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 ...

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