26例有偿献血员HIV-1基因分型与变异特征

目的分析某地区有偿献血人员中流行的人免疫缺陷病毒1型（HIV-1）gag、pol、env基因亚型及基因变异特征。方法提取HIV-1感染者外周血单核细胞（PBMC）DNA,经巢式PCR（NestedPCR）扩增gag（p17-p24）、pol（PR＊RT）、env（C2-V5）基因片段,纯化测序后用MEGA5．0等生物学软件对核苷酸序列进行分析。结果23份样本为B亚型,2份为B亚型与C亚型重组,1份为CRF01-AE与B亚型重组。PR区未发现蛋白酶抑制剂主要耐药性突变,RT区检测到核苷类逆转录酶抑制剂耐药性突变M184V和非核苷类逆转录酶抑制剂耐药性突变K101E,G190A。结论流行于该地区的HIV-1毒株以B亚型为主。大多数毒株对常规抗病毒药物仍然敏感,使用HARRT治疗方案依然有效。CXCR4型辅助受体的毒株顶端四肽多为GPGR（91．7％）,提示GPGR可能与疾病的进展有关。     

1990～1997年云南IDUs HIV-1 B亚型分离株膜蛋白V3环顶端四肽基因序列的变化趋势

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～ 1997年间 6 2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 6 2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 ,主要表现在第四位氨基酸的改变 ,变异呈现一定的趋向性。 6 2株云南标本中有 46例(74 2 % )为GPGR ,10例 (16 1% )为GPGQ ,4例 (6 4% )为GPGK ,另有 2例 (3 3% )为GPER且四肽基序编码核苷酸的变异主要表现为A→G的改变。结论　 1990～ 1997年这一时期内 ,云南IDUs特定感染者范围内HIV 1分离株膜蛋白V3环顶端四肽序列已表现出较大的趋向性 ,符合HIV 1国际B亚型SF2株的GPGR模式     

26例有偿献血员HIV-1基因分型与变异特征

目的 分析某地区有偿献血人员中流行的人免疫缺陷病毒1型( HIV-1) gag、pol、env基因亚型及基因变异特征.方法 提取HIV-1感染者外周血单核细胞(PBMC) DNA,经巢式PCR( Nested PCR)扩增gag(p17-p24)、pol( PR-RT)、env(C2-V5)基因片段,纯化测序后用MEGA5.0等生物学软件对核苷酸序列进行分析.结果 23份样本为B亚型,2份为B亚型与C亚型重组,1份为CRF01_AE与B亚型重组.PR区未发现蛋白酶抑制剂主要耐药性突变,RT区检测到核苷类逆转录酶抑制剂耐药性突变M184V和非核苷类逆转录酶抑制剂耐药性突变K101E,G190A.结论 流行于该地区的HIV-1毒株以B亚型为主.大多数毒株对常规抗病毒药物仍然敏感,使用HARRT治疗方案依然有效.CXCR4型辅助受体的毒株顶端四肽多为GPGR(91.7％),提示GPGR可能与疾病的进展有关.     

山东省部分HIV-1流行株的亚型分析和序列特征研究

目的　对山东省HIV 1流行毒株进行亚型分析 ,并研究其变异特征。方法　采集 2 6份HIV 1感染者的外周静脉抗凝血 ,提取前病毒DNA进行体外扩增 ,获得包膜蛋白 (env)基因的核酸片段 ,并对其C2 V3及邻区的核苷酸进行测定和分析。结果　基因和氨基酸序列分析表明 ,2 6份标本中存在 4种亚型和重组毒株 (B′、C、A、A/E) ,其中B′ 17株 ,其组内基因距离为 11.6 9± 4 .19。V3环顶端四肽有 6种形式 ,最多的是GPGQ(15株 )、GPGR(6株 )。V3环第 11、2 5位氨基酸出现变异 ,并有 1株呈电荷双阳性。结论　山东省HIV 1流行株亚型较多 ,有重组毒株出现的可能 ,基因发生较大变异 ,HIV 1传播在山东省有加快的趋势。     

32例有偿献血感染者HIV-1膜区序列测定及V3环氨基酸变异特点分析

目的 了解我国有偿献血人群中HIV-1B'亚型流行株的膜蛋白V3环序列特征.方法 巢式PCR扩增前病毒DNAc2-c3区后测序,进行病毒亚型鉴定和V3环序列特点分析.结果 所检的32个HIV-1B'亚型株V3环顶端四肽有五种形式.AIDS组与无症状感染组相比前者呈现更多的T/SI型毒株V3环变异特点.结论 我国有偿献血人群中HIV-1 B'亚型流行株V3环顶端四肽有多种形式.处于病程不同时期的患者体内病毒V3区呈现不同的氨基酸变异特点和电荷积累趋势,提示可能具有不同的生物学表型特征.     

