Name: a simple flow cytometry based assay to determine in vitro antibody dependent enhancement of dengue virus using zika virus convalescent serum
Uploaded: 2018-04-10T14:00:00
Description: 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

Beskrevet projektet var støttet af midler fra Uniformed tjenester University of Health Sciences at JJM. Udtalelser eller påstande heri er privat dem af forfatterne og ikke skal fortolkes som officielle eller reflekterende visninger af Department of Defense, Uniformed tjenester University of Sundhedsvidenskab eller ethvert andet agentur i USA Regeringen.
WGV udførte alle eksperimenter og analyseret data; JJM udviklet og overvåget undersøgelse; WGW og JJM skrev avisen.

Serumprøver bruges til at påvise protokollen beskrevet her var fremstillet af rhesus makakaber, der var opstaldet og plejet i overensstemmelse med lokale, statslige og føderale politik i en forening for evaluering og akkreditering af Laboratory Animal Care International (AAALAC)-akkrediteret facilitet. Alle dyreforsøg blev gennemgået og godkendt af institutionelle Animal Care og brug udvalget og prøver blev anskaffet gennem en væv deling protokol.
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<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>

Flavivira som DENV og ZIKV dele omfattende dokumentation af homologi i begge deres strukturelle og non-strukturelle proteiner, der genererer antistoffer, der krydsreagere med hinanden13,14. Disse cross-reactive antistof reaktioner har vist sig at øge infektion både i vivo og in vitro- med enten en heterolog serotype eller andre relaterede flavivira1,2. Potentiale til at krydse øge inf...

Antistof afhængig enhancement (ADE) af infektion er en proces, hvorved delvist cross-reactive antistof svar induceret af en serotype af virus øger optagelsen af en anden serotype af virus, fører til øget viral replikation og viræmi. ADE er blevet udførligt dokumenteret i Dengue virus (DENV) infektioner hvor fire store serotyper er fremherskende. I en delmængde af patienter er ADE forbundet med Dengue hæmoragisk feber (DHF). Vi har for nylig vist, at Zika virus (ZIKV) infektion induceret betydeligt høje niveauer af DENV cross-reactive antistof reaktioner, der forårsagede ADE af DENV in vitro og sandsynligvis bidraget til en forbedring af DENV viræmi i vivo1 , 2. antistof afhængig enhancement assays er et værdifuldt redskab til at vurdere kapaciteten af antistoffer til at forbedre sekundær infektion med relaterede virus og give værdifuld indsigt i patogenesen af flavivirus infektioner og informere den udviklingen af vacciner.
Analysen beskrives bruger her DENV RVP sammen med K562 celler, der normalt utilladelig til infektion. RVP er strukturelt intakt replikering inkompetente DENV virale partikler at indkode en sub-genomisk grøn fluorescerende proteiner (NGL) replikon, der kommer til udtryk efter en enkelt runde af replikering3. Som sådan er celler, der bliver smittet med RVP fluorescerer grønt og let kan påvises ved hjælp af flowcytometri eller mikroskopi. RVP bruges i denne analyse blev indhentet fra kommercielle kilder. De kan dog være genereret mod andre vira og brugt i analysen beskrives i dette håndskrift. I mellemtiden, K562 celler er en FcγIII-receptor-udtrykker leukæmi cellelinie der binder til regionen Fc af antistoffer og blive smittet i overværelse af sub neutralisere koncentrationer af antistof4,5.
ADE assays har været flittigt brugt i studier undersøger risikofaktorer for svær denguefeber og afgrænse mekanismerne af in vitro- ADE6,7,8. ADE analysen beskrives her kan hurtigt og nemt bruges til at bestemme kapaciteten af serum til at styrke in vitro- infektion ved hjælp af RVP og flow flowcytometri, sammenlignet med andre assays anvendes i øjeblikket, som kræver enten bestemmelse af plak danner enheder (pfu) i Vero celler eller antistoffarvning af inficerede celler6,7,8,9,10,11, som begge er tidskrævende og labor intensiv.

<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

Antistof afhængig af forbedring af infektion har vist sig at spille en større rolle i Dengue viral patogenese. Traditionelle assays, der måler antistoffer eller serum evne til at øge infektion i utilladelig cellelinjer har påberåbt sig bruger viral output i medierne efterfulgt af plaque assays til at kvantificere infektion. For nylig, har disse assays undersøgt Dengue virus (DENV) infektion i celle linjerne ved hjælp af fluorescently mærket antistoffer. Begge disse tilgange har begrænsninger, der begrænser den udbredte brug af disse teknikker. Her, beskriver vi en simpel i vitro assay ved hjælp af Dengue virus reporter viruspartikler (RVP), express grøn fluorescerende proteiner og K562 celler til at undersøge antistof afhængig enhancement (ADE) af DENV infektion ved hjælp af serum, der var fremstillet af rhesus makakaber 16 uger efter infektion med Zika virus (ZIKV). Denne teknik er pålidelige, involverer minimal manipulation af celler, indebærer ikke anvendelse af levende replikering kompetente virus og kan udføres i en høj overførselshastighed format til at få en kvantitativ udlæsning bruger flowcytometri. Derudover kan dette assay tilpasses nemt for at undersøge antistof afhængig enhancement (ADE) af andre flavivirus infektioner som gul feber virus (YFV), japansk Equine Encephalitis virus (Kathrine VINDUM), West Nile-virus (VNV) etc. hvor RVP er tilgængelige. Lethed af opsætning af analysen, analysere data og fortolkning af resultater gør det meget åbne over for de fleste laboratorium indstillinger.

Sera fra 4 rhesus makakaber blev indsamlet 16 uger efter infektion med ZIKV og testet for deres potentiale til at forbedre DENV-1, 2, 3 og 4 infektion i K562 celler. Dyrene havde peak viræmi ~ 1 x 105 eksemplarer af ZIKV RNA/mL af plasma af dag 3 efter infektion, der faldt til et niveau, der lå under detektionsgrænsen ved 7 dage efter infektion. Ingen viræmi blev fundet i plasma på 16 uger efter infektionen. Derefter RVP analyse blev udført, og de indsamlede data blev ana...

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

Vi beskriver en enkel og nem protokol til måling af antistof afhængig af forbedring af infektion af Zika virus rekreationshjem serum ved hjælp af Dengue virus Reporter viruspartikler.

En simpel flowcytometri baseret analyse for at bestemme In Vitro antistof afhængig af forbedring af Dengue Virus ved hjælp af Zika Virus rekreationshjem Serum

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

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

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