Vanessa Garcia is funded by a Queen Elizabeth II Ontario Graduate Scholarship in Science and Technology and Cory Batenchuk by a Natural Sciences and Engineering Research Council fellowship.

1. Sample Preparation
<ol>
	<li>Obtain samples containing luciferase-expressing virus (herein VSV&#8710;51-Fluc as an example) for titration and transfer into 96-well plates. Alternately, perform infections in 96-well tissue culture plates and use supernatants directly.</li>
	<li>Leave two columns untreated for the inclusion of standard curves. For experiments done directly in 96-well plates, titer at the end of the experiment (40 hr&#160;post-infection) or store at -80 &#176;C and titer at a later date.</li>
</ol>
2. Preparation of Permissive Cells for Virus Titration
<ol>
	<li>24 hr&#160;prior to titering, prepare a suspension of Vero cells at a concentration of 2.5 x 105 cells/ml&#160;in Dulbecco&#8217;s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 30 mM HEPES, and 1% penicillin/streptomycin. NOTE: Although this protocol uses Vero cells, any suitable permissive cell line for VSV infection could be used.</li>
	<li>Seed 2.5 x 104 cells (100 &#956;l) in 96-well white solid flat-bottom plates using a microplate dispenser.
	<ol>
		<li>Prepare 12 ml&#160;of cell suspension per plate plus 5 ml&#160;for priming.</li>
		<li>Clean microplate dispenser cassette by flushing the tubing with 50 ml&#160;of sterile water.</li>
		<li>Fill the microplate dispenser lines with cell suspension and let 5 ml&#160;of cell suspension flow through.</li>
		<li>Select a program that will dispense 100&#160;&#956;l in each well of a white-walled 96-well plate. Dispense, and repeat as necessary for additional plates. Also seed a few wells in a 96-well clear flat-bottom plate if opaque-bottom white-walled plates are used for verification of cell health and density.</li>
		<li>When finished, flush cells back into original container. Clean the cassette by running 50 ml&#160;of 70% ethanol followed by 50 ml&#160;of warm sterile water through the tubing.</li>
		<li>Make sure cassette and tubing are appropriately cleaned between uses.</li>
	</ol>
	</li>
	<li>Incubate cells for 24 hr at 37 &#176;C in a humidified 5% CO2 incubator.</li>
</ol>
3. Preparation of Viral Standard Curve
<ol>
	<li>Prepare a standard curve of VSV&#8710;51-Fluc in serum-free DMEM such that the final concentration of plaque forming units (pfu) per ml&#160;after transfer onto Vero cells is as follows: 108 pfu/ml, 107 pfu/ml, 106 pfu/ml, 105 pfu/ml, 104 pfu/ml, 103 pfu/ml, 102 pfu/ml, and 101 pfu/ml. Prepare 50 &#956;l of each concentration per plate of Vero cells plus an extra 10%. 
	NOTE: Titer of the luciferase virus stock will need to be assessed in a classical way10 in order to generate a standard curve that will allow for absolute quantification. Otherwise relative quantification can be achieved without precise titer information by arbitrarily setting viral titer based on dilution steps. For example the first dilution of the standard curve may be set to 108 viral units and the following 1/10 dilution to 107 and so on. In this context, one should express values as a fold-change compared to a pre-determined sample as absolute quantification will not be accurate.</li>
</ol>
4. Transfer of Sample Supernatants onto Permissive Cells
<ol>
	<li>Check Vero cells plated in the clear-bottom 96-well plate under a light microscope to confirm that monolayers are at least 95% confluent.</li>
	<li>Transfer 25 &#956;l of sample supernatant onto Vero cells seeded in white-walled plates. Do not transfer supernatants into the 2 columns designated for standard curves. NOTE: This can be done simultaneously for all wells on a single plate using a 96-channel liquid handler.</li>
	<li>Using an 8- or 12-channel multi-channel pipettor, add 25 &#956;l of each dilution of the standard curve prepared in Step 3 to the Vero cells in the 2 designated columns.</li>
	<li>Centrifuge plates for 5 min at 430 x g at RT.</li>
	<li>Incubate for 5 hr at 37 &#176;C in a humidified 5% CO2 incubator.</li>
</ol>
5. Assessment of Cell Viability
<ol>
	<li>NOTE: When starting from supernatants obtained from infection experiments done in clear 96-well plates, sample viability can be assessed prior to quantification with a cell viability indicator dye such as resazurin.</li>
	<li>Add resazurin in an amount equal to 10% of the volume in each well of the 96-well plate of samples containing virus and&#160;cells. Include cell-only controls as well as media-only control to determine values for 100% and 0% viability respectively.</li>
	<li>After 2-4 hr (incubation time will vary depending on cell type and on the concentration of commercially available or reconstituted powder of the dye), read and record the signal using a fluorescence plate reader (530-560 excitation, 590 emission). Report cell viability for a sample according to the formula Relative Metabolic Activity = ((Test sample signal - negative control signal) / (Cell only control signal - negative control signal)) x 100%</li>
</ol>
6. Preparation of the Luciferin Substrate
<ol>
	<li>30 min&#160;before the end of the 5 hr incubation, prepare the luciferin to obtain a 2 mg/ml&#160;solution in sterile phosphate buffered saline (PBS). Prepare 2.5 ml&#160;per plate plus an extra 2 ml. Protect solution from light. NOTE: The luciferin may also be prepared earlier in the day and stored at 4 &#176;C until use.</li>
</ol>
7. Reading Bioluminescence
<ol>
	<li>After the 5 hr incubation period, add 25 &#956;l of luciferin to each well of Vero cells in the white solid plates. Add luciferin manually or with an automated dispenser integrated in the luminometer. NOTE: The use of an instrument with only single and end point reads may require the addition of a luciferase compatible lysis buffer prior to adding the luciferin substrate to improve consistency.</li>
	<li>Read the plates with the following parameters:
	<ol>
		<li>Shake for 5 sec.</li>
		<li>Wait 30 sec.</li>
		<li>Read luminescence at an appropriate fixed sensitivity / exposure value. If the option is available on the instrument, use multi-point reads (e.g., 3 x 3 matrix) to improve accuracy.</li>
	</ol>
	</li>
	<li>Record and save the quantified bioluminescent intensity.</li>
	<li>If an automated dispenser was used to add luciferin purge the luciferin and prime the lines with warm sterile water.</li>
</ol>
8. Data Analysis
<ol>
	<li>For accurate results, solve the non-linear regression to generate a Hill equation from the standard curves. Apply this equation to the titered samples to calculate viral expression units. Some viruses or situations may produce a standard curve with a linear relationship; in this case solve for a linear equation.</li>
</ol>

