Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase

In 1998, an enterovirus 71 (EV71) epidemic in Taiwan resulted in 78 deaths; however, the molecular basis of EV71 pathogenicity remains poorly understood. Comparison of the deduced amino acid sequences in 3D polymerases of EV71clinical isolates showed the T251V or T251I substitution from 1986 and 1998 outbreaks. An EV71 replicon system showed that introducing an I251T mutation did not affect luciferase activities at 35 °C when compared with wild type; however, lower luciferase activities were observed when they were incubated at 39.5 °C. In addition, the I251T mutation in the EV71 infectious clone not only reduced viral replication at 39.5 °C in vitro but also decreased the virulence of the mouse adaptive strain MP4 in neonatal mice in an i.p. infection model. Therefore, these results suggested that the threonine at position 251 results in a temperature sensitivity phenotype of EV71 which may contribute to the attenuation of circulating strains.     

肠道病毒71型山东临沂分离株全基因组序列分析

目的 自1例死亡患儿的标本中分离EV71,并分析其全基因组序列特点,研究其基因序列的改变是否与其神经毒力有关.方法 咽拭子标本采自山东临沂市人民医院1例死亡患儿,在人横纹肌瘤细胞(RD)上分离EV71,分段扩增获得EV71的全序列,用BLAST、Bioedit和MEGA 4进行序列分析.结果 获得1株EV71分离株SDLY107,基因组全长7405 bp,全基因组核苷酸序列与2008年的阜阳株Fuyang.Anhui.P.R.C/17.08/2同源性最高,为98.6%,与原型株BrCr/70的同源性为80.0%,与神经毒型株MS/87的同源性为86.5%.系统进化分析表明,SDLY107与中国大陆的北京株、河南株、广西株、深圳株、兰州株、阜阳株、重庆株、浙江株的亲缘关系较近,按照传统的VP1基因分型方法,可归为C4亚型,是近年来我国大陆流行的主要基因亚型.氨基酸序列分析发现,SDLY107与其他毒株相比,有2个特有的突变(E947D,K1873R).结论 SDLY107分离株属于C4亚型,氨基酸突变E947D和K1873R可能与EV71的致病性有关.

Abstract：

Objective To isolate enterovirus 71 from a death children,and analyze whether the neurovirulence was related to the variation of nucleotide and amino acid. Methods Enterovirus 71 was isolated from throat swabs which were colleted from Shandong Linyi People's Hospital. The full length genome was sequenced by amplification with RT-PCR and sequencing of 9 overlapped gene fragments covering full length of the genomes. The nucleotide and amino acid sequenced was aligned by BLAST, Bioedit and MEGA 4. Results A strain of enterovirus 71 was isolated and named as SDLY107. The full length was 7405 bp. The results of homology analysis of overall nucleotide sequence showed that strain Fuyang. Anhui. P. R. C/17.08/2 had highest homology (98.6%)with strain SDLY107, and the homology was 80.0% between strain SDLY107 with prototype strain BrCr/70,and 86. 5% between strain SDLY107 with nerve strain MS/87. Phylogenetic analysis showed that the phylogeny was close between SDLY107 with some isolated strains from Chinese Mainland, such as Beijing, Henan, Guangxi, Sbenzhen, Lanzhou, Fuyang, Chongqing and Zhejiang strains, which was clustered for C4 subtype. The results of amino acid sequence analysis showed that there were 2 mutations, E947D and K1873R, for strain SDLY107. Conclusion SDLY107 belonged to C4 subtype, amino acid mutations E947D and K1873R of which may be relevant to the pathogenicity of EV71.     

Clinical features of severe cases of hand,foot and mouth disease with EV71 virus infection in China

Introduction

Hand, foot and mouth disease (HFMD) caused by EV71 infection has become one of the major public health issues in China, which deeply affects children''s health. The prevention and control of EV71 is a challenge currently because there is no safe and effective vaccine or antiviral medications available.

