采用异源双链泳动法进行HIV-1亚型分析的研究

目的 探索一种简单、敏感、特异和成本低的HIV-1亚型亚型测定方法。方法 采用改良的异源双链泳动法(HMA)与DNA序列分析法对14份HIV-1抗体阳性者样本进行亚型测定、分析和比较。结果 用HMA改良法明确测定了14份样本中的13份样本的亚型。在14份样本中对13份样本进行DNA序列分析有12份的结果与HMA的相符,符合率达92.3％。结论 改良的HMA测序法是一种简单、敏感、特异和经济的HIV-1亚型测定方法,可在广大的基层单位推广应用。     

采用异源双链泳动法进行HIV—1亚型分析的研究

目的 探索一种简单,敏感,特异和成本低的ＨＩＶ－１亚型亚型测定方法。方法 采用改良的异源双链泳动法（ＨＭＡ）与ＤＮＡ序列分析法对１４份ＨＩＶ－１抗体阳笥者样本进行亚型测定,分析和比较。结果 用ＨＭＡ改良法明确测定了１４份样本中的１３份样本的亚型。在１４份样本中对１３份样本进行ＤＮＡ序列分析有１２份的结果与ＨＭＡ的相符,符合率达９２．３％。结论 改良的ＨＭＡ测序法是一种简单,敏感,特异和经济的ＨＩＶ     

持续中度的HIV-1病毒血症并不一定作为临床衰退的信号

据Medscape．com9月24日报道(原载J Acquir Immune Defic Syndr 2004；37：1147—1154．)，一项研究结果提示，在高活性抗逆转录病毒治疗期间，持续中度病毒血症不一定增加临床疾病进展的危险。     

HIV表型耐药及检测方法的研究进展

高效抗逆转录病毒治疗（HAART）的应用有效地控制了艾滋病患的病情，延长了患的生命，大大地降低了HIV／AIDS相关的发病率和死亡率。但是耐药性的出现却极大地限制了HIV抗病毒治疗效果。选择一个能最大抑制HIV-1复制的方案成为一个极具挑战性的任务。对于接受过抗病毒治疗的病人，不但可能对使用的药物产生耐药，对其它药物亦会产生潜在的交叉耐药，因此耐药性的产生对救援治疗方案的效力亦具有很大的影响。即使未经抗病毒治疗的患，耐药病毒的感染也会限制初期联合抗病毒方案的病毒学和免疫学效力。研究证明根据耐药检测结果选择抗病毒治疗方案可有效地降低病毒载量，升高CD4细胞，从而极大地改善HIV一1感染抗病毒治疗的效果，因此许多专家组织，如IAS—USA、DHHS及EuroGroup已提供耐药检测的推荐和指南。目前HIV耐药性检测方法主要有3种：基因型耐药检测、表型耐药检测和虚拟表型耐药检测。[第一段]     

HIV-1耐药性产生机制及检测方法

HIV-1耐药性的产生是导致艾滋病(AIDS)抗病毒治疗失败的重要原因，目前HIV-1耐药性产生机制及检测方法已成为HIV／AIDS研究领域的热点之一。随着高效抗逆转录病毒联合疗法(highly active antiretroviral therapy，HAART)在我国的展开，对HIV-1耐药株进行监测以指导临床用药及预防耐药株的迅速上升和扩散日显其重要性。本文将对HIV-1耐药性产生机制及当前国际上应用的耐药性检测方法进行综述。     

HIV-1耐药产生机制及其临床应用

自1996年高效抗逆转录病毒治疗(highly ac-tive antiretroviral therapy,HAART)应用于艾滋病(acquired immunodeficiency syndrome,AIDS)临床治疗已有10年时间。10年来 AIDS 的发病率和病死率明显下降,患者生存质量得到了显著改善,但病毒耐药性的出现则大大削弱了 HAART 抑制病毒复制的作用,降低了抗病毒治疗效果。目前,已通过美国食品和药品管理局(Food and     

2011年广西部分地区HIV耐药株发生情况分析

目的了解广西部分地区已接受高效抗反转录病毒治疗(HAART)的病人中,艾滋病病毒(HIV)耐药株的发生情况。方法对南宁市和崇左市2011年已接受HAART6个月的病人,进行横断面调查并采集血液样本检测HIV-1病毒载量,对病毒载量1000拷贝/mL的样本进行耐药基因型分析。结果两市共调查并成功采集了1472例病人的血液样本,病毒未抑制率为4.6%(68/1472),最终获得64份序列用于耐药分析。发现对非核苷类反转录酶抑制剂(NNRTIs)和核苷类反转录酶抑制剂(NRTIs)最主要耐药突变位点分别为G190A和M184V,未发现蛋白酶类抑制剂(PIs)有耐药突变,3类药物的耐药率分别为1.9%(28/1468)、1.6%(24/1468)和0.0%(0/1468),总耐药率为3.5%(52/1468)。52例病人出现了耐药,其中43.8%(28/64)病毒抑制失败病人对NNRTIs耐药,37.5%(24/64)对NRTIs耐药,29.7%(19/64)对NNRTIs和NRTIs双重耐药,81.3%(52/64)至少对某类药物有耐药。结论两个地区2011年已接受HAART人群中,HIV耐药株发生率较低,但出现了对NNRTIs和NRTIs双重耐药,值得关注。     

