The described project was supported by funds from the Uniformed Services University of the Health Sciences to JJM. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences or any other agency of the U.S. Government.
WGV performed all the experiments and analyzed the data; JJM designed and supervised the study; WGW and JJM wrote the paper.

The serum samples used to demonstrate the protocol described here were obtained from rhesus macaques that were housed and cared for in accordance with local, state and federal policies in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility. All animal experiments were reviewed and approved by Institutional Animal Care and Use Committee and samples were acquired through a tissue sharing protocol.
1. Day 1
NOTE: Perform all the steps described below in a sterile laminar flow biosafety cabinet used for tissue culture in a BSL-2 laboratory.
<ol>
	<li>Thaw the serum samples at room temperature and transfer 100 &#181;L of each serum sample to a sterile tube. Heat inactivate for 30 min at 56&#176;C either in a water bath or a temperature-adjustable thermomixer. Make 10-fold serial dilutions of the serum sample ranging from 1:1 to 1:1,000 using cold RPMI-10 (RPMI with 10% fetal bovine serum).</li>
	<li>Transfer 10 &#181;L of serially diluted serum sample to each well of a sterile 96-well V-bottom plate. Include two sets of control wells, RPMI-10 with RVPs only and without serum sample, and RPMI-10 only without RVPs and without serum sample. Set up each serum sample and RPMI-10 controls in triplicates.</li>
	<li>Remove the Dengue-1, 2, 3 and 4 RVPs from the -80 &#176;C freezer and thaw them in a 37 &#176;C water bath. Transfer the thawed RVPs immediately to ice. Obtain approximately 170 &#181;L RVPs for each serum sample (10 &#181;L of RVPs x 3 wells for each serum dilution (1:1, 1:10, 1:100, 1:1,000) and 10 &#181;L of RVPs x 3 wells for each of the RPMI-10 with RVP only control wells).</li>
	<li>Pipette 10 &#181;L of RVPs into each well of the 96-well V-bottom plate containing the serum sample and to the RPMI-10 with RVPs (no serum) control wells. Do not add RVPs to the RPMI-10 only (no serum sample and no RVP) wells. Mix thoroughly by pipetting up and down 5&#8211;10 times. Add 10 &#181;L cold RPMI-10 in lieu of RVPs to each cold RPMI-10 only (no serum sample and no RVP) control wells.</li>
	<li>Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hour at 37 &#176;C in the presence of 5% CO2. While the 96-well V-bottom plate is incubating, clean the surface of the biosafety cabinet with 70% ethanol and run the UV light for 15 min.</li>
	<li>Remove a fully confluent T75 flask of K562 cells from the incubator, mix the cells well using a sterile 5 mL pipet, and transfer 5 mL of cells to a sterile 15 mL conical tube. 
	NOTE: The K562 cells are maintained in RPMI-10 and subcultured at 1 x 106/mL.</li>
	<li>Count the number of cells by removing 10 &#181;L cells from the sterile 15 mL conical tube and mix with 10 &#181;L tryphan blue. Count the cells using a hemocytometer to determine the total number of cells in the 15 mL conical tube.</li>
	<li>Centrifuge the 15 mL conical with cells at ~1,200 x g for 10 minutes, decant the supernatant, and resuspend the cells in warm RPMI-10 at a concentration of 80,000 cells/30 &#181;L of media (2.66 x 106 cells /mL).</li>
	<li>Remove the 96-well V-bottom plate from the incubator (step 1.6), transfer 30 &#181;L of K562 cells to each well of the 96-well V-bottom plate, and mix thoroughly by pipetting up and down 5&#8211;10 times. Transfer the 96-well V-bottom plate to an incubator and incubate the plate for 1 hr at 37 &#176;C in the presence of 5% CO2.</li>
