广西西部地区吸毒人群HIV-1毒株基因序列测定和亚型分析

目的了解吸毒人群HIV-1流行毒株的亚型类型和变异特征，为艾滋病的疫苗预防、诊治提供依据。方法采集血清和外周血单个核细胞，用ELISA法检测HIV-1的抗体；PEIA检测HIV-1血清亚型；PCR扩增HIV-1膜蛋白基因片段，荧光标记未端终止物循环测序法进行测序反应，自动DNA序列分析仪测定产物序列。结果HIV-1毒株至少有C、E两个亚型。C为优势亚型，其株间基因离散率最大为2．93％，与A亚型等其他9个亚型参考株相比较，基因离散率为12．42～19．05％，gp120 V3环多肽序列相似。还可能存在其它亚型或混合感染。结论吸毒人群HIV-1流行毒株亚型呈现多样性，应加强对HIV-1亚型及其变异的监测和研究，指导疫苗预防、诊断和治疗。     

河南省HIV流行毒株env膜蛋白基因C2-V3区序列特征和亚型研究

目的 了解河南省内艾滋病病毒Ⅰ型(HIV-1)流行株的亚型及序列变异特征。方法 用套式聚合酶链反应(nested-PCR)，对河南省内28份HIV-1感染者外周血单个核细胞(PBMc)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350～450个核苷酸序列进行测定和分析。结果 28份样品间的基因离散率为5．72％，与流行于云南省的(泰国B)毒株基因距离为5.80％，与欧美B亚型基因距离为18．05％，而与其它A、C、D、F、G、H、J、O等亚型的基因距离均在24％以上。系统树分析显示，28份样本与泰国B亚型聚在一起而远离其它国际亚型。对于其V3环四肽序列的分析表明，具有泰国B亚型基因型GPGQ的8例，占28．57％；具有欧美B亚型基因型GPGR的13例，占46．4％。结论 河南省流行的HIV株属B‘亚型，其毒株来自与泰国接壤的云南省，在河南省流行的时间约在6～10年，其V3环顶端四肽序列特征以GPGR为主。     

北京市同性恋HIV-1感染者的包膜基因C2-V3区序列测定和亚型分析

目的 了解北京市同性恋HIV-1感染者HIV-1的亚型类型及传播来源和流行时间。方法 应用套式聚合酶链式反应（PCR）对12份1993～2001年北京市HIV-1阳性同性恋者外周血单个核细胞（PBMC）的核酸样品进行扩增,并对其包膜区的C2-V3段的306个核酸序列进行测定和分析。结果12份样品全部是B亚型的HIV-1毒株序列,其亚型内的基因离散率为 10.35± 2.06,与国际 A-E亚型共享序列比较后发现其与 A、C、D、E亚型的共享序列的基因离散率均大于 25％,而与国际 B亚型共享序列的基因离散率仅为11.25±3.60。系统树分析显示,12个毒株与B亚型共享序列聚在一起并远离其它国际亚型,并且12个毒株与SF162紧密相连,而与国际B亚型共享序列和泰国B亚型代表株TH14可以分开。对gp120中最重要的中和抗体决定簇V3环序列进行对比分析发现,12毒株在V3环中变化较大,其中4毒株带有GPGR这一欧美B亚型V3环顶端四肽序列特征,占33.33％,1个毒株带有GLGR,占8.33％,而其它7个毒株为GWGR,占58.34％。结论HIV-1在北京市同性恋人群中流行的为B亚型,流行来源为欧美,流行时间10年左右,V3环顶端四肽序列特征以GWGR为主。     

深圳市HIV—1B亚型感染毒株env基因序列测定和分析

应用巢式-PCR方法,对深圳检出的2份HIV-1感染者的外周血单核细胞样本进行扩增,获得HIV-1膜蛋白(env)基因核酸片段,并对其C_2-V_3区及邻区350～450个核苷酸序列进行测定和分析.结果表明这2份血样为HIV-1B亚型毒株感染(sz-B),彼此间的基因离散率为10.2%;与A-E国际参考亚型序列比较,sz-B与A及C-E参考亚型共享序列间的基因离散率均大于22%,而与欧美B亚型(Boon)间的基因离散率仅为7.5%;与国内部分地区流行的B亚型代表株比较其基因离散率为11%～12%.提示HIV-1B亚型在深圳的流行时间约为10年,主要经性途径由境外传入,且经性传播途径在人群中流行.     

