Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

Published: March 29, 2024

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. 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. Aliquot 500 µL of serum…

Divulgaciones

The authors have nothing to disclose.

Materials

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

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Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay. J. Vis. Exp. (Pending Publication), e22007, doi: (2024).

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