Name: High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses (Video) | JoVE Journal
Uploaded: 2014-05-05T16:15:00.000+00:00
Duration: 11 min 34 s
Description: Discover a high-throughput screening pipeline to isolate broad-spectrum antivirals from chemical libraries using recombinant measles and chikungunya viruses expressing luciferase. This method combines antiviral efficacy and toxicity assessments.

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5 minutes

We thank Dr. Yves L. Janin for his fruitful comments and suggestions. We would like to thank H.H. Gad for CHIK/Ren and C. Combredet for her technical support. This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Sant&#233; Et de la Recherche M&#233;dicale (INSERM), the Institut Carnot - Pasteur Maladies Infectieuses (Programme STING to P.O.V and H.M.-L), the Agence Nationale pour la Recherche (ANR-RPIB, Programme STING 2.0 to P.O.V), and the &#34;Conseil R&#233;gional d'Ile-de-France&#34; (Chemical Library Project, grants n&deg; I 06-222/R and I 09-1739/R to H.M.-L.). The work on CHIKV/Ren was supported by the project ArbOAS (ANR grant 2010-INTB-1601-02).

1. Preparation of 96-well Daughter Plates with Compounds
<ol>
	<li>Move 96-well mother plates containing 10 mM stock solutions of chemical compounds in DMSO from -20 &#176;C to room temperature. Dilute compounds 5x&#160;in DMSO to obtain intermediate 96-well dilution plates at 2 mM. Dilution is achieved by pipetting 5 &#956;l&#160;into 20 &#956;l of DMSO and mixing.</li>
	<li>Pipette 1 &#956;l from dilution plates into dry wells of white, bar-coded tissue culture 96-well plates. This first set of daughter plates (D1) will be used to evaluate the toxicity of compounds at a final concentration of 20 &#956;M. Store daughter plates at -20 &#176;C until use.</li>
	<li>Dilute compounds again 1:10 by pipetting 4 &#956;l from dilution plates into 36 &#956;l of DMSO and mixing. Pipette 1 &#956;l into dry wells of white, flat bottom, bar-coded tissue culture 96-wells plates. This second set of daughter plates (D2) will be used to evaluate the inhibition of MV replication by compounds at a final concentration of 2 &#956;M. Store daughter plates at -20 &#176;C until use.</li>
</ol>
2. Preparation of Cell Cultures to Determine Compound Toxicity
<ol>
	<li>Grow HEK-293T cells at 37 &#176;C and 5% CO2 in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with stabilized L-glutamine and supplemented with 10% fetal calf serum (FCS), streptomycin (100 &#956;g/ml) and penicillin (100 IU/ml). Cells are passaged by trypsinization every 4-5 days, and diluted 1:10 into a fresh flask. Cells should be in log phase for experiments.</li>
	<li>On the day of the experiment, recover cells by trypsinization and perform cell counting. Pellet cells by centrifugation, and resuspend them at 3 x 105 cells/ml in culture medium. Consider that 12.5 ml of cell suspension are required for each screening plate.</li>
	<li>Transfer cells to a trough, and dispense 100 &#956;l of cell suspension in D1 plates containing spiked chemical compounds. Cell suspension within the trough is regularly agitated to avoid sedimentation.</li>
	<li>Spike control wells A1, C1, E1, G1, A12, C12, E12 and G12 with 1 &#956;l of DMSO. Corresponding wells will be used as reference for living cells (no toxicity). Spike control wells B1, D1, F1, H1, B12, D12, F12 and H12 with 1 &#956;l of DMSO and 2.5 &#956;l of a 0.5% IGEPAL solution to kill cells. Corresponding wells will be used as a reference for dead cells (high toxicity).</li>
	<li>Incubate cells for 24 hr&#160;at 37 &#176;C and 5% CO2.</li>
</ol>
3. Preparation of Cell Cultures Infected by rMV2/Luc
<ol>
	<li>Grow and recover cells as described in 2.1 and 2.2. Again, resuspend cells at 3 x 105 cells/ml in culture medium. Consider that 12.5 ml of cell suspension are required for each screening plate.</li>
	<li>Optional: supplement culture medium with uridine at 20 &#956;g/ml. 
	Note: When screening chemical libraries for viral replication inhibitors, it seems that compounds targeting early steps of pyrimidine biosynthesis pathway are frequently isolated13,16,27,28. The addition of uridine to culture medium is used to filter out such antiviral molecules. Indeed, this efficiently restores viral replication when upstream enzymes of pyrimidine biosynthesis pathway are blocked.</li>
	<li>Save 1:10 of cell suspension for control wells with non-infected cells, and proceed to infection with remaining volume.</li>
