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...     

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.     

2011年广州市手足口病病原谱分析

目的了解引起2011年广州市手足口病流行的病原体,确定其型别分布特征。方法收集手足口病疑似病例标本,用Real-time RT-PCR、巢式RT-PCR方法对其部分VP1基因进行扩增并测序,确定其病原谱构成,构建系统发育树。结果共收集2 067例疑似手足口病病例标本,其中实验室确诊肠道病毒阳性1 113份,包括EV71、CoxA16、CoxA6、CoxA10、CoxA9、HEV96、埃可病毒和柯萨奇B组等15种肠道病毒,其中EV71、CoxA16和CoxA6是主要病原体,分别占32.6%、39.2%和18.5%;死亡病例3例,全部由EV71引起;重症病例9例,6例由EV71引起,3例由CoxA6引起。VP1基因构建进化树分型发现,广州市2011年手足口病病原体属于HEV-A（97.2%）、HEV-B（2.5%）、HEV-C（0.3%）3个型别,未发现HEV-D型毒株。结论广州市2011年手足口病的流行主要由HEV-A型的EV71、CoxA16和CoxA6病毒引起,同时还存在HEV-B型和HEV-C型的CoxA9、EC、CoxB、CoxA24多种病毒。     

江苏省2008年手足口病病原分析

肠道病毒71型(EV71)和柯萨奇病毒A16(CoxA16)为手足口病的主要病原体.江苏与山东、河南、安徽交界的区域是我国两大手足口病高发地区之一,本文对2008年4月上旬至6月中旬江苏12个市城乡63批次355份临床诊断为手足口病病人的咽拭子标本进行了病原分离,并对全部182个分离毒株进行鉴定,以了解江苏手足口病流行的病原特征.     

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.     

实时RT-PCR检测159例手足口病患儿样本中肠道病毒的结果分析

目的 了解2009年4-8月首都儿科研究所附属儿童医院手足口病患儿肠道病毒的感染状况,为临床诊治提供参考.方法 采集首诊手足口病159例患儿的咽拭子和疱疹液标本,以肠道病毒(EV)通用型、柯萨奇病毒A16(CA16)型、肠道病毒71(EV71)型核酸检测试剂盒,应用实时RT-PCR法检测标本中的肠道病毒.选取阳性标本扩增VP1区,产物进行序列测定和分析.结果 (1)EV、CA16、EV71的阳性病例数分别为152、102、43;阳性率为95.6%、64.2%、27.0%.(2)CA16占EV阳性的67.3%,EV71占EV阳性的28.3%,非CA16和EV71的EV病例7例,占EV阳性的4.6%.CA16:EV71为2.37:1.(3)部分阳性标本经测序验证与此法结果一致.结论 2009年我院手足口病患儿以EV71和CA16感染为主,EV71感染的手足口病比例较2007年出现明显上升.     

2009-2011年上海市手足口病病原学检测及其临床特点分析

目的 了解上海市手足口病不同病原体的感染现状以及所致的手足口病的临床特点之间的差异,为制定观察、治疗方案,做好手足口病防控工作提供参考.方法 采集2009年3月至2011年12月在本院传染科门诊诊断为手足口病的357例病例的咽拭子、疱疹液、粪便共551份标本进行病原学检测.用RT-PCR法对肠道病毒71型(EV71)、柯萨奇病毒A组16型(CoxA16)、肠道病毒通用型(PE)进行鉴别.结果 357例手足口病患儿共采集到551份标本,检测病毒总阳性率为79.7％,2009年手足口病以CoxA16流行为主,2011年以EV71流行为主,三年病毒流行株差异有统计学意义(P =0.005);疱疹液病毒检出率最高;EV71、CoxA16、PE病毒感染的手足口病患儿在发热、易惊的发生比例比较差异有统计学意义(P＜0.05);EV71感染的患儿外周血白细胞计数和中性粒细胞比例要高于CoxA16和PE感染的患儿,差异有统计学意义(P＜0.05).结论 上海市手足口病在不同时期病原体呈现交替流行;对不同病原体临床特征的分析比较有助于EV71感染的防控、早期治疗、重症病例的筛查.     

2008年深圳地区手足口病病原学调查

目的 研究2008年深圳地区手足口病病原体EV71和CoxA16感染情况,为手足口病的防治提供依据.方法 应用RT-PCR技术检测307例手足口病患儿标本病原体EV71、CoxA16基因,将PCR产物测序,并应用ChstaIW2在线分析软件进行序列分析、进化树分析.结果 不同标本中EV71的阳性率分别为:大便标本24.4%(75/307)、咽拭子为7.8%(24/307)、外周血12.5%(1/8);CoxA16的阳性率分别为,大便13.8%(28/203),咽拭子11.0%(20/181),其中EV71和CoxA16同时为阳性的有3份(0.98%);脑脊液标本未检测到EV71和CoxA16.与标准株BrCr序列比对分析,测序的14例标本中,8例的EV71新出现多个位点变异,其中2例标本的A2528G变异导致氨基酸改变,即N595D,1例标本的A2714G变异引起氨基酸改变,即1658V.进化树分析显示,有2例标本的病毒株与anhui毒株亲缘关系较近,与标准株BrCr及深圳株SHH02-6、SHZH02-40、SHZH03-58等亲缘关系较远;而其余12株与上述毒株亲缘关系较远.根据文献报道的EV71基因分型方法分析,测序的14株EV71基因型为C型.结论 在引起2008年深圳手足口病病原体中相当的比例是EV71,且可能部分是anhui病毒株的入侵.     

