Name: in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection
Uploaded: 2014-11-27T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

This work was supported by NIH grants to T.W. (R01AI072060 and R01AI099123). G. Xie was supported by a Sealy Center for Vaccine Development Predoctoral Fellowship. We thank Dr. Richard Flavell (Howard Hughes Medical Institute, Yale University School of Medicine, New Haven) and Dr. Shizuo Akira (Osaka University, Japan) for providing the MyD88&#8211;/&#8211; mice and Dr. Y Cong (UTMB, Galveston) for providing OTII transgenic mice.

所有的动物实验获得批准的动物护理和使用委员会在得克萨斯大学医疗分支。 来自非感染者和WNV感染的小鼠1.隔离的DC <ol><li>年龄和性别匹配的6-10周龄，野生型C57BL / 6（B6）和MyD88的- / -小鼠。接种腹腔（IP）500空斑形成的WNV NS4B- P38G突变体单元（PFU）。在第3天感染后，安乐死B6和MyD88的- / -小鼠用CO 2。使用这两个群体为对照组的非感染小鼠。使用三只小鼠从每个组。 </li><li>对处死小鼠用70％的乙醇左侧湿毛皮和切去毛发沿着鼠标的左侧，对正面和背面的腿之间中途。剖开体腔。通过使用镊子取出脾脏。将脾脏在培养皿用RPMI。 </li><li>通过根据使用抗CD11c磁珠分离脾DC制造商的说明。 </li></ol> 2.纯化及的OTII转基因小鼠T淋巴细胞标签<ol><li>从天真OTII鼠标收获1脾如上面的步骤1.2描述和同质化的两个幻灯片磨砂两端之间的脾脏。细胞悬液转移到15毫升锥形管中，加入RPMI培养基高达14毫升让该悬浮液静置5分钟，并转移13毫升它以一个新的15毫升锥形管中。 </li><li>通过使用根据制造商的说明一个CD4 + T细胞分离试剂盒分离脾CD4 + T细胞。 </li><li>转移细胞到50ml锥形管中，用PBS洗两次。重悬的细胞在PBS中与0.5％牛血清白蛋白（BSA）以每毫升1×10 6个细胞，并加入CFSE 0.5微摩尔/毫升。孵育在37℃进行10分钟，避光。 </li><li>加入5毫升冷的完全培养基（RPMI-1640，用10％热灭活的胎牛血清，1/100体积抗生素/抗真菌剂，1/100体积L-谷氨酰胺和1 / 1,000（体积）的1000×2-巯基乙醇）。 </li><li>在冰上孵育5分钟。洗涤细胞一次，用PBS，固定0.2×10 6个标记的CD4 + T细胞在2％PFA中并获取关于流式细胞仪。使用该样品，以确定CFSE的基础水平。重新暂停标记的细胞在完全培养基中的其余部分。 </li></ol> 3.共培养OTII T细胞与WNV感染小鼠树突状<ol><li>培养2×10 OTII小鼠5纯化CD4 + T细胞单独或与野生型或MyD88的纯化的DCs - / -小鼠（2×10 4）在一个24孔板有或没有残余的OVA 323-339（1克/毫升）的1毫升每孔完全培养基。 </li><li>孵育细胞在37℃5天。收获细胞，在FACS缓冲液洗两次（PBS中的1％FBS）和0.25ml的2％PFA固定。涡马上。 </li></ol> 体外 4.流式细胞仪分析&lt;/ EM&gt; T细胞活化<ol><li>对于收购，双击流式细胞仪软件图标的流量。对于线性FSC-A 与线性SSC-A的密度图，选择通道0到20万FSC-A和0至20万SSC-A。然后创建活细胞上的栅极（P2;参见图1A）。 </li><li>分析CFSE荧光强度，打开直方图为FL1通道并获取关于门控人口20000事件。上设置的样品的标志物OTII细胞单独培养（ 图1A）。 </li><li>以类似的方式的DC（ 图1C） -采集样品OTII细胞共培养与野生型的DC（ 图1B）或MyD88的- /。 </li></ol> 5.分离的脾细胞为刺激细胞因子测定<ol><li>如STE描述的小鼠与用同样的步骤西尼罗病毒NS4B-P38G突变体-感染B6和MyD88的- /P 1.1。在第30天感染后，再挑战存活小鼠与2000 PFU野生型WNV菌株的ip在天8和21后的主WNV感染或在4天之后继发感染，安乐死的小鼠用CO 2收集脾脏。 注意:我们使用每组2-3只小鼠每个时间点。 </li><li>使脾细胞的单细胞悬浮液如在步骤1.2和步骤2.2，如上所述。计数使用血球和细胞在10ml完全培养基中重新悬浮它们。 </li><li>稀释细胞用完全培养基，以2.5×10 6个细胞/ ml。加入1毫升脾细胞在1.5ml微离心管中。 </li><li>以模拟的CD8 + T细胞，稀释的WNV特异性NS4B和E肽（SSVWNATTA和IALTFLAV）至1mg / ml，在DMSO中。对于CD4 + T细胞的刺激，稀的WNV特异性NS3和E肽（RRWCFDGPRTNTILE和PVGRLVTVNPFVSVA）至1mg / ml，在DMSO中。添加10微升肽至followe细胞ð1微升的布雷菲德菌素一个解决方案。用无细胞治疗肽作为对照。的细胞混合。 </li><li>冲两个孔与18G的针头，在管的盖子。孵育细胞在37℃5小时。 </li></ol> 6.内细胞因子染色<ol><li>转移细胞到新的微量离心管中。旋转细胞5分钟在300-400 XG和倒出上清液。加入1毫升流式细胞仪缓冲，旋转5分钟在300-400 XG和移液用120微升FACS缓冲液重新悬浮细胞。 </li><li>添加2微升的Fc受体阻滞剂孵育细胞，在室温下放置10分钟。加入1 ml FACS缓冲液到每个管中。旋转5分钟，在300 - 400×g的。 </li><li>在300微升FACS再悬浮细胞缓冲剂和它们分成三个管（每管约0.8×10 6个细胞）。如表1所示，保留的各培养条件大鼠IgG-PE染色第一管。对于第二个管中，加入3微升抗CD4的APC与CD4肽处理的细胞，3μ升抗CD8 FITC对CD8肽处理的细胞，或两种抗体对细胞无肽治疗。使用第三管补偿染色。对于每个培养条件，使用细胞染色与APC结合的CD4，FITC结合的CD8或PE-标​​记CD3抗体单独（每管3微升）作为FL1，FL2和FL4渠道补偿控制的三级管。 </li><li>涡细胞短暂。置于冰上20分钟，避光。添加1.4毫升FACS缓冲。旋转5分钟在300-400 XG和倒出上清液。 </li><li>煽动扰乱细胞沉淀（或简称涡）。再暂停250微升固定/通透solutionby吹打细胞沉淀。在室温下孵育20分钟，并避光。 </li><li>添加1.2毫升FACS缓冲。旋转5分钟在300-400 XG和悬浮细胞在300微升FACS缓冲。 注意:在这一点上，细胞可以转移到BL2实验室用于进一步处理或贮存在冰箱中和避光为üp来三天。 </li><li>加入500μlFACS缓冲。旋转5分钟在300-400 XG和悬浮细胞在500微升1个通透/洗涤缓冲液中。旋转5分钟在300-400×g的。 </li><li>重新暂停细胞在100微升1个烫发/洗净，加3.5微升抗IFNγ-PE先前与抗体的CD4和/或CD8 + T细胞标记步骤6.3和3.5微升大鼠IgG-PE预留管加管对于IgG对照在冰上25分钟，避光。将1.4毫升1×通透/洗涤缓冲液洗涤细胞。旋转5分钟在300-400×g的。通过加入1.4毫升1X通透/洗涤缓冲液重复洗涤步骤。 </li><li>旋转5分钟在300-400 XG，倒出上清液。将1.4毫升的FACS缓冲液，并重新悬浮于400μlFACS缓冲液进行最后采集洗涤细胞。 </li></ol>内细胞因子染色7.流式细胞仪分析<ol><li>使用相同的采集的设置步骤4.1。 CREA德活细胞上的栅极（P2，参见图2A）。调整使用补偿管的每个组的电压。 </li><li>打开两个新的散点图，一个用于显示收集FL1 VS数据。 FL2（CD8 VS IFN-γ），另一种是显示收集FL4 VS的数据。 FL2（CD4 VS IFN-γ）。获得50,000个事件对于各样品的门控群体。 </li></ol>

