一名HIV/TB双重感染者的HIV分离和鉴定

1996年8月,我们采用改良的正常人PBMCs与病人PBMCs共培养法,从一名HIV/TB双重感染者的PBMCs中分离出1株HIV—1病毒,命名为GD—3.该毒株毒力强,在正常人PBMCs中培养能引起明显的细胞病变,其HIV—1p24抗原滴度超过阈值,但对H_9细胞不敏感.该毒株经基因序列分析为HIV-1E亚型,与广东省其它经性途径感染HIV人群中的HIV-1亚型相同.     

我国部分地区HIV-1流行株的分离培养

目的分离得到能够稳定传代的我国艾滋病病毒Ⅰ型（HIV-1）流行毒株,并进行生物学表型鉴定。方法分离、活化正常人的外周血单核细胞（PBMC）,分离病人的PBMC,采用PBMC共培养和全血共培养的方法分离病人的HIV-1,通过培养上清内P24抗原含量检测病毒的复制情况。同时,标本采集后测定CD产T淋巴细胞计数和病毒载量。结果从270例HIV-1标本中,分离到可稳定传代的强阳性毒株58株,总分离率为21．48％。病毒载量与分离率呈正相关;CD4^＋T细胞计数对分离率的影响没有显著性差异。病毒载量高的标本比病毒载量低的标本分离株的快高型比例大。提示毒株的生长动力学特征与病人的病毒载量相关。结论从我国5个地区的270份HIV-1感染者样本中,分离出58株原代分离毒株,丰富了我国HIV-1毒种库,为进一步开展相关的艾滋病防治研究提供了重要的材料,奠定了坚实的基础。     

福建艾滋病病毒的分离和微量全血分离法的建立

目的从HIV/AIDS病例血液中分离HIV并进行微量全血分离方法的研究.方法采集20份在福建发现的HIV1感染者肝素抗凝血,分离外周血单核细胞(PBMC),与健康人PBMC共培养,使用含神经氨酸酶(NA)的细胞培养液进行HIV-1的分离,建立微量全血分离法,通过检测p24抗原、间接免疫荧光试验(IFA)等确定病原分离结果.用MT4细胞感染试验分析病毒表型,并通过测定病毒分离株DNA的C2-V3区的核苷酸序列鉴定亚型.结果从20例HIV/AIDS病例PBMC标本中分离到18株HIV-1,分离率达90%,其中对HIV感染者的分离率为84.6%(11/13),对AIDS患者的分离率为100%(7/7).预刺激和同时刺激PBMC共培养对分离结果没有明显影响,可从10～125μl感染者全血中分离出病毒,与大量法分离比较,结果没有明显差异.18株病毒分离株只有1株可在MT4细胞中稳定传代,其它为M嗜性株;HIV-1A亚型1株,B亚型3株,E亚型13株,E亚型基因离散率为13.724±3.595.结论添加NA可能有助于提高HIV的分离率,微量全血分离法可用于病毒的分离,福建病毒分离株主要为HIV-1E亚型的M嗜性慢低复制株.     

有限稀释病人PBMCs共培养法分离培养HIV-1 CRF07_BC生物性克隆病毒

目的通过有限稀释艾滋病病毒(HIV)感染者外周血单个核细胞(PBMCs)与正常供体PBMCs共培养,分离培养HIV-1生物性克隆毒株。方法选择6位新疆HIV-1CRF07_BC感染者,分离病人PBMCs,以不同浓度的病人PBMCs细胞数梯度(推荐浓度分别为5×103、2×104、4×104/孔)与健康供体的1×105PBMCs共培养,每个浓度设置32个复孔,每周检测HIV p24抗原。当某一浓度的复孔中p24阳性率低于33%时,可认为在这些阳性孔中的病毒是起源于同一个感染细胞。同时进行env区V1-V5基因扩增、测序和分析。结果经过有限稀释法共培养后,样本XJZK007在1×104细胞/孔的浓度下,各复孔HIV-1p24阳性率为31.3%;样本CBJB539在4×104细胞/孔的浓度下,得到9.4%分离阳性率;样本CBJB540在2×104细胞/孔的浓度下,得到25%分离阳性率;样本CBJB541在2×104细胞/孔的浓度下,得到12.5%分离阳性率;样本CBJB543在1×104细胞/孔的浓度下,得到21.9%分离阳性率;样本CBJB544在2×104细胞/孔的浓度下,得到25%分离阳性率。同一样本分离的不同生物克隆表现出不同的生物表型。对样本XJZK007分离得到的12株生物克隆的序列分析显示,各序列相互之间存在氨基酸的变异、插入及缺失,从而验证了生物性克隆方法的正确性与可行性。结论有限稀释病人PBMCs共培养方法,以常规分离方法十分之一的细胞数即可分离得到个体内多样性的病毒,即生物克隆病毒。     

