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

注:工作流程的示意图外形如图1所示。 1.细胞<ol><li>使用Vero细胞为寨卡病毒生产和病毒复制周期的分析。 </li><li>制备含有10％牛胎儿血清（FBS），2mM的L-谷氨酰胺，青霉素（100单位/ ml），链霉素完全生长培养基（100单位/ ml），和10mM HEPES。 </li><li>培养Vero细胞在37℃，5％CO 2的指定的完全生长培养基。 </li></ol> 2.兹卡病毒生产<ol><li>在收获80％的细胞密度用0.25％的胰蛋白酶在T-75烧瓶中Vero细胞和细胞计数。 <ol><li>吸从T-75培养烧瓶中的介质并用2ml磷酸盐缓冲盐水（PBS）漂洗细胞。 </li><li>加2ml的0.25％胰蛋白酶的孵育在37℃的烧瓶5分钟。 </li><li>加入8毫升血清的含有生长培养基以灭活胰蛋白酶。吸取上下SUS挂起细胞和细胞转移到15毫升管。 </li><li>取出10微升细胞与等量0.4％台盼蓝混合。加载准备组合，以细胞计数室幻灯片，并获得活细胞使用自动细胞计数器计数。 </li></ol></li><li>种子共有7百万个细胞在30ml的量成T-160瓶。 </li><li>第二天，在每烧瓶10ml的无血清培养基制备感染的寨卡病毒接种物的适当复数（0.01到0.1的MOI）。 </li><li>从T-160烧瓶中取出用过的培养基，然后添加新鲜制备的病毒接种物（10毫升）中。 </li><li>用5％的CO 2孵育接种的烧瓶在37℃4-6小时。轻轻地在每一个小时横盘倾斜瓶传播接种。 </li><li>在温育完成后，取代对每个烧瓶暖血清补充的生长培养基（30毫升）的接种物。然后继续为下96小时的病毒培养。 </li><li>验证的进展通过使用相差显微镜，并采取必要的40X和200X放大倍率的图像（ 图2）观察对细胞单层的病毒斑的出现染。 </li></ol><img alt="图2" src="/files/ftp_upload/54767/54767fig2.jpg" /> 图2:不同放大倍率的由寨卡病毒在Vero细胞上的单层形成斑块明场图像显示在48 HPI寨卡病毒噬斑。注意圆形细胞灶上单层的存在。 （比例尺= 50微米）， <a href="https://www.jove.com/files/ftp_upload/54767/54767fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="8"><li>在96小时的时间点和离心上清在300×g离心10分钟，在4℃下收获细胞培养物上清液以除去细胞碎片。 </li><li>小心取出上清液，而不干扰沉淀杂物和转让ŤO 15毫升管。可以进行超速离心24,000 XG集中的病毒颗粒（可选）。 </li><li>在-80℃保存在多个等份病毒培养上清液。 </li></ol>斑块检测3.测量兹卡病毒滴度<ol><li>种子幼稚Vero细胞以每孔1×10 5个细胞于2ml体积使用12孔板中。 </li><li>次日，制备10倍使用无血清培养基的T-160烧瓶中收集病毒培养上清的系列稀释液。从各取出废传播媒介，一式三份添加400微升准备接种到Vero细胞上。 </li><li>孵育37接种的烧瓶 °下用5％CO 2的4-6小时。轻轻在每1小时侧身倾斜板蔓延的接种。 </li><li>在孵育结束时，替换与血清补充培养基的接种物（2毫升，每孔）。 </li><li>在48小时后接种，使用相对照计数病毒灶AST显微镜。计算病毒滴度为每毫升空斑形成单位（PFU）。 见图3斑块检测。 </li><li>固定和染色在20％乙醇中制备用于噬斑清晰的​​可视化在4％甲醛将细胞与0.1％的结晶紫溶液。 </li></ol> 4.兹卡病毒基因组复制测定<ol><li>种子幼稚Vero细胞以每孔1×10 5个细胞于2ml体积使用12孔板中。一式三份使用无血清培养基低和高滴度;第二天，制备病毒接种物（400微升/孔的0.01和0.1的MOI）。对于模拟感染，只使用无血清培养基（400微升/孔）。 </li><li>重复步骤3.2至3.4。 </li><li>在48和96小时的时间点，收集RNA和免疫细胞化学（ICC）的样品。 <ol><li>收获的RNA的样品，取出介质，然后添加400微升裂解液直接向每个孔和收集裂解物。 </li><li>对于ICC，删除媒体和T母鸡加入1毫升甲醇到每个孔中固定细胞，并在4℃下孵育该板30分钟。 </li><li>从细胞裂解物中，分离使用根据制造商的指令的RNA分离试剂盒提取总RNA。量化使用分光光度计的RNA。 </li></ol></li><li>执行两步逆转录定量PCR（RT-qPCR的），以确定从收获的RNA中ZIKV基因组的内容。 <ol><li>为了扭转转录RNA，首先建立在0.2毫升管组成的分离RNA的5微克，1微升的dNTP（10毫米），1微升随机六聚体（250毫微克）13微升反应混合物。孵育管中，在65℃进行5分钟，然后将其放置在冰上一分钟。接着，加入1微升反转录酶，4μl的5×链合成缓冲液，1微升的0.1M二硫苏糖醇，和1微升RNA酶抑制剂的反应混合物。孵育在25℃的管进行额外的5分钟，随后60分钟在50℃和inactiv吃加热15分钟，反应至70℃。 </li><li>使用qPCR 1微升产生的cDNA的12.5微升2个绿色染料混合超级含有DNA聚合酶和1微升10毫具体为兹卡病毒[寨卡病毒泛基因引物（对于单个引物进行:5&#39;-AARTACACATACCARAACAAAGTGGT-3&#39; ;启:5&#39;-TCCRCTCCCYCTYTGGTCTTG-3&#39;），寨卡病毒亚基因型PRVABC59菌株的引物（5&#39;-AAGTACACATACCAAAACAAAGTGGT-3&#39;;启:5&#39;-TCCGCTCCCCCTTTGGTCTTG-3&#39;）]，或丙型肝炎病毒（JFH RTQ F:5 &#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R:5&#39;CCTATCAGGCAGTACCACA-3&#39;），或细胞持家基因GAPDH（有关:5&#39;-CCACCTTTGACGCTGGG-3&#39;;启:5&#39;-CATACCAGGAAATGAGCTTGACA-3&#39;）在25微升反应体积。 </li><li>使用运行条件95℃15秒和60℃下，在一个实时PCR系统30秒（40个循环）进行PCR。 </li><li>使用GAPDH表达水平的基础上周期阈值（Ct）正常化在ZIKA病毒基因组的测量。计算相比，GAPDH的基因组兹卡的三角洲的Ct（ΔCT）值并获得2ΔCT值。然后，通过利用被感染的和未感染的细胞之间规格化孜卡基因组含量比在指定的时间点计算倍数变化。参见图4 ZIKV基因组复制的结果。 </li></ol></li><li>使用甲醇固定的细胞进行ICC。 <ol><li>洗固定的细胞三次用1×PBS和与ICC封闭缓冲液阻断（3％山羊血清，3％的BSA，0.1％的Triton-X的PBS中100）。 </li><li>以1:1的稀释度使用小鼠单克隆抗dsRNA的抗体J2（1微克/毫升）:100在封闭缓冲液并在4℃下孵育过夜。 </li><li>洗用1×PBS的细胞中并在一个1添加二级抗体的山羊抗小鼠IgG-594（1微克/毫升）:1000稀释在封闭缓冲液孵育在室温下1小时。 </li><li>洗用1×PBS和染色细胞用Hoechst的染料细胞核和观察使用放大100倍的荧光显微镜（ 图4）的细胞。 </li></ol></li></ol> 5.细胞因子筛选对图书馆兹卡病毒感染<ol><li>种子幼稚Vero细胞以1×10 5细胞每孔用12孔板中。一旦细胞附着（6小时后播种），在生物重复所示浓度（2毫升体积/孔）添加每种细胞因子。包括车辆（PBS）单独控制。 </li><li>在12小时的后处理，执行寨卡病毒感染（每孔0.1于400μl的MOI）。包括阴性对照孔，无感染（模拟）。 </li><li>在4小时后感染，替换细胞因子处理的介质（2ml）中的病毒接种物。孵育细胞在37℃下额外44小时。 </li><li>在48小时后感染，计数用相差显微镜病毒噬斑和在40X放大倍率（ 图5）获得感染的细胞的代表性图像。 </li></ol>