	<li>After incubating for 1 h, remove the 96-well V-bottom plate from the incubator and centrifuge at ~1,200 x g for 5 minutes. After centrifugation, decant the media from the wells by turning the plate upside down into a container containing 10% bleach.</li>
	<li>Wash the cells in each well by resuspending them in 125 &#181;L of warm RPMI-10, mix thoroughly with a pipette, and centrifuge the plate at ~1,200 x g for 5 min. Decant the media by turning the plate upside down into a container containing 10% bleach. Repeat this wash step two times.</li>
	<li>After washing, add 100 &#181;L of warm RPMI-10 to each well, mix up and down with the pipet, and incubate the plate for 48 h at 37 &#176;C in the presence of 5% CO2.</li>
</ol>
2. Day 3
<ol>
	<li>Remove the 96-well V-bottom plate containing 100 &#181;L of cells and media/well from the incubator and move it to a biosafety cabinet . Using a multichannel pipette, mix the contents of each well and transfer the cells and media to a 96 well U-bottom plate or to pre-labeled 5 mL polypropylene tubes.</li>
	<li>Rinse each well in the 96-well V-bottom plate with 100 &#181;L of 1% Paraformaldehyde (PFA) in 1x Phosphate Buffered Saline (PBS) and transfer to the respective wells in the 96 well U-bottom plate or labeled 5 mL polypropylene tubes from step 2.1 to yield a final concentration of 0.5% PFA/well or tube. Mix thoroughly using a multichannel pipette, cover the plate with aluminum foil, and let it sit in the incubator for 30 minutes to fix the cells.</li>
	<li>Prepare the flow cytometer (see Table of Materials for example) by running unstained K562 cells to calibrate the side and forward scatter along with the fluorescence settings. The only fluorescence channel required is FL1, as GFP is the only fluorescence emitted from the cells. Acquire ~30,000&#8211;50,000 cells from each sample. 
	NOTE: Any flow cytometer capable of reading a single fluorescence can be used for acquiring the data, as cells infected with RVPs will only emit green fluorescence due to the presence of GFP, and no other fluorescence channel are needed.</li>
</ol>
3. Data Analysis
<ol>
	<li>Analyze data acquired on the flow cytometer using any flow cytometry analysis software (see Table of Materials).
	<ol>
		<li>Set the first gate based on FSC-A (forward scatter &#8211; area) vs FSC-H (forward scatter &#8211; height) to include single cells and exclude auto-fluorescent doublets from analysis12. Then, gate singlet-gated cells based on SSC-A (side scatter &#8211; area) vs FSC-A to exclude any autofluorescent debris.</li>
		<li>Analyze SSC-A vs FSC-A gated cells for the expression of GFP based on SSC-A vs GFP-A. Determine the percentage of GFP+ cells for each dilution of the serum and control samples by setting a gate around GFP+ cells.</li>
	</ol>
	</li>
	<li>Determine the average percentage of GFP+ cells for each serum sample dilution and control wells by dividing the total percentage of GFP+ cells in the triplicate wells by 3.</li>
	<li>Calculate fold-enhancement of infection by dividing the average percentage of GFP+ cells in the serum samples for each dilution divided by the average percentage of GFP+ cells in the RPMI-10 with RVPs (no serum sample) control wells.</li>
	<li>Graph fold-enhancement (y-axis) versus dilution (x-axis), and perform statistical analysis using ANOVA followed by Tukey&#39;s post-hoc test for multiple comparisons.</li>
</ol>

