在FDA批准的Vero细胞疫苗开发产生重组沙粒
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Material and methods | |||
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Cell lines Vero E6 (African green monkey kidney epithelial cells) are maintained in a 37 °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 °C, for 16-18 hr. We recommend that cultures of the hpol-I L be grown at 30 °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 °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 °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 °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 °C. This is used as a blocking solution for IFA. |
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