Ávila-Pérez Ginés, Park Jun-Gyu, Nogales Aitor, Almazán Fernando, Martínez-Sobrido Luis
Department of Microbiology and Immunology, University of Rochester Medical Center.
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid.
J Vis Exp. 2019 Jun 24(148). doi: 10.3791/59537.
The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial propagation.
在最近的全球疫情中，寨卡病毒（ZIKV）感染与神经并发症相关联，且缺乏获批的疫苗和/或抗病毒药物，这凸显了开发ZIKV反向遗传系统以促进ZIKV生物学研究以及治疗和/或预防方法开发的迫切需求。然而，与其他黄病毒一样，由于病毒序列在细菌中扩增时的毒性，ZIKV全长感染性cDNA克隆的产生受到了阻碍。为克服这一问题，我们开发了一种基于细菌人工染色体（BAC）使用的非传统方法。利用这种方法，寨卡病毒北里奥格兰德·纳塔尔毒株（ZIKV-RGN）的全长cDNA拷贝由四个合成DNA片段生成，并在人巨细胞病毒（CMV）立即早期启动子的控制下组装到单拷贝pBeloBAC11质粒中。组装后的BAC cDNA克隆在细菌繁殖过程中是稳定的，并且在转染BAC cDNA克隆后，在Vero细胞中可回收感染性重组（r）ZIKV。本文所述方案为包括ZIKV在内的黄病毒以及其他正链RNA病毒，特别是那些在细菌繁殖过程中存在稳定性问题的大基因组病毒的感染性克隆的产生提供了一种强大的技术。
