我们感谢过去和现在的成员JCT和LM-S实验室的沙粒病毒反向遗传学技术和质粒的​​发展和完善。我们也感谢Snezhana的季米特洛娃技术支持。单克隆抗体到JUNV NP（SA02-BG12）获得从BEI资源研究所（NIAID生物防御和新兴传染病研究资源库）。 BYHC支持由格兰特GM068411机构露丝属Kirschstein国家研究服务奖。 EO-R是一种的富布赖特CONICYT（BIO 2008）和罗切斯特疫苗院士（2012年）收件人。 EO-R目前支持从Office在美国罗切斯特大学教师发展与多样性的多样性与学术卓越的一个博士后奖学金。 LM-S实验室研究资助由NIH资助RO1 AI077719，R21NS075611-01，R03AI099681-01A1，NIAID的卓越中心的研究和监测流感（HHSN266200700008C），中心罗切斯特大学生物防御免疫建模（HHSN272201000055C）的。 研究在JCT实验室的补助RO1 RO1 AI077719，从NIH RO1 AI079665 AI047140支持。

1。沙粒病毒救援转我们的救援体系的基础上使用两个聚合酶II蛋白表达质粒编码的核蛋白（NP）和RNA依赖的RNA聚合酶（L），病毒所需的沙粒病毒基因组RNA的复制和基因表达的反式作用因子7，19，和能够直接细胞内合成，通过细胞的RNA聚合酶I（聚合酶-I）中，将S和L反基因组RNA的物种7的质粒。在我们的研究中，我们使用pCAGGS中的蛋白表达载体，使用的鸡β-肌动蛋白启动子和polyadenylanation（PA）的信号序列，和人的聚合酶我的质粒，使用人的聚合酶I启动子和小鼠的终止子序列（ 图2） 。的S和L RNA片段被克隆到HPOL-I方向反基因的质粒HPOL-I的转录产生基因组RNA片段时允许。为抢救野生-TYPE重组LCMV（rLCMV）和坦诚的＃1（rCandid＃1）将pCAGGS NP和L每个病毒共转染连同各自的人力POL-I S和L RNA片段（ 图3）。重组trisegmented LCMV（r3LCMV）的和坦诚的＃1（r3Candid＃1）的产生遵循了类似的协议，但在S段被分成两个不同的POL-I S质粒，每个编码鲜明的报告基因于一体，NP开放阅读框（ORF）的绿色荧光蛋白（GFP）的报告基因（HPOL-I S GFP / GP）和病毒糖蛋白前体（GPC），在另一方面，Gaussia荧光素酶（GLUC）报告基因的ORF被替换为替换（HPOL-I S NP / GLUC的）。 图4中所示的协议的示意图。在6孔板中已建立以下的转染和感染协议。 <ol><li> 的OptiMEM脂质体2000（LPF2000）混合物：准备250微升的OptiMEM媒体，，并根据关于病毒的救援，10 - 12微克每转染LPF2000（1微克/微升）（ 表1）。建议2.5微克LPF2000每微克DNA的比率。 5 - 10分钟，在RT下孵育。同时，准备在一个单独的离心管（1.2）的质粒转染混合物。 </li><li> 质粒DNA转染混合物：准备质粒转染鸡尾酒离心管中，使用每种病毒救援的建议金额（ 见表1）。使终体积为50微升的OptiMEM。 </li><li> 的OptiMEM LPF2000-DNA质粒混合物：加入250μl的OptiMEM LPF2000混合成的质粒DNA转染混合物（1.2）（1.1）。孵育该混合物在RT为20 - 30分钟。同时准备和计数1×10 6每股Vero细胞转染反应。将被转染Vero细胞在悬浮液中。 </li><li> Vero细胞制备：维罗CELLS 100 mm培养皿中培养。