上海地区人类免疫缺陷病毒1型感染/艾滋病患者病毒耐药基因突变及亚型分布

目的 研究上海地区人类免疫缺陷病毒1型(HIV-1)感染／艾滋病(AIDS)患者中HIV-1耐药株出现的情况及亚型分布。方法 对33例HIV-1感染／AIDS患者的血浆HIV-1分离株,进行抗HIV-1药物(核苷类反转录酶抑制剂、非核苷类反转录酶抑制剂和蛋白酶抑制剂)的基因型耐药检测和亚型分析。结果 33例的HIV-1均未检出对PI的耐药突变;10例高效抗反转录病毒疗法(HAART)治疗失败或抑制病毒复制不完全者中,检出的耐药突变为70％,过渡型耐药突变为20％;23例未经抗HIV-1治疗者中,耐药突变为4．3％,过渡型耐药突变为13％。所有过渡型耐药突变均为T215S。15例经血制品传播的HIV- 1均为B亚型;18例经吸毒和性传播的HIV-1中,B和CRF01-AE亚型分别为39％,和33％,此外,还有C、D、G、K和CRF02-AG亚型。结论 上海地区HIV-1感染／AIDS患者中,HAART治疗失败或复制抑制不完全者HIV-1的NRTI和NNRTI耐药突变率高;吸毒和性传播者的HIV-1中,除主要为B和CRF01-AE亚型外,尚有其他少见的亚型。     

耐药相关性的人类免疫缺陷病毒基因变异

随着高效抗反转录病毒治疗的广泛应用,艾滋病的治疗已获得实质性的进展。现已有23种抗病毒药物,但耐药性的出现使治疗效果下降。回顾性研究显示药物耐药性可以先于新的药物治疗而存在。前瞻性研究显示若事先掌握病人的基因耐药资料,抗病毒治疗的效果将好于未掌握资料者。本文将详细综述人类免疫缺陷病毒耐药机制和临床意义以指导抗病毒治疗。     

西门子医疗诊断产品——病毒耐药检测及基因分型系统TRUGENE介绍

在现今的分子生物学实验室病毒学领域中,基因分型及耐药检测已成为临床科研的重要组成部分,准确可靠的基因型鉴定结果及耐药相关突变报告可以为临床提供制定和修改治疗方案的参考,另一方面还能够对病毒基因型或亚型与抗病毒药物疗效之间相关性研究提供证据,在我国的慢性乙型肝炎及丙型肝炎的治疗指南中,均提及在治疗过程中需要对病毒进行基因分型和耐药突变分析检测.     

柳州和南宁HIV-1亚型分布及耐药情况调查

为了解柳州和南宁两市HIV-1亚型分布和耐药情况,在柳州和南宁招募HIV感染者和AIDS患者共304名,采集外周静脉血,从血浆中提取HIVRNA,扩增HIVpol基因并测序。将获得的序列进行系统进化树分析,结果表明柳州的HIV-1毒株中存在CRF01_AE和CRF07_BC两种亚型,其中CRF01_AE毒株占75.2%,CRF07_BC毒株占24.8%;南宁的HIV-1毒株中存在CRF01_AE、CRF08_BC、B亚型和C亚型共4种亚型,其中CRF01_AE和CRF08_BC仍是南宁最主要的亚型,CRF01_AE占85.8%,CRF08_BC占11.5%。根据所得的序列资料进行HIV-1耐药性分析,计算耐药率。计算结果表明,柳州未治疗和治疗研究对象的耐药率分别为3.3%和8.7%,南宁未治疗和治疗研究对象的耐药率分别为1.4%和27.5%。     

HCMV感染的抗氧化治疗与洁罗维注射液临床预防与治疗应用

人巨细胞病毒(HCMV)是疱疹病毒中最大也是最常见的一种,HCMV感染危害性大,亚洲与非洲地区的人群感染率高,目前临床仍缺乏专属性强的治疗药物。在其治疗过程中,抗病毒药物长期应用导致耐药问题存在,而机体免疫功能抑制与病毒耐药发生率关系密切,因此HCMV防治过程中,抗病毒抗氧化协同治疗势在必行。洁罗维注射液(阿昔洛韦氯化钠注射液Ⅱ)是一种"抗病毒+抗氧化+营养支持"三重作用机制的新型复方抗病毒输液,可提高机体免疫功能,降低病毒耐药性,有利于临床诸多科室HCMV感染的预防与治疗,具有极高的临床推广价值。     

