2009-2011年广东EV71致手足口病临床病原特征及EV71衣壳蛋白P1单克隆抗体制备

发布时间：2018-05-10 08:43

本文选题：手足口病 + EV71　；参考：《南方医科大学》2012年博士论文

【摘要】：手足口病(hand,foot,mouth disease, HFMD)是以发热和手、足、口腔等部位出现皮疹、疱疹为特征的儿童常见病。绝大部分病情较轻,少数可发生严重神经系统并发症并致相当数量儿童死亡,是中国及亚洲的主要公共卫生问题之一。 HFMD由人类肠道病毒(human enterovirus, HEV)感染引起。其中肠道病毒71型(EV71)是最重要的病原体,因其更易导致神经系统感染甚至死亡。EV71属小RNA病毒科,成熟EV71病毒颗粒外壳由60个拷贝的P1蛋白组成,P1又分为VP1-VP4四种衣壳蛋白。按照VP1区不同EV71分A、B、C三个基因亚型,中国大陆流行C4亚型。临床实验室HFMD病原学诊断以分子生物学和血清学检测为主。目前,尚无有效的抗病毒药物和疫苗防治EV71感染。对于EV71外壳抗原结构、中和性表位及其在病毒致病机制中的作用的认识还很有限。已证实VP1、VP2、VP3具中和活性抗原位点,VPl是最主要的抗原决定簇,VP4虽位于病毒表面内侧,但能产生相应抗体,推测其可能参与抗体依赖感染增强效应(ADE)而加强病毒对机体的攻击。本研究对广东地区发生的HFMD进行病原学监测：收集了2009-2011年EV71流行株及所致疾病的临床特征、基因分型及其来源、变迁及突变等信息,为控制和预防HFMD提供预警及对策的参考资料；采用多种目前实验室检测病毒的方法对临床常用的捕获ELISA发检测抗EV71-IgM用于EV71病原诊断进行评估,使临床更合理使用；在此基础上,采用载体pQE30a和大肠杆菌M15系统重组表达了EV71临床分离株的VP1-4蛋白,通过免疫小鼠获得针对这些蛋白的单克隆抗体,为研究EV71病毒衣壳的抗原结构及功能、揭示EV71致病机制以及疫苗制备提供实验依据。本研究包含三个部分内容：第一部分2009-2011年EV71致手足口病临床及病原学特征 2009～2011年来自广东各地在我院就(转)诊的临床诊断HFMD(含疱疹性咽峡炎)分别有107、553、579例,实时荧光RT-PCR对肠道病毒通用型核酸检出率分别为87.9%(94/107)、91.2%(505/553)和88.8%(515/579),EV71占所有病原的比例分别为28.1%(30/107)、45.6%(252/553)和38.7%(221/579),显示EV71仍为广东手足口病最主要病原。 1239例患者中男性813例,女性426例,男女比1.91,EV71感染性别比与全部EV感染无差异(P=0.765)。患者年龄最小1个月,最大35岁,中位数分别是2.08岁、2.58岁和2.41岁；1岁和2岁是发病最多的两个年龄段,占73.24岁以下儿童占患者总数85.0%；6个月以下发病者21例,EV71感染8例；各年龄段感染EV71与EV感染比例相似。诊断为重症手足口病81例,年龄最小4个月,最大10岁,中位数2.0岁,1～2岁是出现重症最多的年龄段,各年龄段发生重症手足口病例与普通手足口病例相似；男女比：2.24:1,略(?)性别比,但无统计学差异(P=0.62),提示重症并不倾向于男孩。监测显示2009-2011年广东以散发流行为主。每年出现两个发病高峰：初夏(5、6月间)和秋季(10、11月),2010年5月中旬达最高峰,持续约1个月,10月出现小高峰；2011年发病高峰期推迟至半个月,春夏季EV71导致的HFMD较秋季略高。临床表现以手掌、臀部、足底斑疹,口腔疱疹为主：90%上的(?),60%以上发病初期有发热伴出疹,顺序不一。少数(7.2-11.3%)表现呼吸道感染症状。