Development of Assays for Testing Compounds Against Human Corona Viruses in BSL2 Conditions

Abstract

RationaleCOVID-19 caused by the SARS-CoV-2 virus has become a global pandemic emphasizing importance of anti-viral drug discovery process. Testing compounds in cellular systems for potential anti-viral properties against human corona viruses in BSL2 conditions may enable faster and more efficient drug discovery. The aim of this study was to develop cellular assays for testing compounds against three human corona viruses in BSL2 conditions. MethodsHuman corona virus 229E (HCoV-229E), Betacoronavirus 1 (HCoV-OC43) and Human Coronavirus strain NL63 (HCoV-NL63) were used as representative human corona viruses. Viruses were propagated and anti-viral assays developed in MRC5, human lung fibroblast cells, and Vero, monkey lung epithelial cells. Cellular viability, affected by the presence of the viruses, was used as primary read out. Cell viability was assessed by measuring ATP content, dead-cell protease activity, cell proliferation (MTS) and membrane integrity (adenylate kinase AK). Viral inoculum size, incubation time, cell number per well and different cell viability read outs were tested during assay optimization. Assays were considered valid when signal ratio between infected and non-infected cells (S:B) was higher than 3 and Z' was higher than 0.35. Chloroquine and remdesivir, reported as COVID-19 relevant, were tested in the assays. ResultsHuman corona viruses OC43 and 229E were successfully propagated in MRC-5 while NL63 strain was propagated in Vero cell line. Different cell seeding densities (10000 - 30000 cells/well), durations of incubation (3-6 days) and cell viability readouts (ATP content, dead-cell protease activity, MTS reduction, AK release) were assessed. The final assay was done with 10000 MRC-5 cells/well and 20000 Vero cells/well incubated for 4 days with the viruses. Optimal cell viability read out for HCoV-OC43 and HCoV-229E virus-induced cell toxicity was ATP content measurement, while for HCoVNL63 virus, it was protease activity measurement. Chloroquine and remdesivir were active against all three viruses with potencies similar to activities against SARS-CoV-2 virus reported in literature. ConclusionsCellular assays for testing compounds against three human corona viruses in BSL2 conditions were developed. Human corona viruses OC43 and 229E effects on MRC-5 cell viability can be assessed by measuring ATP content. Human corona virus NL63 effects on Vero cells viability can be determined by measuring dead-cell protease activity. In these assay settings, chloroquine and remdesivir were active against all three viruses with the potencies similar to the ones against SARS-CoV-2 virus. Therefore, these assays may be used for testing compounds against human corona viruses in BSL2 conditions.