Powassan virus LB neuroinvasion and lethality are determined by envelope protein domain III residues

Powassan viruses (POWVs) are tick-borne orthoflaviviruses that cause lethal encephalitis and long-term neurologic sequelae. POWVs are divided into two lineages that share 86% genetic identity. POWV-LB (Lin I) was isolated from a fatal case of encephalitis, while POWV-LI9 (Lin II) is a tick-derived isolate that causes age-dependent lethality in mice. Domain III of orthoflavivirus envelope proteins (EDIII) directs cell attachment and determines murine neuropathology and lethality. Here, we developed POWV LB reverse genetics, analyzed brains of LB- and recLB mutant-infected mice, and assessed the role of EDIII residues in LB pathogenesis. LB infection of 50-week-old B6 mice focally activated microglia in the midbrain and cerebellum, discrete from and independent of the location of envelope protein in the cerebral cortex, hippocampus, and thalamus. Reverse genetic analysis of LB EDIII residues revealed that mutating D308N only partially reduced LB neuropathology and lethality. However, mutating D308N along with an adjacent EDIII residue, A310T, completely abolished LB-D308N/A310T neuroinvasion and lethality, yet elicited neutralizing antibody responses that protected mice from a lethal WT LB challenge. Signifying a change in neuroinvasion, LB-D308N/A310T failed to activate microglia or yield detectable POWV RNA and antigen in the CNS. Overall, recombinant LB-D308N/A310T mutants fail to induce neurologic symptoms or neuropathology while eliciting a protective immune response. These findings reveal that EDIII residues determine the neuroinvasion and lethality of lineage I POWVs and that genetically modifying EDIII provides a mechanism for POWV attenuation and vaccine development.IMPORTANCEPowassan virus causes lethal encephalitis and long-term neurological sequelae in survivors; however, there are currently no licensed POWV vaccines or therapeutics. POWVs are comprised of two genetic lineages (LB-Lin I and LI9 Lin II) that are derived from discrete ticks and small mammal hosts. Orthoflavivirus envelope proteins determine cell attachment, viral tropism, and neurovirulent spread and lethality in mice. Here, we created a POWV LB reverse genetics system and assessed mutations that determine LB neuroinvasion in 50-week-old mice. Studies reveal novel LB neuropathology with divergent patterns of gliosis and POWV antigen expression within the CNS. Mice subcutaneously infected with an LB-D308N/A310T envelope mutant elicited responses that protect mice from a lethal LB challenge and failed to enter the CNS or cause neurologic symptoms, CNS pathology, or lethality. LB reverse genetics permits defining the determinants of LB neuroinvasion that are required for rational vaccine development.