St. Louis encephalitis virus (SLEV) is a mosquito borne pathogen that causes febrile illness and sometimes fatal encephalitis and was a leading cause of infectious encephalitis in the US during the 1970s. After an 11- year absence in activity in California, SLEV reemerged and reestablished in CA from 2015-2021, and caused an outbreak in nearby Phoenix, AZ. SLEV reemergence is marked by increasing human cases and positive mosquito pools across more counties each year, including areas with concurrent activity of West Nile virus (WNV), a related flavivirus that invaded CA in 2003 and which shares avian reservoirs and Culex mosquito vectors with SLEV. Genetic tracing by our team showed that reemerging (‘contemporary’) SLEV in the Western US is genetically distinct from pre-2003 (‘historical’) SLEV and that it likely originated in South America. However, other than these studies, drivers of SLEV reemergence, including into WNV-endemic areas, have not been examined. Spread of WNV across the US was facilitated by augmented avian reservoir infection and vector competence. We propose that similar fitness gains may have enabled SLEV reemergence. The objective of this project is to assess the extent by which SLEV reemergence is promoted by augmented infectivity and transmissibility in mosquito and avian cells and mosquito vectors. Higher SLEV activity in different regions may be explained by differential vector competence for 3 primary Culex vectors (pipiens, tarsalis, and quinquefasciatus) that favor different environments across the state. We hypothesize that SLEV reemergence in the Western US since 2015 was mediated by augmented fitness of the introduced genotype in cells and vectors and that geographic localization is influenced by relative vector competence of 3 primary Culex species. Viral fitness can be assessed experimentally by comparing SLEV strains using vector competence and competition assays. The project hypotheses will be tested in 2 project Aims: 1) Determine transmission competence of contemporary versus historical SLEV in Culex vectors, and 2) Compare relative fitness of contemporary versus historical SLEV in avian and mosquito cells and vectors. This project is significant in that it will provide a unique opportunity to compare viral factors involved in the sequential invasion and spread of 2 Culex-borne flaviviruses, where understanding co-circulation dynamics can be applied to other sympatric flaviviruses. By defining the role Culex vector species play in SLEV transmission this project will determine whether augmented infectivity and transmissibility is a factor enhancing reemergence and spread. To reduce disease, vector control districts can allocate resources towards spatial targeting of the Culex vector species that exhibit high SLEV competence.

St. Louis encephalitis virus (SLEV) is a mosquito borne pathogen that causes febrile illness and sometimes fatal encephalitis that reemerged and spread in the Western US from 2015-2021. Given that reemergence can be facilitated by changing viral fitness, the goal of this project is to assess the extent by which SLEV reemergence is promoted by augmented infectivity and transmissibility compared to historical strains in avian and mosquito cells and Culex mosquito vectors. The project is significant in that it will define how SLEV has reemerged in a novel landscape where related West Nile virus is now endemic and information learned from it can be used to improve targeted vector control to reduce SLEV disease in people.