Friday, May 9, 2025
5/9/2025
Mechanisms of Age Dependent Powassan Virus Neurovirulence - Powassan virus (POWV) is a neurovirulent tick-borne flavivirus (FV) emerging in the N.E. US. POWV is present in tick saliva, injected into the skin during a 15-minute tick bite and 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, CNS cell targeting and neuroinflammatory pathology, virtually unknown. To reflect POWV spread from skin to the CNS, POWV (LI9) was isolated from deer ticks directly in epithelial cells. Mice s.c. inoculated with POWV LI9 develop lethal neurovirulent disease with overt murine brain damage. Noting lethality in older mice, neurovirulence was assessed as a function of age and found LI9 to be lethal in 90% of 50 wk, and <10% of 10 wk, old C57/Bl6 mice, and consistent with POWV neurovirulence in the elderly. Brains of 50 wk old mice had dramatically increased viremia, chemokines, immune cell infiltrates and histopathology. LI9 was passage attenuated in IFN deficient VeroE6 cells and the resulting virus, LI9-P, fails to cause murine disease and protects mice from LI9 challenge. This POWV pair provides a means of defining neurovirulence mechanisms. Mechanisms of POWV entry into the CNS remain to be revealed but are likely to be via hematogenous blood-brain-barrier (BBB) or blood-CSF-barrier (BCSFB) portals. BBB and BCSFBs are distinct and respectively formed by brain microvascular endothelial cells (BMECs) and choroid plexus epithelial cells (CPEpCs). CPEpCs are uniquely responsive to IFNλ, and the BCSFB is the main immune cell gateway into the CNS. In aged mice upregulated inflammatory IL-1β and IL1R responses are found in choroid plexus cells and suggest potential mechanisms of age-dependent neuroinflammation. POWV LI9 infects CPEPCs and BMECs and regulates IFN responses in vitro. In mice LI9 directed CNS viremia was dramatically inhibited by post-infection IFNλ treatment. In contrast to LI9, LI9-P highly induces IFNβ/λ in infected CPEpCs, and failure to regulate IFN responses may explain LI9-P attenuation in mice. These findings rationalize POWV entry into the CNS via BBB or BCSFB portals, and IFNλ as a key regulator of LI9 neuroinvasion and pathogenesis. To define mechanisms of LI9 neurovirulence and LI9-P attenuation a recombinant approach for generating infectious POWVs and chimeras of LI9 and LI9-P was developed. Studies proposed use single cell RNA sequencing (scRNAseq), model BBB and BCSFBs and brain slice culture to define CNS cells targeted by POWVs and mechanisms of LI9 neuroinvasion. Proposed studies analyze cell responses that permit POWV neuroinvasion and define age-dependent neurovirulence mechanisms using neurovirulent LI9, attenuated LI9- P and POWV mutants. Findings are likely to reveal responses that direct or restrict CNS damage, and targets for preventing POWV neurovirulence that may be broadly applicable to other neurovirulent viruses.
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