This research received support from the Ministry of Higher Education Malaysia under the Long-Term Research Grant Scheme (LRGS MRUN Phase 1: LRGS MRUN/F1/01/2018) and funding for the Higher Institution Centre of Excellence (HICoE) program (MO002-2019). Figure 3 in this study that shows the workflow of staining for the foci forming assay is adapted from &#34;DAB Immunohistochemistry&#34; by BioRender.com (2022). Retrieved from&#160;https://app.biorender.com/biorender-templates/t-5f3edb2eb20ace00af8faed9-dab-immunohistochemistry.

1. Virus propagation
<ol>
	<li>Cell preparation
	<ol>
		<li>Grow Vero cells in a 75 cm2 cell culture flask containing 12 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (see Table of Materials). Incubate the cells in a cell culture incubator at 37 &#176;C with 5% CO2.</li>
		<li>Monitor the cells under a microscope; once the cells reach 70%-90% confluency, they are ready to be used (Figure 1A) . 
		NOTE: The Vero cells double approximately every 24 h17. Dilutions of 1:10 should take 3 to 4 days to reach 80%-90% confluency in a 75 cm2 flask. Monitor the cells daily or every other day.</li>
	</ol>
	</li>
	<li>Infection of the cell monolayer
	<ol>
		<li>Remove the growth medium from the cell culture flask using a 10 mL serological pipette. Rinse the flask with 3 mL of Dulbecco&#39;s phosphate buffered saline (dPBS)&#160;2x&#160;using a 5 mL serological pipette. 
		NOTE: A beaker with 10% sodium hypochlorite must be prepared to disinfect any discarded infectious liquid waste from flasks/plates.</li>
		<li>Add 2 mL of serum-free DMEM (DMEM supplemented with 2 mM L-glutamine without FBS) and 20 &#181;L of ZIKV inoculum into the cell culture flask. Incubate the flask with gentle rocking for 1 h at room temperature to allow virus adsorption. 
		NOTE: Titration of the initial ZIKV stock will be helpful to determine the volume of ZIKV inoculum addition (step 2). 
		CAUTION: ZIKV is classified as a biosafety level 2 (BSL-2) pathogen. All procedures with materials containing ZIKV must be conducted in a certi&#64257;ed biosafety cabinet and follow all the laboratory standard operating procedures (SOPs) with appropriate personal protective equipment.</li>
	</ol>
	</li>
	<li>Addition of the overlay medium
	<ol>
		<li>After the 1 h incubation, remove and discard the diluted virus inoculum using a 5 mL serological pipette. Rinse the cell culture flask with 3 mL of dPBS 2x.</li>
		<li>Add 12 mL of maintenance media (DMEM supplemented with 2% FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin) into the cell culture flask to maintain the infected cells.</li>
		<li>Incubate the infected Vero cells for 3 days in a cell culture incubator at 37 &#176;C with 5% CO2. 
		NOTE: Monitor daily for the CPE of infected Vero cells under a microscope.</li>
	</ol>
	</li>
	<li>Harvesting of cell culture supernatant containing ZIKV
	<ol>
		<li>On day 3 post-infection, cell rounding and detachment (CPE) can be observed (Figure 1B-D). Harvest the cell culture supernatant containing the ZIKV into a 50 mL centrifuge tube using a 10 mL serological pipette.</li>
		<li>Use a 0.22 &#181;m Polyethersulfone syringe filter (see Table of Materials) to filter the cell culture supernatant. Aliquot 500 &#181;L of the filtered cell culture supernatant into 2.0 mL screw cap tubes and store at -80 &#176;C until further use. 
		NOTE: Harvested ZIKV can be stored at -80 &#176;C for long-term storage. 
		&#8203;CAUTION: Extra precautions must be taken if a syringe needle is used, as the needle can puncture the skin. Laboratory SOPs need to be established.</li>
	</ol>
	</li>
</ol>
2. Virus quantification
<ol>
	<li>Cell preparation
	<ol>
		<li>Seed Vero cells in designated plates (24-well plate: 0.5 mL/well, 7.5 x 104 cells/well; 96-well plate: 0.1 mL/well, 1.5 x 104 cells/well) and incubate the plate overnight in a cell culture incubator at 37 &#176;C with 5% CO2. 
		NOTE: There are five different time points to be tested (24-well plate: 48 h, 60 h, 72 h, 84 h, and 96 h post-infection; 96-well plate: 24 h, 36 h, 48 h, 60 h, and 72 h post-infection); therefore, prepare one plate for each time point.</li>
		<li>After the 24 h incubation and once the cells reach 70%-90% confluency (Figure 1E), the plate is ready for infection. Proceed to prepare the diluted virus stock (step 2.2) prior to infection of the cell monolayer.</li>
	</ol>
	</li>
	<li>Virus dilution
	<ol>
		<li>For 24- and 96-well plates, prepare six sterile 1.5 mL microcentrifuge tubes for each plate to carry out a tenfold dilution (10-1 up to 10-5), including a negative control. For the experiment setup for a 24-well plate, add 450 &#181;L of serum-free DMEM into all six microcentrifuge tubes. For the experiment setup for a 96-well plate, add 135 &#181;L of serum-free DMEM into six tubes. 
		NOTE: Label each tube clearly to indicate the dilution of its contents before performing the tenfold serial dilution.</li>
		<li>To perform serial dilution, for the 24-well plate experiment setup, add 50 &#181;L of ZIKV stock that was harvested from step 1 into the 10-1 tube that contains 450 &#181;L of serum-free DMEM. For the 96-well plate experiment setup, add 15 &#181;L of ZIKV stock into the 10-1 tube that contains 135 &#181;L of serum-free DMEM.</li>
		<li>Vortex each tube to mix the virus and medium. This is the first tenfold dilution. 
		NOTE: Proper mixing of virus and culture medium in each dilution tube is required to ensure accurate serial dilution.</li>
		<li>For the 24-well plate experiment setup, use a fresh pipette tip to resuspend the 10-1 tube and transfer 50 &#181;L of diluted ZIKV into the 10-2 tube as a second tenfold dilution. For the 96-well plate experiment setup, use a fresh pipette tip to resuspend the 10-1 tube and transfer 15 &#181;L of diluted ZIKV into the 10-2 tube as a second tenfold dilution.</li>
		<li>Repeat step 2.2.4 four times until the fifth tube (exclude negative control). 
		NOTE: Use a fresh pipette tip after each pipetting step to avoid cross-contamination.</li>
	</ol>
	</li>
	<li>Infection of the cell monolayer
	<ol>
		<li>Remove and discard the conditioned medium from each well (24-well plate: 0.5 mL/well; 96-well plate: 0.1 mL/well).</li>
		<li>Rinse each well with dPBS 2x (24-well plate: 300 &#181;L/well; 96-well plate: 60 &#181;L/well).</li>
		<li>Working from the highest to the lowest dilution, add each serially diluted virus inoculum into the well (24-well plate: 200 &#181;L/well; 96-well plate: 40 &#181;L/well). 
		&#8203;NOTE: The infection of each dilution will be performed in duplicated wells. Maintain two uninfected wells as the negative control. Label the plate and wells clearly to avoid confusion. Change the pipette tips after each pipetting step to prevent cross-contamination.</li>
		<li>Incubate the plate with gentle rocking for 1 h at room temperature to allow virus adsorption.</li>
	</ol>
	</li>
	<li>Addition of the overlay cell culture medium
	<ol>
		<li>After 1 h of incubation, remove and discard the virus suspension from the lowest concentration to the highest.</li>
		<li>Wash the infected cells with dPBS 2x (24-well plate: 300 &#181;L/well; 96-well plate: 60 &#181;L/well).</li>
		<li>Overlay the well with DMEM (supplemented with 2% FBS, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin) and 1.5% low viscosity carboxymethyl cellulose (CMC; see Table of Materials) (24-well plate: 1 mL/well; 96-well plate: 0.2 mL/well).</li>
		<li>Incubate the plate in a cell culture incubator at 37 &#176;C with 5% CO2. 
		NOTE: Do not disturb the plate during this incubation period.</li>
		<li>Take the plate out from the cell culture incubator for staining at different time points (24-well plate: 48 h, 60 h, 72 h, 84 h, and 96 h post-infection; 96-well plate: 24 h, 36 h, 48 h, 60 h, and 72 h post-infection).</li>
	</ol>
	</li>
</ol>
3. Staining
<ol>
	<li>Cell fixation
	<ol>
		<li>After the specific hours of incubation (24-well plate: 48 h, 60 h, 72 h, 84 h, and 96 h post-infection; 96-well plate: 24 h, 36 h, 48 h, 60 h, and 72 h post-infection), remove and discard the overlay medium and wash the cells 3x with 1x PBS. For the 96-well plate, remove and discard the overlay medium and wash the cells 3x with 60 &#181;L/well of 1x PBS with a multichannel pipette.</li>
		<li>Add 4% paraformaldehyde to fix the cells (24-well plate: 300 &#181;L/well; 96-well plate: 60 &#181;L/well). Incubate the plate at room temperature for 20 min. 
		CAUTION: Paraformaldehyde is a solid polymerized formaldehyde. It is a flammable, solid, white crystalline powder that releases formaldehyde gas when mixed with water or heated. All procedures involving the use of paraformaldehyde must be conducted in a certified chemical fume hood or approved exhausted enclosures following laboratory SOPs specific to formaldehyde-containing chemicals.</li>
		<li>After 20 min, discard the paraformaldehyde and wash the cells 3x with 1x PBS. Perform permeabilization, blocking, and antibody incubations following steps 3.2-3.5. 
		NOTE: When discarding 4% paraformaldehyde, follow the university or institute&#39;s waste disposal procedure.</li>
	</ol>
	</li>
	<li>Cell permeabilization
	<ol>
		<li>Add 1% octylphenoxy poly(ethyleneoxy)ethanol (see Table of Materials) to permeabilize the cells (24-well plate: 200 &#181;L/well; 96-well plate: 40 &#181;L/well). Incubate the plate at room temperature for 15 min.</li>
		<li>After 15 min, discard the octylphenoxy poly(ethyleneoxy)ethanol and wash the cells 3x with 1x PBS.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Add 3% skim milk to reduce the nonspecific binding in the sample (24-well plate: 500 &#181;L/well; 96-well plate: 100 &#181;L/well). Incubate the plate at room temperature for 2 h.</li>
		<li>After 2 h, discard the 3% skim milk and wash the cells 3x with 1x PBS. 
		&#8203;NOTE: This is a stopping point. After adding 3% skim milk, the plates can be stored at 4 &#176;C up to 2 days before primary antibody insertion.</li>
	</ol>
	</li>
	<li>Primary antibody incubation
	<ol>
		<li>Add anti-flavivirus monoclonal antibody, 4G2 (clone D1-4G2-4-15; primary antibody, see Table of Materials) diluted in 1% skim milk (1:1,000 ratio) (24-well plate: 200 &#181;L/well; 96-well plate: 40 &#181;L/well). Incubate the plate at 37 &#176;C for 1 h.</li>
		<li>After 1 h, remove the solution containing the monoclonal antibody and wash the cells 3x with 1x PBS.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation
	<ol>
		<li>Add goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (HRP) (see Table of Materials) diluted in 1% skim milk (1:1,000 ratio) (24-well plate: 200 &#181;L/well; 96-well plate: 40 &#181;L/well). Incubate the plates at 37 &#176;C for 1 h.</li>
		<li>After 1 h, remove the solution containing the secondary antibody and wash the cells 3x with 1x PBS.</li>
	</ol>
	</li>
	<li>Substrate incubation
	<ol>
		<li>Add 3,3&#39;diaminobenzidine (DAB) peroxidase substrate (see Table of Materials) and incubate the plate for 30 min in the dark (24-well plate: 200 &#181;L/well; 96-well plate: 40 &#181;L/well). After 30 min, stop the reaction by washing the wells with water.</li>
		<li>Air-dry the plates overnight and proceed to foci enumeration after the plates are completely dry.</li>
	</ol>
	</li>
</ol>
4. Determination of the virus titer
<ol>
	<li>Manual foci counting process (24-well format)
	<ol>
		<li>Foci can be visualized under a stereo microscope. Select the dilution that produces 50-200 foci.</li>
		<li>Count the foci for each replicate of the selected dilution. Calculate the average number of foci for each of the selected dilutions.</li>
		<li>Calculate the foci forming unit per mL (FFU/mL) for each sample with the formula below: FFU/mL = average number of foci counted/(dilution x volume of diluted virus added).</li>
	</ol>
	</li>
	<li>Automated foci counting process (96-well format)
	<ol>
		<li>Scan the 96-well plate on a commercial software analyzer.
		<ol>
			<li>Double click on the software (see Table of Materials) to open.</li>
			<li>Click on Switch Suites and ensure that the suite is switched to any one that is applicable (Supplementary Figure 1A). Click OK.</li>
			<li>Begin the scan by clicking Scan &#62; Full plate scan (Supplementary Figure 1B).</li>
			<li>Click on Dashboard to ensure the capture format is the 96-well format (Supplementary Figure 2A).</li>
			<li>Select the centering mode as &#34;Auto Prealignment User Verified&#34; (Supplementary Figure 2B).</li>
			<li>Click on Eject to load the plate. 
			NOTE: Open the lid of the plate and place it upward with Row A at the top (Supplementary Figure 3A).</li>
			<li>Enter the plate name and click on OK. Click on Load to load the plate.</li>
			<li>Click on Start to start the scan. After that, calibrate the positions for A1, A12, and H1 using the buttons &#34;Up, Down, Left, Right&#34; and click on Confirm (Supplementary Figure 3B). 
			NOTE: The software will start scanning each well.</li>
			<li>Once the scanning is complete, an overview image will pop up. Click on Close.</li>
			<li>Exit the scan by clicking on Back to switchboard.</li>
		</ol>
		</li>
		<li>Count the 96-well plate using the software.
		<ol>
			<li>Click Count &#62; Smart Count.</li>
			<li>On the software, it will show &#34;Step 1 of 5: Select plates to count&#34;. Click on Load plate(s). Tick the whole folder and click on Select to import the scanned plate (Supplementary Figure 4A).</li>
			<li>On the software, it will show &#34;Step 2 of 5: Define counting parameters&#34;. Click on Adjust parameters, and the parameters will be displayed (diffuse process; small/normal/large/largest/largest +1; spot separation; counted area; background balance; fiber removal; gating). 
			NOTE: Start with one well, and check back and forth to find the best settings for all the wells.</li>
			<li>Once done, click on Next (Supplementary Figure 4B).</li>
			<li>On the software, it will show &#34;Step 3 of 5: Select/unselect wells&#34;. Once the selection of the well to be counted is complete, click on Next to proceed. 
			NOTE: The wells selected to be counted will appear with a &#34;C&#34; at the top right corner of each well (Supplementary Figure 5A).</li>
			<li>On the software, it will show &#34;Step 4 of 5: Output Settings&#34;. Click on View/Modify Output Settings to check if the settings (such as image format) are suitable. Click on Save and Exit &#62; Next (Supplementary Figure 5B).</li>
			<li>On the software, it will show &#34;Step 5 of 5: Start AutoCount&#34;. Click on Start AutoCount (Supplementary Figure 6A).</li>
			<li>Once complete, click on Exit AutoCount.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform quality control (QC) in the commercial software analyzer.
	<ol>
		<li>On the switchboard page, click on Quality Control &#62; Add plate(s). Tick the whole folder to import the counted plate, click on Select &#62; Start QC (Supplementary Figure 6B).</li>
		<li>Double-click on each well to audit the spots. Click on Count to remove any spots. The software will start counting. 
		NOTE: Ensure that &#34;Spots: Remove&#34; is ticked. Completion of the counting can be indicated by the counted number of foci, for example &#34;30&#34; (Supplementary Figure 7A).</li>
		<li>Once the counting ends (indicated by the counted number of foci, for example &#34;30&#34;), click &#34;Yes&#34; to remove spots (Supplementary Figure 7B). Triple left-click on the spot to be removed. Right-click to finish, then click on Yes.</li>
		<li>Click on Finish Plate and Go to Project Overview once the QC is complete.</li>
	</ol>
	</li>
	<li>Perform imaging using the software analyzer.
	<ol>
		<li>Check the scanned, counted, and QC plate image (Figure 2).</li>
	</ol>
	</li>
</ol>

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J., 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=Zika+virus+infection+during+pregnancy+in+mice+causes+placental+damage+and+fetal+demise.">Zika virus infection during pregnancy in mice causes placental damage and fetal demise.</a> Cell. 165 (5), 1081-1091 (2016).</li><li>Kang, W., Shin, E. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+focus-forming+assay+with+automated+focus+counting+by+image+analysis+for+quantification+of+infectious+hepatitis+C+virions.">Colorimetric focus-forming assay with automated focus counting by image analysis for quantification of infectious hepatitis C virions.</a> PLoS One. 7 (8), e43960 (2012).</li><li>Brien, J. D., 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=Isolation+and+quantification+of+Zika+virus+from+multiple+organs+in+a+mouse.">Isolation and quantification of Zika virus from multiple organs in a mouse.</a> Journal of VIsualized Experiments. (150), e59632 (2019).</li><li>Payne, A. F., Binduga-Gajewska, I., Kauffman, E. B., Kramer, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+flaviviruses+by+fluorescent+focus+assay.">Quantitation of flaviviruses by fluorescent focus assay.</a> Journal of Virological Methods. 134 (1-2), 183-189 (2006).</li><li>Moser, L. A., 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=Growth+and+adaptation+of+Zika+virus+in+mammalian+and+mosquito+cells.">Growth and adaptation of Zika virus in mammalian and mosquito cells.</a> PLoS Neglected Tropical Diseases. 12 (11), e0006880 (2018).</li><li>Ishimine, T., Tadano, M., Fukunaga, T., Okuno, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+micromethod+for+infectivity+assays+and+neutralization+tests+of+dengue+viruses.">An improved micromethod for infectivity assays and neutralization tests of dengue viruses.</a> Biken Journal. 30 (2), 39-44 (1987).</li><li>Leiva, 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=Application+of+quantitative+immunofluorescence+assays+to+analyze+the+expression+of+cell+contact+proteins+during+Zika+virus+infections.">Application of quantitative immunofluorescence assays to analyze the expression of cell contact proteins during Zika virus infections.</a> Virus Research. 304, 198544 (2021).</li><li>Lee, E. M., Titus, S. A., Xu, M., Tang, H., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+Zika+viral+titer+assay+for+rapid+screening+of+antiviral+drugs.">High-throughput Zika viral titer assay for rapid screening of antiviral drugs.</a> ASSAY and Drug Development Technologies. 17 (3), 128-139 (2019).</li></ol>

The authors declare that they have no competing interests.

There are several assays to determine virus titer; the PFA has a similar virus quantitation protocol as the FFA, in which the virus inoculum is diluted to allow individual plaques or foci to be distinguished. After staining, each plaque or foci indicates a single infectious particle in the inoculum19. The PFA is stained with crystal violet to visualize plaque formation caused by cell lysis or death. Hence, the PFA is more time-consuming, as it requires a longer time for the virus to cause CPEs, and it is only restricted to the viruses that induce cell lysis or death. Many laboratories have successfully used FFAs to determine infectious virus titers for flaviviruses, including ZIKV19,20,21,22,23,24,25. The FFA is a cell-based colorimetric immunodetection method that detects the viral antigen with antibodies and, therefore, identifies the foci areas of infected cells using the immunostaining technique, but not plaques12,26. The FFA offers some advantages for ZIKV over the PFA. One clear advantage is that it is based on antibody recognition of viral components without discernable CPEs. In addition, using virus-specific antibodies is also useful in identifying different viruses or viral serotypes in mixed populations27. Therefore, the FFA is more specific than the PFA as it measures all the virus infections and is not dependent on the viruses that cause enough cell death to form a visible plaque28. The FFA also has a shorter incubation period than the PFA, which requires an obvious CPE that signifies cell lysis and death. Finally, using a 96-well plate, the FFA method provides the advantage of using more replicates and larger dilutions to detect and titrate the virus27,29. In addition, the reported FFA assay can be easily adapted for virus neutralization tests and methods to screen for antiviral compounds. Incorporating commercial or free automated cell counting instruments or software can further improve the usability (consistency, accuracy, throughput) of the FFA and its associated methods.
When comparing the 24-well plate to the 96-well plate, it is important to note that the 24-well format requires a larger number of cells to be grown in monolayer cultures, as well as larger amounts of media and virus stock. As a result, the 24-well plate format may not be suitable for use with samples with limited volumes. This limitation can be overcome by using the 96-well format, as described in this paper. Firstly, the 96-well format requires less material, making it a more cost-effective option. Additionally, the automated counting of stained virus foci in the 96-well format allows high throughput and rapid analysis, which is not possible in the 24-well format, which requires manual counting. This automated counting process also increases the reproducibility and reduces subjectivity compared to manual counting, leading to more accurate and reliable results of the FFA. Therefore, the 96-well format with automated imaging systems is highly recommended for laboratories that require high throughput and reliable virus quantification.
On the other hand, some people use the immunofluorescent focus assay to detect ZIKV25,27,28,30,31. The immunofluorescence focus assay parallels the FFA, except that it is performed by immunostaining the antigens with fluorochrome-conjugated specific antibodies, followed by the counting of infection foci under a fluorescence microscope25. This assay uses more costly reagents and needs fluorescence microscopy to complete the assay. Therefore, the immunofluorescence focus assay is limited to better-equipped laboratories, such as referral laboratories. It is not easy to use this assay in a resource-challenging setting.
Although the FFA has many advantages, it also has some limitations. Compared to other assays, such as the PFA, the FFA involves more steps during the staining processes. While the steps after fixation are flexible and allow for overnight or longer pauses, the staining of the foci still takes longer to complete. Furthermore, the FFA requires specific or cross-reacting antibodies in the staining process, which limits its applicability for identifying and quantifying new or novel viruses. Moreover, the cost of the FFA is higher compared to the PFA, which only requires crystal violet for staining. In addition to that, it is worth noting that the automated counting process (96-well format) can only be performed in a laboratory with the appropriate equipment; hence, it will be not suitable for laboratories without the necessary equipment.
In conclusion, we report detailed protocols for the propagation and quantification of ZIKV using the cell-based colorimetric immunodetection assay or the FFA in 24-well plate and 96-well plate formats. The method offers a number of practical advantages over the classical PFA. It is faster and amenable for high throughput applications when applied with an automated imaging system for foci counting. The standardization of reliable protocols for this study will greatly contribute to Zika research and can also be broadly adapted to quantify other clinically important viruses.

Zika virus (ZIKV) infection is an emerging mosquito-borne viral disease. The first isolation of ZIKV was in Uganda in 19471,2; it remained neglected from 1947 to 2007, as the clinical symptoms are most commonly asymptomatic and characterized by self-limiting febrile illness. In 2007, the Zika epidemic began in the Yap islands3,4, followed by larger epidemics in the Pacific regions (French Polynesia, Easter Island, Cook Islands, and New Caledonia) from 2013 to 20145,6,7,8, where the severe neurological complication Guillain-Barr&#233; syndrome (GBS) was reported in adults for the first time9. During 2015 and 2016, the first widespread ZIKV epidemic swept across the Americas after the emergence of the Asian genotype of ZIKV in Brazil in as early as 201310. During this outbreak, 440,000 to 1.3 million cases of microcephaly, and other neurological disorders, were reported in newborn babies11. There is currently no specific cure or treatment for ZIKV infection; hence, there is an urgent medical need for ZIKV vaccines capable of preventing infections, particularly during pregnancy.
Virus quantification is a process to determine the number of viruses present in a sample. It plays an important role in research, and academic laboratories involve many fields, such as medicine and life sciences. This process is also important in commercial sectors, such as the production of viral vaccines, recombinant proteins, viral antigens, or antiviral agents. Many methods or assays can be used for virus quantification12. The choice of methods or assays normally depends on the virus characteristics, desired level of accuracy, and the nature of the experiment or research. In general, methods of quantifying viruses can be divided into two categories: molecular assays that detect the presence of viral nucleic acid (DNA or RNA) and assays that measure virus infectivity in vitro12. Quantitative polymerase chain reaction (qPCR, for DNA) or quantitative reverse transcription polymerase chain reaction (qRT-PCR, for RNA)13 and digital droplet PCR14 are examples of common molecular techniques used to quantitate the viral nucleic acid in a given sample15. However, these highly sensitive molecular techniques cannot differentiate between viable and non-viable viruses15. Therefore, research that requires information on biological features, such as virus infectivity on cells, cannot be completed using the abovementioned molecular techniques; assays that can measure and determine the presence of viable viruses are needed. Assays that measure virus infectivity include the plaque forming assay (PFA), 50% tissue culture infectious dose (TCID50), the fluorescent focus assay, and transmission electron microscopy (TEM)12.
The PFA, developed by Renato Dulbecco in 1952, is one of the most commonly used methods for virus titration, including for ZIKV16. It is used to directly determine the viral concentrations for infectious lytic virions. The method is based on the ability of a lytic virus to produce cytopathic effects (CPEs; zones of cell death or plaques, an area of infection surrounded by uninfected cells) in an inoculated cell monolayer after viral infection. However, there are several drawbacks to the assay that affect its utility. The assay is time-consuming (takes approximately 7-10 days, depending on viruses), CPE-dependent, and prone to errors. In the present study, we report an immunocolorimetric technique, the focus forming assay (FFA), for detecting and quantifying ZIKV in 24-well plate and 96-well plate formats.

<table><tbody><tr><td>0.22 &amp;micro;m Polyethersulfone syringe filter</td><td>Sartorius</td><td>S6534-FMOSK</td><td></td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>Nest</td><td>615601</td><td></td></tr><tr><td>10 mL sterile serological pipette</td><td>Labserv</td><td>14955156</td><td></td></tr><tr><td>1x Dulbecco&amp;rsquo;s phosphate-buffered saline (dPBS)</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>2.0 mL Screw cap tube&amp;nbsp;</td><td>Axygen</td><td>SCT-200-SS-C-S</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>25 mL Sterile serological pipettes</td><td>Labserv</td><td>14955157</td><td></td></tr><tr><td>3,3&#039;Diaminobenzidine (DAB) peroxidase substrate</td><td>Thermo Scientific</td><td>34065</td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sanyo</td><td>MCO-18AIC</td><td></td></tr><tr><td>5 mL sterile serological pipette</td><td>Labserv</td><td>14955155</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Falcon</td><td>LAB352070</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask&amp;nbsp;</td><td>Corning</td><td>430725U</td><td></td></tr><tr><td>96-well plate</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Anti-flavivirus monoclonal antibody, 4G2 (clone D1-4G2-4-15)</td><td>MilliporeSigma</td><td>MAB10216</td><td></td></tr><tr><td>Autoclaved 20x Phosphate buffered saline (PBS)</td><td>N/A</td><td>N/A</td><td>22.8 g of 8 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 4.0 g of 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 160 g of 0.14 M NaCl, 4.0 g of 2.7 mM KCl, 1 L of MilliQ H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Biological safety cabinet, Class II</td><td>Holten</td><td>HB2448</td><td></td></tr><tr><td>CTL S6 Universal ELISpot/FluoroSpot Analyzer</td><td>ImmunoSpot, Cellular Technology Limited (CTL)</td><td>CTL-S6UNV12</td><td>Commercial software analyzer</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>12800-017</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Bovogen</td><td>SFBS</td><td></td></tr><tr><td>Goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (HRP)</td><td>MilliporeSigma</td><td>12-349</td><td></td></tr><tr><td>Hemacytometer</td><td>Laboroptik LTD</td><td>Neubauer improved</td><td></td></tr><tr><td>IGEPAL CA-630 detergent</td><td>Sigma-Aldrich</td><td>I8896</td><td>Octylphenoxy poly(ethyleneoxy)ethanolIGEPAL&amp;nbsp;</td></tr><tr><td>Inverted microscope</td><td>ZEISS</td><td>TELAVAL 31</td><td></td></tr><tr><td>Laboratory rocker</td><td>FINEPCR</td><td>CR300</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Low viscosity carboxymethyl cellulose (CMC)</td><td>Sigma-Aldrich</td><td>C5678</td><td></td></tr><tr><td>Multichannel micropipette (10 - 100 &amp;micro;L)</td><td>Eppendorf</td><td>3125000036</td><td></td></tr><tr><td>Multichannel micropipette (30 - 300 &amp;micro;L)</td><td>Eppendorf</td><td>3125000052</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Single channel pipettes (10 - 100 &amp;micro;L)</td><td>Eppendorf</td><td>3123000047</td><td></td></tr><tr><td>Single channel pipettes (100 - 1000 &amp;micro;L)</td><td>Eppendorf</td><td>3123000063</td><td></td></tr><tr><td>Single channel pipettes (20 - 200 &amp;micro;L)</td><td>Eppendorf</td><td>3123000055</td><td></td></tr><tr><td>Skim milk</td><td>Sunlac Low Fat</td><td>N/A</td><td>Prepare 3% Skim milk in 1x PBS for blocking stage in staining</td></tr><tr><td>Sodium Hypochlorite</td><td>Clorox</td><td>N/A</td><td>To disinfect any discarded infectious liquid waste from flasks/plates</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1000</td><td></td></tr><tr><td>Syringe disposable, Luer Lock, 10 mL with 21 G Needle</td><td>Terumo</td><td>SS10L21G</td><td></td></tr><tr><td>Vero African green monkey kidney cells&amp;nbsp;</td><td>-</td><td>ECACC 88020401</td><td>Received from collaborator. However, Vero cells obtained from other suppliers should be able to be used with some optimization.</td></tr></tbody></table>

