2.7.3. Plaque Reduction Assay

The antiviral activity was evaluated by plaque reduction assay. The Vero cells were seeded on 24-well plates and infected with the virus inoculum for 1 h at 37 °C with gentle shaking. The virus was diluted to yield 60 plaques/100 µL. A time-of-addition approach was used: (i) the virus inoculum was added on Vero cells and, after the incubation time, the monolayers were covered with a medium containing 0.8% methylcellulose in the presence of OESA and OESY extracts; and (ii) the virus inoculum was added on Vero cells pre-treated with OESA and OESY extracts and, after infection, the monolayers were covered with a medium containing 0.8% methylcellulose. The concentrations used for the antiviral assay were as follows: 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL, and 1 mg/mL. Acyclovir was included as the control at various concentrations (1, 10, and 20 µM). After three days, the cells were fixed, stained with crystal violet, and visualized with an inverted microscope (Leica DMIL, Nuloch, Germany) for plaque detection. After the incubation time, the inoculum was removed, and the monolayers were overlaid with Dulbecco’s Modified Eagle’s Medium containing 0.8% methylcellulose in the presence of the extracts. The plates were incubated at 37 °C with 5% CO₂ for 72 h, and the plaques were visualized by staining the cells with crystal violet.