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

Abstract

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.

An automated high-throughput enterovirus D68 microneutralization assay platform

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Small Molecule Inhibitor of ATPase Activity of HSP70 as a Broad-Spectrum Inhibitor against Flavivirus Infections

“…We established a cytopathic effect (CPE)-based antiviral drug screening system and performed antiviral screening against ZIKV. , As shown in Figure A–C, AZ exerted a steady CPE protection potential at both 10 and 3 μM in multiple ZIKV infected cell lines. In terms of other flaviviruses, including JEV, YFV, and DENV, AZ could protect the virus-induced CPE in a dose-dependent manner (Figure D–F).…”

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“…[56] Luminescence data were normalized to the average readout from the vehicle control cells on the same plates. Cytotoxicity and viral CPE inhibition were calculated as following: [57,58] % , = IDentif.AI Analysis: IDentif.AI, a dynamic optimization AI-based platform, identifies the drug-dose parameter space by harnessing the quadratic relationship between biological responses to external perturbations, such as drug/dose inputs. [59] IDentif.AI analysis of the…”

“…56 Luminescence data were normalized to the average readout from the vehicle control cells on the same plates. Cytotoxicity and viral CPE inhibition were calculated as follows 57,58 :…”