极少数出现神经性症状,如呕吐、抽搐、惊跳或头痛等。EV71感染临床表现与EV所致HFMD相似,无统计学差异。2010～2011年分别有89和144例患儿经ICU治疗,其中EV感染分别是75例(14.9%)和128例(24.9%)。EV71感染较EV感染更多需要ICU治疗(P值分别为0.037和0.000)。所有诊断为重症手足口病者均有肠道病毒相关性脑病发生,如病毒性脑炎等,呼吸道并发症主要是神经源性肺水肿。扩增测序33株2009-2010年临床分离EV71(2009年5株,2010年28株)的部分VP1区,经比对分析,确定均属C4a亚型,与广东历年分离毒株序列比较未见明显差异；与2008年阜阳EV71流行株比较亦未见明显差异,表明为国内循环流行株；与台湾近年流行株的基因亚型不同,表明两岸毒株进化来源不同。通过Clustal W软件对临床分离株与标准株BrCr(U22521).台湾1998年流行株(Taiwan98)比对VPl区序列：核苷酸相似度93.7%-100%,氨基酸相似度97.0%-100%。第22、98号位氨基酸与台湾98年流行株和标准株BrCr略有不同。第二部分实时荧光RT-PCR和EV71-IgM捕获ELISA法对EV71致手足口病诊断的评估为评估捕获ELISA (?)法检测EV71-IgM,我们收集了从发病第1天到158天的134例EV71感染血清256份,发现发病第1天即可检出EV71-IgM,随发病天数增多检出率升高,第5天达100%；发病3个月后血清EV71-IgM检出率明显下降。急性期(发病7天的(?)检测敏感度90.2%(138/153),发病90天内敏感度93.6%(233/249)。与CA16-IgM检测存在交叉反应。实验显示122份CA16感染血清中38份EV71-IgM阳性,49份其它肠道病毒感染血清中14份EV71-IgM阳性,105份其它呼吸道病毒感染血清有2份(来自同一呼吸道合胞病毒A和B型双重感染患者)EV71-IgM阳性对于其它肠道病毒,EV71-IgM捕获法ELISA法检测特异性为69.6%(52171.95。95(?):65.2-72.4%),对于其它呼吸道病毒特异性98.1%(2/105,950%可信区间：96.5-99.5%),检测阳性预测值81.3%(95%可信区间：76.9-85.1%),阴性预测值91.0%(95%可信区间：87.5-93.8%)。206份EV71感染血清检出EV71-IgM阳性199份(95.7%)、CA16-IgM阳性58份(28.2%),其中EV71-IgM的OD值高于CA16-IgM者56份(占96.6%)；119份CA16感染血清CA16-IgM阳性83份(69.7%),EV71-IgM阳性36份(30.3%),其中CA16-IgM的OD值高于EV71-IgM者33份(占91.7%),实际感染的病原产生的IgM检测OD值更高,可正确区分大部分感染病原体。 111例同日收集的肛拭子和血清标本分别用ELISA和实时荧光RT-PCR两种方法检测,发现两种方法诊断EV71感染无显著性差异(McNemar's χ2检验P=0.648),一致性较好(Kappa值0.729)。第三部分EV71病毒衣壳蛋白P1单抗的制备依据EV71基囚序列信息(FJ194965.1)设计VP1～VP4基因全长序列引物,经PCR扩增,Kpn Ⅰ和Hind Ⅲ双酶切PCR产物,连接载体pQE30a,经测序确认与原始序列完全一致后将该重组质粒转化大肠杆菌M15。经诱导表达VP1～VP4蛋白,均以包包涵体形式存在,以8M尿素变性及梯度复性,复性蛋白经HisBind Resin层析柱纯化.获得纯化VP1～4蛋白。以重组VP4蛋白与纯化EV71病毒交替免疫小鼠,经小鼠脾细胞与杂交瘤细胞融合获得杂交瘤细胞克隆,分别采用重组蛋白VP1～4和病毒液包被ELISA、间接免疫荧光法筛选克隆,采用免疫印迹鉴定针对的靶蛋白。