Nonconsensus West Nile Virus Genomes Arising during Mosquito Infection Suppress Pathogenesis and Modulate Virus Fitness In Vivo

Abstract

West Nile virus (WNV) is similar to other RNA viruses in that it forms genetically complex populations within hosts. The virus is maintained in nature in mosquitoes and birds, with each host type exerting distinct influences on virus populations. We previously observed that prolonged replication in mosquitoes led to increases in WNV genetic diversity and diminished pathogenesis in mice without remarkable changes to the consensus genome sequence. We therefore sought to evaluate the relationships between individual and group phenotypes in WNV and to discover novel viral determinants of pathogenesis in mice and fitness in mosquitoes and birds. Individual plaque size variants were isolated from a genetically complex population, and mutations conferring a small-plaque and mouse-attenuated phenotype were localized to the RNA helicase domain of the NS3 protein by reverse genetics. The mutation, an Asp deletion, did not alter type I interferon production in the host but rendered mutant viruses more susceptible to interferon compared to wild type (WT) WNV. Finally, we used an in vivo fitness assay in Culex quinquefasciatus mosquitoes and chickens to determine whether the mutation in NS3 influenced fitness. The fitness of the NS3 mutant was dramatically lower in chickens and moderately lower in mosquitoes, indicating that RNA helicase is a major fitness determinant of WNV and that the effect on fitness is host specific. Overall, this work highlights the complex relationships that exist between individual and group phenotypes in RNA viruses and identifies RNA helicase as an attenuation and fitness determinant in WNV.West Nile virus (WNV; Flaviviridae: Flavivirus) is a positivesense, single-stranded RNA (ssRNA) virus belonging to the Japanese encephalitis virus (JEV) serological complex of the flaviviruses. WNV perpetuates in nature in enzootic transmission cycles through alternating replication in (mainly avian) vertebrates and mosquitoes. The specific mosquito and avian hosts that are most important in a particular locality differ but tend to include Culex species mosquitoes and passerine birds (2, 10). WNV infection of mammals, including humans and horses, occurs via spillover from this enzootic cycle. Since its introduction into North America in 1999 (20), molecular epidemiologic studies have clearly demonstrated that the virus has evolved to maximize its transmission potential within local transmission cycles (8,11,23). This finding stimulated subsequent efforts to understand the details of the underlying evolutionary mechanisms that lead to population-level genetic and phenotypic changes in the virus. In brief, these studies demonstrated that WNV populations are genetically diverse within hosts (15), that genetic diversity may be shared between hosts (15), and that mosquito infection drives genetic diversification of the virus population both through relaxation of purifying selection and through selection of rare genotypes resulting from RNA interference (RNAi) (4,16,17). Thus, in the WNV transmission cycle, different...

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model

“…Chickens were inoculated by the IC route for the sake of comparability with our previous in vivo characterization of the Is98 virus and other WNVs (Dridi et al, 2013), showing a better pathotype discrimination upon IC inoculation in day-old chickens. However, previous studies on WNV quasispecies dynamics used SC inoculation (Deardorff et al, 2011;Jerzak et al, 2007Jerzak et al, , 2008. For bioethical reasons, and to simulate a more natural infection route, crows were infected subcutaneously.…”

“…However, it should be noted also that previous studies comparing WNV diversity in different hosts were not always able to account for inoculation route differences. For instance, WNV diversity in SC-inoculated chickens and intrathoracically infected mosquitoes was compared (Deardorff et al, 2011;Jerzak et al, 2007Jerzak et al, , 2008.…”

“…Using HeLa cells and a model of susceptible mice, it was demonstrated that the pathogenicity of poliovirus is not only determined by the selection of adaptive mutations, but that quasispecies diversity per se is a major determinant of virulence (Vignuzzi et al, 2006). However, in another study (Deardorff et al, 2011) the reduction in the genetic diversity during twenty consecutive passages of WNV in SPF chickens resulted in an increased viral replication in that host, while the enlarged genetic diversity generated by twenty consecutive passages of WNV in mosquitoes of the Culex genus led to a decreased viral replication in SPF chickens.…”

“…So far, intrahost WNV population dynamics has only been investigated in mosquitoes and chickens (Deardorff et al, 2011;Jerzak et al, 2005Jerzak et al, , 2007Jerzak et al, , 2008. From those studies, mosquitoes are vectors that contribute to WNV genetic diversity by relaxing purifying selection, while the more restrictive birds, modelled by young chickens, act as 'selective sieves' that maintain a strong purifying selection and shrink the spectrum of WNV genetic variants.…”

“…It is not known whether all WNV avian host species are equally restrictive for WNV genetic expansion. Furthermore, the available studies on WNV mutant spectrum diversification relied on Sanger sequencing methods (Deardorff et al, 2011;Jerzak et al, 2005Jerzak et al, , 2007Jerzak et al, , 2008, whereby a limited set of consensus sequences was obtained after a plaque-purification or molecular cloning step. Nextgeneration sequencing (NGS) technologies present an opportunity to deepen the analysis of RNA virus genetic variants (Barzon et al, 2011;Van Borm et al, 2015).…”