Wang Min, Yan Jingjing, Zhu Liuyao, Wang Meng, Liu Lizhen, Yu Rui, Chen Ming, Xun Jingna, Zhang Yuling, Yi Zhigang, Zhang Shuye
Shanghai Public Health Clinical Center and Institute of Biomedical Sciences, Fudan University, Shanghai, 201508, China.
Virol Sin. 2020 Aug;35(4):426-435. doi: 10.1007/s12250-020-00198-2. Epub 2020 Mar 6.
Coxsackievirus A10 (CVA10) is one of the major etiological agents of hand, foot, and mouth disease. There are no vaccine and antiviral drugs for controlling CVA10 infection. Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals. Here, two infectious clones for the prototype and a Myc-tagged CVA10 were constructed. Viable CVA10 viruses were harvested by transfecting the viral mRNA into human rhabdomyosarcoma (RD) cells. Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally. We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter, respectively. Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293T (HEK293T) cells led to the production of single round infectious particles (SRIPs). Based on CVA10 replicon RNA, SRIPs with either the enterovirus A71 (EVA71) capsid or the CVA10 capsid were generated. Infection by EVA71 SRIPs required SCARB2, while CVA10 SRIPs did not. Finally, we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme (HHRib) before the 5'-untranslated region (UTR). In summary, reverse genetic tools for prototype strain of CVA10, including both the infectious clone and the SRIPs system, were successfully established. These tools will facilitate the basic and translational study of CVA10.
柯萨奇病毒A10（CVA10）是手足口病的主要病原体之一。目前尚无用于控制CVA10感染的疫苗和抗病毒药物。CVA10的反向遗传工具将有助于其机制研究以及疫苗和抗病毒药物的开发。在此，构建了原型CVA10和带有Myc标签的CVA10的两个感染性克隆。通过将病毒mRNA转染到人横纹肌肉瘤（RD）细胞中收获了有活力的CVA10病毒。通过下一代测序进一步确认了拯救的CVA10并进行了实验表征。我们还分别构建了带有荧光素酶报告基因的CVA10亚基因组复制子载体和带有EGFP报告基因的病毒衣壳载体。在人胚肾293T（HEK293T）细胞中共转染病毒复制子RNA和衣壳表达载体导致产生单轮感染性颗粒（SRIPs）。基于CVA10复制子RNA，产生了带有肠道病毒A71（EVA71）衣壳或CVA10衣壳的SRIPs。EVA71 SRIPs感染需要SCARB2，而CVA10 SRIPs则不需要。最后，我们通过在5'-非翻译区（UTR）之前插入顺式活性锤头状核酶（HHRib）显示出复制子活性和SRIPs产生的极大改善。总之，成功建立了CVA10原型株的反向遗传工具，包括感染性克隆和SRIPs系统。这些工具将促进CVA10的基础研究和转化研究。
