湘潭地区2013-2014年手足口病病原监测结果分析

目的:分析2013-2014年湖南省湘潭市手足口病(HFMD)病原体型别及分布特征,为进一步完善HFMD的预防和治疗提供依据。方法:采用RT-PCR方法,对571例HFMD患儿进行肠道病毒通用型核酸(EV-RNA)、EV71及柯萨奇病毒A16(CA16)检测。结果:2013年除EV71及CA16外其他EV核酸阳性率75.30%,明显高于EV71的17.65%和CA16的7.05%(P0.05),2014年EV71型核酸阳性率为50.68%,明显高于CA16的19.93%和其他EV的29.39%(P0.05)。重症HFMD患儿EV71感染率明显高于轻症。结论:2013年湘潭地区HFMD患儿以除EV71及CA16外的其他EV病毒感染为主,2014年湘潭地区HFMD患儿以EV71感染为主。     

手足口病防控策略探讨

肠道病毒71型(enterovirus 71,EV71)全病毒灭活疫苗的研发成功,对严重影响婴幼儿健康的手足口病(hand,foot and mouth disease,HFMD)的流行提供了防控手段。在婴幼儿目标人群中大量推广疫苗接种,有望降低HFMD的重症和死亡病例的发生率,具有重大的公共卫生意义。本文旨在分析中国EV71疫苗上市后所产生的结果和影响、HFMD的防控形势及疫苗应用策略等,从而对有关HFMD流行的防控策略进行探讨。     

肠道病毒71型疫苗质量控制研究进展

手足口病(Hand foot and mouth disease,HFMD)已成为西太平洋区严重的公共卫生问题之一,肠道病毒71型(Enterovirus 71,EV71)是引起HFMD的主要病原体,研发疫苗是控制HFMD流行的有效手段。目前世界范围已有5个EV71全病毒灭活疫苗进入临床试验,其中中国研制的3种EV71疫苗已分别完成III期临床试验,结果表明3种疫苗都具有良好的安全性和保护效果。在EV71疫苗研发中,抗原质控的方法和标准是研发的瓶颈,其中抗原活性测定是工序工艺优化、免疫原性评价和剂量确定的关键指标。综述了EV71疫苗抗原质控的研究进展。     

2009年上海市手足口病流行病学和病原学特征

本研究对上海市手足口病(Hand-foot-and-mouth disease,HFMD)的流行病学和病原学特征进行了分析。从国家疾病监测信息报告管理系统获取上海市2009年HFMD的流行病学数据;采用荧光定量RT-PCR方法对来自上海15个区县的799例HFMD进行肠道病毒(Enterovirus,EV)核酸检测;对部分肠道病毒71型(Enterovirus71,EV71)的VP1区和部分其它EV的VP4区序列进行测定和分析。采用Office excel软件进行简单数据统计分析,用BioEdit和MEGA软件进行病毒基因特征分析,通过BLAST服务器的在线比对进行EV型别鉴定。分析显示:上海市18个区县均有病例报告,地区分布无显著特点;小于6岁的婴幼儿期为疾病高发年龄段;4～7月为发病高峰期;EV71和柯萨奇病毒A16(Coxsackie virus A16,CA16)为主要病原,不同地区、不同月份的病原构成各不相同,CA16为轻症病例的主要病原,EV71则为重症病例的主要病原;基于VP1区序列分析,上海EV71毒株与C4a亚型毒株具有最近的亲缘性和最高的同源性;2株其它肠道病毒经鉴定属于CA2和CA10型。结果表明:EV71和CA16为2009年上海HFMD流行的主要病原,EV71属于C4a亚型;除EV71和CA16外,还存在小部分其它EV(如:CA2和CA10)引起的HFMD。     