河南省艾滋病病毒感染者HIV毒株膜蛋白基因序列研究

对河南省部分地区HIV-1感染者提取外周血单核细胞(PBMC)DNA,经套式PCR扩增env基因片断,对C2—V3区350—450核苷酸序列进行测定和分析.结果表明所采样品均属HIV—1B亚型,与其它国际亚型的基因离散率在20%以上;与流行于云南省的B亚型毒株基因离散率为2.8%左右,样品间的基因离散率很小.结果提示,流行于河南省的HIV属B亚型,并与云南省的流行株密切相关;其在河南省的流行时间在3年左右.     

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。     

深圳地区HIV-1母婴垂直传播实验室诊断方法探索及其分子流行病学研究

目的 探索艾滋病病毒Ⅰ型(HIV-1)阳性孕产妇新生婴儿核酸检测的辅助性诊断方法,了解深圳地区母婴传播所感染的HIV-1阳性婴儿HIV-1毒株的分子流行情况,帮助分析HIV-1在该人群中传播的危险因素.方法 收集深圳市2000-2007年19例HIV-1阳性孕产妇的血样,她们所生新生儿中28人的血样,应用套式聚合酶链式反应(Nested-PCR),对该样本膜蛋白基因(env基因)和核心蛋白(gag基因)进行扩增,并对其各基因区核苷酸序列进行测定和分析.结果 28例婴儿中共得到6例PCR诊断为阳性的婴儿样本,6例样本中共存在CRF01_AE和CRF07_BC两种重组毒株以及B一种亚型,其在所有分析样本中的比例分别为4/6、1/6和1/6;在env基因区与对应的流行代表株01_AE.TH.90.CM240的基因离散率为(9.200±1.600)%;CRF01_AE重组株组内离散率为(11.700±4.000)%;在gag基因区与对应的流行代表株01_AE.TH.90.CM240、07_BC.CN.97.97CN001和B.CN.RL42的基因离散率分别为(4.075±0.763)%、(5.500±0.566)%和(7.150±1.485)%;CRF01_AE重组株、CRF07_BC重组株和B亚型组内离散率分别为(4.033±1.692)%、0.800%和1.900%.结论 该方法有望成为HIV母婴垂直传播中新生婴儿的早期辅助性诊断方法.在深圳地区HIV-1阳性婴儿中,HIV-1流行株以CRF01_AE重组亚型为主,其次是B亚型.并且在母婴传播过程中,HIV-1 env和gag基因变异性较小.     

1996～1998年中国流行的E亚型艾滋病病毒1型毒株的分子流行病学研究

目的通过对1996～1998年采集的艾滋病病毒1型(HIV-1)毒株样本的env基因的序列分析,阐明在中国流行的E亚型HIV-1毒株的特点、来源和传播方式。为中国E亚型HIV-1疫苗的研制和应用提供基础资料。方法 从HIV感染者淋巴细胞(PBMC)中提取前病毒DNA.使用嵌套式聚合酶链反应(PCR)方法扩增HIV-1的env基因的C2V5区。PCR产物不经克隆直接测序并使用GCG软件包进行序列分析。结果样品采自1996～1998年中国29个省(自治区,直辖市),总共发现37个E亚型HIV-1感染者。他们中大部分是通过性途径感染(23人,占62.2％);部分在静脉吸毒人群中发现(10人,占27.0％);少数是在职业献血员中发现(4人,占10.8％)。经C2-V3区序列分析发现,大部分中国E亚型HIV-1毒株与泰国株很相近,而与非洲株相差很大。而来自广西壮族自治区的毒株与越南吸毒人群中的流行株U48720相一致;系统树分析结果发现,中国的E亚型HIV-1株与泰国(CM240X、H93TH966)、越南(U48720)的代表株聚在一起。结论 中国E亚型HIV-1毒株目前仅在东南沿海地区流行,涉及静脉吸毒、输供血和性乱等各种人群,通过env区的序列分析发现其主要来源于泰国,部分来源于与中国接壤的越南。     

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.     

