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目的 引起手足口病(Hand, foot, and mouth disease, HFMD)的肠道病毒(Enteroviruses)病原组成复杂多样，其中柯萨奇A组病毒(Coxsackievirus A group)的毒株分离鉴定和感染致病特征对于HFMD的研究和疫苗研制显得越来越重要，因此，本研究旨在阐明柯萨奇病毒A组4型(Coxsackievirus A4,CVA4)的分离鉴定过程并对其生物学特征进行分析，为后续疫苗研制提供参考。方法 通过KMB17细胞分离CVA4病毒，同时利用PCR扩增CVA4 VP1基因序列以鉴定基因型别，在电镜下观察病毒颗粒形态，利用Western Blot(WB)和银染实验分析主要结构蛋白。在KMB17细胞上系统探究CVA4增殖动力学以及乳鼠体内的感染特征，最后结合中和抗体实验和ELISpot实验对CVA4病毒免疫原性进行分析。结果 本研究从手足口病患儿粪便样本中分离获得3株CVA4毒株，接种人二倍体细胞KMB17后，可观察到皱缩、变圆和肿胀等致细胞病变(Cytopathic effect, CPE)特征，主要结构蛋白基因VP1测序鉴定毒株均属CVA4病毒D2基因亚型。经KMB17细胞扩增培养，随后分析发现，透射电子显微镜下可观察到CVA4病毒呈现典型的二十面体的颗粒形状，以实心颗粒为主，病毒颗粒直径约为30nm。经WB和银染实验分析发现，扩增病毒纯化液中可见VP1、VP3的完整结构蛋白条带，并为特异性抗-CVA4血清所识别。以KMB17细胞为基质进行噬斑克隆纯化，我们筛选获得E-4、E-2和B-2克隆株，增殖动力学特征显示三株病毒均能够在MOI=0.05条件下实现较快增殖，并在96h以内达到增殖高峰，感染性滴度分别为107.5 CCID50/mL、105.75 CCID50/mL、107.75 CCID50/mL。通过颅内注射2000 CCID50/只病毒的方式感染2日龄乳鼠后，3株CVA4病毒株均能引起乳鼠麻痹症状，并在感染后3～5d全部死亡。初步免疫原分析结果提示，上述3株毒株经腹腔注射BALB/c小鼠，能诱导较好的体液免疫和细胞免疫应答，两次免疫后中和抗体几何平均滴度分别达到2622.2、1456.1、5045.1。结论 结合病毒增殖特征和免疫原性，本研究提示E-4和B-2在增殖特性和免疫原性方面的表现相对优于E-2，未来研究中可进一步探索其作为潜在疫苗候选株的可能性。
Abstract：Objective Hand, foot, and mouth disease(HFMD) is caused by a diverse group of enteroviruses. The isolation, identification, and characterization of Coxsackievirus A(CVA) strains are increasingly important for understanding HFMD pathogenesis and for vaccine development. This study aimed to isolate and identify Coxsackievirus A4(CVA4) strains and to analyze their biological characteristics to provide a basis for future vaccine development. Methods CVA4 was isolated using KMB17 cells. The VP1 gene of the isolates was amplified by PCR and sequenced for genotyping. Viral morphology was examined by transmission electron microscopy. Major structural proteins were analyzed by SDS-PAGE, Western blotting(WB), and silver staining. Viral replication kinetics in KMB17 cells and infection characteristics in suckling mice were investigated. Immunogenicity was further evaluated using neutralization assays and ELISpot assays. Results Three CVA4 strains were isolated from fecal samples of HFMD patients. Typical cytopathic effects(CPE), including cell shrinkage, rounding, and swelling, were observed after infection of KMB17 cells. VP1 gene sequencing confirmed that all isolates belonged to the D2 subgenotype of CVA4. Transmission electron microscopy showed typical icosahedral viral particles with diameters of approximately 30 nm, predominantly appearing as solid particles. Western blot and silver staining analysis demonstrated the presence of structural proteins VP1 and VP3 in purified viral preparations, which were recognized by specific anti-CVA4 sera. Plaque purification yielded three clonal strains designated E-4, E-2, and B-2. Growth kinetics analysis showed that all three strains replicated efficiently in KMB17 cells at a multiplicity of infection(MOI) of 0.05 and reached peak titers within 96 h, with infectious titers of 107.5, 105.75, and 107.75 CCID50/mL, respectively. Intracranial inoculation of 2-day-old suckling mice with 2000 CCID50 per mouse resulted in paralysis and death within 3–5 days post-infection for all three strains. Immunogenicity analysis indicated that intraperitoneal immunization of BALB/c mice with the three strains induced strong humoral and cellular immune responses. After two immunizations, the geometric mean neutralizing antibody titers reached 2622.2, 1456.1, and 5045.1, respectively.Conclusion Based on viral replication characteristics and immunogenicity, strains E-4 and B-2 exhibited relatively superior biological properties compared with E-2. These strains may represent promising candidates for further evaluation as potential vaccine strains.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.260003
中图分类号:R373.23
引用信息:
[1]任潇虹,廖芸,王晶晶,等.柯萨奇病毒A4型毒株的分离鉴定和生物学特征分析[J].病毒学报,2026,42(02):381-392.DOI:10.13242/j.cnki.bingduxuebao.260003.
基金信息:
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~~
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