柯萨奇病毒A组16型分子流行病学和疫苗研究进展

柯萨奇病毒A组16型(Coxsackievirus A16,CVA16)是引起手足口病(Hand,foot,and mouth disease,HFMD)的重要致病原,常与另一重要病原体肠道病毒71型(Enterovirus 71,EV-A71)共同或交替流行。该病毒在世界多个国家和地区引起过暴发流行,成为重要的公共卫生问题。CVA16可分为A和B两个基因型;其中基因型B可以分成B1和B2两个基因亚型;B1亚型又可进一步分成B1a、B1b和B1c三个进化分支。基因型A与B2在世界范围内已不再流行;我国大陆分离到的CVA16毒株均属B1a和B1b进化分支。CVA16感染多为自限性轻症,但亦可引起严重的并发症,甚至引起患者死亡。目前尚无针对该病毒的有效药物或治疗手段,研发安全有效的疫苗成为控制该病毒的重要措施。随着EV-A71疫苗三期临床试验的成功完成,CVA16疫苗的研究也显得更加迫切。多家机构正在研究各种类型的CVA16疫苗,包括灭活疫苗、基因工程疫苗及DNA疫苗等。本文就CVA16的分子流行病学及其疫苗研究进展进行了综述。     

EV-A71感染导致的重症手足口病的机制及防控研究进展

手足口病(Hand,foot and mouth disease,HFMD)是一种具有高度传染性的病毒性传染病,通常夏秋季高发于幼儿和儿童;若患儿并发呼吸和循环功能障碍、神经系统受累等临床症状称为重症HFMD。少数重症病例可出现肺水肿、脑炎和急性弛缓性麻痹等罕见的神经或循环系统并发症,甚至导致患儿死亡。引起HFMD最常见的病原是肠道病毒A71型(EV-A71)和柯萨奇病毒A16型(CV-A16),而EV-A71是引起重症HFMD的主要病原体。EV-A71导致的重症HFMD已成为全球重要的公共卫生问题,减少EV-A71流行范围和预防EV-A71重症HFMD非常重要。EV-A71疫苗是目前最有效的预防重症HFMD发生的措施;中国已经批准了三个厂家的灭活EV-A71疫苗上市并已开展适龄儿童接种,以期预防EV-A71感染引起的重症HFMD,但这种疫苗不能预防其它肠道病毒如CV-A16,CV-A6和CV-A10等引起的HFMD。据文献报道,小分子抑制剂芦平曲韦可以通过阻止EV-A71的3C pro蛋白活性来抑制EV-A71复制;小干扰RNA和单克隆抗体也可抑制EV-A71复制。研制EV-A71和CVA16双价灭活疫苗是防控HFMD的策略之一,而小分子抑制剂、小干扰RNA和单克隆抗体的研制和应用等也是临床防治HFMD的探索。     

肠道病毒71型疫苗的研究进展

近年来,手足口病(Hand foot mouth disease,HFMD)在中国多次爆发流行,严重威胁公众健康,尤其是5岁以下的婴幼儿。而肠道病毒71型(Enterovirus 71,EV71)是引起手足口病的主要病原体之一,由于目前还无针对该疾病有效的抗病毒药物,研制疫苗是控制EV71流行最为有效的措施。目前EV71疫苗及相关研究均取得重大突破,本文就近年来关于EV71疫苗研发、动物模型等的研究进展作一综述。     

肠道病毒71型的研究进展

肠道病毒71型(enterovirus71,EV71)是小RNA病毒科(Picornaviridae)肠道病毒属(Enterovirus)成员,其感染主要引起患者手足口病(hand-foot-and-mouth disease,HFMD).通常情况下,EV71感染引起的HFMD在临床症状等方面与柯萨奇病毒A16(Coxsackie A16,CA16)引起的手足口病难以区别,但EV71感染除了引起HFMD以外,还能够引起无菌性脑膜炎(aseptic meningitis)、脑干脑炎(brainstem encephalitis)和脊髓灰质炎样的麻痹(poliomyelitis-like paralysis)等多种与神经系统相关的疾病[1].自1974年首次报道[2]以来,EV71已在世界范围内引起十多次爆发与流行[3-6].近年来,EV71病毒的流行在亚太地区呈上升趋势[7-9].根据病毒衣壳蛋白VP1核苷酸序列的差异,可将EV71分为A、B、C 3个基因型,其中,B型和C型又进一步分为B1、B2、B3、B4以及C1和C2亚型[10-12].     

