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目的 手足口病(Hand, foot, and mouth disease, HFMD)是一种在儿童中高发的病毒性传染病，主要病原体为肠道病毒71型(Enterovirus A 71, EVA71)和柯萨奇病毒A16型(Coxsackievirus A16, CVA16)，其中EVA71与严重神经系统并发症及死亡密切相关。随着EVA71疫苗的广泛应用，CVA16逐渐成为主要致病因子，亟需建立高效的大规模疫苗生产工艺。方法 本研究在不同温度(33℃、37℃、38.5℃)及感染复数(Multiplicity of infection,MOI:0.01、0.1、0.5)条件下，将CVA16在KMB17二倍体细胞中连续传代30次，系统评估病毒生长动力学、病毒颗粒完整性、遗传稳定性，并结合单细胞转录组分析宿主细胞应答机制，旨在为优化培养工艺参数、提升病毒产量与质量、保障疫苗生产过程提供科学依据。结果 结果显示，37℃、MOI=0.1为最佳扩增条件，病毒在24～36 h达到增殖高峰，滴度可达108.0 CCID50以上，且完整病毒颗粒达78%。基因组测序证实前10代VP1突变率低于0.1%，至P20和P30仍低于0.2%。单细胞分析提示接种病毒后，宿主细胞CXCL8、IER3等生长因子及内质网蛋白加工通路的应答利于促进病毒复制，后期IL6、LDHA等基因进一步上调表明病毒通过调控宿主翻译与细胞周期实现高效复制。结论 本研究表明，在37℃和MOI=0.1条件下有利于CVA16毒株在KMB17二倍体细胞的复制，为大规模生产培养CVA16提供了关键工艺参数。
Abstract：Objective Hand, foot, and mouth disease(HFMD) is a common viral infectious disease in children, primarily caused by Enterovirus A71(EV-A71) and Coxsackievirus A16(CVA16). Among them, EV-A71 is closely associated with severe neurological complications and death. With the widespread use of the EV-A71 vaccine, CVA16 has gradually become a major causative pathogen, highlighting the urgent need to establish an efficient large-scale vaccine production process. Methods In this study, CVA16 was serially passaged 30 times in KMB17 diploid cells under different temperature conditions(33℃, 37℃, and 38.5℃) and multiplicities of infection(MOIs: 0.01, 0.1, and 0.5). Viral growth kinetics, virion integrity, and genetic stability were systematically evaluated. In addition, single-cell transcriptomic analysis was performed to characterize host cell response mechanisms, with the aim of providing a scientific basis for optimizing culture parameters, improving viral yield and quality, and supporting vaccine production. Results The results showed that 37℃ and an MOI of 0.1 were the optimal amplification conditions. Under these conditions, viral replication peaked at 24 – 36 h, with titers exceeding 108.0 CCID50, and intact virions accounting for 78% of total viral particles. Genomic sequencing confirmed that the mutation rate in the VP1 region remained below 0.1%involving growth factor-related genes such as CXCL8 and IER3, as well as the endoplasmic reticulum protein-processing pathway, were conducive to viral replication. At later stages, further upregulation of genes such as IL6 and LDHA suggested that CVA16 achieved efficient replication by modulating host translation and the cell cycle. Conclusion This study demonstrates that culture at 37℃ with an MOI of 0.1 is favorable for CVA16 replication in KMB17 diploid cells and provides key process parameters for the large-scale production of CVA16.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.260011
中图分类号:R373.2
引用信息:
[1]龙佳卿,李盈妍,于丽,等.不同培养条件下CVA16病毒在KMB17细胞上的增殖特性及宿主细胞的相关转录特征分析[J].病毒学报,2026,42(03):727-739.DOI:10.13242/j.cnki.bingduxuebao.260011.
基金信息:
云南省科技计划-生物医药专项(项目号:202502AS100023),题目:手足口病联合疫苗-EV71/CA16二价灭活疫苗I,II期临床研究;云南省科技计划-基础研究专项(项目号:202401AS070047),题目:EV-A71和CV-A16抗原预存免疫对CVA10和CV-A6抗原诱导的B细胞和T细胞免疫的影响;云南省科技计划-生物医药专项(项目号:202402AA310018),题目:EV71-CA16-CA10-CA6灭活疫苗的制备、评价及多价疫苗关键抗原表位筛选; 云南省科技人才与平台计划(项目号:202305AC160008),题目:中青年学术和技术带头人后备人才项目~~
2026-04-23
2026-04-23
2026-04-23