Lu Baojing, Tang Qi, Wang Qianyun, Liu Xuejuan, Peng Hui, Zhu Binbin, Xie Li, Li Zeng, Wang Hanzhong, Zheng Zhenhua, Wang Linding, Li Bao
The Key Laboratory of Microbiology and Parasitology of Anhui Province, The Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Microbiology and Parasitology, School of Basic Medical Sciences, Anhui Medical University, Hefei, China.
Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Hefei, China.
Front Microbiol. 2022 Feb 25;13:825111. doi: 10.3389/fmicb.2022.825111. eCollection 2022.
Enterovirus 71 (EV71) is one of the most important etiological agents for hand-foot-mouth disease. Compared with coxsackievirus A16 infection, EV71 infection is often associated with severe central nervous system complications, such as encephalitis, encephalomyelitis, and acute flaccid paralysis in infants and young children. In this study, we constructed a recombinant baculovirus with T7 ribonucleic acid polymerase under the control of a cytomegalovirus promoter and simultaneously engineered the T7 promoter upstream of a full-length EV71 complementary deoxyribonucleic acid. After transduction into mammalian cells, typical cytopathic effects (CPEs) and VP1 signals were detected in cells transfected with recombinant baculovirus. Additionally, viral particles located in the cytoplasm of human rhabdomyosarcoma cells (Rd) and Vero cells were observed by electron microscope, indicating that EV71 was recovered using a Bac-to-Bac expression system . After four passages, the rescued virus had a growth curve and plaque morphology similar to those of the parental virus. Furthermore, the gene and the protein from the mouse brain were detected by reverse transcription polymerase chain reaction and immunohistochemistry after intracerebral injection of purified recombinant baculovirus. Typical CPEs were observed after inoculation of the supernatant from mouse brain to Rd cells, revealing a reconstruction of EV71 . Thus, we established a new approach to rescue EV71 based on a baculovirus expression system and , which may provide a safe and convenient platform for fundamental research and a strategy to rescue viruses that currently lack suitable cell culture and animal models.
肠道病毒71型（EV71）是手足口病最重要的病原体之一。与柯萨奇病毒A16感染相比，EV71感染常伴有严重的中枢神经系统并发症，如婴幼儿的脑炎、脑脊髓炎和急性弛缓性麻痹。在本研究中，我们构建了一种在巨细胞病毒启动子控制下带有T7核糖核酸聚合酶的重组杆状病毒，并在全长EV71互补脱氧核糖核酸的上游同时设计了T7启动子。转导至哺乳动物细胞后，在用重组杆状病毒转染的细胞中检测到典型的细胞病变效应（CPE）和VP1信号。此外，通过电子显微镜观察到位于人横纹肌肉瘤细胞（Rd）和Vero细胞胞质中的病毒颗粒，表明使用杆状病毒表达系统恢复了EV71。经过四代传代后，拯救的病毒具有与亲本病毒相似的生长曲线和噬斑形态。此外，在脑内注射纯化的重组杆状病毒后，通过逆转录聚合酶链反应和免疫组织化学检测了小鼠脑中的基因和蛋白质。将小鼠脑的上清液接种到Rd细胞后观察到典型的CPE，揭示了EV71的重建。因此，我们基于杆状病毒表达系统建立了一种新的拯救EV71的方法，这可能为基础研究提供一个安全便捷的平台，并为拯救目前缺乏合适细胞培养和动物模型的病毒提供一种策略。
