A positively selected mutation in the WNV 2K peptide confers resistance to superinfection exclusion in vivo

•WNV mutant bearing 2K-V9M disseminates more than wildtype in C. quinquefasciatus.•WNV 2K-V9M replicates to lower titers than WT in mosquitoes.•WNV 2K-V9M overcomes superinfection exclusion in mosquitoes.•WNV 2K-V9M fitness advantage only apparent as a superinfecting virus.

Molecular epidemiologic studies of North American (NA) West Nile virus (WNV; Flaviviridae, Flavivirus) have documented the displacement of the introduced NY99 genotype with WN02. In addition, these studies have shown that particular substitutions are under positive selection. One occurs in the C-terminus of the NS4A coding sequence and results in a valine to methionine substitution at position nine of the 2K peptide. 2K-V9M confers the ability to overcome superinfection exclusion in vitro. We hypothesized that WNV strains bearing 2K-V9M have higher fitness than wildtype in Culex quinquefasciatus mosquitoes. Although infection rates and viral titers were not significantly different, virus dissemination rates were significantly higher with WNV 2K-V9M. As a super-infecting virus, WNV 2K-V9M was more successful than wildtype, however, in a mixed infection, 2K-V9M was not. These data support observations that 2K-V9M confers a context-specific selective advantage in mosquitoes and provides an in vivo mechanism for its positive selection.