抗病毒治疗对HIV-1准种进化的影响研究

发布时间：2018-05-28 08:46

本文选题：人类免疫缺陷病毒 + 抗病毒治疗　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：背景HIV感染的自然过程人为划分为急性感染期、无症状期以及艾滋病期。HIV急性感染期(Acute HTV infection,AHI)是指从病毒感染到可检测抗体出现的一段时期。HIV感染早期(Early HIV infection,EHI)包括急性感染期,其特征是病毒快速复制、广泛的免疫应答和免疫破坏及病毒多样性增加。另外,急性感染期HIV病毒载量较高却没有明显临床症状,HIV传播风险更高。急性感染期不仅仅是感染的早期阶段,在流行病学上也有重要意义。因此,要加强对急性感染期及早期感染者的监测和管理。男男性行为者(men who have sex with men,MSM)是HIV感染的主要高危人群之一。有研究报道,在急性期感染监测中,男男性行为者占很大比例。急性感染期无典型临床表现加之男男性行为者的行为特点,如性伴复杂、安全套使用率低等,使得HIV感染和艾滋病从高危人群向普通人群传播的可能性大大增加。人类免疫缺陷病毒 Ⅰ 型(human immunodeficiency virus type 1,HIV-1)病毒复制过程中具有极高的变异性。HIV-1感染人体后,变异株在体内大量累积,最终以一群密切相关但又不完全相同的复杂病毒混合种群形式存在,即准种(quasispecies)。准种的变化受宿主内复制的病毒数量所影响,HIV病毒群在个体内会出现异质性以及动态的变化。HIV-1感染者接受抗病毒治疗后,在不同种类和强度药物的选择压力下,病毒、宿主和药物相互影响和作用,HIV-1病毒准种群的组成以及分布也一直处于动态变化的过程之中。探究HIV-1准种群进化受抗病毒治疗的影响,对了解治疗对病毒进化、病毒与宿主免疫作用关系、与抗病毒药物选择关系、分子溯源准确性及疾病进展均有重要意义。急性期感染启动抗病毒治疗,在病毒抑制后可以有较小的病毒储存库,然而,早期感染的前病毒DNA的动力学及准种群变化规律仍然不清楚。本课题组前期成功地将深度测序技术(IlluminaMiseq平台)用于HIV-1暴露后感染溯源调查、准种传播及进化规律的研究,并将通量更高的IlluminaHiseq平台用于丙型肝炎病毒感染溯源调查。为了探讨抗病毒治疗对HIV-1准种群进化规律的影响,本研究旨在优化针对HIV-1衣壳蛋白(gag)、聚合酶(pol)和包膜蛋白(env)基因区片段的IlluminaHiseq深度测序(以下简称Hiseq测序)方法,并尝试用于分析抗病毒治疗急性期感染者的HIV-1准种群变化规律。目的优化针对HIV-1gag、pol、env基因区的Hiseq测序技术并应用于准种群规律分析以探讨HIV-1急性期感染者准种群随抗病毒治疗时间的变化规律。材料和方法1.研究对象在北京佑安医院男男性行为者(menwhohavesexwithmen,MSM)急性期感染者抗病毒治疗队列中选取8人作为研究对象。8人在被诊断为HIV-1急性期感染后立即启动抗病毒治疗,对基线及治疗后第2、4、8、12、24、36、48、72、84、96周的血液样本(用乙二胺四乙酸二钾抗凝剂抗凝)进行随访检测。每次取部分血液样本进行CD4细胞、HIV-1病毒载量等检测,其余的分离外周血单个核细胞(Peripheral Blood Mononuclear Cells,PBMC)和血浆保存备用。2.实验方法从基线血样PBMC样本中提取DNA,进行HIV-1基因亚型分析:按常规方法分别对HIV-1gag、pol、env基因区片段进行PCR扩增,产物经纯化后测序,进行基因亚型分析。优化Hiseq测序目的片段PCR扩增所需引物以及反应条件,选择尽可能覆盖多种基因亚型的引物,如有需要可以设计多套引物。从随访血样PBMC样本中提取DNA,进行Hiseq测序目的片段(400bp左右)扩增,分别以HIV-1衣壳蛋白(gag)基因区(910～1294 nt)、聚合酶(pol)基因区(2610～2997 nt)和包膜蛋白(env)基因区(6984～7360 nt)为目的片段进行巢式聚合酶链反应(PCR)。产物经电泳及紫外成像确认,并在一代测序检验无误后,将扩增产物纯化然后混合建立DNA文库,再进行Hiseq测序。Hiseq测序数据进行初期处理后,分别统计出每条独特准种群序列所出现的频数,探讨HIV-1准种群分布情况;剔除频数50以下及重复序列之后分析HIV-1准种群分布,计算基因离散率,并做系统进化树分析。结果1.病毒载量自治疗第4周起开始有样本无法检出,到第24周后全部无法检出。除样本0087直接于治疗第4周无法检出外,其他样本均呈现出快速下降、继而相对稳定、随后再次快速下降的规律。CD4细胞数在治疗第2、4周时有所下降,随后回升。2.基线血样的HIV-1基因亚型分析结果显示,HIV-1基因亚型为CRF01_AE、CRF07_BC亚型的样本分别有3、5份。3.Hiseq测序用目的片段技术优化,env基因区下游设计为两套引物:V3-D1(TGATGTATTACAATAGAAAAATTCTCCTC)和 V3-D2(TGTATTGCAATAGA AAAATTCCCCTC)。4.8份样本在基线、治疗第2～12周gag、pol、env三基因区均扩增成功出Hiseq测序的目的片段(长度均在400bp左右);随治疗时间延长,扩增成功率降低。第84周仅1份样本(0081)三基因区扩增成功,4份样本(0074、0084、0087、0088)三基因区均扩增失败。第96周仅1份样本(0086)三基因区扩增成功,6份样本(0074、0081、0084、0087、0088、0090)三基因区均扩增失败。5.核酸扩增成功的样本全部成功进行了 Hiseq测序,每个目的基因区片段都获得了十万数量级的序列数。6.在基线点,频数最高(最优势)的第一个准种群序列的频数占全部序列频数的比例均高于50%。抗病毒治疗第2～72周,准种群的分布发生不同程度的变化,其中gag、pol基因区频数第一准种群频数所占比例先减小,从第12周起多数增大,而后维持,个别样本于治疗第48周或第72周再次减小。多数样本env基因区第12周以前变动幅度较小,12周以后变化幅度较大,且规律不一。7.样本内平均基因离散率整体稳定,小范围波动。8.各随访时间点与基线点之间的平均基因离散率在前2周增大,随后保持稳定。9.分别选取每份样本三基因区基线及治疗后不同时间点随访样本中频数最高的前20、50及100个准种群序列做系统进化树分析,不同数量准种群序列所显示的规律一致。系统进化树显示,同一 HIV-1感染者不同时间点的准种群序列散在分布且距离接近。结论1.针对HIV-1gag、pol和env基因区的Hiseq测序方法可有效用于分析HIV-1准种群变化规律。2.急性期抗病毒治疗能降低HIV-1病毒载量调定点且延长准种群分散时间。3.在HIV-1病毒载量无法检出时,通过提取前病毒DNA可以获得有效的核酸信息,用于准种群变化规律研究。
[Abstract]:The natural process of background HIV infection is divided into acute infection period, asymptomatic phase and.HIV acute infection period of AIDS (Acute HTV infection, AHI), which refers to the early stage of.HIV infection (Early HIV infection, EHI) from virus infection to detectable antibody (Early HIV infection, EHI) including rapid infection period, which is characterized by rapid replication of the virus. The immune response, immune damage and viral diversity are increased. In addition, there are no obvious clinical symptoms and higher risk of HIV transmission in acute infection period of HIV virus. Acute infection period is not only an early stage of infection, but also important in epidemiology. Therefore, the monitoring and management of acute infection and early infection should be strengthened. Male male actor (men who have sex with men, MSM) is one of the major high-risk groups of HIV infection. Studies have reported that male male actors are in a large proportion in