An estimated 71 million people worldwide are infected with hepatitis C virus (HCV). The lack of small animal models has impeded studies of antiviral immune mechanisms. Here we show that an HCV-related hepacivirus discovered in Norway rats can establish high titer hepatotropic infections in laboratory mice with immunological features resembling those seen in human viral hepatitis. While immune-compromised mice developed persistent infection, immune-competent mice cleared the virus within 3–5 weeks. Acute clearance was T cell dependent and associated with liver injury. Transient depletion of CD4+ T cells prior to infection resulted in chronic infection, characterized by high levels of intrahepatic regulatory T cells and expression of inhibitory molecules on intrahepatic CD8+ T cells. Natural killer cells controlled early infection but were not essential for viral clearance. This model may provide mechanistic insights into hepatic antiviral immunity, a prerequisite for the development of HCV vaccines.

Equine hepacivirus (EqHV); hepatitis C virus (HCV); GB-virus B (GBV-B); non-primate hepacivirus, NPHV; untranslated region (UTR); peripheral blood mononuclear cell (PBMC); interferon (IFN); genome equivalents (GE); RNA-sequencing (RNA-seq); single cell RNA-sequencing (scRNA-seq); luciferase immunoprecipitation system (LIPS); equine pegivirus (EPgV); Theiler's disease associated virus (TDAV); human pegivirus (HPgV/GBV-C); differentially expressed (DE); IFN-stimulated genes (ISGs); rodent hepacivirus (RHV); simian pegivirus (SPgV); simian immunodeficiency virus (SIV); lower level of quantification (LLOQ); fold change (FC); spot forming units (SFU)

Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis, and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed but would enable detailed studies of the virus life cycle crucial for designing animal experiments to model HCV infection. Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liver-derived cells did not readily support the complete RHV life cycle, but replicon-containing cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bicistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHV-rn1 NS3-4A protease cleaves a human mitochondrial antiviral signaling protein reporter, providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals but not by NS3 protease inhibitors. The microRNA-122 antagonist miravirsen inhibited RHV-rn1 replication, demonstrating the importance of this HCV host factor for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology, and characterization of virus-host interactions, thereby providing important complements to in vivo systems. IMPORTANCE A vaccine against hepatitis C virus (HCV) is crucial for global control of this important pathogen, which induces fatal human liver diseases. Vaccine development has been hampered by the lack of immunocompetent animal models. Discovery of rodent hepacivirus (RHV) enabled establishment of novel surrogate animal models. These allow robust infection and reverse genetic and immunization studies of laboratory animals, which develop HCV-like chronicity. Currently, there are no RHV in vitro systems available to study tropism and molecular virology. Here, we established the first culture systems for RHV, recapitulating the intracellular phase of the virus life cycle in vitro. These replicon systems enabled identification of replication-enhancing mutations and selection of cells highly permissive to RHV replication, which allow study of virus-host interactions. HCV antivirals targeting NS5A, NS5B, and microRNA-122 efficiently inhibited RHV replication. Hence, several important aspects of HCV replication are shared by the rodent virus system, reinforcing its utility as an HCV model.