长春地区肠道病毒71型小鼠模型的致病性及对候选疫苗的保护性评价

发布时间：2018-06-03 15:09

本文选题：肠道病毒71型 + 柯萨奇病毒A16型　；参考：《吉林大学》2015年博士论文

【摘要】：手足口病(Hand, Foot and Mouth Disease, HFMD)是由肠道病毒引起的全球性传染病，易发生于5岁以下儿童，多数患者症状轻微，以发热和手、足、口腔等部位皮肤黏膜皮疹、疱疹、溃疡为主要症状，一般10-14天可自愈。少数病例（尤其是小于3岁者）病情进展迅速，可出现脑膜炎、肺水肿、心肌炎、弛缓性麻痹等并发症，极少数病例病情危重，可导致死亡，存活病例留有后遗症。20世纪90年代后期，手足口病开始在东亚地区流行。我国于1981年上海首次报道本病，1983年和1986年在天津2次爆发流行；1998年的台湾地区和2008年安徽省阜阳市均有过手足口病的大范围流行。根据国家卫生和计划生育委员会数据显示，近几年手足口病的发病率一直呈上升趋势。目前该病尚无特异的治疗性药物和预防性疫苗，主要是对症治疗。引起手足口病的肠道病毒有20多种，包括肠道病毒71型，柯萨奇病毒A组2、4、5、7、9、10、16型，B组1、2、3、4、5型以及埃可病毒，其中以肠道病毒71型(EV71)和柯萨奇病毒A16型(CA16)最常见。在历次手足口病大爆发中，EV71与CA16或交替感染或同时出现引起混合感染，成为引发手足口病流行的最主要病原体。 EV71病毒属于小核糖核酸病毒科（小RNA病毒科，Picornavirdae），肠道病毒属（Enterovirus，EV），归属于人类肠道病毒A组，病毒核酸为单股正链RNA，基因组约含7411个核苷酸，基因组两端分别为5′端和3′端非编码区(Untranslated Region, UTR)，中间有一个开放读码框架（Open reading frame，ORF），编码2194个氨基酸组成的无活性多聚蛋白，多聚蛋白被进一步水解成P1、P2、P3三个前体蛋白；其中P2和P3分别裂解成共7个非结构蛋白(2A、2B、2C和3A、3B、3C、3D)，参与调控病毒多聚蛋白的加工、RNA的复制和宿主细胞蛋白合成，与病毒的复制及病毒毒力有关；前体蛋白P1在3C、3D的作用下被切割为结构蛋白VP0、VP1和VP3，VP0进一步裂解为VP2和VP4；VP1（）、VP2（β）、VP3（γ）、VP4（）共同组装形成病毒衣壳。EV71病毒衣壳蛋白VP1是该病毒主要的毒力和中和决定因子，决定病毒的感染性和免疫原性。VP1基因具有与病毒血清型完全对应的遗传多样性，VP1基因序列不仅可作为肠道病毒属内不同血清型分类的依据,并可作为小RNA病毒科内不同属的分类参考，因此VP1基因是EV71基因分型和遗传进化分析的最重要的对象。基于VP1核苷酸序列的差异，，可将EV71分为A、B、C等三个基因型，A型仅包括原型株BrCr；B型和C型分别分为B1、B2、B3、B4、B5以及C1、C2、C3、C4和C5亚型。病毒感染过程是病毒通过与位于细胞表面的特异性受体结合而吸附于被感染的细胞的表面，然后利用细胞的内吞作用进入细胞或将病毒的核酸释放进细胞。本研究从近年收集的手足口病患者咽拭子标本分离出EV71病毒，经细胞适应培养，传代及克隆后建立EV71病毒株，经分子生物学特性分析，揭示其均为EV71病毒C4亚型的重组病毒。以分离的EV71病毒感染乳鼠模型，系统研究了长春地区流行性EV71病毒分离株对ICR乳鼠模型的感染机理，筛选出具有广谱保护性的疫苗候选株。在对EV71病毒致病机理的研究中，观察到乳鼠感染后的发病时间、平均临床症状、死亡时间等与攻毒剂量具有明显的相关性；EV71长春分离株毒力较原型株BrCr、阜阳分离株FY0805、深圳分离株SHZH98明显增强。对感染后乳鼠各组织器官进行病理分析、免疫组织化学染色、病毒动态载量测定等研究，掌握了EV71病毒在乳鼠模型的感染特点和规律。利用筛选到的疫苗候选株，制备了一种EV71全病毒灭活疫苗，用该疫苗免疫小鼠，微量细胞病变法测定其血清可以中和多株EV71病毒。该疫苗免疫母鼠，分娩后，母鼠及其乳鼠血清均具有EV71的广泛中和特性；用致死剂量EV71病毒攻击分娩的1日龄乳鼠，发现疫苗能对乳鼠起到广泛的保护性作用，并且乳鼠组织器官中未能检测到病毒复制。该动物模型可以对候选疫苗株的有效性和广泛保护性做出评价，作为筛选疫苗株的依据。
[Abstract]:Hand (Foot and Mouth Disease, HFMD) is a global infectious disease caused by enterovirus, which occurs easily in children under 5 years of age. Most of the patients have mild symptoms. The main symptoms are skin rash, herpes, and ulcer in fever and hands, feet, and mouth. A few cases (especially those less than 3 years old) can be healed. Rapid progress can occur, such as meningitis, pulmonary edema, myocarditis, flaccid paralysis and other complications, very few cases are critical, can lead to death, surviving cases have sequelae in the late.20 century in the late 90s, and hand foot and mouth disease began to prevail in East Asia. In 1981, Shanghai first reported this disease in Shanghai and Tianjin in 1983 and 1986. The prevalence of hand foot and mouth disease in 1998 in Taiwan and Fuyang in Anhui, Anhui Province, in 2008. According to the national health and family planning committee data, the incidence of hand foot and mouth disease has been on the rise in recent years. There is no specific therapeutic and preprotective vaccines, mainly symptomatic treatment.
There are more than 20 enteroviruses that cause hand foot and mouth disease, including enterovirus 71, Coxsackie virus A group 2,4,5,7,9,10,16, B group 1,2,3,4,5 and exovirus, among which, enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the most common. In the major outbreak of hand foot and mouth disease, EV71 and CA16 or alternate infection or simultaneous occurrence Co infection has become the main pathogen causing hand foot mouth disease.
