Development of a Norway rat hepacivirus reporter for high-throughput quantification of neutralizing antibodies

Hepatitis C virus (HCV) remains a major global health challenge, with vaccine research hindered by a lack of robust immunocompetent animal models. Infection with the HCV-related Norway rat hepacivirus 1 (NrHV) in rats or mice shares HCV-defining characteristics, including chronicity, delayed immune responses, and liver disease, thereby providing immunocompetent small animal models for immune responses and pathology. Infectious NrHV cell culture systems allow studies of host factors and neutralizing antibodies; however, quantification relies on laborious manual counting of infected cells. To permit high-throughput quantification, we engineered culture-adapted NrHV to encode firefly luciferase (FLuc), green fluorescent protein (GFP), or the 11 amino acid HiBiT tag at sites corresponding to those permissive for HCV. While FLuc and GFP constructs were not viable, a reporter with a HiBiT insertion in the NS5A domain III was replication competent and produced infectious virus in rat hepatoma cells. Two mutations in NS5A adapted this virus to levels comparable to wild type, enabling robust luciferase readouts. The NrHV-HiBiT-NS5A reporter was stable, viable in rats, and permitted quantification of luciferase from liver homogenates. Luciferase-based quantification of viral infection after antibody neutralization correlated well with manual quantification. High-throughput luciferase quantification was also used to confirm dependency on the known host factors SR-BI and miR-122 and to assess sensitivity to antivirals. Finally, sensitivity to a specific inhibitor suggested that cyclophilin A, similarly to HCV, may be a host factor for NrHV. This novel reporter system could accelerate characterization of the NrHV model as well as ongoing vaccine studies for HCV.IMPORTANCEDirect-acting antiviral therapy provides an effective cure for chronic hepatitis C virus (HCV) infection, but the infection burden remains high due to cost and availability of therapy and low diagnosis rates. A vaccine, therefore, is critical for global control of viral transmission; however, vaccine development is constrained by a lack of robust immunocompetent animal models. Norway rat hepacivirus 1 (NrHV) infection of rats and mice serves as an effective small animal model for HCV, permitting evaluation of vaccine platforms and candidates. Neutralizing antibody responses are considered crucial for protection and can be quantified using the NrHV cell culture system, however, only through laborious low-throughput methods. We here present an NrHV reporter virus allowing high-throughput quantification of antibody neutralization, virus-host interactions, and antiviral efficacy via luciferase readout. This reporter will facilitate characterization of NrHV and support ongoing vaccine research with the potential for global control of HCV.