Name: zika virus infectious cell culture system and the in vitro prophylactic effect of interferons
Uploaded: 2016-08-23T15:00:00.000+00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons at JoVE.com

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.bdigitaluss.remotexs.co/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。寨卡病毒已从感染胎儿3的脑组织中分离出，并已显示出感染皮层神经祖细胞14。因此，在神经元细胞评估ZIKV感染的病理生理学可以提供额外的特定于小区的见解。在非洲和亚洲基因型的神经毒性表型差异可以调查使用这种传染病的文化体系。这里所描述的病毒生产过程也将是对在非人类灵长类动物或啮齿动物模型系统的体内致病研究产生高滴度病毒是有用的。 虽然，本研究仅限于Vero细胞的用途，高滴度生产人及蚊起源的细胞系可以进行测试。期间的病毒的生产，如果CPE在只有10-30％的细胞观察到的第3天，在受感染的细胞可以在1分裂:4的密度和培养可以继续另外4-5天。在第 7天结束时，一个完整的CPE可以观察到。 ZIKV泛基因型和菌株特异性的引物的有效性进行比较，并确定两者都是在低和高滴度的病毒感染的细胞进行定量寨卡病毒基因组敏感。另外，在这项研究中，基于抗体的探针被验证，以确定感染的细胞。这种特定的双链RNA抗体成功RECOgnized细胞质病毒基因组中的寨卡病毒感染的细胞。这些优化的试剂将解剖病毒复制的不同阶段，如翻译，基因组复制，组装和病毒形态的有用资源。 该体外感染性细胞培养体系是适用于使用由互补DNA（cDNA）克隆产生寨卡病毒特异性亚基因组复制子和感染性病毒的将来的研究。兹卡病毒感染性cDNA克隆的发展将结合遗传和分子生​​物学技术加速病毒基因，包括NS1和NS4B的功能研究。寨卡病毒标记荧光或发光基于报告基因的建立将是作出进一步使用高内涵筛选试验的细胞培养系统的宝贵工具。 I型和II型干扰素表现出抗寨卡病毒活性。 IFN-α，IFN-β和IFN-γ可以进一步EValuated在临床环境作为预防，曝光后的预防性和治疗对抗寨卡病毒感染和相关疾病。高危人群包括孕妇阳性寨卡病毒，可以与干扰素作为治疗的第一线治疗。 α干扰素治疗已被证明是对孕妇18,19安全。 总之，可以用于研究寨卡病毒生长的各种方面的感染细胞培养系统中描述和概括。该协议与此处描述的试剂是为那些谁研究寨卡病毒和神经学研究界的重要资源。

