柯萨奇病毒A组天津分离株的分型鉴定及分子特征分析

目的 鉴定天津市手足口病病原体柯萨奇病毒A组2、4、5、6和10型,并分析其VP1区基因及分子流行病学特征.方法 提取45株非EV71非CV-A16肠道病毒分离株核酸,利用RT-PCR法扩增其VP1基因并测序,然后根据VP1区基因核酸序列,进行肠道病毒型别鉴定和同源性分析,构建种系发生树.结果 45株非EV71非CV-A16肠道病毒天津分离株分别为6株柯萨奇病毒A组2型(Coxsackie virus A2,CV-A2),14株CV-A4,3株CV-A5,8株CV-A6和14株CV-A10.柯萨奇病毒各型的株间核酸序列同源性均在80％以上,各型分离株与原型株的同源性均在71.2％ ～ 85.8％之间.天津各型分离株在种系发生树上均聚集于各自相对独立的分支,与国内流行株处在同一分支内,而与国外原型株处于不同的进化分支.结论柯萨奇病毒A组2、4、5、6和10型成为天津市手足口病的流行病原体,各型天津分离株株间核酸序列同源性较高,呈现一定的区域聚集性.     

2001年徐州地区暴发性病毒性脑膜炎病原的研究

目的 确定引起2001年江苏省徐州地区无菌性脑膜炎流行的病原体。方法 组织培养法从患者脑脊液分离病毒，标准血清中和试验鉴定分离毒株；中和试验检测双份血清中和抗体效价；逆转录聚合酶链反应（RT-PCR）检测肠道病毒特异性基因片段。结果 22份脑脊液中分离出4株柯萨奇B5型、2株柯萨奇B3型、1株艾可7型肠道病毒，分离阳性率31．8％。RT-PCR检测脑脊液21份，肠道病毒阳性11份。阳性率52．4％。19例双份血清中11例中和效价呈4倍以上增长或转阳，阳性率57．9％。结论 此次江苏省徐州地区无菌性脑膜炎流行的病原体是以柯萨奇B5为主要血清型的肠道病毒。     

柯萨奇病毒B5引起济南儿童病毒性脑炎流行的血清流行病学调查

2003年夏秋季山东济南地区发生不明原因的病毒性脑炎(VE)流行,仅济南市儿童医院就收入院病人150例.从本次脑炎流行患儿脑脊液标本中分离得到5株病毒,以分子生物学方法鉴定其为肠道病毒--柯萨奇病毒B5(CVB5).为进一步验证CVB5与本次脑炎流行的因果关系,我们用分离所得病毒株制备抗原,通过间接ELISA对本次脑炎流行进行血清流行病学调查.     

心肌炎和克山病患者分离的肠道病毒核酸序列分析

目的 分析自心肌炎和克山病患者分离的肠道病毒的核酸序列,探讨病毒病因的分子基因。方法 使用肠道病毒特异引物对从心肌炎和克山病患者分离的肠道病毒RNA进行RT－PCR扩增,PCR产物经纯经后分别经不同的引物正反向直接核酸循环测序,并应用SeqEd和DNA软件及AssemblyLIGN软件对测序结果进行分析。结果 确定7株病毒5′端从核苷酸位点40到750之间710bp基因序列;序列分析结果发现,尽管为同血清型病毒,但其5′非编码区基因变异率仍在15％左右,而血清型为柯萨奇B5病毒则高达34．08％,与肠道病毒各血清型之间5′端非编码区基因变异率无明显差异;对血清型为CVB3的两分离株病毒5′端基因特殊位点分析,发现与CVB3标准株（Nancy株）比较234位由T→C、690位由C→A。结论 确定了所分离到的一些肠道病毒5′端非编码区序列;肠道病毒5′端非编码区基因与血清型关系不大;CVB3分离株5′端基因特殊位点变异与致病的关系值得进一步探讨。     

应用随机PCR方法鉴定一株肠道病毒

目的 从基因水平对一肠道病毒分离株进行分析鉴定,方法 提取病毒RNA,以锚定随机引物反转录合成双链cDNA,用锚定特异引物进行随机扩增,扩增产物进行克隆,测序和序列分析。结果 随机扩增的病毒基因在电泳上呈典型的smear带型;11个随机克隆经GenBank检索,与肠道病毒成员具有最高的同源性且同源率超无穷大0%;11个克隆随机分布于病毒的基因组中,排定后的序列形成4个毗连序列群,共计2261个碱基,涵盖病毒基因组的30%;部分序列与柯萨奇病毒B6型氨基酸同源率达97.3%,参照测定序列合成的引物能够特异性地扩增病毒基因。结论 经序列分析证实小RNA病毒XJ90株为肠道病毒科成员。     