	<li>Thaw the appropriate volume of rMV2/Luc stock solution. MV stock solutions are usually at 106-108 infectious particles per ml. Culture must be infected with 0.1 infectious particles of rMV2/Luc per target cells, which corresponds to 0.1 multiplicity of infection (MOI). Note: rMV2/Luc is derived from MV vaccine (Schwarz strain) and is manipulated in a BSL2 environment.</li>
	<li>Add the virus to cell suspension and mix gently. Transfer infected cells to a trough, and dispense 100 &#956;l of infected cell suspension in columns 2 to 11 of D2 plates containing spiked chemical compounds. Cell suspension within the trough is regularly mixed gently.</li>
	<li>In columns 1 and 12, dispense 100 &#956;l of non-infected cells a well on two. Infected cells are dispensed in the others wells.</li>
	<li>Incubate cells for 24 hr&#160;at 37 &#176;C and 5% CO2.</li>
</ol>
4. Luciferase Activity Measures and Data Analysis
<ol>
	<li>Remove D1 plates from the incubator, and determine cellular viability by adding 50 &#956;l of luciferase-based viability reagent directly to culture wells. Mix and incubate for 10 min at room temperature. Read plates with a luminometer. Integration time is set at 100 msec per well.</li>
	<li>Remove D2 plates from the incubator, and determine luciferase activity by adding 50 &#956;l of luciferase substrate directly to culture wells. Incubate for 6 min at room temperature, and read plates with a luminometer as described above.</li>
	<li>Calculate a Z&#39;-factor for each D1 plate of the toxicity assay to warrant the quality of the screen29. Z&#39; = 1-3*(&#963;+ + &#963;-)/(&#181;+ - &#181;-), where &#181;+ and &#963;+ correspond to means of luminescence and standard deviations for HEK-293T cells with DMSO alone (no toxicity), and &#181;- and &#963;- are means of luminescence and standard deviations for culture wells treated with IGEPAL to kill cells. Z&#39; is expected to be above 0.5, or the corresponding plate is discarded.</li>
	<li>Set toxicity threshold at &#181;+/2. Discard compounds with luminescence values below this cut-off (Figure 3A).</li>
	<li>Calculate a Z&#39;-factor for each D2 plate of the antiviral assay. Here, &#181;+ and &#963;+ correspond to means of luminescence and standard deviations for infected cells cultured with DMSO alone, and &#181;- and &#963;- are means of luminescence and standard deviations for non-infected cells. Again, plates with Z&#39; values below 0.5 are discarded.</li>
	<li>Calculate the inhibition of viral replication as follow: percentage of inhibition = (&#181;+ -&#160;luciferase activity for compound X)/(&#181;+ - &#181;-)*100. Set inhibition threshold at 75%, and select compounds that both reduce luminescence values below this cut-off (Figure 3B) and are not toxic according to criteria described in 4.4.</li>
</ol>
5. Secondary Screen for Toxicity and Broad-spectrum Antiviral Activity
<ol>
	<li>Make 1:2 serial dilutions for each hit compound in DMSO, starting from 500 &#181;M to 4 &#181;M (8 dilutions). Fill white, bar-coded tissue culture plates with 50 &#181;l of culture medium. Add 1 &#181;l of each compound dilution in culture wells. Repeat twice to have 3 culture plates with duplicate serial dilutions for each hit compound.</li>
	<li>Prepare 37.5 ml of a HEK-293T cell suspension at 6 x 105 cells/ml in culture medium as described above (in 2.1 and 2.2). Dispense 2x 12.5 ml in falcon tubes and infect cells with either rMV2/Luc at MOI = 0.1 or recombinant CHIKV expressing Renilla luciferase (CHIKV/Ren) at MOI = 0.2. Mix by inverting. 
	Note: CHIKV/Ren is derived from a wild-type strain of CHIKV and therefore should be manipulated in a BSL3 environment. Plates can be moved from BSL3 to BSL1 once Renilla substrate has been added to culture wells (see below).</li>
	<li>Dispense 50 &#181;l of uninfected, rMV2/Luc-infected, or CHIKV/Ren-infected cells in culture plates containing serial dilutions of hit compounds. Incubate 24 hr at 37 &#176;C.</li>
	<li>Add 50 &#181;l of firefly luciferase substrate to determine firefly luciferase activity in rMV2/Luc-infected wells. Add 50 &#181;l of Renilla luciferase substrate to determine Renilla luciferase activity in CHIKV/Ren-infected wells. Finally, add 50 &#181;l of luciferase-based viability assay reagent to uninfected cells to determine cellular viability.</li>
	<li>Plot data (Figure 4) and determine hit compound concentrations that inhibit MV and CHIKV replication by 50%. Disregard compound showing some toxicity in this assay.</li>
</ol>

