We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.

该协议的一个大致轮廓示于图1。 1。细胞<ol><li>制备包含10〜15％的胎牛血清（FBS），10mM的非必需氨基酸，10mM的HEPES，青霉素（100单位/ ml），链霉素（100毫克/毫升），和2mM L-谷氨酰胺的完全生长培养基。 </li><li>在包含用于体外分析丙型肝炎病毒复制周期的上述补充完全生长培养基维持咦-7.5.1细胞13。 </li><li>培养的病毒株在Huh-7.5.1细胞用指定的补充生长培养基，在37℃，5％的CO 2。 </li></ol> 2，病毒和质粒构建<ol><li>产生的互补DNA（cDNA）形式的HCV RNA，并克隆至为便于基因操作的质粒​​载体。注：日本的暴发性肝炎1的发现，JFH-1（基因型2a丙型肝炎病毒），隔离一直为HCV研究的关键，并且是主要用于研究丙型肝炎病毒复制周期11-13。根据JFH-1 HCV间和intragenotypic嵌合病毒常用于研究14-18。合成intragenotype 2A嵌合病毒，FNX-HCV，和一个单顺反子嵌合记者株的构建先前19和施工细节超出了本协议的范围被描述。 </li><li>使用pFNX - HCV intragenotype 2a中的病毒，因为它包含一个5&#39;NTR，结构区和P7和NS2的非结构区的一部分（核苷酸1到2878）的J6CF亲本菌株（NCBI登录号AF177036）的，如以及JFH-1病毒株（NCBI登录号AB047639）的非结构部件。注：合成FNX-HCV的基础上，JC1 intragenotype 2A嵌合HCV以前的序列15日报道。 </li><li>产生单顺反子的嵌合记者病毒构建体中，pFNX-的Rluc的基础上，pFNX-HCV株（以上步骤2.2）通过插入A R5&#39;NTR和核心基因（新台币之间的388和389）之间enilla荧光素酶基因。接着，用口蹄疫病毒2A（F2A）的肽序列，这将作为一个卵裂信号连接的荧光素酶基因和该构建体的核心基因。 </li><li>工程师RNA聚合酶空（POL-）病毒构建使用无论是pFNX-HCV或pFNX，在Rluc病毒的背景。更换NS5B聚合酶催化残基，GDD（AA 2759年至2761年; nt的8615-8623）任一这些病毒骨架与AAG氨基酸残基的。 </li></ol> 3， 在体外 HCV RNA转录<ol><li>使用intragenotype 2A嵌合病毒FNX-HCV和FNX-HCV聚合酶空（POL-）对丙型肝炎病毒复制（ 图2A）的评价。 </li><li>线性化病毒质粒用XbaI限制性内切酶，然后用绿豆核酸酶，产生平末端处理。通过阴离子交换层析纯化经消化的质粒。验证完整性Øf通过对DNA的琼脂糖凝胶电泳（ 图2B）的线性化的质粒。 </li><li>用T7-RNA聚合酶转录的线性化病毒质粒。 </li><li>净化使用RNA纯化试剂盒新合成的DNA酶处理的RNA。 </li><li>通过琼脂糖凝胶电泳（ 图2C）验证RNA的生产。 </li><li>用分光光度法定量的RNA。 </li><li>存储所生成的RNA在-80℃下在10微克工作等分试样。注意：要通过转录RNA的一切对每个病毒样本和控制，同时每个实验的设计中尽量减少RNA质量的变化。 </li></ol> 4，丙型肝炎病毒RNA的转染和样品采集<ol><li>使用胰蛋白酶收获的Huh-7.5.1细胞。 </li><li>冲洗细胞，离心并用冷低血清培养基悬浮两次暂停。重悬的细胞在低血清培养基中以每毫升1×10 7个细胞。 </li><li>混合总共10＆＃956;克的转录病毒RNA在一个0.4厘米电穿孔杯中加入400μl重悬的细胞（4×10 6个细胞）。通过电穿孔在270 V转染的细胞中，100Ω和950μF。 </li><li>重悬的电穿孔的细胞在10ml完全生长培养基含15％FBS中。注：电穿孔的细胞的生存力增加被认为是当的Huh-7.5.1细胞在15％胎牛血清中培养。 </li><li>镀在两个T-25培养瓶中（〜1.2×10 6每烧瓶中的细胞）和48孔板（1×10 4个，每孔细胞）为下列每个时间点的细胞：4，48和96小时。 </li><li>有10％FBS更换介​​质在4-8小时转染后用新鲜补充的生长介质，以从培养瓶和板去除死细胞碎片。 </li><li>收获细胞培养物上清液在48中，96小时的时间点，并从采集的样品通过细胞离心4去除细胞碎片在1,500 rpm离心10分钟 ºC。 </li><li>存储该无细胞上清液于-80℃。裂解细胞用于蛋白和RNA的分析用Western印迹和逆转录 - 定量聚合酶链反应在指定的时间点。 </li></ol> 5，反转录定量PCR（RT-qPCR的），以评估丙型肝炎病毒基因组复制<ol><li>执行两步RT-qPCR的确定HCV基因组RNA的拷贝数。 </li><li>反转录1使用逆转录酶和其结合5&#39;NTR（JFH RTQ注：5&#39;CCTATCAGGCAGTACCACA-3&#39;）具体为HCV义链的引物细胞总RNA微克或特异于持家基因肽基异构酶G（PPIG R A引物：5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;）。