Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

Abstract

Source: Yadavalli, T., et al. Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals. J. Vis. Exp. (2021)

This video demonstrates the method to perform a plaque assay using a virus obtained from infected porcine cornea. The porcine cornea is used to test the antiviral efficacy of experimental drugs.

Protocol

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 nee....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30 G hypodermic needles.	BD	305128
500 mL glass bottle.	Thomas Scientific	844027
Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification.	Thermofisher Scientific	Herasafe 2030i
Calgiswab 6" Sterile Calcium Alginate Standard Swabs.	Puritan	22029501
Cell scraper - 25 cm	Biologix BE	70-1180 70-1250
centrifuge tube
Crystal violet	Sigma Aldrich	C6158	Store the powder in a dark place
Dulbecco's modified Eagle's medium - DMEM	GIBCO	41966029	Store at 4 °C until use
Ethanol	Sigma Aldrich	E7023
Fetal bovine serum -FBS	Sigma Aldrich	F2442	Aliquot into 50 mL tubes and keep frozen until use
Vero cells	American Type Culture Collection ATCC	CRL-1586
Vortex machine
Methanol
Methylcellulose
Microcentrifuge tube
Porcine Corneas	Park Packaging Co., Chicago, IL		Special order by request
Procedure bench covers - as needed.	Thermofisher Scientific	S42400
Serological Pipettes	Thomas Scientific	P7132, P7127, P7128, P7129, P7137
Serological Pipetting equipment.	Thomas Scientific	Ezpette Pro
Stereoscope	Carl Zeiss	SteREO Discovery V20
Tissue culture flasks, T175 cm2.	Thomas Scientific	T1275
Tissue culture incubators which can maintain 5% CO2 and 37 °C temperature	Thermofisher Scientific	Forma 50145523
Tissue culture treated plates (6-well).	Thomas Scientific	T1006
Trypsin