肠道病毒71型分子流行病学研究进展

肠道病毒71型(Enterovirus type71,EV71),自1974年首次报道以来,在世界范围内引起多次爆发与流行[1].EV71感染主要引起患者手足口病(hand,foot and mouth disease,HFMD),在临床上与柯萨奇病毒A16(Coxsakie A16,CA16)感染所引起的手足口病难以区别,但EV71还能够引起多种与神经系统相关的疾病[2].近年来,EV71病毒的流行在亚太地区呈上升趋势[3～5],其中最令人关注的是在该地区的EV71感染引起越来越严重的中枢神经系统症状.     

肠道病毒71型北京分离株VP4基因序列及蛋白抗原性分析

为探讨肠道病毒71型(EV71)VP4基因序列与手足口病(HFMD)不同临床类型之间的关系,分析重组蛋白EV71 VP4的抗原性,并初步探讨其与柯萨奇病毒A16(CA16)重组蛋白VP4是否存在交叉反应性,对2007~2009年从北京患HFMD儿童标本分离到的10株EV71的VP4基因进行克隆测序,运用生物学软件对测序结果进行比较分析,并选择其中1株与1株同期分离的CA16的VP4分别进行原核表达;用表达产物对189份正常体检的成人及来首都儿科研究所就医的非HFMD患儿血清中的IgG进行Western Blot检测,并分析14份确诊为EV71感染和12份CA16感染患者急性期血清中的IgM抗体。分析表明这10株EV71 VP4基因核苷酸同源性为94.20%~100.00%,所推导的氨基酸序列则完全相同,从重症与轻症患儿分离的毒株之间VP4的核苷酸序列未见一致性的差异,基于EV71 VP4基因核苷酸序列的进化树分析表明2007~2009年北京地区所流行的毒株均属于C4亚型;本研究中EV71和CA16的VP4核苷酸序列的同源性为69.60%,所推导的氨基酸序列的同源性为78.60%,在运用Western Blot检测189份血清中的VP4特异性IgG时,EV71 VP4的血清阳性率为38.10%,说明其具有良好的抗原性,CA16 VP4的血清阳性率为58.20%,两者差别具有显著统计学意义(2χ=15.30,P<0.01),提示EV71 VP4与CA16 VP4没有交叉反应性;在用表达的VP4检测已确诊为相应病毒的特异性IgM时,两者皆为阴性,提示感染后机体对VP1和VP4产生不同的反应。     

EV71与CA16双价灭活疫苗诱导小鼠免疫原性研究

目的评价EV71和CA16双价灭活疫苗免疫小鼠诱导的体液免疫和细胞免疫应答。方法分别应用EV71和CA16双价疫苗、EV71和CA16单价疫苗按单针、两针(0,14 d)的程序免疫小鼠,采用细胞病变法检测血清第1针免疫后21 d时中和抗体效价,应用LUMINEX液相芯片技术检测小鼠脾单个核细胞体外经抗原刺激分泌细胞因子的水平。结果双价疫苗与EV71单价疫苗单针、两针免疫相比,EV71中和抗体几何平均值相近(118.8∶84.0;159.5∶156.8,P0.05)。双价疫苗与CA16单价疫苗单针免疫诱导的CA16中和抗体均为阴性;两针免疫CA16中和抗体几何平均值一致(30.0∶26.9,P0.05)。加强免疫7 d后,小鼠脾MNC经EV71抗原刺激,双价疫苗较EV71单价疫苗诱导Th2类细胞因子IL-4、5、6、10和炎症因子TNF-α的分泌水平显著增高(P=0.020,P=0.027,P=0.038,P=0.019,P=0.026);MNC经CA16抗原刺激,双价疫苗与CA16单价疫苗相比诱导细胞因子水平差异无统计学意义(P0.05)。结论双价疫苗可诱导与单价疫苗相似的中和抗体应答水平,并可提高Th2类细胞因子应答,研究为EV71和CA16双价灭活疫苗的研发提供了实验依据。     

Characterization of full-length enterovirus 71 strains from severe and mild disease patients in northeastern China