<ol>
	<li>Grigorov, B., Rabilloud, J., Lawrence, P., Gerlier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+titration+of+measles+and+other+viruses:+optimization+with+determination+of+replication+cycle+length.">Rapid titration of measles and other viruses: optimization with determination of replication cycle length.</a> PLoS One. 6 (e24135), (2011).</li><li>Lizee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum Gene Ther. 14, 497-507 (2003).</li><li>McSharry, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+of+flow+cytometry+in+virology.">Uses of flow cytometry in virology.</a> Clin Microbiol Rev. 7, 576-604 (1994).</li><li>Watcharatanyatip, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispecies+detection+of+antibodies+to+influenza+A+viruses+by+a+double-antigen+sandwich+ELISA.">Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.</a> J Virol Methods. 163, 238-243 (2010).</li><li>Dawson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+Direct+Quantification+of+Viruses+in+Solution+Using+the+ViroCyt+Virus+Counter.">Rapid, Direct Quantification of Viruses in Solution Using the ViroCyt Virus Counter.</a> Journal of Biomolecular Techniques. 23 (S10), (2012).</li><li>Snyder, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+and+RT-PCR:+true+or+false.">AAV and RT-PCR: true or false.</a> Mol Ther. 1, 389-390 (2000).</li><li>Diallo, J. S., Roy, D., Abdelbary, H., De Silva, N., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+infection+of+live+tissue+with+oncolytic+viruses.">Ex vivo infection of live tissue with oncolytic viruses.</a> J Vis Exp. 52, (2011).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells.">Replication and plaque assay of influenza virus in an established line of canine kidney cells.</a> Appl Microbiol. 16, 588-594 (1968).</li><li>Green, M., Loewenstein, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adenoviruses:+propagation,+purification,+quantification,+and+storage.">Human adenoviruses: propagation, purification, quantification, and storage.</a> Curr Protoc Microbiol. 14 (14C 11), (2006).</li><li>Diallo, J. S., Vaha-Koskela, M., Le Boeuf, F., Bell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+purification,+and+in+vivo+testing+of+oncolytic+vesicular+stomatitis+virus+strains.">Propagation, purification, and in vivo testing of oncolytic vesicular stomatitis virus strains.</a> Methods Mol Biol. 797, 127-140 (2012).</li><li>Hoenen, T., Groseth, A., Callison, J., Takada, A., Feldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Ebola+virus+expressing+luciferase+allows+for+rapid+and+quantitative+testing+of+antivirals.">A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals.</a> Antiviral Res. 99, 207-213 (2013).</li><li>Diallo, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+pharmacoviral+approach+identifies+novel+oncolytic+virus+sensitizers.">A high-throughput pharmacoviral approach identifies novel oncolytic virus sensitizers.</a> Mol Ther. 18, 1123-1129 (2010).</li></ol>

The authors have nothing to disclose.

