We would like to thank Dr. Aaron Brault and Dr. Brandy Russell of the Centers for Disease Control and Prevention (CDC), USA for providing Zika viral strain PRVABC59. We thank Nicholas Ten of Yale University for copy-editing this manuscript. This work was supported by the Cedars-Sinai Medical Center Institutional Programmatic Research Award to V.A.

Note: A schematic outline of the work flow is presented in Figure 1.
1. Cells
<ol>
	<li>Use Vero cells for Zika virus production and analysis of viral replication cycle.</li>
	<li>Prepare complete growth media containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 units/ml), and 10 mM HEPES.</li>
	<li>Culture Vero cells with the specified complete growth medium at 37 &#176;C with 5% CO2.</li>
</ol>
2. Zika Virus Production
<ol>
	<li>Harvest the Vero cells at 80% cell density using 0.25% trypsin in T-75 flask and count the cells.
	<ol>
		<li>Aspirate the media from the T-75 culture flask and rinse the cells with 2 ml phosphate buffered saline (PBS).</li>
		<li>Add 2 ml of 0.25% trypsin and incubate the flask at 37 &#176;C for 5 min.</li>
		<li>Add 8 ml of serum containing growth medium to inactivate trypsin. Pipet up and down to suspend cells and transfer the cells to a 15 ml tube.</li>
		<li>Remove 10 &#181;l of cells and mix with equal amount of 0.4% trypan blue. Load the prepared mix to the cell counting chamber slide and obtain viable cell counts using an automated cell counter.</li>
	</ol>
	</li>
	<li>Seed a total of 7 million cells in a 30 ml volume into a T-160 flask.</li>
	<li>The next day, prepare an appropriate multiplicity of infection (0.01 to 0.1 MOI) of Zika virus inoculum in a 10 ml serum free culture media per flask.</li>
	<li>Remove the spent media from the T-160 flask and then add the freshly prepared viral inoculum (10 ml).</li>
	<li>Incubate the inoculated flasks in 37 &#176;C with 5% CO2 for 4-6 hr. Spread the inoculum by gently tilting the flasks sideways at every hr.</li>
	<li>At the completion of incubation, replace the inoculum with warm serum-supplemented growth media (30 ml) for each flask. Then continue the viral culture for next 96 hr.</li>
	<li>Verify the progression of infection by observing the appearance of viral plaques on the cell monolayer using a phase contrast microscope and take images (Figure 2) as needed at 40X and 200X magnifications.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54767/54767fig2.jpg" /> 
Figure 2: Plaques formed by Zika virus on a monolayer of Vero cells. Bright field images of various magnifications show Zika viral plaques at 48 hpi. Note the presence of rounded cell foci on the monolayer. (Scale bar = 50 &#181;m) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54767/54767fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="8">
	<li>Harvest cell culture supernatants at the 96 hr time point and centrifuge the supernatant at 300 x g for 10 min at 4 &#176;C to remove cell debris.</li>
	<li>Carefully remove the supernatant without disturbing pelleted debris and transfer to 15 ml tubes. Ultracentrifugation at 24,000 x g can be performed to concentrate the viral particles (optional).</li>
	<li>Store the viral culture supernatants in multiple aliquots at -80 &#176;C.</li>
</ol>
3. Measuring Zika Virus Titer by Plaque Assay
<ol>
	<li>Seed naive Vero cells at 1 x 105 cells per well in 2 ml volume using a 12-well plate.</li>
	<li>The following day, prepare 10-fold serial dilutions of viral culture supernatants collected from the T-160 flask using serum-free media. Remove the spent media from each well and add 400 &#181;l of prepared inoculum onto Vero cells in triplicate.</li>
	<li>Incubate the inoculated flasks in 37 &#176;C with 5% CO2 for 4-6 hr. Spread the inoculum by gently tilting the plate sideways at every one hr.</li>
	<li>At the end of incubation, replace the inoculum with serum supplemented media (2 ml per well).</li>
	<li>At 48 hr post-inoculation, count the viral foci using a phase contrast microscope. Calculate the viral titer as plaque forming units (PFU) per ml. See Figure 3 for plaque assay.</li>
	<li>Fix and stain the cells in 4% formaldehyde and 0.1% crystal violet solution prepared in 20% ethanol for clear visualization of plaques.</li>
</ol>
4. Zika Viral Genome Replication Assay
<ol>
