title: Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model.
abstract: West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal Middle-East WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 700 ×  in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.
authors: Operational Direction of Viral Diseases, Veterinary and Agrochemical Research Center (CODA-CERVA-VAR), 99 Groeselenberg, 1180 Brussels, Belgium.
authors: M
authors: M
authors: Dridi
authors: Operational Direction of Viral Diseases, Veterinary and Agrochemical Research Center (CODA-CERVA-VAR), 99 Groeselenberg, 1180 Brussels, Belgium.
authors: T
authors: T
authors: Rosseel
authors: Boyd Orr Centre for Population and Ecosystem Health, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.|MRC-University of Glasgow Centre for Virus Research, Institute of Infection Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, UK.
authors: R
authors: R
authors: Orton
authors: Boyd Orr Centre for Population and Ecosystem Health, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors: P
authors: P
authors: Johnson
authors: UMR1161 Virologie INRA, ANSES, ENVA, French Agency for Food, Environmental and Occupational Health & Safety (Anses), 23 avenue du Général De Gaulle, 94706 Maisons-Alfort, France.
authors: S
authors: S
authors: Lecollinet
authors: Integrated Veterinary Research Unit, University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium.
authors: B
authors: B
authors: Muylkens
authors: Operational Direction of Viral Diseases, Veterinary and Agrochemical Research Center (CODA-CERVA-VAR), 99 Groeselenberg, 1180 Brussels, Belgium.
authors: B
authors: B
authors: Lambrecht
authors: Operational Direction of Viral Diseases, Veterinary and Agrochemical Research Center (CODA-CERVA-VAR), 99 Groeselenberg, 1180 Brussels, Belgium.
authors: S
authors: S
authors: Van Borm
mesh_terms: D000818:Animals; D002645:Chickens; D046368:Crows / Q000821:virology*; D004195:Disease Models, Animal; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D048348:Reverse Transcription; D017422:Sequence Analysis, DNA; D014901:West Nile Fever / Q000821:virology*; D014902:West Nile virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D012367:RNA, Viral