在开始之前，将1倍PBS，DMEM培养液10％FBS的1％PS媒体，和胰蛋白酶-EDTA混合物到37℃。 <ol><li>洗涤细胞两次，用5ml的1×PBS。 </li><li>胰蛋白酶消化细胞用1毫升的胰蛋白酶-EDTA，并等待，直到细胞分离（约23分钟）。轻轻拍打可能需要完全分离的细胞从菜肴。 </li><li>小心地重悬细胞，在10毫升的DMEM 10％FBS的1％PS在一个15毫升的离心管中。 </li><li>在离心机中以1,000 rpm的5分钟，离心细胞。 </li><li>在10毫升的DMEM 10％FBS的1％PS和重悬细胞，用血细胞计数器计数细胞，并调整至1×10 6细胞/ ml的浓度。据观察，0.5 - 1×10 6个每转染Vero细胞中产生最好的结果。 </li></ol></li><li> 混合LPF2000/DNA细胞后20 - 30分钟，室温孵育（ 步骤1.3），加1毫升Vero细胞（1×10 6）（ 步骤1.4）LPF2000的OptiMEM质粒DNA混合物在室温下孵育5分钟。 </li><li> 种子LPF2000/DNA-cell到6孔板的组合：（ 步骤1.5）的DNA LPF2000维罗混合物加入到6孔板的孔中。轻轻摇动6孔板，让转染孵育过夜孵化器在37°C和5％的CO 2。 </li><li> 更改转染培养基：约16 - 24小时转染后，除去转染介质，加2毫升感染媒体，培育转染细胞增设48个小时。 </li><li> 细胞通过：感染媒体孵化48小时后，取出组织培养上清（TCS）和转染细胞传递到100毫米的菜。 TCS很少含有转染细胞感染病毒，因为需要额外的孵化产生的病毒颗粒。对于小区通道： <ol><li>洗涤细胞两次，用2毫升的1X PBS中。 </li><li>胰蛋白酶消化细胞<html>第i个0.5毫升的胰蛋白酶-EDTA和，等到细胞分离。 </li><li>在1毫升的DMEM 10％FBS的1％PS 1.5 ml离心管中重悬细胞。 </li><li>离心5分钟，在5,000 rpm，4℃下用微量细胞。 </li><li>小心地重悬细胞，在1毫升的感染媒体，将细胞转移到100毫米的菜。带来的总体积中板，8毫升，有足够的体积，以防止干燥的细胞，以及浓缩病毒，传染性媒体。 </li></ol></li><li>轻轻摇动100毫米盘，并继续培养Vero细胞在孵化器在37℃和5％CO 2 72小时。 </li><li> 收集的TCS：培养72小时后，收集TCS在15毫升离心管。在离心机中以2,500 rpm的转速下离心分离5分钟，以除去细胞碎片。发现传染性沙粒在TCS，所以去除细胞碎片颗粒和存储TCS在-80°C。 </li></ol>乐“&gt; 2。确认重组沙粒病毒救援实现通过一个焦点形成单元（FFU）免疫荧光法（IFA）检测抢救成功rLCMV rCandid＃1。对于野生型病毒拯救，我们使用病毒的NP特异性抗体。的r3LCMV和r3Candid的第1编码的两个报告基因（GFP和GLUC），从而使病毒的抗体，而不需要通过荧光显微镜（GFP）和/或发光（GLUC）的检测和滴定。 <ol><li> 确认野生型重组沙粒病毒救援：滴定前一天，Vero细胞洗两次，用PBS，胰蛋白酶消化并准备96孔板中，达到80 - 90％汇合第二天（4×10 4个细胞/孔）。轻轻摇动板手细胞获得一个穿制服的分布。过夜培养细胞，在37℃培养箱中，用5％的CO 2。在显微镜下检查细胞单层确认之前，继续感染： <ol><li>串行稀释（10倍稀释）的含病毒的回收的TCS从转染的细胞在100mm皿（ 步骤1.10）的OptiMEM。 </li><li>从种子的Vero细胞中取出介质，用50μl的1×PBS中洗两次，并用50μl的系列稀释的病毒（ 步骤2.1）感染细胞。感染细胞1.5小时，在37℃和5％CO 2。 </li><li>感染后，删除病毒接种，加100微升感染媒体/室温孵育16 - 18小时。病毒再感染后可能会出现18小时孵化，这可能会导致高估病毒滴度。 </li><li>于16 - 18小时，感染后，从各孔中取出的TCS，在1x PBS稀释，在RT下15分钟，用4％甲醛固定细胞。 </li><li>接着，去除固定溶液和通透细胞用0.1％Triton X-100在1x PBS稀释，在室温下10分钟。 </li><li>吸出通透的解决方案，然后洗细胞3次无线日1X PBS。 </li><li>座的细胞用2.5％牛血清白蛋白（BSA）在1x PBS（封闭溶液），在RT下进行1小时。另外，细胞可在4℃下封闭过夜，并继续与抗体孵育翌日。 </li><li>同时准备主抗体。用封闭液稀释LCMV和偷拍＃1特异性第一抗体，然后再离心抗体/封闭溶液在3,500 rpm转速下持续15分钟。可用于检测的rLCMV和rCandid＃1的单克隆抗LCMV NP抗体克隆BEI的资源（1:500稀释），1.1.3（1:30稀释）16和单克隆抗JUNV的NP抗体SA02-BG12元。 </li><li>孵育1小时后，吸去封闭液，加入​​50μl的第一抗体的细胞，并温育1小时，在37℃下</li><li>在这个时候，用封闭液稀释的二级抗体，抗体/封闭溶液，在3,500 rpm转速的15分钟，然后离心。的聚克隆的兔抗小鼠IgG抗体FITC标记的第二抗体的初级单克隆抗体，用于检测用于取得在图5中观察到的图像。 </li><li>孵育1小时后，吸出一次抗体，用1×PBS洗涤3次，添加二次抗体30分钟，在37℃下</li><li>孵育30 min后，吸二次抗体和1X PBS洗3次。现在已经准备好待观察细胞用荧光显微镜，同时确定成功的病毒救援和滴定形成单位每毫升（FFU /毫升）计数的焦点。 </li></ol></li><li> r3LCMV和r3Candid＃1病毒救援：由于这些病毒表达基因的两个记者，他们的救助，可以监视通过荧光显微镜检测GFP的表达或TCS GLUC表达。救援也可以证实表达GLUC的TCS。要滴定trisegmented的病毒，我们按照步骤2.1至2.1.4，但细胞并不需要到bE固定，以确定救援，因为一个成功的病毒GFP的表达。病毒滴定将由计数FFU /毫升。要确定病毒救援成功的的GLUC表达，从TCS GLUC表达可以使用GLUC检测试剂盒（碧路Gaussia荧光素酶的检测试剂盒，New England Biolabs公司）测量。为此： <ol><li>到一个96孔白色板（平底微量滴定板中，Fisher Scientific公司）的移液管将100μl的TCS。 </li><li>设置的的光度计（LumiCount，惠普公司）。 </li><li>添加50 - 100微升GLUC测定溶液（制造商建议的）每个样品测量的的记者GLUC基因表达与光度计。作为阴性对照，血糖测定从野生型病毒感染的细胞表达的TCS。 </li></ol></li></ol> 3。组织培养上清液通道病毒救援取决于转染效率。 Vero细胞已被证明具有较低的转染效率比其它细胞系14。如果在TCS的病毒滴度低，0.01（LCMV）或0.1（坦率＃1）的感染复数（MOI）感染新鲜的Vero细胞72小时，以扩增病毒。

