Identification of host factors required by the tick-borne Powassan virus

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PROJECT SUMMARY Flaviviruses cause significant morbidity and mortality worldwide. The sudden emergence and spread of viruses such as Zika illustrate our vulnerability and emphasize the need for preparedness against related emerging viruses. In addition to mosquito-borne flaviviruses, members of tick-borne flaviviruses pose an increasing global threat for which we lack effective antivirals. Powassan virus (POWV) is an emerging virus transmitted by the bite of infected Ixodes ticks. The seroprevalence of POWV in humans is mostly unknown as infections may often be asymptomatic. However, symptomatic POWV infections may progress in to severe and sometimes fatal encephalic disease with mortality rates of ~10% and surviving patients often suffer from debilitating long-term neurologic symptoms. The severe clinical consequences of infections with POWV and other tick-borne flaviviruses and the lack of specific treatments highlight an urgent need for a better understanding of these viruses in their tick and mammalian hosts so that preventative and therapeutic treatments can be developed. The goal of this project is to identify host factors required by POWV that may also impact a wide range of existing and emerging flaviviruses. These host proteins may represent suitable targets for antiviral interventions, and we hypothesize that impaired virus replication due to their inhibition may allow innate and adaptive immunity to control the infection. Further, we propose to compare a panel of flaviviruses, including POWV, and their requirements for the identified host factors in both vertebrate and invertebrate cells. We believe these approaches will allow us to prioritize candidate genes for future drug development studies. The proposed studies will utilize CRISPR/Cas9 screening technology to knock out every gene in the human genome with the goal of identifying host factors that facilitate POWV infection. We will use a similar approach in a targeted, arrayed format to evaluate the identified host factors in various human and tick cell lines during infection with diverse flaviviruses. We expect that this work will broaden our understanding of tick- and mosquito-borne flaviviruses, specifically in terms of clinical phenotype (encephalitic vs hemorrhagic disease) and the shared need for host factors. In addition, it will help to define which genes govern host species and tissue tropism. Together, this work will provide a wealth of new insights into many aspects of flavivirus biology.