The authors are grateful to Dr. Joshua Beckham at the University of Texas at Austin for technical support with molecular docking. This study was partly supported by funding from the Ministry of Science and Technology of Taiwan (MOST107-2320-B-037-002 to C.-J.L. and L.-T.L.; MOST106-2320-B-038-021 and MOST107-2320-B-038-034-MY3 to L.-T.L.).

NOTE: All cell culture and virus infections must be conducted in certified biosafety hoods that are appropriate for the biosafety level of the samples being handled. The two tannin-class of small molecules chebulagic acid (CHLA) and punicalagin (PUG), that were observed to efficiently block CVA16 infection9, are used as examples of candidate inhibitory agents. For basic principles in virology techniques, virus propagation, determination of virus titer, and concepts of plaque forming units (PFU) or multiplicity of infection (MOI), the reader is referred to reference10.
1. Cell Culture, Virus Preparation, Compound Preparation, and Compound Cytotoxicity
<ol>
	<li>Human rhabdomyosarcoma (RD) cells are host cells permissive to CVA16 infection11. Grow the RD cells in 10 mL of Dulbecco&#8217;s modified Eagle&#8217;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 in T-75 flasks at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Prepare CVA16 by propagating the virus in RD cells and determine viral titer in PFU/mL. For optimized protocol, please refer to reference11.</li>
	<li>Prepare the test compounds and controls using their respective solvents: for example, dissolve CHLA and PUG in dimethyl sulfoxide (DMSO). For all infection steps, the basal medium consisted of DMEM plus 2% FBS and antibiotics. 
	NOTE: The final concentration of DMSO in the test compound treatments is equal to or below 0.25% in the experiments; 0.25% DMSO is included as a negative control treatment in the assays for comparison.</li>
	<li>Perform cytotoxicity assay of the test compounds on the RD cells using a cell viability determining reagent such as XTT ((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-phenylamino)-carbonyl]-2H-tetrazolium hydroxide). For detailed protocol, please refer to reference12. Determine the cytotoxicity concentrations of the test compounds using an analytical software such as GraphPad Prism according to manufacturer&#8217;s protocol. Drug concentrations that do not significantly influence the cell viability (&#160;&#8805;95% viable cells) are used for the remainder of the study.</li>
</ol>
2. Time-of-drug-addition Assay
<ol>
	<li>To evaluate the influence of drugs on host cells prior to viral infection (pretreatment)
	<ol>
		<li>Seed RD cells in 12-well plates at a seeding density of 2 x 105 cells/well. Incubate overnight at 37 &#176;C in a 5% CO2 incubator to obtain a monolayer.</li>
		<li>Treat RD cells with test compounds at non-cytotoxic concentrations (determined from step 1.4) in 1 mL of basal media volume for 1 h or 4 h.</li>
		<li>Wash cells with 1-2 mL of PBS before adding 50 PFU/well of virus in basal medium (final volume of the inoculum is 300 &#956;L) for 1 h. Rock the plate every 15 min.</li>
		<li>Following the infection, wash the monolayers again using PBS, then overlay with 1 mL of basal media containing 0.8% methylcellulose for further incubation at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>After 72 h of incubation, remove the overlay media and wash the wells using 2 mL of PBS.</li>
