我们感谢他们周到的建议，有助于改善这个手稿的评论。 MVC（NS21072，HD23315）的ACW（GM61139，S10RR017970）和IM（AI073898，GM056927）从美国国立卫生研究院的赠款，以支持这项工作。旺角是支持部分由美国国立卫生研究院培训资助（5T32 AI007180）。

1。从大鼠胚胎SCG神经元的分离和培养 提供有用的背景下，为了解该协议建立的早期文学的全面讨论，上海建工神经元培养方法，包括在体外培养，涂层板，基板，无血清培养基的成分SCG的基础上，读者可以参考参考2-4。 <ol><li>按照NIH的指引下进行的机构动物照顾及使用委员会 （IACUC）批准了一项活动的协议，利用大鼠SCG神经元来源。 </li><li>前开始清扫，准备胶原蛋白和层粘连蛋白涂层的96孔组织培养皿。使用多通道移液设备，填补所有96口井含有0.66毫克/毫升鼠尾胶原溶液。立即删除的胶原蛋白，它可以回收和使用长达8解剖。在去除T他的胶原蛋白，它是非常重要的，让干井层流罩下。干燥所花费的时间取决于该井在菜的数量。例如，它通常需要大约5-10分钟。在96菜干井，但可能需要长达30 - 40分钟，如果使用较大的格式以及菜24。如果不能正确地在上海建工附件差干井。然后重复使用2微克/毫升粘连的解决方案的过程。在37°Ç在加湿的CO 2培养箱孵育至少2小时的粘连的解决方案，直到您准备好你的神经元（步长1.14）板。 </li><li>商业上获得的怀孕雌性大鼠使用二氧化碳安乐死。喷药后用70％乙醇的尸体，一个U形切口在腹部。皮肤脱皮后，第二个U形切口，通过腹部肌肉壁。子宫后，抬起腹部肌肉层是可见的。在15中取出子宫和地方厘米菜。小心地打开使用钝的剪刀，以避免损害的幼仔在子宫。每个小狗必须从它的胚胎囊释放，脐带切断，小狗擦拭干净，用70％乙醇和Kimwipes。 </li><li>在解剖罩工作，牺牲剪从头部，躯干，腹中E21号幼鼠。剪刀在脖子基地的目标，只是上面的肩膀。揭露节，牵制（头颈部的一面朝上）使用23政针在三个地点：一）脊髓;二）前皮肤拉起了鼻子和固定;及iii）食道/气管必须固定距离从颈部基地。 </li><li>寻找*定位颈动脉分叉（两届胚胎SCGS）两侧的两个上海建工。总公司是位于下方的分支。从动脉分离，由除了拉动分叉，上海建工。放入12毫升L15及媒体（0.4％，D-（+）葡萄糖补充）15毫升锥形管冰核。 </li></ol>*上海建工是无色半透明和不透明的，黄色的脂肪组织，围绕分岔相比。尝试以消除残余的脂肪组织和血管的多少，前收集的节。 <ol start="6"><li>重复1.4和1.5，直到所有SCGS是从胚胎收获。 </li><li>轻轻离心节为1分钟600转，吸去多余的介质。 </li><li>悬浮在L15及媒体含有胰蛋白酶（0.25％，无EDTA）的胶原酶（1毫克/毫升）1毫升的节。孵育30分钟，在37°C，搅拌每10分钟。 </li><li>添加10毫升的C-媒体（1X的MEM，0.4％，D-（+）葡萄糖，L-谷氨酰胺2毫米，10％FBS）灭活的胰蛋白酶和1分钟600转的离心机。 </li><li>删除尽可能*胰蛋白酶/胶原酶媒体和洗的C-媒体10毫升节。离心1分钟600转。重复此步骤一次。 </li></ol> *残余的胶原酶将会interfe重新与胶原基质细胞附着在培养皿。 <ol start="11"><li>取出洗净媒体和重新暂停在1毫升的C-媒体节和磨碎使用21政针5毫升注射器分离的细胞*大的团块。完成23政针的研制过程，直到可见的团块是分离的。 </li></ol> *小心，不要过度，磨碎，因为这会损害细胞。如果胰蛋白酶和胶原酶治疗是成功的，最大的十五岁上下周期21Ğ针的;和三个周期的23政针应该足够了。 <ol start="12"><li>通过70微米尼龙过滤器[BD生物科学细胞过滤过滤游离神经元到50毫升锥形管，丢弃任何剩余的团块。 </li><li>撤回10μL的过滤细胞悬液，台盼蓝的组合，以确定活细胞的数量等分。计数积极不含细胞的数量UDE的染料使用血球。 </li><li>删除从96在37℃培养箱（见第1.2步）等待菜粘连的解决方案。