rologie i - European Congress of Virology

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