Development of a Norway Rat Hepacivirus Reporter for High-Throughput Quantification of Neutralising Antibodies

Hepatitis C virus (HCV) remains a major global health challenge, with vaccine research hindered by lack of 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 neutralising antibodies, however, quantification relies on laborious manual counting of infected cells. To permit high-throughput quantification, we engineered culture adapted NrHV to encode green fluorescent protein (GFP), firefly luciferase (FLuc), or the 11 amino acid HiBiT tag at sites corresponding to those permissive for HCV. While GFP and FLuc constructs were not viable, a reporter with a HiBiT insertion in 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 wildtype, enabling robust luciferase readouts and stability. The NrHV-HiBiT-NS5A reporter was viable in rats and permitted quantification of luciferase from liver homogenates. Luciferase-based quantification of viral infection after antibody neutralisation 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 also be a host factor for NrHV. This novel reporter system could accelerate characterisation of the NrHV model as well as ongoing vaccine studies for HCV. ImportanceDirect acting antiviral therapy provides an effective cure for hepatitis C virus (HCV) infection, but the infection burden remains high due to cost, availability of therapy, and low diagnosis rates. A vaccine therefore is critical for global control of transmission, however development is constrained by a lack of 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. Neutralising antibody responses are considered crucial in 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 neutralisation, virus-host interactions, and antiviral efficacy via luciferase readout. This reporter will facilitate characterisation of NrHV and support ongoing vaccine research with the potential for global control of HCV.