Hyde, JenniferHickson, Sarah2025-01-232025-01-232025-01-232024Hickson_washington_0250E_27802.pdfhttps://hdl.handle.net/1773/52835Thesis (Ph.D.)--University of Washington, 2024Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne positive-sense single-stranded RNA genome virus belonging to the Togaviridae. Present throughout Central and South America, VEEV is responsible for significant outbreaks of epidemic/epizootic VEEV causing febrile disease and encephalitis in both equids and humans. While endemic/enzootic VEEV persists in nature and circulates between reservoir host rodents and mosquitoes, periodic mutation of enzootic VEEV gives rise to the emergence of epizootic VEEV. Using equines as amplification hosts, epizootic VEEV can have devastating outcomes for equine populations as well as cause large spill over events and disease in humans. The main mutations linked to epizootic VEEV emergence involve amino acid mutations within the E2 glycoprotein, which enhance viral entry and equine amplification. Interestingly, the majority of mutations found within epizootic strains are synonymous, indicating that other viral factors, such as RNA secondary structure, may play a critical role in their emergence. Understanding these mechanisms is crucial for predicting and mitigating future outbreaks.In this study, we discovered novel RNA structures within the E1 coding sequence that specifically affect VEEV replication in macrophages, which are critical early targets during infection. Using mass spectrometry and targeted gene knockdown, we identified several RNA-binding proteins essential for the altered macrophage phenotype, none of which had previously been associated with VEEV replication. Our findings also revealed the conservation of single-nucleotide polymorphisms (SNPs) within epizootic VEEV lineages, as well as the preservation of RNA structures across all lineages. Taken together, these findings suggest a previously unrecognized role for RNA secondary structure in the emergence of epizootic VEEV.application/pdfen-USCC BY-NC-SAVirologyMicrobiologyMicrobiologyThesis