作者要感谢詹姆斯博士（张志坚）提供必要的试剂，包括小牛- / -小鼠，并任日（美国加州洛杉矶大学，药理学和分子医学博士陈（UT西南，分子生物学） ）提供的细菌人工染色体的γHV68本研究。

1。滑鼠感染γHV68功能<ol><li>六到八周，性别相匹配的旧窝小鼠（8〜12只/组），用于病毒性感染。让小鼠适应环境装船后四天（96小时）。 </li><li>使用病毒协议的步骤应进行在一个内阁生物安全2级（BSL2）的标准BSL2注意事项。 </li><li>准备病毒悬浮液（40〜1×10 5个噬斑形成单位[PFU]）在30μl的无菌PBS中每只小鼠的γHV68刚刚之前的实验。保持病毒悬挂在冰上。 </li><li>准备氯胺酮/甲苯​​噻嗪溶液（1.5毫克氯胺酮和0.15mg xylazine/20的g体重，100微升/鼠标）鼠标镇静。确保通过执行脚趾夹，老鼠被注射镇静剂。 </li><li> 1.5毫克的氯胺酮和0.15毫克每20毫克体重（100微升/小鼠）腹膜内注射甲苯噻嗪稳重的小鼠。 </li><li>提供病毒悬浮液（30μL/鼠标）滴鼻，在逐滴时尚，镇静小鼠的一个鼻孔。 </li><li>莱小鼠一侧为5 - 10分钟，以方便进入肺部的气道传送的病毒。 </li><li>地点小鼠背部在笼子和显示器小鼠，直到他们完全恢复镇静。 </li><li>在感染后不同天数，牺牲小鼠CO 2窒息和收获后肺保证死亡/亏损的深层意识。将肺部进入无菌1.5毫升含500微升1.0毫米氧化锆/二氧化硅珠旋盖管。保持管在冰上。店内样品在-80°C，如果不是在同一天进行下一步。脾或肝组织可以被收集在这个时候，根据实验的时间帧的分析病毒潜伏期。 </li><li>到管中加入1毫升的冷的无血清DMEM，通过珠跳动30秒的肺部均匀。冷藏至少1 min，管在冰上。一旦重复此过程。 </li><li>裂解液在4℃下以16,000相对离心力离心机肺，持续1分钟，并使用supernatant，以确定病毒滴度的空斑法，使用NIH3T3或BHK21单层（有关详细信息，参见第5章）。 </li></ol> 2。确定γHV68多步生长动力学，在小鼠胚胎成纤维细胞<ol><li>生长的野生型小鼠胚胎成纤维细胞（MEF中）和在宿主基因子汇合电镀前细胞（约80％）密度的缺陷。 </li><li>斯普利特的MEF到24孔板上以10,000细胞/孔的低multipilicity感染复数（MOI）感染的前一天。实验通常进行重复三次，以MOI之间0.001 - 0.05通常使用。 </li><li>准备γHV68悬浮液所需的病毒量（0.5毫升每口井）。 </li><li>取出介质并覆盖的MEF用0.5毫升的悬浮液，每孔γHV68。 </li><li>在组织培养的孵化器，岩石，每30分钟孵育板，并允许潜伏期为2小时进行。 </li><li>删除病毒悬浮液，并覆盖细胞用0.5毫升新鲜的Çomplete含有8％胎牛血清的DMEM培养基中。 </li><li>在不同的感染后，收获的介质和细胞进入无菌1.5毫升的离心管中。立即冻结管在-80°C。 </li><li>发布γHV68由MEF在-80°C冷冻，解冻，在37℃水浴和涡旋。冷冻和解冻的三个周期通常是施加到样品。 </li><li>确定病毒滴度的空斑法，使用NIH3T3或BHK21单层（有关详细信息，参见第5章）。 </li><li>病毒滴度和情节γHV68多步生长曲线的MEF中。 </li></ol> 3。 γHV68在小鼠胚胎成纤维细胞的裂解性复制的分子剖析<ol><li>执行病毒感染中描述的步骤2.1到2.6的第2条。 </li><li>在不同的感染后，弃去上清液。冲洗，与冷PBS和trypsinize的细胞的细胞。离心机在1000相对离心力在室温1分钟沉淀细胞。弃去上清并存储细胞在-80℃下</li><li>总DNA的提取（主机和病毒基因组），和总RNA，按照原样的方法5,6。 </li><li>执行实时PCR使用的总DNA和病毒裂解转录，如RTA（ORF50），ORF57和ORF60的特异性引物。确定细胞内γHV68基因组的参考到主机看家基因（例如，β-肌动蛋白）的相对数量。 </li><li>若要去除基因组DNA污染，用无RNA酶的DNA酶处理的总RNA和寡聚（dT）11-19引物的cDNA制备的关键。无RNA酶的DNA酶和RT-PCR的RNA提取，包括处理有关详细信息，请参阅参考文献5。执行实时PCR用cDNA和引物与上述确定参照主机管家基因的病毒转录物的相对数量。 </li></ol> 4。 ，产生重组γHV68使用细菌人工染色体该methoð这里描述的是用来引入突变成γHV68涉及的基因，该基因在宿主 - 病毒相互作用。 <ol><li>准备的野生型序列或序列中的中央区域通过PCR携带所需突变的DNA片段（约1.5 kb）。 </li><li>准备的细菌人工染色体（BAC），其中包含一个转座子插入7周围的突变位点，其中特别感兴趣的基因的失活的基因，通过的MIDI规模纯化（OriGene）。商店BAC DNA在4°C（避免冻结/解冻的BAC DNA）。 </li><li> BAC DNA转染和PCR产物的细胞（例如，NIH3T3或BHK21），它是高度宽容到γHV68裂解性复制，与Lipofectamine 2000（Invitrogen公司）的感兴趣的基因的含有所需的突变。 </li><li>查看分裂细胞直到细胞病变效应（CPE）显示在单层。收集含病毒上清液，如果有必要，在NIH3T3或BHK21细胞扩增病毒。 </li><li>感染NIH3T3细胞与重组病毒通过离心转速为1,800 rpm，30℃30分钟。 </li><li>收获γHV68感染的NIH3T3细胞和准备使用希尔特的协议8,9内，病毒基因组。 </li><li>变换电MAX DH10B感受态细胞（Invitrogen公司），通过电穿孔和屏幕为是抗氯霉素（Cm），但对卡那霉素（Kan）的（Cm的抗性基因是在BAC骨干，而侃抗性基因是在转座子敏感的菌落插入）。 </li><li>成长厘米ŕ简小号的殖民地的培养基中chlorophenicol，准备BAC DNA与小或MIDI规模的净化（OriGene）。 </li><li>通过PCR验证所需的靶基因中的突变，使用特异性引物侧翼区的突变位点，和测序。 </li><li>在BAC与选定的限制性内切酶消化和脉冲场凝胶电泳确认没有染色体重排。 </li><li>用脂质体转染到NIH 3T3 BHK21细胞重组BAC2000（Invitrogen公司）准备重组γHV68的。 </li><li>继续传代的细胞，直到CPE在单层。收集含有病毒的上清和扩增重组γHV68在NIH3T3或者BHK21细胞。 </li><li>确定重组病毒的滴度和表征病毒的生长特性体外和体内部分1和2中所描述。 </li></ol> 5。确定病毒滴度的空斑法<ol><li>子汇合（约80％）密度电镀前细胞NIH3T3细胞成长。 </li><li>斯普利特NIH3T3到24孔板上，以20,000细胞/孔感染的前一天。 </li><li>准备10倍系列稀释的病毒上清液的DMEM培养基中含有8％新生小牛血清（NCS）。 </li><li>取出介质并覆盖NIH3T3细胞用0.5毫升γHV68悬浮。 </li><li>组织培养箱中孵育板，岩石，每30分钟，让孵化进行2小时。 </li><li>删除病毒悬浮液和盖细胞，用0.5毫升的DMEM培养基中含有2％NCS和0.75％的甲基纤维素（Sigma公司）。 </li><li>在组织培养箱孵育板。在感染后第6天，计算斑块。染色的单层，例如用结晶紫，促进斑块计数。 </li><li>计算在未稀释的组织裂解液中的病毒滴度或细胞裂解物使用下面的公式：效价（PFU /毫升）= D×N个×1000微升/毫升÷V微升。 N，斑数目的平均值，在适当的稀释，D，稀释倍（如5，10，100 .....）V（微升），串联稀释的上清液的体积每孔添加。 </li></ol> 6。代表性的成果： 三个有代表性的数字显示在这里，包括裂解γHV68复制在肺中的野生型和小牛- / -小鼠10，γHV68裂解复制在小鼠胚胎成纤维细胞（MEF）的表型，和尿激酶原蚂蚁γHV68内是调制的的MAVS依赖的IKKβ的磷酸化位点，携带突变。这三个确凿的实验构成了一项计划，定义角色的先天免疫成分的γHV68 在体内和体外的裂解性复制。 <img alt="图1" src="/files/ftp_upload/3140/3140fig1.jpg" /> 图1， 小牛+ / +和小牛- / -小鼠的肺中。γHV68负载。 A）的两个主要与生俱来的信号通路下游的MAVS。 MAVS的的接头分子继电器信号从胞质RIG-I样受体激活NFκB和干扰素调节的因素（IRFS）的，反过来，上调控基因表达的炎性细胞因子和干扰素。 B） 小牛+ / +和小牛- / -小鼠40 PFUγHV68鼻内和病毒载量在肺感染在指定的时间点被阻止<html>的斑块检测NIH3T3单层开采。每个符号代表一个鼠标。 <img alt="图2" src="/files/ftp_upload/3140/3140fig2.jpg" /> 图2。γHV68裂解性复制动力学在小鼠胚胎成纤维细胞（MEF中）。对小牛队的裂解性复制的γHV68+ / +和小牛- / - MEFs细胞多步生长曲线（A）和实时定量PCR（B）进行了评估。对于这两个实验中，MEFs和相等数量的量γHV68用于病毒感染的多重感染（MOI）为0.01。 （A）在指定的时间点收获细胞和上清液的牌匾试验，以确定病毒滴度。 （B）从γHV68感染的MEF中提取总RNA，实时定量PCR为选定的裂解成绩单（RTA，ORF57和ORF60）的特异性引物，进行分析。 ig3.jpg“/&gt; 图3。裂解性复制动力学重组γHV68RTA的转录域的突变，取消磷酸化IKKγ。 （A）多步生长曲线的重组野生型病毒（γHV68.wt）和突变型病毒（γHV68.mut）在小牛队+ / +和小牛- / -的MEF细胞（MOI = 0.01）。的MEF感染γHV68MOI为0.01。收集细胞和上清液，在指定的时间点，和由使用NIH 3T3细胞单层中的噬斑分析测定病毒滴度。 （B）γHV68RTA mRNA水平在的NIH3T3细胞γHV68感染（MOI = 0.01）。在感染后30小时，总RNA，从γHV68感染的NIH 3T3细胞中提取，并通过实时定量PCR分析。

