Cytopathic effect (CPE) inhibition assay

Based on the results of cell cytotoxicity assay, the pleconaril at concentrations 7.5, 5 and 2.5 μg/50 μL and, ribavirin at 15, 10 and 5 μg/50 μL were tested for their antiviral activities. Each concentration of the drugs was tested in triplicate with logarithmic dilutions of FMDV O, A and Asia 1 serotypes. For pleconaril treatment, two 96 well cell culture plates, one each for 24 and 48 h were maintained and for ribavirin, separate plates were maintained to harvest the samples at 2, 4, 6, 12, 24 and 48 h intervals. The drug, virus and healthy cell control wells were also set up in each plate to validate the assay. At the end of each incubation period, plates were visualized under light microscope (20x) for the appearance of virus induced CPE, if any, and the virus titers were calculated as median tissue culture infective dose (TCID₅₀) [35] for each concentration of drugs at different time periods for both pleconaril and ribavirin. Then the cells were frozen at − 80 °C. After 24 h, the plates were thawed at room temperature and the contents of each well of the plates was harvested and mixed with 350μL of maintenance media and the samples were used for sandwich ELISA, PCR and real time PCR. The antiviral activity of pleconaril and ribavirin was expressed as percentage inhibition (PI) using the formula, Percent Inhibition (PI) = [1 − (Antilog of test titer/Antilog of control titer)] × 100. A drug is considered as biologically effective, when the PI is > 80%, at its MNTC [27]. Fold change values were considered as significant, if it is more than tenfold on account of differences between dilutions.