Summary

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Published: June 07, 2017
doi:

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;家庭病毒科: 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

传染性克隆质粒的转化和恢复 使用商业转化方案( 例如 ,NEB 5分钟转化协议)转化两种质粒(单独)并进行一些修改。两种质粒含有编码氨苄青霉素抗性的基因,因此使用氨苄青霉素或羧苄青霉素进行选择。碳青霉素是较为稳定的。 从-80°C冰箱中取出细胞(参见材料表),并在冰上解冻5-10分钟。在37℃下,预热溶血培养液(LB)(含有10g / L NaCl和25μg/ mL羧苄青霉…

Representative Results

这里描述的方案允许恢复感染性克隆衍生的Zika病毒。与高度不稳定的全长版本(数据未显示)相比,操作双质粒感染性克隆系统在小心执行时是简单的。在消化和连接两个不同的片段后,使用T7聚合酶的体外转录产生加帽的RNA,然后将其电穿孔至Vero细胞( 图1 )。在使用RCA或maxiprep进行大规模DNA生产后,可以使用限制性消化作为代谢物监测正?…

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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Citazione di questo articolo
Weger-Lucarelli, J., Duggal, N. K., Brault, A. C., Geiss, B. J., Ebel, G. D. Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone. J. Vis. Exp. (124), e55857, doi:10.3791/55857 (2017).

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