北京市男男性接触HIV-1感染者膜蛋白基因序列测定及亚型分析

目的 了解北京市男男性接触人群(MSM)中HIV-1的最新流行趋势及膜蛋白V3环序列特征.方法 巢式聚合酶链式反应(n-PER)扩增2007年提取的北京市男男性接触HIV感染者基因组DNA样品,对膜蛋白基因C2-V3区测序,进行病毒亚型及V3环序列特点分析.结果 11例样本中,4例是欧美B亚型,5例是AE重组亚型,1例是BC重组亚型,1例是01B重组亚型.V3环顶端四肽以GPGQ和GPGR为主.结论 北京市男男性接触HIV-1感染者中重组亚型呈蔓延流行趋势.     

广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型的关系

目的研究广西HIV-1 CRF01-AE重组毒株env基因V3环序列变异及其与生物表型间的关系。方法从广西主要流行区收集来的50份HIV-1感染者血液样本中提取前病毒DNA，使用巢式聚合酶链反应（nested-PCR）扩增HIV-1 env基因片段并进行亚型鉴定，选择38份CRF01-AE重组型HIV-1毒株env，基因V3环及邻近区域的序列进行系统树和氨基酸变异分析。结果38份CBF01-AE重组毒株中36份与分离于广西地区的CRF01-AE.97CNGX2f和泰国代表毒株THCM240接近，另外2份与中非共和国代表株90CF402聚成一簇；CRF01-AE重组毒株V3环顶端四肽存在着4种类型：CPCQ、GPGR、GPGH和GPGA；根据V3环关键氨基酸推测辅助受体使用情况，结果显示：71．05％的CRF01-AE重组毒株可能使用CCR5作为辅助受体，28．95％不能对其辅助受体的使用情况做出预测。结论广西HIV-1 CRF01-AE重组毒株V3顶端四肽变异较大，而且大部分毒株可能为NSI型。这可为广西该毒株的防治和诊断试剂的更新提供参考。     

黑龙江省17例HIV/AIDS患者的病毒亚型及基因序列特征分析

目的了解黑龙江省内部分人免疫缺陷病毒1型（HIV-1）的亚型及基因序列特征。方法用巢式聚合酶链反应（nested-PCR），对黑龙江省内17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的膜蛋白（env）基因进行扩增，并对C2-V3的核苷酸序列进行测定和分析。结果系统树分析显示，17份样本中病毒与HIV泰国B（B’）亚型聚在一起，基因离散率为（6．94±1．01）％，与欧美B亚型基因离散率为（12．94±2．19）％，与其他亚型的离散率大于20％。对于其V3环四肽序列的分析表明，具有GPGQ的8例，占47．06％；具有GPGR的7例，占41．18％；1例为GQGR；1例为GPGH。通过序列分析预测，大部分利用CCR5辅助受体。结论所检测的黑龙江省17例HIV-1均为B’亚型，提示黑龙江省的HIV-1流行株可能以B’亚型为主，其V3环顶端四肽序列特征以GPGQ和CP（承为主。     

HIV1膜蛋白PND编码基因自然变异株的发现

目的 对1例来自华东地区HIV1分离株（WWBH7）的前病毒env基因C2-V3区进行序列分析。方法 以HIV1感染者外周血单个核细胞基因组为模板进行套式PCR扩增HIV1env基因C2-V3区片段,将此扩增产物插入T-Vector,酶切鉴定重组质粒,使用AB1737自动DNA序列测定仪测定序列并用DNASIS软件进行分析。结果 DNA序列资料显示该毒株属于HIV1B亚型衍生株,但与HIV1B亚型的标准株如SF2株相比,该HIV1毒株的env基因V3区下游有192bp的重复插入突变,使得该毒株的膜蛋白PND编码基因呈现双V3区的变异该段DNA序列已登录于GenBank(AF220245)。结论 该分离株是1个在膜蛋白PND编码基因有大片段插入突变的HIV1变异株。     

