我国西南西北地区吸毒人群重组人类免疫缺陷病毒1？…

目的 寻找人类免疫缺陷病毒１型在中国可能的重组。方法从流行２种以上ＨＩＶ－１亚型的地区收集ＨＩＶ感染者的血样。从ＰＭＣｓ中应用套式聚合酶链反应方法,对ＨＩＶ病毒的ｔａｔ和ｅｎｖ基因进行扩增,ＰＣＲ产物直接测序并进行序列分析。结果 对中国Ｂ’亚型和Ｃ亚型浒区域收集的１４个ＨＩＶ－１毒株进行序列分析,对ｅｎｖ基因进行测序后没有发现重组毒株的证据。     

山东省某些高危人群HIV-1感染者分子流行病学研究

目的 了解流行于山东境内ＨＩＶ－１毒株的亚型分布及变异情况,分析其来源并推测其流行趋势。方法 采集了２５份ＨＩＶ－１抗体阳性感染者的全血分离单核细胞（ＰＢＭＣ）,提取前病毒ＤＮＡ,经ｎｅｓｔｅｄ－ＰＣＲ扩增ＨＩＶ－１膜蛋白（ｅｎｖ）基因的Ｃ２－Ｖ３区并进行序列测定和亚型分析。结果 ２５例ＨＩＶ－１阳性感染者的ＰＢＭＣ样品中扩增到２４份可用于序列测定的ＨＩＶ－１ｅｎｖ基因片段,经序列测定和基因分析鉴     

我国西南西北地区吸毒人群重组人类免疫缺陷病毒1型毒株的发现

目的寻找人类免疫缺陷病毒１型（ＨＩＶ１型）在中国可能的重组。方法从流行２种以上ＨＩＶ１亚型的地区收集ＨＩＶ感染者的血样。从ＰＭＣｓ中应用套式聚合酶链反应（ＰＣＲ）方法,对ＨＩＶ病毒的ｔａｔ和ｅｎｖ基因进行扩增,ＰＣＲ产物直接测序并进行序列分析。结果对中国Ｂ′亚型和Ｃ亚型流行区域收集的１４个ＨＩＶ１毒株进行序列分析,对ｅｎｖ基因进行测序后没有发现重组毒株的证据。对ｔａｔ基因的第一外显子进行序列分析时,１４个样品中的１０个样品发现了Ｂ′亚型和Ｃ亚型重组的ＨＩＶ１毒株。此外,四川省静脉吸毒者中发现了３个非重组的Ｂ′亚型毒株和１个非重组的Ｃ亚型毒株。结论首次在中国西南部的四川省和西部的新疆维吾尔自治区发现了Ｂ′亚型和Ｃ型的重组ＨＩＶ１毒株。相同的序列和重组方式,表明重组毒株具有相同的起源,同时表明这两个艾滋病流行区域密切相关。由于在新疆只发现了重组毒株,而在四川省则发现了Ｂ亚型、Ｃ亚型和Ｂ′／Ｃ重组毒株,重组很有可能发生在四川而不是新疆。     

克隆中国B,C,E亚型的HIV—1代表株建立适于我国流行株的 …

目的 构建我国人免疫缺陷病毒１型（ＨＩＶ－１）Ｂ、Ｃ和Ｅ各亚型代表株ｅｎｖ基因质粒,用于对中国ＨＩＶ－１进行分型。方法 将与我国ＨＩＶ－１ Ｂ,Ｃ和Ｅ各亚型共享序列最接近的毒株用套式聚合酶链反应（ＰＣＲ）技术扩增包膜（ｅｎｖ）基因,克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中,构建成用于异源双链泳动分析（ＨＭＡ）分型的中国标准亚型质粒,并进行了序列分析和分型的敏感性分析。结果 构建的中国ＨＩＶ－１ Ｂ,Ｃ和Ｅ     