		<li>Fix the wells using 0.5 mL of 37% formaldehyde for 15 min.</li>
		<li>Remove the supernatant and wash again using PBS.</li>
		<li>Stain the wells using 0.5 mL of 0.5% crystal violet solution. Then remove the stain solution within 2 min and wash the wells with a gentle stream of water before air drying.</li>
		<li>Count the viral plaques by placing the plate on a white-light box. Calculate the percent (%) CVA16 infection as follows: (Mean # of plaque virus+drug / Mean # of plaque virus+DMSO control) &#215; 100%.</li>
	</ol>
	</li>
	<li>To evaluate the effect of adding the drugs and the virus concurrently (co-addition)
	<ol>
		<li>Seed RD cells in 12-well plates at a seeding density of 2 x 105 cells/well. Incubate overnight at 37 &#176;C in a 5% CO2 incubator to obtain a monolayer.</li>
		<li>Treat RD cells with the test compounds at the appropriate concentrations and 50 PFU/well of CVA16 (final volume of the inoculum is 300 &#956;L) simultaneously for 1 h. Rock the plate every 15 min.</li>
		<li>Wash cells with 1 mL of PBS and then overlay with 1-2 mL of basal media containing 0.8% methylcellulose for further incubation at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>Stain viral plaques with crystal violet after 72 h post infection and determine the% CVA16 infection as described above.</li>
	</ol>
	</li>
	<li>To evaluate drug treatment effect after viral entry (post-infection)
	<ol>
		<li>Seed RD cells in 12-well plates at a seeding density of 2 x 105 cells/well. Incubate overnight at 37 &#176;C in a 5% CO2 incubator to obtain a monolayer.</li>
		<li>Inoculate the RD cells with 50 PFU/well of CVA16 (final volume of the inoculum is 300 &#956;L) for 1 h. Rock the plate every 15 min.</li>
		<li>Wash the wells with 1-2 mL of PBS and overlay the cells with basal media containing 0.8% methylcellulose and the appropriate concentrations of test compounds.</li>
		<li>Stain viral plaques with crystal violet and count after 72 h post infection and determine the% CVA16 infection incubations described above. 
		NOTE: Perform all PBS washes gently to avoid lifting the cells.</li>
	</ol>
	</li>
</ol>
3. Flow Cytometry-based Binding Assay
<ol>
	<li>Seed RD cells in 12-well plates at a seeding density of 2 x 105 cells/well. Incubate overnight at 37 &#176;C in a 5% CO2 incubator to achieve a monolayer.</li>
	<li>Pre-chill the cell monolayer at 4 &#176;C for 1 h.</li>
	<li>Infect RD cells with CVA16 (MOI = 100) in the presence and absence of the test compounds for 3 h at 4 &#176;C. 
	NOTE: Perform the viral inoculation on ice and the ensuing incubation in a 4 &#176;C refrigerator to maintain the temperature at 4 &#176;C, which permits viral binding but not entry.</li>
	<li>Remove virus inoculum and wash once with 1-2 mL of ice-cold PBS.</li>
	<li>Lift cells by adding 1 mL of ice-cold dissociation buffer to the wells on ice for 3 min, before collecting the cells and resuspending them in ice-cold flow cytometry buffer (1x PBS plus 2% FBS).</li>
	<li>Wash cells twice using the ice-cold flow cytometry buffer and fix the cells with 0.5 mL of 4% paraformaldehyde for 20 min on ice.</li>