南宁市HIV-1/AIDS人群治疗前pol区遗传特性及蛋白结构分析

为探讨南宁市某县艾滋病病毒1型(HIV-1)感染人群中治疗前pol区遗传特性及蛋白结构变化情况,本研究通过RT-PCR扩增pol区部分序列并进行测序,将序列同源比对构建系统进化树;分型确定毒株亚型和斯坦福大学HIV耐药性数据库比对,分析耐药相关位点;SWISS-MODEL蛋白质同源数据库进行建模分析氨基酸的突变对蛋白质结构和功能的影响。本研究在90份HIV-1标本中获得46个pol区有效序列,共发现4种亚型,其中CRF01_AE占76.08%(35/46)、CRF08_BC占15.22%(7/46)、CRF07_BC占(3/46)6.52%、CRF59_01B1占2.17%(1/46);46个序列中有4例(8.69%)出现耐药突变位点,没有针对核苷酸反转录酶抑制剂(NRTI)的耐药突变;针对蛋白酶类抑制剂(PIs)1例,PR蛋白酶的柔性部位I47V位点发生突变,β折叠结构的I84V位点发生突变,都是异亮氨酸突变为缬氨酸;针对非核苷酸反转录酶抑制剂(NNRTI)有3例,2例位于活性中心的Y181C位点由酪氨酸突变为半胱氨酸,1例位于转角处的E138G位点由谷氨酸突变为甘氨酸。研究表明,南宁市某县HIV-1病毒CRF01_AE重组亚型比例最大,未经抗病毒治疗HIV1感染者中已经出现pol区耐药突变株,突变位点主要位于活性中心及柔性部位,传播水平已经处于中等流行状态。深入分析蛋白质与抑制剂相互作用机制,有助于为艾滋病抗病毒及耐药性监测方案提供科学依据。     

上海地区HIV毒株基因亚型分布及原发基因耐药的分子流行病学研究

本文通过对未经抗病毒治疗患者的人类免疫缺陷病毒1型（HIV-1）毒株进行检测,了解上海地区HIV-1的亚型分布及原发耐药基因变异现状。对118例未经治疗的HIV感染者其标本中HIV蛋白酶全长和部分反转录酶基因进行反转录-聚合酶链反应（RT-PCR）扩增,经DNA测序后进行系统进化树分析和重组分析,以确定HIV-1基因亚型和重组体,并与斯坦福耐药数据库比对,了解耐药性突变位点。使用斯坦福REGA HIV亚型分型工具和美国国立生物技术信息中心（NCBI）HIV亚型分析工具分析亚型,获得118例患者的HIV基因序列,基因分型分别为CRF01_AE重组体57例(48.3%)、B亚型36例(30.5%)、CRF07_BC 15例 (12.7%)、CRF08_BC 7例(5.9%)、C亚型2例(1.7%),亚型间或重组体间二重重组体(B/CRF01_A E)1例(0.8%)。蛋白酶抑制剂(PI)和反转录酶抑制剂相关的耐药基因突变率达54.2%（64/118）,其中2例（1.7%）发生PI耐药,基因突变位点：M46L、Q58E。5例(4.1%)对反转录酶抑制剂产生耐药,其中对核苷类反转录酶抑制剂（NRTI）和非核苷类反转录酶抑制剂（NNRTI）的耐药率分别为3例(2.4%)和5例(4.1%)。基因突变位点：NRTI为M41L、D67N、T69I/N/S、K70L、L74V、V75L、V118I、M184V、L210W/F/M/S和T215F;NNRTI为V90I、L100V、K103R/N、V106M/P/I/G、E138G/A、V179E/D/T、Y181C、G190A、H221Y、F227L、K238S和Y318F。结果提示,上海地区HIV毒株以CRF01_AE重组亚型为主,且发现新的重组体,可能出现新重组体流行的趋势。PI和反转录酶抑制剂相关的耐药基因突变率较高,且存在高度原发耐药毒株,应加强HIV-1耐药基因变异监测,科学、合理地给予抗病毒治疗。     

2020年银川市HIV-1感染者基因亚型及耐药分析

为了解银川市接受抗病毒治疗的HIV-1感染者基因亚型和耐药突变特征。收集2020年银川市接受抗病毒治疗的HIV-1感染者血样,其中病毒载量大于400拷贝/mL的样本进行基因型耐药检测,通过巢式聚合酶链反应（PCR）扩增HIV-1 pol基因区片段并测序,对所得序列进行基因分型和耐药突变分析。收集642例血浆样本,50例病毒载量大于400拷贝/mL,基因型耐药检测后获得29例pol区基因序列。17例耐药,3例潜在耐药,总体耐药率为2.65%(17/642)。CRF07＿BC和CRF01＿AE是耐药株的主要基因亚型。NRTIs类耐药突变中,M184V/I(31.03%)突变发生率最高,其次为K65R(17.24%)。3TC、FTC和ABC耐药发生率最高,均为37.93%(11/29)。NNRTIs类耐药突变中,G190A/S/E、V179D/DE/E突变发生率最高,均为24.14%;K103N/KN(17.24%)次之。EFV和NVP耐药发生率最高,均为58.62%(17/29)。2020年银川市新型重组亚型增加,首次检出CRF79＿0107。总体耐药率处于低水平,以NNRTIs耐药为主,...     