<ol>
	<li>Albarino, C. G., Bird, B. H., Chakrabarti, A. K., Dodd, K. A., Flint, M., Bergeron, E., White, D. M., Nichol, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+determinant+of+attenuation+in+mice+of+the+Candid1+vaccine+for+Argentine+hemorrhagic+fever+is+located+in+the+G2+glycoprotein+transmembrane+domain.">The major determinant of attenuation in mice of the Candid1 vaccine for Argentine hemorrhagic fever is located in the G2 glycoprotein transmembrane domain.</a> Journal of Virology. 85, 10404-10408 (2011).</li><li>Barton, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytic+choriomeningitis+virus:+a+neglected+central+nervous+system+pathogen.">Lymphocytic choriomeningitis virus: a neglected central nervous system pathogen.</a> Clinical Infectious Diseases: an official publication of the Infectious Diseases Society of America. 22, 197 (1996).</li><li>Buchmeier, M. J., Peter, C. J., de la Torre, J. C., Fields, B. N., 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=Arenaviridae:+The+viruses+and+their+replication.">Arenaviridae: The viruses and their replication.</a> Fields' Virology. 2, 179201827 (2007).</li><li>Emonet, S. E., Urata, S., de la Torre, 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=Arenavirus+reverse+genetics:+new+approaches+for+the+investigation+of+arenavirus+biology+and+development+of+antiviral+strategies.">Arenavirus reverse genetics: new approaches for the investigation of arenavirus biology and development of antiviral strategies.</a> Virology. 411, 416-425 (2011).</li><li>Emonet, S. F., Garidou, L., McGavern, D. B., de la Torre, 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=Generation+of+recombinant+lymphocytic+choriomeningitis+viruses+with+trisegmented+genomes+stably+expressing+two+additional+genes+of+interest.">Generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 3473-3478 (2009).</li><li>Fischer, S. A., Graham, M. B., Kuehnert, M. J., Kotton, C. N., Srinivasan, A., Marty, F. M., Comer, J. A., Guarner, J., Paddock, C. D., DeMeo, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+lymphocytic+choriomeningitis+virus+by+organ+transplantation.">Transmission of lymphocytic choriomeningitis virus by organ transplantation.</a> The New England Journal of Medicine. 354, 2235-2249 (2006).</li><li>Flatz, L., Bergthaler, A., de la Torre, J. C., Pinschewer, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+an+arenavirus+entirely+from+RNA+polymerase+I/II-driven+cDNA.">Recovery of an arenavirus entirely from RNA polymerase I/II-driven cDNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 4663-4668 (2006).</li><li>Gunther, S., Lenz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lassa+virus.">Lassa virus.</a> Critical reviews in clinical laboratory sciences. 41, 339-390 (2004).</li><li>Harrison, L. H., Halsey, N. A., McKee, K. T., Peters, C. J., Barrera Oro, J. G., Briggiler, A. M., Feuillade, M. R., Maiztegui, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+case+definitions+for+Argentine+hemorrhagic+fever.">Clinical case definitions for Argentine hemorrhagic fever.</a> Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 28, 1091-1094 (1999).</li><li>Isaacson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+hemorrhagic+fever+hazards+for+travelers+in+Africa.">Viral hemorrhagic fever hazards for travelers in Africa.</a> Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America. 33, 1707-1712 (2001).</li><li>Kerber, R., Rieger, T., Busch, C., Flatz, L., Pinschewer, D. D., Kummerer, B. M., Gunther, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+analysis+of+the+replication+complex+of+Old+World+arenaviruses+reveals+two+nucleoprotein+sites+involved+in+L+protein+function.">Cross-species analysis of the replication complex of Old World arenaviruses reveals two nucleoprotein sites involved in L protein function.</a> Journal of Virology. 85, 12518-12528 (2011).</li><li>Lee, K. J., Novella, I. S., Teng, M. N., Oldstone, M. B., de La Torre, 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=NP+and+L+proteins+of+lymphocytic+choriomeningitis+virus+(LCMV)+are+sufficient+for+efficient+transcription+and+replication+of+LCMV+genomic+RNA+analogs.">NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs.</a> Journal of Virology. 74, 3470-3477 (2000).</li><li>Mets, M. B., Barton, L. L., Khan, A. S., Ksiazek, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytic+choriomeningitis+virus:+an+underdiagnosed+cause+of+congenital+chorioretinitis.">Lymphocytic choriomeningitis virus: an underdiagnosed cause of congenital chorioretinitis.</a> American Journal of Ophthalmology. 130, 209-215 (2000).</li><li>Murakami, S., Horimoto, T., Yamada, S., Kakugawa, S., Goto, H., Kawaoka, Y. <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+canine+RNA+polymerase+I-driven+reverse+genetics+for+influenza+A+virus:+its+application+for+H5N1+vaccine+production.">Establishment of canine RNA polymerase I-driven reverse genetics for influenza A virus: its application for H5N1 vaccine production.</a> Journal of Virology. 82, 1605-1609 (2008).</li><li>Palacios, G., Druce, J., Du, L., Tran, T., Birch, C., Briese, T., Conlan, S., Quan, P. L., Hui, J., Marshall, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+arenavirus+in+a+cluster+of+fatal+transplant-associated+diseases.">A new arenavirus in a cluster of fatal transplant-associated diseases.</a> The New England Journal of Medicine. 358, 991-998 (2008).</li><li>Rodrigo, W. W., de la Torre, J. C., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+single-cycle+infectious+lymphocytic+choriomeningitis+virus+to+study+hemorrhagic+fever+arenaviruses.">Use of single-cycle infectious lymphocytic choriomeningitis virus to study hemorrhagic fever arenaviruses.</a> Journal of Virology. 85, 1684-1695 (2011).</li><li>Rojek, J. M., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+entry+by+human+pathogenic+arenaviruses.">Cell entry by human pathogenic arenaviruses.</a> Cellular Microbiology. 10, 828-835 (2008).</li><li>Rojek, J. M., Sanchez, A. B., Nguyen, N. T., de la Torre, J. C., Kunz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+mechanisms+of+cell+entry+by+human-pathogenic+Old+World+and+New+World+arenaviruses.">Different mechanisms of cell entry by human-pathogenic Old World and New World arenaviruses.</a> Journal of Virology. 82, 7677-7687 (2008).</li><li>Sanchez, A. B., de la Torre, 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=Rescue+of+the+prototypic+Arenavirus+LCMV+entirely+from+plasmid.">Rescue of the prototypic Arenavirus LCMV entirely from plasmid.</a> Virology. 350, 370-380 (2006).</li></ol>