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作者什么都没有透露。

WNV是BL3病原体。由于安全法规，免疫学检测与WNV感染的样本通常是由设备的可用性BL3设施或者更加漫长和繁琐的进行限制。在最近的研究中，我们使用了两种流式细胞仪为基础的方法西尼罗病毒感染5时，研究细胞介导的免疫反应。在这两个试验中，西尼罗病毒感染的细胞用直接或用含有4％PFA固定/透化溶液1-2％的PFA。众所周知，病毒感染的细胞的1％PFA中固定可有效降低到低于检测限1...

西尼罗河病毒（WNV），神经营养，加黄病毒检测到，是一个新兴的公共健康威胁。目前，还没有疫苗已被批准供人类使用1。减毒WNV菌株，其具有P38G取代的非结构（NS）蛋白4B，被称为诱导小鼠没有杀伤力但较高先天细胞因子和T细胞应答的小鼠比野生型WNV NY99菌株2。免疫小鼠的NS4B-P38G突变均免受致命野生型WNV次要挑战。这表明，NS4B-P38G突变体具有合适特征的理想的候选疫苗。通过该NS4B-P38G突变导致的高保护性免疫机制尚不清楚呢。 toll样受体（TLR），其识别病原体相关分子模式，发挥在先天免疫病毒感染的启动中起重要作用。核心TLR信号通路利用骨髓分化初级反应GENE 88（MyD88的）作为主适配器3,4。在最近的一项研究中，MyD88信号被证明在小鼠5 WNV NS4B- P38G突变感染在细胞介导的免疫发展具有重要作用。树突状细胞（DC）是最重要的抗原呈递细胞表现出病毒感染6,7中以引发初级T细胞反应的独特能力之一。 CD4 +和CD8 + T-细胞既有助于长期的保护性免疫，并且下面的野生型WNV感染8,9-重要寄宿存活。两种免疫测定法在本研究中使用，以评估这些细胞在NS4B-P38G突变感染的小鼠的功能。 首先， 在体外 T细胞吸测定用于比较的DC西尼罗病毒感染的野生型和MyD88的的抗原呈递能力- / -小鼠。为了提高检测，天真CD4 &lt;灵敏度SUP&gt; + T细胞从OTII的转基因小鼠，其表达Vα2/Vβ5TCR特异性鸡卵白蛋白（OVA）肽323分离- 339.议会西尼罗病毒感染小鼠进行纯化，并共培养以羧基琥珀酰亚胺复活节（CFSE ）标记的CD4 + T细胞在OVA肽存在下进行。经过5天共培养，收获细胞并固定用多聚甲醛（PFA）和通过流式细胞术分析。增殖测定法已被传统上进行的，通过将5-溴-2&#39;-脱氧尿苷（BrdU）标记或氚化胸腺嘧啶脱氧核苷（3 HTdr）10掺入。然而，这些测定是要么放射性和/或需要在生物安全等级（BL）的3设施，其中，西尼罗病毒的研究进行的专用设备。的淋巴细胞增殖的活体染料CFSE标记已经变得更加普遍的免疫学测定法中使用的荧光强度的串行减半的流式细胞仪分析的染料更稳定地并均匀地掺入到细胞中，用流式细胞容易检测仪，并且非放射性11。该测定法还具有以评估细胞分裂的数目的能力。使用这种测定法中的WNV研究的一个主要优点是，感染的细胞以1-2％的PFA的固定可以灭活西尼罗病毒12，这将使样品采集在一个BL2实验室流式细胞仪。 接着，经修饰的细胞内细胞因子染色（ICS）的步骤，用于研究的MyD88的信令中的WNV特异性T细胞应答调节NS4B-P38G突变感染的小鼠中的作用。在该测定中，脾细胞从感染小鼠分离的处理在体外用WNV特异性肽。布雷菲德菌素A加入到保持在细胞内的细胞因子。后5小时温育后，收获细胞，洗涤并染色T细胞亚群。然后将细胞固定在PFA，透化染色对干扰素（IFN）-γ和通过流式细胞术分析。与其他术为基础的流测定，一旦细胞被用固定和含有PFA透化缓冲液处理过的，被感染的样品可以被转移到一个BL2实验室用于进一步处理和分析。在一些发表的研究中，我们使用的ICS测量在WNV感染的小鼠13,14的T细胞的效应子功能。虽然它的确定无疑的，此测定中的一个主要缺点是，该过程是非常冗长并且可能是更多的时间内BL3设施执行时费时。这里，微离心管系的ICS方法被证明是更可行的，更容易进行，以及更短的时间时内BL3实验室进行耗时。

