{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T17:02:01Z","timestamp":1774890121151,"version":"3.50.1"},"posted":{"date-parts":[[2026,3,27]]},"group-title":"Microbiology","reference-count":52,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T00:00:00Z","timestamp":1774569600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Powassan Viruses (POWV) are tick-borne Flaviviruses 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 Flavivirus envelope proteins (EDIII) directs cell attachment, and determines murine neuropathology and lethality. Here we developed POWV LB reverse genetics, analyzed the CNS 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 neurovirulence, neuroinvasion and lethality, yet elicited neutralizing antibody responses that protected mice from a lethal WT LB challenge. Signifying changes in neurotropism, 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 neurovirulence and lethality of lineage I POWVs and that genetically modifying EDIII provides a mechanism for POWV attenuation and vaccine development.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>Powassan virus causes lethal encephalitis and long-term neurological sequalae in survivors; however, there are currently no licensed POWV vaccines or therapeutics. POWVs are comprised of two genetic lineages (LB-Lin I; LI9 Lin II) that are derived from discrete ticks and small mammal hosts. Flavivirus 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 neurovirulence in 50 wk old mice. Studies reveal novel LB neuropathology with divergent patterns of gliosis and POWV antigen expression within the CNS. Mice infected with an LB-D308N\/A310T envelope mutant elicited responses that protect mice from a lethal LB challenge and failed to enter the CNS, cause neurologic symptoms, CNS pathology or lethality. LB reverse genetics permits defining determinants of LB neurovirulence that are required for rational vaccine development.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.26.714546","type":"posted-content","created":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T01:05:26Z","timestamp":1774659926000},"source":"Crossref","is-referenced-by-count":0,"title":["Powassan Virus LB Neurovirulence and Lethality is Determined by Envelope Protein Domain III Residues"],"prefix":"10.64898","author":[{"given":"Marissa R.","family":"Lindner","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Infectious Disease, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena E.","family":"Gorbunova","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Infectious Disease, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marcos Romario Matos","family":"de Souza","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Infectious Disease, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Grace E.","family":"Himmler","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Infectious Disease, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2095-9705","authenticated-orcid":false,"given":"Hwan Keun","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Infectious Disease, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9156-1222","authenticated-orcid":false,"given":"Erich R.","family":"Mackow","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Center for Infectious Disease, Renaissance School of Medicine, Stony Brook University, Stony Brook, New York, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026033009050929000_2026.03.26.714546v1.1","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1038\/s41564-020-0714-0","article-title":"The continued threat of emerging flaviviruses","volume":"5","year":"2020","journal-title":"Nature Microbiology"},{"key":"2026033009050929000_2026.03.26.714546v1.2","doi-asserted-by":"publisher","DOI":"10.1128\/cmr.00106&ndash;17"},{"key":"2026033009050929000_2026.03.26.714546v1.3","doi-asserted-by":"crossref","unstructured":"Sanchez-Vicente S , Tagliafierro T , Coleman JL , Benach JL , Tokarz R . 2019. Polymicrobial Nature of Tick-Borne Diseases. mBio 10.","DOI":"10.1128\/mBio.02055-19"},{"key":"2026033009050929000_2026.03.26.714546v1.4","first-page":"708","article-title":"Powassan virus: isolation of virus from a fatal case of encephalitis","volume":"80","year":"1959","journal-title":"Can Med Assoc J"},{"key":"2026033009050929000_2026.03.26.714546v1.5","first-page":"1926","article-title":"Potential Role of Deer Tick Virus in Powassan Encephalitis Cases in Lyme Disease\u2013endemic Areas of New York","volume":"19","year":"2013","journal-title":"USA. Emerging Infectious Disease journal"},{"key":"2026033009050929000_2026.03.26.714546v1.6","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.19-0281"},{"key":"2026033009050929000_2026.03.26.714546v1.7","unstructured":"CDC. 2026. Powassan virus: historic data (2004\u20132024). 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