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We describe the development, optimisation, and validation of an automated, cell-based and high-throughput screening assay using existing luminescence-based ATPlite reagents for identifying antiviral compounds that inhibit enterovirus replication. Antiviral efficacy was determined by measuring the ATP levels in cells that were protected from the viral cytopathic effect (CPE) by the antiviral compounds pleconaril and rupintrivir. CPE-based assay conditions were optimised at a cell density of 5000 cells/well and a viral infection dose of 100 CCID 50  in 384-well plates. The assay exhibited excellent robustness, with Z′-factor values between 0.75 and 0.82, coefficients of variation between 0.33% and 1.45%, and signal-to-background ratios ranging from 6.92 to 22.6 when testing three enterovirus A71 isolates circulating in China. The assay was also suitable for screening other picornaviruses, such as poliovirus, coxsackievirus, echovirus, and parechovirus.

Automated cell-based luminescence assay for profiling antiviral compound activity against enteroviruses