江西省HIV-1基因序列流行特征分析

目的对江西省艾滋病病毒1型(HIV1)感染者进行基因亚型分析,了解HIV1的流行情况、亚型种类、毒株来源及其变异特征等,为政府部门预防控制决策提供技术资料。方法传统流行病学与分子流行病学相结合,对江西省27例HIV1感染者进行流行病学相关因素分析和基因序列、系统进化树分析。结果江西省HIV1感染人群中流行的毒株主要为HIV1CRF01AE,占6897%(20/29),其次为泰国B(B’)、CRF07BC、C三种亚型。序列分析表明,20份样品与泰国代表株相近,与CRF01AETH90CM240平均基因距离为947±271;3份样品与我国BC重组代表株相近,与BC重组代表株(CRF07BCCN97C54A)平均基因距离为566±241;3份样品与BCNRL42相近,与代表株BCNRL42平均基因距离为502±103;1份样品与印度代表株(CIN95IN21068)相近,基因距离为1299。而与已知的其它亚型国际参考株间的平均基因距离都在20%以上。系统进化树分析表明,27份样品与其相应的亚型共享序列聚集在一起,并远离其它国际参考株。吸毒人群中几乎全为HIV1CRF01AE,组内基因距离293±140(n=13),经性传播的四种亚型均有。结论目前江西省HIV1感染者中流行毒株为CRF01AE、CRF07BC、泰国B(B′)、C四种亚型,以CRF01AE为主。CRF01AE主要在吸毒人群中传播,局部爆发或流行时间大约有2年半左右,江西省HIV流行已     

湖北地区人类免疫缺陷病毒-1主要流行株外膜蛋白基因V3-V4区序列变异分析

目的 分析湖北地区HIV-1主要流行株外膜蛋白V3-V4区序列特征,了解其流行特点和变异规律.方法 对湖北地区HIV-1主要流行区域进行流行病学调查,应用套式PCR对102例HIv-1感染者的env基因V3-V4区进行扩增,对阳性扩增样本进行基因测序和序列分析.比较基因距离的差异用卡方检验;基因距离的变异性分析使用描述性分析法.结果 湖北地区共发现4种HIV亚型和重组亚型,其中B'亚型占82.69%,B'/C重组毒株、CRF01_AE重组毒株各占7.69%,C亚型占1.92%.湖北地区HIV-1B'亚型与来源于云南和河南等地的HIV-1B'亚型代表株之间的基因距离分别为7.08±2.19和7.88±2.28.其流行时间约为10年;氨基酸序列变异分析显示,HIV-1B'亚型毒株env基因V3、C3、V4区域均发生不同程度变异,其中以V4区的变异程度最大,V3环顶端四肽表现为GPGR、GPGK、GPGQ和GQGR,分别占46.5%、30.2%、13.6%和9.3%;V3环辅助受体预测显示,其中16.28%为CCR5型,13.95%为CXCR4型,69.77%无法预测;糖基化位点分析显示.湖北地区HIV-1主要流行株env V3-V4区9个糖基化位点有8个存在不同程度丢失.结论 B'亚型仍是湖北地区HIV优势流行株,与来源于云南和河南等地的毒株有较高同源性.     

中国首例HIV-1 J亚型毒株的鉴定

目的通过对1例来自刚果的人类免疫缺陷病毒Ⅰ型（HIV-1）感染者样品进行序列分析，鉴定其亚型种类。方法从该感染者血浆样品中提取RNA，经逆转录后采用套式PCR扩增HIV env、gag及pol基因的部分区段，PCR产物直接测序，将所获序列与参考序列进行比对分析，计算基因距离及构建系统进化树。结果该感染毒株样品序列与HIV-1J亚型国际参考株J．SE．93及J．SE．94聚在一起，与两参考株序列在env基因区距离均为8．2％，pol基因区距离分别为6．4％和6．3％，gag基因区距离分别为8．9％和8．0％，证实其为HIV-1J亚型。这是国内首次报道检出HIV-1J亚型。结论HIV-1J亚型毒株已随国际旅行者传人我国，加强对国际旅行人员中HIV毒株亚型的监测具有重要意义。     

Wide distribution of two subtypes of HIV-1 in Thailand.