<ol>
	<li>George, 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=Prior+exposure+to+zika+virus+significantly+enhances+peak+dengue-2+viremia+in+rhesus+macaques.">Prior exposure to zika virus significantly enhances peak dengue-2 viremia in rhesus macaques.</a> Sci Rep. 7 (1), 10498 (2017).</li><li>Stettler, K., 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=Specificity,+cross-reactivity,+and+function+of+antibodies+elicited+by+Zika+virus+infection.">Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection.</a> Science. 353 (6301), 823-826 (2016).</li><li>Mattia, K., 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=Dengue+reporter+virus+particles+for+measuring+neutralizing+antibodies+against+each+of+the+four+dengue+serotypes.">Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.</a> PLoS One. 6 (11), e27252 (2011).</li><li>Chiofalo, M. S., Teti, G., Goust, J. M., Trifiletti, R., La Via, ., F, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subclass+specificity+of+the+Fc+receptor+for+human+IgG+on+K562.">Subclass specificity of the Fc receptor for human IgG on K562.</a> Cell Immunol. 114 (2), 272-281 (1988).</li><li>Klein, 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=Properties+of+the+K562+cell+line,+derived+from+a+patient+with+chronic+myeloid+leukemia.">Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia.</a> Int J Cancer. 18 (4), 421-431 (1976).</li><li>Kliks, S. C., Nisalak, A., Brandt, W. E., Wahl, L., Burke, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-dependent+enhancement+of+dengue+virus+growth+in+human+monocytes+as+a+risk+factor+for+dengue+hemorrhagic+fever.">Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever.</a> Am J Trop Med Hyg. 40 (4), 444-451 (1989).</li><li>Littaua, R., Kurane, I., Ennis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+IgG+Fc+receptor+II+mediates+antibody-dependent+enhancement+of+dengue+virus+infection.">Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection.</a> J Immunol. 144 (8), 3183-3186 (1990).</li><li>Wang, T. T., 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=IgG+antibodies+to+dengue+enhanced+for+FcgammaRIIIA+binding+determine+disease+severity.">IgG antibodies to dengue enhanced for FcgammaRIIIA binding determine disease severity.</a> Science. 355 (6323), 395-398 (2017).</li><li>Dejnirattisai, 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=Cross-reacting+antibodies+enhance+dengue+virus+infection+in+humans.">Cross-reacting antibodies enhance dengue virus infection in humans.</a> Science. 328 (5979), 745-748 (2010).</li><li>Kawiecki, A. B., Christofferson, R. C. <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-induced+antibody+response+enhances+dengue+virus+serotype+2+replication+in+vitro.">Zika virus-induced antibody response enhances dengue virus serotype 2 replication in vitro.</a> J Infect Dis. 214 (9), 1357-1360 (2016).</li><li>Morens, D. M., Halstead, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+antibody-dependent+infection+enhancement+of+four+dengue+virus+serotypes+by+monoclonal+and+polyclonal+antibodies.">Measurement of antibody-dependent infection enhancement of four dengue virus serotypes by monoclonal and polyclonal antibodies.</a> J Gen Virol. 71 (Pt 12), 2909-2914 (1990).</li><li>Perfetto, S. 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=Amine+reactive+dyes:+an+effective+tool+to+discriminate+live+and+dead+cells+in+polychromatic+flow+cytometry.">Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.</a> J Immunol Methods. 313 (1-2), 199-208 (2006).</li><li>Priyamvada, 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=B+Cell+responses+during+secondary+dengue+virus+infection+are+dominated+by+highly+cross-reactive,+memory-derived+plasmablasts.">B Cell responses during secondary dengue virus infection are dominated by highly cross-reactive, memory-derived plasmablasts.</a> J Virol. 90 (12), 5574-5585 (2016).</li><li>Priyamvada, 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=Human+antibody+responses+after+dengue+virus+infection+are+highly+cross-reactive+to+Zika+virus.">Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.</a> Proc Natl Acad Sci U S A. 113 (28), 7852-7857 (2016).</li></ol>