	<li>Seed na&#239;ve Vero cells at 1 x 105 cells per well in 2 ml volumes using a 12-well plate. The following day, prepare viral inocula (MOI of 0.01 and 0.1; 400 &#181;l/well) of low and high titer using serum free media in triplicate. For mock infection, use serum free media (400 &#181;l/well) only.</li>
	<li>Repeat the steps 3.2 to 3.4.</li>
	<li>At the 48 and 96 hr time points, collect the samples for RNA and immunocytochemistry (ICC).
	<ol>
		<li>For harvesting RNA samples, remove the media and then add 400 &#181;l of lysis solution directly to each well and collect the lysates.</li>
		<li>For ICC, remove the media and then fix the cells by adding 1 ml of methanol to each well and incubate the plate at 4&#160;&#176;C for 30 min.</li>
		<li>From the cell lysate, isolate the total RNA using a RNA isolation kit per the manufacturer&#39;s instructions. Quantify the RNA using a spectrophotometer.</li>
	</ol>
	</li>
	<li>Perform a two-step reverse transcription quantitative PCR (RT-qPCR) to determine the ZIKV genome content from harvested RNA.
	<ol>
		<li>To reverse transcribe the RNA, first set up a 13 &#181;l reaction mix comprised of 5 &#181;g of isolated RNA, 1 &#181;l of dNTPs (10 mM), and 1 &#181;l random hexamer (250 ng) in a 0.2 ml tube. Incubate the tube at 65 &#176;C for 5 min and then place it on ice for one minute. Subsequently, add 1 &#181;l of reverse transcriptase, 4 &#181;l of 5x strand synthesis buffer, 1 &#181;l of 0.1 M dithiothreitol, and 1 &#181;l of RNase inhibitor to the reaction mix. Incubate the tube for an additional 5 min at 25 &#176;C, followed by 60 min at 50 &#176;C and inactivate the reaction by heating to 70 &#176;C for 15 min.</li>
		<li>Perform qPCR using 1 &#181;l of the resulting cDNA with 12.5 &#181;l of 2x green dye super mix containing DNA polymerase and 1 &#181;l of 10 mM individual primers specific for Zika virus [Zika virus pan-genotype primers (For: 5&#39;-AARTACACATACCARAACAAAGTGGT-3&#39;; Rev: 5&#39;-TCCRCTCCCYCTYTGGTCTTG-3&#39;), Zika virus Asian genotype PRVABC59 strain primers (5&#39;-AAGTACACATACCAAAACAAAGTGGT-3&#39;; Rev: 5&#39;-TCCGCTCCCCCTTTGGTCTTG-3&#39;)], or hepatitis C virus (JFH RTQ F: 5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3&#39;), or cellular housekeeping gene GAPDH (For: 5&#39;-CCACCTTTGACGCTGGG-3&#39;; Rev: 5&#39;-CATACCAGGAAATGAGCTTGACA-3&#39;) in a 25 &#181;l reaction volume.</li>
		<li>Perform PCR using the run condition 95 &#176;C for 15 sec and 60 &#176;C for 30 sec (40 cycles) in a real-time PCR system.</li>
		<li>Use GAPDH expression level based on cycle threshold (Ct) value to normalize the Zika viral genome measurement. Calculate the delta Ct (&#916;Ct) value of Zika genome compared to that of GAPDH and obtain the 2&#916;Ct value. Then, calculate the fold change by taking the ratio of normalized Zika genome contents between infected and uninfected cells at indicated time points. See Figure 4 for ZIKV genome replication results.</li>
	</ol>
	</li>
	<li>Perform ICC using the methanol fixed cells.
	<ol>
		<li>Wash the fixed cells three times with 1x PBS and block with ICC blocking buffer (3% goat serum, 3% BSA, 0.1% Triton-x 100 in PBS).</li>
		<li>Use mouse monoclonal anti-dsRNA antibody J2 (1 &#181;g/ml) at a dilution of 1:100 in blocking buffer and incubate overnight at 4 &#176;C.</li>
		<li>Wash the cells with 1x PBS and add secondary antibody goat anti-mouse IgG-594 (1 &#181;g/ml) at a 1:1,000 dilution in blocking buffer and incubate for one hour at room temperature.</li>
		<li>Wash cells with 1x PBS and stain for nuclei using Hoechst dye and observe the cells using a fluorescent microscope at 100X magnification (Figure 4).</li>
	</ol>
	</li>
</ol>
5. Screening Cytokine Library against Zika Virus Infection
<ol>
	<li>Seed naive Vero cells at 1 x 105 cells per well using a 12-well plate. Once the cells are attached (6 hr post-seeding), add each cytokine at indicated concentrations in biological duplicates (2 ml volume/well). Include vehicle (PBS) alone control.</li>
	<li>At 12 hr post-treatment, perform Zika viral infection (MOI of 0.1 in 400 &#181;l per well). Include negative control wells without infection (mock).</li>
	<li>At 4 hr post-infection, replace the viral inoculum with cytokine treated media (2 ml). Incubate the cells at 37 &#176;C for additional 44 hr.</li>
	<li>At 48 hr post-infection, count viral plaques using a phase contrast microscope and acquire representative images of infected cells at 40X magnification (Figure 5).</li>
</ol>

