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…
Divulgations
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 |
Tags
Citer Cet Article
Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay. J. Vis. Exp. (Pending Publication), e22007, doi: (2024).