蕨麻提取物的体外抗HIV-1活性及毒性研究

目的研究蕨麻提取物的抗艾滋病病毒Ⅰ型(HIV-1)活性及毒性。方法用HIV-1实验室适应株SF33、临床分离株XJDC257和020100968、假病毒颗粒9-14,18-36,74-2和Z20-11分别感染TZM-bl、MT-4,利用基于TZM-bl细胞的荧光素酶检测体系和基于MT-4细胞的P24抗原检测方法,观察蕨麻提取物抑制HIV-1病毒复制的活性;用不同稀释度的蕨麻提取物与TZM-bl.MT-4.PBMCs共培养,使用Cell Counting Kit-8检测活细胞的数量,观察蕨麻提取物对相应细胞的毒性作用。结果利用MT-4细胞和TZM-bl细胞测得蕨麻提取物对SF33的半数抑制浓度(IC50)及选择性指数(SI)分别为6.2μg/mL,26.4和4.7μg/mL,73.5。蕨麻提取物对HIV-1临床分离株XJDC257和020100968的IC50和治疗指数(TI)分别为2.1μg/mL,70.6和1.9μg/mL,77.6;对HIV-1假病毒颗粒9-14、18-36、74-2和Z20-11的IC50分别为1.8μg/mL、1.0μg/mL、3.4μg/mL和3.5μg/mL,TI分别为81.9、147.5、43.4和42.1。结论蕨麻提取物在体外具有一定的抑制HIV-1复制活性。     

R5嗜性的人类免疫缺陷病毒-1在疾病不同阶段的生物学特性

目的 研究HIV-1感染者不同时期分离的R5毒株的生物学特性.方法 采用传统的共培养方法分离并培养HIV-1,用表达CD4和CC趋化因子受体5(CCR5)或CXC趋化因子受体4(CXCR4)的GHOST细胞系,通过流式细胞仪测定病毒辅助受体的利用和感染性,从而判断所分离毒株的CCR5嗜型(R5型毒株);使用2 ng P24病毒量感染正常人分离的外周血单个核细胞(PBMC),ELISA法检测第1、3、5、7、10、15天的HIV-1 P24抗原,反映病毒复制能力;采用HIV-1核酸荧光定量检测试剂盒测定血浆病毒载量.数据分析采用t检验.结果 HIV-1B'亚型感染者22例,其中CD4+细胞>0.2×109/L和CD4+细胞≤0.2×109/L各11例;所分离的病毒仅利用CCR5辅助受体,均为R5型毒株,感染性的结果显示,来自CD4+细胞≤0.2×109/L的11株R5毒株的感染性为(7.392 7±4.584 2)%,而CD4+细胞>0.2×109/L的为(2.613 6±1.610 5)%,差异有统计学意义(t=3.262,P<0.05);两组病毒复制滴度在第7天开始明显上升,培养第7、10、15天,两组病毒复制动力学差异有统计学意义(t值分别为3.771、2.509和2.260;P<0.05),CD4+细胞≤0.2×109/L的R5毒株的复制能力较CD4+细胞>0.2×109/L的明显增强;CD4+细胞≤0.2 × 109/L R5型毒株的病毒载量的对数值为(5.606 8±0.815 1)拷贝/mL,CD4+细胞>0.2 × 109/L的为(4.729 8±0.431 6)拷贝/mL,两组差异有统计学意义(t=3.771,P<0.05).结论 疾病进展过程中,即使病毒的辅助受体利用未从CCR5转变为CXCR4,但病毒的感染性和复制能力已有明显改变.     

重庆市HIV-1流行毒株的基因序列测定和亚型分析

目的对重庆市流行的艾滋病（AIDS）病毒1型（HIV-1）进行分子流行病学调查,为艾滋病预防控制策略的制定提供依据.方法用套式聚合酶链反应（nested-PCR）对31份重庆市HIV-1抗体阳性者外周血单个核细胞（PBMC）中前病毒DNA的膜蛋白基因的C2-V3区,及其邻区300个核苷酸序列进行测定和分析.结果31份标本中有21份扩增阳性并得到相应序列,扩增率为67.6%.经过基因离散率计算和系统树分析后证实：1份标本为CRF01-AE流行重组毒株,20份标本为HIV-1 BC重组毒株.结论目前重庆市HIV-1 BC重组病毒为优势流行株,为有效控制HIV流行,需要重点在吸毒人群中开展行为干预措施.     

九江市HIV-1流行毒株基因序列测定和亚型分析

目的了解九江市艾滋病病毒1型(HIV-1)流行毒株的亚型分布情况。方法对采集的6份九江市HIV-1感染者的血浆样品进行RT-PCR扩增,获得gag基因的核酸片断进行核苷酸序列分析。结果在6份样品中发现B’亚型HIV-1毒株4株,B亚型和A/E重组亚型HIV-1毒株各1株。结论HIV-1流行毒株亚型在九江市分布情况复杂,防控形势严峻。     

HIV-1感染、致病机理和人体CCR5、CCR2等基因多态性之间的相互关系研究进展

1 概述 HIV-1感染人体后引起进行性的AIDS病变,通常经历三个典型的时期,第一为急性感染期,表现为广泛的病毒血症;接着是无症状期,此时在淋巴器官中大多可检测出病毒的复制;最后是免疫系统破坏,并伴随病毒血症的再次出现,并产生继发性感染等而死亡。引起AIDS的病毒株有以下几种:(1)嗜巨噬细胞HIV-1株:它主要通过性传播,从初期感染者体内分离到的原代病毒,它感染细胞后不诱生合胞体(syncytia);(2)嗜T细胞HIV-1病毒株:是从发病的AIDS病人身上来源的或实验室培养的毒株。它的毒性很强,感染后,病情呈进行性发展;(3)双嗜性HIV-1病毒株,即感染巨嗜细胞,又感染T细胞。     

甘肃省HIV-1毒株基因序列测定和亚型分析

目的对甘肃省流行的艾滋病病毒（HIV）开展分子流行病学调查.方法用套式聚合酶链反应对23份甘肃省HIV-1感染者外周血淋巴细胞中前病毒脱氧核糖核酸（DNA）的膜蛋白基因进行扩增,并对C2-V3及其邻区300个核苷酸序列进行测定和分析.结果甘肃省流行的HIV-1分属B＇亚型（6例）、C亚型（1例）、CRF-BC亚型（16例）.其中CRF-BC亚型（70%）为主要流行毒株.结论甘肃省HIV-1流行亚型以重组毒株CRF-BC为主,在吸毒人群中广为流行;加强对高危人群监控,是遏制艾滋病在甘肃省蔓延的重中之重.     

CCR5 is the major coreceptor used by HIV-1 subtype C isolates from patients with active tuberculosis.

Tuberculosis (TB) is the major opportunistic infection of HIV-infected patients in developing countries and is associated with activation of the immune system and increased HIV-1 expression. The aim of this study was to explore the biological properties of HIV-1 isolates from patients with active TB. Ten HIV-1 subtype C isolates were analyzed for biological phenotypes, using MT-2 cells, and for coreceptor usage, using coreceptor-transfected cell lines. All isolates were nonsyncytium inducing (NSI) in the MT-2 assay and replicated in CCR5-expressing cells. None of the isolates used CXCR4 or any of the minor coreceptors (CCR1, CCR2b, or CCR3) efficiently. Analysis of the V3 region showed that all isolates contained the GPGQ motif characteristic of subtype C and also had a sequence profile typical of NSI viruses. These data indicate that despite their advanced disease state, patients with TB harbor viruses that use the CCR5 coreceptor. It is possible that activation of monocytes and macrophages during TB infection results in the expansion of macrophage-tropic isolates that preferentially use CCR5.     

