利用反向遗传学操作的裂谷热病毒MP - 12菌株，以提高疫苗的安全性和有效性的NSS基因
Summary
裂谷热病毒的MP – 12疫苗株反向遗传学系统是一个有用的工具，用于创建附加的MP – 12突变体具有更高的衰减性和免疫原性。我们描述了协议的产生和表征NSS突变株。
Abstract
裂谷热病毒（RVFV），从而导致出血热，神经系统疾病，或在人类中的盲目性，以及高利率流产和反刍动物的胎儿畸形1，已被列为作为HHS /美国农业部重叠选择代理和风险组3病原体。它属于在家庭Bunyaviridae属Phlebovirus，是这个家庭的最致命的成员之一。自2006年以来已开发的RVFV MP – 12疫苗株2,3以及野生型RVFV 4-6株，包括ZH548和ZH501，几个反向遗传学系统。 MP – 12株（这是一个危险群2病原体和非选择代理）是由几个突变，其M -和L段高度减毒，但仍带有剧毒分部的S – 3，其编码的RNA功能的毒力因素，NSS。 rMP12 C13type（C13type）进行帧删除NSS的ORF 69％缺乏所有已知的NSS功能，而它复制为efficient作为MP – 12在VeroE6细胞缺乏I型干扰素。 NSS包括干扰素（IFN）-βmRNA的7,8诱导的转录主机关闭，并促进在翻译后水平降解双链RNA依赖的蛋白激酶（PKR）9,10 IFN -β转录干扰素调节因子3（IRF – 3），NF – kB和激活蛋白-1（AP – 1），IFN -β结合IFN-alpha/beta受体（IFNAR）上调刺激IFN -α的转录虽然IRF – 3，NF – kB和激活的基因或其他干扰素刺激基因（ISGs）11，诱导宿主的抗病毒活性，而主机包括NSS IFN -β基因的转录抑制阻止病毒复制的基因upregulations这些ISGs蛋白1（AP – 1），可以通过RVFV7激活。 。因此，NSS是一个很好的的目标，以进一步削弱MP – 12，并取消IFN -β的抑制功能，以提高主机的先天免疫反应。这里，我们描述了产生重组MP – 12的编码突变NSS的协议，并提供一种检查方法，找出缺乏的功能，抑制IFN -βmRNA的合成NSS突变体的一个例子。除了 其在先天免疫系统的重要作用，I型干扰素是重要的树突状细胞诱导适应性免疫反应 12-14的成熟。因此，NSS突变体诱导I型干扰素的进一步减弱，但在同一时间在刺激宿主的免疫反应更有效率比野生型MP – 12，这使得它们接种疫苗的方法理想人选。
Protocol
1。 2质粒DNA的重组的MP – 12编码NSS突变（S）的回收传播幼仓鼠肾（BHK）/ T7 – 15 9细胞，稳定表达T7 RNA聚合酶，到6厘米的菜最起码的基本介质中（MEM）-α（Invitrogen公司，CAT＃32561037），含10％胎牛血清（ FBS ），青霉素，链霉素（青霉素100 U /毫升，链霉素100微克/毫升）（Invitrogen公司，CAT＃15140122），和600微克/ ml的潮霉素B（Cellgro，猫＃30 – 240 – CR）。 *病毒回收效率在6厘…
Discussion
为RVFV反向遗传学系统已经开发了几个小组利用T7启动子2,4,5或鼠标3人4 POL -我子。在这个手稿中，我们描述了一个协议， 使用15个稳定表达T7 RNA聚合酶BHK/T7-9细胞产生重组RVFV MP – 12菌株。不同的病毒回收效率取决于BHK/T7-9细胞的条件，质粒量，转染细胞和数量等。我们总是放大的P0病毒在Vero E6细胞，获得高滴度实验的病毒储存。 I型干扰素主管的细胞，如人肺二倍体?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由赠款5号U54通过西方优秀的区域中心（WRCE）AI057156 – 07，R01从国家过敏和传染病研究所AI08764301 – A1，西利疫苗开发中心，在大学内部资金德克萨斯州医学科。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Minimum Essential Medium (MEM)-alpha | Invitrogen | 32561037 | |
| Dulbecco’s modified minimum essential medium | Invitrogen | 11965092 | |
| Modified Eagle Medium (MEM 2x) | Invitrogen | 11935046 | |
| Penicillin-Streptomycin | Invitrogen | 15140122 | |
| Hygromycin B | Cellgro | 30-240-CR | |
| Tryptose phosphate broth | MP biomedicals | 1682149 | |
| Noble agar | VWR | 101170-362 | |
| TransIT-LT1 | Mirus | MIR2300 | |
| Opti-MEM | Invitrogen | 31985070 | |
| Aerosol tight lid | Eppendorf | C-2223-25 | |
| 0.33% neutral red solution | Sigma Aldrich | N2889-100ML | |
| C57/WT MEF cells | InvivoGen | mef-c57wt | |
| Blasticidin S | InvivoGen | Ant-bl-1 | |
| Zeocin | InvivoGen | ant-zn-1 | |
| QUANTI-Blue | InvivoGen | rep-qb1 | |
| BHK/T7-9 cells15 | Gifu university, Japan | ||
| Vero E6 cells | ATCC | CRL-1586 |
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Tags
Reverse GeneticsNSs GeneRift Valley Fever VirusMP-12 StrainVaccine SafetyVaccine EfficacyHemorrhagic FeverNeurological DisordersBlindnessAbortionFetal MalformationHHS/USDA Overlap Select AgentRisk Group 3 PathogenPhlebovirusBunyaviridae FamilyVirulentRVFV MP-12 Vaccine StrainWild-type RVFV StrainsZH548ZH501M-segmentL-segmentS-segment RNA3Functional Virulence Factor NSsRMP12-C13typeIn-frame Deletion Of NSs ORFHost Transcription Shut-offInterferon (IFN)-beta MRNA78Double-stranded RNA-dependent Protein Kinase (PKR)Post-translational Level Degradation Of PKR
Cite This Article
Kalveram, B., Lihoradova, O., Indran, S. V., Ikegami, T. Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy. J. Vis. Exp. (57), e3400, doi:10.3791/3400 (2011).