【作者】 周海卫；

【导师】 邢辉；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2008， 硕士

【摘要】 自20世纪90年代以来,抗逆转录病毒治疗（ART）以其良好的病毒抑制效果,作为治疗艾滋病的最主要手段在全世界范围内被广泛应用。但ART并不能把病人体内的HIV彻底清除,在药物选择压力下耐药性病毒株迅速产生,这被认为是影响抗病毒治疗效果的主要障碍。HIV基因型耐药性检测方法是检测病毒耐药情况,进而指导临床治疗及治疗失败后更换治疗方案的重要技术手段。在艾滋病的传播过程中,HIV-1 M组不同亚型的基因组存在着10%-30%的差异,在pol基因的差异为10%-15%,这会导致HIV-1不同亚型的耐药进化模式存在差别。目前用于解释基因型耐药结果的耐药解释系统大多基于欧美B亚型的研究结果建立,而我国主要流行的为中国B、CRF01_AE和CRF07_BC亚型,直接利用国际上已有的解释方法评价我国的基因型耐药检测结果是否可行,需要进一步明确。为使基因型耐药性检测方法更好地应用于抗病毒治疗,对我国流行的主要HIV-1亚型进行基因型耐药研究及对基因型耐药解释系统的解释效力进行评价显得非常必要。本研究采用基因型耐药性检测方法,对采集自七个省/自治区（河南、安徽、四川、湖南、广西、云南及新疆）的中国B（122例）、CRF01_AE（117例）和CRF₀7BC亚型（107例）HIV-1未治疗患者和采集自河南、新疆的中国B（107例）、CRF₀7BC亚型（67例）HIV-1治疗后患者进行了横断面研究。研究发现,相对于欧美B亚型,中国B、CRF01_AE及CRF07_BC亚型在蛋白酶基因上各存在5个（E35D、L63P、I72V、V77I和I93L）、5个（I13V、E35D、M36I、H69K和L89M）和4个（E35D、D60E、L63P和I93L）氨基酸替换率大于50%的位点;在逆转录酶基因区各存在3个（I135V、S162C和T200A）、12个（E6D、T39K、K43E、D123S、Q174K、D177E、I178M、T200I、Q207A、R211S、K238R和V245E）和8个（V35T、E36A、T39D、S48T、V60I、D121Y、K122E和V245Q）氨基酸替换率大于50%的位点。在我国三种主要流行亚型中存在3个PIs次要耐药突变,分别存在于PR区的第10、第58及第71位氨基酸位置。PIs次要耐药突变不会影响病毒株对PIs的敏感性,但是这些突变可能影响耐药性的发生或随后耐药突变的进化从而影响抗病毒治疗方案的选择。结合主要耐药突变遗传障碍的研究结果,CRF01_AE亚型中K238R替换可能会影响到RT基因第238位氨基酸耐药突变的进化。对所有主要耐药突变遗传障碍的研究还显示,我国三种主要流行亚型中只有3个耐药突变的遗传障碍与欧美B亚型存在差别,分别是具有较高遗传障碍的V75I（CRF01_AE）和K/R238N（CRF01_AE）及具有较低遗传障碍的V106M（CRF07_BC）,其它44个耐药突变的遗传障碍与欧美B亚型之间没有差异,这说明不同亚型之间已知耐药突变位置的基因多态性对耐药突变的进化起次要作用。我国目前只有两种非核苷类逆转录酶抑制剂NVP和EFV在使用,在CRF07_BC亚型中更容易被诱导的V106M突变能够导致对NVP和EFV两种药物的耐药,而发生在B亚型中的V106A突变只导致对NVP的耐药,一旦感染CRF07_BC毒株的病人发生V106M突变,将导致对NVP和EFV均耐药,致使病人无有效的NNRTIs可用。本研究在我国B亚型及CRF07_BC亚型治疗后人群中发现了三个新的耐药相关突变:H221Y、L228R和L228H。三个突变经常伴随Y181C出现。它们可能由NVP诱导产生。三种基因型耐药解释系统（HIVDB,ANRS和Rega）对我国抗病毒治疗后B亚型及CRF07_BC亚型患者基因型耐药检测结果的解释存在差别,对NNRTIs相关耐药突变解释效力的一致性高于NRTIs相关耐药突变。一些耐药突变如E44A/D、V118I、T69N、K238T、Y18lC、M184V的存在是导致差别的原因,这些耐药突变单独或与其它突变共同出现时对药物敏感性的影响还没有定论,应加强对这些耐药突变及与其它突变之间相互作用的深入研究。目前对我国基因型耐药检测结果的解释仍建议继续以HIVDB基因型耐药解释系统为主,并参考Rega系统的结果;长期的目标是要建立一种适合于我国主要流行亚型的基因型耐药解释方法。更多还原