江苏省2017年新报告HIV感染者治疗前耐药分析及基因亚型分布

基于大样本量的亚型及治疗前耐药分析,更准确地掌握江苏省HIV-1流行亚型,更真实地评估江苏省治疗前耐药水平并为抗病毒治疗的开展提供依据收集2017年全年全江苏省新报告感染者治疗前血样.逆转录PCR扩增HIV-1蛋白酶区(pol)基因并送测序.ChromasPro剪辑,拼接,合成序列.MEGA7做序列比对并构建进化树分析亚型.序列在线比对耐药位点.治疗前耐药分析,耐药率达16.3％.蛋白酶区(PR)主要耐药位点是L33F,占44.4％.核苷酸逆转录酶区(NRTI)主要耐药位点是M184位点变异,占35％.非核苷酸逆转录酶区(NNRTI)耐药位点主要是V179位点变异,占59.2％.CRF01_AE亚型(456,37.7％)和CRF07_BC亚型(381,31.6％)仍为江苏省主要流行亚型;不同的是复杂重组体(CPXs)和独特重组体(URFs)数量极速上升至第三位(241,19.9％).江苏省HIV-1复杂重组体大量出现,提示新人病毒或病毒交叉重组频繁,艾滋病的防制依然任重道远.在治疗前耐药比例大幅上升的情况下,治疗前耐药检测成为个体选择抗病毒治疗方案的关键.     

基于RNA的抗HIV-1基因治疗方法研究进展

艾滋病自发现以来在全球范围内迅速蔓延,危害性极高,目前广泛采用的高效抗逆转录病毒疗法(HAART)虽能够显著提高HIV-1感染者生活质量,但存在着价格昂贵,耐药和副作用的问题经常会导致HAART治疗的中断。要获得长期持续的抗病毒治疗效果还有待于研发新的抗病毒药物和治疗方法。近年来随着分子生物技术、干细胞研究、纳米技术等相关技术的发展,关于抗HIV-1基因治疗方法的研究受到了广泛关注。主要针对基于RNA的抗HIV-1基因治疗方法,包括反义RNA、核酶、RNA诱饵以及RNA干扰技术在抗HIV-1基因治疗方面进行综述。研究表明,以RNA为基础的抗HIV-1基因治疗方法有望成为传统治疗方法的一种有效辅助手段。     

Taking aim at a moving target: designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases

HIV undergoes rapid genetic variation; this variation is caused primarily by the enormous number of viruses produced daily in an infected individual. Because of this variation, HIV presents a moving target for drug and vaccine development. The variation within individuals has led to the generation of diverse HIV-1 subtypes, which further complicates the development of effective drugs and vaccines. In general, it is more difficult to hit a moving target than a stationary target. Two broad strategies for hitting a moving target (in this case, HIV replication) are to understand the movement and to aim at the portions that move the least. In the case of anti-HIV drug development, the first option can be addressed by understanding the mechanism(s) of drug resistance and developing drugs that effectively inhibit mutant viruses. The second can be addressed by designing drugs that interact with portions of the viral machinery that are evolutionarily conserved, such as enzyme active sites.     

Complex patterns of viral load decay under antiretroviral therapy: influence of pharmacokinetics and intracellular delay

We present a model of HIV dynamics under antiretroviral therapy that combines drug pharmacokinetics and intracellular delay. A two compartment pharmacokinetic model is employed to determine the time evolution of the intracellular concentrations of the active forms of drugs, and thereby drug efficacy. The viral replication period is divided into pre- and post-drug action parts, allowing for the introduction of an intracellular delay in drug action. The standard model of viral dynamics is modified to account for the drug dependence of intracellular delay and continuously varying drug efficacy. Model calculations reveal that viral load decay in HIV infected patients under monotherapy can exhibit remarkably complex patterns depending on the relative magnitudes of the pharmacokinetic, intracellular, and intrinsic viral dynamic time-scales. The commonly assumed exponential decay is only a special case. However, uncertainties in measurement and the low sampling frequencies employed in present clinical studies preclude the identification of these patterns from existing clinical viral load data.     

