Mutso Margit, Saul Sirle, Rausalu Kai, Susova Olga, Žusinaite Eva, Mahalingam Suresh, Merits Andres
Institute of Technology, University of Tartu, Tartu, Estonia.
Institute for Glycomics, Griffith University, Gold Coast Campus, Southport, 4222, Queensland, Australia.
J Gen Virol. 2017 Nov;98(11):2712-2724. doi: 10.1099/jgv.0.000938. Epub 2017 Oct 12.
Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.
寨卡病毒（ZIKV，黄病毒属）已成为一种主要通过蚊子传播的人类病原体，近期的疫情爆发与神经并发症发病率的增加有关，尤其是小头畸形和吉兰 - 巴雷综合征。由于该病毒直到最近才成为一种重要的病原体，缺乏可靠的分子工具阻碍了研究进展。在此，我们报告了来自巴西的寨卡病毒分离株BeH819015的感染性cDNA（icDNA）克隆，该克隆被选为在疫情爆发早期分离的南美寨卡病毒的代表。icDNA克隆由与BeH819015分离株的共有序列相对应的合成DNA片段组装而成。从icDNA克隆拯救出的病毒具有与来自南美的天然寨卡病毒分离株相同的特性。获得了克隆衍生病毒的变体，这些变体在哺乳动物和昆虫细胞中表达纳米荧光素酶、增强型绿色荧光蛋白或mCherry标记蛋白，并且在多次体外传代中基因稳定。构建了一个寨卡病毒亚基因组复制子，其缺少编码prM和E糖蛋白的区域并表达高斯荧光素酶标记，结果表明该复制子在哺乳动物和昆虫细胞中均能复制。在表达寨卡病毒结构蛋白的塞姆利基森林病毒复制子存在的情况下，寨卡病毒复制子被包装成病毒 - 复制子颗粒。高效的反向遗传系统、基因稳定的标记病毒和包装的复制子为寨卡病毒感染和疾病的生物学研究以及抗病毒方法的开发提供了显著的改进。
