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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>

a viral attachment assay for screening of antiviral compounds

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.&#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...

A Viral Attachment Assay for Screening of Antiviral Compounds

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.

a-viral-attachment-assay-for-screening-of-antiviral-compounds

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

207 ビュー. 出典: Tai, C. et al., 早期ウイルスエントリーアッセイ for the Identification and Evaluation of Antiviral Compounds. J. Vis. Exp.(2015年) このビデオでは、抗ウイルス化合物をスクリーニングするためのウイルス付着アッセイを実演しています。遺伝子組み換えC型肝炎ウイルスとルシフェラーゼを用いて、ウイルス単独のウェルと比較して、共治療したウェルの光強度が低いことからわかるよ?...

ビデオ: 抗ウイルス化合物のスクリーニングのためのウイルス付着アッセイ - 概念

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

C型肝炎ウイルスに対するヒトラクトフェリンの細胞取り込みおよびそれに関連する抗ウイルス活性を監視するために、免疫蛍光

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

JoVE Journal

免疫・感染学

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

高スループットの Cre Lox 活性化ウイルスの膜融合アッセイの HIV-1 ウイルス膜融合阻害剤を識別するために

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

ウイルス感染のライブイメージングと定量化のためのルシフェラーゼ蛍光レポーターインフルエンザウイルス

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

A Viral Attachment Assay for Screening of Antiviral Compounds

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A Viral Attachment Assay for Screening of Antiviral Compounds