Short CommunicationDevelopment of a multiplex phenotypic cell-based high throughput screening assay to identify novel hepatitis C virus antivirals

•We developed a phenotypic high-throughput assay.•We combined genotype 1 replicon with the genotype 2 HCVcc in single well.•Our phenotypic strategy supports multi-parameter data acquisition from a single well.•Our assay allows to access cross-genotypic activity and compound cytotoxicity.

Hepatitis C virus (HCV) infection is a global health concern with chronic liver damage threatening 3% of the world's population. To date, the standard of care is a combination of pegylated interferon-alpha with ribavirin, and recently two direct acting antivirals have entered the clinics. However, because of side effects, drug resistance and viral genotype-specific differences in efficacy current and potentially also future therapies have their limitations. Here, we describe the development of a phenotypic high-throughput assay to identify new cross-genotype inhibitors with novel mechanism of action, by combining a genotype (gt) 1 replicon with the infectious HCV gt2 cell culture system. To develop this phenotypic multiplex assay, HCV reporter cells expressing RFP-NLS-IPS and gt1b replicon cells expressing NS5A-GFP were co-plated and treated with compounds followed by inoculation with gt2a HCV. At 72 h post treatment, RFP translocation as a marker for HCV infection and GFP fluorescence intensity as a marker for gt1 RNA replication were measured. Additionally, the total cell number, which serves as an indicator of cytotoxicity, was determined. This phenotypic strategy supports multi-parameter data acquisition from a single well to access cross-genotypic activity, provides an indication of the stage of the viral life cycle targeted, and also assesses compound cytotoxicity. Taken together, this multiplex phenotypic platform facilitates the identification of novel compounds for drug development and chemical probes for continuing efforts to understand the HCV life cycle.