云南省陇川县HIV-1流行毒株膜蛋白基因C2-V3区序列测定和分析

使用ＰＣＲ对２１份采集于１９９５年中的云南陇川县ＨＩＶ－１阳性静脉吸毒者外周血单个核细胞（ＰＢＭＣｓ）样品进行扩增，从１７份样品中获得了ＨＩＶ－１膜蛋白（ｅｎｖ）基因的核酸片段，并对其Ｃ２－Ｖ３及邻区４５０个核苷酸序列进行了测定和分析。研究结果表明，１７份陇川样品中存在Ｂ和Ｃ两种亚型的ＨＩＶ－１毒株序列，各亚型内的基因离散率分别为４．７％和３．３％。与Ａ～Ｅ参考亚型及部分Ｂ和Ｃ亚型代表株序列相比较，属陇川Ｂ亚型的１４个毒株与包括泰国、缅甸及云南瑞丽在内的Ｂ亚型毒株序列十分接近，基因离散率在３．９％～４．５％的范围内；属陇川Ｃ亚型的３个毒株则与代表印度毒株的Ｃ亚型共享序列及瑞丽Ｃ亚型毒株序列十分相似，其基因离散率均为２．５％。以上数据提示，ＨＩＶ－１在陇川的流行时间不长，且Ｂ和Ｃ亚型毒株的传入时间相差１年左右。对Ｂ亚型毒株Ｖ３环序列的分析还发现，位于Ｖ３环顶端的四肽序列中ＧＰＧＱ占６４．３％，ＧＰＧＲ则仅占２８．６％，且编码其精氨酸（Ｒ）的密码子均为ＣＧＡ而不是ＡＧＡ。此结果与作者根据早期瑞丽ＨＩＶ－１毒株序列研究结果得出的推测相吻合。     

云南省陇川县HIV—1流行毒株膜蛋白基因C2—V3区序列测定…

使用ＰＣＲ对２１份采集于１９９５年中的云南陇川县ＨＩＶ－１阳性静脉吸毒者外周血单个核细胞（ＰＢＭＣｓ）样品进行扩增，从１７份样品中获得了ＨＩＶ－１膜蛋白（ｅｎｖ）基因的核酸片段，并对其Ｃ２－Ｖ３及邻区４５０个核苷酸序列进行了测定和分析。研究结果表明，１７份陇川样品中存在Ｂ和Ｃ两种亚型的ＨＩＶ－１毒株序列，各亚型内的基因离散率分别为４．７％和３．３％。与Ａ￣Ｅ参考亚型及部位Ｂ和Ｃ亚型代表株序列相比较     

深圳市HIV-1 E亚型感染毒株的分子流行病学分析

目的 了解人类免疫缺陷病毒１型Ｅ亚型毒株在深圳市不同人群中流行传播情况、流行时间和传播规律。方法 应用聚合酶链反应（ＰＣＲ）方法对１９９６年深圳市检出的３份人类免疫缺陷病毒１型（ＨＩＶ－１）感染者外周血单个核细胞样本进行扩增,获得ＨＩＶ－１膜蛋白（ｅｎｖ）基因片段,并对Ｃ２－Ｖ３及其邻区３５０～４５０个核苷酸序列进行测定和分析。结果 这３份血样为ＨＩＶ－１Ｅ亚型毒株感染（ｓｚ－Ｅ）,彼此间的基因离散率为２．６％;与Ａ－Ｅ国际参考亚型及国内部分地区流行的ＢＥ型代表株比较,ｓｚ－Ｅ与Ａ－Ｄ参考亚型共享序列及国内Ｂ亚型代表株间的基因离散率均大于２４％,而与主要代表泰国Ｅ亚型（Ｅｃｏｎ）间的基因离散率仅为６．２％。系统树分析显示,ｓｚ－Ｅ与Ｅｃｏｎ聚集在一起,远离其他国际亚型毒株序列。结论 ＨＩＶ－１Ｅ亚型在深圳的流行     

