Weger-Lucarelli James, Duggal Nisha K, Brault Aaron C, Geiss Brian J, Ebel Gregory D
Department of Microbiology, Immunology, and Pathology, Colorado State University.
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention.
J Vis Exp. 2017 Jun 7(124):55857. doi: 10.3791/55857.
Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.
感染性 cDNA 克隆可对病毒进行基因操作，从而有助于开展疫苗、发病机制、复制、传播及病毒进化等方面的研究。在此，我们描述了寨卡病毒（ZIKV）感染性克隆的构建，该病毒目前正在美洲地区引发爆发性疫情。为防止黄病毒衍生质粒常见的对细菌的毒性，我们构建了一个双质粒系统，该系统在 NS1 基因处将基因组分开，比全长构建体更稳定，全长构建体若无突变则无法成功回收。经消化和连接使两个片段相连后，可通过 T7 RNA 聚合酶体外转录生成全长病毒 RNA。将转录的 RNA 电穿孔导入细胞后，分别在小鼠和蚊子中回收了具有相似体外生长动力学以及体内毒力和感染表型的病毒。
