Experimental and virus-specific drivers of EC₅₀ variability in antiviral assays evaluated using mathematical modeling

The half-maximal effective concentrations (EC₅₀) reported for specific antiviral agents often vary widely across in vitro studies. The cause for this may be due to differences in viral strains, cell types, and experimental design choices. Variability in EC₅₀ estimates complicates potency interpretation and cross-study comparison. In this study, we applied a mechanism-based mathematical target cell-limited model to examine the impact of virus-specific parameters and different experimental design choices on EC₅₀ estimation. Empirical EC₅₀ values were estimated from simulated concentration-response experiments for different virus-specific properties and experimental conditions. Among viral parameters, EC₅₀ changed more than 20-fold across the range of infection rate (β) and over 50-fold across viral production rate (ρ), whereas clearance rate had only minor effects. Among experimental factors, higher initial cell number (T₀) and later sampling time changed EC₅₀ by more than 15-fold, while delayed time of adding drug increased EC₅₀ by approximately 50 %. When both β and ρ were high, EC₅₀ changed nearly 900-fold. Overall, our findings demonstrate that EC₅₀ is highly sensitive to several virus-specific parameters and experimental design choices, and provides quantitative directions which may guide the design and interpretation of antiviral in vitro assays evaluating pharmacological activity.