本文選題：人類免疫缺陷病毒 + 抗病毒治療�。� 參考：《中國疾病預防控制中心》2017年碩士論文

【摘要】：背景HIV感染的自然過程人為劃分為急性感染期、無癥狀期以及艾滋病期。HIV急性感染期(Acute HTV infection,AHI)是指從病毒感染到可檢測抗體出現(xiàn)的一段時期。HIV感染早期(Early HIV infection,EHI)包括急性感染期,其特征是病毒快速復制、廣泛的免疫應答和免疫破壞及病毒多樣性增加。另外,急性感染期HIV病毒載量較高卻沒有明顯臨床癥狀,HIV傳播風險更高。急性感染期不僅僅是感染的早期階段,在流行病學上也有重要意義。因此,要加強對急性感染期及早期感染者的監(jiān)測和管理。男男性行為者(men who have sex with men,MSM)是HIV感染的主要高危人群之一。有研究報道,在急性期感染監(jiān)測中,男男性行為者占很大比例。急性感染期無典型臨床表現(xiàn)加之男男性行為者的行為特點,如性伴復雜、安全套使用率低等,使得HIV感染和艾滋病從高危人群向普通人群傳播的可能性大大增加。人類免疫缺陷病毒 Ⅰ 型(human immunodeficiency virus type 1,HIV-1)病毒復制過程中具有極高的變異性。HIV-1感染人體后,變異株在體內(nèi)大量累積,最終以一群密切相關但又不完全相同的復雜病毒混合種群形式存在,即準種(quasispecies)。準種的變化受宿主內(nèi)復制的病毒數(shù)量所影響,HIV病毒群在個體內(nèi)會出現(xiàn)異質性以及動態(tài)的變化。HIV-1感染者接受抗病毒治療后,在不同種類和強度藥物的選擇壓力下,病毒、宿主和藥物相互影響和作用,HIV-1病毒準種群的組成以及分布也一直處于動態(tài)變化的過程之中。探究HIV-1準種群進化受抗病毒治療的影響,對了解治療對病毒進化、病毒與宿主免疫作用關系、與抗病毒藥物選擇關系、分子溯源準確性及疾病進展均有重要意義。急性期感染啟動抗病毒治療,在病毒抑制后可以有較小的病毒儲存庫,然而,早期感染的前病毒DNA的動力學及準種群變化規(guī)律仍然不清楚。本課題組前期成功地將深度測序技術(IlluminaMiseq平臺)用于HIV-1暴露后感染溯源調查、準種傳播及進化規(guī)律的研究,并將通量更高的IlluminaHiseq平臺用于丙型肝炎病毒感染溯源調查。為了探討抗病毒治療對HIV-1準種群進化規(guī)律的影響,本研究旨在優(yōu)化針對HIV-1衣殼蛋白(gag)、聚合酶(pol)和包膜蛋白(env)基因區(qū)片段的IlluminaHiseq深度測序(以下簡稱Hiseq測序)方法,并嘗試用于分析抗病毒治療急性期感染者的HIV-1準種群變化規(guī)律。目的優(yōu)化針對HIV-1gag、pol、env基因區(qū)的Hiseq測序技術并應用于準種群規(guī)律分析以探討HIV-1急性期感染者準種群隨抗病毒治療時間的變化規(guī)律。材料和方法1.研究對象在北京佑安醫(yī)院男男性行為者(menwhohavesexwithmen,MSM)急性期感染者抗病毒治療隊列中選取8人作為研究對象。8人在被診斷為HIV-1急性期感染后立即啟動抗病毒治療,對基線及治療后第2、4、8、12、24、36、48、72、84、96周的血液樣本(用乙二胺四乙酸二鉀抗凝劑抗凝)進行隨訪檢測。每次取部分血液樣本進行CD4細胞、HIV-1病毒載量等檢測,其余的分離外周血單個核細胞(Peripheral Blood Mononuclear Cells,PBMC)和血漿保存?zhèn)溆谩?.實驗方法從基線血樣PBMC樣本中提取DNA,進行HIV-1基因亞型分析:按常規(guī)方法分別對HIV-1gag、pol、env基因區(qū)片段進行PCR擴增,產(chǎn)物經(jīng)純化后測序,進行基因亞型分析。優(yōu)化Hiseq測序目的片段PCR擴增所需引物以及反應條件,選擇盡可能覆蓋多種基因亞型的引物,如有需要可以設計多套引物。