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The authors declare that they have no competing financial interests.

The screening pipeline described here aims at selecting compounds with a suitable profile as broad-spectrum inhibitors of RNA viruses. When a library of 10,000 compounds was screened with this protocol and aforementioned filtering criteria were applied, i.e. inhibition of MV replication superior to 75%, 40 compounds (0.4%) scored positive. Besides, about half of them showed some toxicity in the luciferase-based viability assay, and were disregarded for this reason. Finally, a dozen of hits readily available in l...

RNA viruses are responsible for a large variety of human infections, and have a huge impact on populations worldwide both in terms of public health and economical cost. Efficient vaccines have been developed against several human RNA viruses, and are widely used as prophylactic treatments. However, there is still a critical lack of therapeutic drugs against RNA virus infections. Indeed, efficient vaccines are not available against major human pathogens such as dengue virus, Hepatitis C virus or human respiratory syncytial virus (hRSV). Besides, RNA viruses are responsible for a majority of emerging diseases, which have increased in frequency because of global exchanges and human impact on ecological systems. Against this threat that represent RNA viruses, our therapeutic arsenal is extremely limited and relatively inefficient1-3. Current therapies are essentially based on recombinant type I interferons (IFN-&#945;/&#946;) to stimulate innate immunity, or the administration of ribavirin. Although the mode of action of this ribonucleoside analog is controversial and probably relies on various mechanisms, the inhibition of cellular IMPDH (inosine monophosphate dehydrogenase), which depletes intracellular GTP pools, is clearly essential4. Ribavirin, in combination with pegylated IFN-&#945;, is the main treatment against Hepatitis C virus. However, IFN-&#945;/&#946;&#160;and ribavirin treatments are of relatively poor efficacy in vivo against most RNA viruses as they efficiently blunt IFN-&#945;/&#946;&#160;signaling through expression of virulence factors5 and often escape ribavirin3. This added to the fact that ribavirin treatment is raising important toxicity issues, although it was recently approved against severe hRSV disease with controversial benefits6. More recently, some virus-specific treatments have been marketed, in particular against influenza virus with the development of neuraminidase inhibitors3. However, the large diversity and permanent emergence of RNA viruses precludes the development of specific treatments against each one of them in a relatively close future. Altogether, this stresses the need for efficient strategies to identify and develop potent antiviral molecules in the near future.
It is trivial to say that a broad-spectrum inhibitor active against a large panel of RNA viruses would solve this problem. Although such a molecule is still a virologist&#39;s dream, our better understanding of cellular defense mechanisms and innate immune system suggest that some possibilities exist7,8. Several academic and industrial laboratories are now seeking molecules that stimulate specific facets of cellular defense mechanisms or metabolic pathways to blunt viral replication. Although such compounds will probably show significant side effects, treatments against acute viral infections will be administered for a relatively short time, making them acceptable despite some potential toxicity on the long term. Various strategies have been developed to identify such broad-spectrum antiviral molecules. Some research programs aim at finding molecules that target specific defense or metabolic pathways. This includes, for example, pathogen recognition receptors to elicit antiviral gene expression9 and activate antiviral factors such as RNaseL10, autophagy machinery to promote virus degradation11, nucleoside synthesis pathways12,13, or apoptotic cascades to precipitate death of virus-infected cells14. Other groups have developed phenotypic screens that are not target-based13,15-17. In that case, antiviral molecules are simply identified by their capacity to block viral replication in a given cellular system. The general assumption is that a compound inhibiting 2-3 unrelated RNA viruses would have a suitable profile for a broad-spectrum antiviral molecule. The mode of action of hit compounds selected with such an empirical approach is only determined in a second time and eventually, may lead to the identification of novel cellular targets for antivirals. Interestingly, a retrospective analysis of new drugs approved by the US Food and Drug Administration between 1999 and 2008 has shown that in general, such phenotypic screenings tend to perform better than target-based approaches to discover first-in-class small-molecule drugs18.