The authors have nothing to disclose.

Flaviviruses such as DENV and ZIKV share significant evidence of homology in both their structural and non-structural proteins that generate antibodies that cross-react with each other13,14. These cross-reactive antibody responses have been shown to enhance of infection both in vivo and in vitro with either a heterologous serotype or other related flaviviruses1,2. The potential to cross enhance infection has significant implications for management of the disease and also for vaccine development.
Traditional culture based assays use non-permissive cells that are infected with infectious live DENV in the presence of serum. Enhancement of infection is measured by quantifying the amount of virus in the supernatant using standard plaque assays11. This assay requires culture of virus in two cell lines and requires significantly long periods of time to perform. Additionally, not all the DENV serotypes plaque the same way, adding another level of complexity to these assays.
Other assays have modified the plaque based assay protocol to measure infection of cell lines by combining intracellular staining for DENV using fluorescently labeled antibodies and flow cytometry9,10,14. Though this reduces the time required to determine the level of infection as compared to plaque based assays there are a number of optimization steps required for these assays to work consistently. Intracellular staining protocols are time consuming, as they require fixing and permeabilizing of cells followed by intracellular staining and require well-titrated DENV specific antibodies that can clearly discriminate each serotype. Additionally, these assays are not easily amenable to a high throughput format and suffer from high levels of intra-assay variability.
In contrast to the above described assays, the DENV RVPs based assay is easy to set up, does not use infectious virus, and is highly amenable to high throughput that can be readily used to measure the potential of serum to enhance infection. Additionally, this assay is not as labor intensive as other assays, and can be completed within 2 - 3 days, which offers a major advantage over current ADE assay protocols. Though the assay described in the manuscript examined the capacity of ZIKV infected serum to enhance infection of DENV-1, 2, 3 and 4 serotypes, the assay can be readily adapted to examine serum from both experimental and clinical samples obtained from other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), and West Nile virus (WNV) if RVPs are available, and with other cell lines. It is, however, critical to optimize the assay by titrating the volume of RVP, number of cells/well, and the duration of incubation to allow optimal staining of cells without background staining.
Titrations need to be performed by incubating a defined number of target cells (for example, 80,000 K562 cells or a cell line being tested) with variable volumes of RVPs (for example, 2.5 &#181;L, 5 &#181;L, 7.5 &#181;L, 10 &#181;L, 20 &#181;L, etc.). Once the correct titer for each RVPs is obtained, the duration of incubation can be determined using the obtained titer along with the defined number of cells and incubating them as described in the protocol for variable periods of time (for example, 24 h, 48 h, 72 h, etc.).
We observed ADE of infection by all 4 serotypes of DENV at a dilution of 1:10, suggesting that a antibody levels in the serum needed to be diluted 10-fold to detect enhancement of infection. On the other hand, other studies have reported enhancement at lower dilutions, which would indicate that the concentrations of antibody in the serum sample were likely too high, needing lower dilutions to detect ADE in vitro. As ADE depends on the right concentration of antibody in the serum sample, most samples are likely to show ADE at dilutions lower than the undiluted serum.
In conclusion, the protocol described in this manuscript is simple and easy to adopt, and allows for scaling up to enable high throughput screening of a large number of samples in a short period time frame to yield high quality data that can easily be analyzed and interpreted.

Antibody dependent enhancement (ADE) of infection is a process whereby partially cross-reactive antibody responses induced by a serotype of virus enhances uptake of another serotype of virus, leading to increased viral replication and viremia. ADE has been extensively documented in Dengue virus (DENV) infections where four major serotypes are prevalent. In a subset of patients, ADE is associated with Dengue hemorrhagic fever (DHF). We have recently shown that Zika virus (ZIKV) infection induced significantly high levels of DENV cross-reactive antibody responses that caused ADE of DENV in vitro and likely contributed to the enhancement of DENV viremia in vivo1,2. Antibody dependent enhancement assays are a valuable tool to assess the capacity of antibodies to enhance secondary infection with related viruses and provide valuable insights into the pathogenesis of flavivirus infections and inform the development of vaccines.
The assay described here uses DENV RVPs along with K562 cells that are normally impermissible to infection. RVPs are structurally intact replication incompetent DENV viral particles that encode a sub-genomic green fluorescent protein (GFP) replicon that is expressed after a single round of replication3. As such, cells that become infected with RVPs fluoresce green and can be readily detected using flow cytometry or microscopy. The RVPs used in this assay were obtained from commercial sources. They can, however, be generated against other viruses and used in the assay described in this manuscript. Meanwhile, K562 cells are an Fc&#947;III-receptor-expressing leukemia cell line that bind to the Fc region of antibodies and become infected in the presence of sub-neutralizing concentrations of antibody4,5.
ADE assays have been extensively used in studies investigating the risk factors for severe dengue and to delineate the mechanisms of in vitro ADE6,7,8.The ADE assay described here can be quickly and easily used to determine the capacity of serum to enhance in vitro infection using RVPs and flow cytometry, as compared to other assays used currently, which require either the determination of plaque forming units (pfu) in Vero cells or antibody staining of infected cells6,7,8,9,10,11, both of which are time consuming and labor intensive.

