Innate Immune Regulation of 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. There are currently no approved POWV vaccines or prophylactics and the role of innate immunity in regulating POWV neuroinvasion, CNS cell targeting and neuroinflammatory pathology is unknown. To reflect POWV spread from skin to the CNS, POWV LI9 was isolated from deer ticks directly in epithelial cells. Mice inoculated s.c. with POWV LI9 develop lethal neurovirulent disease with overt murine brain damage. Noting lethality in older mice, LI9 was found to be lethal in 90% of 50 week (wk), and <10% of 10 wk, old C57/Bl6 mice. Consistent with age-dependent LI9 lethality, brains of 50 wk old mice had dramatically increased CNS virus, chemokines, immune infiltrates and histopathology. Persistent passage of LI9 in IFN deficient VeroE6 cells, resulted in an attenuated POWV (LI9-P), that fails to cause disease and instead protects mice from lethal LI9 challenge. LI9-P is lethal if i.c. injected into mice, implying a block to CNS entry after peripheral inoculation. LI9-P and LI9 differ by 9 residues in proteins associated with cell attachment, spread and IFN regulation. In vitro, IFN prevents LI9/LI9-P infection, while LI9, but not LI9-P, inhibit IFNβ/λ induction. In vivo, IFNλ treatment reduced LI9 levels by 2 logs in mouse brains. These findings suggest innate immune regulation is a key determinant of LI9 neurovirulence and LI9-P attenuation, and rationalize analyzing neuroinvasive mechanisms and IFNλ as a potential inhibitor of POWV spread to the CNS. Entering the CNS is critical for POWV neurovirulence, and the blood-brain-barrier (BBB) and the blood- cerebrospinal-fluid-barrier (BCSFB) are distinct defenses that protect the CNS. The BBB is formed by brain microvascular endothelial cells (BMECs), while the BCSFB is created by tight junctions of epithelial cells (CPEpCs) in the choroid plexus. BBBs and BCSFBs are uniquely regulated by IFNs with IFNλ receptors (IFNLRs) expressed on CPEpCs, not BMECs, and IFNλ responses are linked to CNS repair. In vitro, LI9 and LI9-P infect CPEpCs and BMECs, suggesting that BBB or BCSFBs could function as CNS entry portals in vivo, and that IFNλ may selectively block POWV neuroinvasion via BCSFBs to prevent lethal neurovirulent disease. In mice, IFN regulation by LI9 vs LI9-P in BBB, BCSFB cells, and the role of IFNs in restricting POWV spread to the CNS are unknown. Proposed studies use a novel reverse genetics system to produce infectious recombinant POWVs, chimeras and mutants that uniquely permit analysis of POWV IFN regulation mechanisms and neurovirulence determinants in vivo. Studies define IFN responses that determine LI9 and LI9-P infection and spread across neurovascular barriers in vitro and in vivo. Studies evaluate the therapeutic potential of IFNλ inhibiting POWV neuroinvasion through BCSFBs in a lethal murine model.