Viral replication in high-throughput cell-based assays is usually determined from virus cytopathic effects. Cells are infected and cultured in 96- or 384-well plates in the presence of tested compounds. After few days, cellular layers are fixed and stained with dyes such as crystal violet. Finally, absorbance is determined with a plate reader and compounds inhibiting viral replication are identified by their capacity to preserve cellular layers from virus-induced cytopathic effect. Alternatively, virally-mediated cytopathic effects are assessed using standard viability assays such as MTS reduction. Such assays are highly tractable and cost-effective, but suffer from three major limitations. First, they require a virus-cell combination where viral replication is cytopathic in only few days but this is not always possible, thus calling for alternative approaches19. Second, they are poorly quantitative since they are based on an indirect measure of viral replication. Finally, toxic compounds can be scored as positive hits, and therefore must be eliminated with a counter screen measuring cellular viability. To overcome some of these hurdles, recombinant viruses or replicons have been engineered by reverse genetics to express reporter proteins, such as EGFP or luciferase, from an additional transcription unit or in frame with viral protein genes (few examples are 20-23). When these viruses replicate, reporter proteins are produced together with viral proteins themselves. This provides a very quantitative assay to measure viral replication and evaluate the inhibitory activity of candidate molecules. This is particularly true for recombinant viruses expressing luciferase (or other enzymes capable of bioluminescence) since this reporter system exhibits a wide dynamic range with a high sensitivity and virtually no background. Furthermore, there is no excitation light source, thus preventing interference with compound fluorescence24.
Here, we detail a high-throughput protocol to screen chemical libraries for broad-spectrum inhibitors of RNA viruses. Compounds are tested first on human cells infected with a recombinant measles virus (MV) expressing firefly luciferase25 (rMV2/Luc,&#160;Figure 1, primary screen). MV belongs to Mononegavirales order, and is often considered as a prototypical member of negative-strand RNA viruses. As such, MV genome is used as a template by the viral polymerase to synthesize mRNA molecules encoding for viral proteins. In the recombinant MV strain called rMV2/Luc, luciferase expression is expressed from an additional transcription unit inserted between P and M genes (Figure 2A). In parallel, compounds are tested for their toxicity on human cells using a commercial luciferase-based reagent that evaluates, by ATP quantification, the number of metabolically active cells in culture (Figure 1, primary screen). Entire chemical libraries can be easily screened with these two assays in order to select compounds that are not toxic and efficiently block MV replication. Then, hits are retested for dose-response inhibition of MV replication, the lack of toxicity as well as for their capacity to impair chikungunya virus (CHIKV) replication (Figure 1, secondary screen). CHIKV is a member of Togaviridae family and its genome is a positive single-strand RNA molecule. As such, it is completely unrelated to measles and compounds inhibiting both MV and CHIKV stand a great chance to inhibit a large panel of RNA viruses. CHIKV nonstructural proteins are directly translated from the viral genome, whereas structural proteins are encoded by transcription and translation of a subgenomic mRNA molecule. Our in vitro replication assay for CHIKV is based on a recombinant strain called CHIKV/Ren, which expresses Renilla luciferase&#160;enzyme as a cleaved part of the nonstructural polyprotein through an insertion of the reporter gene between nsP3 and nsP4 sequences26 (Figure 2B). The measure of Renilla luciferase activity allows the monitoring of viral replication at the early stage of CHIKV life cycle.
This high-throughput protocol was used to quickly identify compounds with a suitable profile for broad-spectrum antivirals in a commercial library of 10,000 molecules enriched for chemical diversity. Compounds were essentially following Lipinski&#39;s rule of five, with molecular weights ranging from 250 to 600 daltons, and log D values below 5. Most of these molecules were new chemical entities not available in other commercial libraries.

