In Vitro Antiviral Screening assay Against Viruses of Pandemic Potential v1

This protocol details a primary cytopathic effect (CPE) reduction assay for evaluating antiviral compound efficacy. Vero 76 cell monolayers are prepared in 96-well plates and exposed to eight serial half-log10 concentrations of test compounds, along with infected and uninfected controls, and a known active drug. After virus inoculation and incubation until >80% CPE is observed in virus controls, cell viability is quantified using neutral red staining and spectrophotometric analysis at 540 nm. The 50% effective (EC50) and 50% cytotoxic (CC50) concentrations are determined by regression analysis, and the selectivity index (SI50) is calculated to identify compounds with antiviral activity (EC50 <10 μM, SI50 > 5).