云南省抗病毒治疗失败的人类免疫缺陷病毒感染/艾滋病患者中人类免疫缺陷病毒1型pol区多态性位点的分布

目的探讨人类免疫缺陷病毒感染/艾滋病(HIV/AIDS)患者接受高效抗反转录病毒治疗(HAART)过程中耐药的发生与多态性位点分布规律间的关系。方法纳入2015年6月至2021年12月云南省16个地级行政区成功扩增得到pol区基因序列的HAART失败HIV/AIDS患者, 采用人类免疫缺陷病毒(HIV)局部序列排比检索基本工具(BLAST)对序列进行亚型鉴定, MEGA 6.0软件进行亚型验证。将样本序列提交至美国斯坦福大学HIV耐药数据库进行耐药位点比对。分析不同耐药程度、治疗方案和HIV-1亚型患者的多态性位点分布情况。采用趋势χ2检验分析不同耐药程度患者多态性位点出现频率变化趋势。统计学比较采用χ2检验并作事后两两比较。结果 1 453例患者扩增获得序列, 耐药检测结果为敏感者954例, 潜在或低度耐药者224例, 中度耐药者189例, 高度耐药者86例。随着HIV-1耐药程度的增加, HIV-1蛋白酶区(PR区)I15V、L19I、D60E和反转录酶区(RT区)E36A、T39D、S48T位点出现频率呈下降趋势(χ2趋势=19.86、9.16、13.66、37.64、18.44...     

树突状细胞亚群参与HIV-1感染和免疫发病机制的研究新进展

经过高效抗逆转录病毒(highly active antiretrovirial therapy，HAART)治疗的艾滋病(AIDS)患者，艾滋病病毒HIV载量明显减少，外周血中CD4^4T淋巴细胞计数增加。然而，临床治疗中发现仅仅HARRT难以完全恢复正常淋巴细胞数量和功能，尤其是针对HIV-1的特异性免疫功能。树突状细胞(dendritic celts，DC)作为人体中最有效的抗原提呈细胞，在HIV感染中也出现了数量和功能上变化。针对HIV-1感     

18例抗病毒治疗失败儿童艾滋病患者耐药基因突变规律研究

目的对儿童艾滋病抗病毒治疗失败病例的耐药基因突变规律及特点进行研究。方法分析18例接受过高效抗逆转录病毒治疗并已经出现病毒学及免疫学失败儿童艾滋病患者的横断面临床及实验室资料,对其耐药突变结果进行分析。结果患者年龄11.6±2.4岁,治疗的时间为36±12个月,其中15例患者曾经接受过成人抗病毒药物治疗,患者的 CD4~ T 淋巴细胞为34±30个/μl(3～96个/μl),病毒载量为5.23±0.57 log10 copies/ml(4.27～6.53 log10 copies/ml)。耐药发生率为100%。对非核苷类逆转录酶抑制剂(NNRTIs)耐药突变结果分析显示:所有患者均对奈韦拉平(NVP)产生高度耐药,对依非韦伦(EFV)高度耐药的有16例。对核苷类逆转录酶抑制剂(NRTIs)耐药突变结果分析显示:对拉米夫定(LAM)高度耐药的有14例,对去羟肌苷(ddI)高度耐药的有11例,对齐多夫定(AZT)高度耐药的有14例,对司他夫定(d4T)高度耐药的有16例。重要突变位点包括 Y181C(9例)、K103N(7例)、G190A(8例)、TAMs(17例)、M184V(10例)、K65R(5例)、Q151M(2例)。结论该组治疗失败儿童患者对正在使用的 NRTIs 及 NNRTIs 均已产生高度耐药,须考虑更换新的抗病毒治疗方案。     

Highly active antiretroviral therapy used to treat concurrent hepatitis B and human immunodeficiency virus infections

