Retrospective West Nile Virus Lineage 3 Surveillance in Culex Mosquitoes: Nebraska, USA 2012-2024.

West Nile virus lineage 1 (WNVL1) has been the only lineage known to circulate within the United States; however, multiple other lineages have been identified around the world. West Nile virus lineage 3 (WNVL3) was previously only isolated from mosquitoes in Czechia, but no human cases had been observed. West Nile virus lineage 3 was identified alongside WNVL1 in 2023 in a severely ill Nebraska patient with no history of international travel. To determine the extent of WNVL3 transmission, 1,199 historical mosquito pools collected from the state of Nebraska from 2012 through 2024 totaling >40,000 Culex spp. mosquitoes were tested. All pools tested negative for WNVL3 RNA. This study suggests that WNVL3 may circulate at very low levels or in alternative vector species in Nebraska.