不涸的菜，它可以用于电镀细胞的粘连后立即被删除。免除* 5000-6000 总活细胞（50 - 100μL每口井）的胶原蛋白和粘连预涂96以及组织培养皿。包括在C-媒体50毫微克/毫升的神经生长因子（NGF）。 </li></ol> *它采用良好的无菌组织培养技术是至关重要的，因为抗生素是从生长介质中省略。此外，还有可以是在从不同供应商的胶原蛋白的显著变异。我们不得不使用Millipore公司从鼠尾胶原跨批次一致的结果。 <ol start="15"><li>在DIV（ 体外日 ）1，更换用于板与50μL的NBM（0.4％，D-（+）细胞的C-媒体-葡萄糖，2毫米L-谷氨酰胺的B-27补充剂含有50 ng / ml的神经生长因子， 5μM的aphidicolin和20μM的5 - 氟脲嘧啶5 - 氟-2&#39;-脱氧。孵育5天的文化。在这一点上，trypsinizing几个井剩余的细胞计数确定幸存的抗有丝分裂剂治疗的神经元数目。通常情况下，大约1000名神经元保持在96孔板，每孔。 </li></ol> *真菌或细菌污染的问题通常是明显的文化在最初24小时。检查每个微生物生长取代电镀媒体前仔细。如果只有一个井的数量有限的影响，他们可以用稀释漂白液，用PBS冲洗和干燥，以进行实验，治疗。我们建议重复任何实验，甚至数量有限的油井微生物污染。如果广泛的微生物污染，在96盘观察，实验必须终止。 2。 SCG的邪教感染URES与HSV-1 Us11-EGFP和建立延迟 病毒学技术，包括基本的病毒传播，确定病毒滴度和感染（MOI），多元化的有用的背景，读者可以参考文献5。疱疹病毒生物学的讨论，读者可以参考参考文献6。最后，读者可以参考参考之前的例子，其他的协议，以及关于SCG神经元的α-疱疹病毒裂解感染更多的背景，并与本协议的研究延迟和重新适应的比较7-10。 <ol><li>在6格，添加阿昔洛韦（ACV），终浓度为100微米，在每口井*现有的媒体。例如，如果每个包含50μL，加入25μl300微米阿昔洛韦股票的。通常情况下，阿昔洛韦是增加了感染前的晚上，但也可以增加6-8个小时之前感染。 </li></ol> *这是前ceedingly重要的神经元在任何时候，以尽量减少不必要的物理处理。简单地拆除和更换媒体或感染病毒股票必须非常轻轻地，慢慢地做。否则，由此产生的机械应力产生不利影响后，神经元存活率。 <ol start="2"><li>格7，感染HSV-1 Us11-EGFP（11号描述）SCG的文化在感染复数（MOI），1 -2之间（上看到以下重要意见，确定最优内政部）*。新增摊薄病毒直接以及现有的媒体。包括一个模拟感染控制和媒体缺乏阿昔洛韦裂解感染阳性对照。允许感染进行2-3小时，在37°C。 </li></ol> *教学语言使用病毒滴度执行斑块检测Vero细胞12和1.14步，每孔接种于镀金，活细胞的数量决定的计算方法。这种计算是有用的，只有在操作感，作为种子细胞总数包含污染的神经胶质细胞，成纤维细胞和神经元在一起。由于这些污染的细胞分裂反分裂剂治疗死亡，存活神经元有效的教学语言实际上是更大。从最初的起点数量为5000 - 6000活细胞，约1000神经元抗有丝分裂剂（由trypsinizing和直接细胞计数评估）后与治疗。最佳的教学语言使用感染神经细胞时，从一个病毒备料到下可以有所不同。随着每一个新的病毒的准备，我们建议测试了不同的教学语言，从1到2的范围有限。这里的目标是要找出一个具有神经细胞的活力和诱导激活事件（第3条中定义）的最大数量的结果后，至少影响。 Us11-EGFP病毒是在迅速优化的条件，特别是有帮助的，但也可以使用其他如斑块检测或定量读数tative聚合酶链反应。它可以帮助使用通过蔗糖坐垫纯化，除去杂质，降低细胞活力，虽然这是没有必要的，而不是进行例行的病毒。 <ol start="3"><li> 小心地更换新鲜的NBM含有50 ng / ml的NGF和100微米阿昔洛韦*感染媒介。 </li></ol> *这是极为重要的改变感染媒体时，是非常温和的。在井墙上，而不是在井底对准枪头，并允许媒体轻轻地滑入细胞。