acute phase infection monitoring. There is no typical clinical manifestation in acute infection and the behavior point of male male actors, such as complicated sex companion, low use rate of condom, so HIV The possibility of infection and HIV transmission from high risk population to the general population is greatly increased. Human immunodeficiency virus type I (human immunodeficiency virus type 1, HIV-1) virus replication process with high variability.HIV-1 infection of the human body, the mutant strain in the body, eventually in a group of closely related but incomplete phase The mixed population of the same complex virus exists, that is, the quasi species (quasispecies). The variation of the quasi species is affected by the number of viruses replicating in the host. The HIV virus group is heterogeneous and dynamic in the individual. After the.HIV-1 infection is treated with antiviral therapy, the virus, host and drug under the pressure of different types and intensity drugs, viruses, hosts and drugs. The composition and distribution of HIV-1 virus quasi population have always been in the process of dynamic change. To explore the effects of HIV-1 quasi population evolution on virus evolution, the relationship between virus and host immunity, the selection of antiviral drugs, the accuracy of molecular traceability and the progress of disease. It is of great significance. Acute phase infection starts antiviral therapy and can have a smaller virus store after virus suppression. However, the kinetics of the early infection of the virus DNA and the law of quasi population change are still unclear. This group has successfully applied the deep sequencing technology (IlluminaMiseq platform) to the tracing investigation of infection after HIV-1 exposure. In order to explore the effect of antiviral therapy on the evolution of HIV-1 quasi population, the aim of this study was to optimize the IlluminaHiseq of HIV-1 capsid protein (GAG), polymerase (POL) and envelope protein (Env) gene region fragments in order to investigate the effect of antiviral therapy on the evolution of the quasi population of hepatitis C virus (HCV). The method of deep sequencing (hereinafter referred to as Hiseq sequencing) was used to analyze the HIV-1 quasi population changes in the patients with acute infection of antiviral therapy. Aim to optimize the Hiseq sequencing technology for HIV-1gag, pol, env gene region and apply to the quasi population rule analysis to explore the changes of the quasi population with the antiviral treatment time in the acute phase of HIV-1 infection. Rules. Materials and methods 1. subjects were selected as subjects in the antiviral treatment cohort of menwhohavesexwithmen (MSM) in the menwhohavesexwithmen, MSM. The.8 people began to start the antiviral treatment immediately after the diagnosis of acute HIV-1 infection, and the baseline and after 2,4,8,12,24,36,48,72,84,96 week after treatment. Blood samples were followed up with two potassium anticoagulant anticoagulants. Some blood samples were taken for CD4 cells, HIV-1 viral load, and the rest of the isolated peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cells, PBMC) and plasma preservation reserve.2. test methods from the baseline blood sample PBMC samples extracted DNA, Analysis of the subtype of HIV-1 gene: PCR amplification of HIV-1gag, pol, env gene region fragments according to conventional methods. The products are sequenced and sequenced, and the gene subtype analysis is carried out. The primers and the reaction conditions for PCR amplification in Hiseq sequencing are optimized and the primers to cover a variety of gene subtypes as much as possible are selected as possible. If necessary, many sets of primers can be designed. Primers. DNA was extracted from blood sample PBMC samples and amplified by Hiseq sequencing target fragments (about 400bp), respectively using HIV-1 capsid protein (GAG) gene region (910~1294 NT), polymerase (POL) gene region (2610~2997 NT) and