Project summary Hepatitis C virus (HCV) is an important and underreported infectious disease, causing chronic infection in ~58 million people worldwide. The CDC estimates there are ~67,000 new cases of HCV every year in the US and about 20,000 deaths annually, making HCV more deadly than 60 other infectious diseases combined, including HIV. However, the underlying mechanisms that lead to chronic HCV infection followed by end-stage liver disease are poorly understood. Although chronic hepatitis C can now be effectively treated with direct-acting antivirals, a vaccine to prevent transmission remains a high priority due to extreme treatment costs (up to $50,000 for a 12- week course) and the irreversibility of virally induced liver damage even in “cured” individuals. Efforts to better understand HCV and develop effective vaccines have been limited by the lack of small animal models for this virus, which only robustly infects humans and chimpanzees. In the proposed work, we will build on our substantial research findings to meet this need for an immunocompetent animal model that will allow us to not only systematically analyze the molecular basis of barriers to interspecies HCV transmission but also how such a model could be leveraged for assessing vaccine candidates. Towards this end, we recently identified a highly murine-adapted HCV variant, coined Mad18, with increased replicative capacity in primary murine hepatocytes, which historically only poorly support HCV replication. Through continued passaging of Mad18 in HCV entry factor transgenic (EFT) mice with impaired antiviral signaling (EFT Stat1-/-), we aim to further adapt this variant to a murine physiological environment and define the underpinning biological mechanism (Aim 1). Our considerable experience with studying viral infections in genetically humanized mice will be utilized to also study Mad18 and its derivatives in a variety of new mouse lines, including immunocompetent models, with targeted changes to increase their support of HCV infection (Aim 2). Our observations of persistent HCV infection in hepatocytes from immunocompetent HCV entry factor knock-in mice that also express human TRIM26 (EFT TRIM26[h/h]) put us in a strong position for these in vivo studies. The Ploss lab has made many seminal contributions to the field and will be aided in this important work by our long-standing collaborators Drs. Schwartz (Weill Cornell) and Pietschmann (Twincore, Germany). Our work will advance the field of HCV research by making progress in deciphering the mechanism underlying the exquisitely narrow host tropism and the development of small animal models suitable for studying HCV infection and immune responses, a necessary precursor to improving treatment and developing vaccines.