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目的 阐明柯萨奇病毒A16(Coxsackievirus A16, CVA16)在跨宿主适应过程中毒力增强的分子基础，并构建从母株（P0株）到小鼠适应株（P5株）的表型与遗传演化框架，为解析其宿主适应性机制提供理论依据。方法 以临床分离的CVA16母株（P0株）为起始毒株，通过在ICR乳鼠体内连续传代构建小鼠适应性毒株，建立P0株至P5株的完整演化谱系。利用全基因组测序技术比较P0株与P5株的遗传变异差异，结合RD细胞感染实验与乳鼠攻毒实验，系统评估不同代次毒株的体外复制能力、体内致病性及靶器官组织病理学改变。通过病毒结合试验与内化试验，解析CVA16毒力增强的可能分子机制。结果 基因组比对分析明确了P0株与P5株间的变异特征，其中VP1的关键突变定位于病毒入侵相关功能区域。体内外表型验证显示，P5株相较于P0母株呈现显著增强的复制能力与致病性：在RD细胞中可诱导更高水平的病毒颗粒产生及病毒RNA复制；对乳鼠攻毒后，能引发快速体重下降、后肢麻痹等典型症状，且致死率达100%；在乳鼠骨骼肌、肺和脊髓中，病毒滴度显著升高，并造成更严重的组织炎性浸润与结构损伤。机制研究证实，P5株毒力升高主要与病毒内化效率及后续胞内复制能力的提升有关，而病毒与宿主细胞的初始结合能力无显著差异。结论 本研究揭示CVA16在小鼠适应过程中，其毒力增强是由多层次遗传变异驱动的结果，其中病毒进入效率和胞内复制能力的提升是关键调控因素。本研究为理解CVA16跨宿主适应机制及神经系统致病机理提供了试验证据，也为靶向病毒结构功能域的抗病毒药物研发及疫苗设计奠定了基础。
Abstract：Objective To elucidate the molecular basis underlying virulence enhancement of coxsackievirus A16(CVA16) during cross-species adaptation and to establish a phenotypic and genetic evolutionary framework from the parental strain(P0) to the mouse-adapted strain(P5), thereby providing insights into the mechanisms of host adaptation.Methods A clinically isolated CVA16 parental strain(P0) was subjected to serial passages in ICR neonatal mice to generate a mouse-adapted lineage, establishing a complete evolutionary series from P0 to P5. Whole-genome sequencing was performed to compare genetic variations between P0 and P5. RD cell infection assays and neonatal mouse challenge experiments were conducted to systematically evaluate in vitro replication capacity, in vivo virulence, and target-organ histopathological changes across different viral passages. Viral binding and internalization assays were further employed to investigate the molecular mechanisms underlying CVA16 virulence enhancement. Results Comparative genomic analysis identified distinct genetic variations between P0 and P5, including a critical mutation in the VP1 region located within a functional domain associated with viral entry. Phenotypic characterization revealed that the P5 strain exhibited significantly enhanced replication capacity and virulence compared with the parental P0 strain. In RD cells, P5 produced higher levels of viral RNA and increased yields of infectious virions. In neonatal mice, P5 infection resulted in rapid body weight loss, hind-limb paralysis, and 100% mortality. Moreover, viral titers were markedly elevated in skeletal muscle, lung, and spinal cord tissues, accompanied by more severe inflammatory infiltration and structural tissue damage. Mechanistic analyses demonstrated that virulence enhancement of the P5 strain was primarily associated with increased viral internalization efficiency and subsequent intracellular replication, whereas no significant difference was observed in initial virus– cell attachment compared with P0.Conclusion This study demonstrates that virulence enhancement of CVA16 during mouse adaptation is driven by multilayered genetic variations, with increased viral internalization and intracellular replication serving as key determinants. These findings provide experimental evidence for understanding CVA16 cross-species adaptation and neuropathogenesis and establish a foundation for the development of antiviral agents and vaccines targeting structural and functional domains of the viral capsid.
[1]冀天娇，居昱，程文隽，等.2018-2020年广西边境地区手足口病流行特征及病原学分析[J].病毒学报，2022,38(3):591-599.DOI:10.13242/j.cnki.bingduxuebao.004109.
[2]黄京京，韦欢欢，陈飞，等.2018-2019年北京市柯萨奇病毒A组16型分子流行病学研究[J].微生物学免疫学进展，2023,51(1):40-48.DOI:10.13309/j.cnki.pmi.2023.01.007.
[3]王越，郎兴莹，肖冰，等.大连市2022-2023年手足口病柯萨奇病毒A组16型分子流行特征分析[J].病毒学报，2024,40(6):1404-1410.DOI:10.13242/j.cnki.bingduxuebao.004606.
[4]Bifani AM,Tee HK,Antanasijevic A,et al.Enterovirus A71 priorities,challenges,and future opportunities in humoral immunity and vaccine development[J].NPJ Vaccines,2025,10(1):194.DOI:10.1038/s41541-025-01184-z.
[5]Li Y,Gao F,Wang Y,et al.Immunogenicity and safety of inactivated enterovirus A71 vaccines in children aged 6-35 months in China:a non-inferiority,randomised controlled trial[J].Lancet Reg Health West Pac,2021,16:100284.DOI:10.1016/j.lanwpc.2021.100284.
[6]靳卫平，钱莎莎，申硕.手足口病相关肠道病毒及疫苗的研究进展[J].中国生物制品学杂志，2021,34(11):1273-1283.
