Project Summary Virus infection is a dynamic process. Response to infection causes profound phenotypic changes within infected and uninfected cells within the host. Simultaneous changes also occur in the viral population due to within-host selection and evolution. The links between virus evolution, spread, and pathogenesis are complex, but have profound effects on the development of viral disease. Recent advances in single-cell RNA sequencing now allow the measurement of these dynamic cellular phenotypes and viral genotypes in complex tissues with unprecedented detail. This proposed research is focused on the development and use of these technologies to characterize virus evolution and host responses during enterovirus infection in an established murine model of enterovirus pathogenesis. Aim 1 focuses on the characterization of single-cell transcriptomes from healthy and infected mice to characterize the host response to infection in complex tissues. Aim 2 will use of emerging high-resolution deep sequencing approaches to characterize within-host viral population dynamics within infected mice. Aim 3 will explore the links between viral and host genotype to the kinetics of within-host spread and pathogenesis of enteroviruses. This work will quantify the evolutionary trajectories of infecting viral populations in vivo and correlate this with cell- and tissue-specific gene expression responses to infection. In the mentored phase, this work will focus on the viral and host dynamics of poliovirus infection, and later, in the independent phase, on the emerging pathogenic enterovirus EV71. The proposed research and training goals will build on the strengths of the candidate in systems biology and evolutionary biology, augmenting it with training in animal handling and single-cell sequencing from infected animals. During completion of this award, Dr. Dolan will pursue his transition to independence by participating in local seminars and international conferences, by advising graduate students, and by conducting a search to identify and obtain an independent faculty position, mentored by his career development committee. The result of the proposed aims will be a new paradigm for future investigations of viral infection and pathogenesis.