CNS Senescence and Immunotherapy in Lethal POWV Infection of Aged Mice

Powassan virus (POWV) is a neurovirulent tick-borne flavivirus (FV) emerging in the N.E. US. POWV causes a 10% fatal encephalitis resulting in severe long-term neurologic damage in 50% of patients. The incidence of POWV encephalitis in the elderly remains an enigma with mechanisms of POWV neuroinvasion, neuroinflammation and senescence that contribute to pathology virtually unknown. Mice s.c. inoculated with POWV LI9 develop lethal neurovirulent disease with overt spongiform brain damage similar to Alzheimer's disease. Neurovirulence assessed as a function of murine age, revealed POWV LI9 to be lethal in 82% of 50 wk, and 7.8% of 10 wk old C57/Bl6 mice, and consistent with severe POWV neurovirulence in the elderly. Concomitant with lethality brains of 50 wk old mice revealed higher viral load and cytokine response than 10 wk old mice 15 dpi in addition to microglial activation throughout the CNS, and persistent in survivors. A passage attenuated LI9-P strain was generated which replicates like LI9 in vitro, but fails to enter the CNS or cause lethal disease, and protects mice from WT LI9 challenge. This POWV lethal and attenuated POWV pair, provides a means of defining neurovirulence mechanisms and determinants of POWV entry into the CNS. Mechanisms of POWV entry into the CNS remain to be revealed. Initial LI9 and LI9-P comparisons found that both POWVs productively infect primary human neurovascular barrier cells: brain microvascular endothelial cells (BMECs) and choroid plexus epithelial cells (CPEpCs). These findings rationalize POWV entry into the CNS via BBB or BCSFB portals. In contrast to LI9, LI9-P highly induces IFNß/? responses in BMECs and CPEpCs, that may inhibit POWV neuroinvasion. These findings are consistent with mutations in Envelope and NS3/4A/4B proteins restricting LI9-P neuroinvasion and neurovirulence. A POWV reverse genetics system uniquely permits us to modify infectious POWVs. Using LI9/LI9-P mutants, single cell sequencing, immunohistochemistry, and flow, studies proposed analyze POWV neuroinvasion, CNS cell tropism and roles for CNS immunopathology and cell senescence as mechanisms of age-dependent POWV lethality in mice. Temporal scRNAseq analysis of brains, neuroinflammatory responses and senescent microglial and neuronal progenitor cells (NPCs) will be assessed for roles in POWV directed spongiform damage and lethality in aged versus young mice. Studies are aimed at revealing age-dependent differences in CNS regeneration, microglial and T cell responses and POWV clearance from the CNS that distinguish protective responses of young mice, from lethal POWV infection and long-term CNS damage in aged mice. Studies are likely to reveal defensive CNS responses, and therapeutic approaches for resolving viral encephalitis that are applicable to other neurodegenerative diseases of the elderly. Studies proposed evaluate: 1) Mechanisms of early POWV CNS entry and neuronal depletion; 2) POWV clearance from the CNS by microglia and T cells; and 3) Roles for CNS cell senescence in age-dependent POWV lethality, and senolytics for preventing severe POWV disease.