还逆转录使用HCV义链引物的已知基因组拷贝（10月1日至十月九日标准）的FNX-HCV RNA（在步骤3中生成的）。 </li><li>开展定量PCR用50纳克导致转录的cDNA用HCV特异性引物（JFH RTQF：5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ记：5&#39;CCTATCAGGCAGTACCACA-3&#39;）和DNA结合含有定量PCR超级混合绿色染料。进行定量PCR的看家基因PPIG以及（PPIG引物F：5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3&#39;; PPIG R：5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;）注：为了计算准确的细胞内HCV RNA水平跨越样品，用细胞看家基因，PPIG ，表达水平扫描（CT周期）进行归一化。使用FNX-HCV基因组的拷贝数作为拷贝数测定标准。 </li><li>使用以下条件时，运行定量PCR来确定HCV RNA拷贝数：95ºC为15秒和60 &#160; ºC为30秒（40个循环），使用实时PCR系统。 </li><li>参见图3A和图3B为基因组复制的结果。 </li></ol> 6，Western印迹分析检测的HCV蛋白的表达（图3C） <ol><li>用细胞裂解液FROM病毒RNA转染96小时后转染蛋白免疫印迹分析。 </li><li>使用SDS-PAGE，并转​​移到聚偏二氟乙烯（PVDF）膜，解决了细胞裂解物。 </li><li>用含5％脱脂乳，0.2％吐温20的磷酸盐缓冲盐水（PBS）封闭液阻断膜。 </li><li>孵育膜与初级小鼠单克隆抗体NS3（1千稀释）和β-肌动蛋白（1 5000稀释）。 </li><li>添加山羊抗小鼠IgG缀合至辣根过氧化物酶（1 5000稀释）和通过化学发光检测。 </li></ol> 7，免疫荧光法（IFA） <ol><li>用甲醇30分钟-20ºC的免疫荧光法修复HCV感染和转染的细胞。 </li><li>用PBS洗细胞3次，用IFA阻断缓冲液阻断。 </li><li>用兔多克隆抗NS5A主要抗体或鼠单克隆抗-DSR（由达斯古普塔博士，加州大学洛杉矶分校友情提供）NA抗体J2的稀释度为1:200（1微克/毫升）并孵育5小时至过夜，4 ºC。 </li><li>初级抗体后，3次，用PBS洗涤细胞。 </li><li>添加山羊抗兔IgG-488多克隆第二抗体或山羊抗小鼠IgG-594多克隆第二抗体以1:1000的稀释度（1微克/毫升），并孵育1小时，在室温下进行。 </li><li>用PBS使用荧光显微镜（ 图3D）应用Hoechst染料和视图3倍和染色细胞核洗细胞。 </li></ol> 8，测量病毒滴度<ol><li>板幼稚的Huh-7.5.1细胞以约3×10 3个细胞/井使用一个96孔板中。第二天，进行无细胞培养上清液中使用的生长培养基的HCV RNA转染的细胞中收获的10倍系列稀释液，并一式三份接种到的Huh-7.5.1细胞。 </li><li>用甲醇（30分钟，在-20℃固定细胞72小时后的感染</suP&gt;ºC）和在第7节所列免疫染色为HCV NS5A蛋白。 </li><li>使用的最高稀释度来计算的NS5A阳性病灶和每毫升计算焦点形成单元的平均数目（FFU）。参见图4为HCV滴度。 </li></ol> 9。 海肾萤光素酶报告基因检测为病毒基因组复制和传染性<ol><li>用于评价病毒基因组的复制，板的HCV RNA电穿孔的Huh-7.5.1细胞一式三份在48孔板（1×10 4个细胞/孔）。 </li><li>裂解的细胞与75微升被动裂解缓冲液中在6小时，48小时和96小时后转染。 </li><li>摇动板轻轻地为15分钟，在室温下储存于-80℃。 </li><li>来测量海肾荧光素酶活性，解冻的蛋白质裂解物和海肾萤光素酶测定试剂至室温。 </li><li>加入20微升每孔蛋白裂解液96-WEL的<html> L白色发光板并将板光度计。 </li><li>分配100μl的海肾荧光素酶测定试剂（腔肠素底物+缓冲液中），每孔并经过2秒预读取的延迟;结合所发出的光10秒。 </li><li>从一式三份生物的萤光素酶值计算出平均值和标准偏差对每个样品和经受的数据进行统计分析（ 图5）。 </li><li>用于评估感染性，接种500微升无细胞上清液从HCV RNA转染的细胞在指定的时间点（48小时和96小时），一式三份在48孔板中获得到幼稚的Huh-7.5.1细胞。 </li><li>用500μl新鲜培养基后，每井6小时后更换感染病毒接种。 </li><li>溶胞物与步骤8.2至8.7（ 图5）中所述的细胞在48小时后的感染和受海肾萤光素酶测定。 </li></ol>书名“&gt; 10。统计分析<ol><li>在该图中的误差棒表示标准偏差（SD）。对P值的计算采用非配对t检验。 </li></ol>

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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=Hepatitis+C+virus+p7+and+NS2+proteins+are+essential+for+production+of+infectious+virus.">Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus.</a> J Virol. 81 (16), 8374-8383 (2007).</li><li>Steinmann, 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=Hepatitis+C+virus+p7+protein+is+crucial+for+assembly+and+release+of+infectious+virions.">Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.</a> PLoS Pathog. (7), (2007).</li></ol>

The authors have nothing to disclose.

此图描述了用于分析丙型肝炎病毒复制循环的方法。丙型肝炎病毒是一种人类病原体和规定的生物安全议定书将必须严格遵守。感染性HCV细胞培养系统之前已经11-13,16,17说明。有下列图示的协议，当我们实现几个关键点。首先，它是非常重要，以有完整的全长病毒基因组RNA质量好于下游的研究。输入质粒携带的病毒cDNA要进行线性化处理，并仔细减弱。的绿豆核酸酶来生硬的Xba I位悬...

丙型肝炎病毒（HCV）导致肝硬化和肝癌。它影响1.7亿人在全球拥有35万人，每年死于1-3。 HCV是正链RNA病毒为9.6 kb的基因组大小。 HCV基因组被翻译成的〜3000氨基酸残基被蛋白水解由各种细胞和病毒蛋白酶裂解成10多肽的单个蛋白。 HCV是原型病毒属中的肝炎病毒，属于黄病毒科 4。曝光后，建立了丙型肝炎病毒慢性感染者中80％的个体。这种感染大多是无症状的，及时的诊断可以允许治疗性干预，以防止肝恶化。目前的治疗是不理想的，没有疫苗可用5,6。 丙型肝炎的病因在1989年7年首次描述。学习丙型肝炎病毒复制是丙型肝炎的疫苗和治疗研究的重要，但它一直由缺少一种有效的病毒培养体系的长期阻碍。丙型肝炎病毒的分子克隆被证明是在感染后肝内接种8只黑猩猩。随后，HCV亚基因组复制子进行了说明，其中允许解剖病毒基因组的复制阶段中的细胞培养系统9,10。一个基因型2a丙型肝炎病毒的发现隔离JFH-1（日本暴发性肝炎-1），能够感染细胞培养中打开丙型肝炎病毒复制的研究11-13新途径。基因型2a株JFH-1基于跨内和基因型嵌合病毒基因型1丙型肝炎病毒感染的基础培养体系可作为以及14-18。 我们已成功地使用JFH-1株和HCV intragenotype 2a中的嵌合病毒，得到蛋白质结构域和顺式作用RNA元件19,20的高分辨率官能剖析图。根据这一点，我们在这里描述的有效培养系统经常使用的，允许研究HCV复制周期和宿主 - 病原体相互作用的不同阶段。我们目前的病毒学检测，以评估病毒基因组的复制和intragenotype 2A丙型肝炎病毒和海肾萤光素酶报告基于 ​​丙型肝炎病毒新发感染。