envelope protein (Env) gene region (6984~7360 NT) for nested polymerase chain reaction (PCR). After swimming and ultraviolet imaging confirmed, after a generation of sequencing tests, the amplified products were purified and mixed to establish DNA library, and then Hiseq sequencing.Hiseq sequencing data were carried out for initial processing. The frequency of each unique quasi population sequence was calculated, and the distribution of HIV-1 quasi population was discussed, and the frequency of frequency 50 and repeated sequence were eliminated. Then we analyzed the distribution of HIV-1 quasi population, calculated the rate of gene dispersion, and did the phylogenetic tree analysis. Results 1. virus load was not detected since fourth weeks, and all could not be detected after twenty-fourth weeks. Except the sample 0087 was directly not detected for fourth weeks, the other samples showed a rapid decline, then relatively stable, and then again. The number of.CD4 cells with rapid descent decreased at the time of the treatment of week 2,4, and then the HIV-1 subtype analysis of.2. baseline blood samples showed that the HIV-1 gene subtype was CRF01_AE, and the CRF07_BC subtype samples were optimized with 3,5 portions of.3.Hiseq sequencing, and the downstream of the env gene region was designed to be two sets of primers: V3-D1 (TGATGTA) TTACAATAGAAAAATTCTCCTC) and V3-D2 (TGTATTGCAATAGA AAAATTCCCCTC).4.8 samples were in the baseline, the target fragment of Hiseq sequencing was amplified successfully from second to 12 weeks gag, pol, env three gene region (length is around 400bp); with the prolonged treatment time, the amplification success rate decreased. Eighty-fourth weeks only 1 samples (0081) three gene region expanded successfully, 4 samples. The amplification of this (0074008400870088) three gene region was unsuccessful. Only 1 samples (0086) three gene regions were amplified successfully in ninety-sixth weeks, and all the samples of 6 samples (007400810084008700880090) three gene region were all successfully amplified successfully by Hiseq sequencing, and each target gene fragment was sequenced with an one hundred thousand order of magnitude. The frequency of the first quasi population sequence of the highest frequency number (the most dominant) at the baseline.6. was higher than that of the 50%. antiviral therapy for second to 72 weeks, and the distribution of the quasi population occurred in different degrees, in which the frequency of the first quasi population of the gag, the pol region frequency decreased first, and the majority increased from the twelfth week. Maintenance, the individual samples decreased again at forty-eighth or seventy-second weeks. Most of the samples of env gene region changed slightly before twelfth weeks, and the change range was larger after 12 weeks, and the average gene dispersion rate in the.7. sample was stable, and the average gene dispersion rate between the follow-up time points and the baseline points in the small range of.8. was increased in the first 2 weeks. And then keep stable.9. to select the baseline of three gene region of each sample and the first 20,50 and 100 quasi population sequences in the follow-up samples at different time points after treatment, and make the phylogenetic tree analysis. The rules of the different number of quasi population sequences show the same rules. The population sequence is scattered and the distance is close. Conclusion the Hiseq sequencing method for HIV-1gag, pol and env gene regions can be effectively used to analyze the quasi population variation of HIV-1.2. in the acute phase of.2., which can reduce the HIV-1 virus load regulation and prolong the quasi population dispersion time.3. when the HIV-1 virus load can not be detected, by extracting the predisease. Virus DNA can obtain effective nucleic acid information for the study of quasispecies variation.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

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