Material and methods

A case control study was conducted in a designated hospital to compare severe and mild cases of patients infected with the EV71 virus. Demographic information along with clinical features of HFMD was collected through a standardized questionnaire. Multi-factorial logistic regression was used to analyze independent associations between potential risk factors and severe HFMD.

Results

There were 120 cases (60 cases and 60 controls) collected. The male-to-female ratio was 1.3: 1 in the case group and 1.7: 1 in the control group. Multi-factorial logistic regression revealed that the main risk factors for severe cases were highest body temperature being ≥ 38.5°C (OR = 9.45, 95% CI: 2.07–43.11, p < 0.05), first visited a village level clinic (OR = 4.72, 95% CI: 1.15–19.45, p < 0.05), etc.

Conclusions

Close surveillance combined with laboratory testing should be in place during the epidemic period of HFMD. Grass root level medical facilities and training of clinical and laboratory staff should be reinforced so that the diagnostic and treatment capacity can be improved.     

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.     

柯萨奇病毒A组6型与肠道病毒71型感染所致手足口病临床特征比较

目的 了解柯萨奇病毒A组6型、肠道病毒71型手足口病临床特点,为指导临床诊断,调整手足口病防控策略提供科学依据.方法 采用前瞻性调查的方法,收集2013至2014年在北京市西城区手足口病临床确诊病例信息,将手足口病原核酸检测阳性的病例分为CA6、EV71组,分析比较2组的临床特点、流行特征、预后等.结果 CA6组130例,EV71组121例.2组的年龄、性别分布差异无统计学意义.CA6组发热比例高达82.3％,明显高于EV71组(34.7％,P＜0.05),其中大于等于38℃占85.0％.CA6组出现大疱疹(7.4％)、恢复期脱皮(16.9％)、脱甲(10.0％)的比例高于EV71组(P＜0.05).不同年份流行主要毒株不同.结论 与EV71感染相比,CA6感染引起的高热比例较高,部分病例可表现为大疱疹、脱皮、脱甲,整体预后较好.CA6和EV71感染所致的HFMD发病高峰季节不一致.     

肠道病毒71型手足口病的疫苗筛选和观察

目的 筛选安全有效新型EV71候选疫苗,为将来EV71疫苗开发应用奠定基础.方法 以重组蛋白VP1为疫苗设计靶点,不同候选疫苗包括灭活疫苗、DNA疫苗、VP1蛋白疫苗在0、2周、4周分别按照不同剂量和肌内注射途径免疫BALB/c雌性小鼠,在0、2周、4周、6周、8周、10周、16周分别采集小鼠尾静脉血,16周处死小鼠,收集小鼠脾脏细胞,检测小鼠体液免疫和细胞免疫功能,筛选评价候选疫苗的疗效和安全性.结果 灭活病毒疫苗、VP1 DNA质粒疫苗、VP1蛋白疫苗免疫小鼠2周后IgG抗体滴度即开始升高,4周后明显升高,8周后达高峰,至少维持16周以上;IgG亚类以IgG2a和IgG1为主.三种疫苗能诱导以γ-IFN和IL-4为主的细胞免疫反应.灭活疫苗疗效优于其他候选疫苗,未发现疫苗相关不良反应.结论 灭活病毒疫苗、VP1 DNA质粒疫苗、VP1蛋白疫苗均能诱导持久的特异性细胞免疫和中和抗体反应,以灭活病毒疫苗疗效较好,需要将来攻毒实验可进一步验证其免疫力.     