1990～1997年云南IDUsHIV—1B亚型分离株膜蛋白V3环 …

目的 观察云南静脉药瘾（ＩＤＵｓ）ＨＩＶ－１分离株ｅｎｖ基因Ｖ３环顶端四肽氨基酸和相应核苷酸序列随时间推移的变化。方法 根据１９９０～１９７年间６２株ＨＩＶ－１分离株ｅｎｖ基因Ｃ２－Ｖ３区ＤＮＡ序列，对ＨＩＶ－１膜蛋白Ｖ３环顶端四肽基因序列（基序）及其编码核苷酸进行分析，并探讨其随时间推移的变化趋势。结果 １９９０～１９９７年间６２株ＨＩＶ－１毒株膜蛋白Ｖ３环顶端四肽基序有程度不同的氨基酸变异，主     

应用PRPL串联启动子表达HIV-2gag蛋白

探讨ＨＩＶ－２ｇａｇ非融合蛋白的表达。方法：应用ＤＮＡ重组技术,将ＨＩＶｇａｇ基因全序列（ｇａｇ）和部分（ｇａｇ’）ｃＤＮＡ片段克隆到ｐＢＶ２２０载体ＰＲＰＬ串联启动子下游,构建成ＨＩＶ－２ｇａｇ重组表达载体ｐＢＶ－ｇａｇ和ｐＶＢ－ｇａｇ’,在大肠杆菌中表达。结果：ＳＤＳ－ＰＡＧＥ显示,ＰＢＶ－ｇａｇ’在２１ｋＤ处可见一明显的客外蛋白带,经薄层扫描分析占菌体总蛋白的６．１％,蛋白印迹结果证明,表达     

我国云南省艾滋病病毒感染的长期不进展者人免疫缺陷 …

目的 人类免疫缺陷病毒１型（ＨＩＶ－１）ｔａｔ基因是该病毒的调控基因之一。本研究是探讨ｔａｔ基因变异是否影响ＨＩＶ－１感染者的病程进展。方法 从云南ＨＩＶ流行区的２２例感染后临床进程不同的人抽取外周血,提取核酸,用套式聚合酶链反应（ＰＣＲ）扩增ＨＩＶ－１的ｔａｔ基因,并进行了核酸序列测定和分析。     

High genetic diversity of HIV-1 strains in Chad,West Central Africa

The genetic diversity of HIV-1 strains in Chad was documented with a total of 107 samples from patients attending the general hospital in N'Djamena, the capital city of Chad. The genetic subtypes were identified in the V3-V5 env and p24 gag regions by sequence and phylogenetic tree analyses. Of the 107 strains, 78 had the same subtype/CRF designation between env and gag. Four subtypes and three CRFs were found to cocirculate: subtype A, 20.5%; subtype D, 18.7%; CRF02_AG, 13.1%; CRF11_cpx, 13.1%; subtype G, 3.7%; CRF01_AE, 2.8%; and subtype F1, 0.9%. The remaining 29 strains (27%) had discordant subtypes or CRF designations between env and gag; in 15 of these 29 strains, a CRF was involved in the recombination event, and 10 were subtype G in gag and subtype A in env, forming a separate subcluster within subtypes G and A. Subtype D strains represent almost 20% of the HIV-1 strains circulating in Chad and form a separate subcluster in gag and env. Nearly full-length genome sequencing for two such strains (99TCD-MN011 and 99TCD-MN012) revealed that they represent nonrecombinant subtype D variants. Compared with neighboring countries, the genetic subtype distribution of HIV-1 strains in Chad is unique for several reasons: lower prevalence of CRF02, high prevalence of CRF11 and subtype D, and absence of CRF06. These data clearly show that subtype distribution is very heterogeneous in Africa, probably the result of different founder effects.     

Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus type 1 in South Africa

A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A-D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n = 33); or heteroduplex mobility assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.     

