2.6. SARS-CoV-2 Plaque Reduction Assay

Plaque reduction assay was performed to plot the 50% maximal effective concentration (EC₅₀) as we previously described with slight modifications [17]. Briefly, VeroE6 cells were seeded at 2 × 10⁵ cells/well in 24-well tissue culture plates on the day before carrying out the assay. After 24 h of incubation, 50 plaque-forming units (PFU) of SARS-CoV-2 were added to the cell monolayer with or without the addition of antiviral agents and the plates were further incubated for 1 h at 37 °C in 5% CO₂ before removal of unbound viral particles by aspiration of the media and washing once with DMEM. Monolayers were then overlaid with media containing 1% low melting agarose (Cambrex Corporation, East Rutherford, NJ, USA) in DMEM and appropriate concentrations of individual compound, inverted and incubated as above for another 72 h. The wells were then fixed with 10% formaldehyde (BDH, Merck, Darmstadt, Germany) overnight. After removal of the agarose plugs, the monolayers were stained with 0.7% crystal violet (BDH, Merck) and the plaques counted. The percentage of plaque inhibition relative to the control (i.e., without the addition of compound) wells were determined for each antiviral agent concentration. EC₅₀ was calculated using a sigma plot (SPSS) in an Excel add-in ED50V10. The plaque reduction assay experiments were performed in triplicate and repeated twice for confirmation.