M, REF 386Zacher T, REF 451, REF 452Zaitceva I, REF 037, REF 120Zajonc D, REF 048Zakay Rones Z, REF 590Zaki A M, REF 249Zaki A, REF O83Zakovcik V, REF 327Zaky A, REF O17Zangheri M, REF 186Zangrillo M S, REF 004Zanzottera M, REF 573Zappa A, REF 357Zavattaro E, REF O152Zawilinska B, REF 495Zdzalik M, REF 228Zecca M, REF O148Zeichhardt H, REF 460Zevenhoven J, REF O115Zhang H M, REF O102Zhang J, REF 340Zhirakovskaya E, REF 589Zhong H, SP5_4Zhou H, K13Zhu H, SP5_4Ziegler U, REF 287Zielecki F, REF 249Zientara S, REF O68, REF 295Zimmer G, REF O64Zingali R, REF 285Ziv N, REF 175Zlatkovic J, REF 043, REF 054Zonta W, REF O69Zoppi F, REF 234Zou P, REF 216Zoulim F, REF 036, REF O109, REF 144Zoz O, REF 297Zrein M, REF 562Zuercher S, REF 516Zurbriggen A, REF 149, REF 152, REF 156Zurkova K, REF 539Zuwala K, REF 228Zvirbliene A, REF 389Zybin A, REF O174Virologie, Vol. 17, supplément 2, septembre 2013S303

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