Identification of HIV type 1 intersubtype recombinants in South Africa using env and gag heteroduplex mobility assays
Identification of HIV type 1 intersubtype recombinants in South Africa using env and gag heteroduplex mobility assays
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DOI：
10.1089/088922200309151
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发表时间：
2000-03-20
影响因子：
1.5
通讯作者：
Morris, L
中科院分区：
文献类型：
--
作者：
Bredell, H;Hunt, G;Morris, L
遗传多样性是 1 型人类免疫缺陷病毒 (HIV-1) 的主要特征。根据核苷酸序列分析，HIV-1 M 组病毒株在系统发育上分为 10 个不同的亚型，几乎所有亚型都在中非发现。 1 不同亚型之间的重组是 HIV-1 产生遗传多样性的重要手段。数据库中超过 10% 的完整基因区域或完整 HIV-1 基因组序列似乎是亚型间重组体，2 尽管研究表明非洲一些国家的流行率可能高达 25%。 3 在南非，该流行病以 C 亚型病毒为主，主要通过异性接触传播。 4, 5 一种规模较小的流行病，在 20 世纪 80 年代中期在同性恋/双性恋男性中达到顶峰，与 B 亚型和少数 D 亚型感染有关。 6 最近记录了两种 A 亚型感染。 5 此前的研究尚未揭示南非是否存在重组病毒。 7 在本研究中，开发了 gag 异源双链体迁移率测定 (HMA) 来补充 env HMA，以促进基因组两个区域中的 HIV 亚型确定并识别亚型间重组病毒。我们描述了南非 HIV-1 亚型 A、B 和 C 的鉴定以及相对较低的亚型间重组体流行率。 1995 年至 1998 年间，从代表不同 HIV 危险群体的 172 名 HIV-1 血清阳性个体采集了血液。除了来自夸祖鲁-纳塔尔省的七名商业性工作者外，所有样本均来自约翰内斯堡。在采集血液之前，所有研究对象都签署了知情同意书，并且对大多数患者进行了采访，以获取人口统计信息，包括传播途径和地点。具体来说，这包括患有肺结核和其他艾滋病定义疾病的患者、前往血友病或产前诊所就诊的个人、使用城市艾滋病诊所的移民和流动人员，以及接受私人医生护理的个人。该队列中男性 (n 5 84) 和女性 (n 5 88) 的人数相似，年龄从 18 岁到 59 岁不等。大多数人是通过异性接触感染的，其次是输血、同性性传播、静脉吸毒和职业接触。对 117 名个体进行了 CD41 淋巴细胞计数测定，其中 52% 的 CD41 T 细胞少于 200 个/ml，表明他们患有晚期疾病。该队列并不反映不同风险群体的 HIV 感染率，而是尝试对不同风险群体进行抽样。分离外周血单核细胞 (PBMC)，并用于扩增 gp120 V3-V5 区域中的 700 bp 产物，以便用于 env HMA，如前所述。 4 使用相同的细胞裂解物来扩增 520 bp 的产物，其中包含 100 bp 的长末端重复 (LTR) 区域和整个 p17 gag 基因。为此，外引物MSF12（59 AAATCTCTAGCAGTGGCGCCCGAACAG，位于HXB2基因组的622至648位）8和SK431（59 AGAGAACCAAGGGGAAGTGACATAGCAGG，位于1475至1503位）9和内引物LTR236（59 CGCAGGACTCGGCTTGC，位于688至648位）使用gag 778（位置1231至1254处的59 CACCTAGAACTTTAAATGCATGGG）10。在第一轮和第二轮扩增中，反应混合物中使用了 5 微升 DNA 输入，其中包含 10 pmol 的每种引物、200 m M dNTP、1.5 mM MgCl2 和 0.625 U 的 Super-Therm DNA 聚合酶以及提供的 103 缓冲液（Advanced Biotechnologies ...
GENETIC DIVERSITY is a major characteristic of the human immunodeficiency virus type 1 (HIV-1). On the basis of nucleotide sequence analysis the HIV-1 group M srains are phylogenetically grouped into 10 distinct subtypes, almost all of which have been found in Central Africa. 1 Recombination between different subtypes respresents an important means by which HIV-1 generates genetic diversity. More than 10% of full gene regions or full HIV-1 genome sequences in the database appear to be intersubtype recombinants, 2 although studies indicate that the prevalence in some countries in Africa may be as high as 25%. 3 In South Africa, the epidemic is dominated by subtype C viruses transmitted mainly through heterosexual contact. 4, 5 A smaller epidemic, which peaked in the mid-1980s among homosexual/bisexual men, is associated with subtype B and a few subtype D infections. 6 More recently two subtype A infections have been documented. 5 Previous studies have not revealed the existence of recombinant viruses in South Africa. 7 In this study a gag heteroduplex mobility assay (HMA) was developed to complement the env HMA to facilitate HIV subtype determination in two regions of the genome and to identify intersubtype recombinant viruses. We describe the identification of HIV-1 subtypes A, B, and C, and the relatively low prevalence of intersubtype recombinants, in South Africa. Between 1995 and 1998 blood was collected from 172 HIV-1-seropositive individuals representing different HIV risk groups. Except for seven commercial sex workers from KwaZulu-Natal, all samples were from Johannesburg. Signed informed consent was obtained from all study subjects prior to collection of blood and the majority of patients were interviewed for demographic information including route and place of transmission. Specifically, this included patients admitted with tuberculosis and other AIDS-defining illnesses, individuals attending hemophiliac or antenatal clinics, immigrant and mobile persons using urban AIDS clinics, as well as individuals under the care of private physicians. There was a similar number of men (n 5 84) and women (n 5 88) in the cohort, with ages ranging from 18 to 59 years. The majority of individuals were infected through heterosexual contact, followed by blood transfusion, homosexual transmission, intravenous drug use, and occupational exposure. CD41 lymphocyte counts were determined in 117 individuals, 52% of whom had fewer than 200 CD41 T cells/m l, indicating that they had advanced disease. This cohort does not reflect the HIV infection rates across the different risk groups, but rather an attempt to sample different risk groups.Peripheral blood mononuclear cells (PBMCs) were isolated and used to amplify an, 700-bp product in the gp120 V3–V5 region for use in an env HMA as described previously. 4 The same cellular lysates were used to amplify an, 520-bp product comprising 100 bp in the long terminal repeat (LTR) region and the whole p17 gag gene. For this, outer primers MSF12 (59 AAATCTCTAGCAGTGGCGCCCGAACAG at positions 622 to 648 of the HXB2 genome) 8 and SK431 (59 AGAGAACCAAGGGGAAGTGACATAGCAGG at positions 1475 to 1503) 9 and inner primers LTR236 (59 CGCAGGACTCGGCTTGC at positions 688 to 704) and gag 778 (59 CACCTAGAACTTTAAATGCATGGG at positions 1231 to 1254) 10 were used. Five microliters of DNA input was used in the firstand second-round amplification in a reaction mixture containing 10 pmol of each primer, 200 m M dNTPs, 1.5 mM MgCl2, and 0.625 U of Super-Therm DNA polymerase with supplied 103 buffer (Advanced Biotechnologies …