PROJECT SUMMARY Varicella zoster virus (VZV) is an exclusively human, double-stranded DNA alphaherpesvirus that produces varicella (chickenpox) on primary infection then establishes life-long latency in ganglionic sensory neurons in >95% of adults. With aging and immunosuppression, VZV reactivates in at least 1 in 3 individuals to produce zoster rash (shingles) that can be further complicated by vasculitis (inflammation of the vasculature), myelitis (inflammation of the spinal cord), or chronic pain (post-herpetic neuralgia, PHN). Diagnosing VZV as a causative agent of disease is typically restricted to the detection of viral antigens and/or nucleic acids, however, the disproportionate and sometimes lack of viral nucleic acid/antigen detection in tissues where inflammation persists following acute infection has puzzled clinicians and researchers. Therefore, we suggest a viral-induced, non-infectious “soluble factor” can drive pathology in tissues distant from viral replication in the ganglionic sensory neurons. Exosomes are small extracellular vesicles (~40-200 nm in diameter) of endosomal origin that carry cargo (proteins, nucleic acids) to cells for communication during normal and pathological states, regulating biological processes and response to disease. Using mass spectrometry (MS) and next- generation sequencing (NGS), our preliminary data shows that VZV-infected human sensory neurons (huSNs) release non-infectious exosomes containing unique proteins and miRNAs as well as a single VZV protein (immediate early 62) compared to uninfected neuronal exosomes. These proteins and miRNAs have been shown to suppress the host’s innate antiviral pathway. Indeed, when applied to naïve vascular cells, VZV exosomes induced robust transcriptional changes indicative of suppression of antiviral responses and increased permissiveness to subsequent VZV infection. Compared to mock, VZV exosomes also induced elevated proinflammatory cytokine release in these cells. Given the extensive sensory innervation of multiple organs throughout the body, how these exosomes alter multiple disease-relevant central nervous system (CNS), and non-nervous system cell types remains an important and unexplored area of research. We hypothesize that VZV infected huSNs release non-infectious pathogenic exosomes that contribute to the suppression of innate antiviral responses in cells, resulting in the enhanced permissibility to direct infection, as well as induce a proinflammatory environment. To test this hypothesis, we will: (Aim 1) Determine the content of pathogenic exosomes from VZV-infected huSNs throughout the course of infection and if antiviral treatment alters the pathogenic contents; and (Aim 2) Determine the altered transcriptional and proteomic profile of exosome-exposed primary human spinal cord astrocytes and brain vascular adventitial fibroblasts in the context of innate antiviral immune evasion and inflammation. Understanding how virus-associated, non- infectious e...