<table><tbody><tr><td>Freedom EVO platform</td><td>TECAN</td><td></td><td>Robotic platform</td></tr><tr><td>96-well polystyrene cell culture microplates, white</td><td>Greiner Bio One</td><td>655083</td><td></td></tr><tr><td>CellTiter-Glo Luminescent Cell Viability Assay</td><td>Promega</td><td>G7570</td><td>Luciferase-based viability assay</td></tr><tr><td>Bright-Glo Luciferase Assay System</td><td>Promega</td><td>E2610</td><td>Reagent containing firefly luciferase substrate</td></tr><tr><td>Renilla-Glo luciferase Assay System</td><td>Promega</td><td>E2710</td><td>Reagent containing Renilla luciferase substrate</td></tr><tr><td>Britelite plus Reporter Gene Assay System</td><td>Perkin-Elmer</td><td>6016761</td><td>Reagent containing firefly luciferase substrate. Can be used as an alternative to Brigh-Glo reagent to determine luciferase activity.</td></tr></tbody></table>

high-throughput screening for broad-spectrum chemical inhibitors of rna viruses

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.

This screening pipeline relies first on the selection of compounds that inhibit MV replication and do not show any significant cellular toxicity (Figure 1, primary screen). It takes advantage of luciferase-based assays to determine cellular toxicity and the inhibition of viral replication. All pipetting steps and data acquisition can be performed in a high-throughput setting with a robotic platform.
Cellular toxicity of compounds is assessed using a luciferase-based viability ...

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 ...

