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 suggestingAedes aegyptiare more competent thanAe.albopictusfor American Zika virus (ZIKV) strains and demonstrate that U.S.Ae.albopictushave 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 inAe.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 inAe.albopictusbut had no effect on competence or replication inAe.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 inAe.albopictus. Lastly, experimental infections ofIfnar1(-/-)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.albopictuscould emerge. Author summary The factors that facilitated the emergence and rapid dissemination of ZIKV in the Americas, and the specific role of strain variation and adaptive evolution in ZIKV transmission, are not fully understood. It has been shown that an amino acid substitution in the NS1 gene acquired prior to the American outbreak confers increased infectivity inAe.aegypti. Therefore, we hypothesized that variability in the NS1 could contribute to species-specific differences. To investigate the role of the ZIKV NS1 in competence of bothAe.aegyptiandAe.albopictuswe utilized reverse genetics to revert to the ancestral NS1 residues and characterized the resultant mutant strains. Reversions increased infectivity, transmissibility and viral loads inAe.albopictusbut had no effect on competence or replication inAe.aegypti. Additionally, we assessed the effect of these mutations on NS1 secretion and both virulence and viremia kinetics using a ZIKV mouse model. NS1 secretion, as well as vertebrate morbidity or mortality, were similar among strains, yet strain-specific differences in viremia levels were identified. 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 inAe.albopictus. Moreover, contrasting these data with previous studies suggests a role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts.