Establishment of severe <em>in vitro</em> and <em>in vivo</em> infection models for coxsackievirus B4 and their application in identifying viral virulence determinants

Haoyi Liu; Xianglin Xuan; Bizhen Tang; Huan Wang; Li Zhang; Shuyan Qiu; Chuncong Mo; Xiao Li; Xingui Tian; Rong Zhou; Qian Liu; Wenkuan Liu

doi:10.1016/j.virs.2026.04.003

April 2026

. doi: 10.1016/j.virs.2026.04.003

Citation: Haoyi Liu, Xianglin Xuan, Bizhen Tang, Huan Wang, Li Zhang, Shuyan Qiu, Chuncong Mo, Xiao Li, Xingui Tian, Rong Zhou, Qian Liu, Wenkuan Liu. Establishment of severe in vitro and in vivo infection models for coxsackievirus B4 and their application in identifying viral virulence determinants .VIROLOGICA SINICA, 2026, 41(2) : 343-354. http://dx.doi.org/10.1016/j.virs.2026.04.003

柯萨奇病毒B4体内外重症感染模型的建立及其在鉴定病毒毒力决定因素中的应用

刘好怡 ^a,b ,
玄祥林 ^c ,
唐碧珍 ^c ,
王欢 ^c,d ,
张丽 ^a ,
邱淑燕 ^a ,
莫纯聪 ^a,b ,
李潇 ^a,b ,
田新贵 ^a,b ,
周荣 ^a,b ,
刘倩 ^c,, ,
刘文宽 ^a,b,,

a 呼吸疾病全国重点实验室, 国家呼吸系统疾病临床医学研究中心, 国家呼吸医学中心, 呼吸健康国际合作联合实验室, 广东省高等学校呼吸医学基础研究卓越中心, 广州呼吸健康研究院, 广州医科大学附属第一医院, 中国广州;
b 中国-葡萄牙人工智能与公共卫生问题"一带一路"联合实验室, 粤港澳呼吸系统传染病联合实验室, 广东省呼吸疾病研究重点实验室, 广州医科大学, 中国广州;
c 广东药科大学第一附属医院, 科研中心, 广东药科大学, 中国广州;
d 昆明医科大学海源学院, 中国昆明

通讯作者： 刘倩, qianliu_ln@163.com; 刘文宽, ahlwk2000-2004@163.com
收稿日期： 2025-12-10
录用日期： 2026-04-02

摘要

柯萨奇病毒B4（CVB4）是一种致病性高的肠道病毒，与重症神经系统疾病及死亡病例密切相关。为构建能够模拟重症CVB4 感染的研究模型，本研究选用了两株神经毒力存在显著差异的临床分离株—高毒力毒株GZ-HFM01 和低毒力毒株GZ-R6。基于人神经母细胞瘤SH-SY5Y 细胞的体外神经细胞毒性模型实验显示，GZ-HFM01 毒株形成的噬斑面积显著大于GZ-R6 毒株，表明其对神经细胞的损伤能力更强。同时，本研究通过腹腔接种的方式，在3 日龄ICR 小鼠体内成功建立了优化的重症感染动物模型，该模型能够复现重症病例的核心临床特征。与GZ-R6 毒株感染组相比，GZ-HFM01 毒株感染导致小鼠存活率显著下降，出现进行性神经功能损伤，脑组织内病毒载量呈时间依赖性累积，同时伴随明显的组织病理学损伤，以及血清促炎细胞因子水平升高。为定位调控神经毒力的基因组决定簇，通过将GZ-HFM01 毒株基因组的单个区段（5' 非翻译区[5′UTR]、P1、P2、P3 或3' 非翻译区[3′UTR]）替换为GZ-R6 毒株的对应区段，构建了一系列嵌合病毒。利用已建立的体内外模型对嵌合病毒进行评估，结果显示，P2 区段的替换可显著减弱其在SH-SY5Y 细胞中的致细胞病变效应及对3 日龄ICR 小鼠的致病性。动物实验进一步表明，5′UTR 或P1 区段的替换同样会降低病毒毒力，而这一表型在细胞模型中并未体现，这一结果凸显了CVB4 致病机制的多因素调控特性。综上，本研究成功建立并验证了CVB4 重症感染的体内外研究模型，同时鉴定出参与调控病毒神经毒力的关键基因组区段，为进一步解析其致病机制，以及研发重症感染的靶向干预策略奠定了基础。
- 柯萨奇病毒B4
- , 重症感染
- , 感染模型
- , 神经毒力
- , 病毒毒力决定因子

