Xu Lin-Lin, Shan Chao, Deng Cheng-Lin, Li Xiao-Dan, Shang Bao-Di, Ye Han-Qing, Liu Si-Qing, Yuan Zhi-Ming, Wang Qing-Yin, Shi Pei-Yong, Zhang Bo
Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China.
Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China; Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
J Virol Methods. 2015 Jul;219:62-66. doi: 10.1016/j.jviromet.2015.03.020. Epub 2015 Apr 2.
We report a stable Gaussia luciferase enterovirus 71 (Gluc-EV71) reporter virus to facilitate drug discovery. The Gluc-EV71 reporter virus was generated by engineering the Gaussia luciferase (Gluc) gene between the 5' untranslated region and VP4 gene of the EV71 genome. We could recover Gluc-EV71 after transfection of Vero cells with the cDNA clone-derived RNA. The reporter virus efficiently infects and replicates in various cell types (Vero, human rhabdomyosarcoma, and HeLa cells), producing robust luciferase activity. The Gluc-EV71 virus replicates slower than the wild-type virus in cell culture. The reporter virus is stable in maintaining the Gluc gene after five rounds of continuous passaging in Vero cells. Using known EV71 inhibitors, we demonstrate that the reporter virus can be used for antiviral testing. However, the Gluc-EV71 infection assay cannot be adapted to a homogenous format for high throughput screen, mainly due to the secreted nature of the Gluc protein and the short half-life of the Gluc luminescence signal. The Gluc-EV71 and its infection assay could be useful for antiviral drug discovery as well as for studying EV71 replication and pathogenesis.
我们报道了一种稳定的高斯荧光素酶肠道病毒71型（Gluc-EV71）报告病毒，以促进药物研发。Gluc-EV71报告病毒是通过在肠道病毒71型基因组的5'非翻译区和VP4基因之间构建高斯荧光素酶（Gluc）基因而产生的。用cDNA克隆衍生的RNA转染Vero细胞后，我们能够回收Gluc-EV71。该报告病毒能在多种细胞类型（Vero细胞、人横纹肌肉瘤细胞和HeLa细胞）中有效感染和复制，产生强大的荧光素酶活性。在细胞培养中，Gluc-EV71病毒的复制速度比野生型病毒慢。在Vero细胞中连续传代五次后，该报告病毒在维持Gluc基因方面是稳定的。使用已知的肠道病毒71型抑制剂，我们证明该报告病毒可用于抗病毒测试。然而，Gluc-EV71感染检测法无法适用于高通量筛选的均相形式，主要是由于Gluc蛋白的分泌性质和Gluc发光信号的半衰期较短。Gluc-EV71及其感染检测法可用于抗病毒药物研发以及研究肠道病毒71型的复制和发病机制。
