HCV准种变异特性及其免疫逃逸机制初步研究

发布时间：2018-03-20 00:30

本文选题：丙型肝炎病毒　切入点：准种　出处：《昆明理工大学》2011年硕士论文　论文类型：学位论文

【摘要】：丙型肝炎病毒(Hepatitis C virus, HCV)为单股正链RNA病毒,属于黄病毒科,肝炎病毒属,基因组全长约9.6kb。HCV在复制过程中,由于RNA依赖的RNA聚合酶缺乏校对功能,导致HCV高度变异。病毒在宿主体内存在的多种变异株(同源性95%)称为准种。感染者体内存在的HCV准种反映在特定环境下以病毒复制、变异为基础的突变株竞争性选择的结果。准种的复杂情况与疾病进展、病程转归密切相关。病毒CTL抗原表位编码基因发生变异产生的HCV准种,是病毒逃避宿主免疫监控及药物治疗不反应的主要原因。本研究首先根据HCV病毒载量等临床指标,从50例候选者中筛选出16名HCV单独感染者,经5NCR区和NS5B区基因扩增及核酸序列分析,确定研究入选者感染HCV为4种基因型/亚型,分别为1b、3a、3b和6n,以1b(41.25%)和3b(36.75%)型为主。16名入选者均接受不同程度的药物治疗,经长期(1.5年)病程跟踪和临床指标检测发现,8例入选者继续维持慢性感染,另外8例病程转归,病毒RNA检测为阴性。进而建立了E2区和NS5A区准种检测方法-—多克隆测序分析法,以准种Neighbor-joining进化树、标准化熵值、平均遗传距离和dN/dS值等指标评价准种特性与变化情况。研究发现,慢性感染者和治疗病程转归者在入选初期,体内E2区准种平均遗传距离波动分别为0.0097～0.0625和0.0037～0.0269,熵值波动分别为0.9063～0.9786和0.5769～0.8849；NS5A区准种平均遗传距离波动分别为0.0050～0.0113和0.0022～0.0095,熵值波动分别在0.8847～0.9734和0.5187～0.8007。两个区段HCV准种的平均遗传距离和熵值,慢性感染者均大于治疗病程转归者,表明HCV准种异质性程度越低的患者,更易于产生治疗应答。对入选初期E2区和NS5A区准种dN/dS比值分析发现,所有慢性感染者和6例病程转归者准种dN/dS比值都小于1,其体内准种变化主要由遗传漂变引起。在病程转归者中,有2例患者dN/dS比值大于1,其准种变异是由达尔文进化压力造成的。进而,研究对入选者病程跟踪,多个时间点采集的样本,使用BEAST软件估算患者体内HCV准种的起源时间和进化速率,构建准种群体数量变化的BSP图,并对HCVE2区准种变化指标(多样性和复杂性)与临床指标(ALT值和AST值)相关性进行分析。发现8名慢性感染者随着病程进展,E2区和NS5A区准种存在渐变和彻变两种进化模式,这两个基因区段准种变异呈现出协同进化的特点,即在同一个病人体内E2区和NS5A区段进化模式相同。治疗病程转归者HCV准种进化速率(5.84×10-3 sub/site/month)明显高于持续感染者(0.61×10-3～1.31×10-3sub/site/month)。HCV E2区准种的多样性和复杂性与血清ALT、AST值之间无显著相关性(p0.05)。最后,研究采用Elispot方法对一例慢性感染者(KM10)病程进展过程中,不同时间采集的PBMC中效应性CTL数量进行了测定。经HCV主要准种氨基酸序列分析发现,该慢性感染者在入选观察初期(T1期)病毒E2区CTL抗原表位(LLFTPGSKQNV)随着病程进展(T4期)1年内发生了较大变化(SFFTSGPKQNI),利用合成的T1期主要准种抗原多肽(NH2-SLLFTPGSKQNVQ-COOH),分别刺激T1和T4期采集的PBMC细胞,发现此抗原多肽的效应性CTL在观察初期(T1期)为307个,病程后期(T4期)只有21个,由于抗原表位编码基因变异,CTL明显不能免疫识别。本研究对经过治疗的HCV单独感染者进行随访研究,使用多克隆测序法分析HCV准种变异规律,并建立了Elispot方法分析HCV免疫逃逸,初步分析了CTL抗原表位变异的免疫逃逸能力及其对病程进展的影响,为丙型肝炎慢性化致病机制及疫苗研制等方面的研究提供直接依据。
[Abstract]:Hepatitis C virus (Hepatitis C, virus, HCV) is a single stranded RNA virus belonging to the Flaviviridae family, hepatitis virus, genome 9.6kb.HCV during replication, the RNA dependent RNA polymerase lacks proofreading function, resulting in HCV height variation. Different variants of the virus in the host body (95% homology) called quasispecies. Infection in the presence of HCV quasispecies is reflected in the specific environment for virus replication, mutation based mutant competitive selection results. With the development of complex quasi disease species, disease outcome closely related. CTL virus epitope encoding gene mutation of HCV quasispecies. The main reason is the virus to escape the host immune response is not monitoring and treatment.
Firstly, according to the HCV virus load and other clinical indicators, 50 cases from the candidates identified 16 patients infected with HCV alone, by the analysis of 5NCR and NS5B region gene amplification and DNA sequencing, identified as 4 genotypes / subtypes of HCV infection were enrolled in this study, respectively 1b, 3a, 3b and 6N. 1B (41.25%) and 3b (36.75%) type.16 patients received drug treatment in different degree, the long-term (1.5 years) to detect disease tracking and clinical indicators, 8 patients were maintained in 8 cases of chronic infection, the outcome of the diseases, viral RNA detection was negative. Then set up E2 and NS5A area quasispecies detection method of multiple cloning and sequencing analysis, the phylogenetic tree of Neighbor-joining quasispecies, standard entropy, average genetic distance and the dN/dS value evaluation of quasispecies characteristics