Name: a simple flow cytometry based assay to determine in vitro antibody dependent enhancement of dengue virus using zika virus convalescent serum
Uploaded: 2018-04-10T14:00:00.000+00:00
Duration: 7 min 6 s
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

所述项目得到了来自卫生科学制服服务大学的资金的支持, JJM。本文所载的意见或断言是作者的私人观点, 不应被解释为官方的, 也不反映国防部、卫生科学系制服服务大学或美国任何其他机构的意见。政府。
WGV 进行了所有实验, 并对数据进行了分析;JJM 设计和监督研究;WGW 和 JJM 写了这篇论文。

用于证明此处描述的议定书的血清样本取自恒河猴, 这些猕猴是根据当地、州和联邦政策在实验室动物护理评估和鉴定协会中所安置和照顾的。国际 (AAALAC) 认证的设施。所有动物实验都经过机构动物护理和使用委员会的审查和批准, 并通过组织分享协议获得样本。
1. 1 天
注: 执行下面描述的所有步骤, 在一个不育层流生物安全柜用于组织培养在 BSL-2 实验室。
<ol><li>在室温下解冻血清样品, 将每个血清样本的100µL 转移到无菌管。在56°C 中, 无论是在水浴中还是在温度可调的 thermomixer 中, 热灭活30分钟。使10倍的血清标本序列稀释从1:1 到 1:1, 000 使用冷 RPMI-10 (RPMI 与10% 胎牛血清)。</li>
	<li>将10µL 的连续稀释血清样品转移到无菌96井 V 型底板的每一个井中。包括两套控制井, RPMI-10 只 RVPs, 无血清样本, RPMI-10 无 RVPs, 无血清标本。在 triplicates 中设置每个血清样本和 RPMI-10 控制。</li>
	<li>从-80 °c 冰箱中取出 Dengue-1、2、3和 4 RVPs, 并在37摄氏度水浴中解冻。将解冻的 RVPs 立即转移到冰上。获得大约170µL RVPs 为每个血清样品 (10 µL RVPs x 3 井为每个血清稀释 (1:1, 1:10, 1:100, 1:1, 000) 和10µL RVPs x 3 井为每个 RPMI-10 与仅 RVP 控制井)。</li>
	<li>吸管10µL RVPs 入96井 V 底板的每个井中, 含有血清样本, 并与 RVPs (无血清) 控制井 RPMI-10。请勿将 RVPs 添加到 RPMI-10 (无血清样本和 RVP) 井。通过吹打上下5–10时间彻底混合。添加10µL 冷 RPMI-10 代替 RVPs 每冷 RPMI-10 (无血清样本和无 RVP) 控制井。</li>
	<li>将96井 V 型底板转移到孵化器中, 在 5% CO2的存在下, 在37摄氏度处孵化出1小时的板材。当96井 V 型底板正在孵化时, 用70% 乙醇清洁生物安全柜的表面, 并运行紫外光15分钟。</li>
	<li>从孵化器中移除一个完全汇合的 T75 瓶 K562 细胞, 用不育的5毫升吸管混合细胞, 并将5毫升细胞转移到无菌15毫升锥形管。 注意: K562 细胞保持在 RPMI-10 和传代 1 x 106/毫升。</li>
	<li>通过从无菌15毫升圆锥管中取出10µL 细胞, 并与10µL tryphan 蓝混合, 计算细胞数。使用 hemocytometer 计算单元格, 以确定15毫升圆锥管中的细胞总数。</li>
	<li>离心机15毫升圆锥细胞在 ~ 1200 x g 10 分钟, 醒酒上清, 并并用重悬在温暖 RPMI-10 的细胞集中 8万 cells/30 µL 的媒体 (2.66 x10 6 细胞/毫升)。</li>
	<li>从孵化器 (步骤 1.6) 卸下96井 V 型底板, 将30µL 的 K562 细胞转移到96井 V 底板的每一个井中, 并通过吹打上下5–10时间彻底混合。将96井 V 底板转移到孵化器, 并在 5% CO2的存在下, 在37摄氏度的情况下孵化出1小时的板材。</li>
	<li>孵化1小时后, 从孵化器和离心机中取出96井 V 型底板, 在 1200 x g 处进行5分钟。离心后, 醒酒介质从井中翻转到一个装有10% 漂白剂的容器中。</li>
	<li>通过在125µL 的温暖 RPMI-10 中 resuspending 它们, 将它们洗净, 并将其与吸管完全混合, 并将盘子在 1200 x 克上离心5分钟. 醒酒介质时, 将盘子翻转成含有10% 漂白剂的容器。重复这个洗涤步骤两次。</li>
	<li>洗涤后, 添加100µL 的温暖 RPMI-10 的每一个井, 混合上下与吸管, 并孵化在37摄氏度48小时的板存在 5% CO2。</li>
</ol>2. 3 天
<ol><li>从孵化器中取出含有100µL 细胞和介质的96井 V 型底板, 并将其移到生物安全柜中。使用多通道吸管, 混合每个井的内容, 并将细胞和介质转移到96井 U 型底板上, 或预先标记5毫升聚丙烯管。</li>
	<li>在1x 磷酸盐缓冲盐水 (PBS) 中, 用100µL 1% 多聚甲醛 (煤灰) 的96井 V 底板冲洗每一口井, 并将其转移到96井 U 型底板上的相应井中, 或从步骤5中标记为2.1 毫升聚丙烯管, 从而产生最终浓度为0.5% 粉煤灰/井或管。彻底混合使用多通道吸管, 用铝箔盖住盘子, 让它在孵化器里坐30分钟来修复细胞。</li>
	<li>准备流式细胞仪 (例如, 请参见材料表), 方法是运行无瑕 K562 单元格来校准侧面和正向散射以及荧光设置。唯一需要的荧光通道是 FL1, 因为 GFP 是唯一从细胞中发出的荧光。从每个样品中获取 ~ 30,000–50,000 细胞。 注: 任何能够读取单个荧光的流式细胞仪都可用于获取数据, 因为感染 RVPs 的单元格只会由于 GFP 的存在而发出绿色荧光, 并且不需要其他荧光通道。</li>
</ol>3. 数据分析
<ol><li>
使用任何流式细胞仪分析软件 (见材料表) 分析在流动检测器上获取的数据。
	<ol>
<li>设置第一个门基于 FSC-A (正散射–区域) vs FSC H (正向散射–高度), 以包括单个单元格, 并排除从分析12自动荧光双峰。然后, 基于 SSC (侧向散射-区域) 与 FSC a 的门单线态门控单元, 以排除任何 autofluorescent 碎片。</li>
		<li>分析 ssc-a vs FSC-一个门控细胞的表达的 gfp 基于 SSC-a 与 gfp a。通过在 gfp + 单元格周围设置一个门, 确定 gfp + 细胞对每个稀释血清和控制样本的百分比。</li>
	</ol>
</li>
	<li>通过将三人三井中 gfp + 细胞的总百分比除以 3, 确定每种血清样品稀释和控制井的 gfp + 细胞的平均百分比。</li>
	<li>通过除以血清中 gfp + 细胞的平均百分比来计算感染的褶皱增强, 每个稀释除以 RPMI-10 中 gfp + 细胞的平均百分比与 RVPs (无血清样本) 控制井。</li>
	<li>图形折叠增强 (y 轴) 与稀释 (x 轴), 并进行统计分析使用方差后, Tukey 的特殊测试的多比较。</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>

