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

Mingyu Zhang,Yan Wang,Wanyu Lv,Yong Zhang,Yanyang Zhang

doi:10.1038/s41598-019-42160-7

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 CCID50 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.

Highlights

We describe the development, optimisation, and validation of an automated, cell-based and highthroughput screening assay using existing luminescence-based ATPlite reagents for identifying antiviral compounds that inhibit enterovirus replication
adenosine triphosphate (ATP) levels in cells that were protected from the viral cytopathic effect (CPE) by the antiviral compounds pleconaril and rupintrivir
Outbreaks of HFMD caused by enterovirus A71 (EV-A71) or coxsackievirus A16 have occcured in China and several countries in Southeast Asia[2,3,4,5,6]

Summary

Introduction

Optimisation, and validation of an automated, cell-based and highthroughput screening assay using existing luminescence-based ATPlite reagents for identifying antiviral compounds that inhibit enterovirus replication. Viral cytopathic effect (CPE) inhibition assays use uptake reagents, such as neutral red, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), or crystal violet[15]. Limitations of these colorimetric assays include low throughput, requirement for washing steps, low dynamic range, and low signal-to-noise ratio. Cell debris that remains from infected cells can adhere to the microplate, which can result in high background and low signal-to-noise[15] To overcome these disadvantages, we implemented an alternative assay format using an adenosine triphosphate (ATP) luminescence readout previously developed and validated for a panel of positive-strand RNA viruses[15].

Methods

Results

Conclusion