摘 要：手足口病（HFMD）和疱疹性咽峡炎（HA）是儿童中常见的疾病，主要由肠道病毒（EVs）感染引起。明确HFMD和HA暴发期间EVs的分子流行病学特征，对于理解疾病的病因学特征并促进治疗具有重要意义。在2021年的一次暴发期间，本研究从中国湖南省长沙市湘雅三医院收治的54名平均年龄为（3.40±1.67）岁的HFMD或HA患者中收集了咽拭子样本，通过病毒宏基因组测序（VMGS）评估样本中EVs的流行情况，并通过针对vp1区域的PCR扩增及桑格测序验证这些EVs的血清型。对代表性EVs毒株的全基因组进行测序，并用于系统发育分析。就本次暴发中的主要血清型柯萨奇病毒A4（CVA4），本研究进一步对其VP1蛋白的氨基酸序列进行了突变分析与免疫表位预测。VMGS结果显示，EVs基因组的序列读数占所有读数的98.29%。在54份样本中，通过PCR检测，有47份可确认EVs的存在，阳性率为87.04%。研究共鉴定出5种EVs血清型，包括CVA2（7例，14.89%）、CVA4（22例，46.81%）、CVA10（4例，8.51%）、CVA16（7例，14.89%）和CVB3（3例，6.38%）。此外，有4例EVs阳性样本（8.51%）的肠道病毒血清型未确定。HFMD病例仅检测到CVA4和CVA16阳性，而HA病例中包含所有5种血清型。对11株代表性EVs基因组进行分析，与最接近的参考毒株相比，它们的核苷酸序列一致性范围为96.57%至99.04%。CVA4的VP1蛋白中存在8个氨基酸变异位点，其中2个位于预测的MHC I类表位内。本研究揭示了2021年长沙HFMD和HA暴发期间儿童感染EVs的血清型和突变特征，为长沙市的EVs流行病学监测提供了重要数据。通过对CVA4的VP1蛋白氨基酸序列进行突变分析，发现其免疫表位仍在不断变化，提示该病毒可能正在适应免疫压力，这对于疫苗研发具有重要意义。因此，在疾病暴发期间对病毒分子流行病学进行持续监测是十分必要的，以便为应对这些疾病制定防控策略。
关键词：肠道病毒；手足口病；疱疹性咽峡炎；分子流行病学；血清学
中图分类号：Q939.4                        文献标志码：A                    DOI：10.3969/j.issn.1007-7146.2025.04.007
（Acta Laser Biology Sinica, 2025, 34(4): 340-353）

Abstract: Hand-foot-and-mouth disease (HFMD), and herpangina (HA) are common pediatric diseases mainly caused by infection of enteroviruses (EVs). Therefore, clarifying the molecular epidemiology of EVs in HFMD and HA outbreaks is important for understanding the etiological characteristics of the diseases and facilitating the treatment. During an outbreak in 2021, throat swab samples were collected from 54 HFMD or HA patients aged (3.40±1.67) years in the Third Xiangya Hospital, Changsha, Hunan Province, China. The prevalence of EVs in the samples was evaluated by viral metagenomic sequencing (VMGS), and the serotypes of these EVs were verified by PCR amplification of vp1 region followed by Sanger sequencing. Complete genomes of representative EV strains were further sequenced for phylogenetic analyses. For coxsackievirus (CV) A4, the predominant serotype in this outbreak, the amino acid sequences of VP1 were subjected to mutation analysis and immune epitope prediction. VMGS results revealed that sequence reads of EV genomes accounted for 98.29% of all the reads. PCR testing confirmed EV in 47 out of the 54 samples, representing an EV positive rate of 87.04%. Five EV serotypes were identified, including CVA2 (7, 14.89%), CVA4 (22, 46.81%), CVA10 (4, 8.51%), CVA16 (7, 14.89%), and CVB3 (3, 6.38%). Additionally, EV serotypes were undetermined in 4 EN-positive samples (8.51%). HFMD cases were tested positive for CVA4 and CVA16 only, while HA cases included all five serotypes.11 representative EV genomes revealed nucleotide sequence identities ranging from 96.57% to 99.04%, compared to the closest reference strains. VP1 of CVA4, the predominant serotype, harbored 8 amino acid variation sites among which 2 were located within the predicted MHC class-I epitopes. This study characterized the serotype distribution and mutation profiles of EVs in pediatric HFMD and HA cases during the 2021 Changsha outbreak, providing critical data for local epidemiological surveillance. Amino acid sequence analysis of CVA4’s VP1 protein revealed persistent changes in immunogenic epitopes, suggesting viral adaptation to immune pressure — a finding with significant implications for vaccine development. Consequently, sustained molecular surveillance during outbreaks is essential to formulate effective prevention and control strategies.
Key words: enterovirus; hand-foot-and mouth disease; herpangina; molecular epidemiology; serotype
CLC number: Q939.4                     Document code: A                  DOI：10.3969/j.issn.1007-7146.2025.04.007