No abstract

SummaryBirds play a central role in WNV epidemiology by spreading and amplifying the virus. Increasing numbers of WNV isolates are detected in Europe, and the virulence of these genetically variable isolates is not well characterized for birds. Therefore, we investigated whether SPF chickens could be a valuable avian model for the pathotyping of WNV strains. One-day-old SPF chickens were inoculated subcutaneously (SC) or intracerebrally (IC) with four lineage 1 WNV strains (Is98, It2008, Fr2000 or Kunjin) and were daily clinically monitored for 2 weeks after infection. Additionally, one-day-old SPF chickens were SC inoculated, and one-week-old SPF chickens were SC or IC inoculated with two Euro-Mediterranean isolates, Is98 and Fr2000, to sample blood and feathers at regular time points. These samples were analysed by WN NS2a-specific rRT-PCR and WN NS1 antigen-capture ELISA that were developed for the purpose of this study. Differences in strain virulence were evidenced after IC inoculation of oneday-old SPF chickens, with Is98 eliciting the highest mortality rates and Kunjin the lowest ones, while lethality of Fr2000 and It2008 was intermediate. Neither viral load in sera and feathers nor NS1 antigen in the serum correlated with the differential pathogenicity of Is98 and Fr2000. However, irrespective of the inoculated strain, younger chickens showed higher and longer-lasting viremias than older chickens. In all experimental groups, the detection window for viral RNA in feathers lasted up to 14 dpi. Altogether, the data presented in this study show that WNV strain virulence can be discriminated in a one-day-old SPF chicken model on the basis of mortality rates, while viremia and viral load in feathers appear to be age dependent rather than strain dependent.

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 MiddleEast 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 7006 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.

West Nile virus (WNV) is amplified in an enzootic cycle involving birds as amplifying hosts. Because they do not develop high levels of viremia, humans and horses are considered to be dead-end hosts. Mosquitoes, especially from the Culex genus, are vectors responsible for transmission between hosts. Consequently, understanding WNV epidemiology and infection requires comparative and integrated analyses in bird, mammalian, and insect hosts. So far, markers of WNV virulence have mainly been determined in mammalian model organisms (essentially mice), while data in avian models are still missing. WNV Israel 1998 (IS98) is a highly virulent strain that is closely genetically related to the strain introduced into North America in 1999, NY99 (genomic sequence homology > 99%). The latter probably entered the continent at New York City, generating the most impactful WNV outbreak ever documented in wild birds, horses, and humans. In contrast, the WNV Italy 2008 strain (IT08) induced only limited mortality in birds and mammals in Europe during the summer of 2008. To test whether genetic polymorphism between IS98 and IT08 could account for differences in disease spread and burden, we generated chimeric viruses between IS98 and IT08, focusing on the 3′ end of the genome (NS4A, NS4B, NS5, and 3′UTR regions) where most of the non-synonymous mutations were detected. In vitro and in vivo comparative analyses of parental and chimeric viruses demonstrated a role for NS4A/NS4B/5′NS5 in the decreased virulence of IT08 in SPF chickens, possibly due to the NS4B-E249D mutation. Additionally, significant differences between the highly virulent strain IS98 and the other three viruses were observed in mice, implying the existence of additional molecular determinants of virulence in mammals, such as the amino acid changes NS5-V258A, NS5-N280K, NS5-A372V, and NS5-R422K. As previously shown, our work also suggests that genetic determinants of WNV virulence can be host-dependent.

Lineage 2 West Nile virus (WNV) strains were reported for the first time in Europe in 2004. Despite an almost silent circulation around their entry point in Hungary, an upsurge of pathogenicity occurred in 2010 as 262 people suffered from neuroinvasive disease in Greece. This increase in virulence was imputed to the emergence of a His249Pro mutation in the viral NS3 helicase, as previously evidenced in American crows experimentally infected with the prototype lineage 1 North-American WNV strain. However, since 2003, WNV strains bearing the NS3Pro genotype are regularly isolated in Western-Mediterranean countries without being correlated to any virulent outbreak in vertebrates. We thus sought to evaluate the weight of the NS3249Pro genotype as a virulence marker of WNV in an in vivo avian model of WNV infection. We therefore characterized three genetically-related Eastern-Europe lineage 2 WNV strains in day-old specific pathogen-free (SPF) chickens: Hun2004 and Aus2008 which are both characterized by a NS3249His genotype, and Gr2011 which is characterized by a NS3249Pro genotype. Unlike Hun2004 and Aus2008, Gr2011 was weakly virulent in SPF chicks as Gr2011-induced viremia was lower and waned quicklier than in the Hun2004 and Aus2008 groups. Overall, this study showed that the presence of a proline residue at position 249 of the viral NS3 helicase is neither sufficient nor necessary to confer pathogenicity to any given lineage 2 WNV strain in birds.