<ol>
	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124, 783-801 (2006).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+microorganisms+and+activation+of+the+immune+response.">Recognition of microorganisms and activation of the immune response.</a> Nature. 449, 819-826 (2007).</li><li>Speck, S. H., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+viral+genetics+of+chronic+infection:+a+mouse+model+of+gamma-herpesvirus+pathogenesis.">Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.</a> Curr. Opin. Microbiol. 2, 403-409 (1999).</li><li>Speck, S. H., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+latency+and+its+regulation:+lessons+from+the+gamma-herpesviruses.">Viral latency and its regulation: lessons from the gamma-herpesviruses.</a> Cell Host Microbe. 8, 100-115 (2010).</li><li>Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+gamma-herpesvirus+68+hijacks+MAVS+and+IKKbeta+to+initiate+lytic+replication.">Murine gamma-herpesvirus 68 hijacks MAVS and IKKbeta to initiate lytic replication.</a> PLoS Pathog. 6, e1001001-e1001001 (2010).</li><li>Strauss, W. M., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+genomic+DNA+from+mammalian+tissues.">Preparation of genomic DNA from mammalian tissues.</a> Current Protocols in Molecular Biology. , 2-2 (1998).</li><li>Song, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+viral+genes+essential+for+replication+of+murine+gamma-herpesvirus+68+using+signature-tagged+mutagenesis.">Identification of viral genes essential for replication of murine gamma-herpesvirus 68 using signature-tagged mutagenesis.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 3805-3810 (2005).</li><li>Hirt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extraction+of+polyoma+DNA+from+infected+mouse+cell+cultures.">Selective extraction of polyoma DNA from infected mouse cell cultures.</a> J. Mol. Biol. 26, 365-369 (1967).</li><li>Eva-Maria Borst, E., Crnkovic-Mertens, I., Messerle, M., Zhao, S., Stodolsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+&#946;-herpesvirus+genomes+as+bacterial+artificial+chromosomes.">Cloning of &#946;-herpesvirus genomes as bacterial artificial chromosomes.</a> Methods in Molecular Biology. , 256-256 (2004).</li><li>Sun, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specific+and+essential+role+of+MAVS+in+antiviral+innate+immune+responses.">The specific and essential role of MAVS in antiviral innate immune responses.</a> Immunity. 24, 633-642 (2006).</li><li>Seth, R. B., Sun, L., Ea, C. K., Chen, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+MAVS,+a+mitochondrial+antiviral+signaling+protein+that+activates+NF-kappaB+and+IRF+3.">Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.</a> Cell. 122, 669-682 (2005).</li><li>Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPS-1,+an+adaptor+triggering+RIG-I-+and+Mda5-mediated+type+I+interferon+induction.">IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.</a> Nat. Immunol. 6, 981-988 (2005).</li><li>Meylan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardif+is+an+adaptor+protein+in+the+RIG-I+antiviral+pathway+and+is+targeted+by+hepatitis+C+virus.">Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.</a> Nature. 437, 1167-1172 (2005).</li><li>Xu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VISA+is+an+adapter+protein+required+for+virus-triggered+IFN-beta+signaling.">VISA is an adapter protein required for virus-triggered IFN-beta signaling.</a> Mol. Cell. 19, 727-740 (2005).</li></ol>