以FV71病毒液、溶于8M尿素的重组蛋白VP1、VP2和VP4分别包被板条,ELISA检测融合细胞克隆上清,挑选OD值高的克隆,进一步间接免疫荧光法对EV71感染和未感染RD细胞反应,筛选仅对EV71感染RD细胞有阳性荧光的克隆。克隆上清采用纯化EV71病毒液和重组VP1-VP4蛋白经SDS-PAGE胶分离的蛋白组分进行免疫印迹反应鉴定抗体针对的靶蛋白。初步获得25株针对P1蛋白的单抗：6株针对EV71病毒VP1蛋白(天然蛋白),其中4株对重组VP1蛋白有反应；3株仅针对重组VP2蛋白;2株针对病毒VPl和VP2蛋白,其中针对重组VP1和VP2各一株；1株针对重组蛋白VP4；另有13株目前尚未鉴定出针对的靶蛋白。小结根据以上三个部分的研究成果,本研究小结如下： 1.2009-2011年监测EV感染手足口病患者病原,EV71感染分别是：30、252和221例,分别占28.1%(30/107)、45.6%(252/553)和38.7%(221/579),EV71仍为广东手足口病最主要病原。 2.2009-2011年广东以散发为主,每年出现一大一小两个发病高峰,即初夏(5、6月之间)和秋季(10、11月)；1、2岁是发病最多的年龄段,占73.2%,4岁以下儿童占总数85.0%；EV71感染致重症手足口病患者年龄分布与此相似,无统计学差异。 3.2009～2010年广东EV71临床分离株属C4a亚型,与广东历年分离株和08年安徽阜阳流行株序列比较未见差异,为国内循环流行毒株；与台湾近年流行株比较,为不同基因亚型,提示两地毒株进化来源不同；与标准株BrCr、台湾1998年流行株Taiwan98VP1区核苷酸相似度93.7%-100%,氨基酸相似97.0%-100%。第22、98号位氨基酸于台湾98年流行株和标准株BrCr略有不同。 4.捕获ELISA法EV71-IgM检测诊断EV71致手足口病急性期敏感度为90.2%(138/153),特异性分别为69.6%(52/171,相对其他肠道病毒)和98.1%(2/105,相对其他呼吸道病毒),检测阳性预测值81.3%(95%可信区间：76.9-85.1%),阴性预测值91.0%(95%可信区间：87.5-93.8%)。与实时荧光RT-PCR方法诊断EV71感染无显著性差异(McNemar's χ2检验P=0.648),一致性较高。该方法适用于临床。 5.捕获ELISA法EV71-IgM检测诊断EV71虽然对CA16感染HFMD血清有交叉反应性(28.2%和30.3%),但较高的OD值可以反映感染病毒的类型。 6.采用EV71纯化病毒和重组VP4蛋白免疫小鼠,初步获得25株针对P1蛋白的单克隆抗体,经EV71病毒液、VP1-VP4重组蛋白ELISA、间接免疫荧光筛选和免疫印迹鉴定分别是：针对VP1蛋白6株,VP2蛋白3株、同时针对VP1和VP22株和VP4蛋白1株,尚未确定13株。
[Abstract]:Hand (foot, mouth disease, HFMD) is a common disease in children with fever and hand, foot and mouth and other parts of the mouth. Most of the disease is common in children. The vast majority of the disease is mild. A few serious neurological complications and a considerable number of children are killed. It is one of the major public health problems in China and Asia.
