Project Summary Coxsackievirus is a common infection that causes viral myocarditis, meningoencephalitis, and hand, foot, and mouth disease in children and adults. Coxsackievirus is transmitted by the fecal-oral route and initiates infection in the gastrointestinal tract. Currently, there is a lack of effective prevention and treatment options for Coxsackievirus infections. Using a mouse model, recent data demonstrate that Coxsackievirus B3 (CVB3) utilizes bacteria to aid viral replication and pathogenesis in vivo. However, the mechanism for this observation is unclear. In preliminary data, we have demonstrated that bacteria can enhance CVB3 infectivity and thermostability in vitro. We also found that Gram-negative Salmonella enterica binds to CVB3 and can enhance infectivity and stability of CVB3 while Escherichia coli does not. We also found that structures in the major cell wall component of Salmonella, lipopolysaccharide (LPS), can impact viral stability. These data suggest that specific bacteria and bacterial cell wall components are required to enhance CVB3. Therefore, this study aims to elucidate the bacterial structure and site of binding to CVB3, and investigate the mechanisms by which bacteria aid viral thermostability. This goal will be accomplished in three aims. In Aim 1, we will determine the bacterial cell wall components that interact with CVB3. Using Salmonella LPS mutants, we will characterize the critical elements of LPS required to interact with CVB3. In Aim 2, we will identify the virion binding site between bacteria and Coxsackievirus. Using a combination of state-of-the-art cryogenic electron microscopy and CVB3 mutants, we will elucidate the LPS footprint on the CVB3 virion. Finally, in Aim 3, we will determine the effect of lipopolysaccharide on CVB3 infectivity and viral stability. Overall, this project will significantly impact our understanding of viral-microflora interactions and aid future studies to identify antiviral targets for the development of novel therapeutics.