The overall goal of this procedure is to isolate broad spectrum antivirals from chemical libraries using a combination of in vitro cell based assays. This is accomplished by first selecting compounds that inhibit the in vitro replication of a recombinant strain of measles virus that encodes luciferase as a reporter. In parallel, the compound's toxicity is estimated using a luciferase based viability assay.The combined phenotypic data from both assays is used to isolate HIT molecules. The final steps are to retest the HIT compounds for dose response replication-inhibition of measles and chikungunya virus, and to measure in vitro toxicity more accurately. Ultimately, broad spectrum antiviral compounds are identified and validated by the screen.The main advantage of this technique over existing methods like standard virus replication assays based on cytopathic effects, is that it uses luminescence as a readout and combines two highly divergent viruses expressing luciferase as a reporter. Demonstrating the procedure will be Olivier Helynck, a technician from Helene Munier-Lehmann's team, and Marianne Lucas-Hourani, an engineer working with me in Frédéric Tangy's research unit. Begin by warming to room temperature the 96 well mother plates containing 10 millimolar stock solutions of chemical compounds in DMSO. Transfer five microliters of each compound to new 96 well plates already with 20 microliters of DMSO in each well, thus creating intermediate dilution plates. Now pipette one microliter of these dilution plates into dry wells of white barcoded tissue culture 96 well plates.These are the D one plates and are used to evaluate the toxicity of compounds at 20 micromolar. Store them at minus 20 degrees Celsius. Now make new plates from the intermediate dilution plates that are further diluted 10 times.Take four microliters of each diluted compound and mix them into wells with 36 microliters of DMSO. From these plates, transfer one microliter per sample into dry wells of white flat bottom barcoded tissue culture 96 well plates.These are the D two plates and they are used to evaluate inhibition of MV replication. Store them at minus 20 degrees Celsius as well. Grow HEK-293-T cells in supplemented DMEM with stabilized L-glutamine. Every four to five days, passage the cells by trypsin and make one 10th dilution into fresh flasks.During their logarithmic growth phase, collect and count the cells for the experiment. Resuspend the cells at 300, 000 cells per milliliter. 12.5 milliliters of cells at this dilution are needed for each plate that will be screened.Now, prepare the plates. Spike one set of control wells with one microliter of DMSO, spike a second set of control wells with one microliter of DMSO and 2.5 microliters of a 0.5% IGEPAL solution to kill the cells. Transfer the cell suspension to a trough and dispense 100 microliters to each well of the D-one plates.Regularly agitate the suspension to avoid sedimentation. Then incubate the prepared plates for 24 hours. Prepare the cells for the plates as done in the previous section. The culture medium can be supplemented with uridine to filter out antiviral molecules that target the early steps of pyrimidine biosynthesis, as these are fairly common. Save one 10th of the cell suspension for the control wells with non-infected cells and infect the remaining volume. Thaw enough rMV2-luc stock solution to infect the culture with 0.1 infectious particles per target cell for a 0.1 multiplicity of infection. Add the calculated volume of virus to the cell suspension and mix it gently. Transfer the infected cells to a trough and dispense 100 microliters of infected cells to the D-two plates. Target columns two through 11, which contain the spiked chemical compounds and positive control wells in columns one and 12.Regularly, gently mix the cells in the trough. In alternate rows of columns one and 12, dispense 100 microliters of non-infected cells. Now incubate the plates for 24 hours.After the incubation time, determine the viability of the D-one plate cells. Mix 50 microliters of luciferase based viability reagent into each well and wait 10 minutes. Then read the plates with a luminometer. Set the integration time to 100 milliseconds per well. Next, determine MV replication in the D-two plates by mixing 50 microliters of firefly Luciferase substrate into each well and waiting six minutes at room temperature. Then consult a Z factor for each D-one and D-two plate of the toxicity assay.Continue by calculating the inhibition of viral replication. HIT compounds are those that reduce viral replication to below the applied cutoff value and are not toxic. For each HIT compound, make serial dilutions in DMSO starting at 500 micro molars and moving in half steps down to four micro-molars along one column of a 96 well plate. Add one microliter from the compound dilution plate to the white barcoded tissue culture plate. Repeat this process twice to create a triplicate set of test plates. Then fill tissue culture plates with 50 microliters of culture medium. Now prepare 37.5 milliliters of HEK-293T cells, suspended in supplemented DMEM at 600, 000 cells per milliliter.Load two 15 milliliter tubes with 12.5 milliliters of the cell suspension each. Infect one tube of cells with rMV2/luc at a multiplicity of infection of 0.1. Infect the second tube of cells with recombinant chikV expressing renilla luciferase. Use a multiplicity of infection of 0.2. Mix both tubes of virus infected cells using inversion. This should be performed in a biosafety level three laboratory because of the chikV pathogenicity. Now load the uninfected cells and the sets of infected cells into their own set of HIT compound plates.Dispense 50 microliters of cells per well and incubate the plates at 37 degrees Celsius for 24 hours. The next day, add 50 microliters of firefly luciferase to each well of rMV2/luc infected cells. Similarly, add 50 microliters of renilla Lucifer substrate to the ChikV-Ren infected cells. To the uninfected control cells add 50 microliters of luciferase based viability reagent. Proceed by determining the HIT compound concentrations that inhibit MV and ChikV replication by 50%. In addition, disregard compounds showing cell toxicity. This screening pipeline relies on selecting compounds that do not show any significant cellular toxicity and inhibit both MV and ChikV replication as determined in the primary and secondary screens respectively. It takes advantage of recombinant viruses expressing firefly or renilla luciferase as a reporter.Compound toxicity is assessed using a commercial luciferase based assay where luminescence correlates to living cells. On this plate 21 compounds were scored as toxic based on a threshold set by the positive control wells. Antiviral activity was first measured using rMV2/ luc. Luminescence was compared to the limit set by control wells. Here, four compounds were found able to inhibit viral replication by more than 75%. In the other assay, two of these were found to be toxic. For all the HIT compounds the half maximal inhibitory concentration was determined on both MV and ChikV RNA viruses expressing luciferase. These two viruses are unrelated and thus make the screen more robust. In parallel, the lack of cellular toxicity of selected compounds was confirmed in a dose response experiment.In all 13 non-toxic compounds were identified from a chemical library of 10, 000 molecules. These compounds were novel in terms of both chemical structure and biological activity. Based on structural similarities, the compounds fell into three chemical families. For reference, half maximal inhibitory concentration values were obtained with Brequinar a substance with potent antiviral activity. When looking at these values, less than an order of magnitude, separated the reference molecule from most active HITs identified through the screen. This demonstrates the relatively strong antiviral activity of the selected compounds Once mastered, this technique can be used to screen large chemical libraries in a high throughput setting and select sets of small molecules enriched for broad spectrum antivirals.

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Research

JoVE Journal

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Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

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High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

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Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Discover a high-throughput screening pipeline to isolate broad-spectrum antivirals from chemical libraries using recombinant measles and chikungunya viruses expressing luciferase. This method combines antiviral efficacy and toxicity assessments.

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

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