HFMD is caused by the human enterovirus (human enterovirus, HEV) infection. Among them, the enterovirus 71 (EV71) is the most important pathogen, because it is more susceptible to the infection of the nervous system and even the death of.EV71 family in the small RNA virus family. The mature EV71 virus particle shell is composed of 60 copies of the P1 protein, and P1 is divided into VP1-VP4 four capsid proteins. Three subtypes of EV71 A, B, C, and C4 subtypes in mainland China. The diagnosis of HFMD in clinical laboratories is based on molecular biology and serological detection. At present, there are no effective antiviral drugs and vaccines for the prevention and treatment of EV71 infection. The understanding of the antigen structure of EV71, the neutralization epitopes and their role in the pathogenesis of the virus It has been limited. VP1, VP2, VP3 have been proved to have neutralization active antigen sites, and VPl is the most important antigenic determinant. Although VP4 is located inside the surface of the virus, it can produce corresponding antibodies. It is presumed that it may participate in the antibody dependent infection enhancement effect (ADE) to strengthen the attack of the virus to the body.
In this study, the etiology of HFMD in Guangdong was monitored. The clinical features, genotyping, its origin, changes and mutations were collected for 2009-2011 years of EV71 epidemic strains and the disease, and the reference materials for the prevention and control and prevention of HFMD were provided. The use of ELISA hair detection to detect anti EV71-IgM for the diagnosis of EV71 pathogens and to make the clinical use more reasonable. On this basis, the carrier pQE30a and Escherichia coli M15 system were used to restructure the VP1-4 protein of the EV71 clinical isolates, and the monoclonal antibodies against these proteins were obtained by immunizing mice to study the resistance of the EV71 virus capsid. The original structure and function provide an experimental basis for revealing the pathogenic mechanism of EV71 and the preparation of vaccine. This study contains three parts:
Part 1 clinical and etiological characteristics of hand foot mouth disease caused by EV71 in 2009-2011 years
In 2009~2011 years, there were 107553579 cases of HFMD (herpetic angina) from all over Guangdong in our hospital. The detection rate of real time fluorescent RT-PCR for enterovirus general nucleic acid was 87.9% (94/107), 91.2% (505/553) and 88.8% (515/579), and EV71 accounted for 28.1% (30/107), 45.6% (252/553) and 3, respectively. 8.7% (221/579) showed that EV71 is still the most important pathogen of HFMD in Guangdong.
Among the 1239 patients, 813 were male and 426 women were women, and the ratio of male and female was 1.91. The sex ratio of EV71 infection was not different from that of all EV infection (P=0.765). The age was 1 months, the maximum was 35 years, the median was 2.08, 2.58 and 2.41, and 1 and 2 years were the two age groups, which accounted for the total number of patients under the age of 73.24. There were 21 cases of disease and 8 cases of EV71 infection. The proportion of EV71 and EV infection in all ages was similar. The diagnosis was 81 cases of severe hand foot and mouth disease, the minimum age of 4 months, the maximum 10 years, the median 2 years, and 1~2 years of age, similar to those of the ordinary hand foot and mouth cases in each age group; the ratio of men and women: 2.24:1, slightly (?) Sex ratio, but no statistical difference (P=0.62), suggesting that severe illness is not inclined to boys.
The monitoring showed that Guangdong was mainly sporadic in 2009-2011 years. There were two peaks of onset each year: early summer (5,6 months) and autumn (10,11 month), the highest peak in mid May 2010, lasting about 1 months, and a small peak in October; the peak period in 2011 was delayed to half a month, and the HFMD in spring and summer season was slightly higher than that in autumn.
The clinical manifestations were mainly palms, buttocks, plantar macula and oral herpes: 90% (?), more than 60% of the early onset of fever accompanied by a rash, and a few (7.2-11.3%) symptoms of respiratory tract infection. The clinical manifestations of.EV71 infection, such as vomiting, convulsions, startle or headache, were similar to those caused by EV caused by HFMD. In.2010 to 2011, 89 and 144 children were treated with ICU, of which 75 cases (14.9%) and 128 cases (24.9%) had more.EV71 infection than EV infection (P value 0.037 and 0 respectively). All patients with severe hand, foot and mouth disease had enterovirus related encephalopathy, such as viral encephalitis, and respiratory complications. It is mainly neurogenic pulmonary edema.
