肠道病毒71型VP1及2A基因特征研究

目的 检测临床分离的肠道病毒71型(EV71)轻型、重型毒株VP1基因、2A基因的核苷酸序列,进行遗传进化途径分析.方法 将50份临床手足口病患者粪便标本处理后分离病毒,用EV71特异性引物进行RT-PCR扩增,获得EV71毒株,针对不同临床背景的标本,分别选取轻型、重型的毒株,用特异性引物分别对VP1基因及2A基因进行RT-PCR扩增,对其产物进行测序及遗传进化分析,并探讨它们之间的差别.结果 50份临床手足口病标本中有30份检测是EV71,其中轻型(手足口)13份,重型(手足口并发脑炎或心肌炎)17份.轻型和重型中各自选取5株对VP1基因和2A基因进行测序和遗传学分析,通过同源性比较和构建系统发生树发现,此10株EV71病毒和中国大陆已发表的5株EV71病毒(fuyangEU703814.1、xi_anHM003207.1、shandongEU753418.1、shenzhen-FJ607337.1、henanGU366191.1)全部属于C基因型,且核苷酸同源性较高,VP1基因和2A基因的同源性分别在94.7%～99.4%和93.6%～99.3%范围内.本次分离的10株EV71病毒与A、B基因型代表株比较,核苷酸同源性分别为81.0%～84.6%和78.4%～82.2%,差异较大.与已知的C1、C2、C3亚型代表株比较,核苷酸同源性在87.8%～90.2%,差异≥10%,与已知的C4亚型代表株比较,核苷酸同源性在96.8%～99.6%,因此认为可将这10株病毒划分为C4亚型.在系统发生树上,这10株病毒形成一个较独立的分支.结论 EV71 C4亚型病毒在中国大陆有较广泛的传播,且毒株之间VP1基因的遗传关系紧密;2A基因在轻型和重型病例毒株之间遗传差异不大.

Abstract：

Objective To detect VP1 and 2A genes of Enterovirus type 71 (EV71) isolated from clinical specimens of patients with light or heavy symptoms and analyze the homogeneity and phylogenetic tree. Methods Fifty clinical specimens of children with hand-foot-and-mouth disease ( HFMD) were dealed with, which were tested by RT-PCR assay with specific primer pairs for EV71. EV71 isolates from patients with light or heavy clinical symptoms were tested by RT-PCR assay with two specific primer pairs for VP1 and 2A genes of EV71 respectively. All of the PCR products were sequenced and compared with that of previously isolated EV71 isolates available from GenBank by homogeneity and phylogenetic tree analyses. Results The RT-PCR results indicated that 30 isolates were EV71, 13 of 30 isolates were from clinical specimens of patients with light symptoms of hand-foot and mouth, the other were from clinical specimens of patients with heavy symptoms of complications. VP1 genes and 2A genes of 10 EV71 isolated strains including 5 light strains and 5 heavy strains were sequenced and compared with that of previously isolated 5 EV71 Chinese isolates available from GenBank (fuyangEU703814.1, xi_anHM003207. 1, shandongEU753418.1, shenzhenFJ607337.1, henanGU366191. 1) by homogeneity and phylogenetic tree analyses. The homogeneity of VP1 and 2A genes of the 10 EV71 isolated strains and 5 previously isolated strains were between 94.7% -99.4% and 93.6% -99.3% respectively, with the representative isolates of A and B genotypes was between 81.0%-84. 6% and 78. 4%-82. 2% respectively. The data suggested that all of the 10 Chinese isolates belong to EV71 genotype C. There were only 87.8% -90.2% homology among these 10 strains and the representative strains of C1, C2, C3 sub-genotypes of EV71 but 96. 8% -99.6% homology among these 10 strains and the representative strains of C4 sub-genotypes of EV71, this suggested that these 10 Chinese isolates composed the C4 sub-genotype, of the C genotype, that formed a single branch in the phylogenetic tree. Conclusion EV71 of sub-genotype C4 distributed in Mainland China, and VP1 genes have close genetic relationship between isolated strains. There is no obvious difference in 2A genes between clinical specimens of patients with light or heavy symptoms by homogeneity and phylogenetic tree analyses.     

