determining antiviral efficacy of experimental drugs through a plaque assay

Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

To quantify the amount of virus collected from each cornea, prepare a log10 one-fold dilution of the virus in serum-free medium in microcentrifuge tubes until a dilution of 1 x 10-8 is reached.
After aspirating the growth medium from each well of cells, transfer 1 milliliter of each virus dilution, starting at 1 x 10-3 to the plated cell monolayers, and place the infected cells in the cell culture incubator for two hours. At the end of the incubation, gently wash each well 2 times with PBS before adding 2 milliliters of methylcellulose-laden medium to each well for a 72-hour incubation, or until the formation of plaques can be observed.
Upon plaque observation, slowly add 1 milliliter of methanol to the corner of each well for a 15-minute incubation at room temperature. At the end of the incubation, slowly aspirate the contents from each well without disturbing the cell monolayer.
Next, label each well with 1 milliliter of crystal violet working solution, taking care that all of the cells are covered for a 30-minute incubation protected from light. At the end of the incubation, discard the solution and dry the wells on a sheet of absorbent paper. Then, count the number of plaques at the highest dilution well to quantify the total virus content in the starting solution 3 times.

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1. Virus infection quantification
NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.
<ol>
	<li>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.</li>
	<li>Aliquot 500 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the microcentrifuge tube containing the swabbed cotton tip on a vortex machine and vortex at high speed for 1 min.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Add 1 mL of 0.05% Trypsin to the cell monolayer. Incubate the flask at 37 &#176;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.</li>
	<li>Collect all the cells from the flask along with the whole media and place them in a 15 mL centrifuge tube.</li>
	<li>For every well of the 6 well plates that are used for plaque assay, use 300 &#181;L 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.</li>
	<li>In order to perform a plaque assay, perform a serial dilution of samples needs to be conducted before the quantification of the virus.</li>
	<li>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 &#176;C incubator for 2 h.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G hypodermic needles.</td>
			<td>BD</td>
			<td>305128</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL glass bottle.</td>
			<td>Thomas Scientific</td>
			<td>844027</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification.</td>
			<td>Thermofisher Scientific</td>
			<td>Herasafe 2030i</td>
			<td></td>
		</tr>
		<tr>
			<td>Calgiswab 6&#34; Sterile Calcium Alginate Standard Swabs.</td>
			<td>Puritan</td>
			<td>22029501</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper - 25 cm</td>
			<td>Biologix BE</td>
			<td>70-1180 70-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Sigma Aldrich</td>
			<td>C6158</td>
			<td>Store the powder in a dark place</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium - DMEM</td>
			<td>GIBCO</td>
			<td>41966029</td>
			<td>Store at 4 &#176;C until use</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum -FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td>Aliquot into 50 mL tubes and keep frozen until use</td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>American Type Culture Collection ATCC</td>
			<td>CRL-1586</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex machine&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine Corneas</td>
			<td>Park Packaging Co., Chicago, IL</td>
			<td></td>
			<td>Special order by request</td>
		</tr>
		<tr>
			<td>Procedure bench covers - as needed.</td>
			<td>Thermofisher Scientific</td>
			<td>S42400</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Thomas Scientific</td>
			<td>P7132, P7127, P7128, P7129, P7137</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipetting equipment.</td>
			<td>Thomas Scientific</td>
			<td>Ezpette Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Carl Zeiss</td>
			<td>SteREO Discovery V20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks, T175 cm2.</td>
			<td>Thomas Scientific</td>
			<td>T1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture incubators which can maintain 5% CO2 and 37 &#176;C temperature</td>
			<td>Thermofisher Scientific</td>
			<td>Forma 50145523</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture treated plates (6-well).</td>
			<td>Thomas Scientific</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Yadavalli, T., et al. <a href="https://app.jove.com/v/62195">Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals.</a> 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.

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

	To ...

Begin with a virus-containing sample obtained from an infected porcine cornea treated with an antiviral drug.
Perform a serial dilution of the sample to create a sequence of decreasing virus concentrations.
Optimum dilution prevents single-cell infection by multiple viruses.
Introduce the diluted samples to a host-cell monolayer and incubate.
The virus attaches to the host-cell receptor, enters the cell, and initiates its replication. The infected cell lyses, releasing virions.
Add methylcellulose, a viscous substance that confines the viruses to the nearby cells.
Subsequently, the viruses cause cell death and plaque formation in the infected region.
Add methanol to fix the cells.
Use crystal violet to stain the live cells.
Observe plaques as clear zones indicating infected cells and cell death, surrounded by crystal violet-stained uninfected cells.
Finally, count the number of plaques at the highest dilution to quantify the virus titer in the starting solution to determine the efficacy of the antiviral drug.

94 visualizações. Determinando a eficácia antiviral de drogas experimentais por meio de um ensaio de placa

Vídeo: Determinando a eficácia antiviral de drogas experimentais por meio de um ensaio de placa - Experimento

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Concentração Determinação Viral Através de placa Ensaios: Usando tradicionais e novos Sistemas de sobreposição

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Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

The goal of the protocol is to illustrate the different assays relating to viral entry that can be used to identify candidate viral entry inhibitors.

Uso de ensaios de entrada viral e análise de ancoragem molecular para a identificação de candidatos antivirais contra coxsackievirus a16

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for ...

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

Plaquing de Vírus Herpes Simplex

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

Transcrição

Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay