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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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+the+focus-forming+assay+and+digital+automated+imaging+analysis+for+the+detection+of+dengue+and+Zika+viral+loads+in+cultures+and+acute+disease.">Combination of the focus-forming assay and digital automated imaging analysis for the detection of dengue and Zika viral loads in cultures and acute disease.</a> Journal of Tropical Medicine. 2022, 2177183 (2022).</li><li>Dangsagul, W., 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+isolation,+propagation,+and+quantification+using+multiple+methods.">Zika virus isolation, propagation, and quantification using multiple methods.</a> PLoS One. 16 (7), e0255314 (2021).</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=Genotype-specific+neutralization+and+protection+by+antibodies+against+dengue+virus+type+3.">Genotype-specific neutralization and protection by antibodies against dengue virus type 3.</a> Journal of Virology. 84 (20), 10630-10643 (2010).</li><li>Lazear, H. M., 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+mouse+model+of+Zika+virus+pathogenesis.">A mouse model of Zika virus pathogenesis.</a> Cell Host &amp; Microbe. 19 (5), 720-730 (2016).</li><li>Miner, J. 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, an...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;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&#8217;s phosphate-buffered saline (dPBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL Screw cap tube&#160;</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&#39;Diaminobenzidine (DAB) peroxidase substrate</td>
			<td>Thermo Scientific</td>
			<td>34065</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C incubator with 5% CO2</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 cm2 tissue culture flask&#160;</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 Na2HPO4, 4.0 g of 1.5 mM KH2PO4, 160 g of 0.14 M NaCl, 4.0 g of 2.7 mM KCl, 1 L of MilliQ H2O</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&#39;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&#160;</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 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3125000036</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel micropipette (30 - 300 &#181;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 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000047</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes (100 - 1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes (20 - 200 &#181;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&#160;</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 (<strong class="xfig"...

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

Research

JoVE Journal

Immunology and Infection

592 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

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa (X. fastidiosa), a xylem-limited bacterium that is the causal agent of Pierce&#39;s disease (PD) of grapevine. Pierce&#8217;s disease is economically damaging; thus, H. vitripennis have become a target for pathogen management strategies. A dicistrovirus identified as Homalodisca coagulata virus-01 (HoCV-01) has been associated with an increased mortality in H. vitripennis populations. Because a host cell is required for HoCV-01 replication, cell culture provides a uniform environment for targeted replication that is logistically and economically valuable for biopesticide production. In this study, a system for large-scale propagation of H. vitripennis cells via tissue culture was developed, providing a viral replication mechanism. HoCV-01 was extracted from whole body insects and used to inoculate cultured H. vitripennis cells at varying levels. The culture medium was removed every 24 hr for 168 hr, RNA extracted and analyzed with qRT-PCR. Cells were stained with trypan blue and counted to quantify cell survivability using light microscopy. Whole virus particles were extracted up to 96 hr after infection, which was the time point determined to be before total cell culture collapse occurred. Cells were also subjected to fluorescent staining and viewed using confocal microscopy to investigate viral activity on F-actin attachment and nuclei integrity. The conclusion of this study is that H. vitripennis cells are capable of being cultured and used for mass production of HoCV-01 at a suitable level to allow production of a biopesticide.

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Here we present a protocol to propagate Homalodisca vitripennis cells and HoCV-1 in vitro. Medium was removed from HoCV-1 positive cultures and RNA extracted every 24 hr for 168 hr. Cell survivability was quantified by trypan blue staining. Whole virus particles were extracted post-infection. Extracted RNA was quantified by qRT-PCR.

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...