我们感谢彼得Staeheli提供B6.A2G-MX1小鼠和Semra Ozcelik酒店的技术咨询。 这项工作是由德国研究基金会通过SFB829和KN536 / 16，和科隆财富计划/科隆大学医学院，支持。

伦理声明。 表皮片从处死的动物的制备是严格按照Landesamt献给NATUR，UMWELT和Verbraucherschutz，北莱茵 - 威斯特法伦（德国）的指南的建议。该研究是通过LANUV NRW（编号8.84-02.05.20.13.018）。 1.准备仪器仪表及文化传媒<ol><li>培养表皮片在Dulbecco改良的Eagle培养基（DMEM）/高葡萄糖含谷氨酰胺二肽，10％FCS，100IU / ml青霉素，和100μg/ ml链霉素（以下简称"DMEM"）。 </li><li>对表皮片的制备中，仔细地溶解分散酶II粉末（5毫克/毫升）中加热的PBS（高达37℃）。过滤通过0.22微米的过滤器将溶液直接将其用于治疗。 </li><li>对于表皮整体编制机构 13，准备拦截的buff尔用0.5％奶粉，从冷水鱼皮和0.5％的Triton X-100的TBS 0.25％明胶。允许物质通过至少2小时温育该混合物在RT下在旋转混合器溶解。还在PBS制备0.2％吐温20洗涤缓冲液。为固定，制备3.4％和在PBS中的4％甲醛。 </li><li>用于冷冻切片的免疫染色，制备封闭缓冲液，用5％正常山羊血清和0.2％吐温20在PBS中。对于固定，准备0.5％甲醛的PBS。 </li></ol>从鼠皮2制备表皮片的<ol><li>从新生儿背部皮肤感染的研究表皮的制备<ol><li>放置一个断头小鼠（出生后1至3天）成夹层菜和切断四肢和尾靠近躯干，以允许从完全躯干有效地除去皮肤的。在去除四肢，尽量保持切口尽可能小的直径。 </li><li>修正了曲细镊子一躯干ND轻轻移动钝头剪刀皮肤下面从颅沿后尾鳍到皮肤从躯干分离。狭缝沿背部皮肤。 </li><li>对于FACS分析，角质形成细胞或准备RNA的分离，用钳子固定的组织和钝头剪刀推下躯干的躯干剥离完整的皮肤。对于冰冻切片或整个坐骑的准备只拿个背部皮肤。 </li><li>印章的背部皮肤，用手术刀为1-2枚约10×10毫米。把碎片在一个6孔板以〜1毫升无菌PBS中的一个孔中。确保真皮侧面的底部。 </li><li>更换PBS 1.5毫升分散酶II（5毫克/毫升的PBS）和孵育在37℃或者30分钟（对于整个坐骑）或O / N在4℃。用PBS洗3次。轻轻地取出表皮为使用一个镊子来解决下面的真皮和其他钳解除表皮完整片。用双眼做这一步EAS年。转移表皮片成DMEM符合其对底部的基底的一面。立即感染实验使用的床单。 注:使用从新生儿皮肤的表皮准备主要为FACS分析，角质形成细胞或准备RNA的隔离。由于其脆弱性，编制感染薄板的冰冻切片或整个坐骑不太适合。 </li></ol></li><li>成人尾部皮肤感染研究表皮的制备<ol><li>安乐死的小鼠在1至3个月的颈椎脱臼的年龄。取尾巴，而不是背部皮肤，因为在背面的长嵌入毛囊阻碍完好表皮片的分离。切断从处死小鼠的尾部，用手术刀切开纵向它。 </li><li>剥去骨头的皮肤，用手术刀把它切成约5×5mm的碎片。竖起来4件于一体的，并在2.1.5所述继续进行孵化分散酶II 注:使用成人泰l湿准备冰冻切片或整个坐骑。可替代地，以立即使用，尾部可以存储或在4℃下输送大约24小时，而不会失去显著感染效率。如果尾巴跟上至48小时，表皮片可以几乎不感染HSV-1。 </li></ol></li><li>表皮用于FACS分析的解离 注:检测通过FACS分析表面蛋白质的表达需要适当的细胞解离技术不损害细胞表面和所关心的蛋白。 <ol><li>孵育1.5毫升/孔的重组胰蛋白酶的基于细胞解离15分钟，室温溶液和15分钟，在37℃的新生表皮片在一个6孔板与基底侧。 </li><li>通过加入3ml DMEM停止孵育。