肠道病毒71型致神经系统感染的影响因素初步分析

发布时间：2018-09-14 17:13

【摘要】：一、研究背景和目的肠道病毒71型(Human Enterovirus 71,简称EV71)属于小核糖核酸病毒科、肠道病毒A属,是引起婴幼儿手足口病(Hand Foot Mouth Disease, HFMD)的主要病原体之一,其感染常引起儿童手足口病、疱疹性咽峡炎等较轻的临床症状。然而,越来越多的报道揭示EV71的感染可引起严重的神经系统综合征,包括无菌性脑膜脑炎、脑干脑炎、脊髓灰质炎样麻痹、神经源性肺水肿,严重者甚至死亡。自从1969年首次在美国加利福尼亚州分离该病毒,此后在保加利亚、马来西亚、新加坡、日本、越南、中国均引起过大规模流行。据广东省卫计委5月17日发布的手足口病预警信息,截止5月16日,全省今年共报告手足口病88366例,其中死亡3例,较2015年同期病例数(51327例)上升72%,以EV71病毒致死率最高。作为手足口病的主要病原之一,EV71已经成为目前全球范围内取代脊髓灰质炎病毒,对公共卫生安全威胁最严重的中枢神经毒性病原。因此,探讨EV71致神经系统感染的影响因素对EV71的防治意义重大。从分子生物学的角度来看,EV71为RNA病毒,在复制过程中缺乏核酸外切酶的校对功能而导致较高的出错率,约每复制一个基因就有一个自发突变。VP1区由于其高度多样性和较少参与重组而被认为是肠道病毒进化研究中最有意义的基因区域。有研究表明,VP1的变异与神经系统的感染可能存在相关关系,决定EV71神经毒性可能是单一位点的突变,也有可能是多个位点突变共同作用的结果。有研究在分析具有神经毒性的EV71分离株时发现,VP1上有一个重要的氨基酸变异Alal 70Val(丙氨酸170颉氨酸),猜测该变异使得病毒蛋白结构发生变化,改变了病毒与受体的结合能力。然而,此研究只是基于少量的样本发现的突变位点,突变位点少,且突变的位点对人群发病的影响有多大意义并不清楚。因此,我们旨在通过更大样本的人群来筛选,以验证VP1蛋白变异是否与EV71神经系统感染有关。此外,一项关于大脑组织TLR-5和TLR-6受体的表达与EV71感染致神经系统损伤的研究发现,脑组织病毒受体的高表达与神经系统损伤有关。另有研究也证实EV71受体对于病毒感染细胞和组织至关重要。病毒进入细胞的第一步便是与细胞表面的病毒受体结合,病毒进入细胞的主要途径通过病毒受体介导的RNA释放或者胞吞作用,因此猜测病人自身的受体数量、病毒蛋白与受体的相互作用可能也与神经系统的感染有关。目前已知的EV71的受体有：P-selectin glycoprotein ligand-1 (PSGL-1)、 Human scavenger receptor class B2(SCARB2), Vimentin(VIM)。近年来,越来越多研究报道了VIM在病原体吸附、胞吞、迁移过程中的重要作用。VIM是新近发现的EV71受体,在人脑微血管细胞中表达丰富并介导大肠杆菌细菌侵袭,EV71感染与相类似。因此猜测EV71通过VIM受体介导进入人脑微血管内皮细胞而感染神经系统。根据已有文献报道我们提出：EV71病毒的变异和患者自身受体的差异可能是影响EV71感染神经系统的因素,二者或独立作用或存在交互作用。本文主要从广东省2008年～2010年EV71的流行病学分析、VIM在EV71感染HBMEC中的作用两方面来探究EV71所致神经系统的影响因素,以确定EV71病毒VP1蛋白的A289T变异,以及VIM受体与EV71神经系统感染的关系；并明确EV71病毒感染血脑屏障细胞是神经系统感染的一条重要途径。本研究不但有助于分析EV71的流行病特征、病毒变异情况；也将为EV71病毒的神经系统感染建立有意义的预警信号,及早发现易感人群,及时采取有效的防范措施,对于减少神经系统感染的发病率具有重要的意义。二、研究方法1.广东省2008～2010年EV71的流行病学分析收集2008～2010年广东省多个市区送至广东省疾病预防控制中心检验的样本(包括粪便、咽拭子、脑脊液等),采用荧光定量PCR技术和病毒分离培养技术确诊为EV71感染的共194例样本。根据症状的差异分成EV71神经系统感染组和非神经系统感染两组。分别提取病人的RNA,逆转录合成cDNA,并扩增全长的病毒VP1序列。通过比对病毒VP1序列和构建系统进化树确定病毒的分型；采用Logistic回归分析影响患者致神经系统感染的因素；病毒的VP1基因序列用DNAman软件翻译成蛋白质序列,定义在同一个位点上,出现两种以上氨基酸为一个变异。2.人脑微血管内皮细胞VIM敲除细胞系的建立在美国国立生物技术信息中心(NCBI)的数据库中找到VIM的基因序列,找到VIM基因CDS区,分析其基因结构,明确CDS的外显子部分。按照基因本身的性质,选择候选的待敲除位点后设计并合成识别靶位点的一对DNAOligos。把sgRNA连接到Lenti CRISPRv2载体中并构建Cas9质粒。将构建好的目的质粒及空白对照分别包装成慢病毒再感染HBMEC,筛选出稳定表达VIM-KO的细胞株以及正常对照株。3.VIM在EV71感染HBMEC中的作用分析在RD细胞中扩增并滴定的病毒分别感染正常和VIM-KO的HBMEC,观察HBMEC的病变效应。采用荧光定量PCR技术检测EV71病毒在正常和VIM-KO的细胞中吸附能力、复制能力和病毒核酸释放水平的差异。同时检测在HBMEC正常和VIM-KO细胞中病毒VPl蛋白合成的差异。三、结果1.广东省2008～2010年EV71的主要型别为C4a型在2008～2010年期间,广东省各个地区流行的EV71病毒株为C4a型,也是目前我国主要的流行株。同时发现此型在广东省流行过程中已经开始演化,增加了三个族,即C4a2-C4a4族。2.EV71病毒株型别的时间、地区分布从各病毒株的分布时间来看：2008年四种均有,但主要集中在C4a1型；2009年的流行株为C4a1和C4a2型；而在2010年,除湛江的一株属于C4a4型,其余均为C4a1型。从地区分布来看：大部分地区的病毒株型别为C4a1和C4a2型；只有2008年东莞分株009-08-M单独构成了C4a3型；C4a4也仅分别出现在佛山和湛江。综合来看各病毒株的流行并没有地区和时间集中趋势。3.病毒A289T变异与神经系统感染有关随着年龄的增加,神经系统感染患者的发病率明显下降(P0.05)。对患者进一步用Logistic回归法分析其性别、年龄、病毒型别、VP1变异对患者临床症状轻重有无神经系统症状的影响。结果表明EV71患者的临床症状与性别、病毒的型别与有无神经系统症状没有无统计学相关性：年龄和病毒A289T变异与患者的临床表现相关,病毒289A(289丙氨酸)容易致EV71神经系统感染(P0.05,OR=2.360,95% CI为1.163-4.659)。4.人脑微血管内皮细胞VIM敲除细胞系的成功建立采用CRISPR-Cas9技术及慢病毒载体包装系统包装出VIM-KO和正常对照的病毒后,感染HBMEC 48 h后观察到GFP的表达接近70%-80%。