Passage-attenuated Powassan virus LI9P protects mice from lethal LI9 challenge and links envelope residue D308 to neurovirulence.

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes lethal encephalitic disease and chronic neurologic deficits in surviving patients. POWV-LI9 is a tick-derived isolate that causes neurovirulent disease and age-dependent lethality in mice. Serial passage of VeroE6 cells infected with LI9 resulted in eight amino acid changes in a POWV strain LI9P. LI9P fails to cause neurological sequelae, or lethality in C57BL/6 mice yet elicits neutralizing POWV antibody responses and protects mice from lethal LI9 challenge. Analysis revealed that LI9, but not LI9P, is present at high levels in the CNS, suggesting that LI9P is restricted from neuroinvasion or CNS replication. LI9 and LI9P are distinguished by a D308N envelope change within a domain associated with cell attachment. We evaluated the roles of Env-Domain III residue changes in LI9 virulence and LI9P attenuation using recombinant POWVs (recPOWVs) generated by reverse genetics. Remarkably, mutating D308N in LI9 completely abolished viral lethality and neuroinvasion in 50-week-old mice, reflecting the avirulent phenotype of LI9P. Analysis of the reciprocal N308D change in LI9P only partially restored neuroinvasion and lethality to the LI9P-N308D mutant, indicating that further LI9P residue changes contribute to LI9P attenuation. Consistent with differences in neuroinvasion, we found that rapid LI9P RNA synthesis and corresponding early IFN induction may contribute to LI9P clearance. Collectively, these findings define D308 as a determinant of POWV neuroinvasion and lethality, suggest potential mechanisms for restricted LI9P CNS entry, and reveal passage-attenuated LI9P as a candidate POWV vaccine platform. IMPORTANCE: Powassan virus (POWV) infection causes a 10% lethal encephalitis, resulting in chronic neurological symptoms in half of survivors. POWV is transmitted in as short as 15 min following tick attachment, demonstrating the need for the development of POWV vaccines and therapeutics. Mechanisms of POWV neurovirulence remain to be defined to inform vaccine and therapeutic design. Cell culture passage has successfully been used to generate live-attenuated flavivirus vaccines. Accordingly, we serially passaged POWV LI9-infected VeroE6 cells and isolated an attenuated POWV strain, LI9P, that fails to cause neurologic sequelae or murine lethality. LI9P elicits neutralizing antibody responses, protects mice from a lethal WT POWV challenge, and is a potential POWV vaccine. Analysis of attenuating mutations in LI9P revealed that changing envelope residue D308N alone in LI9 prevents POWV neurovirulence and lethality in immunocompetent mice. Altogether, this study defines viral determinants of POWV pathogenesis and attenuating mutations that inform the development of live-attenuated POWV vaccines.