<ol>
	<li>Brasil, P., 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+in+Pregnant+Women+in+Rio+de+Janeiro+-+Preliminary+Report.">Zika Virus Infection in Pregnant Women in Rio de Janeiro - Preliminary Report.</a> N Engl J Med. , 1-11 (2016).</li><li>Lucey, D. R., Gostin, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emerging+Zika+Pandemic:+Enhancing+Preparedness.">The Emerging Zika Pandemic: Enhancing Preparedness.</a> JAMA. 315 (9), 865-866 (2016).</li><li>Mlakar, 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+Associated+with+Microcephaly.">Zika Virus Associated with Microcephaly.</a> N Engl J Med. 374 (10), 951-958 (2016).</li><li>Schuler-Faccini, L., 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=Possible+Association+Between+Zika+Virus+Infection+and+Microcephaly.">Possible Association Between Zika Virus Infection and Microcephaly.</a> MMWR Morb Mortal Wkly Rep. 65 (3), 59-62 (2015).</li><li>Foy, B. 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=Probable+non-vector-borne+transmission+of+Zika+virus,+Colorado,+USA.">Probable non-vector-borne transmission of Zika virus, Colorado, USA.</a> Emerg Infect Dis. 17 (5), 880-882 (2011).</li><li>Kuno, G., Chang, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+sequencing+and+genomic+characterization+of+Bagaza,+Kedougou,+and+Zika+viruses.">Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.</a> Arch Virol. 152 (4), 687-696 (2007).</li><li>Dick, G. W. <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> Trans R Soc Trop Med Hyg. 46 (5), 521-534 (1952).</li><li>Dick, G. W., 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> Trans R Soc Trop Med Hyg. 46 (5), 509-520 (1952).</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.">Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia.</a> Euro Surveill. 19 (9), 1-3 (2013).</li><li>Baronti, C., 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=Complete+coding+sequence+of+zika+virus+from+a+French+polynesia+outbreak+in+2013.">Complete coding sequence of zika virus from a French polynesia outbreak in 2013.</a> Genome Announc. 2 (3), e00500-e00514 (2014).</li><li>Lanciotti, R. 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=Genetic+and+serologic+properties+of+Zika+virus+associated+with+an+epidemic,+Yap+State,+Micronesia,+2007.">Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007.</a> Emerg Infect Dis. 14 (8), 1232-1239 (2008).</li><li>Lanciotti, R. 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=Phylogeny+of+Zika+virus+in+Western+Hemisphere,+2015+[Letter].">Phylogeny of Zika virus in Western Hemisphere, 2015 [Letter].</a> Emerg Infect Dis. 22 (5), (2016).</li><li>Musso, D., Nilles, E. J., Cao-Lormeau, V. 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The authors have nothing to disclose.

