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. However, laboratory studies show that MAYV can also be transmitted by mosquitoes of the Aedes genus, 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 the risk of existing adaptive variations and interactions with hosts may lead to the emergence of alphaviruses. My central hypothesis is that mutations in the MAYV nsP3 gene can lead to efficient transmission by Aedes mosquitoes by promoting viral-host interactions. I will address this hypothesis through three specific aims. In specific aim 1, I will determine nucleotide variations in the genome of MAYV that function in the vector competence of Aedes mosquitoes. To find these nucleotide variations, I have performed high throughput sequencing of salivary glands of infected Aedes mosquitoes. I will then determine at which step of mosquito infection they are important through 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 by spurring novel surveillance strategies and countermeasures targeting virus-host interactions. This project and career development award will be paramount in my continued growth as an independent researcher.