作者没有什么可透露的。

Flaviviruses, 如 DENV 和 ZIKV, 在其结构和非结构蛋白中都有明显的同源性, 它们产生的抗体相互相互反应13,14。这些交叉反应抗体反应已被证明, 以增强感染的体内和体外与异种血清或其他相关 flaviviruses1,2。交叉增强感染的潜力对疾病的管理和疫苗的开发有着重要的影响。
传统的...

抗体依赖性增强 (艾德) 感染是一个过程, 其中部分交叉反应抗体的反应, 由一个血清型的病毒增强吸收另一种血清型病毒, 导致增加病毒复制和病毒血症。艾德已广泛记录在登革热病毒 (DENV) 感染, 其中四主要血清型流行。在患者的一小部分, 艾德与登革热出血热 (DHF) 有关。我们最近表明, Zika 病毒 (ZIKV) 感染引起明显的 DENV 交叉反应抗体应答, 导致 DENV体外, 并可能有助于增强 DENV 病毒血症在体内 1,2. 抗体依赖性增强检测是评估抗体增强继发感染相关病毒能力的宝贵工具, 并为黄感染的发病机制提供有价值的见解, 并告知疫苗的研制。
这里描述的化验使用 DENV RVPs 和 K562 细胞通常不允许感染。RVPs 是结构上完整的复制不称职的 DENV 病毒粒子, 编码一个亚基因组绿色荧光蛋白 (GFP) 传代, 在一轮复制3后表示。因此, 感染 RVPs 的细胞荧光绿色, 可以通过流式细胞术或显微镜随时检测到。本试验中使用的 RVPs 是从商业来源获得的。但是, 它们可以生成针对其他病毒, 并用于本手稿中描述的化验。同时, K562 细胞是一种 FcγIII 受体表达的白血病细胞系, 与抗体 Fc 区结合, 并在抗体4,5的亚中和浓度存在时感染。
在调查严重登革热危险因素的研究中广泛使用了艾德测定法, 并描述了体外的机制6,7,8。在这里描述的艾德化验可以快速和容易地用来确定血清的能力, 以提高体外感染使用 RVPs 和流式细胞术, 与目前使用的其他化验相比, 这需要确定的斑块形成单位 (pfu) 在维罗细胞或抗体染色的感染细胞6,7,8,9,10,11, 这两个都是时间消耗和劳动力密集。

