Virus Plaque Assay: A Technique to Measure Infectious Herpes Simplex Virus via Quantification of Plaques Formed in Monolayer of Virus-Infected Cells

To begin, prepare serial dilutions of herpes simplex virus, an enveloped infectious virus, in buffer.

Obtain a multi-well plate containing a monolayer of virus-susceptible epithelial cell culture. Replace specific volume of media with diluted viral suspensions, ensuring even monolayer coverage. Allow the virus to adsorb onto the cell surface.

Remove any unadsorbed viruses. Cover the cell monolayer with methylcellulose-containing media, forming a semisolid overlay.

Upon incubation, the viral envelope and cell membrane fuse, releasing the viral nucleocapsid enclosing double-stranded genome into the cytoplasm. Further, viral genome gets released into the cell nucleus.

Using the host's cellular machinery and virion protein, viral genome expresses immediate early viral proteins, which regulate the expression of early genes. Early viral proteins mediate viral DNA replication.

Then, late viral genes express structural proteins, which along with viral genome assemble to produce progeny virion particles. The virus-infected cells lyse, releasing virions.

Methylcellulose restricts virus movement, causing virions to only infect the neighboring cells and subsequent lysis, creating holes in the monolayer.

Post-incubation, remove methylcellulose overlay. Add ethanolic crystal violet solution. Ethanol fixes cells and inactivates viruses, while crystal violet stains the cells. As a result, clear lysed zones or plaques appear on the purple cell monolayer.

Count the plaques in each well at each virus dilution to determine the infectious virus titer or concentration.