Arthropod-borne viruses (arboviruses) adapt to local conditions, maximizing their potential to perpetuate and emerge as health threats. The adaptive potential of arboviruses is driven by error-prone replication, which creates a genetically diverse pool of competing virus genotypes within each host. This proposal examines some of the ways that temperature may impact arbovirus evolutionary biology. Our previous research has allowed us to make clear predictions about the outcome of each proposed aim, and generated molecular and computational tools, and methodological approaches that we propose to combine in this project. Global temperatures are changing at an unprecedented rate, and RNA viruses such as WNV continue to emerge at a frightening pace. Our preliminary studies have shown quite clearly that temperature is a key factor that dictates how natural selection affects arboviruses within mosquitoes. Therefore, Aim 1 will address how temperature, both constant and fluctuating, with varying means and amplitudes, alters natural selection on WNV within mosquitoes and the strength of bottlenecks. Our predictions (in general) are that fluctuating temperatures will increase the strength of purifying selection, that diversity will be maximized at optimal constant temperatures, and that bottlenecks will become wider as temperature increases. Our results have demonstrated that flavivirus infections are most frequently initiated by aggregates of virus particles. Aim 2 will address the extent that this occurs in a host- and temperature-dependent manner, bringing our previous work into a more ecologically relevant, realistic context. In the second phase of Aim 2, we will ask whether these genome aggregates can help to facilitate the maintenance of genetic diversity in the WNV population. Birds that generate high WNV viremia and are highly infectious to mosquitoes have significantly more unique WNV genomes per cell than those that have lower viremias. Aim 3 will assess whether something similar may occur in mosquitoes. We will use barcoded WNV to infect mosquitoes with a range of vector competence and assess the number of unique WNV genomes per cell. As above, we also will assess the degree to which this phenomenon may allow for the maintenance of low fitness viral genotypes while preventing those of high fitness from gaining dominance. This work will provide comprehensive data on the ways that changing environmental conditions may alter the fundamental population biology of arboviruses. Our work is also significant because it will provide mechanistic data on how viruses may maintain genetic diversity in the face of both selective and stochastic reductions in genetic diversity. The proposed studies are technically and conceptually innovative because of the ways that we can combine realistic transmission systems in the lab with barcoded viruses, single cell approaches, and other new molecular tools.