广州地区健康儿童肠道病毒71型抗体水平调查

目的调查广州地区健康儿童肠道病毒71型抗体IgG（EV71-IgG）的水平,分析与EV71所致手足口病（HFMD）发病的关系。方法以来源于广州市妇女儿童医疗中心（我院）中心实验室分离到的EV71 C4亚型标本,制备EV71诊断抗原,建立EV71-IgG抗体诊断试剂盒方法,并进行实验室评价。收集2010年1~3月在我院儿保科抽血进行常规保健检测的标本,筛选出无发热、无HFMD症状的健康儿童血清标本,检测EV71-IgG抗体。选取我院2010年全年门诊及住院HFMD患儿的咽拭子标本进行EV71特异性核酸检测。结果①本研究建立的试剂盒EV71-IgG抗体检测临界值为0.148;与细胞中和实验方法阳性符合率为100%,阴性符合率为92%,与柯萨奇A16病毒抗体无交叉反应。②819名健康儿童血清标本中,男481名,女338名。〈1岁组（60%,60/100名）和~2岁组（39.3%,72/183名）EV71-IgG抗体阳性率显著高于其他各年龄组[~3岁、~4岁、~5岁和~14岁组分别为12.9%（15/116名）、27.1%（38/140名）、14.2%（16/113名）和22.8%（38/167名）]。EV71-IgG抗体阳性率无显著性别差异。③4 780例EV71阳性HFMD患儿中,男3 070例,女1 718例。〈1岁310例（6.5%）,~2岁1 157例（24.2%）,~3岁1 337例（28.0%）,~4岁1 094例（22.9%）,~5岁450例（9.4%）,~14岁432例（9.0%）。④各年龄组EV71-IgG抗体阳性率与EV71阳性年龄构成比呈相反趋势,〈1岁组EV71-IgG抗体阳性率最高,EV71阳性年龄构成比最低;~3岁组EV71-IgG抗体阳性率最低,EV71阳性年龄构成比最高。结论 〈1岁健康儿童EV71-IgG阳性率最高,EV71阳性年龄构成比与EV71-IgG阳性率呈相关现象。     

Molecular epidemiology analysis of enterovirus 71 strains isolated in Dak Lak,Vietnam, 2011-2016

Human enterovirus 71 (EV71) is the major etiologic agent of hand, foot, and mouth disease (HFMD). EV71 outbreaks have been reported in Dak Lak in recent years, however, the genotypes/subgenotypes information and phylogeny of circulating EV71 strains are limited. The objectives of this study were to determine the genotypes/subgenotypes and investigate the phylogeny of EV71 isolates in Dak Lak over a 6-year period. Viruses were isolated from clinical samples from patients with HFMD. In total, 43 EV71 isolates circulated in Dak Lak during 2011-2016 were used for the phylogenetic analysis using complete VP1 gene. The phylogenetic analysis of the VP1 gene revealed that two major genotypes, B and C, were found. Among the 43 EV71 strains, 29 belonged to subgenotype C4, 2 belonged to subgenotype C5, and 12 belonged to subgenotype B5. Of these, the subgenotype C4 was predominant in 2011-2013 and this was later replaced by the subgenotype B5 in 2014. The subgenotype B5 was dominant between 2014 and 2015, and then C4 recirculated in 2016. Our study also indicated that the subgenotypes C4 and B5 emerged into Dak Lak were closely related to variants causing epidemics of HFMD in the southern and central region of Vietnam and Thailand. Sequence analysis showed that nine amino acid mutations were detected in the VP1 region. Our results identified two significant amino acid substitutions (D31N and E145G/Q) associated with enhancing EV71 virulence.     

感染肠道病毒71型尸检病例的分子病原学诊断

目的 探讨感染肠道病毒71型(EV71)的尸检病例石蜡包埋组织病原学分子检测的应用价值.方法 尸体解剖2例怀疑因EV71感染死亡的患儿,对脑组织进行了组织学观察和免疫组织化学EnVision法标记;应用逆转录聚合酶链反应检测尸检后的石蜡组织中肠道病毒的核酸并测序分析.结果 2例均有中枢神经系统脑干脑炎的病理学特点.坏死神经元周围可见大量的小胶质细胞(CD68阳性)和少量的中性粒细胞(CD15阳性)浸润.2例延髓石蜡组织中均检测出EV71的核酸序列,与GenBank最新公布的安徽阜阳暴发EV71感染的病毒株序列同源性为100%.结论 从甲醛固定、石蜡包埋的EV71感染患儿脑组织中检测出病毒特异性核酸序列,及时明确诊断临床误诊、漏诊的死亡病例,深化认识了手足口病,为公共卫生管理机构及时加强EV71病原学监测提供决策依据.     