Human enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) has been a leading cause of childhood infection in China since 2008. Epidemic and molecular characteristics of HFMD have been examined in many areas of China, including the central and southern regions. However, clinical and genetic characterization of EV71 in the northeastern region of China is scarce. In this study, a series of analyses were performed on seven full-length EV71 sequences from HFMD patients who had either severe or mild disease. We have determined that these seven circulating EV71 viruses from Changchun, China are actually complex recombinant viruses involving multiple type A human enterovirus (HEV). Classified as EV71 subtype C4 (EV71 C4), these Changchun EV71 viruses contain genetic recombination events between the CA4, CA5, EV71B4 and EV71C1 strains. Most of the structural protein region (P1) of these viruses resembled that of the prototype EV71 C1 strains. The non-structural protein domains (P2 and P3) showed a high degree of similarity with CA4, CA5 and EV71 B4 in different regions. The 5'UTR had unclassified recombination,while partial 3D region of these viruses showed a high degree of similarity to CA16. Phylogenetic analysis of full-length or partial sequences of isolates from severe or mild disease patients in Changchun always formed a single cluster in various phylogenetic analyses of different genomic regions, suggesting that all seven strains originated from one single common ancestor. There was no correlation between viral genomic sequence and virulence. Thus, we found that circulating recombinant forms of EV71 are prevalent among HFMD patients in Northeastern China. The existence of a unique cluster of EV71 related viruses in Northeast China has important implications for vaccine development that would address the increasing prevalence of HFMD.     

A One-Step,Triplex, Real-Time RT-PCR Assay for the Simultaneous Detection of Enterovirus 71, Coxsackie A16 and Pan-Enterovirus in a Single Tube

The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001–0.04 TCID₅₀/ml, 0.02 TCID₅₀/ml, and 0.001 TCID₅₀/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.     

Seroprevalence of Human Enterovirus 71 and Coxsackievirus A16 in Guangdong,China, in Pre- and Post-2010 HFMD Epidemic Period

Background

Human Enterovirus 71 and Coxsackie A16 have caused many outbreaks in the last decade in mainland China, resulting in thousands of fatal cases. Seroepidemiology which provides important information to document population immunity is rare in China.

Methodology/Principal Findings

A cross sectional study of Enterovirus 71 (EV71) and Coxsackie A16 (CA16) seroprevalence was carried out in Guangdong, China, pre- and post- the 2010 hand, foot and mouth disease (HFMD) epidemic period. The levels of EV71 and CA16 specific antibodies were evaluated by a microneutralization test and the geometric mean titer (GMT) was calculated and compared. Our results indicated frequent infection by EV71 and CA16 in Guangdong before the 2010 epidemic. Only EV71 neutralizing antibody but not CA16 seroprevalence was significantly increased after the 2010 HFMD epidemic. Children less than 3 years old especially those aged 2 years showed the lowest positive rates for EV71 and CA16 NA before epidemic and the most significantly increased EV71 seroprevalence after epidemic. CA16 GMT values declined after the 2010 epidemic.

Conclusions

These results indicate EV71 was the major pathogen of HFMD in Guangdong during the 2010 epidemic. The infection occurs largely in children less than 3 years, who should have first priority to receive an EV71 vaccine.     

2013年大连市手足口病病原监测结果分析

目的通过对2013年大连市手足口病（HFMD）的病原进行鉴定,以了解其型别分布。方法采用realtime-PCR方法对754份标本进行肠道病毒（EV）通用引物和肠道病毒71型（EV71）、柯萨奇病毒A组16型（CVA16）、柯萨奇病毒A组6型（CVA6）型特异性引物检测,对EV通用引物检测结果为阳性,但EV71、CVA16和CVA6检测结果均为阴性的标本采用巢氏PCR进行肠道病毒VP1基因部分序列的扩增、测序和生物信息学分析以鉴定其型别。结果 2013年引起大连市HFMD的主要病原CVA6占44.30%（334/754）、CVA16占19.12%（114/754）、EV71占11.54%（87/754）,另有少数病例由CVA2、4、5、8型,CVB2、4型,ECHO9型及EV的其他型别引起。结论 CVA6取代EV71和CVA16成为2013年大连市HFMD病原的主要流行型别,后续应加强对HFMD病原的监测,全面了解其型别分布及毒株变异情况以更有效地控制疾病流行。     