The luciferase-based approach described here provides a number of advantages over other existing methods including its ease, quickness, minimal equipment need, and relatively low cost. A key contributor to this is the avoidance of a serial dilution step. Nonetheless, serial dilution-based derivatives of this protocol are certainly feasible and were recently used to assess luciferase-expressing Ebola titers in a high-throughput antiviral screen11. While inherently more time consuming and more expensive, such adaptations may provide greater dynamic range for viral quantification when necessary. In addition to being particularly well suited for evaluating viral titers in the context of high-throughput screens, our one-step luciferase based viral quantification method generates accurate estimates of infectious virions in the case of replicating viruses. Furthermore, readouts of luminescence along with cytotoxicity data from the same experiment give a more complete picture of the effect of the experimental conditions on target cells, which is particularly useful in the context of oncolytic viruses and drug screens.
The example illustrated here uses a replicating negative single strand RNA virus; however, this protocol can be adapted to a number of replicating and non-replicating viruses with a few minor protocol adjustments. This includes DNA viruses such as Vaccinia, HSV, and AAV (see Figure 5). Samples infected with intracellular viruses, such as Vaccinia virus for example, require a virus-release step prior to quantification (e.g., at least one freeze-thaw cycle). When applying this technique to other viruses, it is necessary to optimize the incubation time from the transfer of the viral supernatant or lysate to the reading of the plates by the luminometer. This parameter will depend mainly on the replication cycle of the virus in the permissive cell line and the strength of the promoter driving luciferase expression. This is best done by using the full standard curve in the optimization step. To do this, one must infect the appropriate permissive cell line with various replicates of the prepared standard curve and read each replicate at a different time points post-transfer. Ideally, an incubation time point is chosen that leads to a linear relationship between LOG(RLU) and LOG(titer) spanning the expected sample titer range. For VSV&#8710;51-Fluc, this is typically from 104 pfu/ml&#160;-107 pfu/ml&#160;for a 5 hr incubation time. If lower or higher titers are expected from samples, one can simply increase or reduce the incubation time respectively. Alternately, samples may be diluted to fall within the range much as is done typically for ELISA.
As mentioned above, this method is well suited to perform high-throughput drug screens using drug libraries as most of the steps can be automated. Cells can be plated efficiently using an automated microplate dispenser, the drug library can be added using a 96-channel liquid handler, virus can be added using a microplate dispenser and plates read using an automated luminometer. In theory, this can also be adapted to 384-well or smaller formats; however, the limitation to this end is number of cells that can be plated, given fewer cells leads to a narrower range in the linearity of the LOG(RLU) to LOG(Titer) relationship. Finally, assessment of cell viability using resazurin or other metabolic dyes can be easily incorporated in the workflow, allowing for discrimination of cytotoxic compounds in antiviral screens or identification of compounds that lead to synergistic killing in combination with viruses12. Nevertheless, limitations of this method include the requirement of a luciferase transgene expressing virus, which is not always possible, and the availability of a sufficiently permissive cell line. However, it is likely possible to adapt the method for use with other reporter genes (e.g., GFP) provided the reporter quantification method has a suitable linearity and signal to noise ratio. Overall, the described high-throughput method can be modified to suit many different viruses and tailored to diverse applications.

