assessing the effectiveness of an antiviral test compound through a viral inactivation assay

Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay

To begin the viral inactivation assay, first, plate 1 times 10 to the fourth cells per well in a 96-well plate. Incubate the cells at 37 degrees Celsius with 5% carbon dioxide overnight for attachment. For infection, prepare Gaussia luciferase reporter-tagged hepatitis C virus or HCV particles, as referenced in the text protocol.
In a sterile tube, mix 100 microliters of 100 micromolar CHLA or PUG with 100 microliters of 10 to the fourth focus-forming units or FFU of HCV. For a positive control, mix HCV with heparin at a final concentration of 1,000 micrograms per milliliter. Incubate at 37 degrees Celsius for three hours.
After three hours, dilute the virus compound mixture 50-fold with 9.8 milliliters of basal medium at room temperature. Then, prepare a new virus compound mixture, and immediately dilute this mixture in basal medium for the zero-hour incubation sample. After removing the incubation medium from the cells, add 100 microliters of the diluted virus drug solution per well in triplicate.
The solution now contains 10 to the second FFU per well. Incubate at 37 degrees Celsius and 5% carbon dioxide for three hours to allow viral infection of the cells. After incubation, remove the viral suspension from the wells and gently wash the cells with 200 microliters of PBS twice.
Incubate the cells in 100 microliters of basal medium for 72 hours for viral replication and release of luciferase reporter into the supernatant. Then, collect the supernatants from the cultures in microtubes, and centrifuge at 17,000 times g for 5 minutes at 4 degrees Celsius to remove cellular debris. Mix 20 microliters of each supernatant with 50 microliters of Gaussia luciferase assay reagent, and measure the luminescence from the luciferase reporter activity in a luminometer.

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1. Viral Inactivation Assay
Note: Examples of incubation period and viral dose for various viruses are listed in Figure 1A. Higher concentrations of the virus can also be tested by increasing the MOI/PFU.
<ol>
	<li>Seed Huh-7.5 cells in a 96-well plate (1 &#215; 104 cells per well) and incubate at 37 &#176;C in a 5% CO2 incubator O/N to obtain a monolayer.</li>
	<li>Incubate the test compounds or controls (final concentrations are: CHLA = 50 &#956;M; PUG = 50 &#956;M; heparin = 1,000 &#956;g/ml; DMSO = 1%) with the HCV particles at 37 &#176;C (Figure 1A, &#39;Long-Term&#39;) in a 1:1 ratio. For example, to a 100 &#956;l virus inoculum containing 1 x 104 FFU, add 100 &#956;l of a 100 &#956;M CHLA working dilution; this yields CHLA treatment at a final concentration of 50 &#956;M.</li>
	<li>Dilute the virus-drug mixture to a &#34;sub-therapeutic&#34; (ineffective) concentration of the test compounds. For example, the ineffective concentration of CHLA and PUG against HCV is at 1 &#956;M31; therefore, this requires a 50-fold dilution of the virus-drug mixture which can be accomplished with 9.8 ml of basal medium (cell culturing medium with 2% FBS). 
	Note: The dilution to sub-therapeutic concentration prevents significant interaction between the test compounds and the host cell surface and allows examination of the treatment effect on the cell-free virions. Note that this dilution is dependent on the antiviral dose response of the test compounds against the particular viral infection and is determined prior to performing this particular assay.</li>
	<li>For comparison, mix the virus with the test compounds and immediately dilute (no incubation period) to sub-therapeutic concentration prior to infection (Figure 1A, &#39;Short-Term&#39;).</li>
	<li>Add 100 &#956;l of the diluted HCV-drug mixture onto the Huh-7.5 cell monolayer (the amount of virus is now at 1 x 102 FFU; final MOI = 0.01) and incubate for 3 hr at 37 &#176;C to allow viral adsorption.</li>
	<li>Following the infection, remove the diluted inocula and gently wash the wells with 200 &#956;l of PBS twice. 
	Note: Perform the washes gently to avoid lifting the cells.</li>
	<li>Apply 100 &#956;l of basal medium to each well and incubate at 37 &#176;C for 72 hr.</li>
	<li>Analyze the resulting infection by assaying the supernatant for luciferase activity.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>GIBCO</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>In vitro toxicology assay kit, XTTbased</td>
			<td>Sigma</td>
			<td>TOX2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4</td>
			<td>GIBCO</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Thermo Scientific</td>
			<td>89087-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002420</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLux Gaussia luciferase assay kit</td>
			<td>New England Biolabs</td>
			<td>E3300L</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax-20/20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate, dihydrate</td>
			<td>Sigma</td>
			<td>71402</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Tai, C. et al., <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp. (2015)
This video demonstrates the viral inactivation assay, showcasing the evaluation of the impact of antiviral agents on viral particles.

