来自新世界Zika病毒感染性克隆的重组病毒的救援和表征
Summary
该方案描述了从双质粒感染性cDNA克隆中恢复感染性Zika病毒。
Abstract
传染性cDNA克隆允许病毒的遗传操作，从而有助于疫苗，发病机理，复制，传播和病毒进化的工作。在这里，我们描述了Zika病毒（ZIKV）感染性克隆的构建，目前在美洲引起爆发性爆发。为了防止黄病毒衍生质粒通常观察到的对细菌的毒性，我们产生了一个双质粒系统，其将NS1基因的基因组分离，并且比不能在没有突变的情况下成功恢复的全长构建体更稳定。在消化和连接两个片段后，可以通过T7 RNA聚合酶的体外转录产生全长病毒RNA。在将转录的RNA电穿孔进入细胞后，分别在小鼠和蚊子中显示类似的体外生长动力学和体内毒力和感染表型的病毒。
Introduction
Zika病毒（ZIKV;家庭黄病毒科： 黄病毒属）是一种蚊子传播的黄病毒，于2013-14年抵达巴西，随后发生在美洲蔓延的发热性疾病的大规模爆发1 。此外，ZIKV已经与严重的疾病结果相关联，如成人的吉兰 – 巴雷综合征和胎儿和新生儿的小头症2 。在西半球迅速传播之前，关于ZIKV的知之甚少。这包括缺乏分子工具，从而阻碍机械研究。用于病毒的分子工具，例如感染性cDNA克隆，促进疫苗和抗病毒治疗发展，并允许评估与差异病毒发病机理，免疫应答和病毒进化相关的病毒遗传因素。
已知黄病毒感染性克隆由于cr而在细菌中高度不稳定其基因组中存在的原核启动子3 。已经采用了几种方法来改善这个问题;包括在病毒序列上游插入串联重复4 ，推定的原核启动子序列的突变5 ，将基因组分裂成多个质粒6 ，低拷贝数载体（包括细菌人造染色体） 7,8和内含子在病毒基因组9中的插入。 ZIKV已经描述了一个没有修改的全长系统;然而，该克隆似乎在细胞培养物和小鼠中减弱10 。其他组已经将内含子设计到ZIKV基因组中，允许细菌中不稳定序列的破坏，其可以在体外在哺乳动物细胞中剪接以产生感染性病毒11， 12 。另外，已经成功地使用了一种名为“传染性亚基因组扩增子”的基于PCR的系统来拯救ZIKV13原型MR766菌株。这里描述的方法不需要外来序列，而是通过使用先前已经成功用于黄热病6号 ，登革热14,15和西尼罗河病毒16的多个质粒来破坏高不稳定区域的基因组。此外，在病毒基因组末端添加丙型肝炎核酶序列有助于产生真实的3'端，而不需要添加线性化位点。另外，两种质粒都构建在低拷贝数载体（pACYC177，每个细胞约15个拷贝）中以减轻任何残留毒性17 。恢复的病毒显示出与之相当的增长特征包含来自哺乳动物和昆虫的各种细胞类型的8种细胞系中的亲代病毒在体外生长曲线中，并且在小鼠中表现出相同的致病性谱，并且在蚊子中具有感染，传播和传播率。
在这里，我们详细说明了如何生长感染性克隆质粒， 在体外产生全长病毒RNA（病毒基因组） 的方案，并在细胞培养物中回收感染性病毒。首先，我们描述了使用滚环扩增（RCA）的细菌在细菌或无细菌扩增中的繁殖。接下来，我们展示两种质粒如何消化，然后连接在一起以产生全长病毒。最后，我们描述转录的RNA的生产及其随后的电穿孔进入Vero细胞，然后滴定回收的病毒（ 图1 ）。描述的方法是快速的，允许fo在1-2周内恢复感染性病毒库存。
Protocol
Representative Results
Discussion
Here we describe a method for the recovery of a bipartite infectious cDNA clone system for ZIKV. Previously described clones for ZIKV suffer from either attenuation or require the addition of introns, making plasmids larger and preventing rescue in insect cells. Infectious virus can be recovered using the two-plasmid clone system in either mammalian or insect cells (data not shown). In addition, virus recovered from this system behaves similarly to wild-type virus in several cell lines, in an immunocompromised mouse mode…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢克里斯汀·布拉德·费贝尔曼，米拉娜·维塞利诺维奇和克劳迪娅·鲁克特（ClaudiaRückert）的帮助，以表征克隆衍生的病毒。这项工作部分得到NIH国家过敏和传染病研究所赠款AI114675（BJG）和AI067380（GDE）的支持。
Materials
| NEB Stable CompetentE. coli | New England BioLabs | C3040H | |
| Carbenicillin, Disodium Salt | various | ||
| Zyppy Plasmid Miniprep Kit | Zymo Research | D4036 | |
| ZymoPURE Plasmid Maxiprep Kit | Zymo Research | D4202 | |
| SalI-HF | New England BioLabs | R3138S | 20,000 units/ml |
| NheI-HF | New England BioLabs | R3131S | 20,000 units/ml |
| ApaLI | New England BioLabs | R0507S | 10,000 units/ml |
| EcoRI-HF | New England BioLabs | R3101S | 20,000 units/ml |
| BamHI-HF | New England BioLabs | R3136S | 20,000 units/ml |
| HindIII-HF | New England BioLabs | R3104S | 20,000 units/ml |
| illustra TempliPhi 100 Amplification Kit | GE Healthcare Life Sciences | 25640010 | |
| NucleoSpin Gel and PCR Clean-up | Macherey-Nagel | 740609.5 | |
| Shrimp Alkaline Phosphatase (rSAP) | New England BioLabs | M0371S | 1,000 units/ml |
| Alkaline Phosphatase, Calf Intestinal (CIP) | New England BioLabs | M0290S | 10,000 units/ml |
| T4 DNA Ligase | New England BioLabs | M0202S | 400,000units/mL |
| HiScribe T7 ARCA mRNA Kit | New England BioLabs | E2065S | |
| Vero cells | ATCC | CCL-81 | |
| ECM 630 High Throughput Electroporation System | BTX | 45-0423 | Other machines are acceptable. |
| LB Broth with agar (Miller) | Sigma | L3147 | Can be homemade as well. |
| Terrific Broth | Sigma | T0918 | Can be homemade as well. |
| Petri Dish | Celltreat | 229693 | |
| Culture Tubes | VWR International | 60818-576 | |
| T75 flasks | Celltreat | 229340 | |
| T182 flasks | Celltreat | 229350 | |
| 1x PBS | Corning | 21-040-CV | |
| RPMI 1640 with L-glutamine | Corning | 10-040-CV | |
| DMEM with L-glutamine and 4.5 g/L glucose | Corning | 10-017-CV | |
| Fetal Bovine Serum (FBS) | Atlas Biologicals | FP-0500-A | |
| Tragacanth Powder | MP Bio | MP 104792 | |
| Crystal Violet | Amresco | 0528-1006 | |
| Ethanol Denatured | VWR International | BDH1156-1LP | |
| 6 well plate | Celltreat | 229106 | |
| 12 well plate | Celltreat | 229111 | |
| Sequencing Oligos | IDT | see table 1 | |
| Qubit 3.0 | ThermoFisher | Qubit 3.0 | other methods are acceptable. |
| Qubit dsDNA BR Assay Kit | ThermoFisher | Q32850 | other methods are acceptable. |
| Qubit RNA HS Assay Kit | ThermoFisher | Q32852 | other methods are acceptable. |
| Class II Biosafety Cabinet | Varies | N/A | This is necessary for live-virus work. |
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