Institute of Human Virology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou 510080, China.
Department of Immunology, Zhongshan School of Medicine, and Key Laboratory of Tropical Disease Control of Ministry of Education, Sun Yat-Sen University, Guangzhou 510080, China.
Virus Res. 2018 Sep 15;257:14-24. doi: 10.1016/j.virusres.2018.08.016. Epub 2018 Aug 23.
Zika virus (ZIKV) is an Aedes mosquitoes-transmitted flavivirus, and its infection may cause severe neurological diseases. A genetically stable infectious clone is essential for ZIKV research, however the toxicity and instability of the viral cDNA in bacteria potentially due to its bacterial promoter activity are major challenges. Here, we constructed a full-length cDNA clone for isolate ZG01 by introducing non-coding changes T1865C/A1868G to reduce the bacterial promoter activity. Wild-type and recombinant ZG01 were highly attenuated in Vero cells, thus we serially passaged wild-type ZG01 through neonatal mice and Vero cells to generate high-titer virus, from which four mutations (4m, C2178T/G2913A/T4991C/T10561C) were identified. Addition of 4m greatly enhanced the infectivity, as ZG01_4m released ZIKV of 10-10 plaque-forming unit (PFU)/ml in infected Vero and A549 cells. ZG01_4m resembled the infectivity of high-titer ZG01 in vitro and in vivo. Notably, ZG01_4m plasmid was genetically stable after multiple rounds of transformation-purification in bacteria. Using ZG01_4m, we identified a potential RNA-RNA interaction between 5'UTR and 3'UTR and demonstrated that the nucleotides involved were essential for ZIKV production. The genetically stable ZG01 cDNA clone provides a reliable tool for the study of this important virus, and the strategy used here is feasible for the development of reverse genetics systems for other ZIKV isolates and related flaviviruses.
寨卡病毒（ZIKV）是一种经伊蚊传播的黄病毒，其感染可能导致严重的神经系统疾病。对于寨卡病毒的研究来说，一个遗传稳定的传染性克隆是至关重要的，然而，病毒 cDNA 在细菌中的毒性和不稳定性主要是由于其细菌启动子活性造成的，这是一个重大挑战。在这里，我们通过引入非编码变化 T1865C/A1868G 来降低细菌启动子活性，构建了用于分离株 ZG01 的全长 cDNA 克隆。野生型和重组 ZG01 在 Vero 细胞中高度减毒，因此我们通过新生小鼠和 Vero 细胞连续传代野生型 ZG01 以产生高滴度病毒，从中鉴定出四个突变（4m、C2178T/G2913A/T4991C/T10561C）。添加 4m 极大地增强了感染性，因为感染的 Vero 和 A549 细胞中 ZG01_4m 释放的寨卡病毒滴度为 10-10 噬菌斑形成单位（PFU）/ml。ZG01_4m 在体外和体内与高滴度 ZG01 的感染性相似。值得注意的是，ZG01_4m 质粒在细菌中的多次转化-纯化后遗传稳定。使用 ZG01_4m，我们鉴定出 5'UTR 和 3'UTR 之间存在潜在的 RNA-RNA 相互作用，并证明涉及的核苷酸对寨卡病毒的产生至关重要。遗传稳定的 ZG01 cDNA 克隆为研究这种重要病毒提供了可靠的工具，并且这里使用的策略对于开发其他寨卡病毒分离株和相关黄病毒的反向遗传学系统是可行的。