<img alt="Figure 1" src="/files/ftp_upload/21998/21998_Figure1.jpg" /> 
Figure 1. Inactivation of viral infections by the test compounds CHLA and PUG. Different viruses were treated with the test compounds for a long period (incubated for 1.5 &#8211; 3 hr before titration; light gray bars) or short period (immediately diluted; dark gray bars) at 37 &#176;C before a dilution to sub-therapeutic concentration and subsequent analysis of infection on the respective host c...

1. Viral Inactivation Assay
Note: Examples of incubation period and viral dose for various viruses are listed in Figure 1A. Higher concentrations of the ...

Take tubes with a recombinant hepatitis C virus, or HCV suspension containing RNA encoding a secreted luciferase reporter.
Add an antiviral test compound and solvent to the test and negative control tubes.
The test compounds bind to HCV glycoproteins, inactivating the virus.
Post-incubation, dilute the mixtures to prevent compound-cell interactions in subsequent steps.
Add these diluted mixtures onto HCV-replication-supporting hepatoma monolayers. Incubate.
The compound-inactivated HCV cannot attach to specific cell surface receptors, while active HCV glycoproteins bind to these receptors, facilitating cellular attachment.
Remove unadsorbed HCVs. Wash with buffer and add media. Incubate for a prolonged duration.
The bound HCV is internalized, releasing viral RNA and leading to viral protein and luciferase synthesis, secreted from the cell.
Collect the luciferase-containing culture supernatants. Centrifuge and add a luciferase substrate to the supernatants.
Luciferase oxidizes the substrate, emitting light.
A higher luminescence in the control well than in the test indicates viral inactivation by the compound.

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Determining Viral Disinfection Efficacy of Hot Water Laundering

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol was developed for research during the coronavirus disease 2019 (COVID-19) pandemic, it is intended to be a framework, adaptable to other virus disinfection studies; it demonstrates the steps for preparing the test virus, inoculating the test material, assessing visual and integrity changes to the washed items due to the laundering process, and quantifying the reduction in viral load. Additionally, the protocol outlines the necessary quality control samples for ensuring the experiments are not biased by contamination and measurements/observations that should be recorded to track the material integrity of the personal protective equipment (PPE) items after multiple laundering cycles. The representative results presented with the protocol use the Phi6 bacteriophage inoculated onto cotton scrub, denim, and cotton face-covering materials and indicate that the hot water laundering and drying process achieved over a 3-log (99.9%) reduction in viral load for all samples (a 3-log reduction is the disinfectant performance metric in U.S. Environmental Protection Agency's Product Performance Test Guideline 810.2200). The reduction in viral load was uniform across different locations on the PPE items. The results of this viral disinfection efficacy testing protocol should help the scientific community explore the effectiveness of home laundering for other types of test viruses and laundering procedures.

In response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a laboratory protocol was developed to test the viral disinfection efficacy of hot water laundering of cloth face coverings, cotton scrubs, and denim pants. The Phi6 virus (bacteriophage) was used as the organism to test disinfection efficacy.

温水洗浄のウイルス消毒効果の決定

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol ...

JoVE Journal

工学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

代替体外ウイルスのカプシド構造健全性の定量方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

免疫・感染学

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.

コキサッキーウイルスA16に対する抗ウイルス候補の同定のためのウイルスエントリーアッセイと分子ドッキング解析の使用

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

Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay

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Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay