Zika virus is becoming one of the most widely transmitted arboviruses in the world. Development of antiviral inhibitor and vaccine requires an experimental system that allows rapid monitoring of the virus infection. This is achievable with a reverse genetic system. In this study, we constructed an infectious clone for Zika virus that stably expressing EGFP. A PCR-mediated recombination approach was used to assemble the full-length Zika virus genome containing the CMV promoter, intron, EGFP, hepatitis delta virus ribozyme, and SV40 terminator sequence for cloning into the pBAC11 vector to produce recombinant pBAC-ZIKA-EGFP. ZIKA-EGFP virus was rescued by transfection of pBAC-ZIKA-EGFP into 293T cells. The characterization of ZIKA-EGFP virus was determined by qPCR, plaque assay, CCK-8, and Western blot. Rescued ZIKA-EGFP virus exhibited stable replication for at least five generations in tissue culture. ZIKA-EGFP can effectively infect C6/36, SH-SY5Y and Vero cells, and cause cytopathic effects on SH-SY5Y and Vero cells. The inhibition of ZIKA-EGFP by NF-κB inhibitor, caffeic acid phenethyl ester was observed by fluorescence microscopy. Our results suggested that Zika virus infectious clone with an EGFP marker retained it infectivity as wide-type Zika virus which could be used for drugs screening.

中文翻译：

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在真核表达系统中稳定表达 EGFP 标记的寨卡病毒感染性克隆的构建

寨卡病毒正在成为世界上传播最广泛的虫媒病毒之一。开发抗病毒抑制剂和疫苗需要一个能够快速监测病毒感染的实验系统。这可以通过反向遗传系统来实现。在这项研究中，我们构建了一个稳定表达 EGFP 的寨卡病毒感染性克隆。使用 PCR 介导的重组方法组装包含 CMV 启动子、内含子、EGFP、丁型肝炎病毒核酶和 SV40 终止子序列的全长寨卡病毒基因组，用于克隆到 pBAC11 载体中以产生重组 pBAC-ZIKA-EGFP。通过将 pBAC-ZIKA-EGFP 转染到 293T 细胞中来拯救 ZIKA-EGFP 病毒。ZIKA-EGFP 病毒的表征通过 qPCR、噬斑测定、CCK-8 和蛋白质印迹确定。获救的 ZIKA-EGFP 病毒在组织培养中表现出至少五代的稳定复制。ZIKA-EGFP 可有效感染 C6/36、SH-SY5Y 和 Vero 细胞，并对 SH-SY5Y 和 Vero 细胞造成细胞病变作用。荧光显微镜观察了NF-κB抑制剂咖啡酸苯乙酯对ZIKA-EGFP的抑制作用。我们的研究结果表明，带有EGFP标记的寨卡病毒感染性克隆保留了其作为广泛型寨卡病毒的感染性，可用于药物筛选。