EV71 virus belongs to the family of small ribonucleic acid virus family (small RNA virus family, Picornavirdae), enterovirus (Enterovirus, EV), belonging to the human enterovirus A group, the virus nucleic acid is single strand positive chain RNA, the genome contains about 7411 nucleotides, the two ends of the genome are respectively the 5 'end and the 3' end non coding region (Untranslated Region, UTR), and there is one in the middle. Open reading frame (ORF), which encodes an inactive polyprotein composed of 2194 amino acids, which is further hydrolyzed to P1, P2, and P3 three precursor proteins; P2 and P3 split into 7 non structural proteins (2A, 2B, 2C, etc.), and are involved in the processing of viral polyproteins, replication and host cells Protein synthesis is related to the replication of the virus and virulence of the virus; the precursor protein P1 is cut into structural protein VP0, VP1 and VP3 under the action of 3C and 3D, and VP0 is further cracked into VP2 and VP4; VP1 (), VP2 (beta), VP3 (gamma), etc. are assembled to form the viral capsid virus capsid protein, which is the main virulence and neutralization determinant of the virus. The infectious and immunogenic.VP1 genes of the virus have genetic diversity corresponding to the viral serotypes. The VP1 gene sequence can not only be used as the basis for the classification of different serotypes within the enterovirus genus, but also can be used as a reference for the classification of the different genus of small RNA virus Kone. Therefore, the VP1 gene is the most important analysis of the EV71 genotyping and genetic evolution. Based on the difference in the nucleotide sequence of VP1, EV71 can be divided into three genotypes, such as A, B, C, and A type only BrCr. B and C are divided into B1, B2, B3, etc., and the virus is adsorbed on the surface of infected cells by binding to the specific receptors on the surface of the cell. The endocytosis of cells is then used to enter cells or release the nucleic acids of the virus into cells.
In this study, the EV71 virus was isolated from the swab specimens of patients with hand foot and mouth disease in recent years. The EV71 virus strain was established by cell adaptation culture, generation and clone, and the recombinant virus of EV71 virus C4 subtype was revealed by molecular biological characteristics. The epidemic EV in Changchun region was systematically studied by using the isolated EV71 virus to dye the milk rat model. 71 the virus isolation strains infected the ICR suckling mice model, and screened broad spectrum protective vaccine candidates.
In the study of the pathogenesis of EV71 virus, it was observed that the time after infection, the average clinical symptoms and the time of death had obvious correlation with the attack dose, and the virulence of the EV71 Changchun isolates was more than that of the prototype strain BrCr, the Fuyang isolate FY0805, and the SHZH98 of the Shenzhen isolated strain. The characteristics of infection of EV71 virus in suckling mice were studied by means of theoretical analysis, immunohistochemical staining and dynamic load measurement.
A EV71 total virus inactivated vaccine was prepared by using the selected vaccine candidate. The vaccine was immunized with the vaccine. The serum could neutralize multiple EV71 viruses in mice. The Vaccine Immunized female mice. After delivery, the serum of mother mice and their Lactobacillus mice had the wide neutralization characteristics of EV71; the lethal dose of EV71 virus was used to attack 1 of the labor. This animal model can evaluate the effectiveness and wide protection of candidate vaccine strains as a basis for screening vaccine strains.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R-332;R512.5

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