The partial VP1 region of 33 strains of EV71 (5 in 2009 and 28 in 2010) was amplified and sequenced. By comparison and analysis, the subtype of C4a was determined, and no significant difference was found in the sequence of isolates from Guangdong. Compared with the Fuyang EV71 epidemic strains in 2008, there was no obvious difference, indicating that it was a domestic circulating epidemic strain and the epidemic strain of Taiwan in recent years. Different genetic subtypes showed that the evolutionary origin of the cross straits strains was different. Through Clustal W software, the clinical isolates and the standard strain BrCr (U22521). Taiwan 1998 epidemic strain (Taiwan98) was compared to the VPl region sequence: nucleotide similarity 93.7%-100%, amino acid similarity 97.0%-100%. 22,98 bit amino acid and Taiwan 98 year epidemic strain and standard strain BrCr It's different.
The second part is real-time fluorescence RT-PCR and EV71-IgM capture ELISA method to evaluate the diagnosis of HFMD caused by EV71.
In order to assess the ELISA (?) method for detecting EV71-IgM, we collected 256 sera from EV71 infection from first days to 158 days. It was found that EV71-IgM was detected in first days. The number of cases increased with the number of days increasing and fifth Tianda 100%. After 3 months, the detection rate of serum EV71-IgM was significantly decreased. The acute phase (7 days of onset (?) detection sensitivity) The sensitivity was 90.2% (138/153), and the sensitivity was 93.6% (233/249) within 90 days.
There was a cross reaction with CA16-IgM detection. The experiment showed that 38 EV71-IgM positive in serum of 122 CA16 infection, 14 EV71-IgM positive in 49 Other enterovirus infection sera, 2 sera in 105 other respiratory virus infection sera (from the same respiratory syncytial virus A and B type double infected patients) EV71-IgM positive for other enterovirus, EV The specificity of 71-IgM capture method ELISA was 69.6% (52171.95.95 (?): 65.2-72.4%), for other respiratory virus specific 98.1% (2/105950% confidence interval: 96.5-99.5%), the positive predictive value was 81.3% (95% confidence interval: 76.9-85.1%), negative predictive value 91% (95% confidence interval: 87.5-93.8%).206 EV71 infection serum detected EV71-IgM Positive 199 (95.7%), CA16-IgM positive 58 (28.2%), of which EV71-IgM was higher than CA16-IgM in 56 (96.6%), 119 CA16 infected serum CA16-IgM positive 83 (69.7%), 36 EV71-IgM positive (30.3%), of which CA16-IgM was higher than EV71-IgM (91.7%), the actual infection of the pathogenic IgM detection of higher value, can be correct area Most of the pathogens are infected.
111 cases of anal swabs and serum samples collected on the same day were detected by two methods of ELISA and real time fluorescence RT-PCR. There was no significant difference between two methods to diagnose EV71 infection (McNemar's x 2 test P=0.648), and the consistency was better (Kappa value 0.729).
The third part is the preparation of EV71 virus capsid protein P1 monoclonal antibody.
The full length sequence primers of VP1 ~ VP4 gene were designed according to the EV71 sequence information (FJ194965.1) sequence information. PCR amplification, Kpn I and Hind III double enzyme cut PCR products, connected the carrier pQE30a. The recombinant plasmid was converted into VP1 ~ VP4 protein after the sequencing confirmed that the recombinant plasmid was induced to express the VP1 to VP4 protein, and all were in the form of inclusion body. 8M urea denaturation and gradient refolding were purified by HisBind Resin chromatography column. The purified VP1 ~ 4 protein was purified. The mice were immunized alternately with the recombinant VP4 protein and purified EV71 virus, and the hybridoma cells were cloned through the fusion of murine splenocytes and hybridoma cells. The recombinant egg white VP1 ~ 4 and the virus package were covered with ELISA and indirect immunofluorescence. The target protein was screened by immunoblot.
