發(fā)布時間：2018-02-28 09:01

  本文關(guān)鍵詞： HIV 耐藥相關(guān)突變位點 多態(tài)性位點 適應(yīng)性　出處：《中國疾病預(yù)防控制中心》2015年博士論文　論文類型：學(xué)位論文

【摘要】：HIV目前在世界各地廣泛傳播,抗病毒治療的開展可以有效的提高患者的生命質(zhì)量,延長生存壽命,并能達到減少艾滋病傳播的目的。但是HIV耐藥的出現(xiàn)卻嚴(yán)重影響了抗病毒治療的效果。HIV耐藥的產(chǎn)生是一個極其復(fù)雜的動態(tài)過程,這一過程除了包含多種環(huán)境因素外,還與病毒基因相關(guān),是多個位點共同作用的結(jié)果。參與耐藥發(fā)生的病毒基因位點包括耐藥相關(guān)突變位點和多態(tài)性位點,它們通過改變病毒的復(fù)制能力,進而達到調(diào)整HIV在患者體內(nèi)的準(zhǔn)種分布,實現(xiàn)病毒最優(yōu)適應(yīng)性的目的。大多數(shù)耐藥相關(guān)突變位點可以降低病毒的適應(yīng)性,病毒在進化過程中可能會產(chǎn)生協(xié)同突變的多態(tài)性位點,補償耐藥位點所造成的病毒適應(yīng)性下降,但是這一說法還缺少實驗證據(jù)。本研究通過對我國CRF01_AE亞型、CRF07_BC亞型和B’亞型的HIV序列分析,從生物信息學(xué)的角度證明了多態(tài)性位點與耐藥相關(guān)突變位點的協(xié)同突變現(xiàn)象。進一步通過體外實驗驗證這些耐藥相關(guān)特征性突變位點對于病毒適應(yīng)性的影響,并對其影響適應(yīng)性的機制進行初步的探索。該研究對于理解耐藥發(fā)生的分子基礎(chǔ)有重要意義,而且對于改善抗病毒治療方案,實現(xiàn)對HIV的有效防控提供了重要的基礎(chǔ)數(shù)據(jù)。一、北京市朝陽區(qū)2011-2013年男男性行為人群HIV感染情況及耐藥基因特征分析本部分通過對北京市朝陽區(qū)MSM中未治療HIV感染者的病毒pol基因分析,獲得了該人群的基因亞型分布情況及耐藥基因特征,進一步通過比較攜帶耐藥相關(guān)突變位點的HIV基因序列與不攜帶耐藥相關(guān)突變位點的HIV基因序列,獲得了未治療CRF01_AE亞型中耐藥相關(guān)突變位點與多態(tài)性位點的協(xié)同突變現(xiàn)象。研究結(jié)果顯示,北京市朝陽區(qū)MSM處于HIV高流行水平,流行的主要基因亞型依次為CRF01_AE亞型、CRF07_BC亞型和B亞型。該部分的研究人群為未治療的HIV感染者,其中有25.56%的HIV感染者出現(xiàn)了至少一種相關(guān)耐藥突變位點。該人群中CRF01_AE亞型中,耐藥相關(guān)突變位點V179D/E與7個多態(tài)性位點(R238K、A272P、T11K、I173K、K174Q、S207Q、S211K)之間具有協(xié)同突變關(guān)系。二、CRF07_BC亞型和B,亞型耐藥相關(guān)突變位點與多態(tài)性位點的協(xié)同突變分析本部分利用R語言技術(shù)及相關(guān)的軟件包CorMut,對CRF07_BC亞型和B’亞型的序列進行分析,獲得這兩種基因亞型中耐藥相關(guān)突變位點與多態(tài)性位點的協(xié)同突變現(xiàn)象。本部分通過比較新疆、四川等地的CRF07 BC亞型中600例未治療的HIV-1感染病人和344例接受治療的HIV-1感染病人序列,分析獲得了該亞型治療組中RT區(qū)耐藥相關(guān)突變位點(K103N、M184V、Q197K、G190A、Y181C、L228R、M230L)和多態(tài)性位點(A36E、R135I、R277K、L283I、D291E)具有協(xié)同突變關(guān)系,PR區(qū)耐藥相關(guān)突變位點(L10I)和多態(tài)性位點(I132L)之間具有協(xié)同突變關(guān)系；對600例未治療的HIV-1感染病人進行分析,獲得了未治療組中RT區(qū)耐藥相關(guān)突變位點(Y188C、K103N、E138G、V108I、L74F)和多態(tài)性位點(M16K、K166R、E248V、L283I、E297A、T200M)具有協(xié)同突變關(guān)系,PR區(qū)耐藥相關(guān)突變位點(L10I)和多態(tài)性位點(I64V、A71V)具有協(xié)同突變關(guān)系。本部分通過比較河南、安徽兩地的B’亞型中178例未治療的HIV-1感染病人和327例接受治療的HIV-1感染病人序列,分析獲得了該亞型治療組中RT區(qū)耐藥相關(guān)突變位點(Y181C、L210W、M41L、E44A、E203D、H208Y、T215Y、L228R、K65R、Q151M、V75T)和多態(tài)性位點(S162C、Q207E、R211K、L214F、I293V)之間具有協(xié)同突變關(guān)系；對178例未治療的HIV-1感染病人進行分析,獲得了未治療組中RT區(qū)耐藥相關(guān)突變位點(V106I、M41L、L210W、T215Y、H221Y)與多態(tài)性位點(I135V、S68G、I178L、R277K)具有協(xié)同突變關(guān)系。三、HIV-1耐藥相關(guān)特征性突變位點對病毒復(fù)制適應(yīng)性的影響本部分研究中,我們首次建立了HIV-1 CRF07_BC亞型的體外生長競爭實驗體系,結(jié)合HIV-1 B亞型體外生長競爭實驗體系,通過體外實驗,研究分析第二部分所發(fā)現(xiàn)的CRF07_BC亞型和B’亞型中耐藥相關(guān)特征性突變位點對于病毒適應(yīng)性的影響,并通過檢測P51蛋白相對含量、逆轉(zhuǎn)錄酶活性和蛋白質(zhì)三級結(jié)構(gòu)預(yù)測的方法,對其改變病毒適應(yīng)性的機制進行初步的探討。對于HIV-1 B'亞型未治療組中所發(fā)現(xiàn)的協(xié)同突變位點進行研究發(fā)現(xiàn),HIV-1RT區(qū)T215Y+I178L協(xié)同突變的重組病毒其適應(yīng)性明顯高于T215Y單點突變的重組病毒適應(yīng)性,而且與之相比P51蛋白相對含量和逆轉(zhuǎn)錄酶活性也明顯上升。這提示1178L可能通過影響P51蛋白相對含量及逆轉(zhuǎn)錄酶活性的方式補償耐藥相關(guān)突變位點T215Y所造成的病毒適應(yīng)性下降。對于HIV-1 B'亞型治療組中所發(fā)現(xiàn)的協(xié)同突變位點進行研究發(fā)現(xiàn),在無藥環(huán)境中,HIV-1 RT區(qū)T215Y+R211K協(xié)同突變的重組病毒其適應(yīng)性明顯高于T215Y單點突變的重組病毒適應(yīng)性；在AZT存在的情況下,T215Y+R211K協(xié)同突變的重組病毒的適應(yīng)性要高于野毒株；這提示在治療患者體內(nèi),R211K的出現(xiàn),可以使病毒適應(yīng)性上升,這樣的現(xiàn)象可能會加速耐藥發(fā)展的進程。