通过使用弯曲的细镊子轻轻移动表皮在6孔板使得角质得到溶解解离的角质形成细胞。收集细胞悬液，重复此步骤3次总是用新鲜的DMEM。 注:此分离程序是合适的检测的Nectin-1角质形成细胞的表面上。 </li><li>可替代地，孵育上无酶细胞解离溶液的表皮片（CDS）15分钟，在室温和15分钟，在37℃。孵育另外15分钟，在RT下轻轻冲洗掉角质吹打CDS上下。 </li><li>收集细胞悬浮液，并使用新鲜的DMEM重复冲洗步骤3次。 注:此分解适用于检测HVEM的表面表达。在CDS溶液的缺点是，更少的细胞解离比用重组胰蛋白酶的基于细胞解离溶液。除了感染表皮片用重组胰蛋白酶的基于细胞解离溶液的离解后表面表达，核或细胞质表达如ICP0表达可以通过FACS进行分析。 </li></ol></li><li>表皮解离隔离角质形成细胞或前道RNA <ol><li>放新生儿表皮片在一个6孔板与朝其含有1.5毫升/孔的重组胰蛋白酶的基于细胞解离溶液的培养皿底部的基底侧。孵育在室温30分钟。添加3 ml的DMEM中，并通过使用弯曲的细镊子轻轻移动的菜表皮角化细胞解离。 </li><li>收集细胞悬浮液，并使用新鲜的DMEM重复此步骤3次。使用该细胞悬浮液中提取RNA或文化原代鼠角质形成细胞。 </li></ol></li></ol> 3.感染表皮片与HSV-1 <ol><li>制备的HSV-1重量17株11的溶液在不大于500微升DMEM中以下，由病毒溶液在其上表皮片是浮更换介质。这个时间点被定义为时间0 12。在一个24孔板的一个孔，在37℃下进行表皮片的感染。病毒滴度的计算是基于所估计的数细胞在每表皮片的基底层的（大约每5×5毫米片2×10 5个细胞）。 </li><li>感染了HSV-1在约100噬斑形成单位（PFU）/细胞并更换含病毒介质由DMEM，在1小时圆周率 注:如果从新生儿皮肤表皮片的，记住，张开始孵化过程中解离。 </li></ol> 4.可视化感染的细胞<ol><li>表皮整体机构 13 <ol><li>修复表皮片用3.4％的甲醛无论是2小时在室温或O / N在4℃。洗两次，用PBS和0.5％奶粉，从冷水鱼皮和0.5％的Triton X-100的TBS 1小时在室温14 0.25％明胶阻塞。 </li><li>孵育板O / N小鼠抗ICP0（单克隆抗体11060）15稀释1:60在封闭缓冲液在室温下。以可视化的中间纤维网络中同时孵育兔多克隆抗鼠标角蛋白14（100纳克/毫升）。 </li><li>期间4小时，在室温洗涤4〜5次，用PBS-0.2％吐温20。孵育O / N与AF488标记的抗小鼠IgG（1微克/毫升），AF555缀合的抗兔IgG（1微克/毫升），和DAPI（4&#39;，6-二脒基-2-苯基）（100纳克/毫升）在封闭缓冲液在室温下。 </li><li>用PBS-0.2％Tween 20的4小时在室温洗。山表皮片与他们的标本幻灯片顶底侧，嵌入荧光封固剂，盖上盖玻片。 </li></ol></li><li>表皮冷冻切片<ol><li>成人表皮片材在室温下固定在PBS中的4％甲醛20分钟，洗涤2次，用PBS。在组织中嵌入固定片冷冻介质和冷冻在液氮中。切8-10微米的部分用冷冻切片机。 </li><li>固定再次用在PBS中的0.5％甲醛在室温10分钟的部分中，洗涤2次，用PBS，用30分钟，在室温在PBS 5％正常山羊血清和0.2％吐温20块，然后染色60分钟以米乌斯抗ICP0（单克隆抗体11060）15稀释1:60在封闭缓冲液，随后3次用封闭缓冲液洗涤。 </li><li>然后用AF488标记的抗小鼠IgG（1微克/毫升）和DAPI（100毫微克/毫升）培养在封闭缓冲液在室温45分钟，洗3次。荧光封固剂嵌入部分，盖上盖玻片。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

