Encephalitic flavivirus infections affect thousands of people globally every year causing acute encephalomyelitis and placing significant burden on healthcare systems. Currently, no virus-specific treatments are available for these life-threatening conditions. The central nervous system (CNS) encompasses dozens of cell types with diverse properties and functions. Limited by a lack of adequate tools that combine throughput, depth and resolution, the interactions between viruses and this complex environment remain largely a mystery. To complicate matters, the CNS is also extensively connected to the periphery by both physical neural projections and peripheral immune signaling. Our preliminary studies demonstrate that tropism of the important encephalitic virus West Nile (WNV) within the CNS after direct intracranial inoculation of mice differs from that seen after spread to the CNS following peripheral infection. We hypothesize that CNS tropism is largely determined by resident neural and glial innate immune profiles, which can be readily modified by immune signals generated during peripheral infection. To address this hypothesis, we will utilize WNV to study immune interactions between cell types in an in vivo mouse model and in vitro using human induced pluripotent stem cell (hPSC) models. Tissue clearing techniques and whole mount imaging will be used to visualize viral antigens across the entire brain and spinal cord using light sheet microscopy, creating a complete time-resolved 3-dimensional map of infection. Responses of single cells will be examined using a combination of cutting-edge nuclear RNA sequencing and microscopy-based spatial transcriptomics. Co-cultures of hPSC-derived neurons and glia will be interrogated using high-throughput microscopy and sequencing to identify resistance factors and responses in human cell types. Hits will be mechanistically studied using blocking antibodies and CRISPR-mediated knockouts. Lastly, by using systemic and cell-type specific knock out animals or cytokine neutralizing antibodies, we will investigate the role of type I interferon in modulating CNS tropism and disease. This project will provide new data on flaviviral encephalitis at unparalleled resolution to help bridge current information gaps and improve fundamental knowledge by defining cellular tropism and CNS inflammatory responses at the single cell level and evaluating how changes in peripheral signaling influence infection of the brain. Identified peripheral factors restricting CNS infection are possible targets for immunomodulatory therapy, thus promoting research that may improve treatment for other forms of viral encephalitis. Finally, the resulting experimental pipeline will be broadly applicable to the study of CNS stress and inflammation, with relevance to other diseases like Lyme neuroborreliosis or chronic debilitating conditions like traumatic brain injury.

This project aims to uncover specific host factors and responses that contribute to viral tropism and patterns of neuroinflammation. Through a combination of whole mount and single molecule imaging, sequencing, and classical virology exploiting both murine and human models, the immune reaction to flaviviral encephalitis will be profiled at the single cell level and interrogated as to how it is modulated by autocrine, paracrine, and endocrine immune signaling. This will shed light on the pathogenesis of encephalitic flaviviruses and serve as a blueprint for understanding neuroinflammation for a variety of non- viral CNS conditions.