Assay development and high-throughput antiviral drug screening against Bluetongue virus

Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5000 cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z′-values ≥0.70, Coefficient of Variations ≥5.68 and signal-to-background ratio ≥7.10. This assay was further validated using a 9532 compound library. The fully validated assay was then used to screen the 194 950 compound collection, which identified 693 compounds with >30% CPE inhibition. The 10-concentration dose response assay identified 185 structures with IC50 ≤ 100 μM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease.