5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>assay for BDV antigen, and 11 <strong>of</strong> 327 (3.36%) tissue samples were positive.The BDV genome was detected in 14 <strong>of</strong> 63 (22.2%) sheep leukocytesamples and 11 <strong>of</strong> 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 Council<strong>of</strong> Selcuk University (08102001) and summarized from PhD thesis <strong>of</strong>Oguzhan Avci.Key words: BDV, direct ELISA, immunoperoxidase, one step RT PCR,sheepREF 343Serologic and virologic investigation <strong>of</strong> BHV 1, BVDV and BHV 4 incattle with metritisSibel YAVRU 1 , Oguzhan AVCI 1 , Mehmet KALE 21 University <strong>of</strong> Selcuk, Faculty <strong>of</strong> Veterinary Medicine, Department <strong>of</strong><strong>Virology</strong>, Konya, TURKEY; 2 University <strong>of</strong> Mehmet Akif Ersoy, Faculty<strong>of</strong> Veterinary Medicine, Department <strong>of</strong> <strong>Virology</strong>, Burdur, TURKEYBovine herpesvirus type 1 (BHV 1), Bovine viral diarrhoea virus (BVDV),and Bovine herpesvirus type 4 (BHV 4), are well known, importantpathogens <strong>of</strong> cattle that give rise to substantial economic losses due toreproductive failures and increased calf mortality, as well as enteric andrespiratory disease. The purpose <strong>of</strong> the present study was to evaluate thepossible effects <strong>of</strong> BHV 1, BVDV and BHV 4 involving metritis in theselected unvaccinated dairy cattle herds in Afyon province <strong>of</strong> Turkey byserologically and virologically methods. A total <strong>of</strong> 63 dairy cattle withclinical signs <strong>of</strong> metritis were sampled in order to investigate the presence<strong>of</strong> BHV 1, BVDV and BHV 4 infections. The sera samples weretested for presence <strong>of</strong> antibodies to BHV 1, BVDV and BHV 4 using ancommercially available indirect Enzyme Linked Immunosorbent Assay(ELISA, Biox, Belgium). Leukocyte samples were tested for presence<strong>of</strong> 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%) <strong>of</strong> 63 against BVDV and 51 (80.95%)<strong>of</strong> 63 against BHV 4. All sera samples were negative for BHV 1 antibodies.BHV 4 genome were detected in 8 (12.69%) <strong>of</strong> 63 leukocytesamples while BVDV and BHV 1 genomes could not be found. Presence<strong>of</strong> BVDV and BHV 4 antibodies in unvaccinated animals indicates thatthese cattle had contracted infection. The serologic and virologic results<strong>of</strong> the study strongly suggest that BHV 4 infections may play a director indirect role in causing bovine metritis; therefore their importancein the aetiology <strong>of</strong> metritis and their economical impact needs furtherattention.Key words: BHV 1, BVDV, BHV 4, metritisREF 344Immunomodulatory effects <strong>of</strong> 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 Vi<strong>rologie</strong>, Berlin,GERMANYEquine alphaherpesvirus 4 (EHV 4) was a major cause <strong>of</strong> respiratorydisease in horses throughout the world. It causes significant economicimpact to the equine industry because <strong>of</strong> respiratory problems and losttime for training and performance. Currently, no specific treatment wasavailable in the field to cure these equine infections and the efficacy <strong>of</strong> 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 <strong>of</strong>natural target cells during infection <strong>of</strong> EHV 4. The aim <strong>of</strong> this study wasto 1/determine the immunological pr<strong>of</strong>ile induced by EHV 4 infection onperipheral blood mononuclear cells (PBMC) and 2/develop and characterizea new epithelial cells line to characterize the immunological pr<strong>of</strong>ile..Modulation <strong>of</strong> the expression <strong>of</strong> 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 pr<strong>of</strong>ile during EHV 4 infection in airwayepithelial cells.Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013S215