当从C57BL / 6成人皮肤的表皮片材被感染HSV-1在约100 PFU /细胞，我们观察到感染几乎所有细胞中的基底层中的滤泡表皮而较低的病毒剂量关联与少感染的细胞和较慢的感染的进展。在一般情况下，毛囊显示可变数目的感染的细胞;而大多数的相当小的角化细胞衬显影毛囊感染，成人毛囊仅实施发胚芽被完全感染。取决于如何平缓的表皮由分散酶II处理前进的分离，毛囊的部件会丢失。在大多数情况下?...

单纯疱疹病毒（HSV）可引起一系列疾病从轻度无并发症的皮肤粘膜损伤到危及生命的感染人类。单纯疱疹病毒1型（HSV-1）是显性与口面部感染和脑炎相关，而HSV 2型（HSV-2）更可能导致生殖器感染1。虽然在了解HSV是如何进入细胞的培养，引发感染并产生病毒的后代显著的进步，我们知道一点关于病毒的入侵途径（S）进入组织细胞水平2。单纯疱疹病毒的皮肤或粘膜感染，小鼠的研究，兔和豚鼠已用作动物模型。皮肤感染成立通过皮内注射或通过刮擦中病毒的存在的皮肤，和疾病发展率与病毒的产生。这些方法有助于理解疾病的发病机制的各个方面，并用于评估抗病毒药。研究HSV感染的Tissu酒店E级，人体器官皮肤模型已经应用。作为感染的速率被限制在这些筏培养，研究调查感染，病毒传播和抗病毒成分的影响仅有限数量的已出版3-6。 为了表征细胞的决定因素，在HSV-1感染的完整上皮细胞发挥 ​​作用，我们建立了一个协议，用于小鼠表皮7的体外感染的研究。皮肤从新生儿或从成年小鼠的尾部制备。由于HSV-1能不感染完整的皮肤样本，它被淹没在含病毒的培养基中，我们通过分散酶II处理分离的真皮表皮。含病毒介质上浮动的表皮片材后，感染的细胞可以在不同的时间感染后（PI）的7被可视化的表皮基底层。现有可视感染个体细胞的启动病毒复制一第二病毒产生，我们用针对所述感染的细胞蛋白质0（ICP0），这是在HSV-1感染所表示的第一蛋白之一的抗体。 ICP0的细胞定位在早期感染穿过不同的阶段。而ICP0存在于核灶期间病毒基因表达的早期阶段，ICP0至细胞质的重新定位指示随后感染8的相位。 我们使用表皮片的体外感染测定从不同小鼠模型感染期间测试各种细胞因子的潜在作用。为了解决Rac1的作为肌动蛋白动力学的关键调节的影响，我们感染小鼠的表皮具有 ​​角化细胞特异性缺失RAC1基因9。此模型使我们能够研究对HSV-1感染在表皮角化层中的效率缺陷的Rac1的后果。以对照同窝感染表皮重新比较vealed无显著差异，这表明不存在的Rac1的有感染在表皮7的基底层开始没有影响。使用进一步小鼠模型使我们能够解决该细胞受体介导进入表皮。从任一的Nectin-1-或HVEM缺陷小鼠用HSV-1感染表皮片显露最初的病毒进入组织强烈依赖的Nectin-1 10的存在。此外，我们的结果表明，HVEM也可以有效地少充当受体在鼠表皮，虽然比的Nectin-1 10。 为了解决感染的细胞中的表皮层的空间分布，我们想象在组织切片和表皮全样载（图1）ICP0表达。在完整皮肤的冷冻切片，没有ICP0表达细胞中检测到（ 图1）。与此相反，表皮片的冷冻切片表明细胞质ICP0表达在基底层已经3小时圆周率（ 图1）。在稍后时间，病毒扩散到基底层可以可视化。感染细胞在基底层的空间分布可以很容易地跟随在表皮全样载（ 图1）。在感染了HSV-1在100 PFU /细胞，在滤泡表皮的基底角质形成细胞的大约50％显示在1.5小时丕ICP0表达在这个时间点最感染的细胞表达核ICP0。 ICP0的再定位至细胞质指示早期基因表达的稍后阶段存在于几乎所有细胞在3小时圆周率（ 图1）。在体外 HSV-1感染的可视化感染细胞的这些模式提供了有力的实验来研究对病毒的进入和扩散在组织中删除/突变的细胞成分的抑制剂或效果。

<table><tbody><tr><td>DMEM/high glucose/GlutaMAX</td><td>Life Technologies</td><td>31966047</td><td>needed for cultivation of epidermal sheets</td></tr><tr><td>dispase II powder</td><td>Roche</td><td>4942078001</td><td>has to be solved in heated PBS</td></tr><tr><td>enzyme-free cell dissociation solution</td><td>Sigma</td><td>C5914</td><td>needed for very gentle dissociation of epidermal sheets</td></tr><tr><td>TrypLE select cell dissociation solution</td><td>Life Technologies</td><td>12563-029</td><td>needed for dissociation of epidermal sheets</td></tr><tr><td>chelex 100 resin</td><td>Bio-Rad</td><td>142-2832</td><td>needed for chelation of polyvalent metal ions from the fetal calf serum</td></tr><tr><td>gelatin from cold water fish skin&amp;nbsp;</td><td>Sigma</td><td>G7765</td><td>needed for minimization of non-specific antibody binding</td></tr><tr><td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td><td>Covance</td><td>PRB-155P</td><td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td></tr><tr><td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (conc.: 2 mg/ml)</td><td>Life Technologies</td><td>A-11029</td><td>used as secondary antibodies</td></tr><tr><td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate (conc.: 2 mg/ml)</td><td>Life Technologies</td><td>A-21429</td><td>used as secondary antibodies</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (conc.: 0.1 mg/ml)</td><td>Sigma</td><td>36670</td><td>used to counterstain the nucleus</td></tr></tbody></table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