EV71感染抗病毒药物及疫苗研究进展

手足口病在世界多个地区,尤其是亚洲爆发并流行,且其感染率和死亡率逐年增高,危害十分严重。肠道病毒71(Enterovirus 71,EV71)是手足口病(Hand,foot,andmouth disease,HFMD)的主要病原体,以感染婴幼儿为主,其感染常伴随神经系统并发症,严重可导致儿童死亡。近年来,分子生物学和抗病毒研究方面取得的进展为EV71感染的预防及治疗提供了新的途径。本文对EV71病毒学特点及抗EV71药物的筛选、疫苗开发、RNA干扰等进行了综述,以期为相关研究提供参考。     

Hand foot and mouth disease due to enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth.The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents.Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae.EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD),aseptic meningitis,encephalitis and poliomyelitis-like acute flaccid paralysis.A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997,and caused 41deaths amongst young children.In late 2000,a recurrence of an outbreak of HFMD occurred in Malaysia with S fatalities in peninsular Malaysia.Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities.A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006.The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010.As with other HFMD outbreaks in Malaysia,both EV71 and CA16 were the main aetiological viruses isolated.In similarity with the HFMD outbreak in 2005,the isolation of CA16 preceded the appearance of EV71.Based on the VP 1 gene nucleotide sequences,4 sub-genogroups of EV71 (C1,C2,B3 and B4) co-circulated and caused the outbreak of hand,foot and mouth disease in peninsular Malaysia in 1997.Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak.EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003.In the 2005 outbreak,besides the EV71 strains of sub-genogroup C1,EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003.The four EV71 strains isolated from clinical specimens of patients with hand,foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5.Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years.In each of the past outbreaks,more than one sub-genogroup of the virus co-circulate.     

濮阳市2008—2012年手足口病流行病学特征分析

目的了解濮阳市手足口病流行病学特征,为制定防控策略提供科学依据。方法通过国家疾病监测信息管理系统收集的全市2008—2012年6月6日手足口病疫情资料进行描述和分析,并对部分病例和重症病例标本进行肠道病毒病原学检测。结果全市共报手足口病16 492例,发病高峰是每年的3-5月(第12～20周),呈典型的单峰型曲线;发病年龄以0～4岁居多;男性多于女性;散居儿童多于托幼机构儿童,爆发病例多发生在托幼机构,手足口病病原有EV71、CoxA16和其他肠道病毒,以EV71和CoxA16为主。结论手足口病发病有明显的季节性、年龄和性别差异,小年龄组儿童是手足口病预防控制重点人群,流行年度和流行季节的优势毒株为EV71,重症患者中EV71占到86.35%;非流行年和季节手足病例主要由CoxA16和其他肠道病毒引起。手足口病防控重点应体现在对病例分类管理上,同时应继续加强重症病例疫情监测和爆发控制。     

An overview of the evolution of enterovirus 71 and its clinical and public health significance

Since its discovery in 1969, enterovirus 71 (EV71) has been recognised as a frequent cause of epidemics of hand-foot-and-mouth disease (HFMD) associated with severe neurological sequelae in a small proportion of cases. There has been a significant increase in EV71 epidemic activity throughout the Asia-Pacific region since 1997. Recent HFMD epidemics in this region have been associated with a severe form of brainstem encephalitis associated with pulmonary oedema and high case-fatality rates. The emergence of large-scale epidemic activity in the Asia-Pacific region has been associated with the circulation of three genetic lineages that appear to be undergoing rapid evolutionary change. Two of these lineages (B3 and B4) have not been described previously and appear to have arisen from an endemic focus in equatorial Asia, which has served as a source of virus for HFMD epidemics in Malaysia, Singapore and Australia. The third lineage (C2) has previously been identified [Brown, B.A. et al. (1999) J. Virol. 73, 9969-9975] and was primarily responsible for the large HFMD epidemic in Taiwan during 1998. As EV71 appears not to be susceptible to newly developed antiviral agents and a vaccine is not currently available, control of EV71 epidemics through high-level surveillance and public health intervention needs to be maintained and extended throughout the Asia-Pacific region. Future research should focus on (1) understanding the molecular genetics of EV71 virulence, (2) identification of the receptor(s) for EV71, (3) development of antiviral agents to ameliorate the severity of neurological disease and (4) vaccine development to control epidemics. Following the successful experience of the poliomyelitis control programme, it may be possible to control EV71 epidemics if an effective live-attenuated vaccine is developed.