Establishment of severe in vitro and in vivo infection models for coxsackievirus B4 and their application in identifying viral virulence determinants

Haoyi Liu ^a,b ,
Xianglin Xuan ^c ,
Bizhen Tang ^c ,
Huan Wang ^c,d ,
Li Zhang ^a ,
Shuyan Qiu ^a ,
Chuncong Mo ^a,b ,
Xiao Li ^a,b ,
Xingui Tian ^a,b ,
Rong Zhou ^a,b ,
Qian Liu ^c,, ,
Wenkuan Liu ^a,b,,

a. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Joint International Research Laboratory of Respiratory Health, Guangdong Basic Research Center of Excellence for Respiratory Medicine, Guangzhou Institute of Respiratory Health, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510182, China;
b. China-Portugal Artificial Intelligence and Public Health Technologies Joint Laboratory, Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory Infectious Diseases, Guangdong Provincial Key Laboratory of Respiratory Disease Research, Guangzhou Medical University, Guangzhou 511436, China;
c. Scientific Research Center, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangdong Pharmaceutical University, Guangzhou 510062, China;
d. Kunming Medical University Haiyuan College, Kunming 650106, China

Corresponding author: Qian Liu, qianliu_ln@163.com Wenkuan Liu, ahlwk2000-2004@163.com
Received Date: 10 December 2025
Accepted Date: 02 April 2026

Abstract

Coxsackievirus B4 (CVB4) is a highly pathogenic enterovirus associated with severe neurological disease and mortality. To establish research models that recapitulate severe CVB4 infection, two clinical isolates with distinct neurovirulence—the high-virulence strain GZ-HFM01 and low-virulence strain GZ-R6—were used. An in vitro neurocytotoxicity model with human neuroblastoma SH-SY5Y cells showed that GZ-HFM01 produced significantly larger plaques than GZ-R6, reflecting its increased capacity to damage neuronal cells. Concurrently, an optimized in vivo severe infection model was established in 3-day-old ICR mice through intraperitoneal inoculation, which reproduced key clinical features of severe disease. Compared with GZ-R6, GZ-HFM01 infection resulted in significantly reduced survival, progressive neurological impairment, time-dependent viral accumulation in brain tissue, pronounced histopathological injury, and elevated serum pro-inflammatory cytokine levels. To map genomic determinants of neurovirulence, a chimeric virus panel was generated by replacing individual genome segments (5′untranslated region [UTR], P1, P2, P3, or 3′UTR) of GZ-HFM01 with the corresponding regions from GZ-R6. Evaluation with the established models demonstrated that replacement of the P2 region significantly attenuated both the cytopathic effect in SH-SY5Y cells and pathogenicity in 3-day-old ICR mice. Animal studies further indicated that substitution of the 5′UTR or P1 region also reduced virulence—a phenotype absent from the cell-based model—underscoring the multifactorial regulation of CVB4 pathogenesis. In conclusion, this study provides validated in vitro and in vivo models of severe CVB4 infection and identifies key genomic segments that contribute to neurovirulence, offering a foundation for mechanistic research and the development of targeted interventions against severe CVB4-induced disease.
- Coxsackievirus B4 (CVB4)
- , Severe infection
- , Infection model
- , Neurovirulence
- , Viral virulence determinant

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