Key result
An automated cell-based luminescence assay demonstrated excellent robustness for high-throughput screening of enterovirus inhibitors, yielding Z′-factor values between 0.75 and 0.82.
An automated, cell-based luminescence assay was successfully developed and validated as a robust high-throughput screening tool for identifying antiviral compounds against enteroviruses.
Enables robust HTS for enterovirus inhibitors; leaves open clinical translation pending further studies.
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.
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Zhang et al. (2019) studied Enterovirus infection (in vitro). Pleconaril and rupintrivir vs. Virus control (no drug) was evaluated on Assay robustness (Z′-factor) and antiviral efficacy (EC50). An automated cell-based luminescence assay demonstrated excellent robustness for high-throughput screening of enterovirus inhibitors, yielding Z′-factor values between 0.75 and 0.82.
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