The on-going West Nile virus (WNV) epidemic is the largest mosquito-borne outbreak in California and North American history and the largest WNV outbreak documented globally. WNV has now become endemic throughout California providing a unique natural experiment to determine how this invading virus will persist within different mosquito - avian transmission cycles over varying landscapes, ranging from the southeastern deserts to densely populated maritime Los Angeles to agricultural and rural settings throughout the Central Valley. Our competitive continuation proposal investigates the cascade of early season epidemiological events that determine whether WNV will remain at low persistence levels or amplify to outbreak levels. Quantitative delineation of these events and their precursors may enhance decision support systems and allow skillful forecasting to trigger timely and effective intervention. Concurrent with the WNV invasion has been the elimination of St. Louis encephalitis virus (SLEV), a previously endemic and closely related virus within the Japanese encephalitis virus (JEV) serocomplex. Since the level of WNV transmission presumably dictates the receptivity of California for the re-establishment of SLEV, our planned research also will address our previous hypothesis that similarity in natural history among closely related flaviviruses within the JEV serocomplex precludes concurrent sympatric amplification. We propose to address our hypotheses with the following specific aims: 1) Determine the relative importance of different mechanism[s] of WNV overwintering in different biomes, including continued horizontal transmission at southern latitudes, vertical transmission within mosquito populations, and relapse of chronic avian infection. 2) Delineate the timing of Culex spp. diapause termination, the onset of blood feeding and the initiation of enzootic transmission; and 3) evaluate the impact of vernal avian seroprevalence ['herd immunity'] on WNV amplification and SLEV introduction. Our new research will provide data on whether WNV has overwintered successfully, degree-day functions to anticipate the initiation of virus amplification, and the impact of herd immunity on the rate and level of amplification. Combining these factors into an early season risk models may allow us to forecast outbreaks of WNV in California and perhaps western North America. PUBLIC HEALTH RELEVANCE West Nile virus (WNV), a serious public and veterinary health problem throughout the North America, is now endemic throughout California, but it remains unclear what epidemiological factors will allow it to persist and resurge to outbreak levels. Our competitive continuation proposal investigates the cascade of early season events that determine if WNV will remain at low maintenance levels or amplify to create outbreaks. Quantitative delineation of these events and their precursors may allow skillful forecasting and enhance decision support systems to trigger timely intervention.