用嘌呤霉素筛选出稳定的HBMEC VIM-KO和对照细胞株后,用VIM的特异性抗体分别检测HBMEC对照和VIM-KO中的VIM表达量。结果发现VIM-KO的细胞中VIM的表达量下降了90%以上,说明此次VIM敲除效果较好。5.VIM在EV71感染HBMEC中的作用分析EV71进入细胞后,由于其对细胞的损伤,会导致细胞的形态发生改变。分别用病毒感染对照以及VIM-KO的HBMEC,发现病毒感染HBMEC正常对照细胞24 h后,细胞出现了皱缩,变圆甚至破碎；随着感染时间的延长,到了48h后,部分细胞已经脱落而飘浮在培养中。相比之下,EV71同样的MOI感染VIM-KO,24 h后细胞的形态几乎不变,即使到了48 h,细胞的形态改变很小,只有很小一部分细胞开始变圆。说明EV71感染能引起HBMEC病变效应,但在VIM敲除的细胞中,细胞状态变化不大,病变效应很弱。EV71侵袭细胞的第一步是吸附于细胞表面。用荧光定量PCR检测HBMEC对照和VIM-KO表面的病毒核酸量的差异,结果表明与对照相比,VIM敲除后EV71在细胞表面的吸附下降了40%。EV71感染细胞24 h和48h后,正常的HBMEC中病毒量分别同比增加到3倍和8倍,而VIM-KO细胞复制的非常少,24 h后仅为0 h的1.3倍,48h后病毒量也仅为0 h的2倍左右。EV71感染HBMEC 48 h后,VIM敲除细胞EV71病毒核酸量是对照细胞的1/12。细胞培养液上清EV71病毒核酸量检测结果表明,VIM敲除的细胞比对照细胞少1.4倍。EV71病毒在细胞内的核酸复制和合成蛋白是基本同步的,通过检测EV71在HBMEC对照和敲除的细胞内合成蛋白的差异,发现VIM敲除后病毒VP1蛋白的合成也比HBMEC对照细胞少很多。四、结论1、在2008～2010年期间,广东省各个地区流行的EV71病毒株为C4a型,也是目前我国主要的流行株。同时发现此型在广东省流行过程中已经开始演化,增加了三个族,即C4a2-C4a4族。EV71病毒亚型在时间、地区上的分布无明显趋势。2、病毒的A289T变异与与EV71神经系统感染有关,病毒289A(289丙氨酸)容易致EV71神经系统感染。本次所获得的A289T变异位点是广东省194例EV71感染人群的分子流行病学的研究结果,此位点的进一步研究,对于广东省乃至全国的EV71感染的防治具有实际的指导意义。3、VIM是HBMEC表面EV71的受体,EV71通过与受体VIM的结合而侵袭HBMEC,并且进一步影响EV71在细胞中的复制甚至释放。本课题提出EV71感染神经系统的通路假说：EV71通过VIM受体介导的RNA释放,进入HBMEC细胞,穿越血脑屏障,最终感染神经系统。此项研究将有助于揭示EV71的神经系统感染机制。4、本文将病毒变异研究与受体研究相结合,认为病毒的变异与自身的受体同时影响EV71致神经系统感染。这为EV71致神经系统感染的分析提供一条有意义的线索。
[Abstract]:Background and Objectives: Human Enterovirus 71 (EV71) belongs to the family of small ribonucleoviruses. Enterovirus A (EV71) is one of the main pathogens causing Hand Foot Mouth Disease (HFMD) in infants and young children. Its infection often causes minor clinical symptoms such as hand-foot-mouth disease (HFMD) and herpes angina. A growing number of reports have revealed that EV71 infection can cause severe neurological syndrome, including aseptic meningoencephalitis, brainstem encephalitis, poliomyelitis-like paralysis, neurogenic pulmonary edema, and even death. The virus was first isolated in California, USA, in 1969, and has since been found in Bulgaria, Malaysia, Singapore, etc. As of May 16, 88 366 cases of hand-foot-mouth disease (HFMD) have been reported in Guangdong Province, including 3 deaths, 72% higher than the corresponding number of cases (51 327 cases) in 2015. EV71 is the leading cause of HFMD. First, EV71 has become the most serious central neurotoxic pathogen in the world, replacing poliovirus and threatening public health. Therefore, it is of great significance to explore the influencing factors of nervous system infection caused by EV71 for the prevention and treatment of EV71. VP1 region is considered to be the most significant gene region in the evolution of enterovirus because of its high diversity and less recombination. Studies have shown that VP1 mutation may be associated with nervous system infection. Neurotoxicity of EV71 may be a single site mutation or a combination of multiple site mutations. In the analysis of neurotoxic EV71 isolates, an important amino acid variant Alal 70Val (alanine 170 pi) was found in VP1. It was speculated that the mutation caused structural changes in the protein of