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>11875</td><td>warm up at 37 &amp;deg;C</td></tr><tr><td>anti-CD11c magnetic beads</td><td>Miltenyi Biotec</td><td>130-052-001</td><td>follow the manufacturer&amp;rsquo;s instructions</td></tr><tr><td>anti-CD4 magnetic beads</td><td>Miltenyi Biotec</td><td>130-095-248</td><td>follow the manufacturer&amp;rsquo;s instructions</td></tr><tr><td>CFSE</td><td>Invitrogen</td><td>C34554</td><td></td></tr><tr><td>OVA residue 323-339</td><td>Genscript Corporation</td><td>RP10610</td><td></td></tr><tr><td>Peptides</td><td>Proimmune</td><td>PC0AD-D</td><td></td></tr><tr><td>Brefeldin A solution</td><td>BD Bioscience</td><td>555029</td><td></td></tr><tr><td>Mouse Fc Blocker</td><td>e-Bioscience</td><td>14-0161-85</td><td></td></tr><tr><td>APC-conjugated CD4</td><td>e-Bioscience</td><td>17-0041-81</td><td></td></tr><tr><td>FITC-conjugated CD8</td><td>e-Bioscience</td><td>11-0081-82</td><td></td></tr><tr><td>Fixation/Permeabilization Solution</td><td>BD-Bioscience</td><td>554722</td><td></td></tr><tr><td>Permeabilization/wash buffer</td><td>BD-Bioscience</td><td>554723</td><td></td></tr><tr><td>anti-IFN&amp;gamma;-PE</td><td>e-Bioscience</td><td>12-7311-82</td><td></td></tr><tr><td>Accuri flow cytometer</td><td>BD Bioscience</td><td></td><td></td></tr></tbody></table>

in vitro analysis of myd88-mediated cellular immune response to west nile virus mutant strain infection

减毒西尼罗河病毒（WNV），非结构（NS）的4B-P38G突变，诱发较高先天细胞因子和T细胞应答比野生型WNV小鼠。近日，髓样分化因子88（MyD88的）信号被证明在WNV NS4B-P38G突变感染是初始T细胞吸和记忆性T细胞的发育很重要。在这项研究中，2流式细胞术为基础的方法- 在体外的T细胞引发测定和胞内细胞因子染色（ICS） -被用于评估树突细胞（DC）和T细胞的功能。标记OTII转基因小鼠的CD4 + T细胞-在T细胞吸试验中，细胞增殖通过流式细胞仪从两组小鼠羧基琥珀酰亚胺酯（CFSE）的下列DC的共培养物进行分析。这种方法提供了一种准确测定增殖的CD4 + T细胞的百分比与显著改进比传统的整体灵敏度人检测放射性试剂。微量离心管系统被用来在ICS协议的细胞培养和细胞因子染色程序。相较于传统的组织培养板为基础的系统，这个修改过程是比较容易在生物安全水平（BL）3设施进行。此外，WNV-感染的细胞与多聚甲醛在两种测定中，这使BL3设施以外进一步分析处理。总之，这些体外免疫学测定法可以用来有效地评估WNV感染期间细胞介导的免疫反应。

在T细胞吸试验中，CFSE标记的CD4 + T细胞是从NS4B-P38G突变体感染的野生型和MyD88的纯化的DC培养- / -小鼠中OVA肽的存在或不存在。标记的T细胞单独培养有或无OVA 5天用作阴性对照。 如图1A所示 ，总T细胞被门控为荧光强度对FL1通道分析。标记成立基于所述新鲜标记的CD4 + T细胞在第0天以确定细胞增殖速度，而不DC的共培养物。有这种培养条件下增殖率（1.6％）的低?...

Watch this Scientific Journal Video about In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection at JoVE.com

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

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

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

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

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JoVE Journal

Immunology and Infection

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

8.2K 次观看.  The University of Texas Medical Branch. Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

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

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

γHV68感染小鼠的测量

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

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. ...

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

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

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

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

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

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

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

果蝇病毒感染的建立及宿主病毒相互作用分析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

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 ...

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation of RNA virus transmission using Wolbachia-transfected mosquitoes. Key strategies to control viruses include the manipulation of host reproduction via cytoplasmic incompatibility and the inhibition of viral transcripts via immune priming and competition for host-derived resources. However, the underlying mechanisms of the responses of Wolbachia-transfected mosquitoes to viral infection are poorly understood. This paper presents a protocol for the in vitro identification of Wolbachia infection at the nucleic acid and protein levels in Aedes albopictus (Diptera: Culicidae) Aa23 cells to enhance the understanding of the interactions between Wolbachia and its insect vectors. Through the combined use of polymerase chain reaction (PCR), quantitative PCR, western blot, and immunological analytical methods, a standard morphologic protocol has been described for the detection of Wolbachia-infected cells that is more accurate than the use of a single method. This approach may also be applied to the detection of Wolbachia infection in other insect taxa.

Detecting Wolbachia Strain wAlbB in Aedes albopictus Cell Lines

Four methods were used to detect intracellular Wolbachia, which complemented each other and improved the detection accuracy of Wolbachia infection of Aedes albopictus-derived Aa23 and Aa23-T&#160;cured of native Wolbachia infection using antibiotics.

检测白纹伊蚊细胞系中的沃尔巴克氏体菌株 wAlbB

As a maternally harbored endosymbiont, Wolbachia infects large proportions of insect populations. Studies have recently reported the successful regulation ...