Pathogenesis and neurotropism of a contemporary Powassan virus lineage II strain in mice.

Powassan virus (POWV) is an emerging tick-borne flavivirus that can cause neurologic disease in humans. POWV exhibits substantial genetic and phenotypic diversity, including marked variability in replication in vitro and pathogenesis in mice. However, most experimental studies have relied on historical lineage I and II strains to assess POWV pathogenesis. Here, we characterized a contemporary lineage II strain, NY.19.12, in mice and investigated the potential viral determinants of disease. NY.19.12 caused earlier onset of clinical signs and earlier detection of viral RNA (vRNA) in the spleen and brain compared to mice infected with a widely used Midwest lineage II strain (WI.97.ic). Sequencing of NY.19.12 identified three amino acid differences in the envelope, non-structural 1, and non-structural 5 proteins. These substitutions are also present in other lineage II strains currently circulating in the Northeast United States, suggesting that they are maintained in nature. To assess their contribution to pathogenesis, the three amino acid substitutions were engineered into an infectious clone and evaluated in mice. At early time points post-infection, clinical signs and viral distribution in the brain were similar between NY.19.12 and the mutant clone, suggesting that these amino acid substitutions may contribute to disease progression and neurotropism. However, NY.19.12 vRNA was detected in the spleen at significantly higher rates than both WI.97.ic and the mutant clone, indicating that factors beyond these mutations contribute to persistence in the spleen. Together, these findings highlight the complexity of POWV pathogenesis and suggest that lineage II strains exhibit variable disease phenotypes likely driven by multiple genetic determinants.IMPORTANCETick-borne flaviviruses exhibit considerable genetic and phenotypic diversity in nature, but it is unclear if this influences their transmission and pathogenesis. Defining the mechanisms of pathogenesis requires understanding how inter-lineage variation translates to phenotypic differences in viral dissemination and neuroinvasion. This study demonstrates that even closely related Powassan virus (POWV) lineage II strains display distinct disease phenotypes that are only partially attributable to nonsynonymous consensus mutations within the viral coding sequence. By highlighting the complexity of strain-dependent POWV pathogenesis and neurotropism in mice, these findings provide valuable insights into how POWV lineage II diversity may shape disease progression and severity in humans.