本文選題：腸道病毒71型 + 遺傳進(jìn)化分析　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：手足口病是由多種腸道病毒引起的病毒性傳染病,主要感染五歲及以下嬰幼兒,可導(dǎo)致患者出現(xiàn)發(fā)熱以及手足口周的皰疹等臨床癥狀,嚴(yán)重時(shí)可出現(xiàn)無菌性腦膜炎以及神經(jīng)源性肺水腫等一系列神經(jīng)系統(tǒng)的并發(fā)癥,甚至可以導(dǎo)致患者死亡。目前手足口病在全球尤其是亞太地區(qū)廣泛流行,已成為重要的公共衛(wèi)生焦點(diǎn)問題。腸道病毒71型(Enterovirus 71,EV71)屬于小RNA病毒科腸道病毒屬,是導(dǎo)致手足口病的主要病原體之一,也是目前最主要的導(dǎo)致手足口病重癥或死亡的病原體,已成為目前全球范圍內(nèi)最主要的嬰幼兒中樞神經(jīng)毒性病原。目前,EV71導(dǎo)致患者神經(jīng)系統(tǒng)病變的致病機(jī)制尚不明確,有研究顯示在病毒基因組多個(gè)區(qū)域存在有影響病毒神經(jīng)毒力的位點(diǎn)。本研究通過對(duì)手足口病患者以及密切接觸者的標(biāo)本進(jìn)行病毒分離鑒定和序列分析,捕捉到EV71在自然傳播過程中的變異,在VP1上篩選出2個(gè)毒力候選位點(diǎn),利用反向遺傳技術(shù)構(gòu)建并拯救VP1置換的重組病毒SDLY107-VP1株,研究VP1蛋白毒力候選位點(diǎn)在EV71致病機(jī)制中的作用。目的:1.從手足口病患者以及密切接觸者標(biāo)本中分離EV71,并進(jìn)行序列分析,捕捉EV71在自然傳播過程中的變異,篩選毒力候選位點(diǎn);2.將兩株EV71不同毒力表型株的VP1區(qū)進(jìn)行置換,構(gòu)建并拯救出重組病毒;3.比較親本株與重組病毒之間在復(fù)制能力、致細(xì)胞損傷以及自噬等方面的差異,探究VP1區(qū)的毒力候選位點(diǎn)在EV71致病機(jī)制中的作用。方法:1.將采集自2015年山東省濟(jì)寧市手足口病患者及密切接觸者的標(biāo)本處理后使用RD細(xì)胞進(jìn)行病毒分離,并使用PCR特異性鑒定引物進(jìn)行鑒定;2.使用基于EV71病毒基因組設(shè)計(jì)的全序列擴(kuò)增引物對(duì)新分離到的病毒進(jìn)行全序列擴(kuò)增并測(cè)序,對(duì)獲得的病毒基因組全序列使用DNAstar7.1和MEGA6軟件進(jìn)行序列分析;3.設(shè)計(jì)引物并通過融合PCR的方法獲得弱毒株VP1片段,并將其置換到強(qiáng)毒株SDLY107株中,構(gòu)建并拯救重組病毒SDLY107-VP1株,通過觀察細(xì)胞病變、PCR擴(kuò)增以及間接免疫熒光實(shí)驗(yàn)對(duì)重組病毒進(jìn)行鑒定,使用50%終點(diǎn)法以及空斑試驗(yàn)對(duì)病毒滴度進(jìn)行測(cè)定;4.使用SH-SY5Y細(xì)胞、U87細(xì)胞以及Vero細(xì)胞對(duì)重組病毒及其親本病毒的復(fù)制能力、致細(xì)胞損傷作用以及致細(xì)胞自噬能力進(jìn)行研究。結(jié)果:1.從手足口病患者及其密切接觸者糞便標(biāo)本中分離到兩株EV71病毒,命名為SDJN2015-01株以及SDJN2015-01.1株,序列以及遺傳進(jìn)化分析顯示,兩株病毒同屬于EV71 C4a亞型,是我國(guó)近年來流行的優(yōu)勢(shì)亞型;2.兩株病毒存在6個(gè)氨基酸突變,其中VP1蛋白上存在2個(gè)氨基酸位點(diǎn)的差異,分別為K98E(全編碼區(qū)為K663E)和A133T(A698T);2C蛋白上存在4個(gè)氨基酸的差異,分別為 D48N(D1159N)、V126I(V1237I)、I238V(I1349V)和 N314Y(N1425Y);3.成功拯救出重組病毒SDLY107-VP1株,并對(duì)其滴度進(jìn)行測(cè)定;4.重組病毒SDLY107-VP1株在U87細(xì)胞以及Vero細(xì)胞上的復(fù)制能力、致細(xì)胞損傷作用以及引起細(xì)胞自噬的能力與SDLY107株和SDJN2015-01株沒有明顯的區(qū)別,但在神經(jīng)母細(xì)胞瘤細(xì)胞SH-SY5Y細(xì)胞上,重組病毒SDLY107-VP1株失去了引起細(xì)胞病變的能力,病毒在細(xì)胞能復(fù)制能力大大降低,沒有導(dǎo)致SH-SY5Y細(xì)胞明顯的細(xì)胞損傷,同時(shí)也沒有引起SH-SY5Y細(xì)胞明顯的細(xì)胞自噬發(fā)生,這與強(qiáng)毒株SDLY107株明顯不同,與弱毒株SDJN2015-01株的表現(xiàn)類似。結(jié)論:1.從2015年山東省濟(jì)寧市手足口病患兒及密切接觸者中成功分離到EV71,新分離到的EV71毒株屬于C4a亞型,與我國(guó)近年來流行的優(yōu)勢(shì)型相一致。2.成功構(gòu)建并拯救出重組病毒SDLY107-VP1株,并發(fā)現(xiàn)EV71病毒VP1蛋白區(qū)域存在影響病毒神經(jīng)毒力的氨基酸位點(diǎn),其中第146位(纈氨酸→異亮氨酸)、第147位(纈氨酸→丙氨酸)氨基酸位點(diǎn)的突變影響了病毒對(duì)神經(jīng)細(xì)胞的感染能力和致細(xì)胞損傷能力,突變病毒在神經(jīng)細(xì)胞上的增殖能力和誘導(dǎo)細(xì)胞自噬的水平明顯改變。
[Abstract]:Hand foot and mouth disease (HFMD) is a viral infectious disease caused by a variety of enteroviruses. It mainly infects five years old and below, which can cause fever and herpes and herpes around the hand and mouth of the patient. In serious cases, a series of complications such as aseptic meningitis and neurogenic pulmonary edema can occur, and may even lead to death of the patient. At present, hand foot and mouth disease is widely popular in the world, especially in the Asia Pacific region, and has become an important public health focus. Enterovirus 71 (Enterovirus 71, EV71) belongs to the small RNA virus family, one of the main pathogens causing hand foot and mouth disease, and is also the most important cause of the severe or fatal hand foot and mouth disease. It has become the leading neurotoxic cause of the central nervous system in infants and young children worldwide. At present, the pathogenesis of EV71 in patients with neuropathy is not clear. Studies have shown that there are sites that affect the virulence of the virus in many regions of the virus genome. This study is conducted through specimens of patients with hand foot and mouth disease and close contacts. The virus isolation and sequence analysis were carried out to capture the variation of EV71 in the natural transmission process. 