肠道病毒71型VP1蛋白毒力候选位点的筛选及其对病毒复制能力的影响

发布时间：2018-05-26 10:12

本文选题：肠道病毒71型 + 遗传进化分析　；参考：《山东大学》2017年硕士论文

【摘要】：手足口病是由多种肠道病毒引起的病毒性传染病,主要感染五岁及以下婴幼儿,可导致患者出现发热以及手足口周的疱疹等临床症状,严重时可出现无菌性脑膜炎以及神经源性肺水肿等一系列神经系统的并发症,甚至可以导致患者死亡。目前手足口病在全球尤其是亚太地区广泛流行,已成为重要的公共卫生焦点问题。肠道病毒71型(Enterovirus 71,EV71)属于小RNA病毒科肠道病毒属,是导致手足口病的主要病原体之一,也是目前最主要的导致手足口病重症或死亡的病原体,已成为目前全球范围内最主要的婴幼儿中枢神经毒性病原。目前,EV71导致患者神经系统病变的致病机制尚不明确,有研究显示在病毒基因组多个区域存在有影响病毒神经毒力的位点。本研究通过对手足口病患者以及密切接触者的标本进行病毒分离鉴定和序列分析,捕捉到EV71在自然传播过程中的变异,在VP1上筛选出2个毒力候选位点,利用反向遗传技术构建并拯救VP1置换的重组病毒SDLY107-VP1株,研究VP1蛋白毒力候选位点在EV71致病机制中的作用。目的:1.从手足口病患者以及密切接触者标本中分离EV71,并进行序列分析,捕捉EV71在自然传播过程中的变异,筛选毒力候选位点;2.将两株EV71不同毒力表型株的VP1区进行置换,构建并拯救出重组病毒;3.比较亲本株与重组病毒之间在复制能力、致细胞损伤以及自噬等方面的差异,探究VP1区的毒力候选位点在EV71致病机制中的作用。方法:1.将采集自2015年山东省济宁市手足口病患者及密切接触者的标本处理后使用RD细胞进行病毒分离,并使用PCR特异性鉴定引物进行鉴定;2.使用基于EV71病毒基因组设计的全序列扩增引物对新分离到的病毒进行全序列扩增并测序,对获得的病毒基因组全序列使用DNAstar7.1和MEGA6软件进行序列分析;3.设计引物并通过融合PCR的方法获得弱毒株VP1片段,并将其置换到强毒株SDLY107株中,构建并拯救重组病毒SDLY107-VP1株,通过观察细胞病变、PCR扩增以及间接免疫荧光实验对重组病毒进行鉴定,使用50%终点法以及空斑试验对病毒滴度进行测定;4.使用SH-SY5Y细胞、U87细胞以及Vero细胞对重组病毒及其亲本病毒的复制能力、致细胞损伤作用以及致细胞自噬能力进行研究。结果:1.从手足口病患者及其密切接触者粪便标本中分离到两株EV71病毒,命名为SDJN2015-01株以及SDJN2015-01.1株,序列以及遗传进化分析显示,两株病毒同属于EV71 C4a亚型,是我国近年来流行的优势亚型;2.两株病毒存在6个氨基酸突变,其中VP1蛋白上存在2个氨基酸位点的差异,分别为K98E(全编码区为K663E)和A133T(A698T);2C蛋白上存在4个氨基酸的差异,分别为 D48N(D1159N)、V126I(V1237I)、I238V(I1349V)和 N314Y(N1425Y);3.成功拯救出重组病毒SDLY107-VP1株,并对其滴度进行测定;4.重组病毒SDLY107-VP1株在U87细胞以及Vero细胞上的复制能力、致细胞损伤作用以及引起细胞自噬的能力与SDLY107株和SDJN2015-01株没有明显的区别,但在神经母细胞瘤细胞SH-SY5Y细胞上,重组病毒SDLY107-VP1株失去了引起细胞病变的能力,病毒在细胞能复制能力大大降低,没有导致SH-SY5Y细胞明显的细胞损伤,同时也没有引起SH-SY5Y细胞明显的细胞自噬发生,这与强毒株SDLY107株明显不同,与弱毒株SDJN2015-01株的表现类似。结论:1.从2015年山东省济宁市手足口病患儿及密切接触者中成功分离到EV71,新分离到的EV71毒株属于C4a亚型,与我国近年来流行的优势型相一致。2.成功构建并拯救出重组病毒SDLY107-VP1株,并发现EV71病毒VP1蛋白区域存在影响病毒神经毒力的氨基酸位点,其中第146位(缬氨酸→异亮氨酸)、第147位(缬氨酸→丙氨酸)氨基酸位点的突变影响了病毒对神经细胞的感染能力和致细胞损伤能力,突变病毒在神经细胞上的增殖能力和诱导细胞自噬的水平明显改变。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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