肠道病毒71型VP1及2A基因特征研究

目的 检测临床分离的肠道病毒71型(EV71)轻型、重型毒株VP1基因、2A基因的核苷酸序列,进行遗传进化途径分析.方法 将50份临床手足口病患者粪便标本处理后分离病毒,用EV71特异性引物进行RT-PCR扩增,获得EV71毒株,针对不同临床背景的标本,分别选取轻型、重型的毒株,用特异性引物分别对VP1基因及2A基因进行RT-PCR扩增,对其产物进行测序及遗传进化分析,并探讨它们之间的差别.结果 50份临床手足口病标本中有30份检测是EV71,其中轻型(手足口)13份,重型(手足口并发脑炎或心肌炎)17份.轻型和重型中各自选取5株对VP1基因和2A基因进行测序和遗传学分析,通过同源性比较和构建系统发生树发现,此10株EV71病毒和中国大陆已发表的5株EV71病毒(fuyangEU703814.1、xi_anHM003207.1、shandongEU753418.1、shenzhen-FJ607337.1、henanGU366191.1)全部属于C基因型,且核苷酸同源性较高,VP1基因和2A基因的同源性分别在94.7%～99.4%和93.6%～99.3%范围内.本次分离的10株EV71病毒与A、B基因型代表株比较,核苷酸同源性分别为81.0%～84.6%和78.4%～82.2%,差异较大.与已知的C1、C2、C3亚型代表株比较,核苷酸同源性在87.8%～90.2%,差异≥10%,与已知的C4亚型代表株比较,核苷酸同源性在96.8%～99.6%,因此认为可将这10株病毒划分为C4亚型.在系统发生树上,这10株病毒形成一个较独立的分支.结论 EV71 C4亚型病毒在中国大陆有较广泛的传播,且毒株之间VP1基因的遗传关系紧密;2A基因在轻型和重型病例毒株之间遗传差异不大.     

惠州市重症手足口病病原谱及EV71型VP1区基因特征分析

目的分析惠州市手足口病重症病例的病原谱构成,了解惠州市肠道病毒71型分离株的VPl区基因特征．为科学防治手足口病提供科学依据。方法采集300例重症手足口病（HFMD）患者标本,采用实时荧光RT-PCR检测肠道病毒核酸,并对肠道病毒7l型（EV71）和柯萨奇病毒A16型（CoxA16）进行分型检测;选择8株EV71分离株进行VPl区基因全长序列测定,测序结果利用DNASTAR软件进行核苷酸、氨基酸序列分析和同源性比较。并用Mega5．0软件构建亲缘性进化树。结果通过实时荧光RT-PCR特异性检测,EV71阳性结果154份,阳性率为51．33％;CoxAl6阳性结果38份,阳性率为12．67％。测序结果表明,8株EV71之间的VPl基因核苷酸同源性为96．9％～99．2％。氨基酸同源性为99．3％～100％。VPI区基因遗传进化分析表明。8株EV71分离株与c4基因亚型的代表株处于同一分支,均属于C4基因亚型的C4a进化分支。结论惠州市手足口病疫情主要病原EV71病毒均属于C4a基因亚型,与2004年以来的中国大陆EV71病毒流行的基因型一致,未产生明显的抗原漂移及变异。     

肠道病毒71型外壳蛋白VP1的可溶性表达、纯化及活性鉴定

目的克隆、表达和鉴定肠道病毒71型（EV71）VP1基因,得到可溶性的蛋白VP1,为制备EV71的抗体和诊断试剂的开发打下基础。方法优化EV71VP1蛋白基因,克隆并构建重组表达质粒pET15b/VP1,转化大肠杆菌BL21。使用Ni2＋亲和层析柱对重组蛋白进行纯化,并用Westernblotting检测目的蛋白。以重组蛋白VP1为抗原,ELISA检测抗原活性。结果重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量为36000,与预计大小一致。ELISA实验证实,重组蛋白具有良好的抗原性。结论本研究成功克隆和表达了EV71VP1蛋白,并得到可溶性的蛋白,对肠道病毒71型诊断试剂的开发有进一步潜在的应用价值。     