Independent introduction of transmissible F/D recombinant HIV-1 from Africa into Belgium and The Netherlands

Most HIV-1 subtype F viruses described so far have been isolated from individuals originating in South America, Romania, or Central Africa. Previous studies have shown that subtype F viruses from these three areas can be distinguished by phylogenetic tree analysis of various parts of the HIV genome. Subtype F strains circulating in Central Africa and classified as subgroup F2 and F3 have relatively large nucleotide distances from strains of subgroup F1, which includes some African strains, along with strains from Romania and South America. Subtype F strains have now appeared in Europe. In this study, we analyzed the complete gag gene and a large fragment of the pol gene of seven strains of African origin that represent the three F subgroups. At least five of the seven strains appear to be intersubtype recombinants. Of four strains circulating in Belgium and the Netherlands, three were F/D mosaics and the fourth harboured a G(gag)/GH(pol)/F3(env) recombinant structure. Two of the three F/D mosaics showed identical breakpoints and were independently introduced in Belgium and the Netherlands. At least two of the mosaics were further transmitted. The remaining three strains of the seven we studied were isolated from individuals in Cameroon. Two included large or smaller F1 fragments in gag and pol. The third strain was subtype D along the entire gag and pol fragment. A parental African subtype F that showed no evidence for recombination was not found.     

Determination of human immunodeficiency virus type 1 subtypes in Taiwan by vpu gene analysis

The genetic diversity of human immunodeficiency virus (HIV) type 1 (HIV-1) has been characterized mainly by analysis of the env and gag genes. Information on the vpu genes in the HIV sequence database is very limited. In the present study, the nucleotide sequences of the vpu genes were analyzed, and the genetic subtypes determined by analysis of the vpu gene were compared with those previously determined by analysis of the gag and env genes. The vpu genes were amplified by nested PCR of proviral DNA extracted from 363 HIV-1-infected individuals and were sequenced directly by use of the PCR products. HIV-1 subtypes were determined by sequence alignment and phylogenetic analysis with reference strains. The strains in all except one of the samples analyzed could be classified as subtype A, B, C, E, or G. The vpu subtype of one strain could not be determined. Of the strains analyzed, genetic subtypes of 247 (68.0%) were also determined by analysis of the env or gag gene. The genetic subtypes determined by vpu gene analysis were, in general, consistent with those determined by gag and/or env gene analysis except for those for two AG recombinant strains. All the strains that clustered with a Thailand subtype E strain in the vpu phylogenetic analyses were subtype E by env gene analysis and subtype A by gag gene analysis. In summary, our genetic typing revealed that subtype B strains, which constituted 73.8% of all strains analyzed, were most prevalent in Taiwan. While subtype E strains constituted about one-quarter of the viruses, they were prevalent at a higher proportion in the group infected by heterosexual transmission. Genetic analysis of vpu may provide an alternate method for determination of HIV-1 subtypes for most of the strains, excluding those in which intersubtype recombination has occurred.     

Molecular epidemiology of HIV-1 in Santa Catarina State confirms increases of subtype C in Southern Brazil

Recent studies have demonstrated an increased prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C in southern Brazil. Although Santa Catarina State (SC) is located in this area and presents one of the country's highest incidences of HIV/AIDS, knowledge on the molecular epidemiology of HIV-1 in such State is lacking. The aim of this study was to investigate the HIV-1 molecular diversity and epidemiological profile of HIV-1-infected patients from SC. DNA samples were PCR amplified and HIV-1 subtypes were determined using both env and gag genes by direct sequencing. Phylogenetic analyses revealed that 48% were subtype C and 23% were subtype B. Possible recombinant forms were observed for both B/C (23%) and B/F (6%) subtypes. Our results, for the first time, identifies HIV-1 subtype C as a major clade circulating in SC and contributes to the understanding of HIV epidemics in the country by confirming the epidemic spread of the HIV-1 subtype C in southern Brazil.     