[Abstract]:HIV is currently widely spread all over the world, the development of antiviral therapy can effectively improve the quality of life of patients, prolong the survival life, and can achieve the purpose of reducing the spread of AIDS. But HIV resistance has seriously affected the effect of antiviral therapy resistant.HIV production is a complicated dynamic process, this process in addition to contain a variety of environmental factors, but also related with viral gene, multiple sites is the result of joint action. Viral genes involved in resistance loci including resistance related mutations and polymorphisms, their ability to change through the replication of the virus, and then adjust the HIV quasispecies distribution in the patient's body, to achieve the optimal adaptation of the virus purpose. Most resistance related mutations can reduce the virus virus adaptability, may produce polymorphic loci mutation in the evolutionary process of collaborative, Drop compensation resistance loci caused by virus adaptation, but this argument is the lack of experimental evidence. Through the study on China's CRF01_AE subtype, sequence analysis of HIV CRF07_BC subtype and B subtype ", proved that the polymorphic loci associated with resistance mutation sites collaborative phenomenon from the perspective of bioinformatics further. In vitro experiments verify the characteristics of resistance related mutations in virus affecting the adaptability, and its influence on the adaptability of the mechanism was preliminarily explored. The research has an important significance for understanding the molecular basis of drug resistance, but also for improving the anti viral treatment programs provide important basic data to achieve effective prevention and control of HIV. 1. The analysis of Beijing city Chaoyang District 2011-2013 years of MSM HIV infection and drug resistance characteristics through this part of the untreated HIV Beijing city in Chaoyang District MSM Analysis of virus pol gene infection, the distribution of the population genetic subtype and drug resistance gene characteristics, further by comparing the carrying drug-resistant mutations in HIV gene sequence loci associated with drug-resistant mutations do not carry the HIV gene sequences obtained in untreated collaborative resistant CRF01_AE subtype mutation related mutation and polymorphism the site of Beijing city. The results showed that the Chaoyang District MSM HIV in high prevalence, major gene prevalent subtypes were CRF01_AE subtype, CRF07_BC subtype and B subtype. The study population is not part of the treatment of HIV infection, including 25.56% HIV infected patients had at least one related drug resistance mutations in this population. CRF01_AE subtypes, resistance related mutations V179D/E and 7 polymorphic loci (R238K, A272P, T11K, I173K, K174Q, S207Q, S211K) have a synergistic relationship with mutation. Two, CRF07_BC subtype and B subtype resistance related mutations and polymorphisms of the cooperative mutation analysis of this part of the use of R language technology and related software CorMut sequences of CRF07_BC subtype and B subtype analysis, cooperative mutation phenomenon received the two kinds of genotypes of resistance related mutations loci and polymorphic loci. This part compares Xinjiang, Sichuan and other places of CRF07 BC subtype in patients with sequence and 344 patients received the treatment of HIV-1 infection in 600 cases of infected untreated HIV-1 was obtained RT subtype in the treatment group, the drug resistance related mutations (K103N, M184V, Q197K, G190A Y181C, L228R, M230L), and polymorphic loci (A36E, R135I, R277K, L283I, D291E) has a synergistic relationship between the mutation and PR resistance associated mutation region (L10I) and polymorphic loci (I132L) has a synergistic relationship between mutations; 600 cases of untreated HIV-1 infection Carries on the analysis, obtained from untreated RT resistant mutations were in the group (Y188C, K103N, E138G, V108I, L74F) and polymorphic loci (M16K, K166R, E248V, L283I, E297A, T200M) has a synergistic relationship between the mutation and PR resistance associated mutation region (L10I) and polymorphic loci (I64V A71V) has a synergistic mutation relationship. This part compares Henan, Anhui two B subtypes in patients' sequence and 327 patients received the treatment of HIV-1 infection in 178 cases of infected untreated HIV-1 was obtained RT subtype in the treatment group, the drug resistance related mutations (Y181C, L210W, M41L, E44A. E203D, H208Y, T215Y, L228R, K65R, Q151M, V75T) and polymorphic loci (S162C, Q207E, R211K, L214F, I293V) has a synergistic relationship between mutations; 178 cases of untreated HIV-1 infected patients were analyzed, obtained in untreated RT resistant mutations were in the treatment group (V106I, M41L L210W, T215Y, H221Y). With polymorphic loci (I135V, S68G, I178L, R277K) has a synergistic relationship between the mutation of HIV-1. Three, resistance characteristic effects of mutation site on the viral replication adaptability of this part of the study, we first established the HIV-1 subtype CRF07_BC in vitro competition experiment system, combined with the growth of HIV-1 B subtype in vitro competition experiment system in vitro experiments, research, analysis of drug resistance of CRF07_BC subtype and B subtype found the second part of the relevant features of the virus effects of mutation site adaptability, and by detecting the relative content of P51 protein, prediction method of reverse transcriptase activity and protein three level structure, carry on the preliminary discussion to the mechanism of virus adaptability change for the HIV-1 B'subtype is not found in the treatment group collaborative mutations were found that the recombinant virus HIV-1RT T215Y+I178L cooperative mutation its adaptability is obviously higher than that of T215Y The single point mutation of the recombinant virus and adaptability, and the relative content of the reverse transcriptase activity of P51 protein also increased significantly compared with it. The results suggest that 1178L and reverse transcriptase activity by affecting P51 protein relative content of the compensation of drug resistance related mutations caused by T215Y virus adaptability decreased. For the detection of HIV-1 subtype B'in the treatment group the synergistic mutations were found in the drug free environment, the recombinant virus HIV-1 RT T215Y+R211K mutation was significantly higher than that of the adaptive collaborative T215Y single point mutant recombinant virus adaptability; in the presence of AZT, the recombinant virus T215Y+R211K cooperative mutation adaptability is higher than that of wild strain; this suggests that in the treatment of patients. The emergence of R211K, can make the virus adaptation of this phenomenon may rise, will accelerate the resistance development.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R512.91

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