Development of a cell-based assay for high-throughput screening of inhibitors against HCV genotypes 1a and 1b in a single well

The Hepatitis C (HCV) replicon system is a useful tool for the high-volume screening of inhibitors of HCV replication. In this report, a cell-based assay has been described, which monitors the inhibition of HCV genotypes 1a and 1b as well as cytotoxicity, from a single well of a 96-well plate. A mixture of two stable replicon cell lines was used: one containing a 1a-H77 replicon expressing a firefly luciferase reporter, and the other one containing a 1b-N replicon with a secreted alkaline phosphatase reporter, thus allowing us to monitor replication of two HCV genotypes in the same well. Cytotoxicity was measured using the Resazurin cytotoxicity assay. The assay was validated with known HCV inhibitors and showed that the antiviral activity and cytotoxicity of compounds were reproducibly measured under screening conditions. It was also showed that the assay's signal-to-noise ratio and Z′ coefficient were suitable for high-throughput screening. A panel of HCV inhibitors showed a good correlation between EC50 and TD50 values for 1a and 1b replicon activity and cytotoxicity measured using either a single replicon format or mixed replicon format. Thus, the use of this mixed replicon format provides an economical method for simultaneous measurement of compound activity against two HCV genotypes as well as cytotoxicity, thereby reducing cost of reagents and labor as well as improving throughput.