Cross-clade protection induced by human immunodeficiency virus-1 DNA immunogens expressing consensus sequences of multiple genes and epitopes from subtypes A, B, C, and FGH

The correlate of protection in human immunodeficiency virus (HIV) infection is not known, but preclinical and clinical studies support the involvement of both antibodies and cellular immunity. In addition, the existence of multiple HIV clades makes HIV vaccine design especially challenging. We have constructed a vaccine platform with an HIV-1 subtype B DNA immunogen expressing full length consensus sequences from HIV-1 rev, nef, tat, and gag with additional cellular epitope clusters from the env and pol regions. Furthermore, this platform has been extended to three additional plasmids expressing the same immunogens but originating from subtypes A or C consensus or FGH ancestral sequences. Immunogenicity in BALB/c mice, by gene gun or intramuscular delivery, revealed strong IFN-gamma production in response to in vitro re-stimulation with a H-2d restricted gag peptide (AMQMLKETI) or even stronger toward an env epitope (RGPGRAFVTI). Weak humoral immunity was detected. Gene gun immunization with a cocktail of all four plasmids induced pre-challenge cellular immunity in C57Bl6/A2.01 mice and subsequently a robust frequency of protection (11/12 animals) after experimental challenge with subtype A or B HIV-1/Murine Leukemia Virus (HIV-1/MuLV). The cross-clade protection observed in this challenge experiment demonstrates that these multigene/multiepitope HIV DNA immunogens are likely to be potent immunogens also against the HIV-infection of human beings.     

HIV-1型中国株E、B亚型gag基因的克隆与表达

目的 克隆和表达人免疫缺陷病毒（ＨＩＶ）Ⅰ型中国株Ｅ、Ｂ亚型代表株的结构基因ｇａｇ。方法 在分子流行病学调查的基础上，选择１份经部分ｇｐ１２０基因测序判定为Ｅ亚型和１份经部分ｇｐ１２０基因测序判定为Ｂ亚型的代表性样品。利用套式聚合酶链反应（ＰＣＲ），扩增外周血中的前病毒。获得了结构基因ｇａｇ全长片断，并先后克隆到ｐｌｉｎ８Ｐｒ５５和ｐＦａｓｔＢａｃｌ载体中，我们首次克隆到全长的中国株Ｅ亚型ｇａｇ基     

A gag gene heteroduplex mobility assay for subtyping HIV-1

A heteroduplex mobility assay (HMA) using 753 and 446 base pair (bp) amplicons of the p17/p24 region of the gag gene of HIV-1 has been developed and validated with reference clones and clinical samples representative of subtypes A, B, C, D, E, G, and H. There was complete concordance between the gag HMA assigned subtype and the subtype known from gag or env sequence data or env HMA. The heteroduplexes from both amplicons can be clearly resolved on either MetaPhor XR agarose or MDE polyacrylamide gels. The MetaPhor XR gel system was the more convenient and is the preferred choice for routine HMA subtyping. This gag HMA provides a rapid, simple and inexpensive method for subtyping HIV-1 based on a genomic region other than the commonly used env gene target. The incorporation of gag HMA into subtype determination algorithms should allow the detection of gag/env recombinant strains of HIV-1.     

Serotyping and genotyping of HIV-1 infection in residents of Khayelitsha, Cape Town, South Africa

It is estimated that between 5.5 and 6.1 million people are infected with HIV/acquired immunodeficiency syndrome (AIDS) in South Africa, with subtype C responsible for the majority of these infections. The Khayelitsha suburb of Cape Town has one of the highest HIV prevalence rates in South Africa. Overcrowding combined with unemployment and crime in parts of the area perpetuates high-risk sexual behavior, which increases exposure to infection by HIV. Against this background, the objective of this study was to characterize HIV-1 in residents confirmed to be seropositive. Serotyping was performed through a competitive enzyme-linked immunosorbent assay (cPEIA). Genotyping methods included RNA isolation followed by RT-PCR and sequencing of the gag p24, env gp41 immunodominant region (IDR), and env gp120 V3 genome regions of HIV-1. With the exception of a possible C/D recombinant strain, all HIV-1 strains were characterized as HIV-1 group M subtype C. One individual was shown to harbor multiple strains of HIV-1 subtype C. In Southern Africa, the focus has been to develop a subtype C candidate vaccine, as this is the major subtype found in this geographical area. Therefore, the spread of HIV-1 and its recombinant strains needs to be monitored closely.