Project Summary/Abstract Alphaviruses are human pathogens that represent a global health threat. Mayaro virus (MAYV) and chikungunya virus (CHIKV) are alphaviruses that can cause acute disease with fever, headache, myalgia, chills, and long- term debilitating arthralgia. Adaptation of CHIKV lineages to the urban mosquitoes Aedes aegypti and Aedes albopictus has contributed to its worldwide distribution and led to large outbreaks. MAYV is thought to be restricted to transmission by sylvatic mosquitoes of the genus Haemagogus, but laboratory studies show that MAYV can also be transmitted by Aedes mosquitoes, suggesting the need for improved surveillance and countermeasures. I found that MAYV can infect Aedes mosquitoes from Salvador (Brazil) or Galveston when present at titers found in viremia in humans. This project aims to understand how adaptive mutations and interactions with hosts may lead to the emergence of alphaviruses outbreaks. My central hypothesis is that mutations in the MAYV nsP3 gene can lead to efficient transmission by Aedes mosquitoes through promoting viral-host interactions. I will address this hypothesis through three specific aims. In specific aim 1, I will determine existing and prospective mutations in the genome of MAYV that function in vector competence of Aedes mosquitoes. I have performed next generation sequencing of salivary glands of infected Aedes mosquitoes and found 17 putatively adaptive mutations. I will find and validate MAYV minority variants that arise upon mosquito infection using competition assays, 50% oral infectious dose experiments and dual host models. As a proof-of- principle, I discovered an adaptive mutation in the virus non-structural protein 3 (nsP3). I will then determine at which step of mosquito infection they are important through a series of dissections followed by titrations. In specific aim 2, I will uncover the roles of nsP3 in mosquito infections and vector competence. I will assemble nsP3 chimeras of different MAYV strains to identify regions that are necessary and sufficient for increased fitness in Aedes mosquitoes and study a natural insertion that has evolved at least twice in MAYV strains, suggesting it is adaptive, and its proposed changes in protein phosphorylation. In specific aim 3, I will assess how m6A modifications on MAYV and CHIKV RNA may promote immune evasion and how these modifications in cellular RNAs modulate the immune system. These modifications are thought to have key functions in immune system regulation and immune evasion. I showed that MAYV has m6A modifications on its RNA which are concentrated in the sub genomic RNA and promote viral replication. Completion of this project will have a major impact in the control of alphaviruses by spurring novel surveillance strategies and countermeasures targeting virus-host interactions. This project and career development award aligns well with my current skills and career goals to become an independent principal investigator...