Screening protocol for PEDV inhibitors. A Screening procedure. Vero cells were treated with 10 μM compound for 1 h, then infected with PEDV (0.01 MOI) for 1 h. The cells were washed with PBS, then incubated in medium containing 10 μM compound for another 15 h. B Screening process flowchart. The criteria for passing the primary screening were that the compound must have no apparent cytotoxicity and must reduce CPE by at least 50% compared with the DMSO treatment. The criteria for passing the secondary screening were that the compound must leave cells at least 80% viable and inhibit PEDV by more than 80%. Compounds that passed the third screen inhibited PEDV in a dose-dependent manner and had a selective index (SI) higher than 10. C Each dot represents the percent inhibition of each compound. The dots located above the dotted line indicate 80% or greater inhibition. D IFA of infected cells treated with one of the four designated compounds. PEDV N-protein is colored green; a bright field shows CPE. E IC50 and CC50 curves of the four designated compounds. Cell viability is calculated as a percentage of the viability in the cells treated with the compounds divided by that in the DMSO-treated cells. The structure of each compound is inset. F SIs of the four designated compounds.