Project Summary/Abstract RNA viruses frequently undergo recombination, and the resulting deletions and duplications in both genomic and subgenomic RNA can contribute to the viral lifecycle, pathogenesis, and immune evasion. While recombination has been studied for some viruses, including mosquito-borne flaviviruses, its role in tick-borne flaviviruses is unknown. This project will investigate RNA recombination, deletions, and duplications in Powassan virus, which is an emerging flavivirus that is transmitted by Ixodes scapularis ticks, causes severe encephalitis, and has been detected in the United States with increasing frequency. The long-term objective of this work is to understand the biological role of recombination in tick-borne virus infection. The goals of the current project are to comprehensively characterize the types, genomic locations, and host-specific patterns of RNA recombination in Powassan virus. The first Aim is to map recombination junctions, deletions, and duplications in Powassan virus RNA collected from wild-caught Ixodes scapularis ticks. The investigators will use laboratory and analysis methods specifically designed for detecting recombination: ClickSeq for library preparation and ViReMa for analysis. They will sequence samples from an existing biobank of RNA from over 100 Powassan-positive ticks, representing a range of geography and genetic diversity. Recombination junctions common to multiple samples are hypothesized to represent biologically meaningful RNA species, such as structural variants (functional RNA molecules important in the viral lifecycle) or defective RNAs, dysfunctional RNA molecules that may inhibit wild-type virus replication or modify immune signaling. Evaluating the role of these RNA species in pathogenesis or transmission will be the focus of future projects. The second Aim of the current project is to assess whether there are host-specific patterns in RNA recombination, as vectorborne viruses must replicate in both arthropod and mammalian hosts throughout their lifecycle. First, the investigators will use a Powassan virus infectious clone to infect tick and mammalian cell lines, and carefully measure recombination both intracellularly and extracellularly. They will compare the amount of recombination, including deletions and duplications, and the specific recombination junctions, between host cell types. Finally, the investigators will use the infectious clone of Powassan virus to infect mice and nymphal ticks in order to assess whether observed patterns of recombination are reproducible in vivo. Overall, this work will provide critical insight into the currently-understudied role of recombination in Powassan virus, a representative emerging tick-borne flavivirus. Some recombinant species, such as defective RNAs, are currently being investigated for therapeutic benefit in other viruses, and thus this project may ultimately contribute to antiviral treatment of tick-borne flaviviruses. Important...