<table border="0" cellpadding="0" cellspacing="0" width="1121">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Name of Material/ Equipment</td>
			<td style="width:267px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Non essential amino acid</td>
			<td style="width:267px;">Fisher Scientific</td>
			<td style="width:119px;">MT25025CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">HEPES</td>
			<td style="width:267px;">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:267px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Opti-MEM Reduced Serum Medium,no Phenol Red</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">11058-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Huh-7.5.1</td>
			<td style="width:267px;">The Scripps Research Institute</td>
			<td style="width:119px;"></td>
			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:569px;">Plasmids (pFNX-HCV, pFNX-HCV Pol null, pFNX-Rluc, and pFNX-Rluc Pol null)&#160;</td>
			<td style="width:267px;">Cedars-Sinai Medical Center</td>
			<td style="width:119px;"></td>
			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">XbaI</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Mung Bean Nuclease</td>
			<td>New England Biolabs Inc.</td>
			<td style="width:119px;">M0250S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">T7 RiboMAX Express Large Scale RNA Production System</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">P1320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Rneasy Mini Kit</td>
			<td style="width:267px;">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:267px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Electroporation Cuvette (4 mm)</td>
			<td style="width:267px;">Bioexpress</td>
			<td>E-5010-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Gene Pulser Xcell Total System</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">165-2660</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 488</td>
			<td style="width:267px;">Life Technologies</td>
			<td>A11008</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:267px;">Life Technologies</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVDF membrane package</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">162-0263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Blotting Grade Blocker Non Fat Dry Milk</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6404XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">Tween-20</td>
			<td style="width:267px;">Bio-Rad</td>
			<td style="width:119px;">170-6531XTU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [8 G-2]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab65407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Hepatitis C Virus NS3 antibody [H23]</td>
			<td style="width:267px;">Abcam</td>
			<td style="width:119px;">ab13830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td style="width:119px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Prime Western Blotting Detection Reagents&#160;</td>
			<td style="width:267px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">SUPERSCRIPT III RT&#160;</td>
			<td style="width:267px;">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:267px;">Life Technologies</td>
			<td style="width:119px;">11744500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ViiA 7 real-time PCR system</td>
			<td style="width:267px;">Life Technologies</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla Luciferase Assay System kit</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">RNase-Free DNase</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;">M6101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:569px;">GloMax-Multi Detection System (Luminometer)</td>
			<td style="width:267px;">Promega</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for analyzing hepatitis c virus replication

丙型肝炎病毒（HCV）影响世界人口的3％，并导致严重的肝脏疾病，包括慢性肝炎，肝硬化和肝细胞癌。丙型肝炎病毒是属于黄病毒科的包膜RNA病毒。目前的治疗是不充分有效的，并导致不利的副作用。没有丙型肝炎病毒疫苗可用。因此，需要开发一种疫苗和更好的治疗持续的努力。的HCV细胞培养体系是研究HCV生长的不同阶段，包括病毒进入，基因组复制，包装和出口的关键。在当前的程序提出，我们用野生型intragenotype 2a中的嵌合病毒，FNX-HCV和重组FNX-的Rluc病毒携带的Renilla荧光素酶报道基因来研究病毒的复制。一个人肝癌细胞株（Huh-7型）用于体外转录转染丙型肝炎病毒基因组RNA的。无细胞培养物上清液，蛋白裂解液和叔OTAL核糖核酸收获的不同时间点转染后，以评估HCV生长。丙型肝炎病毒基因组的复制状态是由定量RT-PCR和可视化HCV的存在双链RNA进行评估。丙型肝炎病毒蛋白的表达，用特异性HCV NS3和NS5A蛋白的抗体，Western blot和免疫荧光试验验证。感染性颗粒释放到培养上清液中和病毒滴度的HCV RNA转染的细胞进行测定。荧光素酶检测被用于评估记者丙型肝炎病毒的复制水平和传染性。总之，我们提出的各种病毒学检测表征丙型肝炎病毒复制周期的不同阶段。

丙型肝炎病毒是一种RNA病毒。因此，对于遗传操作的目的，所述HCV基因组cDNA已被克隆到细菌质粒载体。甲T7 RNA聚合酶启动子序列立即被引入HCV基因组的5&#39;末端之前。 HCV的分析工作流程的一般概述示于图1，生成HCV基因组RNA具有精确的3&#39;末端含有质粒HCV基因组被切割用 XbaⅠ限制性内切酶和所产生的单链突出端补平用绿豆核酸酶消化。线性化的质粒的HCV的质量通过琼脂糖凝胶?...

Watch this Scientific Journal Video about A Protocol for Analyzing Hepatitis C Virus Replication at JoVE.com

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

一个协议，用于分析丙型肝炎病毒复制

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

a-protocol-for-analyzing-hepatitis-c-virus-replication

Research

JoVE Journal

Immunology and Infection

23.9K 次观看.  Cedars-Sinai Medical Center. Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle. 

视频: A Protocol for Analyzing Hepatitis C Virus Replication

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

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 ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

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 ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

乙型肝炎病毒感染原代肝细胞和库珀细胞的 "肝片上" 培养

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...