Courtney, S. J. | bioRxiv Science

bioRxiv2025

Investigation of Powassan virus lineage II pathogenesis and neurotropism in mice

Powassan virus (POWV) is an emerging tick-borne flavivirus that causes disease in humans. POWV has considerable genetic and phenotypic diversity, including highly variable replication in vitro and pathogenesis in mice. This study sought to define the extent of variability in pathogenesis within POWV lineage II in mice and investigate possible viral determinants. Relative to other strains, two New York-derived isolates, NY.19.12 and NY.19.32, caused earlier clinical signs and earlier detection of viral RNA (vRNA) in the spleen and brain compared to mice infected with virus derived from a lineage II infectious clone (WI.97.ic). Sequencing revealed these strains share three amino acid substitutions in envelope, NS1, and NS5 compared to other lineage II strains, which were engineered into a mutant infectious clone. At early time points post-infection, clinical signs, vRNA detection in the cerebellum, and viral distribution in the brain were similar between NY.19.12 and the mutant clone, suggesting these mutations may play a role in disease progression and early neuroinvasion. However, NY.19.12 vRNA was detected in the spleen at significantly higher rates compared to both WI.97.ic and the mutant clone, indicating factors other than these mutations are responsible for increased spleen infection. Importantly, this study highlights the complexity of POWV pathogenesis and suggests that POWV lineage II strains have varying disease phenotypes likely driven by multiple genetic differences. ImportanceTick-borne flaviviruses exhibit considerable genetic and phenotypic diversity in nature, influencing their transmission and pathogenesis. Defining the mechanisms of pathogenesis requires understanding how inter-strain 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 POWV infection dynamics in mice, these findings provide valuable insights into how POWV lineage II diversity may shape disease progression and severity in humans.

microbiology↗

bioRxiv2024

Evidence for Powassan virus deletions and defective RNA in field collected ticks

Powassan virus (POWV) is a tick-borne flavivirus in the tick-borne encephalitis virus (TBEV) serogroup endemic to the United States, Canada, and parts of Russia. POWV remains an under-studied pathogen, despite the potential for serious and life-threatening neurologic complications following infection. While prior studies have characterized viral diversity due to single nucleotide polymorphisms, little is known about POWV recombination, defective RNAs (D-RNAs), and functional structural variants (SVs). Understanding POWV recombination in its natural vector can provide important insights into its replication and evolution. Thus, we analyzed POWV sequence data from 51 ticks collected from the Northeast United States to characterize deletion expression levels and patterns in naturally infected ticks, and we compared these results to single-passage isolates. We found that deletions were common in POWV RNA from ticks and that several areas of the genome were enriched for recombination junctions. Deletions were often associated with areas of microhomology. While most deletions were sample-specific, two major deletion archetypes were observed across multiple tick samples. The first consisted of small 19-50 base deletions in the methyltransferase domain of the ns5 RNA-dependent RNA-polymerase gene, resulting in a mixture of putative SVs and D-RNAs. The second consisted of approximately 1600 base deletions spanning the ns2a-ns3 genes, resulting in putative D-RNAs with abrogated viral protease function. Protease deletions were significantly enriched after one passage in baby hamster kidney cells despite a decrease in overall deletion expression. These results demonstrate the proclivity of POWV for recombination, with potential implications for immune evasion and persistence in ticks. IMPORTANCEPowassan virus is a tick-borne flavivirus that can cause serious, life-threatening neurological disease. Understanding how Powassan virus replicates and evolves within its tick vector may elucidate factors important in persistence, transmission, and human disease. Defective RNAs are replication-incompetent viral genomes generated through internal deletions, which have been associated with disease severity and persistent infection in other viruses but have not been described for Powassan virus. Here, we show that Powassan virus produces abundant defective RNAs in field-caught ticks, and that expression patterns of these defective RNAs changes after one passage in mammalian cells. Although the function of these defective RNAs remains unknown, this work establishes a critical framework for investigating the role of defective RNAs in Powassan virus replication and transmission.

genomics↗