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>19. VIRAL EVOLUTION ANDQUASISPECIESPosters: REF 346 to REF 359REF 346Use <strong>of</strong> deep sequencing to evaluate the intrinsic heterogeneity <strong>of</strong>human 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 <strong>of</strong> RNA Viruses, Paris, FRANCE;3 Institut Pasteur, National Influenza Centre, Paris, FRANCE; 4 University<strong>of</strong> Tel Aviv, Sackler Faculty <strong>of</strong> Medicine, Department <strong>of</strong> Cell and DevelopmentalBiology, Tel Aviv, ISRAELIn 2009, a new influenza type A virus <strong>of</strong> H1N1 subtype (H1N1pdm09)entered the human population and caused the first pandemic <strong>of</strong> 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 <strong>of</strong> influenza type A virusesis composed <strong>of</strong> eight single stranded RNA segments <strong>of</strong> negative polarity.So far, the evolutionary potential <strong>of</strong> 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 <strong>of</strong> closely related sequences (referred to as a quasispecies) thatcould influence its fitness and its adaptability. Interest in the quasispeciesnature <strong>of</strong> influenza viruses has only recently increased with the development<strong>of</strong> next generation sequencing (NGS) technologies that allow awider study <strong>of</strong> the genetic variability. NGS deep sequencing methodologieswere developed to determine the whole genome genetic heterogeneity<strong>of</strong> the three subtypes <strong>of</strong> 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 <strong>of</strong> influenza,were selected to cover the different epidemic seasons. We are presentinghere the results <strong>of</strong> the comparison performed to identify subtype/severitysignatures focusing on mutation frequencies and specific nucleotidepolymorphisms.REF 347Isolation and characterization <strong>of</strong> Coxsackie virus B3 RdRp variantsin vitroStéphanie BEAUCOURT, Nina GNÄDIG, Olve PEERSON, MarcoVIGNUZZIInstitut Pasteur, Paris, FRANCEThe crystal structure <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> 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 determinants<strong>of</strong> 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 <strong>of</strong> Anelloviridae: analysis <strong>of</strong> 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 Néph<strong>rologie</strong> etTransplantation Rénale, CHU Conception, Marseille, FRANCEThe Anelloviridae family is composed <strong>of</strong> 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 <strong>of</strong> these circular single stranded DNAviruses are still a matter to debate. In order to tentatively gain insightsabout the evolution rate <strong>of</strong> these viruses, we characterized and analyzedserial sequences belonging to genus TTV, based on the study <strong>of</strong> 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 <strong>of</strong> the viralgenomes. Following cloning and sequencing, sequences analysis (∼2kbeach) demonstrated the presence <strong>of</strong> 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 part<strong>of</strong> DXL2 ORF1. Details <strong>of</strong> the protocol and precise analysis <strong>of</strong> moleculardata obtained are exposed.REF 349Evolution <strong>of</strong> viral RNA dependent polymerasesJiri CERNY 1,2 , Barbora BOFIKOVA 3,4 , Libor GRUBHOFFER 1,2 ,Daniel RUZEK 2,51 Faculty <strong>of</strong> Science, University <strong>of</strong> South Bohemia in Ceske Budejovice,Ceske Budejovice, CZECH REPUBLIC; 2 Institute <strong>of</strong> Parasitology, BiologyCentre <strong>of</strong> the Academy <strong>of</strong> Sciences <strong>of</strong> the Czech Republic, CeskeBudejovice, CZECH REPUBLIC; 3 Faculty <strong>of</strong> Science, Charles Universityin Prague, Prague, CZECH REPUBLIC; 4 Faculty <strong>of</strong> Tropical AgriSciences,Czech University <strong>of</strong> Life Sciences, Prague, CZECH REPUBLIC;5 Veterinary Research Institute, Brno, CZECH REPUBLICRNA viruses evolve rapidly. Fast accumulation <strong>of</strong> many mutations leadsto high diversity <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> evolutionary history <strong>of</strong> all RNA viruses. Toreconstruct evolutionary history <strong>of</strong> viral RNA dependent polymerases, weused both sequence and structural data. We unified sequence data from theS216 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
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