Name: virus propagation and cell-based colorimetric quantification
Uploaded: 2023-04-07T14:55:00
Description: Watch this Scientific Journal Video about Virus Propagation and Cell-Based Colorimetric Quantification at JoVE.com

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 (<stron...

<ol>
	<li>Dick, G. W. A., Kitchen, S. F., Haddow, A. J. <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.+I.+Isolations+and+serological+specificity.">Zika virus. I. Isolations and serological specificity.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 46 (5), 509-520 (1952).</li><li>Dick, G. W. A., Kitchen, S. F., Haddow, A. J. <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.+II.+Pathogenicity+and+physical+properties.">Zika virus. II. Pathogenicity and physical properties.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 46 (5), (1952).</li><li>Duffy, M. R., 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+outbreak+on+Yap+Island,+Federated+States+of+Micronesia.">Zika virus outbreak on Yap Island, Federated States of Micronesia.</a> The New England Journal of Medicine. 360 (24), 2536-2543 (2009).</li><li>Musso, D., Nilles, E. J., Cao-Lormeau, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+spread+of+emerging+Zika+virus+in+the+Pacific+area.">Rapid spread of emerging Zika virus in the Pacific area.</a> Clinical Microbiology and Infection. 20 (10), O595-O596 (2014).</li><li>Cao-Lormeau, V. -. 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=Zika+virus,+French+polynesia,+South+pacific,+2013.">Zika virus, French polynesia, South pacific, 2013.</a> Emerging Infectious Diseases. 20 (6), 1085-1086 (2014).</li><li>Dupont-Rouzeyrol, 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=Co-infection+with+Zika+and+dengue+viruses+in+2+patients,+New+Caledonia,+2014.">Co-infection with Zika and dengue viruses in 2 patients, New Caledonia, 2014.</a> Emerging Infectious Diseases. 21 (2), 381-382 (2015).</li><li>Roth, 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=Concurrent+outbreaks+of+dengue,+chikungunya+and+Zika+virus+infections-an+unprecedented+epidemic+wave+of+mosquito-borne+viruses+in+the+Pacific+2012-2014.">Concurrent outbreaks of dengue, chikungunya and Zika virus infections-an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014.</a> Euro Surveillance. 19 (41), 20929 (2014).</li><li>Tognarelli, 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=A+report+on+the+outbreak+of+Zika+virus+on+Easter+Island,+South+Pacific,+2014.">A report on the outbreak of Zika virus on Easter Island, South Pacific, 2014.</a> Archives of Virology. 161 (3), 665-668 (2016).</li><li>Oehler, E., 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+complicated+by+Guillain-Barre+syndrome-case+report,+French+Polynesia,+December+2013.">Zika virus infection complicated by Guillain-Barre syndrome-case report, French Polynesia, December 2013.</a> Euro Surveillance. 19 (9), 20720 (2014).</li><li>Faria, N. R., 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+in+the+Americas:+early+epidemiological+and+genetic+findings.">Zika virus in the Americas: early epidemiological and genetic findings.</a> Science. 352 (6283), 345-349 (2016).</li><li>Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barr&#233; syndrome - 4th update. European Centre for Disease Prevention and Control Available from: <a target="_blank" href="https://www.ecdc.europa.eu/en/publications-data/rapid-risk-assessment-zika-virus-epidemic-americas-potential-association">https://www.ecdc.europa.eu/en/publications-data/rapid-risk-assessment-zika-virus-epidemic-americas-potential-association</a> (2015)</li><li>Payne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+study+viruses.">Methods to study viruses.</a> Viruses. , 37-52 (2017).</li><li>Bustin, S. A., Mueller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+reverse+transcription+PCR+(qRT-PCR)+and+its+potential+use+in+clinical+diagnosis.">Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis.</a> Clinical Science. 109 (4), 365-379 (2005).</li><li>Sedlak, R. H., Jerome, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+diagnostics+in+the+era+of+digital+polymerase+chain+reaction.">Viral diagnostics in the era of digital polymerase chain reaction.</a> Diagnostic Microbiology and Infectious Disease. 75 (1), 1-4 (2013).</li><li>Bae, H. -. G., Nitsche, A., Teichmann, A., Biel, S. S., Niedrig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+yellow+fever+virus:+a+comparison+of+quantitative+real-time+PCR+and+plaque+assay.">Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.</a> Journal of Virological Methods. 110 (2), 185-191 (2003).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+plaques+in+monolayer+tissue+cultures+by+single+particles+of+an+animal+virus.">Production of plaques in monolayer tissue cultures by single particles of an animal virus.</a> Proceedings of the National Academy of Sciences. 38 (8), 747-752 (1952).</li><li>Nahapetian, A. T., Thomas, J. N., Thilly, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+environment+for+high+density+Vero+cell+culture:+effect+of+dissolved+oxygen+and+nutrient+supply+on+cell+growth+and+changes+in+metabolites.">Optimization of environment for high density Vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites.</a> Journal of Cell Science. 81, 65-103 (1986).</li><li>Agbulos, D. S., Barelli, L., Giordano, B. V., Hunter, F. F. <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:+quantification,+propagation,+detection,+and+storage.">Zika virus: quantification, propagation, detection, and storage.</a> Current Protocols in Microbiology. 43, (2016).</li><li>Brien, J. D., Lazear, H. M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+quantification,+detection,+and+storage+of+West+Nile+virus.">Propagation, quantification, detection, and storage of West Nile virus.</a> Current Protocols in Microbiology. 31, (2013).</li><li>Bol&#237;var-Marin, S., Bosch, I., Narv&#225;ez, C. 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>

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 ...

