Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

1. Virus infection quantification

NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.

1. To quantify virus, seed Vero cells at a density of 5 x 10⁵ of cells per well if using a 6-well plate as done in this experiment. Do this a day prior to the infection. Incubate the plated cells overnight to ensure they are confluent for virus quantification.
2. Aliquot 500 µL of serum-free media into multiple microcentrifuge tubes. Insert sterile cotton-tipped swab dipped into the serum-free media-filled tubes. The cotton swabs need to be dipped and wetted for at least 5 min prior to the use.
3. Without disturbing the underlying media, transport the infected porcine cornea plate into a biosafety cabinet. Using the wet cotton swabs, make 3 revolutions clockwise and 3 revolutions anti-clockwise at a diameter of 5 mm from the center of the infected porcine cornea without applying excessive pressure.
4. Insert back the cotton swab into the serum-free media-filled microcentrifuge tube and rotate it clockwise and anti-clockwise 5 times. The metal tip of the cotton swab should be cut short so that it fits entirely into a microcentrifuge tube and the lid can be closed.
5. Place the microcentrifuge tube containing the swabbed cotton tip on a vortex machine and vortex at high speed for 1 min.
6. Perform virus quantification via a plaque assay on these samples.
   NOTE: This quantification step needs to be performed on days 2, 4, and optionally on 6 days post-infection.

2. Virus quantification by Plaque assay

NOTE: To perform a plaque assay, grow and plate Vero cells into a 6-well plate and ensure 90% confluency of cells before the start of the assay. Use a confluent 75 cm² flask of these cells. All the steps below need to be performed inside a biosafety cabinet.

1. Wash the confluent monolayer of Vero cells in the flask with 10 mL of fresh phosphate buffer saline (PBS) after aspirating the culture medium. Repeat the wash step once again with PBS after aspirating the first set of wash solutions.
2. Add 1 mL of 0.05% Trypsin to the cell monolayer. Incubate the flask at 37 °C for 5 min. With the naked eye, ensure that the cells are detached from the inside surface of the flask. If not, wait for another 5 min before examining the flask again. When cells appear to be detached, add 9 mL of whole media to the monolayer to ensure that the cells dislodge completely from the flask surface.
3. Collect all the cells from the flask along with the whole media and place them in a 15 mL centrifuge tube.
4. For every well of the 6 well plates that are used for plaque assay, use 300 µL from the centrifuge tube. Top up each well of the 6-well plate with 2 mL of whole media. Leave the plates in the incubator overnight to allow them to grow and form a confluent monolayer in each well.
5. In order to perform a plaque assay, perform a serial dilution of samples needs to be conducted before the quantification of the virus.
6. Perform a log101 fold dilution of the virus in micro-centrifuge tubes using serum-free media until a dilution of 10^-8 is reached. When at a 10^-3 dilution, transfer 1 mL of the dilution to the monolayer of plated cells after aspirating the growth media on the cells from the 6-well plate, this will be the infection step. Incubate the infected plate at 37 °C incubator for 2 h.
7. Aspirate the existing infection media, wash with PBS twice gently to coat cells, and add 2 mL of methylcellulose-laden media per well to all 6 wells. Incubate for 72 h or until the formation of plaques can be seen. Plaques can be identified by the formation of small gaps between cells in the cell monolayer.
8. Add 1 mL of methanol slowly to the corner of each well, using the wall as a guiding tip. Incubate the 6-well plate at room temperature for 15 min. Slowly aspirate the contents of each well from the plate without disturbing or agitating the cell monolayer.
9. Add 1 mL of crystal violet working solution to each well of 6-well plate, ensuring all cells are covered. Incubate the 6-well plate in the dark for 30 min. Discard the crystal violet solution by aspirating it and dry the wells on a sheet of absorbent paper.
10. Count the number of plaques at the highest dilution well to quantify the total virus content in the starting solution. Repeat the process twice to confirm the viral titer.