甚至从吸管媒体的快速弹射可以产生足够的力量脱离基板的轴突和导致细胞蜕去了通常作为细胞表。如果只有一个神经元的分离（20-30％）的数量有限，其后果是最小的细胞出现健康。分离的神经元有可能重新连接，随着时间的推移，然而，轴突将不再被很好地伸出d新轴突再生。虽然不是最佳的，可以进行实验，如果只有一个井数量有限的影响。如果有广泛的神经元支队（70-80％），我们不建议，包括受影响以及在实验中（S）。如果大多数井含有70-80％的独立神经元，我们建议终止实验。 虽然仍可能重新连接的神经元，它们通常会形成较大的团块。这个复杂的个体激活神经元的正确评估。我们建议重复任何分离神经元的井，观察实验，以确保激活率没有影响神经元的井坚持差。 此外， 关键是要始终处理轻轻为可能的文化 。从不必要的，突然的动作（包括反复开放和孵化室门关闭）或有力的媒体应用C的机械应力·乌尔德·妥协的文化支持HSV-1的延迟，可能导致高得令人无法接受的自发活化率在一个给定的实验能力。 <ol start="4"><li>维持6天，在此期间，病毒会建立延迟的文化。在此期间，使用荧光显微镜监测神经健康和Us11-EGFP的表达裂解复制的一个指标。 us11-EGFP应只发现在缺乏控制阿昔洛韦治疗井，表示成功的原发性感染和生产的裂解复制。即使在病毒生产增长的情况下，神经元可能显示病变的影响。受感染的文化应密切监测和模拟感染细胞相比。 </li></ol> 3。恢复和评估<ol><li>在14格，仔细取代阿昔洛韦含媒体与新媒体缺乏阿昔洛韦。如果合适的话，包括在这个时候的药理（或生物）的变量。是S余吕杏茜按照2.3所述的预防措施。 </li><li>超过5至6天期间，监测用荧光显微镜检测神经元处于激活的活文化。或者收集其他类型的分析样品（蛋白质，核酸，斑块检测等）。 </li><li>激活频率计数含有绿色荧光蛋白阳性神经元的井数计算，并表示作为样本井总数的比例。请注意，在单个EGFP的表达，不区分主激活事件和随后的感染，由于病毒传播。由于传染病的传播包含一个单一的文化，在一个良好的EGFP阳性神经元的一个或多个操作定义在这些条件下作为一个激活事件。 </li></ol> *多重激活事件可以发生在一个文化，如1所述。从反应来区分从病毒传播的主要激活事件<html> tivati​​on，包壳抑制剂WAY-150138可以加入。阻断包壳，传染性病毒产生和传播从激活是不可能的。因此，绿色荧光蛋白信号检测中存在的方式150138反映在单个文化以及1,13-15独立激活事件。请注意这样的150138是唯一有效的对HSV-1巴顿应变，而不是市售。作为一种替代方式150138，考虑使用以下任何：i）一个突变病毒蔓延到邻近的细胞的能力受损，可利用二）记者病毒EGFP的是从IE的启动表示，在持续存在阿昔洛韦; iii）与PAA或阿昔洛韦治疗，以防止后期的基因表达（和传染性病毒的生产），其次是针对IE基因产物的抗体免疫使用。 4。替代的方法来评估活化使用的检测从一个单一的SCG的准备。内容】“ 从个别的，单一的实验结果应得到重复实验可以进行，只要具有独立的上海建工准备为每个重复使用一个单一的上海建工批次进行。 <ol><li> HSV-1裂解成绩单的评估。阿昔洛韦在不同的实验诱导激活的存在或没有清除，收集后的RNA所需的时间。当使用96孔培养板，我们建议每个样品至少有20口井池（大约10 5个细胞的RNA样品）。另外，SCGS可以镀在较大的井（如24孔板，用4-5×10 4细胞/孔）。虽然检测RNA不到20口井，小体积参与不必要的采样样本变异的结果，当然是可能的。 </li><li> HSV-1裂解蛋白质的检测。收集裂解所需的时间后重新启动感应。加入裂解缓冲液7.5μl到10 W在96孔板的ELLS和SDS-PAGE电泳和免疫印迹分析。另外，病毒的蛋白质可以通过间接免疫荧光显微镜检测。最初的电镀贴装基板上的成像，如无菌盖玻片一直与聚-D-赖氨酸（0.2毫克/毫升，Sigma公司）预涂胶原蛋白和层粘连蛋白的应用前必须做（见1.10） 。 </li><li>传染性微粒的检测。培养上清收集后重新启动。执行斑块检测上采用系列稀释的上清Vero细胞单层。