进入其人类宿主，单纯疱疹病毒1型（HSV-1），必须克服的粘膜表面，皮肤或角膜的屏障。初始进入期间HSV-1的目标的角质，并建立在该上皮，之后是神经元的潜伏感染的主要感染。激活后，病毒可出现皮肤水泡或粘膜溃疡粘膜部位变得明显。如何HSV-1侵入皮肤或黏膜和达到其受体知之甚少。调查入侵路线的HSV-1的成表皮组织在细胞水平上，我们建立鼠表皮的离体感染模型，它代表的原发性和复发性感染中的皮肤的部位。该测定法包括鼠皮肤的制备。表皮从真皮层经分散酶II处理分离。浮动的表皮片含病毒的培养基上后，将组织是固定的，感染后可在不同的时间感染后由可视化用针对HSV-1的立即早期蛋白ICP0的抗体染色感染细胞。 ICP0表达细胞，可以在基底角质形成细胞层在1.5小时感染后观察到了。较长感染次，感染的细胞在基底层层检测，表明感染并不限定于基底角质形成细胞，但病毒扩散到其他的层中的组织。使用各种小鼠模型的表皮片，感染协议允许确定向HSV-1侵入到组织的细胞成分的参与。另外，该测定是合适的，以测试在组织抑制剂，与初始条目的步骤干扰，细胞至细胞的传播和病毒生产。在这里，我们详细描述的体外感染协议和使用的Nectin-1-或HVEM缺陷型小鼠呈现我们的结果。

该方法的挑战是，制备表皮片到其中HSV-1可以从基底层穿透。的关键步骤是从真皮的表皮由分散酶II处理，这取决于该小鼠品系，需要调整的分离。分散酶II的浓度的范围可以从2.5至5毫克/毫升，并孵育20至45分钟的时间。的中间丝蛋白的角蛋白14的染色很容易地允许预测所述基底表皮层是否可以感染或是否会显示出感染的细胞（图2）只有非常有限。理想的是，角蛋白14染色的表皮整体坐...

Watch this Scientific Journal Video about Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1 at JoVE.com

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

ex-vivo-infection-of-murine-epidermis-with-herpes-simplex-virus-type-1

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

11.1K 次观看.  University of Cologne. The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

视频: Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

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 ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

神经科学

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

siRNA 电穿孔以在单纯疱疹病毒类型 1-感染单细胞衍生的丹突细胞中调制自噬

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

猪角膜组织外植研究单纯疱疹病毒-1抗病毒药物的功效

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

单纯疱疹病毒的扁平化

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells

Source: D&#252;thorn, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60190">siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells.</a> J. Vis. Exp. (2019).
This video demonstrates a small interfering RNA (siRNA)-based technique to interfere with autophagic flux. A siRNA targeting FIP200 mRNA is introduced into monocyte-derived immature dendritic cells (iDCs) using electroporation, causing the knockdown of FIP200 expression and leading to the inhibition of nuclear lamin disruption via autophagy. Upon adding herpes simplex virus (HSV), the nuclear egress of viral progeny is inhibited due to the absence of lamin disruption via autophagic flux.

基于 siRNA 的单纯疱疹病毒感染的未成熟树突状细胞自噬抑制

Monocyte-derived DCs were generated from leukapheresis products of healthy donors. For this, a positive vote from the local ethics committee has been ...

鼠表皮离体感染单纯疱疹病毒1型

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体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

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基于 siRNA 的单纯疱疹病毒感染的未成熟树突状细胞自噬抑制

鼠表皮离体感染单纯疱疹病毒1型

摘要

探索更多视频

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

siRNA 电穿孔以在单纯疱疹病毒类型 1-感染单细胞衍生的丹突细胞中调制自噬

猪角膜组织外植研究单纯疱疹病毒-1抗病毒药物的功效

单纯疱疹病毒的扁平化

基于 siRNA 的单纯疱疹病毒感染的未成熟树突状细胞自噬抑制

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色