Scientists wanted to identify the genetic characteristics of 2 HIV-1 subtypes in Thailand. Staff from regional laboratories of the Ministry of Public Health took blood samples from people in various high risk groups and from all regions of the country. Staff at the National Institutes of Health in Bangkok then did lymphocyte separation, DNA extraction, and virus culture. They took the extracted DNA specimens and sent them to the US Centers for Disease Control where scientists did serologic testing, polymerase chain reaction, and sequence determination. They used Kimura's method to study sequence variations. They sequenced 300 nucleotides, including the C2-V3 domains of HIV-1 envelope gene and/or hybridization. Every risk group had HIV-1 subtype A, but subtype B was mostly found in drug users. Subtype A had spread mainly among heterosexuals. The mean intraperson variation for subtypes A and B stood at 2% and 2.7%, respectively, while the interperson variation within subtype A and B stood at 3.8% and 3.7%, respectively. The mean interperson variation between subtypes A and B from different persons was 18.1%. Phylogenetic tree analysis showed that subtype B identified with about 85% of the sequence as that of the North American isolates, making it more closely related to them than to African isolates (about 75% sequence identity). On the other hand, subtype A had a GPGQ motif at the V3 crown which was common among African HIV-1 isolates. Antibodies which usually recognize HIV-1 MN strains (which have the GPGR motif) may not react wholly with the V3 loop from the Thailand subtype A viruses, thus the GPGQ motif at the V3 crown may pose a problem. Now for the first time, scientists can follow the natural history of 2 HIV-1 subtypes and determine their relative pathogenicity and transmission efficiency between adults or from mother to infant. The relative homogeneity of the HIV-1 strains in Thailand presents a theoretical advantage in designing vaccines for potential large-scale clinical trials.     

A comparison of full-length glycoprotein 120 from incident HIV type 1 subtype E and B infections in Bangkok injecting drug users with prototype E and B strains that are components of a candidate vaccine

Complete gp120 sequence information was obtained from eight persons with incident HIV-1 infections (four subtype E and four subtype B) who were part of a prospective injecting drug user (IDU) cohort in Bangkok, Thailand, during 1996-1998. The incident subtype E strains were similar to the prototype subtype E strain CM244 isolated in 1992 in northern Thailand. The incident subtype B strains displayed divergence, in both overall genetic distance and other significant gp120 characteristics, from the prototype North American subtype B strain HIV-MN. Recombinant gp120s derived from CM244 and HIV-MN strains are components of a vaccine that is undergoing phase III efficacy testing, begun in March 1999, among Bangkok area IDUs. The information presented here will be important in the evaluation of any breakthrough HIV-1 infections occurring among vaccinees during the vaccine trial and in ongoing vaccine development efforts in Thailand.     

Apparent founder effect during the early years of the San Francisco HIV type 1 epidemic (1978-1979)

HIV-1 envelope sequence variants were RT-PCR amplified from serum samples cryopreserved in San Francisco in 1978-1979. The HIV-1 subtype B env V3-V5 sequences from four homosexual men clustered phylogenetically, with a median nucleotide distance of 2.8%, reflecting a recent common origin. These early U.S. HIV-1 env variants mapped close to the phylogenetic root of the subtype B tree while env variants collected in the United States throughout the 1980s and 1990s showed, on average, increasing genetic diversity and divergence from the subtype B consensus sequence. These results indicate that the majority of HIV-1 currently circulating in the United States may be descended from an initial introduction and rapid spread during the mid- to late 1970s of subtype B viruses with limited variability (i.e., a founder effect). As expected from the starburst-shaped phylogeny of HIV-1 subtype B, contemporary U.S. strains were, on average, more closely related at the nucleic acid and amino acid levels to the earlier 1978-1979 env variants than to each other. The growing levels of HIV-1 genetic diversity, one of multiple obstacles in designing a protective vaccine, may therefore be mitigated by using epidemic founding variants as antigenic strains for protection against contemporary strains.     

武汉市HIV-1相关基因序列分析及亚型分布

目的研究武汉市HIV感染人群中的HIV毒株的亚型分布特点和流行规律。方法采集武汉市60名已被确认为HIV-1感染者的抗凝全血样品,提取前病毒DNA,用巢式聚合酶链反应方法（nested-PCR）扩增病毒膜蛋白env基因的C2-V5区及gag基因的部分区段,对PCR纯化产物直接测序,并应用GCG软件对序列进行分析。结果通过PCR扩增得到60份样品的结果,其中env基因序列45份、gag基因序列52份。依据env和gag区基因序列,与HIV-1各个亚型国际参考株比较,通过系统进化分析,最后确定武汉市样品分属5个亚型,分别为HIV-1B亚型中的泰国B（B’）亚型32份,流行重组型CRF07-BC12份,流行重组型CRF01-AE9份,A亚型1份和C亚型6份。结论武汉市存在多种HIV-1亚型,应加强对HIV-1毒株亚型变异的监测,及时调整防治策略。     

