本文選題：腸道病毒71型 + 柯薩奇病毒A16型　； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：手足口病(Hand, Foot and Mouth Disease, HFMD)是由腸道病毒引起的全球性傳染病，易發(fā)生于5歲以下兒童，多數(shù)患者癥狀輕微，以發(fā)熱和手、足、口腔等部位皮膚黏膜皮疹、皰疹、潰瘍?yōu)橹饕Y狀，一般10-14天可自愈。少數(shù)病例（尤其是小于3歲者）病情進(jìn)展迅速，可出現(xiàn)腦膜炎、肺水腫、心肌炎、弛緩性麻痹等并發(fā)癥，極少數(shù)病例病情危重，可導(dǎo)致死亡，存活病例留有后遺癥。20世紀(jì)90年代后期，手足口病開始在東亞地區(qū)流行。我國(guó)于1981年上海首次報(bào)道本病，1983年和1986年在天津2次爆發(fā)流行；1998年的臺(tái)灣地區(qū)和2008年安徽省阜陽市均有過手足口病的大范圍流行。根據(jù)國(guó)家衛(wèi)生和計(jì)劃生育委員會(huì)數(shù)據(jù)顯示，近幾年手足口病的發(fā)病率一直呈上升趨勢(shì)。目前該病尚無特異的治療性藥物和預(yù)防性疫苗，主要是對(duì)癥治療。 引起手足口病的腸道病毒有20多種，包括腸道病毒71型，柯薩奇病毒A組2、4、5、7、9、10、16型，B組1、2、3、4、5型以及�？刹《�，其中以腸道病毒71型(EV71)和柯薩奇病毒A16型(CA16)最常見。在歷次手足口病大爆發(fā)中，EV71與CA16或交替感染或同時(shí)出現(xiàn)引起混合感染，成為引發(fā)手足口病流行的最主要病原體。 EV71病毒屬于小核糖核酸病毒科（小RNA病毒科，Picornavirdae），腸道病毒屬（Enterovirus，EV），歸屬于人類腸道病毒A組，病毒核酸為單股正鏈RNA，基因組約含7411個(gè)核苷酸，基因組兩端分別為5′端和3′端非編碼區(qū)(Untranslated Region, UTR)，中間有一個(gè)開放讀碼框架（Open reading frame，ORF），編碼2194個(gè)氨基酸組成的無活性多聚蛋白，多聚蛋白被進(jìn)一步水解成P1、P2、P3三個(gè)前體蛋白；其中P2和P3分別裂解成共7個(gè)非結(jié)構(gòu)蛋白(2A、2B、2C和3A、3B、3C、3D)，參與調(diào)控病毒多聚蛋白的加工、RNA的復(fù)制和宿主細(xì)胞蛋白合成，與病毒的復(fù)制及病毒毒力有關(guān)；前體蛋白P1在3C、3D的作用下被切割為結(jié)構(gòu)蛋白VP0、VP1和VP3，VP0進(jìn)一步裂解為VP2和VP4；VP1（）、VP2（β）、VP3（γ）、VP4（）共同組裝形成病毒衣殼。EV71病毒衣殼蛋白VP1是該病毒主要的毒力和中和決定因子，決定病毒的感染性和免疫原性。VP1基因具有與病毒血清型完全對(duì)應(yīng)的遺傳多樣性，VP1基因序列不僅可作為腸道病毒屬內(nèi)不同血清型分類的依據(jù),并可作為小RNA病毒科內(nèi)不同屬的分類參考，因此VP1基因是EV71基因分型和遺傳進(jìn)化分析的最重要的對(duì)象�；赩P1核苷酸序列的差異，，可將EV71分為A、B、C等三個(gè)基因型，A型僅包括原型株BrCr；B型和C型分別分為B1、B2、B3、B4、B5以及C1、C2、C3、C4和C5亞型。病毒感染過程是病毒通過與位于細(xì)胞表面的特異性受體結(jié)合而吸附于被感染的細(xì)胞的表面，然后利用細(xì)胞的內(nèi)吞作用進(jìn)入細(xì)胞或?qū)⒉《镜暮怂後尫胚M(jìn)細(xì)胞。 本研究從近年收集的手足口病患者咽拭子標(biāo)本分離出EV71病毒，經(jīng)細(xì)胞適應(yīng)培養(yǎng)，傳代及克隆后建立EV71病毒株，經(jīng)分子生物學(xué)特性分析，揭示其均為EV71病毒C4亞型的重組病毒。以分離的EV71病毒感染乳鼠模型，系統(tǒng)研究了長(zhǎng)春地區(qū)流行性EV71病毒分離株對(duì)ICR乳鼠模型的感染機(jī)理，篩選出具有廣譜保護(hù)性的疫苗候選株。 在對(duì)EV71病毒致病機(jī)理的研究中，觀察到乳鼠感染后的發(fā)病時(shí)間、平均臨床癥狀、死亡時(shí)間等與攻毒劑量具有明顯的相關(guān)性；EV71長(zhǎng)春分離株毒力較原型株BrCr、阜陽分離株FY0805、深圳分離株SHZH98明顯增強(qiáng)。對(duì)感染后乳鼠各組織器官進(jìn)行病理分析、免疫組織化學(xué)染色、病毒動(dòng)態(tài)載量測(cè)定等研究，掌握了EV71病毒在乳鼠模型的感染特點(diǎn)和規(guī)律。 利用篩選到的疫苗候選株，制備了一種EV71全病毒滅活疫苗，用該疫苗免疫小鼠，微量細(xì)胞病變法測(cè)定其血清可以中和多株EV71病毒。該疫苗免疫母鼠，分娩后，母鼠及其乳鼠血清均具有EV71的廣泛中和特性；用致死劑量EV71病毒攻擊分娩的1日齡乳鼠，發(fā)現(xiàn)疫苗能對(duì)乳鼠起到廣泛的保護(hù)性作用，并且乳鼠組織器官中未能檢測(cè)到病毒復(fù)制。該動(dòng)物模型可以對(duì)候選疫苗株的有效性和廣泛保護(hù)性做出評(píng)價(jià)，作為篩選疫苗株的依據(jù)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R-332;R512.5

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