We report a case of simultaneous infection with hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) in a 26-year-old Japanese homosexual man. He was admitted to our hospital for acute hepatitis caused by HBV. At that time, HIV-1-antibody (Ab) was not detected in his serum. After 6 months, he was readmitted to our hospital for further examination of his liver because of confined liver enzyme abnormalities. Anti-HIV-1 Ab was detected in his serum by both enzyme immunosorbent assay (EIA) and particle agglutination (PA). His serum HIV-1 RNA level was 50 × 10⁴ copies/ml and serum levels of HBV DNA polymerase (DNA-P) and HBV DNA were 6535 cpm and 3 plus (>1000 copies/ml). His clinical course and laboratory data suggested progression from acute to chronic hepatitis related to coinfection with HIV-1. The diagnosis was chronic active hepatitis caused by HBV as an opportunistic infection due to coinfection with HIV-1. We began highly active antiretroviral therapy (HAART) because interferon (IFN) therapy was ineffective. HAART was started at an initial dosage of 600 mg zidovudine (AZT), 300 mg lamivudine (3TC), and 2400 mg indinavir (IDV) daily. After 4 weeks, the serum level of HBV DNA-polymerase (p) had decreased markedly to 37 cpm and that of HIV-1 RNA had decreased to below the sensitivity threshold, indicating considerable suppression of the replication of these viruses by the treatment. But HBV DNA remained at low levels. Although the incidence of HBV infection in patients with HIV-1 infection has been reported to be high in the United States and Europe, simultaneous HBV and HIV-1 infection leading to persistent HBV infection is rare. (Received Feb. 13, 1998; accepted Sept. 25, 1998)     

Temporal Trends in HIV-1 Mutations Used for the Surveillance of Transmitted Drug Resistance

In 2009, a list of nonpolymorphic HIV-1 drug resistance mutations (DRMs), called surveillance DRMs (SDRMs), was created to monitor transmitted drug resistance (TDR). Since 2009, TDR increased and antiretroviral therapy (ART) practices changed. We examined the changing prevalence of SDRMs and identified candidate SDRMs defined as nonpolymorphic DRMs present on ≥ 1 expert DRM list and in ≥0.1% of ART-experienced persons. Candidate DRMs were further characterized according to their association with antiretrovirals and changing prevalence. Among NRTI-SDRMs, tenofovir-associated mutations increased in prevalence while thymidine analog mutations decreased in prevalence. Among candidate NRTI-SDRMs, there were six tenofovir-associated mutations including three which increased in prevalence (K65N, T69deletion, K70G/N/Q/T). Among candidate NNRTI-SDRMs, six that increased in prevalence were associated with rilpivirine (E138K/Q, V179L, H221Y) or doravirine (F227C/L) resistance. With the notable exceptions of I47A and I50L, most PI-SDRMs decreased in prevalence. Three candidate PI-SDRMs were accessory darunavir-resistance mutations (L10F, T74P, L89V). Adding the candidate SDRMs listed above was estimated to increase NRTI, NNRTI, and PI TDR prevalence by 0.1%, 0.3%, and 0.3%, respectively. We describe trends in the prevalence of nonpolymorphic HIV-1 DRMs in ART-experienced persons. These data should be considered in decisions regarding SDRM list updates and TDR monitoring.     

HIV-1基因变异与耐药之间的关系研究现况

HIV-1以其高突变率、高重组率和高复制率的生物学特性,使得患者体内的病毒成为复杂的准种,给艾滋病的诊断、治疗和疫苗的研制带来众多难题。高效抗逆转录病毒联合疗法的使用降低了艾滋病患者的病死率,然而在高速复制、高度遗传变异和药物选择压力的作用下,日益严重的耐药问题削弱了其抑制病毒复制的作用,降低了抗病毒治疗的效果。本文综述了HIV-1常见的基因变异特点与耐药之间关系的研究进展,以期对临床制订抗HIV-1药物方案、发展新的抗HIV-1策略带来指导意义。     