【Abstract】 Since 1990s,Antiretroviral Therapy(ART) has dramatically changed the clinical outcome of AIDS,leading to decreased AIDS-related mortality and morbidity. However,ART cannot completely eradicate HIV from the body,results in long-term toxicity and eventually leads to the emergence of drug-resistant HIV strains under the drug pressure in vivo,which was considered a major cause of treatment failure.HIV-1 genome differs by 10%to 30%between subtypes of M group.Amino acid sequence diversity in the pol gene is 10%to 15%between different subtypes,which may influence the pattern of drug resistance in different subtypes.HIV-1 genotypic drug resistance testing is an effective tool for the surveillance of HIV-1 drug resistant strains.These testing results can be used in the choice of antiretroviral regimen.However,the benefit of genotyping is limited by the need to interpret the mutation pattern in order to obtain a prediction regarding susceptibility to each antiretroviral drug.There are different genotypic HIV drug resistance interpretation systems currently available,which are mostly constructed based on data for clade B viruses.Subtype B,CRF01_AE and CRF07_BC are prevailing in China but little information is available on non-B subtypes to date.It becomes more and more important to study resistance-associated genetic characteristic of subtype B, CRF01_AE and CRF07_BC isolates in China and evaluate the prediction efficiency of different genotypic drug resistance interpretation systems.In this study,Protease and reverse transcriptase sequences were available from 122 subtype B,117 CRF01_AE and 107 CRF07_BC isolates from drug-naive patients in Henan,Anhui,Sichuan,Hunan,Guangxi,Yunnan and Xinjiang provinces.Protease and reverse transcriptase sequences were also available from 107 subtype B and 67 CRF07_BC isolates from treated HIV-1 infected individuals in Henan and Xinjiang provinces.It showed that of the total 99 amino acids of protease,polymorphisms were found to be more than 50%at 5 codons(E35D,L63P,I72V,V77I and I93L) in subtype B,at 5 codons(I13V,E35D,M36I,H69K and L89M) in CRF01 AE,and at 4 codons (E35D,D60E,L63P and I93L) in CRF07_BC.Of the first 270 amino acids of reverse transcriptase,polymorphisms were found to be more than 50%at 3 codons(I135V, S162C and T200A) in subtype B,at 12 codons(E6D,T39K,K43E,D123S,Q174K, D177E,I178M,T200I,Q207A,R211S,K238R and V245E) in CRF01_AE,and at 8 codons(V35T,E36A,T39D,S48T,V60I,D121Y,KI22E and V245Q) in CRF07_BC.There were 3 secondary mutations in amino acid position 10,58 and 71 of protease in 3 subtypes prevailing in China.Although secondary mutations do not significantly alter the susceptibility of the PIs,these mutations,which exist before the emergence of a primary mutation,may alter the rate of the development of drug resistance or the subsequent evolution of mutations.The amino acid substitution K238R of reverse transcriptase in CRF01_AE may influence the drug resistance evolution.The increased genetic barrier was calculated for V75I(CRF01_AE) and K/R238N (CRF01_AE),and the decreased genetic barrier was calculated for V106M (CRF07_BC) compared with those of subtype B.The same genetic barrier was calculated for the other mutations between subtypes.This study found few differences between subtypes in the calculated genetic barrier for evolution to major drug-resistance-associated substitutions,which imply that subtype diversity might have little influence on the patterns of resistance substitutions that will emerge.The emergence of V106M in patients harboring clade CRF07_BC infections will be a matter of concern because cross-resistance to all NNRTIs occur as a result of this mutation.Only NVP and EFV can be chose in all NNRTIs in China,so we should be aware of the V106M mutation and its potential impact on therapy outcome in CRF07_BC patients.We found that H221Y,L228R and L228H were new resistance-associated mutations in subtype B and CRF07_BC isolates.All of three mutations were correlated with novel Y181C mutation.H221Y was also correlated with novel K103N mutation.The H221Y,L228R and L228H may be associated with the specific use of NVP.Variable prediction of antiretroviral treatment outcome by HIVDB,ANRS and Rega genotypic drug resiatance interpreting systems was observed in subtype B and CRF07_BC patients.Higher levels of interpreting discordance were observed for predictions of NNRTIs than for predictions of NRTIs susceptibility.The discordance could be attributed to specific mutations,including E44A/D,V118I,T69N,K238T, Y181C and M184V,which always have the interactions with other mutations.The mutations found here should be further investigated as to whether they contribute to differences in resistance and therapy response between subtypes prevailing in China.The HIVDB genotypic drug resistance interpreting system can be used to predict therapy response of subtype B and CRF07_BC patients in China.The Rega system should be an important complernent to HIVDB especially the presence of complicated mutation patterns.There exists,therefore,an urgent need for standardization of genotypic drug resistance interpreting system for HIV-1 genotyping.更多还原