江苏省肠道病毒71型分离株的分子遗传演化特征

肠道病毒71型(EV71)是小RNA病毒科肠道病毒属成员,其感染主要引起患者手足口病(handfoot-mouth disease,HFMD),个别患者可引起无菌性脑膜脑炎等并发症.自1998年EV71在中国台湾暴发以来,EV71病毒在亚洲的流行呈上升趋势.流行病学资料表明,不同地区不同症状的EV71病毒性疾病的暴发可能与毒株间的差异等因素有关[1].为了解EV71病毒的遗传和变异特征,本实验室对2009年在江苏手足口病流行地区分离到的一株EV71型病毒株(Jiangsu01,GenBank:FJ600325.1)的基因组核苷酸序列进行了遗传进化分析,从分子水平为EV71病毒的流行以及所致疾病的防控提供理论基础.     

2008-2009 年宁波地区肠道病毒71型的分离及VP1基因分析

肠道病毒71型(EV71)病毒是引起儿童手足口病的重要病原之一.本研究通过MRC-5(人胚肺成纤维细胞)对2008-2009年宁波地区288份手足口病例标本进行病毒分离,分离鉴定后得到6株EV71病毒,分别命名为1/CHNNB/08、2/CHNNB/08、76/CHNNB/09、87/CHNNB/09、103/CHNNB/09 和 107/CHNNB/09.     

河北省手足口病病例的病原构成及EV71病毒基因特征分析

目的了解河北省不同地区、不同严重程度手足口病病例的病原构成情况及EV71病毒的基因特征。方法采集河北省不同地区的HFMD患者粪便、疱疹液、咽拭子标本进行核酸检测和病毒分离,同时结合所收集的HFMD患病例的居住地、疾病严重程度信息加以分析。选取18株EV71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,与其它38株各基因型和基因亚型的EV71代表株构建系统发生树。结果2009年河北省HFMD临床诊断病例的EV阳性率为65．13％,其中以EV71为主,占阳性病例的58．0％（752／1296）。秦皇岛、邯郸、保定、邢台地区手足口病例以EV71感染为主,而衡水、沧州等地区则以CA16为主。轻型病例中EV71阳性率为37．74％,重症病例中EV71阳性率为80．64％,死亡病例检测13例,均为EV71阳性。18株EV71分离株的VP114核苷酸同源性为94．9％～99．8％,与C4亚型代表株的VP1区核苷酸同源性最高,为91．9％～99．6％。进化树结果显示,河北省EV71分离株与c4亚型代表株处于同一分支,并在C4a进化分支的不同簇中。结论2009年引起河北省手足口病流行的病原体主要为EV71和CA16。秦皇岛、邯郸、保定、邢台地区手足口病例以EV7I感染为主,而衡水、廊坊、沧州等地区则以CA16为主。EV71是重症病例的主要致病病原体。河北省EV71分离株为c4亚型C4a进化分支。     

Molecular characterization and complete genome analysis of human enterovirus 71 and coxsackievirus A16 from children with hand, foot and mouth disease in Thailand during 2008-2011

Hand, foot and mouth disease (HFMD) has mostly been caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). CA 16 was the most common cause of HFMD in 2010. EV71 had a high prevalence in 2008-2009 and has been identified with a higher frequency since 2011. Nearly complete genome sequences of three EV71 strains (2008-2009 strains) and two CA16 strains (2010 strains) obtained from outbreaks in Thailand in 2008 to 2010 were characterized. Based on a phylogenetic tree of the complete VP1 region, three EV71 strains grouped into the B5, C1 and C4 genotypes, and two CA16 strains grouped into the C genotype. Based on sequence analysis, nucleotide changes were found to cluster in the internal ribosome entry site (IRES) element of the 5′-untranslated region (5′-UTR). Amino acid differences identified in all strains were located in the non-structural protein. These data also provide the molecular epidemiology of EV71 and CA16 outbreaks in Thailand.     