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this protease–substrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease–substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate residue has distal effects on other residues’ interactions as well, including through the induction of a conformational change in the protease. Additionally, molecular dynamics simulations suggest that restoration of active site dynamics is an additional constraint in the selection of coevolved mutations. Hence, protease–substrate coevolution permits mutational, structural, and dynamic changes via molecular mechanisms that involve distal effects contributing to drug resistance.Resistant pathogens evolve under the selective pressure of drug therapies, commonly by acquiring mutations in the drug target (1–4). Most of these mutations cluster around the drug-binding site and alter key interactions between the drug and its target. Strikingly, mutations in other off-target proteins have also been reported to contribute to drug resistance (5–8) where the mechanism of resistance is not as straightforward to rationalize. In the case of HIV-1, mutations in the target protease gene confer resistance to protease inhibitors (PIs) and correlate with emergence of mutations elsewhere in Gag. There are currently nine protease inhibitors (PIs) that are US Food and Drug Administration (FDA) approved for clinical use including in highly active antiretroviral therapy (HAART) (9), and all are competitive inhibitors binding at the active site.HIV-1 protease is a key antiviral drug target due to its essential function of processing Gag and Gag-Pol polyproteins in viral maturation (10–12). Under the selective pressure of PI-including therapy regimens, viral variants carrying mutations in the protease gene impair the inhibitor efficacy. Although the PIs become weaker binders of these resistant protease variants, the substrates are still hydrolyzed (13, 14), skewing the balance between inhibitor binding and substrate processing in favor of the latter. Earlier work from our group revealed the molecular determinants of this fine balance and formulated the substrate envelope hypothesis to effectively explain the molecular mechanism of resistance due to primary active site mutations (15). Among primary protease mutations, I50V is commonly observed in patients failing therapy with the PIs amprenavir (APV) and darunavir (DRV) (16–18). Residue 50 is located at the flap tip of the flexible loop (50s loop) that controls the access of substrates and competitive inhibitors to the protease active site. In addition to conferring resistance to PIs, the I50V mutation also impairs substrate processing (19). The loss of catalytic efficiency due to I50V is compensated by secondary mutations, in particular A71V (20), which is observed in more than 50% of patient sequences bearing I50V (14).Several mutations in Gag both within cleavage sites and elsewhere coevolve with primary protease mutations contributing to viral fitness and possibly drug resistance (5, 7, 21–25). Particularly, mutations in the p1-p6 cleavage site are statistically associated with I50V protease mutation in the viral sequences retrieved from patients (Fig. 1) (26). The Gag L449F mutation rescues the protease activity by 10-fold, whereas P453L, despite being distal from the catalytic site, causes a 23-fold enhancement (19). However, the molecular basis for the selection advantage of these correlated mutations and the mechanism by which the compensatory mutations restore substrate recognition in drug resistance is not clear. In this study, we report the structural basis for the coevolution of I50V/A71V protease with the p1-p6 substrate. Through a series of cocrystal structures, the Gag mutations L449F and P453L were shown to enhance van der Waals (vdW) interactions between the substrate and mutant protease, whereas R452S results in an additional hydrogen bond. Unexpectedly, the P453L substrate mutation causes a conformational change in the protease flap loop, revealing the molecular mechanism by which this distal substrate mutation is able to enhance substrate–protease interactions. In addition, molecular dynamics simulations suggest that coevolution restores the dynamics at the active site, a key aspect of substrate recognition and turnover that is largely uncharacterized.Open in a separate windowFig. 1.HIV-1 protease and p1-p6 cleavage site coevolution with I50V primary drug resistance mutation. (A) p1-p6 cleavage site sequence and the most common coevolution mutations at P1′, P4′, and P5′ sites. (B) Residues 50 and 71 are indicated as spheres on the homodimeric HIV-1 protease structure. (C) Frequency of mutations in the p1-p6 cleavage site without (dark blue) and with (gray) any mutations at position 50 of the protease. The difference is statistically significant for LP1′, RP4′, and PP5′. Data from ref. 26. (D) Side chains of the substrate residues LP1′, RP4′, and PP5′ and the protease residue I50 are shown as sticks. Monomers of HIV-1 protease are in light purple and green, and the substrate is red in B and D.     

高效联合抗反转录病毒治疗引起血脂异常和脂肪性肝病的临床研究

目的探讨人类免疫缺陷病毒（HIV）/丙型肝炎病毒（HCV）合并感染的艾滋病患者予高效联合抗反转录病毒治疗（HAART）后出现的血脂代谢变化和脂肪肝发病情况及临床对策。方法患者分为治疗组和对照组,治疗组HAART方案为（d4T＋3TC＋EFV）,比较抗病毒治疗前后空腹血脂变化（包括甘油三脂、总胆固醇、高密度脂蛋白、低密度脂蛋白）和脂肪肝发病情况,对照组观察随访指标同治疗组。结果治疗组患者HAART治疗前后血甘油三脂分别为（1.44±0.35）mmol/L和（2.10±0.54）mmol/L（P〈0.01）,总胆固醇分别为（3.91±0.86）mmol/L和（5.45±0.87）mmol/L（P〈0.01）,血高密度脂蛋白分别为（1.26±0.21）mmol/L和（1.22±0.05）mmol/L（P〉0.05）,血低密度脂蛋白分别为（2.29±0.33）mmol/L和（3.11±0.29）mmol/L（P〈0.01）。治疗组49例患者到2006年7—8月B超肝脏检查累计发现脂肪肝9例。对照组随访过程中血脂无明显变化,没有发现脂肪肝。结论治疗组患者HAART后血甘油三脂、总胆固醇、低密度脂蛋白均较抗病毒治疗前显著增高;高密度脂蛋白无显著变化。影像学B超诊断脂肪肝发生率为18.3%（9/49）。提示HAART与血脂代谢异常及脂肪肝发病密切相关。