Drugs of abuse and HIV infection/replication: implications for mother-fetus transmission

Human immunodeficiency virus (HIV) infection and progression of acquired immunodeficiency syndrome (AIDS) can be modulated by a number of cofactors, including drugs of abuse. Opioids, cocaine, cannabinoids, methamphetamine (METH), alcohol, and other substances of abuse have been implicated as risk factors for HIV infection, as they all have the potential to compromise host immunity and facilitate viral replication. Although epidemiologic evidence regarding the impact of drugs of abuse on HIV disease progression is mixed, in vitro studies as well as studies using in vivo animal models have indicated that drugs of abuse have the ability to enhance HIV infection/replication. Drugs of abuse may also be a risk factor for perinatal transmission of HIV. Because high levels of viral load in maternal blood are associated with increased risk of HIV vertical transmission, it is likely that drugs of abuse play an important role in promoting mother-fetus transmission. Furthermore, because the neonatal immune system differs qualitatively from the adult system, it is possible that maternal exposure to drugs of abuse would exacerbate neonatal immunity defects, facilitating HIV infection of neonate immune cells and promoting HIV vertical transmission. The availability and use of antiretroviral therapy for women infected with HIV increase, there is an increasing interest in determining the impact of drug abuse on efficacy of AIDS Clinical Trials Group (ACTG)-standardized treatment regimens for woman infected with HIV in the context of HIV vertical transmission.     

Immune responses and the emergence of drug-resistant virus strains in vivo

The treatment of viral infections using antiviral drugs has had a significant public health benefit in the setting of human immunodeficiency virus (HIV) infection, and newly developed drugs offer potential benefits in the management of other viral infections, including acute self-limiting infections such as influenza and picornaviruses (including the rhinoviruses that are responsible for a large proportion of 'common colds'). A serious concern with such treatments is that they may lead to the selection of drug-resistant strains. This has been a significant problem in the case of HIV infection. Existing mathematical-modelling studies of drug resistance have focused on the interactions between virus, target cells and infected cells, ignoring the impact of immune responses. Here, we present a model that explores the role of immune responses in the rise of drug-resistant mutants in vivo. We find that drug resistance is unlikely to be a problem if immune responses are maintained above a threshold level during therapy. Alternatively, if immune responses decline at a fast rate and fall below a threshold level during treatment (indicating impaired immunity), the rise of drug-resistant mutants is more likely. This indicates an important difference between HIV, which impairs immunity and for which immune responses have been observed to vanish during treatment, and viral infections such as influenza and rhinoviruses, for which such immune impairment is not present. Drug resistance is much more likely to be a problem in HIV than in acute and self-limiting infections.     

An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.     

Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 10⁸ M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.     

Complementary antiviral efficacy of hydroxyurea and protease inhibitors in human immunodeficiency virus-infected dendritic cells and lymphocytes

Dendritic cells are susceptible to human immunodeficiency virus (HIV) infection and may transmit the virus to T cells in vivo. Scarce information is available about drug efficacy in dendritic cells because preclinical testing of antiretroviral drugs has been limited predominantly to T cells and macrophages. We compared the antiviral activities of hydroxyurea and two protease inhibitors (indinavir and ritonavir) in monocyte-derived dendritic cells and in lymphocytes. At therapeutic concentrations (50 to 100 microM), hydroxyurea inhibited supernatant virus production from monocyte-derived dendritic cells in vitro but the drug was ineffective in activated lymphocytes. Concentrations of hydroxyurea insufficient to be effective in activated lymphocytes cultured alone strongly inhibited supernatant virus production from cocultures of uninfected, activated lymphocytes with previously infected monocyte-derived dendritic cells in vitro. In contrast, protease inhibitors were up to 30-fold less efficient in dendritic cells than in activated lymphocytes. Our data support the rationale for testing of the combination of hydroxyurea and protease inhibitors, since these drugs may have complementary antiviral efficacies in different cell compartments. A new criterion for combining drugs for the treatment of HIV infection could be to include at least one drug that selectively targets HIV in viral reservoirs.     

Drug resistance testing in PBMCs of HIV infected patients.

HIV infected patients are considered a sort of reservoir having different genetically distinct viral variants (quasispecies), that evolve from the starting virus inoculum. Frequently, during replication, HIV can generate nucleotide differences in the new viral population; such genetic changes may be uninfluential in viral "fitness" (replication capacity) or give the virus some advantages under a selective pressure, due to immune response or drug treatment. The use of potent combination therapy for the treatment of HIV infections has certainly improved the "quality of life" for patients, decreasing the viral load in the plasma (HIV RNA). In our study, we investigated whether detection of drug resistance-related mutations was possible in circulating PBMCs, which represent a sort of genetic archive of viral drug resistances, when the levels of viral RNA were reduced to below 400 or 50 copies/ml, since, generally, plasma samples with more than 1,000 copies/ml of HIV RNA are needed to generate some results. The study was successfully performed sequencing proviral HIV DNA in PBMCs from 32 samples belonging to 25 patients, using a new modified protocol, that showed a good reproduciblity and very interesting data, also in patients with low or without circulating HIV RNA levels.