山西省临汾市ECHO30病毒所致病毒性脑炎疫情的病原学分析

目的 对山西省临汾市一起病毒性脑炎疫情进行病原学分析.方法 用Real-timePCR方法对9份咽拭子标本进行肠道病毒核酸检测.对肠道病毒核酸阳性的标本,分别扩增肠道病毒5'UTR区和VP2基因区片段,PCR扩增产物测序后,经BLAST比对初步确定病毒型别为ECHO30;使用ECHO30的特异性引物扩增VP1基因部分片段,测序后使用MEGA 4.0软件与NCBI数据库的其他ECHO30毒株进行进化分析.结果 9份病毒性脑炎标本中有4份为肠道病毒阳性;经肠道病毒5'UTR片段、VP2基因和VP1基因测序,确定4条临汾株ECHO30病毒核苷酸序列与2004年浙江ECHO30株和ECHO30标准株Bastianni同源性较高,且4条ECHO30病毒VP1基因的差异性为0.5％.进化分析显示,本地区的4株ECHO30病毒自成一簇,且与中国大陆地区的其他参考株亲缘关系较近.结论 肠道病毒属ECHO30病毒引发山西省临汾市病毒性脑炎疫情,有必要监测本地区的病毒性脑炎病原谱.     

九江地区手足口病病原柯萨奇A6病毒的分离、鉴定及分子流行病学特征

目的探讨九江地区流行手足口病病(HFMD)原体柯萨奇A6(CVA6)的分离、鉴定及分子流行特征。方法取手足口病患儿的咽拭子,将咽拭子病毒悬液接种于人横纹肌肉瘤细胞(RD细胞)。通过CVA6病毒致细胞病变效应(CPE)分析、MTT法检测病毒增殖效果、CVA6病毒特异性核酸聚合酶链反应(PCR)及同源和遗传进化分析等方法检测CVA6的致病效果和分子流行特征。结果咽拭子病毒悬液接种RD细胞盲传5代后出现CPE,收集其病毒培养基上清,再接种RD细胞2次,均出现CPE视为CPE阳性;CVA6病毒VP1区域通过序列比对,以此确认2株病毒为CVA6,分别命名JJ2012037和JJ2012039;CVA6呈时间依赖性抑制宿主细胞增殖;利用CVA6毒株的VP1序列和国内外流行的CVA6序列进行核苷酸同源性分析;该毒株与上海分离到CVA6(JX495144)序列发生的同源性最高(99.2%～99.6%)。结论成功分离出2株九江地区流行的手足口病病原CVA6病毒株,这2株CVA6分离株的分子流行特征接近上海地区的CVA6的流行病原,可能为同一个基因亚型。     

2015—2018年青海省手足口病病原谱变化和流行趋势

目的了解青海省手足口病(hand, foot and mouth disease, HFMD)患者中肠道病毒流行情况和基因型特征。方法采集青海省HFMD患者的咽拭子标本, 进行病毒分离和特异性引物聚合酶链反应(RT-PCR), 对扩增产物进行核苷酸序列测定和分析, 并参考NCBI部分毒株序列构建基因进化树。结果采集临床诊断HFMD阳性病例标本1 738份, 其中人肠道病毒A组71型(EV-A71)型326例, 占18.76%、人肠道病毒柯萨奇A16型(CV-A16)型237例, 占13.64%、人肠道病毒柯萨奇A6型(CV-A6)型628例, 占36.13%, 4年间不同基因型别间存在差异, 具有统计学意义(EV-A71, χ2=245.315,P<0.001; CV-A16, χ2=27.680,P<0.001; CV-A6, χ2=702.713,P<0.001), 用RD细胞分离后共得到到317株细胞培养物, 测序后选取2017年和2018年典型基因型的VP1区核酸序列进行序列分析, 全部基因型VP1区核酸序列间同源性在59%～100%之间, 19株EV-A71...     