肠道病毒71 VP1基因的原核表达及其抗血清的制备

目的克隆并表达重组肠道病毒71(EV71)VP1基因,进行抗血清的制备并检测抗血清效价。方法利用原核表达系统将PCR获得的VP1片段构建成原核表达载体pET32a(+)-VP1,诱导其表达VP1融合蛋白并利用包涵体纯化方法进行重组蛋白的纯化,将纯化的重组蛋白免疫新西兰兔制备VP1抗血清,ELISA检测抗体效价。同时构建真核表达载体,利用其在真核细胞中的瞬时表达检测抗血清的特异性。结果通过克隆获得VP1原核表达载体,IPTG诱导VP1蛋白表达并纯化,SDS-PAGE结果显示重组蛋白表达且纯化浓度较高,将重组蛋白乳化后免疫新西兰兔3次,取血检测抗血清的效价,ELISA结果显示抗体效价为1∶64 000,利用所构建真核表达载体pcDNA3.1(+)-VP1表达VP1对抗血清进行检测,Western blot结果表明抗血清可较好的与VP1特异性结合。结论制备具有免疫原性的VP1蛋白及其效价较高的抗血清,为进一步研究抗EV71诊断方法、血清学诊断试剂盒及抗病毒疫苗的研制奠定基础。     

EV71结构蛋白VP1的原核表达及VP1单克隆抗体的制备

目的:原核表达EV71结构蛋白VP1并制备其单克隆抗体。方法:以肠道病毒71型(EV71)P1基因为模板,设计引物扩增出目的片段VP1,将其连接至大肠杆菌表达载体pET-32a(+)上,转化大肠杆菌TG1,筛选出阳性克隆后进行测序。将重组表达载体pET-32a(+)-VP1转化大肠杆菌表达菌株BLgold(DE3),对该工程菌进行诱导表达,SDS-PAGE电泳分析,表达产物以包涵体的形式存在。包涵体用6 mol/L盐酸胍溶解,经过Ni-NTA亲和层析法纯化,获得了纯度较高的目的蛋白。用纯化的VP1蛋白免疫小鼠,制备单克隆抗体(mAb)。结果:获得了24株mAb,其中1株EV71 Westernblot法鉴定呈阳性,5株EV71间接免疫荧光法(IFA)鉴定阳性。结论:成功地制备了VP1的mAb。     

人肠道病毒71型VP0蛋白的原核表达及多克隆抗体制备

目的:原核表达并纯化人肠道病毒71型(EV71)VP0蛋白,免疫豚鼠制备多克隆抗体,并鉴定其用于EV71检测的反应性和特异性。方法:PCR方法扩增VP0基因,构建原核表达质粒pET-VP0并转化大肠杆菌,诱导表达并纯化VP0重组蛋白,免疫豚鼠制备抗VP0多克隆抗体,ELISA方法检测抗VP0抗体效价,细胞免疫荧光和Western blot方法鉴定抗体特异性。结果:VP0重组蛋白在大肠杆菌BL21中高效表达,制备的抗VP0抗体效价为1∶106。细胞免疫荧光及Western blot检测结果显示,抗VP0多克隆抗体可以识别原核表达及EV71感染细胞中的VP0蛋白。结论:原核表达了EV71的VP0蛋白并制备出抗VP0多克隆抗体,为EV71的临床诊断、疫苗开发和分子病毒学研究提供了新的研究工具和手段。     

2018—2020年丽江市手足口病持续流行及2020年EV-A71分离株VP1区基因特征分析

目的明确2018—2020年丽江市手足口病(hand, foot and mouth disease, HFMD)的流行情况及2020年EV-A71分离株的基因特征。方法对收集到的2018—2020年丽江市HFMD患者粪便标本进行核酸检测, 结果为EV-A71阳性的标本进行病毒分离, 扩增其VP1区, 获得的序列与参考株构建系统进化树。结果 2018—2019年, 丽江市HFMD报告病例以≤5岁儿童为主。历年男性病例数均高于女性, 但男女性病例阳性检出率差异无统计学意义。构成丽江市HFMD主要病原的是EV-A71、CV-A16和其他EV。其中EV-A71阳性病例2018年1份(0.48%, 1/210), 2019年未检出, 2020年159份(66.53%, 159/239)。系统进化分析显示丽江市EV-A71分离株均处于C4a分支, 分离株间核苷酸和氨基酸同源性分别是99.44%～100%和99.65%～100%, 与攀枝花参考株核苷酸和氨基酸同源性分别是99.10%～100%和98.99%～100%。所有丽江分离株在中国大陆EV-A71常见的氨基酸变异位点均发生了S283T和A2...     