Classical viral plaque assays continue to be a mainstay in virus research even though they can be notoriously time-consuming and constitute a significant bottleneck to obtaining results from experiments. More rapid indirect virus quantification methods have emerged including quantitative polymerase chain reaction (qPCR), ELISA, and flow cytometry1-4. Recent innovations such as the Virocyt virus counter can directly count viruses using advanced flow sorting technology and through a combination of protein and DNA/RNA dyes5. While all of these methods have undoubtedly quickened the pace of virus research, each method has its advantages and drawbacks. For example, qPCR can allow for quantification of specific viral genome sequence but cannot effectively discriminate infectious from defective virions6. ELISAs can be very specific however require a suitable antibody against the desired target viral protein and can be very expensive. While flow cytometry technology offers many advantages and has improved significantly, throughput and accessibility to highly specialized equipment nonetheless remains a hurdle. Importantly, all of these techniques are not ideally suited for high-throughput screening, for which the ease and time requirement of the virus quantification step is of critical importance.
Here we describe a high-throughput and easily automatable technique to titer viruses that express a Firefly luciferase (Fluc) transgene. This method generates approximate viral titers in a test sample based on luminescence signal reads through the parallel use of a standard curve of known amounts of virus. Samples containing unknown quantities of luciferase-expressing virus are transferred on to a permissive &#8220;plaquing&#8221; cell line in parallel with the standard virus dilution curve and virus-associated luminescence is read after a few hours incubation time. This allows for rapid, quantitative, often same-day generation of results, unlike classic plaque assay protocols which typically require several days of incubation in order to manually count visible plaques7-9. 
The protocol outlines the steps of our titration method using oncolytic Vesicular Stomatitis Virus encoding a Fluc transgene (VSV&#8710;51-Fluc) as an example and provides an overview of 1. Sample preparation 2. The plating of a permissive cell line for virus titration using an automated dispenser 3. The preparation of the viral standard curve 4. The transfer of the sample supernatants onto the permissive cell line using a 96-well manual pipettor 5. The assessment of sample cytotoxicity using a cell viability reagent 6. The preparation of the luciferin substrate 7. Reading of bioluminescence and 8. Data analysis.

<table><tbody><tr><td>Dulbeccos&#039; Modified Eagle&#039;s Medium (DMEM)</td><td>Corning</td><td>SH30243.01</td><td></td></tr><tr><td>Fetal bovine serum</td><td>NorthBio Inc.</td><td>NBSF-701</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-1</td><td>Prepare a 1 mM solution, pH 7.3</td></tr><tr><td>alamarBlue&amp;nbsp;</td><td>AbD Serotec</td><td>BUF012B</td><td></td></tr><tr><td>D-Luciferin potassium salt</td><td>Biotium</td><td>10101-2</td><td></td></tr><tr><td>96-well solid white flat bottom polystyrene TC-treated microplates</td><td>Corning</td><td>3917</td><td>384-well plates can also be used for higher throughput</td></tr><tr><td>Synergy Mx</td><td>BioTek</td><td>SMTBL</td><td>Monochromator microplate reader</td></tr><tr><td>Liquidator96</td><td>Mettler Toledo</td><td>LIQ-96-200</td><td>96 tip manual pipetting system</td></tr><tr><td>Liquidator96 LTS Tips sterilized with filters</td><td>Mettler Toledo</td><td>LQR-200F</td><td>Any sterile filtered tips compatible with pipettors of choice are appropriate</td></tr><tr><td>Microflo</td><td>BioTek</td><td>111-206-21</td><td>Used to plate cells in a 96-well plate</td></tr><tr><td>Fluoroskan Ascent FL</td><td>Thermo Scientific</td><td>5210450</td><td>Microplate fluorometer</td></tr></tbody></table>

high-throughput titration of luciferase-expressing recombinant viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