The recombinant protein VP1, VP2 and VP4, dissolved in 8M urea, were coated with slate, VP2 and VP4, respectively, to detect the clone supernatant of fusion cells, select the clone with high OD value, and further indirect immunofluorescence to respond to EV71 infection and non infected RD cells, and screen the clone of only EV71 infected RD cells. The clone supernatant was purified with the purified EV71 virus. The target protein was identified by the immunoblotting reaction of the protein components separated by the recombinant VP1-VP4 protein by SDS-PAGE glue. 25 monoclonal antibodies against P1 protein were preliminarily obtained: 6 strains of EV71 virus VP1 protein (natural protein), 4 of which were reacted to the recombinant VP1 protein; the 3 strains were only targeted to the recombinant VP2 protein, and the 2 strains against the virus VPl and VP2 eggs. Among them, one strain was targeted against recombinant VP1 and VP2, the other 1 were directed against recombinant protein VP4, and another 13 strains had not identified target proteins.
Summary
Based on the above three parts, the research summaries are as follows:
In 1.2009-2011, the pathogen of hand foot and mouth disease (HFMD) infected with EV was detected in 1.2009-2011. The EV71 infection was 30252 and 221, respectively, 28.1% (30/107), 45.6% (252/553) and 38.7% (221/579), and EV71 was still the main pathogen of hand foot and mouth disease in Guangdong.
In the year of 2.2009-2011, in Guangdong, there were two major onset peaks, namely, early summer (between 5,6 months) and autumn (10,11 month); 1,2 years were the most common age segments, accounted for 85% of the children under 4 years of age, and the age distribution of patients with severe hand foot and mouth disease caused by EV71 infection was similar to this, and there was no statistical difference.
Guangdong EV71 clinical isolates from 3.2009 to 2010 were C4a subtypes, which were not different from those in Guangdong and Fuyang epidemic in Anhui for 08 years. It was a domestic circulating epidemic strain. Compared with the epidemic strains of Taiwan in recent years, it is different gene subtypes, suggesting that the evolutionary origin of the two strains is different, and the standard strain BrCr, and the 1998 epidemic strain of Taiwan, Taiwan. The nucleotide similarity of 93.7%-100% in 98VP1 region is similar to that of amino acid. The amino acid of 22,98 97.0%-100%. is slightly different from that of Taiwan's 98 year old strain and standard strain BrCr.
4. the sensitivity of EV71 induced ELISA EV71-IgM detection in the diagnosis of EV71 induced hand foot and mouth disease was 90.2% (138/153), the specificity was 69.6% (52/171, relative other enterovirus) and 98.1% (2/105, relative to other respiratory viruses), and the positive predictive value was 81.3% (95% confidence interval: 76.9-85.1%) and negative predictive value 91% (95% confidence interval: 87.5-93.8%). There was no significant difference in the diagnosis of EV71 infection with real-time fluorescence RT-PCR (McNemar's chi square test 2), and the consistency was high. The method is suitable for clinical application. Conclusion: there is no significant difference between the two methods (McNemar's chi square test).
5. ELISA EV71-IgM detection was used to detect EV71, although there was a cross reactivity (28.2% and 30.3%) for CA16 infected HFMD sera, but the higher OD value could reflect the type of infected virus.
6. EV71 purified virus and recombinant VP4 protein were used to immunize mice, and 25 monoclonal antibodies against P1 protein were obtained. EV71 virus liquid, VP1-VP4 recombinant protein ELISA, indirect immunofluorescence screening and immunoblotting identification were respectively: 6 strains of VP1 protein, 3 VP2 protein, 1 strains of VP1 and VP22 strain and VP4 protein with clockwise, and 13 strains were not determined.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392;R725.1

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