This protocol enables identifying and quantifying the Zika virus without relying on cytopathic effect. It depends on antibody recognition of virus component. The use of virus-specific antibody can help in identifying different virus serotypes in mixed populations.This method has advantages over classical plaque-forming assays. It's faster and enabled for high-throughput applications when applying with an automated imaging system for fast cell counting. The proposed protocol is highly adaptable.With appropriate modifications, the proposed protocol can be applied to various cell types and viral targets. Start by growing Vero cells in a 75-square=centimeter cell culture flask that contains 12 milliliters of DMEM supplemented with 10%FBS and two millimoles of L-glutamine. Place the flask in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide.To infect the cell monolayer, use a 10-milliliter serological pipette to remove the growth medium from the cell culture flask. Using a five-milliliter serological pipette, rinse the flask with three milliliters of DPBS twice. Next, add two milliliters of serum-free DMEM and 20 microliters of Zika virus inoculum into the cell culture flask.Let the flask incubate at room temperature for one hour with gentle rocking to encourage virus adsorption. At the end of the incubation, using a five-milliliter serological pipette, carefully remove and discard the diluted virus inoculum from the cell culture flask. Rinse the cell culture flask twice with three milliliters of DPBS.Then, add 12 milliliters of maintenance media into the cell culture flask to maintain the infected cells. Incubate the infected Vero cells for three days in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide. After three days of incubation, use a 10-milliliter serological pipette to harvest the cell culture supernatant containing the Zika virus into a 50-milliliter centrifuge tube.For virus quantification, seed Vero cells in designated plates and allow them to grow overnight at 37 degrees Celsius with a 5%carbon dioxide atmosphere. Prepare six sterile 1.5-milliliter microcentrifuge tubes for each plate to perform a ten-fold serial dilution, including an extra tube for a negative control. For a 24-well plate setup, add 450 microliters of serum-free DMEM to all six microcentrifuge tubes.For a 96-well plate set up, dispense 135 microliters of serum-free DMEM to six additional tubes. To perform serial dilution for the 24-well plate experiment, add 50 microliters of the Zika virus stock into the 10 to minus one tube containing 450 microliters of serum-free DMEM. For the 96-well plate experiment, add 15 microliters of Zika virus stock into the 10 to minus one tube containing 135 microliters of serum-free DMEM.Vortex each tube to thoroughly mix the virus and medium, ensuring an even distribution of virus particles within the dilution. Using a fresh pipette tip, resuspend the 10 to minus one tube and transfer 50 and 15 microliters of diluted Zika virus into the 10 to minus two tube as a second ten-fold dilution for the 24-and 96-well plates, respectively. Remove and discard the condition medium for each well of the appropriate plate.Rinse each well with DPBS twice to remove any residues. Starting with the highest dilution, add the serially diluted virus inoculum into the wells, working towards the lowest dilution. After one hour of incubation, remove and discard the virus suspension from the wells, starting with the lowest to the highest concentration.Wash the infected cells with DPBS twice to remove any traces of virus suspension. Overlay the well with DMEM and 1.5%low-viscosity carboxymethylcellulose. Incubate the plate in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide.After incubation, remove and discard the overlay medium and wash the cells three times with PBS. For the 96-well plate, use a multichannel pipette to remove and discard the overlay medium, and wash the cells three times with 60 microliters of PBS per well. Add 4%paraformaldehyde to fix the cells and incubate the plate at room temperature for 20 minutes.After 20 minutes, discard the paraformaldehyde and wash the cells thrice with PBS. Next, add 3, 3'diaminobenzidine peroxidase substrate and incubate the plate for 30 minutes in the dark. After 30 minutes, stop the reaction by washing the wells with water.Air-dry the plates overnight and proceed to foci enumeration. Count the foci for each replicate of the selected dilution and calculate the average number of foci for each. The infected Vero cells were fixed at different time points post-infection.For the 2-well plate, the first appearance of virus foci was observed 48 hours post-infection, although the foci size was too small to count accurately. After 96 hours post-infection, there were no signs of cell detachment. At 60 hours post-infection, the foci size had increased to an optimal level for counting.Subsequently, as time progressed, the foci became larger and began to merge or overlap with one another in intensity, forming clusters that grew over time. Therefore, foci formed 60 hours after the infection were chosen to determine the Zika virus titre in a 24-well plate. For the 96-well plate, the cells remained intact after 72 hours post-infection.The appearance of virus foci was first observed 24 hours post-infection. However, up to 36 hours post-infection, the foci size was too small. The optimal foci size was achieved at 48 hours post-infection.At the latter time points, overlapped or merged foci were observed, and the number of overlapped foci increased over time. The foci formed 48 hours after the infection was chosen to determine the virus titre of Zika virus isolates. Gently add DPBS down the side of each well and rock the plates backward and forward one to three times to remove cellular debris and excess media.This technique will greatly benefit researchers in Zika research, and can also be broadly adapted to quantify other clinically important viruses, making it a valuable tool for virus surveillance and diagnosis.

Research

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