新型肠道病毒分离株(BJ302)全基因组序列测定及分析

目的 测定并分析新分离肠道病毒基因组序列,从病毒的分子生物学角度研究其分类学位置.方法从1例不明病因的急性肝炎患者粪便标本中提取病毒RNA,以随机引物逆转录PCR法及5'、3'RACE扩增病毒全基因序列.将拼接得到的核苷酸序列及推断的氨基酸序列与GenBank数据库中序列进行比较并建立系统进化树.结果分离病毒基因与肠道病毒76、89型有较高的同源性,尤其是与肠道病毒89型,相应片段的核苷酸序列同源性达83%～94%,推断相应氨基酸序列同源性高达91%～100%,而与其他病毒不存在有意义的同源性;系统进化树分析显示新分离肠道病毒株属于肠道病毒属中新型肠道病毒89型一簇.结论新分离病毒属肠道病毒89型,可能为89型的一个新亚型.     

引起河南省一起病毒性脑炎暴发的柯萨奇病毒B5的鉴定及其序列分析

目的 鉴定2011年引起河南省漯河地区一起病毒性脑炎(VE)暴发的病原体,并对分离的柯萨奇病毒B5(CVB5)进行基因特征分析.方法 采集漯河地区病毒性脑炎暴发期间29例患者的5份咽拭子、21份粪便和14份脑脊液,采用实时荧光RT-PCR方法分别检测总肠道病毒(PE)、EV71及CA16病毒核酸.所有标本均进行病毒分离培养,对其中5例患者的2份粪便和3份脑脊液的阳性分离产物进行VP1和5′-UTR区核苷酸序列测定及亲缘进化分析.结果 实时荧光RT-PCR结果显示,所有临床标本EV71和CA16病毒核酸检测均为阴性,咽拭子、粪便和脑脊液的PE核酸检测阳性率分别为60.0％(3/5)、61.9％(13/21)和85.7％ (12/14).咽拭子、粪便和脑脊液病毒分离率分别为20.0％(1/5)、25.0％(5/21)和29.0％ (4/14).VP1和5′-UTR区核苷酸序列BLAST分析及分子分型结果显示为CVB5,对其中5株分离毒株进行VP1全长基因分析显示彼此之间核苷酸同源性为97.9％ ～ 99.5％.与其同源性最近的毒株是2010年引起长春手足口病暴发的CVB5分离株CC10/10/Changchun,核苷酸同源性为97.1％～98.1％.与2010及2012年河南省平顶山地区的脑炎分离株COXB5/Henan/2010和03001N/HN/CHN/2011/CB5的同源性分别为89.0％～89.6％和91.8％～92.5％.VP1区遗传进化树显示此次分离株为基因型D,且与长春株成一簇.结论 导致此次病毒性脑炎暴发的病原体为肠道病毒CVB5,优势基因型的变迁可能与此次暴发相关.     

Molecular characterization of a coxsackievirus A24 variant that caused an outbreak of acute haemorrhagic conjunctivitis in Spain, 2004

BACKGROUND: Coxsackievirus A24 variant is one of the major etiological agents involved in acute haemorrhagic conjunctivitis. STUDY DESIGN: An outbreak of acute hemorrhagic conjunctivitis occurred in the Southeast of Spain between September and November 2004. Cellular and molecular methods were used to identify and characterize the viral agent associated with the epidemic. RESULTS: Enterovirus was detected in the conjunctival swabs of 35 patients. None of the viruses isolated could be typed by conventional neutralization assays; however, amplification and sequencing of the 3'-end VP1 region of 19 of the samples identified coxsackievirus A24 variant as the serotype causing the outbreak. Phylogenetic analysis of the 5'-half VP1 region of the genome revealed that Spanish sequences, like other strains isolated during outbreaks of hemorrhagic conjunctivitis in American and African countries in 2003 and 2004, were closely related to the isolates detected in Korea (2002), thus proving their worldwide spread. CONCLUSIONS: This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Spain. Molecular typing in combination with phylogenetic analysis is useful to study the enterovirus epidemiology associated with epidemics.     

Enterovirus sequences resembling coxsackievirus A2 detected in stool and spleen from a girl with fatal myocarditis

A 10-year-old girl who died suddenly was found at post mortem to have myocarditis. Virus could not be cultured from post-mortem stool, spleen or heart but enterovirus RNA was detected in stool and spleen by PCR, and the stool caused flaccid paralysis in newborn suckling mice. A 654 base pair (bp) sequence from the capsid-coding region of the viral genome was amplified from an affected mouse and sequenced. Using this sequence, strain-specific nested primers were designed and used to amplify viral sequences directly from stool and spleen. These sequences were identical to each other and to that obtained from the infected mouse, and most closely resembled Coxsackievirus A2, an uncommon serotype rarely associated with myocarditis. Testing spleen tissue may be useful in etiological investigation of suspected viral myocarditis. PCR proved more sensitive than suckling mouse inoculation in detecting this Coxsackievirus, but a combination of both methods was required for genotypic characterization.     

