A Plaque-Based Assay to Assess Antiviral Activity of a Phytochemical Agent

Take epithelial cell monolayers.

Add carboxymethylcellulose-containing media with virus alone as the negative control, virus with an antiviral drug as the positive control, and virus with the phytochemical agent as the test.

Incubate for viral adsorption. Carboxymethylcellulose forms a semisolid overlay that restricts viral diffusion.

In the negative control, the virus enters the cell, delivers its genome into the nucleus, replicates, and produces viral proteins that assemble into new virions.

The infected cells lyse, forming clear zones called plaques.

In the positive control, the antiviral drug blocks viral genome replication, reducing viral spread and plaque formation.

In the test, the phytochemical inhibits viral attachment and entry, lowering infection and plaque numbers.

Treat the monolayers with a fixative. Add a dye that stains intact cells dark blue, leaving plaques clear.

Fewer plaques in the test well, similar to the positive control and lower than in the negative control, confirm the phytochemical’s antiviral activity.