從隨訪血樣PBMC樣本中提取DNA,進行Hiseq測序目的片段(400bp左右)擴增,分別以HIV-1衣殼蛋白(gag)基因區(qū)(910～1294 nt)、聚合酶(pol)基因區(qū)(2610～2997 nt)和包膜蛋白(env)基因區(qū)(6984～7360 nt)為目的片段進行巢式聚合酶鏈反應(PCR)。產(chǎn)物經(jīng)電泳及紫外成像確認,并在一代測序檢驗無誤后,將擴增產(chǎn)物純化然后混合建立DNA文庫,再進行Hiseq測序。Hiseq測序數(shù)據(jù)進行初期處理后,分別統(tǒng)計出每條獨特準種群序列所出現(xiàn)的頻數(shù),探討HIV-1準種群分布情況;剔除頻數(shù)50以下及重復序列之后分析HIV-1準種群分布,計算基因離散率,并做系統(tǒng)進化樹分析。結果1.病毒載量自治療第4周起開始有樣本無法檢出,到第24周后全部無法檢出。除樣本0087直接于治療第4周無法檢出外,其他樣本均呈現(xiàn)出快速下降、繼而相對穩(wěn)定、隨后再次快速下降的規(guī)律。CD4細胞數(shù)在治療第2、4周時有所下降,隨后回升。2.基線血樣的HIV-1基因亞型分析結果顯示,HIV-1基因亞型為CRF01_AE、CRF07_BC亞型的樣本分別有3、5份。3.Hiseq測序用目的片段技術優(yōu)化,env基因區(qū)下游設計為兩套引物:V3-D1(TGATGTATTACAATAGAAAAATTCTCCTC)和 V3-D2(TGTATTGCAATAGA AAAATTCCCCTC)。4.8份樣本在基線、治療第2～12周gag、pol、env三基因區(qū)均擴增成功出Hiseq測序的目的片段(長度均在400bp左右);隨治療時間延長,擴增成功率降低。第84周僅1份樣本(0081)三基因區(qū)擴增成功,4份樣本(0074、0084、0087、0088)三基因區(qū)均擴增失敗。第96周僅1份樣本(0086)三基因區(qū)擴增成功,6份樣本(0074、0081、0084、0087、0088、0090)三基因區(qū)均擴增失敗。5.核酸擴增成功的樣本全部成功進行了 Hiseq測序,每個目的基因區(qū)片段都獲得了十萬數(shù)量級的序列數(shù)。6.在基線點,頻數(shù)最高(最優(yōu)勢)的第一個準種群序列的頻數(shù)占全部序列頻數(shù)的比例均高于50%�？共《局委煹�2～72周,準種群的分布發(fā)生不同程度的變化,其中gag、pol基因區(qū)頻數(shù)第一準種群頻數(shù)所占比例先減小,從第12周起多數(shù)增大,而后維持,個別樣本于治療第48周或第72周再次減小。多數(shù)樣本env基因區(qū)第12周以前變動幅度較小,12周以后變化幅度較大,且規(guī)律不一。7.樣本內(nèi)平均基因離散率整體穩(wěn)定,小范圍波動。8.各隨訪時間點與基線點之間的平均基因離散率在前2周增大,隨后保持穩(wěn)定。9.分別選取每份樣本三基因區(qū)基線及治療后不同時間點隨訪樣本中頻數(shù)最高的前20、50及100個準種群序列做系統(tǒng)進化樹分析,不同數(shù)量準種群序列所顯示的規(guī)律一致。系統(tǒng)進化樹顯示,同一 HIV-1感染者不同時間點的準種群序列散在分布且距離接近。結論1.針對HIV-1gag、pol和env基因區(qū)的Hiseq測序方法可有效用于分析HIV-1準種群變化規(guī)律。2.急性期抗病毒治療能降低HIV-1病毒載量調定點且延長準種群分散時間。3.在HIV-1病毒載量無法檢出時,通過提取前病毒DNA可以獲得有效的核酸信息,用于準種群變化規(guī)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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.91

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