Advancing high-throughput anti-HCV drug screening: a novel dual-reporter HCV replicon model with real-time monitoring.

Background and purpose: Hepatitis C virus (HCV) infection is a global health concern due to its substantial impact on morbidity and mortality. The burden of diseases related to HCV continues to escalate, particularly as infections progress to late-stage liver conditions, resulting in hepatocellular carcinoma on a global scale. Direct-acting antivirals effectively target HCV replication; however, their unreasonable costs and adverse effects emphasize the need for accessible and efficient therapeutic alternatives with minimal side effects. The primary aim of this study was to devise an HCV replicon system featuring a dual-reporter mechanism to facilitate high-throughput screening of potential novel antiviral agents. Experimental approach: The full-length HCV genome (pJFH1) was used to construct an HCV replicon system. The glycoprotein regions (E1 and E2) were substituted with a red fluorescent reporter, mCherry, enabling visualization of protein synthesis within the replicon. In addition, an adjacent green fluorescent reporter, dBroccoli, was strategically introduced in proximity to the NS5B stop codon to serve as a reliable indicator of HCV replication activity by monitoring the fluorescence signals. Findings/Results: The findings of this study unequivocally validated the effectiveness of the novel HCV replicon system for transfecting Huh-7 cells. Furthermore, the replicon system demonstrated a concentration-dependent response to anti-HCV pharmaceutical agents including telaprevir and sofosbuvir. Conclusion and implications: These compelling results underscored the potential utility of the proposed HCV replicon system as an innovative model for the expeditious high-throughput screening of prospective anti-HCV agents within a short timeframe.