Reference methods in antiviral drug discovery, the plaque reduction assay (PRA) and viral yield reduction, are labor-intensive and low-throughput, whereas tetrazolium-based metabolic assays (MTT) may be unsuitable for viruses that sustain host-cell metabolism during productive infection, including poxviruses. We validated a digital densitometric assay (DDA), which quantifies antiviral and cytotoxic effects in crystal violet-stained monolayers using the open-source software Fiji/ImageJ, against three reference methods, with vaccinia virus strain Western Reserve in Vero cells. Across 38 fungal extracts, DDA agreed substantially with PRA for antiviral activity (Lin's concordance correlation coefficient, CCC = 0.869; 95% limits of agreement −31.5 to +21.1 percentage points) and moderately with MTT for cytotoxicity (CCC = 0.677) and was more precise between replicates (median coefficient of variation 2.5% versus 5.5%). For the test compound FAC22, the half-maximal cytotoxic concentration was 238.3 μM by DDA and 46.4 μM by MTT, a five-fold difference between readouts; the half-maximal effective concentration was 8.83 μM by viral yield reduction and 11.9 μM by DDA over its non-cytotoxic range. In infected cultures MTT separated virus and cell controls by only 1.08 to 1.39-fold, precluding estimation of an effective concentration, against 1.8 to 2.3-fold for DDA. DDA met conventional robustness criteria on the screening plate (Z′ = 0.818 antiviral, 0.861 cytotoxicity), comparable to PRA (Z′ = 0.91), cut reagent consumption by 80 to 90%, and shortened analysis from 20 min to under 1 min per plate. DDA is accurate, resource-efficient and scalable for poxvirus screening, and applicable to other cytopathic viruses pending validation