<table border="0" cellpadding="0" cellspacing="0" width="794">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DENV 1-4 RVP</td>
			<td style="width:139px;">Integral Molecular</td>
			<td style="width:119px;">RVP-501</td>
			<td style="width:323px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">K562 cells</td>
			<td style="width:139px;">ATCC</td>
			<td style="width:119px;">CCL-243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">RPMI</td>
			<td style="width:139px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FBS</td>
			<td style="width:139px;">GE lifesceinces</td>
			<td style="width:119px;">SH 30910.03</td>
			<td>10% FBS in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Penicillin-Streptomycin</td>
			<td style="width:139px;">MP biomedicals</td>
			<td style="width:119px;">1670049</td>
			<td>100 IU Pen/mL, 100 ug Strep/mL in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HEPES</td>
			<td style="width:139px;">Cellegro</td>
			<td style="width:119px;">25-060-Cl</td>
			<td>0.025M in RPMI-10</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">MEM Non-essential Amino Acid Solution (100&#215;)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">M7145</td>
			<td>1x in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sodium Pyruvate</td>
			<td style="width:139px;">Cellegro</td>
			<td style="width:119px;">25-000-Cl</td>
			<td>1mM in RPMI-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">L-glutamine</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005CI&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sterile V-bottom plates</td>
			<td style="width:139px;">Thomas Scientific</td>
			<td style="width:119px;">333-8001-01V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">BD Falcon Polypropylene 5 ml FACS tubes</td>
			<td style="width:139px;">VWR</td>
			<td style="width:119px;">60819-728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Non-sterile U-bottom plates</td>
			<td style="width:139px;">Falcon</td>
			<td style="width:119px;">Ref 353910</td>
			<td>A high throughput alternative to FACS tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">5mL, sterile, serological pipette</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P7127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">200uL sterile pipette tips</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P3020 CPS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20uL sterile pipette tips</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P1121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">50-200uL multichannel pipette</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P3975-8-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">5-50uL multichannel pipette</td>
			<td style="width:139px;">Denville</td>
			<td style="width:119px;">P3975-9-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20% formadehyde</td>
			<td style="width:139px;">Tousimis</td>
			<td style="width:119px;">&#160;#1008B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water Bath</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:119px;">TSCOL35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">thermomixer</td>
			<td style="width:139px;">Eppendorf</td>
			<td style="width:119px;">2231000387</td>
			<td>An alternative to a waterbath for heat inactivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CO2 Incubator</td>
			<td style="width:139px;">ThermoFisher</td>
			<td style="width:119px;">13-998-213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:119px;">E7023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Liquid Bleach</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">NC9724348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Flowjo 9.8</td>
			<td style="width:139px;">TreeStar, Inc.</td>
			<td style="width:119px;"></td>
			<td>Flow cytometry analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSDiva 6.1.2</td>
			<td style="width:139px;">Becton Dikinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">BD LSR II flow cytometer</td>
			<td style="width:139px;">Becton Dikinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Liquid Bleach</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">NC9724348</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple flow cytometry based assay to determine in vitro antibody dependent enhancement of dengue virus using zika virus convalescent serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