the virus. However, this study is based on a small number of mutation sites, and the significance of the mutation site on the pathogenesis of the population is not clear. Therefore, we aim to screen a larger sample of people to verify whether VP1 protein mutation is associated with EV71 infection of the nervous system. In addition, a study on the expression of TLR-5 and TLR-6 receptors in brain tissue and neurological damage caused by EV71 infection found that the high expression of virus receptors in brain tissue was associated with neurological damage. Other studies have also confirmed that EV71 receptors are essential for viral infection of cells and tissues. The first step for viruses to enter cells is with the cell surface. Viral receptors bind to the surface of the virus, and the main way the virus enters the cell is through RNA release or cytophagy mediated by the virus receptor. Therefore, it is speculated that the number of receptors in the patient and the interaction between the virus protein and the receptor may also be related to the infection of the nervous system. The known receptors for EV71 are P-selectin glycoprotein ligand-1 (PS-71). GL-1, Human scavenger receptor class B2 (SCARB2), Vimentin (VIM). In recent years, more and more studies have reported that VIM plays an important role in pathogen adsorption, endocytosis and migration. We have reported that the mutation of EV71 virus and the difference of the patient's own receptor may be the factors influencing the infection of EV71 in the nervous system. The two factors either act independently or interact with each other. This paper mainly from 2008 to 2010 in Guangdong Province. Epidemiological analysis of EV71 in 1997 showed that the role of VIM in EV71 infection with HBMEC was related to the factors affecting the nervous system caused by EV71 in order to determine the A289T variation of VP1 protein of EV71 and the relationship between VIM receptor and the infection of EV71 nervous system. This study will not only help to analyze the epidemiological characteristics of EV71 and the variation of EV71 virus, but also establish a meaningful early warning signal for the infection of the nervous system of EV71 virus, early detection of susceptible groups, and timely and effective preventive measures. It is of great significance to reduce the incidence of infection of the nervous system. 2. Research methods 1. Guangdong 200 Epidemiological analysis of EV71 from August to 2010 collected 194 samples (including feces, pharyngeal swabs, cerebrospinal fluid, etc.) from several urban districts of Guangdong Province sent to the Guangdong Center for Disease Control and Prevention for examination (including feces, swabs, cerebrospinal fluid) from 2008 to 2010. Systemic infection group and non-nervous system infection group. RNA was extracted from patients, reverse transcription was used to synthesize cDNA, and full-length VP1 sequence was amplified. Viral typing was determined by comparing VP1 sequence and constructing phylogenetic tree. Logistic regression analysis was used to analyze the influencing factors of nervous system infection. NAman software translation into protein sequence, defined at the same site, there are more than two amino acids for a mutation. 