此外，牙菌斑检测可使用裂解液冻结解冻文化的，包括细胞相关的颗粒，以确定滴度。 </li></ol> 5。代表结果 图2A说明了一个例子，1μM的曲古霉素（TSA），已知的激活16-18诱导，被应用到SCG的潜伏感染的文化，与野生型HSV1的-EGFP Us11记者的应变。与TSA，激活达到2天之内最高的50％，并通过5天的激活高原约60％的水井。底线（“自发的”）激活是在这个实验中约10％，通常范围从10-20％用在体外系统。最大的活化水平各不相同，这取决于激活诱导利用。许多活化诱导剂，可应用于实验的时间，因为他们不干扰病毒复制与生产，但是这需要凭经验确定。其他诱导，包括任何试剂，影响神经细胞的可行性，或阻碍生产力病毒生命周期的完成，可能需要一个短暂的脉冲应用挑起激活。 而积累的-EGFP Us11在每个图2A中所描述的实验以及从延迟激活的指示，不区分是否us11-EGFP在单个神经元的信号来自一个独立的激活事件，或一个激活病毒通过文化传播。为了评估神经元的经历以及在每个独立的激活事件的数量，培养预先处理方式150138，一种化合物，特别是阻止病毒传播，防止病毒DNA基因组13-15包壳。受感染的治疗交感神经文化路150138与TSA诱导激活。 EGFP阳性神经元的重大数字只发现在TSA的处理井相比，二甲基亚砜处理控制文化，展示一些独立激活事件发生每个文化（ 图2B）。 在我们的分析，Us11-EGFP记者表示，从内源性Us11启动11。由于Us11是一个“真实迟到”或γ2病毒裂解基因，病毒DNA复制，需要强大的expressi上。 图3说明了这一点，作为受损是由病毒DNA聚合酶抑制剂phosphonoacetic酸（PAA），在诱导激活PI3激酶抑制剂LY294002的文化-EGFP Us11积累的。 <img alt="图1" src="/files/ftp_upload/3823/3823fig1.jpg" /> 图1。示意图说明了一个典型的实验协议， 在体外系统培养的神经元，研究HSV-1的延迟和重新使用。1）从第E21大鼠颈上神经节（SCG）后解剖，分离神经元接种于96孔的菜和反分裂治疗剂，以消除非神经细胞。 2） 在体外 （DIV），经过6天的文化与重组的HSV-1包含EGFP的融合中存在阿昔洛韦（ACV），抗病毒药物病毒编码Us11晚期基因（EGFP HSV-1）感染阻止裂解复制。 3）第14天（14格），阿昔洛韦被删除和EGFP表达没有检测到。文化可以稳定保持在这种方式为1个月或以上，或可以添加一个测试变量，以评估其能力挑起延迟激活。变量可以是在一个特定基因的shRNA或异位表达的蛋白质的小分子化学抑制剂，针对可溶性营养因子或细胞表面蛋白的抗体，或慢病毒表达形式。然后重新监测得分实时EGFP阳性井。 <img alt="图2" src="/files/ftp_upload/3823/3823fig2.jpg" /> 图2。 TSA的重新SCG的文化HSV-1潜伏感染SCG的文化，在图1中所述。在DIV的14，阿昔洛韦被删除，并含1μM的曲古抑菌素与媒体所取代（TSA）。 （一）文化进行可视化，并取得为期5天的药物治疗后使用荧光显微镜每天。接受reactivat井的百分比在实验过程中的离子显示相比，DMSO对照处理的文化。活化比例计算出的绿色荧光蛋白阳性井96孔培养板20口井的数量。错误的横道平均标准错误。 （二）潜伏感染的文化处理TSA及抑制病毒的DNA包壳，程150138（20微克/毫升）。 EGFP的神经元的数量相比，48小时后，与药物治疗的控制与治疗DMSO和路150138。每个数据点代表的绿色荧光蛋白+神经元以及1000文化中的神经元的比例。栏显示EGFP的平均百分比+良好的神经元。 <img alt="图3" src="/files/ftp_upload/3823/3823fig3.jpg" /> 图3。 EGFP的检测中是依赖于病毒DNA复制。SCG的文化，然后用10 LY29004，称为诱导激活1微米治疗潜伏感染。立法院（蓝色）WA诱导活化病毒DNA合成抑制剂，phophonoacetic酸，聚丙烯酸（300μg/ml，红色），这两种化合物（绿色）治疗的对照。