北京地区与手足口病相关的非EV71、非CoxA16型肠道病毒的分子特征分析

目的 了解北京地区引起手足口病的非EV71、非CoxA16型肠道病毒的病原构成及优势型别VP4区遗传特征分析.方法 收集2009年4月-12月检测为非EV71、非Cox16型肠道病毒的手足口病患者咽拭子标本共45份.提取病毒核酸,用巢式RT-PCR法扩增病毒VP4区序列,对PCR阳性扩增产物进行测序,通过与GenBan...     

New enteroviruses, EV-93 and EV-94, associated with acute flaccid paralysis in the Democratic Republic of the Congo

Surveillance of acute flaccid paralysis often identifies enteroviruses not typeable by virus neutralization in cell culture. During 2000 and 2001, 186 isolates from 138 children with acute flaccid paralysis in the Democratic Republic of the Congo were sent for typing to the National Reference Centre for Enteroviruses in Lyon, France. The 5' UTR of the viral genome could be amplified by PCR for 158 isolates from 114 patients. Isolates from 89 patients were neutralizable, and contained non-polio enterovirus types. Seventeen children were infected with more than one entero- or adenovirus; another three were co-infected with both these viruses. Serological typing failed with 19 isolates from 13 (9%) patients. The VP1 region of these strains could be amplified by PCR and sequenced, which revealed that five children were infected with CV-A17, EV-70, EV-76, EV-77, or CV-A13. Two patients were doubly infected, one with CV-A24 and E-9, and another with E-27 and EV-81. Isolates from six children contained strains with divergent VP1 region. The amino acid sequences of these complete VP1 regions diverged >or=28% from published types indicating that they represented two new enterovirus types, tentatively designated EV-93 belonging to HEV-B and EV-94 within HEV-D. The latter enterovirus has in parallel been isolated from sewage in Egypt. In conclusion, there was a high frequency of "untypable" enterovirus isolates from cases with acute flaccid paralysis in the Democratic Republic of the Congo. Six of these were shown to represent two enteroviruses not previously described.     

Detection of enteroviruses in endomyocardial biopsy by molecular approach.

Enteroviruses are considered to be the most common agents implicated in myocarditis and cardiomyopathy. Recent studies have suggested persistent enterovirus infection in chronic disease showing the presence of enteroviral RNA in the myocardium. We used gene amplification by PCR which can demonstrate directly the presence of enteroviral sequences in endomyocardial biopsies. The primers were chosen in the 5' non-coding region of the genome representing highly conserved sequences among enteroviruses and therefore allowed the amplification of the majority of enteroviruses. The hybridization of the amplified products was effected with specific general riboprobe derived from 5' non-coding sequences internal of the amplified fragments. The results include 105 patients distributed in 6 groups: 45 idiopathic dilated cardiomyopathies with 66.7%, 17 alcoholic cardiomyopathies with 52.9%, 10 myocarditis with 30%, 5 multifactorial cardiomyopathies with 40%, 5 patients with immunosuppressive therapy with 100%, and 23 control group without viral etiology with 39.1% positive samples. The study suggested a positive link between viral infection and cardiomyopathies, but did not allow a direct relation between enterovirus infection and idiopathic dilated cardiomyopathy to be established.     

Rapid diagnosis of acute hemorrhagic conjunctivitis due to coxsackievirus A24 variant by real-time one-step RT-PCR

Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant.     

福建省新分离3株乙型脑炎病毒的分子特征鉴定

目的　确定福建省新分离乙型脑炎病毒 (JEV)的基因分型及其E区段氨基酸序列特征。方法　用逆转录聚合酶链反应 (RT PCR)扩增并克隆新分离JEV(0 2 4 1、0 2 4 3、0 2 10 2 )的PrM、E区段核苷酸序列 ,测序后应用ClustalX软件做碱基配对和比较分析 ,种系发生采用PHYLIP软件包分析。结果　新分离的 3株JEV属于基因Ⅲ型 ,E区段核苷酸和氨基酸与减毒活疫苗株SA 14 14 2株的同源性均在 96 %以上 ,在关键结构域有部分氨基酸差异。结论　福建省新分离的 3株病毒属于基因Ⅲ型JEV ,E蛋白与疫苗株相比有部分氨基酸差异