2. Human brain microvascular endothelial cell VIM knockout cell line established in the United States National Center for Biotechnology Information (NCBI) database to find the VIM gene sequence, find the VIM gene CDS region, analyze its gene structure, clear C According to the nature of the gene itself, a pair of DNA Oligos was designed and synthesized to identify the target site. The sgRNA was linked to the Lenti CRISPRv2 vector and the Cas9 plasmid was constructed. The role of VIM in the infection of HBMEC by EV71 was analyzed. The viruses amplified and titrated in RD cells were infected with normal and VIM-KO HBMEC respectively, and the pathological changes of HBMEC were observed. The adsorption, replication and nucleic acid release of EV71 in normal and VIM-KO cells were detected by fluorescence quantitative PCR. At the same time, the difference of VPl protein synthesis between normal HBMEC and VIM-KO cells was detected. 3. Results 1. The main type of EV71 in Guangdong Province from 2008 to 2010 was C4a. During 2008 to 2010, the EV71 strain prevalent in Guangdong Province was C4a, which was also the main prevalent strain in China. In the course of evolution, three families, C4a2-C4a4.2.EV71 strain, were added, and the distribution time of each strain was as follows: all the four strains in 2008, but mainly concentrated in C4a1; the epidemic strains in 2009 were C4a1 and C4a2; in 2010, except one strain in Zhanjiang, which belonged to C4a4, the others were C4a1. From the regional distribution point of view: most of the virus strains are C4a1 and C4a2; only in 2008 Dongguan ramet 009-08-M constituted C4a3 alone; C4a4 only appeared in Foshan and Zhanjiang. In a comprehensive view, the epidemic of each virus strain has no regional and temporal concentration trend. With age increasing, the incidence of neurological infection decreased significantly (P 0.05). Logistic regression was used to analyze the influence of gender, age, viral type and VP1 mutation on the severity of clinical symptoms of patients with neurological symptoms. There was no statistically significant correlation between age and viral A289T variation. Viral 289A (289 alanine) was associated with EV71 nervous system infection (P 0.05, OR = 2.360, 95% CI was 1.163-4.659). 