陕西省HIV-1流行株膜蛋白基因 C2-V3区序列特征和亚型分析

目的了解陕西省HIV-1毒株的流行情况和亚型特征.方法用套式聚合酶链式反应(Nested-PCR)对6份采集于陕西省经确认为HIV-1感染者或艾滋病病人外周血淋巴细胞(PBMC)提取核酸,从6份样品中获得了HIV-1膜蛋白(ENV)基因的核酸片段进行扩增,并测定和分析了C2-V3及其邻区共312个核苷酸序列.结果 6份血样中,2份为HIV-1A亚型毒株感染(SHX7、SHX8);4份为HIV-1B'亚型(泰国B'亚型)毒株感染(SHX1、SHX5、SHX6、SHX9).SHX1、SHX5、SHX6、SHX9彼此间基因离散率为5.62%,SHX7与来自卢旺达的U08794之间的基因离散率仅为2.41%.结论陕西省目前存在HIV-1 A、B'两种亚型的毒株,HIV-1 B'亚型由邻近地区传入,HIV-1 A亚型为与非洲人接触传入.     

Phenotypic characteristics of human immunodeficiency virus type 1 subtype C isolates of Ethiopian AIDS patients.

It has been estimated that, to date, about 48% of all HIV-infected people in the world carry HIV-1 subtype C virus. Therefore, it is of great importance to gain better knowledge about the genetic and biological characteristics of this virus subtype. In the present study, the biological properties of HIV-1 isolates obtained from nine Ethiopian patients with AIDS were studied. DNA sequencing of the V3 loop of gp120 classified the isolates as subtype C. In primary isolation cultures, virus infection was accompanied by syncytium formation and cell lysis. Interestingly, when examining the growth in primary monocyte-macrophage cultures, initial low-level virus replication was followed by a nonproductive state, from which virus could be rescued by cocultivation with Jurkat(tat) cells. Furthermore, none of the isolates replicated in T cell lines (CEM, MT-2, HuT-78, and H9) or in the promonocytic cell line U937 clone 2. All isolates could use CCR5 as coreceptor, whereas no isolates could use CCR2b, CCR3, CCR5, CXCR4, Bonzo/STRL33, or BOB/GPR15. The genotype of the V3 region correlated with the MT-2 negative/non-syncytium-inducing (NSI) phenotype. Comparative studies revealed that the scarcity of CXCR4 usage as well as other phenotypic characteristics of subtype C isolates distinguish this subtype. On the basis of these data, we suggest that in addition, factors other than viral phenotype may govern the pathogenic potential of subtype C isolates.     

HIV-1 biological phenotype in long-term infected individuals evaluated with an MT-2 cocultivation assay.

OBJECTIVE: We have previously demonstrated that detection of syncytium-inducing (SI) HIV-1 in asymptomatic seropositive individuals is associated with rapid progression to AIDS. In the present study, we sought to develop and evaluate an HIV-1 phenotyping assay for the screening of large numbers of individuals. METHODS: Efficiency of HIV-1 isolation from patient peripheral blood mononuclear cells (PBMC) was studied with donor PBMC or seven different CD4+ T-cell lines as target cells. The biological phenotype of sequential isolates from 20 long-term asymptomatic HIV-1-seropositive individuals was determined by two different assays. RESULTS: Non-SI isolates, efficiently recovered by cocultivation with donor PBMC, were never isolated with T-cell lines as target cells. Direct cocultivation with MT-2 cells, but not with six other CD4+ T-cells, resulted in the efficient recovery of SI isolates. HIV-1 MT-2 tropism and SI capacity were shown to be coupled properties at the clonal level. SI isolates emerged in 10 out of 20 longitudinally-studied individuals. In these long-term infected individuals, appearance of SI isolates was associated with progression to AIDS. CONCLUSIONS: Direct cocultivation of patient PBMC with the MT-2 cell line is a sensitive, specific and convenient method to detect SI isolates. The availability of an assay suitable for the screening of large groups allows further study of the value of HIV-1 biological phenotyping as a prognostic marker.     

Length variation of glycoprotein 120 V2 region in relation to biological phenotypes and coreceptor usage of primary HIV type 1 isolates.

