Development of robust antiviral assays for profiling compounds against a panel of positive-strand RNA viruses using ATP/luminescence readout

The development of antiviral assays using an ATP/luminescence-based readout to profile antiviral compounds against the positive-strand RNA viruses: yellow fever virus (YFV), West Nile virus (WNV), Sindbis virus, and Coxsackie B virus, representing three virus families, is described. This assay readout is based upon the bioluminescent measurement of ATP in metabolically active cells. Antiviral efficacy was determined by measuring the ATP level in cells that were protected from the viral cytopathic effect (CPE) by the presence of antiviral compounds. The antiviral assay parameters were optimized and the assays were validated using a panel of different reference compounds to determine the intra- and inter-assay reproducibility. The signal-to-noise ratios for the yellow fever virus and West Nile virus assays were 7.5 and 36, respectively, comparing favorably with a signal-to-noise ratio of only 1.5 in the yellow fever virus neutral red dye uptake assay, an alternative readout for CPE inhibition. For Coxsackie B and Sindbis viruses, the signal-to-noise ratios were 40 and 50, respectively. These assays are robust, high-throughput, reproducible, and give much improved signal-to-noise ratios than those of dye uptake assays.