Plaque Assay–Based Quantification of Viral Load in an Infected Mouse Tissue Sample

Take epithelial cell monolayers. Add diluted supernatants from a virus-infected mouse tissue homogenate.

Incubate for viral adsorption, then wash.

Cover the cells with agar-containing medium, forming a semisolid overlay, and incubate.

The virus enters cells, replicates, and lyses them, releasing new viruses.

The solidified agar restricts viral spread, forming clear zones called plaques.

Remove the overlay. Treat with a fixative to inactivate viruses.

Add a permeabilization buffer to disrupt cell membranes.

Add a blocking buffer to prevent non-specific interactions.

Add primary antibodies. Incubate to allow their interactions with viral nucleocapsid proteins.

Wash and add biotin-conjugated secondary antibodies that bind to the primary antibodies.

Rinse and introduce avidin-biotin-peroxidase complexes, which bind to biotin on the secondary antibodies.

Add chromogenic substrates that react with peroxidase to produce brown precipitates, which stain the plaques.

Count the plaques at different dilutions to quantify the viral load in the tissue sample.

Utilizing the supernatant obtained from the tissue homogenates, perform tenfold serial dilution and infect confluent monolayers of Vero E6 cells with one milliliter of each supernatant dilution in triplicates.

Let the virus adsorb for one hour at 37 degrees Celsius in a humidified 5% carbon dioxide incubator. Once done, wash the cells with one milliliter of 1X PBS and then incubate the cells in two milliliters of post-infection media containing 1% agar in the humidified 5% carbon dioxide incubator at 37 degrees Celsius for 72 hours. After the incubation, inactivate the plates in 10% neutral buffered formalin for 24 hours at four degrees Celsius, ensuring the entire plate is submerged.

Take plates out of biosafety level three and wash the cells three times with one milliliter of 0.5% Triton X-100 for 10 minutes at room temperature. Next, block the permeabilized cells with one milliliter of 2.5 bovine serum albumin or BSA and PBS for one hour at 37 degrees Celsius, followed by incubation in one milliliter of one microgram per milliliter of the SARS-CoV nucleocapsid protein cross-reactive monoclonal antibody 1C7C7 diluted in 2.5 BSA for one hour at 37 degrees Celsius. Wash the cells three times with one milliliter of 1X PBS and develop the plaques using the ABC and diaminobenzene, or DAB, peroxidase substrate kits according to the manufacturer's instructions, then calculate the viral titers as plaque forming units per milliliter.