	<li>Wash the cells using PBS to remove any unbound or weakly bound viruses, and then stain the cells with 1 mL of anti-VP1 antibody (1:2,000; diluted in PBS containing 3% BSA) on ice for 1 h, followed by incubation with a secondary Alexa 488-conjugated anti-mouse IgG (1:250; diluted in PBS containing 3% BSA) on ice for 1 h. Perform PBS washes (3 times) following each antibody treatment.</li>
	<li>Resuspend the cells in 0.5 mL of the ice-cold flow cytometry buffer and perform flow cytometry analysis on a flow cytometer using standard procedures. Present data in histograms using the associated software and quantitate for bar graph representation.</li>
</ol>
4. Viral Inactivation Assay
<ol>
	<li>Perform the viral inactivation assay as previously described12 using the following conditions: 
	- A starting concentration of 106 PFU/mL of CVA16. 
	- RD cell monolayers in 12-well plates from a seeding density of 2 x 105 cells/well. 
	- 50-fold dilution for titrating out the drug compounds resulting in a final virus concentration of 50 PFU/well. 
	- Wash steps using 1-2 mL of PBS. 
	- Overlay media containing 0.8% methylcellulose.</li>
	<li>Perform final readout of viral infection using the crystal violet staining of viral plaques procedure as detailed above.</li>
</ol>
5. Molecular Docking Analysis
<ol>
	<li>Download 3D molecules of test compounds from PubChem (https://pubchem.ncbi.nlm.nih.gov/). If molecules do not have a 3D structure uploaded, download the 2D structures or use the SMILES string sequence and transform into 3D molecules via a molecular program (e.g. CORINA).</li>
	<li>Download viral biological assembly unit from RCSB Protein Data Bank (https://www.rcsb.org/) and prepare the viral structure model using a biocomputing program (e.g. UCSF Chimera). For example, in the case of CVA16 mature virion crystal structure (PDB: 5C4W)3, delete solvents from the PDB file, replace incomplete side chains using data from the Dunbrach 2010 Rotamer Library, and add hydrogens and charges to the structure as previously reported13. Docking targets can be any relevant viral proteins for the intended analysis with a biological assembly information (Protein Data Bank).</li>
	<li>Dock test compounds onto the prepared virus unit using for example UCSF Chimera, and analyze the output files with a visualization software (e.g., Autodock Vina, PyMol):
	<ol>
		<li>Upload the test compound file into UCSF Chimera as the &#8216;ligand&#8217; and perform blind docking by selecting the whole prepared viral protein as the &#8216;receptor&#8217;. Use the computer mouse or trackpad to resize the search volume to the entire &#8216;receptor&#8217;. In &#8216;Advanced options&#8217;, allow the number of binding modes to be at maximum. Docking frames will be automatically ranked from highest to lowest binding energy.</li>
		<li>(Optional) Further to blind docking, confine the docking site onto the viral protein in regions of interest derived from the blind docking results using the mouse or trackpad again to reduce the search volume (e.g., 100 &#197; x 100 &#197; x 100 &#197;). This step helps confirm the blind docking results and increases specificity.</li>
		<li>Use a molecular graphics system (e.g., PyMol) to analyze the binding modes&#8217; positions by uploading the docking file. Find polar contacts from the compound to the viral protein by selecting the &#8216;ligand&#8217; and identifying polar contacts with the option &#8216;to any atoms&#8217;; examine the results.</li>
	</ol>
	</li>
</ol>