Conflicting data have been published concerning the correlation between the length of the second variable region (V2) in the HIV-1 envelope and the biological phenotype of the virus. Here the V2 region length of primary HIV-1 isolates was compared with biological phenotype and coreceptor usage. The V2 region variation was determined by DNA fragment length analysis, virus biological phenotype by the MT-2 cell assay, and coreceptor usage by infection of U87.CD4 cells expressing CCR3, CCR5, or CXCR4. Ninety-three primary virus isolates from 40 patients were analyzed. This panel of viruses included sequential isolates obtained from patients who progressed to AIDS with or without a virus phenotypic switch. We found that NSI MT-2-negative isolates had significantly shorter V2 regions than SI MT-2-positive isolates. However, when V2 region lengths of viruses were analyzed in more detail, we observed that NSI isolates obtained from patients shortly before the phenotypic switch had V2 region lengths similar to those of SI isolates. V2 regions of NSI isolates obtained from patients who progressed to AIDS without a virus phenotypic switch had, in contrast, shorter V2 region than isolates obtained just before virus phenotypic switch. Coreceptor analysis revealed that CCR5-using (R5) isolates generally had shorter V2 regions than virus isolates with the ability to enter CXCR4-expressing cells. Moreover, no significant difference in V2 region length was observed between monotropic SI isolates, that is, X4 isolates, and multitropic SI isolates, that is, R3R5X4 or R5X4 isolates. Thus, we conclude that R5 NSI isolates obtained from patients with stable virus phenotype through the whole disease course display shorter V2 regions than isolates obtained from patients at switch of virus phenotype, suggesting that V2 region length may influence virus coreceptor usage.     

Biological characterization and chemokine receptor usage of HIV type 1 isolates prevalent in Brazil

The human immunodeficiency virus type 1 (HIV-1), the etiological agent of the acquired immunodeficiency syndrome (AIDS), shows a variety of biological properties, which may constitute an obstacle to development of effective vaccines or antiretroviral therapy. To characterize Brazilian strains of HIV-1, we studied 24 viruses isolated from blood samples of HIV-1-positive patients from different regions of the country. To examine the cell tropism and the virus ability to form syncytia, primary macrophages and the CD4+ T cell line MT-2 were infected with these viruses. We found that 22 isolates replicated well in macrophages (macrophage-tropic isolates), 2 infected only MT-2 cells (T cell line tropic variants), while 6 of them grew in both cells. We found 8 syncytium-inducing (SI) and 16 non-SI (NSI) isolates. Continuous cultures of 18 isolates were established in the CCR5+/CXCR4+ cell line PM-1, and SI/NSI features of these viruses were confirmed by cell fusion assay with uninfected CD4+ T cell lines (PM-1, MT-2, H9, and SUP-T1). The coreceptor usage of 18 isolates was investigated by infecting U87 cells transfected with CD4 and chemokine receptors, and we found that 11 isolates infected only CCR5+ cells, 3 only CXCR4+ cells, whereas 4 used both coreceptors. We also observed that X4 isolates were more sensitive to neutralization by dextran sulfate than R5 or R5X4 viruses. Our findings show that the Brazilian isolates are phenotypically similar to those prevalent in other regions, which could mean that therapeutic strategies based on HIV-1 phenotypic properties would be efficient in Brazil, as in other countries.     

HIV-1 B'/C亚型毒株的复制动力学特点

目的分析不同疾病进展阶段B'/C亚型毒株的复制动力学特点。方法从长期不进展者(long-term nonprogressors,LTNPs)和AIDS期患者体内分离病毒株,用相同量的病毒株去感染正常人的去CD8+T淋巴细胞的外周血单个核细胞,通过检测感染后第0、3、6、9、12、18、21天的上清液中核衣壳蛋白p24(p24)含量,作出折线图来分析该毒株的复制动力学特征。结果从AIDS期患者体内分离的B'/C重组亚型病毒株的复制速度快且p24峰值高,而从LTNPs体内分离的B'/C重组亚型病毒株的复制速度慢且p24峰值低。结论从AIDS期患者体内分离的B'/C重组亚型病毒株复制能力较强,而从LTNPs患者体内分离的B'/C重组亚型病毒株复制能力较弱。