5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>REF O41Clonal expansion <strong>of</strong> 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 <strong>of</strong> Infectious Diseases, University <strong>of</strong> Gothenburg, Gothenburg,SWEDEN; 2 Department <strong>of</strong> 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 <strong>of</strong> Sweden, TickBorne Encephalitis Virus (TBEV) is a recently introduced and emergingzoonotic infection in the region <strong>of</strong> Western Gotaland, Sweden. Almostall <strong>of</strong> the 200 human cases recorded at present are located in the closeproximity <strong>of</strong> lakes or rivers. The aim <strong>of</strong> the current study was to understandthe mechanisms <strong>of</strong> 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 <strong>of</strong> lake Vänern.Here, we performed whole genome sequencing and phylogeny <strong>of</strong> 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 <strong>of</strong> human cases <strong>of</strong> TBE) showed high genetic homology despitethat these viruses were found 100 km apart. The results are compatiblewith a clonal expansion <strong>of</strong> TBEV from a common viral ancestor introducedin the lake Vänern region. Surprisingly, this cluster could not be linkedspecifically to any one <strong>of</strong> the other five viruses collected from the east towest coast <strong>of</strong> southern Sweden. These findings argue against a continuousand consecutive spread westward in Sweden <strong>of</strong> a single strain <strong>of</strong> TBEV.Instead, the phylogenetic analyses are compatible with an introduction <strong>of</strong>multiple TBEV strains to Western Sweden, either from the east <strong>of</strong> 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 <strong>of</strong> Veterinary Studies, University <strong>of</strong> Edinburgh, Edinburgh,UK; 3 Biology Centre, Institute <strong>of</strong> Parasitology and Faculty <strong>of</strong> Science, University<strong>of</strong> South Bohemia, Ceske Budejovice, CZECH REPUBLIC; 4 SaBio.Instituto de Investigación en Recursos Cinegéticos IREC (CSIC UCLMJCCM)s, Ciudad Real, SPAIN; 5 Department <strong>of</strong> Veterinary Pathobiology,Center for Veterinary Health Sciences, Oklahoma State University, Stillwater,USATicks are second only to mosquitoes in their importance in transmittinga wide variety <strong>of</strong> different pathogens including bacteria, protozoa andviruses. Many arboviruses <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> Ixodes sppcell lines and Ixodes ricinus ticks mock infected or infected with TBEV.Homologues <strong>of</strong> several genes known to be effective components <strong>of</strong> vertebrateinnate immunity, including ferritin binding (e.g. a homologue <strong>of</strong>Rattus norvegicus Fth1), calmodulin, heat shock proteins (e.g. HSP70)and components <strong>of</strong> the prophenoloxidase system (e.g. peroxinectin) andthe complement system (e.g. C4b binding protein and complement factorD), were differentially expressed. The roles <strong>of</strong> some <strong>of</strong> 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 <strong>of</strong> tick cells than just RNAi.Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013S53
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>Thursday 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 OPENSHAWPr<strong>of</strong>essor <strong>of</strong> 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 <strong>of</strong> disease induction by respiratorypathogens has undergone radical change over the past two decades. Respiratorysyncytial virus used to be thought <strong>of</strong> a cause <strong>of</strong> serious diseaseonly in infants but is increasingly recognized as a cause <strong>of</strong> 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 <strong>of</strong> disease in the natural host. We have infectedadult volunteers with Memphis 37 strain <strong>of</strong> RSV, causing reproducibledisease without serious adverse events. We have found that viral load isrelated to and severity <strong>of</strong> symptoms, strengthening the case for the development<strong>of</strong> 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 <strong>of</strong> natural infections, but are providing novel insights that areleading to improved understanding <strong>of</strong> viral disease.Studies <strong>of</strong> individual responses to viral infection are also helpful in planningfor outbreaks <strong>of</strong> 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 <strong>of</strong> age. However,many <strong>of</strong> 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 <strong>of</strong> the pathogenesis <strong>of</strong> influenza with intensive collection and analysis<strong>of</strong> 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 cause<strong>of</strong> 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 <strong>of</strong> explaining thevariable response to pH1N1 infection.This unique study is allowing a series <strong>of</strong> 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% <strong>of</strong>our cases but is rarely present in normal individuals. This has been confirmedon other populations in the Far East. The success <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> Geneva, Geneva, SWITZERLAND; 2 Laboratory <strong>of</strong> <strong>Virology</strong>,Division <strong>of</strong> Infectious Diseases and Division <strong>of</strong> Laboratory Medicine, University<strong>of</strong> Geneva Hospitals, Geneva, SWITZERLAND; 3 Epithelix sárl, 14Chemin des Aulx, 1228 Plan les Ouates, Geneva, Switzerland, Geneva,SWITZERLANDThe development <strong>of</strong> new molecular diagnostic tools has recently allowedthe discovery <strong>of</strong> human rhinovirus species C (HRV C). Several reportssuggest that HRV Cs may be overrepresented in children with pneumoniaor acute wheezing and exacerbation <strong>of</strong> asthma. HRV Cs cannot be propagatedin conventional immortalized cell lines, hence unlike HRV As andHRV Bs, the biological properties <strong>of</strong> these species C rhinoviruses havebeen difficult to study. However three groups have recently described thesuccessful amplification <strong>of</strong> 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 <strong>of</strong> clinical HRV C specimensincluding HRV C2, HRV C7, HRV C12, HRV C15 and HRV C29types were all capable <strong>of</strong> 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 <strong>of</strong> HRVCs. Finally, we show that the optimum temperature requirement duringthe growth <strong>of</strong> HRV Cs may be type dependant.REF O44Replication <strong>of</strong> 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 <strong>of</strong>ten associated with more severe infections than those<strong>of</strong> HRV A and –B. Studies <strong>of</strong> HRV C has so far been hampered due to theabsence <strong>of</strong> simple and rapid methods to propagate this virus in standard celllines. Recently Hao et al. reported replicating HRV C after transfection <strong>of</strong>fully 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 replication<strong>of</strong> a strain <strong>of</strong> HRV C in the standard cell line; human rhabdomyosarcoma(RD) cells. In vitro transcribed viral RNA from a cDNA clone <strong>of</strong> HRV C34was transfected into RD cells and viral replication was verified by methodsdetecting accumulation <strong>of</strong> viral genomes. Further characterization <strong>of</strong> 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 Propagation<strong>of</strong> Human Rhinovirus C in Human Airway Epithelial Cells. J Virol 2012;86 (24): 13524-32.S54 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
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