<ol>
	<li>Camarena, V., Kobayashi, M., Kim, J. Y., Roehm, P., Perez, R., Gardner, J., Wilson, A. C., Mohr, I., Chao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+duration+of+growth+factor+signaling+through+receptor+tyrosine+kinases+regulates+HSV-1+latency+in+neurons.">Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons.</a> Cell Host &#38; Microbe. 8, 320-330 (2010).</li><li>Johnson, M. I., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+sympathetic+ganglia.">Primary cultures of sympathetic ganglia.</a> Protocols for Neural Cell Culture. , 71-94 (2001).</li><li>Letourneau, P. C., Fedoroff, S., Richardson, A. <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+substrata+for+in+vitro+culture+of+neurons.">Preparation of substrata for in vitro culture of neurons.</a> Protocols for Neural Cell Culture. , 245-254 (2001).</li><li>Price, J. P., Brewer, G. J., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+media+for+neural+cell+cultures.">Serum-free media for neural cell cultures.</a> Protocols for Neural Cell Culture. , 255-264 (2001).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+family+Herpesviridae:+A+brief+introduction.">The family Herpesviridae: A brief introduction.</a> Fields Virology. 2, 2381-2397 (2001).</li><li>Price, R. W., Rubenstein, R., Khan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+infection+of+isolated+autonomic+neurons+in+culture:+viral+replication+and+spread+in+a+neuronal+network.">Herpes simplex virus infection of isolated autonomic neurons in culture: viral replication and spread in a neuronal network.</a> Arch. Virol. 71, 127-140 (1982).</li><li>Tomishima, M. J., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+alpha-herpesvirus+protein+necessary+for+axonal+localization+of+viral+membrane+proteins.">A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins.</a> J. Cell Biol. 154, 741-752 (2001).</li><li>Ch'ng, T. H., Flood, E. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+primary+and+transformed+neuronal+cells+for+studying+pseudorabies+virus+infection.">Culturing primary and transformed neuronal cells for studying pseudorabies virus infection.</a> Methods Mol. Biol. 292, 299-316 (2005).</li><li>Wang, F., Tang, W., McGraw, H. M., Bennett, J., Enquist, L. W., Friedman, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+glycoprotein+E+is+required+for+axonal+localization+of+capsid,+tegument,+and+membrane+glycoproteins.">Herpes simplex virus type 1 glycoprotein E is required for axonal localization of capsid, tegument, and membrane glycoproteins.</a> J. Virol. 79, 13362-13372 (2005).</li><li>Benboudjema, L., Mulvey, M., Gao, Y., Pimplikar, S. W., Mohr, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+herpes+simplex+virus+type+1+us11+gene+product+with+the+cellular+kinesin+light-chain-related+protein+PAT1+results+in+the+redistribution+of+both+polypeptides.">Association of the herpes simplex virus type 1 us11 gene product with the cellular kinesin light-chain-related protein PAT1 results in the redistribution of both polypeptides.</a> J. Virol. 77, 9192-9203 (2003).</li><li>Blaho, J., Morton, E. R., Yedowitz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus:+propagation,+quantification+and+storage.">Herpes simplex virus: propagation, quantification and storage.</a> Curr. Protoc. Microbiol. Chapter 14, Unit 14E.1 (2005).</li><li>Van Zeijl, M., Fairhurst, J., Jones, T. R., Vernon, S. K., Morin, J., LaRocque, J., Feld, B. L., O'Hara, B. L., Bloom, J. D., Johann, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+class+of+thiourea+compounds+that+inhibit+herpes+simplex+virus+type+1+DNA+cleavage+and+encapsidation:+resistance+maps+to+the+UL6+gene.">Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.</a> J. Virol. 74, 9054-9061 (2000).</li><li>Newcomb, W. W., Brown, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+herpes+simplex+virus+replication+by+WAY-150138:+assembly+of+capsids+depleted+of+the+portal+and+terminase+proteins+involved+in+DNA+encapsidation.">Inhibition of herpes simplex virus replication by WAY-150138: assembly of capsids depleted of the portal and terminase proteins involved in DNA encapsidation.</a> J. Virol. 76, 10084-10088 (2002).</li><li>Pesola, J. M., Zhu, J., Knipe, D. M., Coen, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+1+immediate-early+and+early+gene+expression+during+reactivation+from+latency+under+conditions+that+prevent+infectious+virus+production.">Herpes simplex virus 1 immediate-early and early gene expression during reactivation from latency under conditions that prevent infectious virus production.