Plaque Titration Assay for Quantification of Infectious Respiratory Syncytial Virus

Take serial dilutions of a respiratory syncytial virus (RSV) suspension.

Inoculate each dilution into separate wells of a multi-well plate seeded with RSV-susceptible mammalian cells.

Incubate to allow the virus to bind to cell surface receptors, mediate membrane fusion, and release its genome into the cytoplasm.

Next, apply a semi-solid overlay medium to restrict viral diffusion, and incubate.

The infected cells express the viral fusion protein that mediates cell-to-cell fusion, forming multinucleated structures called syncytia.

As viral replication progresses, these syncytia degenerate and form clear zones known as plaques.

Gently shake the plate to remove the overlay and rinse the cells to eliminate debris.

Apply a dye to stain viable cells, leaving the plaques unstained.

Remove the dye, decontaminate the plate in bleach, and rinse with water.

Allow the plate to air-dry.

Select wells with distinct plaques and count them to determine the viral titer.

To perform a plaque titration assay, first make 2X minimum essential medium by diluting commercial 10X MEM with sterile water and adding L-glutamine, 1,000 units per milliliter penicillin, and one milligram per milliliter streptomycin. Shake th