A summary of the workflow describing the high-throughput method is illustrated in Figure 1. Figure 2 shows the results of a typical standard curve ofVSV&#8710;51-Fluc. Four-parameter non-linear regression analysis generated a Hill plot from which unknown input pfu (estimate of viral titer) can be interpolated. These estimated titers are termed viral expression units (VEU). Figure 3A shows VEUs and titers obtained by performing a standard plaque assay with the same samples10. Samples originated from an experiment where various chemicals were used to enhance the replication and spread of VSV&#8710;51-Fluc in 786-0 cells. VEUs interpolated from the standard curve must be multiplied by a factor which is based on the dilution of sample supernatants being transferred on to Vero cells (in this case, the dilution factor is 5). The linear correlation between titers and VEU is shown in Figure 3B with an R2 of 0.8083 and a Pearson&#8217;s r score of 0.899 (p &#60;0.0001). In Figure 4, typical cell cytotoxicity data obtained from a metabolic assay is shown for 786-0 cells treated with chemicals prior to infection with VSV&#8710;51-Fluc. Finally, Figure 5 shows typical standard curves obtained with various viruses (Herpes Simplex Virus (HSV), Vaccinia virus, and Adeno-Associated Virus (AAV), all expressing Firefly luciferase) and describes incubation times and freeze-thaw cycles, as required.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> 
Figure 1. Workflow of high-throughput virus titering and cytotoxicity assay using VSV&#8710;51-Fluc. (A) Seed 2.5 x 104 Vero cells per well (100 &#956;l) and incubate at 37 &#176;C. (B) 24 hr later transfer 25 &#956;l of standard curve on to Vero cells (2 columns per plate). (C) Transfer 25 &#956;l&#160;of samples to be titered on to remaining Vero cells. Centrifuge plates and incubate at 37 &#176;C. (D) In an amount equal to 10% of the volume in the well, add resazurin reagent to the plate containing the original samples. Incubate plates at 37 &#176;C. (E) After 3 hr, read and record fluorescence and assess cytotoxicity. (F) After 5 hr, add 25 &#956;l&#160;of 2 mg/ml&#160;solution of luciferin to each well of Vero cells. Read luminescence and calculate Viral Expression Units.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> 
Figure 2. Expected standard curve of VSV&#8710;51-Fluc. Luciferase expression was measured 5&#160;hr post supernatant transfer at five different points within a well using a luminometer and bioluminescence was expressed in mean relative light units (RLU). Mean RLU was plotted against known input pfu/ml&#160;to solve the non-linear regression and generate a Hill equation. The average of two replicate curves and standard error bars are shown (r2= 0.9993).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> 
Figure 3. Comparison of standard plaque assay titers with those obtained by high-throughput method. (A) Viral titers in pfu/ml&#160;obtained by standard plaque assay on Vero cells were compared with calculated viral titers (VEU/ml) from the same samples titered using the high-throughput luciferase assay. (B) Linear relationship between VEU/ml&#160;and titer obtained via standard plaque assay. Linear regression curve and coefficient of determination (R2) are shown.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> 
Figure 4. Sample viability. Sample viability prior to supernatant transfer onto Vero cells was determined by assessing cellular metabolic activity using a commercially available resazurin solution. Raw fluorescence values were normalized to that of untreated, uninfected wells.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> 
Figure 5. Expected standard curves with HSV, Vaccinia, and AAV viruses. Luciferase expression was measured at five different points within a well using a luminometer and bioluminescence was expressed in mean relative light units (RLU). (A) HSV standard curve was added onto Vero cells plated 24 hr earlier at a density of 2.5 x 104 cells per well (100 &#956;l) and luciferase measurement was made 17 hr post supernantant transfer (R2= 0.9489, n=1). A Hill equation was generated by solving the non-linear regression. (B) Vaccinia standard curve was added onto Vero cells plated 24 hr earlier a density of 2.5 x 104 cells per well (100 &#956;l) and incubated for 2.5 hr at 37 &#176;C, after which luciferase measurements were taken (R2= 0.9892). A linear equation was generated by solving for the linear regression. (C) AAV standard curve was added onto human lung carcinoma cells (A549) plated 24 hr&#160;earlier at a density of 2.5 x 104 cells per well (100 &#956;l) and luciferase measurement was made 24 hr post infection. A Hill equation was generated by solving the non-linear regression. The average of five replicate curves and standard error bars are shown (R2= 0.9926).

Watch this Scientific Journal Video about High-throughput Titration of Luciferase-expressing Recombinant Viruses at JoVE.com

High-throughput Titration of Luciferase-expressing Recombinant Viruses

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

high-throughput-titration-of-luciferase-expressing-recombinant-viruses

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Biology

12.8K Views.  Ottawa Hospital Research Institute. This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Video: High-throughput Titration of Luciferase-expressing Recombinant Viruses

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Immunology and Infection

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Cancer Research

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

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.

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

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Assessing Serum Neutralization Against SARS-CoV-2 with Pseudotyped Viruses

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Author Spotlight: Studying Host-Virus Interactions with Pseudotyped Viruses 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

The Luciferase Assay: A High-Throughput Assay to Measure Luminescent Signals Using Engineered Nanoluciferase System-Based Bioreporters

Source: Rezaei, R., et al. <a href="https://app.jove.com/v/62488">Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter.</a> J. Vis. Exp. (2021).
The video demonstrates the luciferase assay used to quantify luciferase-tagged proteins. The assay uses an engineered luciferase enzyme composed of a large fragment that binds with the small fragment, which is conjugated with a protein of interest. The bound subunits form an active enzyme that, in the presence of the substrate, releases a luminescent signal. This signal is then quantified using a luminometer.

Source: Rezaei, R., et al. Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter. J. Vis. Exp. (2021).
The video ...