Here, a streamlined protocol for culturing Zika virus in vitro is presented. Critical steps including, identifying optimum end points for expanding virus culture, measuring titer, and quantifying genome replication were provided. Zika virus is a human pathogen, so, while handling infectious agents, biosafety procedures are to be strictly followed. A monkey kidney cell line, Vero, was used for demonstrating various virological assays. Zika viral replication kinetics may differ in cells of various tissues and species. Additional cell lines can be used as described previously15. Zika virus has been isolated from the brain tissues of infected fetuses3 and has been shown to infect cortical neural progenitor cells14. Thus, evaluating the pathophysiology of ZIKV infection in neuronal cells can provide additional cell-specific insights. Differences in the neurovirulent phenotype of African and Asian genotypes can be investigated using this infectious culture system. The viral production procedure described here will also be useful for generating high-titer virus towards in vivo pathogenic studies in non-human primate or rodent model systems.
While, this study is limited to the usage of Vero cells, high-titer producing cell lines of human and mosquito origins can be tested. During viral production, if CPE is observed in only 10-30% of cells on day 3, the infected cells can be split in 1:4 density and culturing can be continued for additional 4-5 days. At the end of the 7th day, a complete CPE can be observed.
The efficiency of ZIKV pan-genotype and strain-specific primers were compared and determined that both are sensitive in quantifying Zika viral genome in low and high titer virus infected cells. Furthermore, in this study, an antibody-based probe is verified to identify infected cells. This dsRNA-specific antibody successfully recognized cytoplasmic viral genomes in the Zika virus-infected cells. These optimized reagents will be a useful resource to dissect various stages of viral replication such as translation, genome replication, assembly and virion morphogenesis.
This in vitro infectious cell culture system is applicable to future studies using Zika virus-specific sub-genomic replicons and infectious virus generated from complementary DNA (cDNA) clones. Development of Zika viral infectious cDNA clones will accelerate the functional study of viral genes including NS1, and NS4B by combining genetic and molecular biological techniques. Establishment of Zika virus tagged with a fluorescent or luminescent based reporter gene would be a valuable tool in making further use the cell culture system in high-content screening assays.
Both type I and type II IFNs demonstrated anti-Zika viral activity. IFN-&#945;, IFN-&#946; and IFN-&#947; can be further evaluated in clinical settings as a prophylactic, post-exposure prophylactic, and treatment against Zika virus infection and associated diseases. High-risk groups including pregnant women positive for Zika virus, can be treated with interferons as a first line of treatment. Interferon-&#945; treatment has been shown to be safe for pregnant women18,19.
In summary, an infectious cell culture system that can be used to investigate various aspects of Zika viral growth is described and outlined. The protocol and reagents described here are important resources for those who study Zika virus and the neurological research community.

