Screening of Natural Extracts for Inhibitors against Japanese Encephalitis Virus Infection

Guo, Jiao; Jia, Xiaoying; Liu, Yang; Wang, Shaobo; Cao, Junyuan; Zhang, Bo; Xiao, Gengfu; Wang, Wei

HCS assay of drug library screening. To reveal novel targets and inhibitors against JEV infection, an immunofluorescence assay (IFA)-based HCS assay was performed by screening a library of 1,034 natural extracts for the anti-JEV activities ( Fig. 1A ; Table S1 ). As shown in Fig. 1B and C, 851 compounds were selected for the second screening because of their solubility in dimethyl sulfoxide (DMSO) at room temperature and absence of any obvious cytotoxicity on Vero cells. Compounds (50 M) that exerted a Ͼ90% inhibition against JEV were defined as prime candidates; based on this criterion, 23 compounds (2.22%) were selected. A screening to reconfirm the results was then carried out using these prime candidates over a broader concentration range (3. 125 to 50 M). Eight hit compounds (0.77%) were selected based on their concentration-dependent inhibitory effects and a cell viability of Ͼ80% ( Fig. 2A and B) . Among the 8 compounds, ouabain and digoxin were subjected to further investigation because of their high selective indexes (SI, which is defined as 50% cytotoxic concentration [CC 50 ]/50% inhibitory concentration [IC 50 ]) and similar mechanisms (namely, they are cardiac glycoside compounds and act as inhibitors of Na ϩ /K ϩ -ATPase) of Ͼ1,917 and Ͼ969.9, respectively.