Characterization of mosquito-adapted West Nile virus.

West Nile virus (WNV), a mosquito-borne flavivirus, has significantly expanded its geographical and host range since its 1999 introduction into North America. The underlying mechanisms of evolution of WNV and other arboviruses are still poorly understood. Studies evaluating virus adaptation and fitness in relevant in vivo systems are largely lacking. In order to evaluate the capacity for host-specific adaptation and the genetic correlates of adaptation in vivo, this study measured phenotypic and genotypic changes in WNV resulting from passage in Culex pipiens mosquitoes. An increase in replicative ability of WNV in C. pipiens was attained for the two lineages of WNV tested. This adaptation for replication in mosquitoes did not result in a replicative cost in chickens, but did decrease cell-to-cell spread of virus in vertebrate cell culture. Genetic analyses of one mosquito-adapted lineage revealed a total of nine consensus nucleotide substitutions with no accumulation of a significant mutant spectrum. These results differed significantly from previous in vitro studies. When St Louis encephalitis virus (SLEV), a closely related flavivirus, was passaged in C. pipiens, moderately attenuated growth in C. pipiens was observed for two lineages tested. These results suggest that significant differences in the capacity for mosquito adaptation may exist between WNV and SLEV, and demonstrate that further comparative studies in relevant in vivo systems will help elucidate the still largely unknown mechanisms of arboviral adaptation in ecologically relevant hosts.

Adaptation of two flaviviruses results in differences in genetic heterogeneity and virus adaptability.

West Nile virus (WNV) is a mosquito-borne flavivirus that was first introduced into the USA in the New York City area in 1999. Since its introduction, WNV has steadily increased both its host and geographical ranges. Outbreaks of the closely related flavivirus, St. Louis encephalitis virus (SLEV), occur in the USA periodically, but levels of activity and host range are more restricted than those of WNV. Understanding the selective pressures that drive arbovirus adaptation and evolution in their disparate mosquito and avian hosts is crucial to predicting their ability to persist and re-emerge. Here, we evaluated the in vivo phenotypes of mosquito cell-adapted WNV and SLEV. Results indicated that in vitro adaptations did not translate to in vivo adaptations for either virus, yet SLEV displayed attenuated growth in both mosquitoes and chickens, while WNV generally did not. In vitro growth analyses also indicated that WNV adaptations could be generalized to cell cultures derived from other mosquito species, while SLEV could not. Analysis of genetic diversity for passaged SLEV revealed a highly homogeneous population that differed significantly from previous results of high levels of diversity in WNV. We hypothesize that this difference in genetic diversity is directly related to the viruses' success in new and changing environments in the laboratory and that differences in a viruses' ability to produce and maintain heterogeneous populations in nature may in some instances explain the variable levels of success seen among arboviruses.

Role of the mutant spectrum in adaptation and replication of West Nile virus.

West Nile virus (WNV) has successfully spread throughout the USA, Canada, Mexico, the Caribbean and parts of Central and South America since its 1999 introduction into North America. Despite infecting a broad range of both mosquito and avian species, the virus remains highly genetically conserved. This lack of evolutionary change over space and time is common with many arboviruses and is frequently attributed to the adaptive constraints resulting from the virus cycling between vertebrate hosts and invertebrate vectors. WNV, like most RNA viruses studied thus far, has been shown in nature to exist as a highly genetically diverse population of genotypes. Few studies have directly evaluated the role of these mutant spectra in viral fitness and adaptation. Using clonal analysis and reverse genetics experiments, this study evaluated genotype diversity and the importance of consensus change in producing the adaptive phenotype of WNV following sequential mosquito cell passage. The results indicated that increases in the replicative ability of WNV in mosquito cells correlate with increases in the size of the mutant spectrum, and that consensus change is not solely responsible for alterations in viral fitness and adaptation of WNV. These data provide evidence of the importance of quasispecies dynamics in the adaptation of a flavivirus to new and changing environments and hosts, with little evidence of significant genetic change.

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occur in the U.S. periodically and are also characterized by limited genetic change overtime. We measured both phenotypic and genotypic changes in WNV and SLEV serially passaged in mosquito cell culture in order to clarify the role of an individual host cell type in flavivirus adaptation and evolution. Genetic changes in passaged WNV and SLEV were minimal but led to increased relative fitness and replicative ability of the virus in the homologous cell line C6/36 mosquito cells. Similar increases were not measured in the heterologous cell line DF-1 avian cells. These phenotypic changes are consistent with the concept of cell-specific adaptation in flaviviruses.