<ol><li>Legay, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fatal+coxsackievirus+A-16+pneumonitis+in+adult%5BTitle%5D)+AND+%22Emerging+Infectious+Diseases%22%5BJournal%5D)">Fatal coxsackievirus A-16 pneumonitis in adult.</a> Emerging Infectious Diseases. 13, 1084-1086 (2007).</li>
<li>Wang, C. Y., Li Lu,, Wu, F., H, M., Lee, C. Y., Huang, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fatal+coxsackievirus+A16+infection%5BTitle%5D)+AND+%22Pediatric+Infectious+Disease+Journal%22%5BJournal%5D)">Fatal coxsackievirus A16 infection.</a> Pediatric Infectious Disease Journal. 23, 275-276 (2004).</li>
<li>Ren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structures+of+coxsackievirus+A16+capsids+with+native+antigenicity%3A+implications+for+particle+expansion%2C+receptor+binding%2C+and+immunogenicity%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Structures of coxsackievirus A16 capsids with native antigenicity: implications for particle expansion, receptor binding, and immunogenicity.</a> Journal of Virology. 89, 10500-10511 (2015).</li>
<li>Nishimura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+P-selectin+glycoprotein+ligand-1+is+a+functional+receptor+for+enterovirus+71%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71.</a> Nature Medicine. 15, 794-797 (2009).</li>
<li>Yamayoshi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scavenger+receptor+B2+is+a+cellular+receptor+for+enterovirus+71%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Scavenger receptor B2 is a cellular receptor for enterovirus 71.</a> Nature Medicine. 15, 798-801 (2009).</li>
<li>Yamayoshi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+SCARB2-dependent+infection+by+coxsackievirus+A7%2C+A14%2C+and+A16+and+enterovirus+71%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Human SCARB2-dependent infection by coxsackievirus A7, A14, and A16 and enterovirus 71.</a> Journal of Virology. 86, 5686-5696 (2012).</li>
<li>Lin, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrolyzable+tannins+%28chebulagic+acid+and+punicalagin%29+target+viral+glycoprotein-glycosaminoglycan+interactions+to+inhibit+herpes+simplex+virus+1+entry+and+cell-to-cell+spread%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread.</a> Journal of Virology. 85, 4386-4398 (2011).</li>
<li>Lin, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Broad-spectrum+antiviral+activity+of+chebulagic+acid+and+punicalagin+against+viruses+that+use+glycosaminoglycans+for+entry%5BTitle%5D)+AND+%22BMC+Microbiology%22%5BJournal%5D)">Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry.</a> BMC Microbiology. 13, 187(2013).</li>
<li>Lin, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecules+targeting+coxsackievirus+A16+capsid+inactivate+viral+particles+and+prevent+viral+binding%5BTitle%5D)+AND+%22Emerging+Microbes+%26+Infections%22%5BJournal%5D)">Small molecules targeting coxsackievirus A16 capsid inactivate viral particles and prevent viral binding.</a> Emerging Microbes & Infections. 7, 162(2018).</li>
<li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFlint%2C+SJ+%22Principles+of+Virology%22">Principles of Virology</a>. , 3rd edn, ASM Press. (2008).</li>
<li>Velu, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BPR-3P0128+inhibits+RNA-dependent+RNA+polymerase+elongation+and+VPg+uridylylation+activities+of+Enterovirus+71%5BTitle%5D)+AND+%22Antiviral+Research%22%5BJournal%5D)">BPR-3P0128 inhibits RNA-dependent RNA polymerase elongation and VPg uridylylation activities of Enterovirus 71.</a> Antiviral Research. 112, 18-25 (2014).</li>
<li>Tai, C. J., Li, C. L., Tai, C. J., Wang, C. K., Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> Journal of Visualized Experiments. , e53124(2015).</li>
<li>Lang, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DOCK+6%3A+combining+techniques+to+model+RNA-small+molecule+complexes%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">DOCK 6: combining techniques to model RNA-small molecule complexes.</a> RNA. 15, 1219-1230 (2009).</li>
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</ol>