Specific primer amplification of the VP1 region directed by 5′ UTR sequence analysis: Enterovirus testing and identification in clinical samples from hand-foot-and-mouth disease patients

Many genotypes of the enterovirus (EV) pathogens can cause clinical hand-foot-and-mouth disease (HFMD). Therefore, rapid identification and monitoring of HFMD pathogens can be difficult, especially from the original clinical specimens. In this study, both universal pan-enterovirus and EV71/CA16 VP1-specific primer sets were designed and used to examine clinical specimens from HFMD patients. Based on the initial sequence analysis of the 5′-untanslated region (5′-UTR) and VP1 amplification products, additional primers for the VP1 region were redesigned for further genotyping of the remaining small portion non-EV71/non-CA16 specimens. With a known panel, it was possible to identify 15 out of 16 members using 5′-UTR sequence typing and VP1 typing, suggesting good detectability and genotyping of this method. One strain that was not typed by 5′-UTR was shown to be a recombinant virus. When this method was applied to examine clinical specimens from 44 suspected HFMD patients, 41 were detected as EV positive. In only one case, the VP1 sequence could not be identified. Four types of EVs, including CA16 (26/41, 63.4%), EV71-C4 (6/41, 14.6%), CA6 (5/41, 12.2%) and CA10 (3/41, 7.3%), were detected. In conclusion, 5′ UTR amplification sequencing and subsequent VP1 specific primer amplification ensures a high detection rate and good genotyping accuracy in the examination of clinical samples. This detection strategy can be used for routine evaluation and monitoring of HFMD to follow local trends of EV infection.     

Cost-effectiveness of bivalent versus monovalent vaccines against hand,foot and mouth disease

ObjectivesEnterovirus 71 (EV71) and coxsackievirus A16 (CA16) were responsible for 43.3% (235 123/543 243) and 24.8% (134 607/543 243) of all laboratory-confirmed hand, foot and mouth disease (HFMD) cases during 2010–2015 in China. Three monovalent EV71 vaccines have been licensed in China while bivalent EV71/CA16 vaccines are under development. A comparative cost-effectiveness analysis of bivalent EV71/CA16 versus monovalent EV71 vaccination would be useful for informing the additional value of bivalent HFMD vaccines in China.MethodsWe used a static model parameterized with the national HFMD surveillance data during 2010–2013, virological HFMD surveillance records from all 31 provinces in mainland China during 2010–2013 and caregiver survey data of costs and health quality of life during 2012–2013. We estimated the threshold vaccine cost (TVC), defined as the maximum additional cost that could be paid for a cost-effective bivalent EV71/CA16 vaccine over a monovalent EV71 vaccine, as the outcome. The base case analysis was performed from a societal perspective. Several sensitivity analyses were conducted by varying assumptions governing HFMD risk, costs, discounting and vaccine efficacy.ResultsIn the base case, choosing the bivalent EV71/CA16 over monovalent EV71 vaccination would be cost-effective only if the additional cost of the bivalent EV71/CA16 compared with the monovalent EV71 vaccine is less than €4.7 (95% CI 4.2–5.2). Compared with the TVC in the base case, TVC increased by up to €8.9 if all the test-negative cases were CA16-HFMD; decreased by €1.1 with an annual discount rate of 6% and exclusion of the productivity loss; and increased by €0.14 and €0.3 with every 1% increase in bivalent vaccine efficacy against CA16-HFMD and differential vaccine efficacy against EV71-HFMD, respectively.ConclusionsBivalent EV71/CA16 vaccines can be cost-effective compared with monovalent EV71 vaccines, if suitably priced. Our study provides further evidence for determining the optimal use of HFMD vaccines in routine paediatric vaccination programme in China.     

A large outbreak of hand, foot, and mouth disease caused by EV71 and CAV16 in Guangdong, China, 2009

A large outbreak of hand, foot and mouth disease (HFMD) occurred in Guangdong, China, in 2009. A total of 92,749 cases were officially reported to the Center for Disease Control and Prevention of Guangdong (GDCDC). To clarify the pathogen causing the outbreak, 600 specimens, including stool, rectal swabs, vesicular swabs, cerebrospinal fluid, and throat swabs, were collected from 541 patients and subjected to one-step RT-PCR. Four hundred eighty-nine of 541 patient samples were positive for enterovirus. All positive samples were cultured on RD and Hep2 cells; 307 specimens displayed CPE. Sequence analysis of PCR fragment and typing real-time PCR indicated that these isolates included EV71 (56%), CAV16 (35.5%), CAV6 (2.0%), CAV10 (1.0%), CAV2 (0.7%), CAV4 (1.3%), Echo30 (0.7%), Echo25 (1.0%), Echo4 (0.3%), CBV5 (1.0%) and human rhinovirus (0.7%). 100% (12/12) of fatal cases and 97.2% (140/144) of severe cases carried EV71 and CAV16. The results implied that EV71 and CAV16 were mainly responsible for the outbreak. Comparison with the three global types of EV71 and the five clusters of genotype C showed that the sequences from mainland China (not including the Hong Kong region) are located in subgenogroup C-4 and originate from isolates from the Shenzhen area of Guangdong Province. Results from this study show that the C-4 genotype has been a prevalent pathogen in mainland China since 1998.