virus propagation and cell-based colorimetric quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barr&#233; syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

ZIKV can be quantified using the FFA, as outlined schematically in Figure 3. For the 24-well plate, the infected Vero cells were fixed at 48 h, 60 h, 72 h, 84 h, and 96 h post-infection. The results showed that the cells remained intact (no cell detachment was observed) after 96 h (4 days) post-infection (Figure 4 and Supplementary Figure 8A-E). The appearance of virus foci was first observed at 48 h (2 days) post-infection (Figure 4A-F). However, the foci size was too small, making it difficult to count the foci accurately. The optimal foci size was achieved at 60 h (2.5 days) post-infection (Figure 4B). At the latter time points (72 h, 84 h, and 96 h post-infection), foci were larger and tended to merge or overlap. The merged or overlapped foci increased over time (Figure 4C-E). Therefore, foci formed at 60 h (2.5 days) after the infection were chosen to determine the ZIKV titer in a 24-well plate (Figure 4B).
For the 96-well plate, the infected Vero cells were fixed at 24 h, 36 h, 48 h, 60 h, and 72 h post-infection. The results showed that the cells remained intact after 72 h (3 days) post-infection (Figure 5A-F and Supplementary Figure 9A-E). The appearance of virus foci was first observed at 24 h (1 day) post-infection (Figure 5A). However, the foci size was too small up to 36 h (1.5 days) post-infection, making it difficult to accurately determine the number of foci (Figure 5A,B). The optimal foci size was achieved at 48 h (2 days) post-infection (Figure 5C). At the latter time points (60 h and 72 h post-infection), overlapped or merged foci were observed, and the number of the overlapped foci increased over time (Figure 5D,E). Therefore, foci formed at 48 h (2 days) after the infection were chosen to determine the virus titer of ZIKV isolates (Figure 5C). The foci formation can be visualized and enumerated using commercial software analyzers as an alternative.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64578/64578fig01.jpg" /> 
Figure 1: Microscopic image of Vero cells. (A)&#160;Vero cells in 40x magnification showed70%-90% confluency in a 75 cm2 cell culture flask for virus propagation. (B) CPE on Vero cells after being infected with ZIKV on day 1 post-infection at 100x magnification. (C) CPE on Vero cells after being infected with ZIKV on day 2 post-infection at 100x magnification. (D) CPE on Vero cells after being infected with ZIKV on day 3 post-infection at 100x magnification. (E) Vero cells at 40x magnification showed 70%-90% confluency in a 24-well plate after 24 h of incubation for virus quantification. Scale bar: 100 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/64578fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64578/64578fig02.jpg" /> 
Figure 2: Image of a 96-well format scanned, counted, and post-quality control plate using a commercial software analyzer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/64578fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64578/64578fig03.jpg" /> 
Figure 3: Workflow of staining for the foci forming assay. The staining processes including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/64578fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64578/64578fig04.jpg" /> 
Figure 4: Foci forming assay for ZIKV P6-740 in 24-well plates at different time points. (A) Foci are less distinct on day 2 (48 h) post-infection. (B) Optimal foci size can be seen on day 2.5 (60 h) post-infection. (C-E) Foci have merged on day 3 (72 h), day 3.5 (84 h), and day 4 (96 h) post-infection. (F) Negative control of the plate. Scale bar: 1,000 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/64578fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64578/64578fig05.jpg" /> 
Figure 5: Foci forming assay for ZIKV P6-740 in 96-well plates at different time points. (A) Foci are less distinct to be counted on the commercial software analyzer on day 1 (24 h) post-infection. (B) Foci are less distinct to be counted on the commercial software analyzer on day 1.5 (36 h) post-infection. (C) Optimal foci size can be seen on day 2 (48 h) post-infection. (D,E) Foci have merged on day 2.5 (60 h) and day 3 (72 h) post-infection. (F) Negative control of the plate. Scale bar: 1,000 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/64578fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Screenshots of the startup page of the commercial software analyzer. (A) Switch the suite to any applicable one on the commercial software analyzer. Click &#34;OK&#34; once done. (B) To begin scanning on the commercial software analyzer, click &#34;Scan&#34; and then click &#34;Full plate scan&#34;. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Screenshots to view &#34;Capture format&#34; and the selected centering mode as &#34;Auto Prealignment User Verified&#34; on the commercial software analyzer. (A) To view the capture format, click &#34;Dashboard&#34; and select the 96-well format as the capture format. (B) For centering mode, select &#34;Auto Prealignment User Verified&#34;. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: 96-well plate placement on the commercial software analyzer plate reader tray and a screenshot to calibrate the positions for wells A1, A12, and H1. (A) Place the plate upward with Row A at the top. (B) To calibrate the positions for wells A1, A12, and H1, use the buttons &#34;Up, Down, Left, Right&#34;. Once done, click &#34;Confirm&#34;. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: Screenshots to select folders containing scanned plates for counting on the commercial software analyzer and &#34;Step 2 of 5: Define counting parameters&#34;. (A) To count the scanned plates, click &#34;Load plate(s)&#34;. Tick the whole folder and click &#34;Select&#34; to import the scanned plates. (B) Adjust the counting parameters. Click &#34;Next&#34; once the parameters are set. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 4.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5: Screenshots of the commercial software analyzer &#34;Step 3 of 5: Select/unselect wells&#34; and &#34;Step 4 of 5: Output Settings&#34;. (A) Select and unselect the wells to be counted and click &#34;Next&#34; once done. (B) For output settings, click &#34;View/Modify Output Settings&#34; to check if the settings are suitable (e.g., image format). Click &#34;Save and Exit&#34; once done and then click &#34;Next&#34;. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 5.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 6: Screenshots of the commercial software analyzer &#34;Step 5 of 5: Start AutoCount&#34; and quality control page. (A) Once ready for plate counting, click &#34;Start AutoCount&#34;. (B) On the quality control page, tick the whole folder to import the counted plate, click &#34;Select&#34;, then click &#34;Start QC&#34;. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 6.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 7: Screenshots of the commercial software analyzer quality control page. (A) Double-click each well to audit spots. Click &#34;Count&#34; to remove any spot. Tick &#34;Spots: Remove&#34;. (B) Triple left-click to remove spots. Right click to finish, then click &#34;Yes&#34;. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 7.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 8: Foci forming assay for ZIKV P6-740 in 24-well plates at different time points. A tenfold serial dilution (10-1 to 10-5) was performed on ZIKV P6-740 and infection was performed on Vero cells in duplicate, including a negative control. A raw data image was taken using a stereo microscope, with the scale bar indicating 1,000 &#181;m. (A)&#160;Day 2 (48 h) post-infection. (B)&#160;Day 2.5 (60 h) post-infection. (C)&#160;Day 3 (72 h) post-infection. (D)&#160;Day 3.5 (84 h) post-infection. (E)&#160;Day 4 (96 h) post-infection. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 8.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 9: Foci forming assay for ZIKV P6-740 in 96-well plates at different timepoints. A tenfold serial dilution (10-1 to 10-5) was performed on ZIKV P6-740 and infection was performed on Vero cells in duplicate, including a negative control. A raw data image was taken using a stereo microscope, with the scale bar indicating 1,000 &#181;m. (A)&#160;Day 1 (24 h) post-infection. (B)&#160;Day 1.5 (36 h) post-infection. (C)&#160;Day 2 (48 h) post-infection. (D)&#160;Day 2.5 (60 h) post-infection. (E)&#160;Day 3 (72 h) post-infection. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64578/Supplementary Figure 9.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Virus Propagation and Cell-Based Colorimetric Quantification at JoVE.com

Virus Propagation and Cell-Based Colorimetric Quantification

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

virus-propagation-and-cell-based-colorimetric-quantification

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

655 Views.  Universiti Malaya. The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

Video: Virus Propagation and Cell-Based Colorimetric Quantification

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Biology

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. ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

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.

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 ...