Liu Zhong-Yu, Yu Jiu-Yang, Huang Xing-Yao, Fan Hang, Li Xiao-Feng, Deng Yong-Qiang, Ji Xue, Cheng Meng-Li, Ye Qing, Zhao Hui, Han Jian-Feng, An Xiao-Ping, Jiang Tao, Zhang Bo, Tong Yi-Gang, Qin Cheng-Feng
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
Anhui Medical University, Hefei, China.
J Virol. 2017 Oct 13;91(21). doi: 10.1128/JVI.00484-17. Print 2017 Nov 1.
Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential -acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the host. Moreover, two crucial -acting replication elements (5'-SLA and 5'-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.
寨卡病毒（ZIKV）最近在美洲引发了大规模疫情，因其能够导致小头畸形和其他神经并发症而引起全球关注。迫切需要一种稳定且高效的寨卡病毒感染性克隆。然而，黄病毒cDNA克隆在宿主中的不稳定性和毒性阻碍了寨卡病毒感染性克隆的开发。在此，我们采用一种基于新型自我剪接核酶的策略，构建了一个2016年从委内瑞拉输入中国的当代寨卡病毒毒株的稳定感染性cDNA克隆。构建的克隆在E基因和NS1基因交界处附近含有II组自我剪接内含子的修饰版本，通过易于建立的剪接反应可将其从RNA转录本中去除。将剪接后的RNA转染至BHK - 21细胞可产生类似于亲本病毒的感染性子代病毒。最后，基于这一新型反向遗传学系统鉴定了寨卡病毒基因组RNA中的潜在顺式作用RNA元件，并通过多种不同实验方法确定了5'-SLA启动子和5'-3'环化序列的关键作用。我们的研究结果为寨卡病毒提供了另一种稳定可靠的反向遗传学系统，有助于研究寨卡病毒感染和发病机制，且基于新型自我剪接内含子的策略可进一步扩展用于构建其他新出现和重新出现的黄病毒的感染性克隆。持续的寨卡病毒（ZIKV）疫情因与胎儿小头畸形及其他严重神经并发症之间意外的因果关系而引起全球关注。寨卡病毒的感染性cDNA克隆对于研究界研究该病毒、了解疾病以及指导疫苗设计和抗病毒筛选至关重要。一系列现有技术已被用于开发寨卡病毒感染性克隆。在此，我们利用一种基于新型自我剪接核酶的技术成功构建了一个2016年寨卡病毒毒株的稳定感染性克隆，该技术消除了寨卡病毒cDNA克隆对宿主的潜在毒性。此外，利用这一新型反向遗传学系统首次鉴定了寨卡病毒的两个关键顺式作用复制元件（5'-SLA和5'-CS）。这种基于新型自我剪接核酶的反向遗传学平台将在未来寨卡病毒研究中得到广泛应用，并为开发其他新出现病毒的感染性克隆提供思路。
