Name: a viral attachment assay for screening of antiviral compounds
Uploaded: 2024-02-29T13:13:41.000+00:00
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Description: NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

a viral attachment assay for screening of antiviral compounds

A Viral Attachment Assay for Screening of Antiviral Compounds

For the viral attachment assay, plate 1 x 104 cells per well in a 96-well plate, and incubate overnight for cell attachment. The next morning, first, chill the 96-well plate containing the cell monolayer at 4 degrees Celsius for 1 hour.
Prepare the test compounds and virus mixture on ice. For example, HCV-CHLA, HCV - 1% DMSO, or positive control HCV-heparin solutions with 102 FFU virus for each treatment well. Aspirate the medium from the cell culture wells gently. Then, immediately add 100 microliters of virus test compound mixture in triplicate wells, taking care not to raise the temperature. Incubate the plate in a refrigerator maintained at 4 degrees Celsius for three hours.
Virus particles will attach on the cell surface at 4 degrees Celsius but will not be internalized. Next, aspirate the supernatants. Gently wash the cells twice with 200 microliters of ice-cold PBS. Add 100 microliters of basal medium to each well, and then incubate at 37 degrees Celsius for 72 hours. Finally, for quantitating the released luciferase reporter from the infected cells, collect the supernatant from each sample well and perform the Gaussia luciferase assay.

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NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the biosafety level of the samples being handled. For the purpose of describing the protocols, Gaussia luciferase reporter-tagged&#160;hepatitis C virus (HCV) is used as a model virus. In the context of the representative results, the compounds chebulagic acid (CHLA) and punicalagin (PUG) are used as candidate antivirals that target viral glycoprotein interactions with cell surface glycosaminoglycans during the early viral entry steps. Heparin, which is known to interfere with the entry of many viruses, is used as a positive control treatment in such a context. For basic background on virology techniques, propagation of viruses, determination of virus titer, and expression of infectious dose in plaque forming units (PFU), focus forming units (FFU), or multiplicity of infection (MOI), the reader is referred to reference. For prior examples and optimized conditions used for viruses shown in the representative results, the reader is referred to references&#160;as well as details listed in&#160;Table 1, Figure 1A.
1. Cell Culture, Compound Preparation, and Compound Cytotoxicity
<ol>
	<li>Grow the respective cell line for the virus infection system to be analyzed (Table 1). For HCV, grow the Huh-7.5 cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin G, 200 &#181;g/ml streptomycin, and 0.5 &#181;g/ml amphotericin B.</li>
	<li>Prepare the test compounds and controls using their respective solvents: for example, dissolve CHLA and PUG in dimethyl sulfoxide (DMSO); prepare heparin in sterile double-distilled water. For all subsequent dilutions, use culture media. 
	NOTE: The final concentration of DMSO in the test compound treatments is less than 1% in the experiments; 1% DMSO is included as a negative control treatment in the assays for comparison.</li>
	<li>Determine the cytotoxicity of the test compounds (e.g., CHLA and PUG) on the cells for the viral infection by using cell viability determining reagent such as XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-phenylamino)-carbonyl]-2H-tetrazolium hydroxide):
	<ol>
		<li>For HCV, seed Huh-7.5 cells in a 96-well plate (1 &#215; 104&#160;cells per well) and incubate at 37 &#176;C in a 5% CO2&#160;incubator O/N to obtain a monolayer.</li>
		<li>Apply DMSO control (1%) or increasing concentrations of the test compounds CHLA and PUG (ex. 0, 10, 50, 100, and 500 &#956;M) to the culture wells in triplicate.</li>
		<li>Incubate at 37 &#176;C for 72 hr, then discard the medium in the plate and wash the cells with 200 &#956;l of phosphate-buffered saline (PBS) twice.</li>
		<li>Add 100 &#181;l of assaying solution from the XTT-based&#160;in vitro&#160;toxicology assay kit to each well and incubate the plates at 37 &#176;C for another 3 hr to allow XTT formazan production.</li>
		<li>Determine the absorbance with a microplate reader at a test wavelength of 492 nm and a reference wavelength of 690 nm.</li>
		<li>Calculate the percentage of surviving cells using the following formula: cell viability (%) = At / As &#215; 100%, where &#39;At&#39; and &#39;As&#39; refer to the absorbance of the test compounds and the solvent control (ex. 1% DMSO) treatments, respectively. Determine the concentration of 50% cellular cytotoxicity (CC50) of the test compounds from an analytical software such as GraphPad Prism according to the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>
2. Readout of Viral Infection
NOTE: The readout of viral infection depends on the virus system used and can involve methods such as plaque assays or measuring reporter signals from reporter-tagged viruses. The method for detecting reporter-HCV infection based on the luciferase reporter activity is described below.
<ol>
	<li>Collect the supernatants from the infected wells and clarify at 17,000 x g in a microcentrifuge for 5 min at 4 &#176;C.</li>
	<li>Mix 20 &#956;l of test supernatant to 50 &#956;l of luciferase substrate from the&#160;Gaussia&#160;luciferase assay kit and directly measure with a luminometer according to the manufacturer&#39;s instructions.</li>
	<li>Express HCV infectivity as log10&#160;of relative light units (RLU) for determining viral inhibition (%) and calculate the 50% effective concentration (EC50) of the test compounds against HCV infection using algorithms from GraphPad Prism software according to the manufacturer&#39;s protocol.</li>
</ol>
3. Viral Attachment Assay
NOTE: Examples of incubation period and viral dose for various viruses are listed in&#160;Figure 1A, &#39;Attachment&#39;. 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&#160;cells per well) and incubate at 37 &#176;C in a 5% CO2&#160;incubator O/N to obtain a monolayer.</li>
	<li>Pre-chill the cell monolayers in plates at 4 &#176;C for 1 hr.</li>
	<li>Co-treat the cells with HCV inoculum (MOI = 0.01) and test compounds or controls (final concentrations are: CHLA = 50 &#956;M; PUG = 50 &#956;M; heparin = 1,000 &#956;g/ml; DMSO = 1%) at 4 &#176;C for 3 hr. For example, to a 90 &#956;l virus inoculum containing 1 x 102&#160;FFU, add 10 &#956;l of a 500 &#956;M CHLA working dilution; this yields CHLA treatment at a final concentration of 50 &#956;M and an infection by HCV at MOI = 0.01 on the cell monolayer. 
	NOTE: It is important to carry out the experiment at 4 &#176;C since it allows for virus binding but precludes entry which most efficiently occurs at 37 &#176;C. Perform the addition of virus and test compounds on ice and the ensuing incubation in a 4 &#176;C refrigerator to ensure that the temperature is maintained at 4 &#176;C.</li>
	<li>Remove the supernatant and gently wash the cell monolayer with 200 &#956;l of ice-cold 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 as described in &#39;2. Readout of Viral Infection&#39;.</li>
</ol>
Table 1: Host cell type for viral infection.&#160;The cell type used for each viral infection system described in the representative results is indicated. Additional details regarding the cells can be found in the reference.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Virus</td>
			<td>Cell Type</td>
		</tr>
		<tr>
			<td>HCMV</td>
			<td>HEL</td>
		</tr>
		<tr>
			<td>HCV</td>
			<td>Huh-7.5</td>
		</tr>
		<tr>
			<td>DENV-2</td>
			<td>Vero</td>
		</tr>
		<tr>
			<td>MV</td>
			<td>CHO-SLAM</td>
		</tr>
		<tr>
			<td>RSV</td>
			<td>HEp-2</td>
		</tr>
	</tbody>
</table>