2008-2009年北京分离的肠道病毒71型基因组3'末端序列分析

目的 了解2008-2009年北京分离的肠道病毒71型(EV71)基因组3'末端的基因特征(未包括多聚腺苷尾),探讨是否与病毒的致病性有关.方法 于2008-2009年手足口病流行期间采集来首都儿科研究所附属儿童医院就诊的普通手足口病患儿和合并重症神经系统并发症患儿的咽/鼻拭子或疱疹液标本.将标本接种Vero细胞进行肠道病毒分离,同时设计肠道病毒通用引物、EV71和柯萨奇病毒A组16型(CA16)特异性引物通过巢式RT-PCR对分离到的肠道病毒进行型别鉴定.对引起不同临床类型感染的10株EV71分离株的基因组3'末端进行序列测定和分析.结果 10株EV71的3'末端均包含1386 nt的3D、终止密码子TGA和81 nt的3'UTR,由3D核苷酸序列所推导的3D蛋白含462个氨基酸.10株EV71的3D和3'UTR核苷酸同源性分别为95.8%～99.6%和96.3%～100%,重症来源的4株毒株中有3株在3Dpol第140位和第263位同时出现了相同的氨基酸变异(R140K和I263V).10株EV71的3D与C4亚型代表株具有最高的核苷酸和氨基酸同源性,分别为92.7%～94.2%和96.8%～97.6%;在3'UTR与CA16/G10具有最高的核苷酸同源性(88.9%～91.4%).3D区的遗传进化分析表明10株EV71在亲缘关系上与C4亚型最密切,与CA16/G10较密切,而与EV71其他基因型和亚型较疏远.结论 基因组3'末端在EV71进化中可能有一定的作用.     

西安地区2008年肠道病毒71型基因特征分析

目的 研究西安地区2008年引起手足口病病原构成及EV71的基因特征.方法 采集124例临床诊断手足口病病例标本,RT-PCR检测肠道病毒血清型别;挑选EV71阳性标本进行病毒分离,扩增7株EV71病毒,扩增其VP1区,测序并与EV71各血清型代表株序列比对,进行进化分析.结果 2008年西安地区手足口病（HFMD）的病原中CA16占49.45%,EV71占30.76%,其他肠道病毒占19.78%.7株EV71 VP1区与标准株序列比对,亲缘进化分析显示本地区EV71与中国大陆其他地区毒株相似.结论 2008年西安地区引起手足口病的病原以CA16为主,而EV71属于C4亚型.     

北京市2008年肠道病毒71型的RT-PCR鉴定及VP1区基因特征分析

目的 研究2008年北京市手足口病（Hand,foot and mouth disease,HFMD）患者中肠道病毒71型（Human enterovirus 71,HEV71）的VP1编码区基因特征.方法 采集北京市朝阳区医院和幼儿园的HFMD患者标本共285份,进行HEV71特异性RT-PCR鉴定和病毒分离,随机选取10株HEV71阳性分离株进行VP1编码区基因扩增和核苷酸序列测定和分析.结果 129份标本RT-PCR鉴定结果 为阳性,阳性率为45.26%.10株HEV71在VP1区核苷酸水平和氨基酸水平上的差异分别在94.6%～99.6%和95.9%～100%之间.北京朝阳HEV71分离株属于C4基因亚型C4a进化分支.结论 RT-PCR法可以快速、准确地鉴定HEV71.引起本次HFMD流行的HEV71为C4基因亚型C4a进化分支,并且存在多个传播链,提示自1998年起,C4亚型的HEV71在中国大陆有较广泛的传播.加强对HEV71的分子流行病学监测,了解HEV71的基因特征,对预防和控制HEV71在中国的暴发具有重要意义.     