我国HIV-1主要流行株外膜蛋白(env)基因V3～V4区变异及其与生物学特性的关系

目的　研究我国HIV 1主要流行毒株亚型的envV3～V4区变异与生物学特性的关系。方法　应用nested PCR对 1 57份获自我国 1 2个省份的HIV 1毒株env区序列进行扩增 ,并使用ABI 377型测序仪测序 ,然后应用BLAST、GCG和MEGA等生物学软件或程序对env基因V3～V4区序列进行分析。结果　B′亚型毒株V3顶端四肽存在着 4种类型 :GPGR ( 54% )、GPGQ ( 2 8% )、GPGK( 1 6 % )和GPGA( 2 % ) ,B′/C重组毒株全部为GPGQ( 1 0 0 % ) ,CRF0 1 AE重组毒株呈现GPGQ( 95% )和GPGR( 5% )两种类型 ;B′/C和CRF0 1 AE重组毒株V3～V4区及其临近区域N 糖基化位点比B′亚型毒株N 糖基化位点保守。而B′亚型毒株V3环的净电荷分别显著高于B′/C和CRF0 1 AE毒株 (P <0 .0 1 ) ;根据V3环关键氨基酸推测辅助受体使用情况的结果显示 :B′亚型毒株有 9.2 6 %可能使用CCR5,7.4 1 %可能使用CXCR4 ,其余 83.33%不能对辅助受体的使用作出预测。所有B′/C重组毒株被预测可能使用CCR5。CRF0 1 AE重组毒株有 90 .4 8%被预测可能使用CCR5,没有被预测为使用CXCR4的序列 ,9.52 %不能作出预测。结论　B′亚型毒株大部分可能为NSI型 ,少部分可能为SI型 ,而B′/C和CRF0 1 AE重组毒株绝大部分为NSI型。我国主要流行株的V3～V4区尤其是V3环的氨基酸     

云南瑞丽人免疫缺陷病毒感染者gp120基因C2—V3区的序…

经套式聚合酶链反应对１７份１９９５年初采集于云南瑞丽市人免疫缺陷病毒１型阳性静脉吸毒者外周血单核细胞的核酸样品进行扩增，从１７份样品中获得了ＨＩＶ－１膜蛋白基因的核酸片段，并对其Ｃ２－Ｖ３及邻区４５０个核苷酸序列进行了测定和分析。     

Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3_B-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.     

High frequency of BF mosaic genomes among HIV-1-infected children from Sao Paulo, Brazil

HIV-1 genetic diversity information from a pediatric population is scarce. This study enrolled 128 children living with HIV/AIDS, 103 antiretroviral-treated and 25 naive, from the Sao Paulo metropolitan area. Gag, pol and env regions were amplified, and drug resistance mutations, V3 loop, tropism and viral clades were evaluated. Drug resistance mutations among naïve children infected by vertical transmission were uncommon (4.2%), whereas most ARV-experienced children showed extensive mutation patterns. Clade B predominated at the pol region, but the analysis of the three regions concatenated showed 28% with BF mosaic structures. The most common V3 motif was GPGR, followed by GWGR in clade B samples and GPGQ in clade F samples. A predicted X4 phenotype was observed in 27%, without correlation to HIV clade. These findings expand the limited information on molecular characteristics of HIV-1 among children living with HIV/AIDS in the area and may provide information useful for monitoring the epidemic.     

Structural studies of human HIV-1 V3 antibodies

X-ray crystallographic structures of two human, anti-V3 HIV-1 neutralizing antibodies, 447-52D and 2219, show how the two antibodies use different strategies for their cross-reactivity with different V3 sequences. 447-52D recognizes V3 primarily through main-chain hydrogen bonds to the N-terminal side of V3, with the GPGR tip region buried in the antigen-combining site. The side chains on the N-terminal side of V3 are exposed to solvent, allowing for sequence changes in this region, thus explaining how 447-52D can neutralize a wide array of viral isolates. Antibody 2219 contacts a more extensive V3 surface, with more side-chain involvement. However, residues at the GPGR tip are exposed to solvent, with no constraints on residue size, so that binding to unusual tip sequences with larger side chains such as RPRQ is possible.     

Characteristic of HIV-1 in V3 loop region based on seroreactivity and amino acid sequences in Thailand

The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.     

V3 loop region of the HIV-1 gp120 envelope protein is essential for virus infectivity.

The mechanism by which HIV-1 mediates cell fusion and penetrates target cells, subsequent to receptor (CD4) binding, is not well understood. However, neutralizing antibodies, which recognize the principal neutralizing determinants of the gp120 envelope protein (the V3 loop region, residues 296 to 331), have been shown to effectively block cell fusion and virus infectivity independent of the initial gp120-CD4 binding. To investigate the role of the V3 loop in an HIV infection, a series of site-specific mutations were introduced into the HIV-1 envelope gene. Specifically, each residue (312 to 315) in the strongly conserved tetrapeptide sequence, GPGR, which is positioned in the center of the V3 loop domain was individually altered. The processing, transport, and CD4 binding properties of the mutant envelope proteins were comparable to those of the wild-type protein, however, none of the mutants were able to form syncytia in the HeLa-T4 assay. Molecular HIV-1 clones containing mutations altering the G312, G314, or R315 residues produced noninfectious virions, whereas a clone with a P313A mutation was found to be infectious. These results demonstrate that certain V3 loop mutations can be lethal and clearly indicate that this region of the HIV-1 gp120 protein is essential for virus infectivity.