[7]Chi F,Zhang X,Zhang D,et al.A nucleoside-modified m RNA vaccine prevents enterovirus A71 infection in mouse model[J].Front Immunol,2025,16:1535758.DOI:10.3389/fimmu.2025.1535758.
[8]Caine EA,Fuchs J,Das SC,et al.Efficacy of a trivalent hand,foot,and mouth disease vaccine against enterovirus 71 and coxsackieviruses A16 and A6 in mice[J].Viruses,2015,7(11):5919-5932.DOI:10.3390/v7112916.
[9]Li D,Sun T,Tao L,et al.A mouse-adapted CVA6strain exhibits neurotropism and triggers systemic manifestations in a novel murine model[J].Emerg Microbes Infect,2022,11(1):2248-2263.DOI:10.1080/22221751.2022.2119166.
[10]Jiang Z,Zhang Y,Lin H,et al.A 10-day-old murine model of coxsackievirus A6 infection for the evaluation of vaccines and antiviral drugs[J].Front Immunol,2021,12:665197.DOI:10.3389/fimmu.2021.665197.
[11]Wu Y,Qu Z,Xiong R,et al.A practical method for evaluating the in vivo efficacy of EVA-71 vaccine using a h SCARB2 knock-in mouse model[J].Emerg Microbes Infect,2021,10(1):1180-1190.DOI:10.1080/22221751.2021.1934558.
[12]Zhang G,Hu B,Huo Y,et al.Amino acid substitutions in VP2,VP1,and 2C attenuate a Coxsackievirus A16 in mice[J].Microb Pathog,2021,150:104603.DOI:10.1016/j.micpath.2020.104603.
[13]宗彦君，孙甜甜，王蕊，等.柯萨奇病毒A组16型VP1-145位氨基酸变异影响对硫酸乙酰肝素结合能力的初步探究[J].病毒学报，2024,40(2):346-356.DOI:10.13242/j.cnki.bingduxuebao.004484.
[14]Cheng D,Chiu YW,Huang SW,et al.Genetic and cross neutralization analyses of coxsackievirus A16circulating in Taiwan from 1998 to 2021 suggest dominant genotype B1 can serve as vaccine candidate[J].Viruses,2022,14(10):2306.DOI:10.3390/v14102306.
[15]Xu L,Cui D,Wang L,et al.Genetic characteristics of the P1 coding region of Coxsackievirus A16 associated with hand,foot,and mouth disease in China[J].Mol Biol Rep,2018,45(6):1947-1955.DOI:10.1007/s11033-018-4345-y.
[16]Chang CK,Wu SR,Chen YC,et al.Mutations in VP1and 5'-UTR affect enterovirus 71 virulence[J].Sci Rep,2018,8(1):6688.DOI:10.1038/s41598-018-25091-7.
[17]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Mol Biol Evol,2016,33(7):1870-1874.DOI:10.1093/molbev/msw054.
[18]Ke X,Zhang Y,Liu Y,et al.A single mutation in the VP1 gene of enterovirus 71 enhances viral binding to heparan sulfate and impairs viral pathogenicity in mice[J].Viruses,2020,12(8):883.DOI:10.3390/v12080883.
[19]Zhang YX,Huang YM,Li QJ,et al.A highly conserved amino acid in VP1 regulates maturation of enterovirus 71[J].PLo S Pathog,2017,13(9):e1006625.DOI:10.1371/journal.ppat.1006625.
[20]Liu X,Zhu H,Wang M,et al.An enterovirus A71virus-like particle with replaced loops confers partial cross-protection in mice[J].Virus Res,2023,337:199235.DOI:10.1016/j.virusres.2023.199235.
[21]Wang H,Guo T,Yang Y,et al.Lycorine derivative LY-55 inhibits EV71 and CVA16 replication through downregulating autophagy[J].Front Cell Infect Microbiol,2019,9:277.DOI:10.3389/fcimb.2019.00277.
[22]史颖颖，陈仕同，胡波，等.柯萨奇病毒A16型3C蛋白对人横纹肌肉瘤细胞凋亡的诱导作用及其机制[J].吉林大学学报（医学版），2021,47(4):874-881.DOI:10.13481/j.1671-587X.20210408.
[23]Tsai YH,Huang SW,Hsieh WS,et al.Enterovirus A71 containing Codon-deoptimized VP1 and highfidelity polymerase as next-generation vaccine candidate[J].J Virol,2019,93(13):e02308-18.DOI:10.1128/JVI.02308-18.
[24]谢星辰，束佳熠，张晓燕，等.手足口病相关肠道病毒疫苗的研究进展与展望[J].传染病信息，2025,38(1):83-88.DOI:10.3969/j.issn.1007-8134.2025.01.016.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250382
中图分类号:R373.23
引用信息:
[1]李慧洁,王蕊,李冀琛,等.柯萨奇病毒A16小鼠适应过程中VP1位点突变与复制动力学的相关性研究[J].病毒学报,2026,42(01):23-36.DOI:10.13242/j.cnki.bingduxuebao.250382.
基金信息:
国家疾病预防控制局公共卫生人才培养支持项目(项目号:GJJKJ-2024-ZY); 传染病溯源预警与智能决策全国重点实验室(NITFID)资助项目(项目号:ZDGWNLJS25-36),题目:未知及新发病原体监测及预测预警技术研究; 国家重点研发计划项目(项目号:2021YFC2302003),题目:病毒监测网络数据标准及数据平台建设~~