Maintenance of a host-specific minority mutation in the West Nile virus NS3.

West Nile virus (WNV), the most prevalent arthropod-borne virus (arbovirus) in the United States, is maintained in a cycle between Culex spp. mosquitoes and birds. Arboviruses exist within hosts and vectors as a diverse set of closely related genotypes. In theory, this genetic diversity can facilitate adaptation to distinct environments during host cycling, yet host-specific fitness of minority genotypes has not been assessed. Utilizing WNV deep-sequencing data, we previously identified a naturally occurring, mosquito-biased substitution, NS3 P319L. Using both cell culture and experimental infection in natural hosts, we demonstrated that this substitution confers attenuation in vertebrate hosts and increased transmissibility by mosquitoes. Biochemical assays demonstrated temperature-sensitive ATPase activity consistent with host-specific phenotypes. Together these data confirm the maintenance of host-specific minority variants in arbovirus mutant swarms, suggest a unique role for NS3 in viral fitness, and demonstrate that intrahost sequence data can inform mechanisms of host-specific adaptation.

Reversion to ancestral Zika virus NS1 residues increases competence of Aedes albopictus.

Both mosquito species-specific differences and virus strain -specific differences impact vector competence. Previous results in our laboratory with individual populations of N. American mosquitoes support studies suggesting Aedes aegypti are more competent than Ae. albopictus for American Zika virus (ZIKV) strains and demonstrate that U.S. Ae. albopictus have higher competence for an ancestral Asian ZIKV strain. A982V, an amino acid substitution in the NS1 gene acquired prior to the American outbreak, has been shown to increase competence in Ae. aegypti. We hypothesized that variability in the NS1 could therefore contribute to species-specific differences and developed a reverse genetics system based on a 2016 ZIKV isolate from Honduras (ZIKV-WTic) to evaluate the phenotypic correlates of individual amino acid substitutions. In addition to A982V, we evaluated G894A, which was acquired during circulation in the Americas. Reversion of 982 and 894 to ancestral residues increased infectivity, transmissibility and viral loads in Ae. albopictus but had no effect on competence or replication in Ae. aegypti. In addition, while host cell-specific differences in NS1 secretion were measured, with significantly higher secretion in mammalian cells relative to mosquito cells, strain-specific differences in secretion were not detected, despite previous reports. These results demonstrate that individual mutations in NS1 can influence competence in a species-specific manner independent of differences in NS1 secretion and further indicate that ancestral NS1 residues confer increased competence in Ae. albopictus. Lastly, experimental infections of Ifnar1-/- mice demonstrated that these NS1 substitutions can influence viral replication in the host and, specifically, that G894A could represent a compensatory change following a fitness loss from A982V with some viral genetic backgrounds. Together these data suggest a possible role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts and demonstrate that strains with increased transmission potential in U.S. Ae. albopictus could emerge.