代重组砂粒病毒使用基于质粒反向遗传学技术已经成为一种广泛使用的方法调查砂粒病毒生物学的许多不同的方面。在这里，我们记录到当前系统中的一个关键的改进，通过执行沙粒救援在Vero细胞中，允许对砂粒病毒和疫苗载体对其他传染病的潜在代FDA批准的候选疫苗。 实验过程涉及的一般完善，应该不会构成重大障碍。然而，有几个因素，应仔细监测，以确保一致的成功?...

与一个bisegmented，负链RNA基因组的3属于沙粒病毒科的的砂粒病毒是有包膜的病毒。沙粒病毒基因组编码的蛋白质在一个的时尚ambisense从两个不同的病毒分部3。大（L）段编码RNA依赖的RNA聚合酶（L）和小环（非常有趣的新基因）指蛋白（Z）作为病毒出芽的主要推动力。小段（S）编码病毒的核蛋白（NP）的表面糖蛋白（GP）（ 图1）。 沙粒病毒科的几位成员负责致命的出血热（HF）在人类3。原则问题是拉沙病毒（LASV）和胡宁病毒（JUNV），这是众所周知的导致高死亡率住院患者8，9。尽管这些病毒的西非和阿根廷农村地区，分别是地方性的，有越来越多的关注进口LASV JUNV的非流行地区由于旅游增加10。此外，尽管通常不伴有严重的人类疾病，被认为是原型沙粒淋巴细胞性脉络丛脑膜炎病毒（LCMV）一个被忽视的病原体已经有致死性感染的情况下，在免疫功能低下的患者6，15，先天性出生缺陷和自然流产负责孕妇中，13。目前，还没有美国FDA批准的疫苗的砂粒病毒和治疗是有限的，以三氮唑核苷核苷类似物，这是只是部分有效，并经常伴有显着的副作用。 基于质粒重组砂粒病毒7,19反向遗传学4代的推出，极大地推进了砂粒病毒研究领域。目前，啮齿动物细胞（如BHK-21）是用于根儿振动性的重组的砂粒病毒由于种属特异性的小鼠RNA聚合酶I（聚合酶-I）启动子指导的S和L段的初始转录。然而病毒拯救BHK-21细胞的代重组砂粒病毒作为潜在疫苗种子候选人未获批准的方法。在这里，我们记录使用人POL-I启动旧世界的原型（OW）LCMV和新世界（NW）JUNV偷拍＃1株在Vero细胞有效的救援。使用类似的方法，我们产生重组trisegmented LCMV（r3LCMV）和坦率＃1（＃1 r3Candid）的砂粒病毒，包含两个额外的编码的外源基因在两个不同的S RNA片段5。这个新的系统，不仅按照高度的重现性和简单的协议，但在生成的重组砂粒病毒作为的潜在疫苗或疫苗载体的种子，可以立即实施。

<table border="1">
	<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material and methods</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
 Cell lines 
Vero E6 (African green monkey kidney epithelial cells) are maintained in a 37 &deg;C incubator with 5 % CO2 in DMEM 10 % FBS 1 % PS. Cells are available from the American Type Culture Collection (ATCC, catalogue number CRL-1586). 
 