Zika Virus (ZIKV) is an important human pathogen associated with microcephaly and poor pregnancy outcomes1,4. ZIKV belongs to the set of medically relevant flaviviruses that can cause neurological defects such as the Dengue, West Nile, and St. Louis encephalitis viruses. The main mode of viral transmission is by the mosquito vector Aedes aegypti, and, in addition, sexual transmission has also been reported5,6. ZIKV has become a major global health issue due to the expanding geographical distribution of the mosquito vector and its strong correlation with birth defects. ZIKV was first isolated in 1947 from a sentinel rhesus monkey in the Zika forest, Uganda and the first human case was reported in 19527,8. Individuals that become infected with ZIKV present with mild symptoms such as fever, rash, headache, conjunctivitis, and muscle/joint pain. Infected pregnant women can transmit ZIKV to the developing fetus1. ZIKV infection has also been linked to Guillain-Barre syndrome, a peripheral nerve auto-immune demyelination disorder9.
The Zika viral genome consists of a positive sense, single-stranded RNA molecule which is about 10.8 kilobases in length. The genome&#39;s structure is organized as 5&#39;NCR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-3&#39;NCR, with non-coding regions (NCR) flanking a protein-coding region6. A single polyprotein (3,419 aa) is translated that is co- and post-translationally cleaved into 10 smaller peptides. Both the 5&#39;NCR and 3&#39;NCR RNA stem-loop structures play a critical part in the commencement of viral genome translation and replication. The structural components of the genome are comprised of the capsid, membrane, and envelope proteins. The non-structural proteins are critical for genome replication.
Currently, Zika viral strains are grouped into three main genotypes: West African, East African, and Asian6,10-13. It has been proposed that the East African lineage spread to West Africa and Asia, where it later further evolved12. The Asian genotype is responsible for the current outbreaks in the Americas. Zika virus can be cultured in both mosquito and mammalian cells. Primary dermal fibroblasts, immature dendritic cells, cortical neural progenitor cells, and Vero cells are susceptible to Zika viral infection10,14,15. Both type I and type II interferons have been shown to restrict ZIKV growth in skin fibroblasts15. The objectives of this study are to provide a step-wise, detailed protocol for the production and assaying of the Asian genotype ZIKA viral strain PRVABC59 in a mammalian cell culture system and to demonstrate the utility of this infectious culture system as a drug development platform. This resource has the potential to greatly benefit the Zika viral and neurological research community to further elucidate its mechanisms of viral pathogenesis and evolution of viral virulence.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:355px;">Sigma Life Science</td>
			<td style="width:119px;">D5796</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Glutamax</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">2.5% Trypsin, 10X [-] Phenol Red&#160;&#160;</td>
			<td style="width:355px;">Corning</td>
			<td style="width:119px;">25-054-C1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Stain 0.4%</td>
			<td style="width:355px;">Life Technologies</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Countess &#8211; Automated Cell Counter</td>
			<td style="width:355px;">ThermoFisher Scientific</td>
			<td style="width:119px;">C10227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Countess-cell counting&#160; chamber slides</td>
			<td style="width:355px;">ThermoFisher Scientific</td>
			<td style="width:119px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:355px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Nanodrop 2000</td>
			<td style="width:355px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-dsRNA antibody J2&#160;</td>
			<td>English &#38; Scientific Consulting Kft.</td>
			<td style="width:119px;">10010200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Goat anti-rabbit IgG Alexa Fluor 594</td>
			<td style="width:355px;">Life Technologies</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">18080085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SYBR QPCR SUPERMIX W/ROX</td>
			<td style="width:355px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantStudio12K Flex Real-Time PCR System</td>
			<td style="width:355px;">Thermo Fischer</td>
			<td style="width:119px;">4471088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:355px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Vero Cell Line&#160;</td>
			<td style="width:355px;">ATCC</td>
			<td style="width:119px;">CCL-81</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">Zika viral strain PRVABC59</td>
			<td style="width:355px;">Centers for Disease Control and Prevention (CDC)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">IL-6</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">200-06&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
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			<td height="22" style="height:23px;width:569px;">IL-1 alpha&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">200-01A&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">TNF-alpha</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">300-01A&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">Interferon alpha A&#160;&#160;</td>
			<td style="width:355px;">R &#38; D Systems</td>
			<td style="width:119px;">11100-1</td>
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			<td height="22" style="height:23px;width:569px;">Interferon beta</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">300-02BC</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:569px;">Interferon gamma</td>
			<td style="width:355px;">Peprotech</td>
			<td style="width:119px;">300-02</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:569px;">Centrifuge 5415R</td>
			<td style="width:355px;">Eppendorf</td>
			<td style="width:119px;">5415R</td>
			<td></td>
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			<td height="21" style="height:21px;width:569px;">Centrifuge 5810R</td>
			<td style="width:355px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:33px;width:569px;">Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI</td>
			<td style="width:355px;">Nikon</td>
			<td style="width:119px;">Visit Nikon for Request</td>
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</table>

zika virus infectious cell culture system and the in vitro prophylactic effect of interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