<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, XTT-based</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>Bio-Tek Instrument, Inc.</td>
			<td>ELx800</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>
	</tbody>
</table>

Source: Tai, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

<img alt="Figure 1" src="/files/ftp_upload/21999/21999_Figure1.jpg" /> 
Figure 1. Evaluation of antiviral activities of the test compounds CHLA and PUG against virus attachment and entry/fusion.&#160;(A) The experimental procedure, virus concentration (PFU/well or MOI), and the time of addition and treatment with the test compounds (i, ii, iii) are presented for each virus in the schematics and the associated tables. In virus attachment analysis (light gray bars), mo...

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...

Begin with a multi-well plate containing a pre-chilled human hepatoma cell monolayer.
Treat test wells with a mix of genetically modified hepatitis C virus carrying the luciferase gene and a target compound; treat control wells with the virus alone.
In the test wells, target molecules interact with viral glycoproteins and cell surface receptors, preventing viral attachment.
However, in control wells, viral glycoproteins interact with cell surface receptors, enabling their attachment. A low temperature inhibits viral entry.
Remove the supernatant, introduce a medium, and re-incubate at the optimum temperature, facilitating viral entry.
Upon entry, the virus releases the genetic material, initiating the synthesis of viral proteins and luciferase enzymes, which are secreted into the medium.
Transfer the luciferase-containing supernatant, and introduce a substrate.
The enzyme-substrate interaction produces light in proportion to luciferase levels.
The lower light intensity in the test wells, compared to the control wells, indicates the antiviral property of the target compound.

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A Viral Attachment Assay for Screening of Antiviral Compounds

Transcripción

A Viral Attachment Assay for Screening of Antiviral Compounds