2008-2009年广东手足口病非CA16非EV71肠道病毒株的鉴定及其VP1基因分型研究

目的 探讨2008-2009年广东省手足口病非CAI6非EV71肠道病毒株的流行情况以及毒株型别.方法 从2008-2009年广东省手足口病粪便样本中采用RD细胞和HEp-2细胞分离病毒毒株,待出现细胞病变后收集上清采用RT-PCR进行鉴定.非CA16非EV71毒株再进行VP1测序分析,序列通过BLAST程序鉴定型别,用MEGA4.0软件进行基因进化分析.结果 共分离到22株非CA16非EV71毒株,通过BLAST程序鉴定犁别.2008年9株非CA16、非EV71的毒株有CA2型、CA4、CB3型;2009年13株有EV80、E13、E30、CB5、E24、PV1、CA10、CA6和CA2.各毒株间同源性低,各毒株分别归属CoxA、CoxB、埃可肠道病毒、新型肠道病毒和脊灰病毒组.结论 广东省2008-2009年两年来,手足口病除了CA16和EV71占主导地位外,并不存在其他的优势株,其他型肠道病毒分布比较广,既有CoxA组病毒、CoxB组,也有E13、E24、E30、新型病毒EV80和脊髓灰质炎病毒PV1,儿组病毒伴随流行.     

济南市手足口病患者肠道病毒71型的分离与鉴定

目的 从13例2008年、2009年济南市HFMD患者的粪便标本中分离获得EV71病毒并对其基因型进行鉴定.方法 按照中国疾病控制预防中心<手足口病标本采集及检测技术方案>的规定对标本中的病毒进行分离和鉴定,对VP1编码基因的部分核苷酸序列进行测序分析.结果 分离到6株EV71病毒,均与EV1 C4亚型处于同一进化树分支上,与C4亚型的核酸同源率均大于96%.结论 济南地区流行的EV71主要为C4亚型,与2008年中国流行的C4亚型EV71相比并未出现明显突变,引起重症的毒株与未引起重症的毒株相比,在VP1蛋白部分片段上没有发现明显的氨基酸差异.     

肠道病毒71型重组VP1蛋白的表达和EV71型手足口病血清学诊断方法的建立

目的 获得纯化的具有免疫活性的肠道病毒71型VP1蛋白,建立EV71感染早期、快速和准确的ELISA血清学诊断方法.方法 通过PCR方法扩增出VP1基因,定向克隆到原核表达载体pET-21b(+),阳性质粒转化入E.coli B121(DE3)感受态细胞经IPTG诱导,SDS-PAGE电泳和蛋白免疫印迹分析目的 蛋白的表达水平.纯化的VPI蛋白用作包被抗原,建立手足口病(HFMD)患者抗-EV71-IgM和IgG的血清学诊断方法.结果 成功表达和纯化了VP1重组蛋白,所表达的蛋白能被EV71型手足口病患者血清所识别.调查发现,与正常人和EV71阴性手足口病患儿比较,EV71阳性手足口病血清中抗-EV71-IgM和IgG中A值显著升高.差异具有统计学意义(P<0.05).与RT-PCR结果比较,发现该方法IgM的诊断敏感性和特异性分别为73%和77%;该IgG诊断方法的敏感性和特异性分别为82%和83%.完成该试验仪需4 h.结论 利用pET原核表达系统成功克隆、表达和纯化了肠道病毒71型重组外壳蛋白VPI,且具有良好的抗原性.该抗原可用于研制EV71血清学诊断试剂盒.     

Identification and characterization of a monoclonal antibody recognizing the linear epitope RVADVI on VP1 protein of enterovirus 71

Several large outbreaks of hand–foot–mouth disease (HFMD) have occurred in the Asian‐Pacific region since 1997, with Enterovirus 71 (EV71) and/or Coxsackievirus A16 (CAV16) as the main causative agents. Despite the close genetic relationship between the two viruses, only EV71 is associated with severe clinical manifestations and deaths. Effective antiviral treatment and vaccines are not available. High‐quality monoclonal antibodies (mAbs) are necessary to improve the accuracy of the diagnosis of EV71. In this study, a mAb (designated 1D9) was generated using EV71 C5 strain virus particles as immunogens. Examined by indirect immunofluorescence assay (IFA) and Western blotting, 1D9 detected successfully all 11 subgenotypes of EV71 and showed no cross‐reactivity to the four selected subgenogroups of Coxsackieviruses CAV4, CAV6, CAV10, and CAV16. A linear motif, R₃VADVI₈, which is located at the N‐terminus of the EV71 VP1 protein, was identified as the minimal binding region of 1D9. Alignment and comparison of the 1D9‐defined epitope sequence against the listed sequences in the NCBI EV71 database indicated that this epitope R₃VADVI₈ was highly conserved among EV71 strains, while no significant similarity was observed when blasted against the Coxsackieviruses. This suggests that the mAb 1D9 may be useful for the development of a cost‐effective and accurate method for surveillance and early differentiation of EV71 from CAV16 infection. J. Med. Virol. 84:1620–1627, 2012. © 2012 Wiley Periodicals, Inc.     

