Temperature influences West Nile virus evolution and adaptation

West Nile virus (WNV), the most common mosquito-borne disease in the continental U.S., is vectored by Culex spp. mosquitoes. Since its introduction to New York State (NYS) in 1999, WNV has become endemic. NYS temperatures have risen by 0.14°C per decade since 1900, with larger increases linked to higher WNV transmission. Using surveillance and sequencing data, we find a significant correlation between rising temperatures, increased WNV genetic diversity, and higher prevalence. Given the experimentally demonstrated role of temperature influencing WNV fitness, we hypothesized that contemporary strains should exhibit greater fitness in mosquitoes at higher temperatures compared to historic strains. To test this, we analyzed genetically distinct WNV strains from mosquitoes collected during recent warm summers (2017 and 2018) and cooler historic summers (2003 and 2004). Assessing Culex pipiens vector competence and calculating the relative R₀ at 20°C, 24°C, and 28°C, we found that contemporary strains exhibit higher transmission potential at increased temperatures. Our results show that contemporary WNV strains possess greater phenotypic and genotypic diversity, facilitating the emergence of strains with enhanced transmission potential in a warming climate.