没有利益冲突的声明。

在对病毒感染的反应，MAVS依赖先天免疫信号通路的激活，促进了生产的抗病毒炎性细胞因子10-14。作为模型病毒对人类的致癌性卡波济氏肉瘤相关疱疹病毒，EB病毒3,4使用鼠γHV68的，我们发现，γHV68侵占MAVS-IKKβ的途径，通过促 ​​进病毒裂解性复制转录激活5。采用转基因MEFs和技术在分子病毒学，该协议可以有效识别的信令组件的一个特定的途径是至关重要的γHV68裂解性?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>脂质体2000 </td><td> Invitrogen公司</td><td> 11668-019 </td></tr><tr><td> ELECTRO-MAX DH10B感受态细胞</td><td> Invitrogen公司</td><td> 18290-015 </td></tr><tr><td>甲基纤维素</td><td>西格玛</td><td> M0512 </td></tr><tr><td> POWERPREP HP质粒小提系统</td><td> OriGene </td><td> NP100004 </td></tr><tr><td> POWERPREP HP质粒Midiprep系统</td><td> OriGene </td><td> NP100006 </td></tr></tbody></table>

dissecting host-virus interaction in lytic replication of a model herpesvirus

在对病毒感染的反应，主机开发各种防御反应，激活先天免疫信号转导途径，导致抗病毒细胞因子的产生1,2等。为了殖民主机，是专病毒逃避宿主抗病毒反应和处理信号转导通路。揭开宿主 - 病毒相互作用，揭示了对抗病毒感染的新的治疗策略的发展。 小鼠γHV68是密切相关的人类致癌卡波济氏肉瘤相关疱疹病毒和Epsten的EB病毒3,4。在实验室老鼠提供了一个听话的小动物模型来研究宿主反应和病毒感染的整个过程，这是不是人类疱疹病毒在体内的 γHV68感染。在这个协议中，我们提出了一个面板的表型特征的方法和分子剖析主机的信号传输元件的在γHV68裂解replic通报BULLETIN无论是在体内和体外 。在γHV68急性感染体内的转基因小鼠品系的可用性允许在审讯主机信号转导通路中的作用。此外，小鼠胚胎成纤维细胞（MEF中），可以使用从这些缺陷的小鼠品系中分离进一步解剖这些分子的作用，在γHV68裂解复制体外 。 使用病毒学和分子生物学分析，我们可以找出宿主 - 病毒相互作用的分子机制，并确定必要病毒裂解复制的宿主和病毒基因。最后，细菌人工染色体（BAC）的系统有利于突变引入到病毒因子（s），专门中断的宿主 - 病毒相互作用。携带这些突变基因重组γHV68可以用来概括MEFs细胞的表型的γHV68裂解性复制缺陷的在关键主机信号组件。此协议提供了一个很好的策略，询问宿主-病原体相互作用， 在体内和体外的多层次的干预。 最近，我们发现，γHV68侵占先天免疫信号转导通路促进病毒裂解性复制。具体而言，γHV68原发感染激酶IKKβ激活免疫激活IKKβ磷酸化的主病毒的转录因子，复制和反式激活（RTA），促进病毒的转录激活。在这样做时，γHV68有效地夫妇的转录激活机体天然免疫激活，从而促进病毒的转录和裂解性复制。该研究提供了一个很好的例子，可以应用到其他病毒询问宿主 - 病毒相互作用。

Watch this Scientific Journal Video about Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus at JoVE.com

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

我们描述了一个协议来识别主机的信号分子裂解性复制的模型疱疹病毒，γ疱疹病毒68（γHV68）的关键角色。利用转基因小鼠品系和胚胎成纤维细胞裂解性复制γHV68，该协议允许表型特征和在病毒裂解复制的病毒 - 宿主相互作用的分子审问。

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 ...

dissecting-host-virus-interaction-lytic-replication-model

Research

JoVE Journal

Immunology and Infection

10.8K 次观看.  UT Southwestern Medical Center. 我们描述了一个协议来识别主机的信号分子裂解性复制的模型疱疹病毒，γ疱疹病毒68（γHV68）的关键角色。利用转基因小鼠品系和胚胎成纤维细胞裂解性复制γHV68，该协议允许表型特征和在病毒裂解复制的病毒 - 宿主相互作用的分子审问。

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

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

科研

免疫与感染

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

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 ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;：主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

为马科动物疱疹病毒2诊断的呼吸道液体定量PCR检测方法的开发和验证

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号：对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.