PROJECT SUMMARY Understanding how viruses circumvent immune defense mechanisms to travel throughout the body is of critical importance to human well-being. The near-eradication of polio, an enteric neurotropic RNA virus that targets the brain causing paralysis, remains one of our most historic victories in the struggle against human pathogens. Recent outbreaks of non-polio enteroviruses that also exhibit neurotropism pose potential threats to public health. However, our understanding of these viruses and their interactions with hosts is still not complete. The zebrafish model system provides a powerful platform for elucidating host immunity and virus virulence mechanisms, given the ease of molecular imaging and the abundance of genetic tools. Our recent discovery of a naturally occurring zebrafish enterovirus (ZfPV) that can easily be transmitted in the lab, provides an unprecedented tool to study enterovirus infection in a living organism, where the complexity of natural host-pathogen co-evolution is represented and the model organism is amenable to genetic manipulation and whole body fluorescence imaging. This novel finding, combined with the fortuitous discovery that an isogenic zebrafish line, CG2, displays neural infection tropism, provides a unique opportunity to study enterovirus infection tropism. By investigating natural differences in viral neurotropism observed between two zebrafish lines, Tübingen and CG2, I will pursue a new understanding of the cellular and molecular processes underlying viral dissemination from enteric tissue to the nervous system and associated host responses. Specifically, I will (1) identify genetic loci associated with enterovirus neurotropic susceptibility, (2) identify, at the single-cell level, which cell populations are infected and the specific cytokine and innate immune signaling triggered in local and distant tissues, and (3) investigate, in real-time and in a whole organism, the timing of innate IFN activation and termination, the source, timing, and activity of recruited neutrophils, and the inflammatory consequences on the local intestinal cell survival. These studies, based on a natural infection model, will advance our understanding of fundamental mechanisms of viral disease development and uncover important features underlying the evolution of viral tropism.