RNAi Targeting of West Nile Virus in Mosquito Midguts Promotes Virus Diversification

Abstract

West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification. West Nile virus (WNV) was introduced into New York state in 1999 and has since spread across the Americas. It is transmitted in nature between adult female mosquitoes and birds and occasionally infects humans and horses. Within the host, WNV exists as a diverse assortment of closely related mutants. WNV populations within mosquitoes are more complex genetically than are those within birds. The reasons for this discrepancy are unknown, but may be related to the host's innate antivirus response. We demonstrate that WNV is targeted by RNA interference, a highly sequence-specific pathway in the mosquito. Further, we present data that correlates the intensity of this targeting with virus mutation under natural conditions. These results provide a mechanistic explanation for the increasead complexity of WNV populations in mosquitoes: the RNAi response creates an intracellular environment where rare genotypes are favored. In addition, our results suggest that genetically diverse WNV populations may have an advantage over less diverse populations because they present a more complex target for the RNAi response. Finally, these data suggest that WNV, and possibly other viruses with high mutation rates, may escape an engineered antivirus intervention that is highly sequence-specific.