and changes. The study found that the outcome of chronic infection and treatment duration in selected early in E2 area The average genetic distance of volatility was 0.0097 ~ 0.0625 and 0.0037 ~ 0.0269, the entropy volatility was 0.9063 ~ 0.9786 and 0.5769 ~ 0.8849; the average genetic distance of NS5A quasispecies fluctuations were 0.0050 ~ 0.0113 and 0.0022 ~ 0.0095, respectively in 0.8847 the average genetic entropy fluctuation of ~ 0.9734 and 0.5187 ~ two 0.8007. section HCV quasispecies the distance and entropy for chronic infection were higher than the treatment outcome of the diseases, showed that the HCV quasispecies heterogeneity is lower in patients are more prone to the response to treatment. The selected initial E2 and NS5A area quasispecies dN/dS ratio analysis showed that the outcome of all chronic infection and 6 cases of course of disease is less than the ratio of dN/dS quasispecies 1, the body is mainly composed of quasispecies changes caused by genetic drift. In the course of the disease outcome in 2 patients with dN/dS ratio greater than 1, the quasispecies variation is caused by Darwin's evolutionary pressure.
Then, the research on the selected course tracking, multiple time samples, the estimated HCV in the patients with quasi origin time and the evolution rate for the use of BEAST software, construction of quasispecies changes in the number of BSP, and a change of index of HCVE2 region (diversity and complexity) and clinical parameters (ALT value and AST) correlation analysis. The discovery of 8 chronic infection with the progression of the disease, which are gradual and fundamentally changed the two patterns of evolution of E2 and NS5A, the two gene quasispecies variation showed the characteristics of co evolution, i.e. in the same patient E2 and NS5A section of the same evolutionary pattern the clinical course of HCV treatment. Quasispecies evolution rate (5.84 x 10-3 sub/site/month) was significantly higher than that of infection (0.61 * 10-3 ~ 1.31 * 10-3sub/site/month).HCV E2 quasispecies complexity and diversity, and serum ALT, no significant correlation between AST values (p0.0 5).
Finally, Elispot method was used on a case of chronic infection (KM10) course, the number of effector CTL PBMC collected in different time were determined by HCV. The main quasispecies amino acid sequence analysis showed that the chronic infection in selected initial observation (T1) virus E2 CTL epitope (LLFTPGSKQNV) with disease progression (T4) changed greatly within 1 years (SFFTSGPKQNI), using T1 synthesis mainly quasispecies antigenic peptides (NH2-SLLFTPGSKQNVQ-COOH), T1 and T4 were used to stimulate acquisition of PBMC cells, found the antigen peptide effector CTL in the observation of early (T1) 307 a later stage (stage T4), only 21, due to the epitope encoding gene mutation, CTL was not immune recognition.
The research on the follow-up study after treatment of HCV infection alone, analysis of HCV quasispecies variation using multi clone sequencing method, and Elispot method was established for analysis of HCV immune escape, a preliminary analysis of CTL epitope variation in immune escape ability and its influence on disease progression, provide direct basis for the study of pathogenic mechanism of hepatitis C hepatitis B vaccine development and other aspects.

【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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