Sera from 4 rhesus macaques were collected 16 weeks after infection with ZIKV and tested for their potential to enhance DENV-1, 2, 3 and 4 infection in K562 cells. The animals had peak viremia of ~1 x 105 copies of ZIKV RNA/mL of plasma by day 3 after infection that declined to levels that were below the limit of detection by 7 days after infection. No viremia was detected in the plasma at 16 weeks post-infection. Then, the RVP assay was performed and the collected data were analyzed, as described in the above protocol.
The gating strategy used to identify GFP+ cells are shown in Figure 1. The initial gate was set to exclude autofluorescent doublets and only include single cells based on FSC-A vs FSC-H. The next plot shows only cells from the single cell gate; there, a gate was drawn based on SSC-A vs FSC-A to exclude any autofluorescent debris. These gated cells were then displayed in a third plot based on SSC-A vs FL1-A to identify GFP+ cells. Cells infected with RVPs express GFP, and the percentage of GFP+ cells was determined by setting a gate around these cells. Approximately 12.2% of GFP+ cells are detectable in the ZIKV infected serum sample, as compared to no GFP+ cells in the control sample. This gating strategy is followed for each serum dilution and control sample included in the assay.
The percentage of GFP+ cells was determined for each sample dilution and control sample as described above (Figure 1). Data from a representative animal using DENV-1 RVPs are shown in Figure 2. As compared to the no serum control sample that had 0.253% GFP+ cells, the percentage of GFP+ cells were 2.58% in undiluted sample, 12.8% at 1:10, 0.735% at 1:100, and 0.022% at 1:1,000 dilution. The percentage of GFP+ cells will change as the serum is diluted due to the decreasing concentrations of antibody in the sample.
As enhancement of infection is associated with the antibody concentration that is ideal to increase uptake of virus, we observed a dramatic increase in the percentage of GFP+ cells at 1:10 dilution as compared to either undiluted sample or other dilutions, suggesting that the undiluted sample had higher concentrations of antibody whereas the 1:100 and 1:1,000 dilutions had lower levels of antibody that was not sufficient to induce ADE of infection. If antibody concentrations are significantly high, then additional dilutions of serum should be included to determine the dilution at which ADE becomes apparent. In the serum samples we examined, we observed ADE at a dilution of 1:10. On the other hand, ADE may be observed at lower dilutions if the antibody concentration in the serum is quite high.
Next, we calculated the fold-enhancement of infection by dividing the average percentage of GFP+ cells in the triplicate wells for each dilution with the average percentage of GFP+ cells in the triplicate wells for no serum control. The kinetics of ADE against DENV-1, 2, 3 and 4 serotypes were determined by graphing fold-enhancement on the y-axis and serum dilution on the x-axis (Figure 3). The data shows that the serum collected at 16 weeks post infection significantly enhanced infection of K562 cells by DENV-1, 2, 3 and 4 serotypes at a dilution of 1:10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57371/57371fig1.jpg" /> 
Figure 1: Gating strategy for acquiring and analysis by flow cytometry. K562 cells infected with DENV RVPs express GFP that was analyzed using a flow cytometer. (A) No serum control and (B) 16 week post-ZIKV infected serum. Cells were first gated based on forward scatter area (FSC-A) vs forward scatter height (FSH-H) to exclude doublets followed by a side scatter (SSC-A) vs FSC-A to exclude debris. The SSC-A vs FSC-A gated cells were then gated based on SSC-A vs GFP-A (Green Fluorescence Protein-area). The black rectangles and ovals represent the gates, and the numbers above the gates are the percentage of cells falling within that gate. The black arrow between the FACS plots shows the sequence of gates used. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57371/57371fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57371/57371fig2.jpg" /> 
Figure 2: Representative dot plots showing frequency of DENV RVP+ cells. The percentage of K562 cells that were infected with DENV-1 RVPs was determined by flow cytometry. Serum collected from a representative Rhesus macaque at 16 weeks post infection was incubated with DENV-1 RVPs either undiluted or at a 1:10, 1:100, and 1:1,000 dilutions and compared to no serum control samples. The percentage of GFP+ cells at each concentration of serum is shown. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57371/57371fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57371/57371fig3.jpg" /> 
Figure 3: Significant enhancement of Dengue-1, 2, 3 and 4 infection is observed at 1:10 dilution of serum. Antibody dependent enhancement of Dengue infection was examined for the 4 DENV serotypes by plotting serum dilutions on the x-axis and fold-enhancement (relative to no serum sample controls) on the y-axis. Sera collected from 4 ZIKV immune rhesus macaques at 16 weeks after infection was used in this assay. Error bars represent standard error and * represents p &#60;0.05. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57371/57371fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum at JoVE.com

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

a-simple-flow-cytometry-based-assay-to-determine-vitro-antibody

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.7K Views.  Uniformed Services University. We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Video: A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

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 ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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.

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

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...