A Zika viral strain (PRVABC59; GenBank accession number KU501215) of the Asian genotype was utilized in this study12. Vero cells at 80% confluency were used for investigating de novo Zika viral infection. For viral production and subsequent virological characterization, an early passage (P3) Zika virus was employed. The viral plaques were observed on the second day of infection. Zika viral progenies released from the initially infected cell can spread to neighboring cells, which result in the formation of visible plaques on the monolayer culture (Figure 2). The cytopathic effect (CPE) of Zika viral infection is characterized by morphological changes such as rounding and detachment of cells as well as lytic cell death.
For measuring viral titer, virus-containing cell-free supernatant harvested from T-160 flasks was subjected to 10-fold serial dilution and added onto the monolayer of Vero cells in a 12-well plate format (Figure 3). The 48 hr post-inoculation time point was used as an endpoint to avoid counting the plaques arising from secondary infection. The wells with 30-100 plaques were chosen for counting to obtain an accurate titer.
RT-qPCR assay was utilized to estimate the genome replication of Zika virus. A pan-genotype primer pair specific for the highly conserved ZIKV NS5 region was included3,16. Zika viral strain PRVABC59-specific primers based on the genomic location of pan-genotypic primers (sequences of primers are given in the protocol section 4.4.2) were also tested. Both of the primer pairs showed similar levels of sensitivity in amplifying Zika viral genome from infected cells (Figure 4). As a negative control, hepatitis C virus-specific primers were also tested17. A significant increase in the ZIKV viral genome content was observed between 48 and 96 hr time points both in the low and high MOI infected cells, suggesting a robust viral infection (Figure 4). During Flavivirus genome replication, double-stranded (ds) RNA replicatory intermediates are generated by the formation of base-pairing between sense and anti-sense genomic RNA. An antibody probe specifically for recognizing dsRNA detected viral infected cells (Figure 4). Viral dsRNA were localized to the cytoplasm suggesting the genome replication compartments. Immunocytochemistry staining revealed expansion of virus infected cells during the 48 and 96 hr time points.
Using the infectious Zika virus culture system, a small library of cytokines was screened to determine antiviral activity (Figure 5). The cells were pre-treated with various cytokines for 12 hr and then infected with Zika virus. Type I interferon, IFN-&#945;, exhibited strong antiviral activity against Zika virus in a broad range of tested doses [10 international units (IU), 100 IU and 1,000 IU]. IFN-&#946; demonstrated a potent inhibitory effect as well. Type II IFN-&#947; also demonstrated anti-Zika viral activity. Cytokines TNF-&#945;, IL-1 and IL-6 did not inhibit ZIKV at the indicated drug concentrations.
<img alt="Figure 1" src="/files/ftp_upload/54767/54767fig1.jpg" /> 
Figure 1: General outline of Zika virus production and assay workflow.&#160; For virus production, ZIKV is inoculated onto Vero cells in T-160 tissue culture flasks. At 48 or 96 hpi, the cell-free virus supernatant is harvested and stored at -80 &#176;C for downstream analysis. Virus titer is measured by a limiting dilution assay. The Zika virus inoculum is subsequently used for studying virus replication kinetics and screening drug compounds. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54767/54767fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54767/54767fig3.jpg" /> 
Figure 3: Plaque assay for measuring Zika virus production.&#160;Naive Vero cells were used for measuring virus titer. Bright field images depict the density of viral plaques at various dilutions (Scale bar = 50 &#181;m). Note: The 1:100 dilution image shows distinct plaques that can be accurately counted. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54767/54767fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54767/54767fig4.jpg" /> 
Figure 4: Assays for evaluating Zika virus replication. (A) Graph shows Zika virus genomic content measured by RT-qPCR. Both pan-genotype (ZIKV GEN) and PRVABC59-strain (ZIKV UNI) primers showed equal sensitivity and specificity. As expected, hepatitis C viral (HCV) primers did not amplify any product. (B) Graph presents the Zika viral growth kinetics at the indicated time points in low and high titer infected cells compared to that of the uninfected mock control. Mean values and standard deviations are given. (C) Immunocytochemistry assay for investigating viral replication. Zika virus infected cells were specifically stained with dsRNA antibody. At 48 hpi, infected cells are in few clusters, whereas at 96 hpi most of the cells are infected. Hoechst stain was used for the visualization of nuclei (Scale bar = 50 &#181;m). BF: Bright field image. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54767/54767fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54767/54767fig5.jpg" /> 
Figure 5: Interferons demonstrate potent anti-Zika viral activity. (A) Graph showing modulation of Zika viral growth by various cytokines compared to that of vehicle control. Mean values and standard deviation are shown in the bar graph. Interferons showed statistically significant inhibition of Zika virus replication (p value &#60;0.05). (B and C) Phase contrast images of Zika virus-infected cells with or without cytokine treatment. Viral plaques formed by infected cells can be seen as foci. Note that interferon-treated wells do not have viral plaques. Mock control without ZIKV infection included as negative control. (Scale bar = 50 &#181;m) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54767/54767fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons at JoVE.com

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

zika-virus-infectious-cell-culture-system-vitro-prophylactic-effect

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

20.7K Views.  Cedars-Sinai Medical Center. Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Video: Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

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 ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...