<ol>
	<li>Brasil, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infection+in+Pregnant+Women+in+Rio+de+Janeiro+-+Preliminary+Report.">Zika Virus Infection in Pregnant Women in Rio de Janeiro - Preliminary Report.</a> N Engl J Med. , 1-11 (2016).</li><li>Lucey, D. R., Gostin, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emerging+Zika+Pandemic:+Enhancing+Preparedness.">The Emerging Zika Pandemic: Enhancing Preparedness.</a> JAMA. 315 (9), 865-866 (2016).</li><li>Mlakar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Associated+with+Microcephaly.">Zika Virus Associated with Microcephaly.</a> N Engl J Med. 374 (10), 951-958 (2016).</li><li>Schuler-Faccini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+Association+Between+Zika+Virus+Infection+and+Microcephaly.">Possible Association Between Zika Virus Infection and Microcephaly.</a> MMWR Morb Mortal Wkly Rep. 65 (3), 59-62 (2015).</li><li>Foy, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probable+non-vector-borne+transmission+of+Zika+virus,+Colorado,+USA.">Probable non-vector-borne transmission of Zika virus, Colorado, USA.</a> Emerg Infect Dis. 17 (5), 880-882 (2011).</li><li>Kuno, G., Chang, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+sequencing+and+genomic+characterization+of+Bagaza,+Kedougou,+and+Zika+viruses.">Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses.</a> Arch Virol. 152 (4), 687-696 (2007).</li><li>Dick, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus.+II.+Pathogenicity+and+physical+properties.">Zika virus. II. Pathogenicity and physical properties.</a> Trans R Soc Trop Med Hyg. 46 (5), 521-534 (1952).</li><li>Dick, G. W., Kitchen, S. F., Haddow, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus.+I.+Isolations+and+serological+specificity.">Zika virus. I. Isolations and serological specificity.</a> Trans R Soc Trop Med Hyg. 46 (5), 509-520 (1952).</li><li>Oehler, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus+infection+complicated+by+Guillain-Barre+syndrome--case+report,+French+Polynesia.">Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia.</a> Euro Surveill. 19 (9), 1-3 (2013).</li><li>Baronti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+coding+sequence+of+zika+virus+from+a+French+polynesia+outbreak+in+2013.">Complete coding sequence of zika virus from a French polynesia outbreak in 2013.</a> Genome Announc. 2 (3), e00500-e00514 (2014).</li><li>Lanciotti, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+serologic+properties+of+Zika+virus+associated+with+an+epidemic,+Yap+State,+Micronesia,+2007.">Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007.</a> Emerg Infect Dis. 14 (8), 1232-1239 (2008).</li><li>Lanciotti, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+Zika+virus+in+Western+Hemisphere,+2015+[Letter].">Phylogeny of Zika virus in Western Hemisphere, 2015 [Letter].</a> Emerg Infect Dis. 22 (5), (2016).</li><li>Musso, D., Nilles, E. J., Cao-Lormeau, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+spread+of+emerging+Zika+virus+in+the+Pacific+area.">Rapid spread of emerging Zika virus in the Pacific area.</a> Clin Microbiol Infect. 20 (10), O595-O596 (2014).</li><li>Tang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+Virus+Infects+Human+Cortical+Neural+Progenitors+and+Attenuates+Their+Growth.">Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth.</a> Cell Stem Cell. , 1-5 (2016).</li><li>Hamel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+Zika+Virus+Infection+in+Human+Skin+Cells.">Biology of Zika Virus Infection in Human Skin Cells.</a> J Virol. 89 (17), 8880-8896 (2015).</li><li>Faye, O., 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=Quantitative+real-time+PCR+detection+of+Zika+virus+and+evaluation+with+field-caught+mosquitoes.">Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes.</a> Virol J. 10, 311 (2013).</li><li>Chu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+enhancer+and+critical+cis-acting+RNA+elements+in+the+protein-encoding+region+of+the+hepatitis+C+virus+genome.">Systematic analysis of enhancer and critical cis-acting RNA elements in the protein-encoding region of the hepatitis C virus genome.</a> J Virol. 87 (10), 5678-5696 (2013).</li><li>Hiratsuka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+interferon-alpha+during+pregnancy:+effects+on+fetus.">Administration of interferon-alpha during pregnancy: effects on fetus.</a> J Perinat Med. 28 (5), 372-376 (2000).</li><li>Ozaslan, 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=Interferon+therapy+for+acute+hepatitis+C+during+pregnancy.">Interferon therapy for acute hepatitis C during pregnancy.</a> Ann Pharmacother. 36 (11), 1715-1718 (2002).</li></ol>