The authors declare that they have no conflict of interest.

In this report, we described the protocols that are useful for the identification of antiviral candidates that target viral entry, in particular against the non-enveloped CVA16. The assays are designed in ways to dissect the early events during viral entry, which is helpful to clarify the mechanism(s) of action and potential target(s) of the test agents&#39; antiviral activity. The &#39;time-of-drug-addition assay&#39; permits to broadly determine the potential target of the test compounds, for instance the uninfected ho...

Hand, foot, and mouth disease (HFMD) is a disease most commonly caused by coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) in young children. Recently across the Asia-Pacific region, there has been a significant uptick in CVA16-induced HFMD. While symptoms can be mild, severe complications can occur that affect the brain and the heart, with potential fatality1,2. At present, there are no licensed antiviral therapies or vaccinations available for CVA16, and thus there is a pressing need to develop antiviral strategies to curb future outbreaks and the associated complications.
CVA16 is a non-enveloped virus which has an icosahedral capsid assembled from pentamers that each contain 4 structural proteins namely VP1, VP2, VP3, and VP4. Encircling each five-fold axis in the pentamer is a &#39;canyon&#39; region that shows as a depression and is noted for its role in receptor binding3. At the bottom of this canyon lies a hydrophobic pocket in the VP1 region that contains a natural fatty ligand named sphingosine (SPH). Cellular receptors, such as human P selectin glycoprotein ligand 1 (PSGL-1) and scavenger receptor class B member 2 (SCARB2), have been suggested to play a role in viral binding by displacing this ligand which results in conformational changes to the capsid and the subsequent ejection of viral genome into the host cell4,5,6. Identifying possible inhibitors that block the successive events in the viral entry process could provide potential therapeutic strategies against CVA16 infection.
The steps in the virus life cycle can be dissected through experimental approaches as targets to help identify mode-specific antiviral agents. A time-of-drug-addition analysis examines the drug treatment effect at different times during the viral infection, including pre-entry (added prior to the virus infection), entry (added concurrent to the virus infection), and post-entry (added following the virus infection)7. The impact can be assessed using a standard plaque assay by quantitating the number of viral plaques formed in each of the treatment conditions. The flow cytometry-based viral binding assay determines if the drug prevents viral attachment to host cells. This is achieved by shifting the temperature from 37 &#176;C, at which the majority of human virus infections occur, to 4 &#176;C, where the virions are able to bind to the host cell surface but are unable to enter the cells7. The cell membrane-bound virus particles are then quantified through immunostaining against viral antigens and assessed by flow cytometry. The viral inactivation assay on the other hand helps to assess potential physical interactions of the drug with free virus particles, either shielding or neutralizing the virions, or causing aggregations or conformational changes that render them inactive for subsequent interactions with the host cell surface during the infection8,9. In this experiment, the viral inoculum is allowed to first incubate with the drug before being diluted to titrate out the drug prior to infecting the host cell monolayer and performing a standard plaque assay8. Finally, molecular docking is a powerful tool to predict potential drug interaction sites on the virion surface, including the viral glycoproteins from enveloped viruses and the viral capsid proteins from non-enveloped viruses, by using computational algorithms. This helps to mechanistically pinpoint targets of the drug&#39;s mode of action and provide useful information that can be further validated by downstream assays.
We recently employed the above described methods to identify antiviral compounds that efficiently blocked infection by the non-enveloped CVA169. Herein, the detailed protocols that were used are described and discussed.

<table><tbody><tr><td>4% Paraformaldehyde</td><td>Sigma</td><td>AL-158127-500G</td><td></td></tr><tr><td>Alexa 488-conjugated anti-mouse IgG</td><td>Invitrogen</td><td>A11029</td><td></td></tr><tr><td>Amphotericin B</td><td>GIBCO</td><td>15290-018</td><td></td></tr><tr><td>Anti-VP1 antibody</td><td>Merck-Millipore</td><td>MAB979</td><td>Anti-Enterovirus 71 Antibody, cross-reacts with Coxsackie A16, clone 422-8D-4C-4D</td></tr><tr><td>Beckman Coulter Cytometer</td><td>Beckman Coulter</td><td>FC500</td><td></td></tr><tr><td>Corina</td><td>Molecular Networks GmbH</td><td></td><td></td></tr><tr><td>Crystal violet</td><td>Sigma</td><td>C3886-100G</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>11995-040</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D5879</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140-079</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F8775</td><td></td></tr><tr><td>Graphpad Prism</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3393</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>Methylcellulose</td><td>Sigma</td><td>M0512-100G</td><td></td></tr><tr><td>PBS pH 7.4</td><td>GIBCO</td><td>10010023</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>GIBCO</td><td>15070-063</td><td></td></tr><tr><td>PyMol</td><td>Schr&amp;ouml;dinger</td><td></td><td></td></tr><tr><td>UCSF Chimera</td><td>University of California, San Francisco</td><td></td><td></td></tr></tbody></table>

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 time-of-drug-addition assay is indicated in Figure 1 and shows the influence from treatment using the small molecules CHLA and PUG on CVA16 infection either pre-viral entry (pretreatment), during viral entry (co-addition), or post-viral entry (post-infection). Both small molecules only produced marginal impact against CVA16 infectivity whether in the pretreatment of the host cells prior to viral infection (Figure 1A) or in th...

Watch this Scientific Journal Video about Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16 at JoVE.com

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

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.