EV71、CA16及通用型肠道病毒实时荧光定量PCR诊断试剂盒的研发

目的 研制一种新的含竞争性内标引物,能同时检测EV71、CA16及通用型肠道病毒的四重实时荧光定量PCR诊断试剂盒,用于手足口病的临床诊断及流行病学监测.方法 设计特异性的EV71型、CA16型及其他型肠道病毒和内参基因的引物,改进病毒核酸提取方法,优化实时荧光定量PCR检测体系,并研究产品的准确度、稳定性、精密度和扩增效率和检测线性范围.结果 本试剂盒的引物和探针特异性好,试剂盒采用的的病毒RNA抽提效果与QIAGEN公司QIAamp Viral RNAmini Kit抽提效果相当,但具有更低的试剂成本.优化引物探针浓度分别为0.2μmol/L的上下游引物浓度、0.06 μmol/L的通用探针浓度、0.08 μmol/L的EV71分型探针浓度和0.08μmol/L的CA16分型探针浓度.产品具有较好的稳定性和良好的准确度、精密度,CA16、EV71和肠道病毒通用型对引物的扩增效率分别是106％、101％和105％,线性检测范围是109拷贝/μl ～ 102拷贝/μl.结论 采用四重荧光定量PCR技术开发的能同时检测EV71、CA16、其他肠道病毒和内标的手足口病诊断试剂盒具有良好的准确性、稳定性、精密度和扩增效率等,具有很好的临床应用价值.     

吉林省2009-2012年肠道病毒71型VP1编码区氨基酸位点的变异分析

目的分析2009--2012年引起吉林省手足口病（handfootandmouthdisease,HFMD）流行的肠道病毒71型（EV—A71）的VP1区氨基酸位点的特征性变异位点。方法对2009--2012年分离于吉林省HFMD病例的70株EV—A71分离株的VPl编码区进行逆转录．聚合酶链反应,然后对扩增产物进行核苷酸序列测定,使用Bioedit软件和MEGA4．0软件分析VPl区氨基酸位点的变异特征以及基因亲缘关系。结果2009--2012年在吉林省分离到的70株EV—A71均属于C4a基因亚型,其中69株与2008年安徽阜阳的分离株亲缘关系最近,并且这69株病毒在VPl编码区第22位氨基酸位点均由谷氨酰胺转变为组氨酸（Q—H）,与2008年安徽阜阳株一致。结论引起2009-2012年吉林省HFMD流行的EV-A71可能：来源于安徽阜阳流行株延续传播。     

EV71受体SCARB2和 PSGL-1在重症手足口病患者肺组织中的表达

目的：探讨人肠道病毒71型(enterovirus 71, EV71)清道夫受体B2(scavenger receptor class B member 2, SCARB2)和人P选择素糖蛋白配体-1(P-selectin glycoprotein ligand-1, PSGL-1)在重症手足口病(hand, foot and mouth disease, HFMD)患者、正常儿童及成人肺组织中的定位和分布。方法运用免疫组化SuperVision两步法检测15例重症HFMD患者、3例正常儿童及8例成人肺组织中SCARB2和PSGL-1的表达及分布。结果 SCARB2分布于重症HFMD患者、正常儿童及成人肺组织支气管上皮、细支气管上皮、肺泡上皮细胞及炎症细胞,其在三组中的阳性率差异无显著性( P>0．05)。 PSGL-1分布于成人肺组织支气管、细支气管上皮,但在重症HFMD患者及正常儿童中不表达,阳性率分别为100%、0、0,三组比较差异均有统计学意义(P<0．05),三组肺组织的炎症细胞PSGL-1均为阳性,阳性率分别为100%、66．7%、100%(P>0．05);PSGL-1在三组的肺泡上皮细胞中均不表达。结论 EV71受体SCARB2分布于重症HFMD患者肺组织支气管上皮、细支气管上皮、炎症细胞及肺泡上皮细胞,PSGL-1仅分布于重症HFMD患者肺组织炎症细胞,提示SCARB2可能在重症HFMD的感染过程中具有一定作用。