Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone.
Rescue of the 1947 Zika Virus Prototype Strain with a Cytomegalovirus Promoter-Driven cDNA Clone.
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DOI：
10.1128/msphere.00246-16
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发表时间：
2016-09
期刊：
影响因子：
4.8
通讯作者：
Evans MJ
中科院分区：
文献类型：
--
作者：
Schwarz MC;Sourisseau M;Espino MM;Gray ES;Chambers MT;Tortorella D;Evans MJ
ZIKV的研究随着最近该病毒与小头畸形和格林-巴利综合征的关联而变得越来越重要，将受益于基因操纵病毒的有效策略。本工作描述了一种在细胞培养中产生感染性病毒的模型系统。我们创建了携带原型1947乌干达MR 766 ZIKV基因组的质粒，该质粒在细菌中稳定，并且可以通过直接递送该DNA在哺乳动物细胞中产生高水平的感染性病毒。此外，该拯救的病毒的生长特性与其来源的病毒分离株的生长特性非常相似。该模型系统将提供一种简单有效的手段来研究ZIKV遗传学如何影响病毒复制和发病机制。最近的寨卡病毒（ZIKV）爆发与严重的发病机制有关。在这里，我们报告了携带巨细胞病毒（CMV）启动子表达的原型1947乌干达MR 766 ZIKV cDNA的质粒的构建，其可以在哺乳动物细胞的直接质粒DNA转染后启动感染。在ZIKV多蛋白的非结构蛋白1（NS 1）区域中掺入合成内含子降低了细菌中病毒cDNA相关的毒性。在转染携带野生型MR 766 ZIKV基因组的质粒后产生高水平的感染性病毒，但不是具有对病毒非结构蛋白5（NS 5）聚合酶活性位点的破坏的质粒。多细胞生长曲线和空斑测定实验表明，由质粒转染产生的MR 766病毒表现出比先前公布的2010年柬埔寨和2015年巴西cDNA拯救的ZIKV更类似于其亲本分离株的生长特征。该ZIKV感染性克隆将可用于研究ZIKV感染和发病机制的遗传决定因素，并且应该适合于构建表达报告蛋白并代表一系列ZIKV分离株的多种感染性克隆。重要性ZIKV的研究随着最近该病毒与小头畸形和格林-巴利综合征的关联而变得越来越重要，将受益于一种有效的遗传操纵病毒的策略。本工作描述了一种在细胞培养中产生感染性病毒的模型系统。我们创建了携带原型1947乌干达MR 766 ZIKV基因组的质粒，该质粒在细菌中稳定，并且可以通过直接递送该DNA在哺乳动物细胞中产生高水平的感染性病毒。此外，该拯救的病毒的生长特性与其来源的病毒分离株的生长特性非常相似。该模型系统将提供一种简单有效的手段来研究ZIKV遗传学如何影响病毒复制和发病机制。
The study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and pathogenesis. The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. This ZIKV infectious clone will be useful for investigating the genetic determinants of ZIKV infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of ZIKV isolates. IMPORTANCE The study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and pathogenesis.