2 virulence loci were screened on VP1, and the reverse genetic technique was used to construct and save the recombinant SDLY107-VP1 strain of VP1 replacement. The role of the VP1 protein virulence candidate loci in the pathogenesis of EV71 was studied. Objective: 1. from the hand foot and mouth disease patients. The EV71 was isolated from the specimens of the close contacts, and the sequence analysis was carried out to capture the variation of EV71 in the natural propagation process and to screen the virulence candidate loci; 2. the VP1 region of the EV71 strains of different virulence was replaced, and the recombinant virus was saved, and 3. compared the replication ability, cell damage and the damage between the parent strain and the recombinant virus. The difference in autophagy, explore the role of the virulence candidate loci in the VP1 area in the pathogenesis of EV71. Methods: 1. the samples were collected from the specimens of hand foot and mouth disease and close contacts in Jining, Shandong Province, in 2015, and the RD cells were used to isolate the virus, and the PCR specific primers were used to identify the virus, and 2. using the EV71 based virus gene. The full sequence amplification primers were designed to amplify and sequence the newly isolated virus in full sequence, and sequence analysis of the complete sequence of the virus genome using DNAstar7.1 and MEGA6 software. 3. designed primers and obtained the VP1 fragment of the weak strain by fusion of PCR, and replaced it to the SDLY107 strain of the virulent strain, and constructed and saved the weight of the virus. The virus SDLY107-VP1 strain was identified by observing cytopathic disease, PCR amplification and indirect immunofluorescence test. The virus titer was measured by 50% end point method and plaque test. 4. the replication ability of SH-SY5Y cells, U87 cells and Vero cells to the recombinant virus and its parent virus caused cell damage. Results: 1. the two EV71 viruses were isolated from the stool specimens of patients with hand, foot and mouth disease and their close contacts, named SDJN2015-01 and SDJN2015-01.1 strains. Sequence and genetic evolution analysis showed that two viruses belong to EV71 C4a subtype, which is the dominant subtype in China in recent years; 2. and two strains in China. There are 6 amino acid mutations in the virus, in which there are 2 amino acid sites on VP1 protein, which are K98E (all coding region K663E) and A133T (A698T), and there are 4 amino acids on 2C protein, D48N (D1159N), V126I (V1237I), I238V (I1349V) and A133T, respectively. The titer was measured; 4. the replication ability of the recombinant virus SDLY107-VP1 strain on U87 and Vero cells, the damage to the cell and the ability to induce autophagy were not distinctly different from that of the SDLY107 and SDJN2015-01 strains, but the recombinant virus SDLY107-VP1 strain lost the cell disease on the SH-SY5Y cell of the neuroblastoma cells. The ability to change the virus is greatly reduced in cell ability to replicate, and does not cause obvious cell damage to SH-SY5Y cells. At the same time, there is no obvious cell autophagy in SH-SY5Y cells, which is obviously different from the SDLY107 strain of the strong strain and similar to the SDJN2015-01 strain of the weak strain. Conclusion: 1. from the hand foot and mouth disease in Jining, Shandong Province in 2015 EV71 was successfully isolated in the children and close contacts, and the newly isolated EV71 strains belonged to the C4a subtype. It was successfully constructed and saved the recombinant virus SDLY107-VP1 strain with the prevailing dominant type in our country in recent years, and found that the EV71 virus VP1 protein region has the amino acid loci that affect the virulence of the virus, 146th of which are valine to different. Leucine (leucine), the mutation of the amino acid loci of 147th sites (valine to alanine) affects the virus's ability to infect the nerve cells and the ability to induce cell damage. The ability of the mutant virus to proliferate on the nerve cells and the level of the induction of autophagy in the cells are obviously changed.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373

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