The authors have nothing to disclose.

这里， 在体外培养寨卡病毒精简协议被提出。关键步骤包括，确定最佳的终点扩大病毒培养，测量滴度，并量化基因组复制被提供。寨卡病毒是一种人类病原体，因此，在处理感染性物质，生物安全程序必须严格遵守。阿猴肾细胞系，Vero细胞，用于展示各种病毒学检测。寨卡病毒复制动力学可以在各种组织和物种的细胞不同。可以如前面15所描述的使用的其他细胞系。寨卡病毒已?...

兹卡病毒（ZIKV）是小头畸形及妊娠结局不佳有关1,4重要的人类病原体。 ZIKV属于集合医学相关的黄病毒可导致神经缺陷，如登革热，西尼罗河，和圣路易脑炎病毒。病毒传播的主要方式是通过蚊子载体埃及伊蚊 ，并且，此外，性传播也有报道5,6。 ZIKV已经成为一个主要的全球性健康问题，由于蚊子的地域扩张分布及其与出生缺陷密切相关。 ZIKV最早是在1947年从兹卡森林定点恒河猴隔离，乌干达和第一个人类病例报告，1952年7,8。个人认为感染了ZIKV存在轻度症状，如发热，皮疹，头痛，结膜炎和肌肉/关节疼痛。受感染的孕妇可以传输ZIKV发育中的胎儿1。 žIKV感染也被链接到吉兰巴雷综合征，外周神经自体免疫脱髓鞘病症9。 的寨卡病毒基因组由一个正感，单链RNA分子，其长度为约10.8千碱基的。基因组的结构被组织成5&#39;NCR-C-的prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-3&#39;NCR，与非编码区域（NCR）侧翼的蛋白质编码区6。一个单一的多聚（3419 AA）被翻译的是合作和翻译后切割成10小肽。无论是5&#39;NCR和3&#39;NCR RNA茎环结构在病毒基因组翻译和复制生效的重要组成部分。基因组的结构部件是由衣壳，膜和包膜蛋白。非结构蛋白是基因组复制的关键。 目前，寨卡病毒株被分成三个主要的基因型:西非，东非和亚洲6,10-13。有人提出，蔓延到西非和亚洲，后来进一步发展12东非血统。亚洲基因型负责在美洲当前疫情。寨卡病毒可以是在这两个蚊子和哺乳动物细胞中培养。初级真皮成纤维细胞，未成熟树突状细胞，皮层神经祖细胞，Vero细胞易受寨卡病毒感染10,14,15。 I型和II型干扰素已显示限制在皮肤成纤维细胞15 ZIKV增长。这项研究的目的是提供用于生产和亚洲基因型ZIKA病毒株PRVABC59的化验在哺乳动物细胞培养系统的逐步，详细的协议，并证明这种传染病文化体系作为药物开发平台的效用。此资源具有对兹卡病毒和神经学研究界进一步阐明我受益非浅的潜力病毒发病机理和病毒的毒性进化TS机制。

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

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

寨卡病毒（ZIKV）是链接到胎儿发育异常，如小头畸形，眼缺陷，和受损生长一个新兴的病原体。 ZIKV是黄病毒科的RNA病毒。 ZIKV主要是由蚊子传播的，但也可以通过母体传播给胎儿的垂直传播和性接触。迄今为止，有可用的，以保护那些被病毒感染没有可靠的治疗或疫苗的选项。可重复的，有效的兹卡病毒感染的细胞培养系统的发展是研究ZIKV复制以及药物和疫苗发展的分子机制的关键。在这方面，描述的哺乳动物细胞为基础的体外寨卡病毒培养病毒的生产和生长分析系统的协议在这里报道。对一个细胞单层和斑块比色法检测病毒滴度形成斑块由寨卡病毒的详细介绍。病毒基因组复制动力学和双链RNA基因组replicatory中间体被确定。此培养平台利用屏幕对一小部分产生的干扰素α（IFN-α）的鉴定细胞因子的文库，IFN-β和IFN-γ作为寨卡病毒生长的有效抑制剂。总之， 在体外感染寨卡病毒培养体系和各种病毒学测定法证明了在本研究中，这也在研究界中进一步阐明病毒致病机制和病毒毒力的演变极大地受益的可能性。抗病毒的IFN-α还可以作为预防，暴露后预防性，和治疗的高危人群寨卡病毒感染，包括病毒感染的孕妇选项进行评估。

一个兹卡病毒株;亚洲基因型（PRVABC59 GenBank登录号KU501215）得到使用，这项研究12。被用于研究从头寨卡病毒感染的Vero细胞在80％汇合。对于病毒生产和随后的病毒学特征，早期通道（P3）兹卡病毒使用。上观察到感染的第二天病毒斑块。孜卡从最初感染的细胞释放的病毒后代可以扩散到相邻细胞，这导致在单层培养物（ 图2）可见斑块的形成。?...

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

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

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

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 ...

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

Research

JoVE Journal

Immunology and Infection

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

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

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

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

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

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

从现场收集到的蚊子传染病毒Vero细胞培养法检测

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

科研

免疫与感染

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.

可视登革病毒通过的Alexa Fluor标签

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

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

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

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

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

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

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

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

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

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

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

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

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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

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

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

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

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.

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 ...

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).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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

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

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

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

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 ...

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

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

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

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

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

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