Plasmids 
All plasmids, with the exception of hpol-I L, can be grown at 37 &deg;C, for 16-18 hr. We recommend that cultures of the hpol-I L be grown at 30 &deg;C for 24 hr. Plasmids are prepared using a plasmid maxi kit (EZNA Fastfilter Plasmid Maxi Kit, Omega Bio-tek) following the manufacturer's recommendations and stored at -20 &deg;C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriate for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel electrophoresis. 
 
Viruses 
The described protocol for rescuing rLCMV (Armstrong 53b) and rCandid#1 can be executed under biosafety level (BSL) 2 conditions. Contaminated material, including TCS and cells should be sterilized before disposal. Rescue of other arenavirus may require higher BSL facilities so proper safety/security measures must be followed. 
 
Tissue culture media and solutions 
DMEM 10 %FBS 1 %PS: 445 ml Dulbecco's modified Eagle's medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4 &deg;C. This media will be used for maintenance of Vero cells. 
 
Infectious Media: 2-to-1 mixture of OptiMEM and DMEM 10 %FBS 1 %PS. Store at 4 &deg;C. This media will be used during viral infections. 
 
10X Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 liter. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
1X PBS: Dilute 10X PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
2.5 % BSA: 2.5 g of BSA in 97.5 ml of 1X PBS. Store at 4 &deg;C. This is used as a blocking solution for IFA.
</td>
 </tr>
 </tbody>
 </table>

generation of recombinant arenavirus for vaccine development in fda-approved vero cells

沙粒病毒反向遗传学的发展和实施代表的一个重大突破在沙粒领域4。沙粒病毒的生命周期的不同步骤的调查病毒的决定因素的贡献，以及病毒 - 宿主细胞为基础的沙粒一起能够产生预定在他们的基因组突变重组传染性砂粒病毒微型基因组系统的使用提供了便利相互作用和机制沙粒病毒发病机制1，3，11。此外，已经允许发展trisegmented砂粒病毒沙粒病毒基因组的表达还额外的外源基因的使用，从而打开的可能性的沙粒病毒为基础的疫苗载体的应用5。同样，单周期能够表达报告基因的传染性砂粒病毒的发展提供了一种新的实验工具，提高了安全性的研究，涉及极力Ÿ人类致病的砂粒病毒16。代重组砂粒病毒质粒为基础的反向遗传学技术至今依赖于啮齿动物细胞线使用7,19，这带来了一些障碍，发展食品和药物管理局（FDA）许可的疫苗或疫苗载体。为了克服这个障碍，我们在这里描述的高效生成重组砂粒病毒在FDA批准的Vero细胞。

抢救成功重组野生型沙粒将被确认使用IFA（ 图5）病毒抗原的存在。成功的病毒拯救的重组trisegmented病毒的情况下，可以评估通过观察GFP的表达，通过荧光显微镜观察（ 图6A）。抢救成功，将进一步证实通过评估GLUC的表达（ 图6B）。代表性的成果，说明抢救成功野生型和trisegmented的沙粒使用上述表示的协议。 ...

Watch this Scientific Journal Video about Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells at JoVE.com

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

救援重组砂粒病毒克隆的cDNA，称为反向遗传学的方法，使研究人员能够调查特定的病毒基因产物的作用，以及其不同的具体领域和残留物的贡献，砂粒病毒的生物学的许多不同方面。同样，反向遗传学技术在FDA批准的细胞系（Vero）疫苗研制的新一代安全有效的疫苗，打击人类致病的砂粒病毒提供了新的可能性。

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

generation-recombinant-arenavirus-for-vaccine-development-fda

Research

JoVE Journal

Immunology and Infection

16.8K 次观看.  University of Rochester School of Medicine and Dentistry. 救援重组砂粒病毒克隆的cDNA，称为反向遗传学的方法，使研究人员能够调查特定的病毒基因产物的作用，以及其不同的具体领域和残留物的贡献，砂粒病毒的生物学的许多不同方面。同样，反向遗传学技术在FDA批准的细胞系（Vero）疫苗研制的新一代安全有效的疫苗，打击人类致病的砂粒病毒提供了新的可能性。

视频: Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

使用黑色素瘤和鳞状癌干细胞生成新型支气干细胞疫苗

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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

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 mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

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 ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

重组呼吸道合胞病毒的生成、扩增和滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.