The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene.
The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene.
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DOI：
10.3389/fmicb.2022.1101850
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发表时间：
2022
影响因子：
5.2
通讯作者：
Zhang, Shuye
中科院分区：
文献类型：
--
作者：
Yu, Rui;Wang, Min;Liu, Lizhen;Yan, Jingjing;Fan, Jun;Li, Xiaohong;Kang, Miaomiao;Xu, Jianqing;Zhang, Xiaoyan;Zhang, Shuye
柯萨奇病毒A16（CA 16）属于人类肠道病毒A型，是引起儿童手足口病的常见病原体。目前，针对CA 16的特异性疫苗和药物不可用，进一步了解该病毒并发明有效治疗方法的需求尚未得到满足。构建带有报告基因的CA 16感染性克隆，将为CA 16的病毒学研究提供极大的便利。在这里，我们首次报道了CA 16感染性克隆（rCA 16）的构建，其后代在乳鼠中具有高度复制性和毒性。在rCA 16的基础上，我们进一步插入NanoLuc（Nluc）报告基因，并制备了rCA 16-Nluc克隆。我们发现rCA 16-Nluc中的Nluc基因在Vero细胞中连续生长期间保持稳定，因此可以在10代内检测到rCA 16-Nluc感染的Vero细胞中稳定的荧光素酶信号。其在抗病毒表征和高通量筛选中的应用通过测量盐酸胍、利巴韦林、氯喹和氯化铵对抗CA 16的IC 50、CC 50和选择指数来举例说明。最后，我们表明基于rCA 16-Nluc的测定大大简化了CA 16中和抗体测试。因此，这两个CA 16感染性克隆将是未来肠道病毒研究和抗病毒药物开发的强大工具。
Coxsackievirus A16 (CA16) belongs to the Human Enterovirus A species, which is a common pathogen causing hand, foot, and mouth disease in children. Currently, specific vaccines and drugs against CA16 are unavailable, and there is an unmet need to further understand the virus and invent effective treatment. Constructing a CA16 infectious clone with a reporter gene will greatly facilitate its virological studies. Here, we first reported the construction of a CA16 infectious clone (rCA16) whose progeny is highly replicative and virulent in suckling mice. On the basis of rCA16, we further inserted a NanoLuc (Nluc) reporter gene and made the rCA16-Nluc clone. We found that the Nluc gene in rCA16-Nluc is stable during continuous growing in Vero cells and thus allowed detection of a steady luciferase signal in rCA16-Nluc-infected Vero cells over 10 passages. Its application in antivirals characterization and high-throughput screening is exemplified by measuring IC50, CC50, and selection index of guanidine hydrochloride, ribavirin, chloroquine, and ammonium chloride against CA16. Finally, we showed that rCA16-Nluc based assay greatly simplified the CA16 neutralizing antibody tests. Thus, these two CA16 infectious clones will be robust tools for future enterovirus studies and antivirals development.
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