Evaluating EGCG's Antiviral Efficacy via Plaque Assay

Vero cells were cultured in 6-well plates until 100% confluent. The virus was treated with 75 µM EGCG or EGCG-S for one hour. The untreated virus served as the control. Virus dilutions ranging from 10⁻³ to 10⁻⁷ were prepared. Cells were infected with the diluted virus for one hour and the unadsorbed virus was aspirated. Plaque assay method is a modification of protocol previously reported in Adams et al. [42 (link)]. After one hour of incubation, cells were overlaid with plaque media consisting of bacteriological agar containing 3X Eagle medium (Gibco Invitrogen Corporation, Grand Island, NY, USA), 1.5 mL of 5% sodium bicarbonate (Gibco Invitrogen Corporation, Carlsbad, CA, USA), 0.5 mL FBS, 0.1 mL DEAE-dextran (ICN Biomedicals Incorporated, Aurora, OH, USA), 0.1 mL penicillin/streptomycin (Cambrex, Walkersville, MD, USA), with 0.05 mL gentamicin and 0.6% bacteriological agar (Oxoid Limited, Baskingstoke, Hampshire, England). Both solutions were placed in a 41 °C water bath, combined 1:1, and cells were overlaid with 3 mL of plaque media.
After 72 h of incubation at 5% CO₂, cells were stained with crystal violet and plaques were counted followed by the calculation of mean and standard deviation.