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><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Sigma Life Science</td><td>D5796</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630080</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>2.5% Trypsin, 10x [-] Phenol Red&amp;nbsp;&amp;nbsp;</td><td>Corning</td><td>25-054-C1</td><td></td></tr><tr><td>Trypan Blue Stain 0.4%</td><td>Life Technologies</td><td>T10282</td><td></td></tr><tr><td>Countess &amp;ndash; Automated Cell Counter</td><td>ThermoFisher Scientific</td><td>C10227</td><td></td></tr><tr><td>Countess-cell counting&amp;nbsp; chamber slides</td><td>ThermoFisher Scientific</td><td>C10283</td><td></td></tr><tr><td>Rneasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Nanodrop 2000</td><td>Thermo Scientific</td><td>Nanodrop 2000</td><td></td></tr><tr><td>mouse monoclonal anti-dsRNA antibody J2&amp;nbsp;</td><td>English &amp;amp; Scientific Consulting Kft.</td><td>10010200</td><td></td></tr><tr><td>Goat anti-rabbit IgG Alexa Fluor 594</td><td>Life Technologies</td><td>A11020</td><td></td></tr><tr><td>SUPERSCRIPT III RT&amp;nbsp;</td><td>Life Technologies</td><td>18080085</td><td></td></tr><tr><td>SYBR QPCR SUPERMIX W/ROX</td><td>Life Technologies</td><td>11744500</td><td></td></tr><tr><td>QuantStudio12K Flex Real-Time PCR System</td><td>Thermo Fischer</td><td>4471088</td><td></td></tr><tr><td>RNase-Free DNase</td><td>Promega</td><td>M6101</td><td></td></tr><tr><td>Vero Cell Line&amp;nbsp;</td><td>ATCC</td><td>CCL-81</td><td></td></tr><tr><td>Zika viral strain PRVABC59</td><td>Centers for Disease Control and Prevention (CDC)</td><td></td><td></td></tr><tr><td>IL-6</td><td>Peprotech</td><td>200-06&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>IL-1 alpha&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Peprotech</td><td>200-01A&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>TNF-alpha</td><td>Peprotech</td><td>300-01A&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Interferon alpha A&amp;nbsp;&amp;nbsp;</td><td>R &amp;amp; D Systems</td><td>11100-1</td><td></td></tr><tr><td>Interferon beta</td><td>Peprotech</td><td>300-02BC</td><td></td></tr><tr><td>Interferon gamma</td><td>Peprotech</td><td>300-02</td><td></td></tr><tr><td>Centrifuge 5415R</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Nikon Eclipse Ti Immunofluorescence Microscope with Nikon Intenselight C-HGFI</td><td>Nikon</td><td>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。寨卡病毒感染的细胞病变效应（CPE）的特征在于，形态学改变，如细胞的舍入和脱离以及裂解细胞死亡。 用于测量病毒滴度，在T-160烧瓶收获含有病毒的无细胞上清液进行10 FOLð系列稀释，并在12孔板形式加入到Vero细胞上的单层（ 图3）。在48小时后的接种时间点作为终点，以避免计数从继发感染引起的斑块。与30-100噬斑的孔被选择用于计数，以获得准确的滴度。 RT-qPCR分析被用于估计寨卡病毒的基因组中复制。具体为高度保守ZIKV NS5区泛基因引物对被列入3,16。基于泛基因型引物的基因组位点兹卡病毒株特异性PRVABC59引物（引物序列在该协议第4.4.2节中给出）进行了测试。两个引物对显示在从感染的细胞（ 图4）放大寨卡病毒基因组的灵敏度水平相似。作为阴性对照，丙型肝炎病毒特异性的引物也进行了测试17。一个显著增量缓解ZIKV病毒基因组的含量无论是在低和高MOI感染的细胞，这表明强大的病毒感染（ 图4）48和96小时时间点之间观察到。在黄病毒基因组的复制，由有义和反义基因组RNA之间形成碱基配对的生成双链（DS）的RNA replicatory中间体。具体地，用于识别双链RNA的抗体探针检测病毒感染的细胞（ 图4）。病毒dsRNA被定位于细胞质暗示基因组复制的室中。免疫组化染色显示在48和96小时的时间点病毒感染细胞的扩张。 使用传染性寨卡病毒培养系统，细胞因子的小文库进行筛选，以确定抗病毒活性（ 图5）。将细胞用各种细胞因子预处理12小时，然后感染寨卡病毒。我型我<html> nterferon，IFN-α，在广泛的范围内进行测试的剂量[10国际单位（IU），100 IU和1,000 IU]的表现出针对寨卡病毒强的抗病毒活性。 IFN-β的表现出强的抑制效果为好。 II型IFN-γ也证明抗寨卡病毒活性。细胞因子TNF-α，IL-1和IL-6的没有在指定的药物浓度抑制ZIKV。 <img alt="图1" src="/files/ftp_upload/54767/54767fig1.jpg" /> 图 1: 寨卡病毒的生产和试验工作流程的一般轮廓对于病毒生产，ZIKV被接种到Vero细胞中的T-160组织培养瓶中。以48或96 HPI，无细胞病毒上清收获并储存在-80℃用于下游分析。病毒滴度通过有限稀释测定法测定的。的寨卡病毒接种物随后用于研究病毒复制动力学和筛选药物化合物。HREF ="htt​​ps://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54767/54767fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54767/54767fig3.jpg" /> 图 3: 用于测定寨卡病毒产生菌斑测定幼稚Vero细胞被用于测量病毒滴度。亮场图像描绘在不同的稀释液（比例尺= 50微米）的病毒噬斑的密度。注:1:100稀释的图像显示，可以准确地计算不同斑块<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54767/54767fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54767/54767fig4.jpg" /> 图4: 用于评估寨卡病毒复制测定法。 </strong&gt;（A）图表显示通过RT-qPCR的测定寨卡病毒基因组的内容。既泛基因型（ZIKV GEN）和PRVABC59应变（ZIKV UNI）的引物显示出相等的敏感性和特异性。正如预期的那样，丙型肝炎病毒（HCV）的引物没有扩增任何产物。 （B）的图形呈现在指定的时间点在低和高滴度感染细胞的寨卡病毒生长动力学相比，在未感染的模拟控制。平均值和标准偏差给出。 （ 三 ）免疫细胞化学法检测调查病毒复制。寨卡病毒感染的细胞特异性地与双链RNA抗体染色。在48 HPI，感染的细胞是在几簇，而在96 HPI大部分细胞被感染。用于核的可视化（比例尺= 50微米）的Hoechst染色。 BF:明亮场图像<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54767/54767fig4large.jpg" target="_blank">，请点击此处查看该图的放大版本</a>。 <img alt="图5" src="/files/ftp_upload/54767/54767fig5.jpg" /> 图 5: 干扰素证明有效的抗寨卡病毒活性 （A）的图表表示寨卡病毒生长的调制通过各种细胞因子相比，车辆控制的。平均值和标准偏差示于条形图。干扰素显示寨卡病毒的复制（P &lt;0.05），统计学显著抑制作用。 （B和C）具有或不具有细胞因子治疗寨卡病毒感染的细胞的相衬图像。由感染细胞形成的病毒噬斑可以被看作是病灶。需要注意的是干扰素处理的孔不具有病毒噬斑。没有ZIKV感染模拟对照包括作为阴性对照。 （比例尺= 50微米）， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54767/54767fig5large.jpg" target="_blank">请点击这里查看大图</a>版本这个数字。

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

Nanyang Technological University

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

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

科研

发育生物学

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 ...

免疫与感染

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 ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

神经科学

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 ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

高通量抗病毒检测筛查寨卡病毒复制抑制剂

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

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://app-jove-com.bdigitaluss.remotexs.co/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 ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

Zika Virus Infection of Fetal Neural Stem Cells

Source: McGrath, E. L., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56917">Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.</a> J. Vis. Exp. (2018)
This video demonstrates a procedure to infect human fetal neural stem cells with Zika virus. The infected cells can be used for further studies.

Source: McGrath, E. L., et al., Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis. J. Vis. Exp. (2018)
...

兹卡病毒感染细胞培养系统和

摘要

探索更多视频

此视频中的章节