</a> J. Virol. 79, 4516-14525 (2005).</li><li>Arthur, J. L., Scarpini, C. G., Connor, V., Lachmann, R. H., Tolkovsky, A. M., Efstathiou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+promoter+activity+during+latency+establishment,+maintenance+and+reactivation+in+primary+dorsal+root+neurons+in+vitro.">Herpes simplex virus type 1 promoter activity during latency establishment, maintenance and reactivation in primary dorsal root neurons in vitro.</a> J. Virol. 75, 3885-3895 (2001).</li><li>Danaher, R. J., Jacob, R. J., Steiner, M. R., Allen, W. R., Hill, J. M., Miller, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+induce+reactivation+of+herpes+simplex+virus+type+1+in+a+latency-associated+transcript-+independent+manner+in+neuronal+cells.">Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript- independent manner in neuronal cells.</a> J. Neurovirol. 11, 306-317 (2005).</li><li>Terry-Allison, T., Smith, C. A., DeLuca, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxed+repression+of+herpes+simplex+virus+type+1+genomes+in+murine+trigenminal+neurons.">Relaxed repression of herpes simplex virus type 1 genomes in murine trigenminal neurons.</a> J. Virol. 71, 12394-12405 (2007).</li><li>Harris, R. A., Preston, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+latency+in+vitro+by+the+herpes+virus+type+1+mutant+in1918.">Establishment of latency in vitro by the herpes virus type 1 mutant in1918.</a> J. Gen. Virol. 72, 907-913 (1991).</li><li>Wagner, E. K., Bloom, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+investigation+of+herpes+simplex+virus+latency.">Experimental investigation of herpes simplex virus latency.</a> Clin. Microbiol. Rev. 10, 419-443 (1997).</li><li>Strelow, L. I., Laycock, K. A., Jun, P. Y., Rader, K. A., Brady, R. H., Miller, J. K., Pepose, J. S., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+and+functional+comparison+of+the+latency-associated+transcript+promoters+of+herpes+simplex+virus+type+1+strains+KOS+and+McKrae.">A structural and functional comparison of the latency-associated transcript promoters of herpes simplex virus type 1 strains KOS and McKrae.</a> J. Gen Virol. 75, 2475-2480 (1994).</li><li>Stroop, W. G., Banks, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+strain+KOS-63+does+not+cause+acute+or+recurrent+ocular+disease+and+does+not+reactivate+ganglionic+latency+in+vivo.">Herpes simplex virus type 1 strain KOS-63 does not cause acute or recurrent ocular disease and does not reactivate ganglionic latency in vivo.</a> Acta Neuropathol. 87, 14-22 (1994).</li><li>Sawtell, N. M., Poon, D. K., Tansky, C. S., Thompson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+latent+herpes+simplex+virus+type+1+genome+copy+number+in+individual+neurons+is+virus+strain+specific+and+correlates+with+reactivation.">The latent herpes simplex virus type 1 genome copy number in individual neurons is virus strain specific and correlates with reactivation.</a> J. Virol. 72, 5343-5350 (1998).</li><li>Thompson, R. L., Cook, M. L., Devi-Rao, G., Wagner, E. K., Stevens, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+analysis+of+the+avirulent+wild-type+herpes+simplex+virus+type+1+strain+KOS.">Functional and molecular analysis of the avirulent wild-type herpes simplex virus type 1 strain KOS.</a> J. Virol. 58, 203-211 (1986).</li><li>Wilcox, C. L., Smith, R. L., Freed, C. R., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor-dependence+of+herpes+simplex+virus+latency+in+peripheral+sympathetic+and+sensory+neurons+in+vitro.">Nerve growth factor-dependence of herpes simplex virus latency in peripheral sympathetic and sensory neurons in vitro.</a> J. Neurosci. 10, 1268-1275 (1990).</li><li>Roehm, P. C., Camarena, V., Gardner, J. B., Wilson, A. C., Mohr, I., Chao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+vestibular+ganglion+neurons+demonstrate+latent+herpes+simplex+type+I+reactivation.">Cultured vestibular ganglion neurons demonstrate latent herpes simplex type I reactivation.</a> Laryngoscope. 121, 2268-2275 (2011).</li><li>Kuhn, M. A., Nayak, S., Camarena, V., Gardner, J., Wilson, A., Mohr, I., Chao, M. V., Roehm, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+culture+model+of+facial+palsy+resulting+from+reactivation+of+latent+herpes+simplex+virus+type+1.">A cell culture model of facial palsy resulting from reactivation of latent herpes simplex virus type 1.</a> Otology &#38; Neurotology. , (2012).</li></ol>

