Figure 2. Flowchart for the plaque assay.

The whole process includes: cell seeding, to prepare cells in a 6-well plate for the experiments; stock virus titration, to optimize the assay conditions; and compound testing, for the evaluation of the antiviral activity of test compounds. Both stock virus titration and compound testing comprise the following steps: dilute the stock virus, to obtain the desired MOI; infect the cells in the 6-well plate with a small volume (500 µL) of the diluted virus; incubate the 6-well plate in the incubator for 1-2 h, to facilitate viral attachment (virus adsorption); add an avicel overlay containing the test compounds (only for the compound testing experiment); incubate the 6-well plate in the incubator, to allow plaque formation; stain the cells with crystal violet; analyze data. A. Preparation of cells in the 6-well plate for the plaque assay; B. Major steps of stock virus titration in the plaque assay; C. Assessment of antiviral activity of test compounds in the plaque assay.