福建省HIV-1流行毒株序列测定与亚型分析

使用PCR技术对 19份福建省HIV - 1阳性感染者或病人的外周血淋巴细胞 (PBMCs)进行扩增 ,获得HIV - 1膜蛋白 (env)基因的部分核酸片段 ,并对其V3及邻区～ 30 0个核酸序列进行测定及分析。结果表明 ,19份样品中 11份为E亚型 ,5份为B亚型 ,1份为A亚型 ,1份为C亚型。上述资料表明 ,到目前为止流行于福建省的HIV - 1毒株的特点为以E、B亚型为主 ,多种亚型并存。两者型内以及与国际标准亚型序列的基因离散率均比较大。与其他省份的比较表明我省的毒株的传入与其他省份没有相关     

Group M-based HIV-1 Gag peptides are frequently targeted by T cells in chronically infected US and Zambian patients

BACKGROUND: The enormous sequence diversity of HIV-1 has been a major obstacle in the development of a globally useful vaccine for AIDS. The consensus and ancestral sequence-based immunogens minimize the genetic distance between contemporary isolates and vaccine strains. Hence these sequences may be promising candidates for HIV vaccines or serve as a universal reagent set for evaluating Gag-specific responses. METHODS: In this study, we measured the T-cell reactivity to consensus (subtype A, B, C and group M), ancestral (group M and subtype B) and HXB2 Gag peptides (15-mers overlapping by 11) in HIV-1-infected subjects from two reference populations. We evaluated the Gag-specific T-cell responses in 43 chronically infected US (subtype B) and 13 Zambian (subtype C) subjects using an interferon-gamma enzyme-linked immunosorbent spot assay. RESULTS: Our findings demonstrate a broad cross-reactivity of nearly 70% among all the seven Gag immunogens evaluated. Consensus M sequences elicited similar levels of responses as did the consensus B, ancestral subtype B and HXB2 peptides in subtype B-infected US patients. In subtype C-infected Zambian subjects, responses of similar breadth and magnitude were elicited by consensus C, consensus M and ancestral M peptides. CONCLUSION: Our data demonstrate that peptide pools based on consensus or ancestral M-based sequences can be used to evaluate Gag-specific responses elicited by subtype B or subtype C-based immunogens.     

Genetic characterization of incident HIV type 1 subtype E and B strains from a prospective cohort of injecting drug users in Bangkok, Thailand

We obtained specimens from 128 HIV-1 seroconverters identified from 1995 through 1998 in a prospective cohort study of 1,209 HIV-negative injecting drug users (IDUs) in Bangkok, Thailand. Epidemiologic data indicated that parenteral transmission accounted for nearly all infections. HIV-1 DNA from the C2-V4 env region was sequenced, and phylogenetic analyses determined that 102 (79.7%) of the specimens were subtype E and 26 (20.3%) subtype B strains. All subtype B strains clustered with strains often referred to in previous studies as Thai B or B'. The interstrain nucleotide distance (C2-V4) within subtype E strains was low (mean, 6.8%), and pairwise comparisons with a prototype subtype E strain, CM244, showed limited divergence (mean, 5.6%). The subtype B stains showed greater interstrain divergence (mean, 9.2%) and were significantly divergent from the prototype B strain HIV-MN (mean, 13.0%; p < 0.0001). The subtype E strains had significantly lower mean V3 loop charge than did subtype B strains (p = 0.017) and, on the basis of analysis of amino acid sequences, were predicted to be predominantly (91%) non-syncytium-inducing (NSI), chemokine coreceptor CCR5-using (CCR5+) viruses. The subtype B strains had a higher mean V3 loop charge, and a smaller proportion (23%) were predicted to be NSI/CCR5+ viruses. This study demonstrates that most incident HIV1 infections among Bangkok IDUs are due to subtype E viruses, with a narrow spectrum of genetic diversity. The characterization of incident HIV-1 strains from 1995 to 1998 will provide important baseline information for comparison with any breakthrough infections that occur among IDUs in Bangkok who are participating in an HIV-1 vaccine efficacy trial initiated in 1999.