Understanding Enterovirus 71 Neuropathogenesis and Its Impact on Other Neurotropic Enteroviruses

Enterovirus A71 (EV‐A71) belongs to the species group A in the Enterovirus genus within the Picornaviridae family. EV‐A71 usually causes self‐limiting hand, foot and mouth disease or herpangina but rarely causes severe neurological complications such as acute flaccid paralysis and encephalomyelitis. The pathology and neuropathogenesis of these neurological syndromes is beginning to be understood. EV‐A71 neurotropism for motor neurons in the spinal cord and brainstem, and other neurons, is mainly responsible for central nervous system damage. This review on the general aspects, recent developments and advances of EV‐A71 infection will focus on neuropathogenesis and its implications on other neurotropic enteroviruses, such as poliovirus and the newly emergent Enterovirus D68. With the imminent eradication of poliovirus, EV‐A71 is likely to replace it as an important neurotropic enterovirus of worldwide importance.     

Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement

Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2₁₄₉ (VP2_149M) or glutamine to glutamic acid substitution at VP1₁₄₅ (VP1_145E) showed greater viral titers and apoptosis. Synergistic effect of VP2_149M and VP1_145E double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2_149M and VP1_145E mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.     

Patterns of polymorphism and divergence in the VP1 gene of enterovirus 71 circulating in the Asia-Pacific region between 1994 and 2013

Enterovirus 71 has been implicated in several outbreaks of hand, foot and mouth disease in the Asia-Pacific region. The present study aimed to achieve comprehensive evolutionary dynamic aspects of EV71 during 1994–2013, based on phylogenetic analyses of the VP1 sequences. The results indicated that 4 genotypes, namely C4, C1, C2 and B4 are the predominant strains, especially in Southeast Asian countries. No common ancestor was shared in different countries. Fourteen sites of substitutions were detected in the VP1 gene sequences; including the most common sites related to neutralization at position V249I [47.1% (189/401)] and A289T [42.6% (171/401)]. However, the sites Q22H and Q22R associated with increased virulence were recognized only in 13.7% (55/401) and 18% (72/401), respectively. None of the above mutations seemed to become fixed because the ratio of Ka/Ks was greater than 1.0. Mutations K43E, A58T, S184T, and T240S could possibly change the spatial structure. Two mutations, G145E and T240S, could obviously affect the hydrophobicity of VP1 and thus alter the EV71 immunoreactivity. In conclusion, the VP1 gene of EV71 strains circulating in the Asia-Pacific region during 1994–2013, showed polymorphisms and divergence with very slow evolution rate, which may be one of the reasons for periodic outbreaks in this area.     

2009年肠道病毒71型广州分离株的全基因组序列分析

目的分析2009年肠道病毒71型广州分离株GZHY09的全基因组序列,并与GenBank中的序列进行分析比对。结论从GenBank上选不同地区的EV71全基因组序列,设计相互重叠的覆盖病毒整个基因组序列的12对引物,采用RT-PCR扩增出12个基因片段,通过测序获得全基因组序列,并参考国内外各EV71基因型的分离毒株,用MEGA4软件进行进化树分析,用DNAStar软件中的MegAlign进行同源性分析。结果 12个重叠基因片段序列拼接获得EV71全基因组序列,共7404个核苷酸,同源性分析结果表明在VP1区,GZHY09株与中国大陆的Anhui08、Zhejiang08、Shenzhen08的核苷酸序列同源性比较高,其中以Anhui08最高（97.7%）。结论 GZHY09属EV71病毒的C4亚型,与近年我国大陆地区流行的EV71株在进化上属于同一基因型,与Anhui08、Zhejiang08、Shenzhen08具有高度的同源性。