Yu Rui, Wang Min, Liu Lizhen, Yan Jingjing, Fan Jun, Li Xiaohong, Kang Miaomiao, Xu Jianqing, Zhang Xiaoyan, Zhang Shuye
Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
Clinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai, China.
Front Microbiol. 2023 Jan 10;13:1101850. doi: 10.3389/fmicb.2022.1101850. eCollection 2022.
Coxsackievirus A16 (CA16) belongs to the , 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 IC, CC, 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.
柯萨奇病毒A16型（CA16）属于 ，是引起儿童手足口病的常见病原体。目前，尚无针对CA16的特异性疫苗和药物，因此迫切需要进一步了解该病毒并研发有效的治疗方法。构建带有报告基因的CA16感染性克隆将极大地促进其病毒学研究。在此，我们首次报道了CA16感染性克隆（rCA16）的构建，其后代在乳鼠中具有高度复制性和致病性。在rCA16的基础上，我们进一步插入了一个纳米荧光素酶（Nluc）报告基因，构建了rCA16-Nluc克隆。我们发现rCA16-Nluc中的Nluc基因在Vero细胞中连续传代过程中稳定，因此在rCA16-Nluc感染的Vero细胞传代10次以上后仍能检测到稳定的荧光素酶信号。通过测定盐酸胍、利巴韦林、氯喹和氯化铵对CA16的半数抑制浓度（IC）、细胞毒性浓度（CC）和选择指数，举例说明了其在抗病毒药物特性鉴定和高通量筛选中的应用。最后，我们表明基于rCA16-Nluc的检测方法大大简化了CA16中和抗体检测。因此，这两个CA16感染性克隆将成为未来肠道病毒研究和抗病毒药物开发的有力工具。
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