Characterization of mosquito-adapted West Nile virus

Abstract

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.

Fig. 1

Virus growth kinetics of WNV in IT-inoculated C. pipiens mosquitoes before (UNP) and after (MP) passage in C. pipiens. Individual points represent mean titres±sd of eight to ten mosquitoes. An asterisk indicates significantly higher titres for MP20 strains relative to UNP virus (t-test, P<0.05).

Fig. 2

Virus growth kinetics of SLEV in IT-inoculated C. pipiens mosquitoes before (UNP) and after (MP) passage in C. pipiens. Individual points represent mean titres±sd of eight to ten mosquitoes. An asterisk indicates significantly higher titres for SLEV UNP relative to MP20 strains (t-test, P<0.05).

Fig. 3

Foci size of WNV MP1 L2 and WNV MP20 L2 identified by fluorescent focus assay in Vero or C6/36 cell culture. Vero measurements were taken at 24 h p.i. and C6/36 measurements at 48 h p.i. Significantly lower foci sizes were found for MP20 relative to MP1 in Vero cell culture, indicated by an asterisk (t-test, P<0.001).

Fig. 4

WNV viraemia in 1–2-day-old chickens following inoculation of WNV before (UNP) and after (MP) C. pipiens passage. Individual points represent the mean titres±sd of five chickens. Statistically significant differences are indicated by an asterisk (t-test, P<0.05).