没有利益冲突的声明。

这主要神经文化和感染系统提供了一个简单而有效的方法来探讨HSV-1潜伏期和活化的分子机制。该系统忠实地概括公认的标志，在人类的感染和在活的动物模型中定义的延迟。当病毒潜伏在SCG的文化，传染性颗粒和病毒裂解基因产物不能被检测出来。在潜伏感染神经细胞的检测是唯一的病毒基因产物的土地增值税的成绩单，非编码核内积累。激活恰逢病毒裂解基因表达和传染性颗粒生...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 产品编号 </td><td> 评论 </td></tr><tr><td>为70μm尼龙过滤器（细胞过滤器） </td><td> BD公司 </td><td> 352350 </td><td></td></tr><tr><td> 1X汉克的平衡盐溶液（HBSS中- / - ） </td><td> Invitrogen公司 </td><td> 14175 </td><td> W / O CaCl 2和MgCl 2的 </td></tr><tr><td> 1X最小（MEM）的主要媒介</td><td> Invitrogen公司 </td><td> 11095-080 </td><td></td></tr><tr><td> 5 - 氟-2&#39;-脱氧尿苷</td><td> 西格玛 </td><td> F0503 </td><td>准备在1x的MEM 20毫米的股票;储存在-20°C </td></tr><tr><td> 96以及平井底TC板</td><td> 康宁 </td><td> 3599 </td><td></td></tr><tr><td>阿昔洛韦</td><td> &lt;EM&gt; Calbiochem公司</td><td> 114798 </td><td>准备31毫米在DMSO股票;商店在-20℃ </td></tr><tr><td> aphidicolin </td><td> Calbiochem公司 </td><td> 178273 </td><td>准备在DMSO 10毫米的股票;存储在-20℃ </td></tr><tr><td> B - 27补编</td><td> Invitrogen公司 </td><td> 17504-44 </td><td></td></tr><tr><td>胶原酶</td><td> 西格玛 </td><td> C2674 </td><td>在-20°C，准备在HBSS 10毫克/毫升股票- / - ;商店</td></tr><tr><td> D - （+）葡萄糖</td><td> 西格玛 </td><td> G6152 </td><td>准备在H 2 O 40％的股票;过滤消毒及储存于4°C间</td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司 </td><td> 25030-081 </td><td></td></tr><tr><td>层粘连蛋白</td><td> 西格玛 </td><td> L2020 </td><td>准备在H 2O 1毫克/毫升的股票;快速冻结20μLaliquats及存储于-80℃;稀释至2μg/ ml的工作浓度。在无菌的H 2 O </td></tr><tr><td> leibovit&#39;z L - 15家媒体</td><td> Invitrogen公司 </td><td> 11415 </td><td></td></tr><tr><td>神经生长因子</td><td> 哈伦实验室 </td><td> BT.5017 </td><td>准备在HBSS 50微克/毫升股票- / -存储在-80°C间</td></tr><tr><td>的Neurobasal培养基</td><td> Invitrogen公司 </td><td> 12348 </td><td></td></tr><tr><td> phosphonoacetic酸（PAA）的</td><td> 西格玛 </td><td> P6909 </td><td>准备在H 2 O 75毫克/毫升的股票;商店在-20℃ </td></tr><tr><td>聚-D-赖氨酸氢溴酸</td><td> 西格玛 </td><td> P0899 </td><td>准备在H 2 O 20毫克/毫升的股票;商店在-20°C </td></tr><tr><td>鼠尾胶原</td><td> Millipore公司 </td><td> 08-115 </td><td>浓度变化与大量供应，储存在4°C和稀释至0.66 mg / ml的工作浓度。在无菌的H 2 O </td></tr><tr><td>曲古菌素A </td><td> 西格玛 </td><td> T8552 </td><td>准备在DMSO 1毫米的股票;存储在-20℃ </td></tr><tr><td>胰蛋白酶2.5％ </td><td> Invitrogen公司 </td><td> 15090-04 </td><td></td></tr></tbody></table>