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

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7.9K Views.  University of Texas at Austin. Dit protocol kan helpen bij de ontdekking van potentiële antivirale kleine moleculen door middel van een mechanisme-gedreven aanpak. De tijd van toevoegingstest bepaalt bij welke stap van de infectie het kleine molecuul zijn antivirale activiteit vertoont. Moleculair docking voorspelt de interactie tussen het kleine molecuul en de virale eiwitten.Om de invloed van geneesmiddelen op gastheercellen voorafgaand aan virale infectie te evalueren, zaad RD cellen in 12-well platen op een twe...

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

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors

The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in Southern Africa. The development of resistance to non-nucleotide reverse transcriptase inhibitors (NNRTIs) threatens the long-term success of antiretroviral therapy. In 2019, antimicrobial resistance directly accounted for an estimated 1.27 million deaths globally. This study employed an in-silico approach to investigate NNTRI drugs and their derivatives. Techniques used included density functional theory calculations, molecular docking, enumeration, quantitative structure-activity relationship (QSAR) analysis, molecular dynamics simulation (MDS), and molecular mechanics with generalized Born and surface area methods. The analysis focused on various pyrimidine derivatives and six NNRTI drugs, examining their interactions with the HIV-1 protein (PDB code 1HQU).
A QSAR model was developed to predict the biological activity of the six NNRTIs under investigation. Using 94 pyrimidine derivatives, the QSAR model achieved an R2 OF 0.822 and a Q2 of 0.815, indicating a high level of predictive accuracy.
MDS were conducted to assess the stability of various ligands and their newly developed alternatives, ensuring they remained bound to the protein&#39;s active site over a 200-nanosecond simulation timeframe. Etravirine exhibited root mean square deviation (RMSD) fluctuations of approximately 4.5 &#197;, while its enumerated derivatives showed RMSD fluctuations of 3.5 &#197;. Through molecular docking, MDS, and free energy computations, enumerated Etravirine demonstrated the best performance, with an activity value of 7.373 and a docking score of -10.517 kcal/mol. Furthermore, the calculated free energy of binding for enumerated Etravirine was -89.684 kcal/mol, outperforming other ligands under investigation. The significant improvement suggests that the modified Etravirine holds promising potential as a novel agent in antiretroviral therapy.
The obtained lower RMSD value, the enhanced amino acid interactions, and the highest binding free energy indicate that enumerated Etravirine could serve as a viable alternative for HIV/AIDS treatment.

This study used in-silico strategies to identify Enumerated Etravirine as a promising therapeutic agent for HIV. Our findings on molecular interactions and dynamics support the rational design of novel NNRTIs as possible HIV treatment alternatives.

The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in ...

Chemistry

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

Bioengineering

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce active enzymes essential for viral replication. Viral proteases are highly specific and recognize conserved cleavage site motif sequences (~6-8 amino acids). In several Group IV viruses, the nsP protease(s) cleavage site motif sequences can be found in specific host proteins involved in generating the innate immune responses and, in some cases, the targeted proteins appear to be linked to the virus-induced phenotype. These viruses utilize short stretches of homologous host-pathogen protein sequences (SSHHPS) for targeted destruction of host proteins. To identify SSHHPS the viral protease cleavage site motif sequences can be inputted into BLAST and the host genome(s) can be searched. Cleavage initially can be tested using the purified nsP viral protease and fluorescence resonance energy transfer (FRET) substrates made in E. coli. The FRET substrates contain cyan and yellow fluorescent protein and the cleavage site sequence (CFP-sequence-YFP). This protease assay can be used continuously in a plate reader or discontinuously in SDS-PAGE gels. Models of the bound peptide substrates can be generated in silico to guide substrate selection and mutagenesis studies. CFP/YFP substrates have also been utilized to identify protease inhibitors. These in vitro and in silico methods can be used in combination with cell-based assays to determine if the targeted host protein affects viral replication.

We present a general protocol for identifying short stretches of homologous host-pathogen protein sequences (SSHHPS) embedded in the viral polyprotein. SSHHPS are recognized by viral proteases and direct the targeted destruction of specific host proteins by several Group IV viruses.

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce ...

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

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.

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

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

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.

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

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

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