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

抗体依赖性增强感染已被证明在登革热病毒发病机制中发挥重要作用。传统的测定抗体或血清能力以增强不允许细胞系感染的方法, 依赖于在培养基中使用病毒输出, 然后用斑块化验来量化感染。最近, 这些化验已经检查了登革热病毒 (DENV) 感染的细胞系使用荧光标记抗体。这两种方法都限制了这些技术的广泛使用。在这里, 我们描述一个简单的体外试验, 使用登革病毒报告病毒微粒 (RVPs), 表达绿色荧光蛋白和 K562 细胞, 以检查抗体依赖性增强 (艾德) DENV 感染的血清, 从恒河猴获得猕猴感染 Zika 病毒后16周 (ZIKV)。这种技术是可靠的, 涉及对细胞的最小操纵, 不涉及使用活复制主管病毒, 并可以执行高吞吐量格式, 以获得定量读数使用流式细胞术。此外, 这种检测可以很容易地适应检查其他黄感染的抗体依赖增强 (艾德), 如黄热病病毒 (YFV), 日本马脑炎病毒 (JEEV), 西尼罗河病毒 (WNV) 等 RVPs 可用。建立测试、分析数据和解释结果的简便性使其对大多数实验室设置非常适合。

4只恒河猴的血清在感染 ZIKV 16 周后被采集, 并经过测试, 以增强 DENV-1、2、3和 4 K562 细胞感染的潜能。这些动物的峰值病毒血症为 1 x10 5 的 ZIKV RNA/毫升血浆在3天后, 感染后, 下降到低于检测限度的水平, 在感染后7天。16周后感染后, 血浆中未发现病毒血症。然后, 对 RVP 进行了分析, 并对所收集的数据进行了研究, 如上述协议所述。
<p class="jove_content" fo:keep-together.wit...

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

我们描述了一个简单和简单的协议, 以测量抗体依赖性增强感染的 Zika 病毒恢复血清使用登革热病毒报告病毒粒子。

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

8.7K 次观看.  Uniformed Services University. 我们描述了一个简单和简单的协议, 以测量抗体依赖性增强感染的 Zika 病毒恢复血清使用登革热病毒报告病毒粒子。

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

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

科研

生物化学

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.

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

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

免疫与感染

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.

Zika 病毒特定诊断表位发现

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

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

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

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

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

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Virus Propagation and Cell-Based Colorimetric Quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barr&#233; syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

病毒传播和基于细胞的比色定量

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

Cell-Based Electrical Impedance Platform to Evaluate Zika Virus Inhibitors in Real Time

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture plate wells embedded with electrodes. The technology can be used to monitor the consequences of Zika virus (ZIKV) infection and adherent cell replication in real time, as this virus is highly cytopathogenic. It is a straightforward assay that does not require the use of labels or invasive methods and has the benefit of providing real-time data. The kinetics of ZIKV infection are highly dependent on the employed cell line, virus strain, and multiplicity of infection (MOI), which cannot be easily studied with conventional endpoint assays. Furthermore, the CEI assay can also be used for the evaluation and characterization of antiviral compounds, which can also have dynamic inhibitory properties over the course of infection. This methods article gives a detailed explanation of the practical execution of the CEI assay and its potential applications in ZIKV research and antiviral research in general.

Here, we demonstrate the use of cell-based electrical impedance (CEI) as a very easy and straightforward method to study Zika virus infection and replication in human cells in real time. Furthermore, the CEI assay is useful for the evaluation of antiviral compounds.

基于细胞的电阻抗平台，用于实时评估寨卡病毒抑制剂

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture ...

An In Vitro Assay for Screening Interferons as Potent Inhibitors of Zika Virus Growth

Source: Contreras, D. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54767">Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons.</a> J. Vis. Exp. (2016)
The video demonstrates an in vitro assay for screening interferons with antiviral activity against the Zika virus, a human pathogen. Within the host cell, the Zika virus undergoes multiplication upon internalization, infecting neighboring cells and creating distinct zones or viral plaques. Type I interferon, acting as an antiviral agent, hinders virus multiplication, thereby preventing the formation of viral plaques in the host cell monolayer.

用于筛选干扰素作为寨卡病毒生长有效抑制剂的体外测定

1. Screening Cytokine Library against Zika Virus Infection

	Seed naive Vero cells at 1 x 105 cells per well using a 12-well plate. Once the cells are ...

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

摘要

探索更多视频

此视频中的章节

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

兹卡病毒感染细胞培养系统和

Zika 病毒特定诊断表位发现

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

从小鼠多个器官分离和定量寨卡病毒

使用寨卡病毒对埃及伊蚊的载体能力分析

病毒传播和基于细胞的比色定量

基于细胞的电阻抗平台，用于实时评估寨卡病毒抑制剂

用于筛选干扰素作为寨卡病毒生长有效抑制剂的体外测定

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

摘要

探索更多视频

此视频中的章节

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

兹卡病毒感染细胞培养系统和

Zika 病毒特定诊断表位发现

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

从小鼠多个器官分离和定量寨卡病毒

使用寨卡病毒对 埃及伊 蚊的载体能力分析

病毒传播和基于细胞的比色定量

基于细胞的电阻抗平台，用于实时评估寨卡病毒抑制剂

用于筛选干扰素作为寨卡病毒生长有效抑制剂的体外测定

使用寨卡病毒对埃及伊蚊的载体能力分析