a primary neuron culture system for the study of herpes simplex virus latency and reactivation

单纯疱疹病毒1型（HSV-1）建立终身潜伏感染在周边神经元。这种潜在的水库是经常激活事件的源，确保传输和临床疾病作出贡献。目前的抗病毒药物不影响潜在的水库，有没有疫苗。而裂解复制的分子细节是良好的特点，在神经元的延迟控制机制仍然是难以捉摸的。我们目前所知的延迟是来自于体内研究使用小动物模型，并已确定病毒基因的要求和免疫反应的作用是不可或缺的。然而，这是无法区分病毒神经元的关系，从更一般的感染，在活的动物免疫或支持非神经细胞介导的​​后果的具体影响。此外，动物实验是昂贵，费时，并在有限的可用选项的操作主机流程。为了克服这些限制，只有一个神经元系统的迫切需要，抄录在体内的潜伏期和活化的特点，但在同质化和无障碍方面提供了组织文化的好处。 在这里，我们提出利用体外原代培养大鼠颈上神经节（SCG）（ 图1）交感神经元模型，研究HSV-1的延迟和重新启动，适合大多数如果不是所有所需的标准。消除非神经细胞后，近同质TrkA的神经元文化与HSV-1感染中存在阿昔洛韦（ACV）的抑制裂解复制。以下阿昔洛韦去除，非生产的HSV-1感染的忠实表现出延迟接受的特点是有效建立。值得注意的是，裂解基因，蛋白质，传染性病毒成为不到，即使在没有选择的情况下，但潜伏相关转录（LAT），快递离子在神经元细胞核坚持。病毒的基因组都保持在25％神经元的平均拷贝数，可以诱导高效复制干扰与PI3激酶/ Akt信号或神经生长因子1的简单撤出。重组HSV-1编码EGFP的融合病毒裂解蛋白Us11提供的复制功能，实时的标记从激活，很容易量化。除了化学处理，如RNA干扰或基因通过慢病毒载体传递基因的方法，可以成功地应用于系统允许是非常困难的，如果不是不可能的，在动物的机理研究。总之，在SCG基于HSV-1的延迟/激活系统提供了一个强大的，必要的工具来解开单纯疱疹病毒潜伏期和活化在神经元，在病毒学的长期难题的解决方案，可提供新的见解，新疗法的开发控制的分子机制目标t他潜伏的疱疹病毒水库。

Watch this Scientific Journal Video about A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation at JoVE.com

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

协议描述了一种高效和可重复性的模型系统研究单纯疱疹病毒1型（HSV-1）延迟和重新启动。该法采用同质交感神经神经元文化，并允许使用各种工具，包括RNA干扰和重组蛋白表达的病毒神经元相互作用的分子解剖。

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 ...

a-primary-neuron-culture-system-for-study-herpes-simplex-virus

Research

JoVE Journal

Immunology and Infection

17.2K 次观看.  New York University School of Medicine. 协议描述了一种高效和可重复性的模型系统研究单纯疱疹病毒1型（HSV-1）延迟和重新启动。该法采用同质交感神经神经元文化，并允许使用各种工具，包括RNA干扰和重组蛋白表达的病毒神经元相互作用的分子解剖。

视频: A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

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

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 during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

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 ...

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 ...

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

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

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

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

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

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

原发性鸡初囊-细胞培养模型研究传染性囊内病病毒发病机制

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

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.