4. The successful establishment of VIM knockout cell lines in human brain microvascular endothelial cells using CRISPR-Cas9 technology and lentiviral vector packaging. After 48 hours of infection with HBMEC, the expression of GFP was nearly 70%-80%. After screening stable HBMEC VIM-KO and control cell lines with purinomycin, the expression of VIM in HBMEC control and VIM-KO was detected with specific antibody of VIM. The results showed that the expression of VIM in VIM-KO cells decreased. More than 90% of the cells were knocked out by VIM. 5. Analysis of the role of VIM in EV71 infection of HBMEC. When EV71 enters the cell, the cell morphology will change due to its damage to the cell. By contrast, the morphology of the cells infected with VIM-KO by the same MOI of EV71 remained almost unchanged 24 hours after infection. Even at 48 hours, the morphological changes of the cells were very small, and only a small number of cells began to become round. The first step of EV71 invasion was to adsorb on the cell surface. The difference in the amount of viral nucleic acid between HBMEC control and VIM-KO surface was detected by fluorescence quantitative PCR. The results showed that the adsorption of EV71 on the cell surface decreased by 40% after VIM knockout. After 24 hours and 48 hours of infection, the viral quantity in normal HBMEC increased to 3 times and 8 times respectively, while the viral quantity in VIM-KO cells was only 1.3 times of that in 0 hours after 24 hours. After 48 hours of infection with HBMEC, the nucleic acid content of EV71 virus in VIM knockout cells was 1/12 of that in control cells. The results of acid assay showed that the number of cells knocked out by VIM was 1.4 times less than that of the control cells. The replication of nucleic acid and the synthesis of protein of EV71 virus in the cells were basically synchronous. During 2008-2010, the EV71 virus strain in Guangdong Province was C4a, which was also the main epidemic strain in China. It was found that the EV71 virus strain had evolved during the epidemic process in Guangdong Province, and three families, C4a2-C4a4, were added. The EV71 virus subtype had no obvious trend in time and region. 2. The A289T variation and distribution of the virus were not significant. Virus 289A (289 alanine) is easy to cause the infection of EV71 nervous system. The A289T mutation locus is the result of molecular epidemiology of 194 EV71 infected people in Guangdong Province. Further study on this locus is of practical significance for the prevention and treatment of EV71 infection in Guangdong Province and even in the whole country. 3. VIM is the receptor of EV71 on the surface of HBMEC. EV71 invades HBMEC by binding to the receptor VIM, and further affects the replication and even release of EV71 in cells. The hypothesis that EV71 infects the nervous system is put forward: EV71 enters HBMEC cells through the release of RNA mediated by VIM receptors, crosses the blood-brain barrier, and eventually infects the nervous system. The study will be helpful to reveal the mechanism of EV71 infection in the nervous system. 4. In this paper, we combine the study of virus mutation with the study of receptors, and conclude that the virus mutation and its receptors affect both the nervous system infection caused by EV71.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R512.5;R742.9

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