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.     

河北省HIV-1流行株基因序列测定及亚型分析

目的 了解河北省HIV-1流行株的亚型分布和流行趋势.方法 从感染者的血浆样品中提取病毒RNA,逆转录后采用套式PCR扩增HIV-1 gag和env基因的部分片段,对PCR产物直接进行核苷酸序列测定,所获序列与各亚型国际参考株序列比对,确定基因型并进行系统进化树分析.结果 对154份HIV-1感染者的样品进行扩增,得到了148份样品的HIV-1基因片段.发现6种HIV-1亚型和重组型,以及2例未定型.其中B'亚型61例(41.2%)、CRF01_AE 59例(39.9%)、CRF07_BC 16例(10.8%)、CRF08_BC 6例(4.1%)、C亚和B01亚型各2例(1.4%).在河北省首次发现了B01亚型.结论 2009年河北省存在多种HIV-1亚型和流行重组型,主要是B'亚型和CRF01_AE重组型,应加强对HIV-1毒株亚型变异的监测,及时调整防治策略.     

湖北省HIV-1流行株gag基因序列测定及亚型分析

目的 了解湖北省HIV-1感染者流行毒株亚型分布和流行趋势.方法 随机采集湖北省HIV-1感染者的抗凝全血标本,分离血浆,提取病毒RNA,用套式聚合酶链反应扩增HIV-1病毒gag基因,并进行序列测定和亚型分析.结果 对80份HIV-1感染者的样品进行扩增,得到了62份样品的HIV-1基因片段.共发现7种HIV-1亚型和流行重组株.泰国B'亚型占11.3％ (7/62),欧美B亚型占4.8％ (3/62),G亚型占4.8％ (3/62),CRF07-BC占22.6％ (14/62),CRF08-BC占6.5％(4/62),CRF01-AE占48.4％(30/62),CRF15-01B占1.6％ (1/62).在湖北省发现了CRF15-01B和G亚型.结论 湖北省存在多种HIV-1亚型和流行重组型,CRF01-AE、CRF07-BC两种重组亚型毒株为目前湖北省HIV-1流行优势毒株,应加强对HIV-1毒株亚型变异的监测,及时调整防治策略.     

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。     

2006年北京市外来人口中HIV-1感染者流行毒株gag基因序列测定和亚型分析

目的了解北京市外来人口中HIV-1亚型的特点和流行规律。方法随机采集北京市2006年外来人口中新确证HIV-1感染者的抗凝全血标本80份，分离血浆，提取病毒RNA，用套式聚合酶链反应扩增病毒gag基因，并进行序列测定和亚型分析。结果系统进化分析确定北京市外来人口HIV-1毒株属于8个亚型，分别为B亚型4份，泰国B亚型15份，C亚型1份，CRF01-AE亚型5份，CRF02-AG亚型1份，CRF07-BC亚型29份，CRF08-BC亚型3份，CRFl5—01B亚型1份。结论北京市外来人口中己存在8种HIV-1亚型和流行重组型，应该加强对HIV-1亚型变异的监测。     

5株不同亚型HIV-1毒株gp120序列特征分析及其蛋白的表达

目的 分析我国HIV-1主要亚型毒株的gp120区域的序列特征,并表达出gp120糖基化蛋白,为研究gp120的致病机制和设计疫苗奠定基础.方法 利用巢式PCR从来自于不同省份的5名HIV-1感染者外周血单个核细胞DNA中扩增全长gp120基因并测序,对序列特征进行分析,并将获得的gp120基因克隆入真核表达载体,体外表达获得gp120糖基化蛋白.结果 序列分析显示,此5条gp120序列分属HIV-1 Thai-B、BC重组和AE重组哑型,虽然亚型不同,但这些gp120序列在相同的位置都存在着一些保守的糖基化位点,且都具备相同的Furin蛋白酶第一切割位点,与参考序列比较发现,不同的HIV亚型序列中,除V3序列长度较为保守外,V1、V2、V4、V5各区域都有不同程度的缺失现象,与BC重组和AE重组亚型相比,Thai-B亚型V3的顶端环呈现出多种组合.最终将这5条gpl20序列都克隆人真核表达载体并表达出糖基化的gp120蛋白.结论 在设计疫苗和检测试剂时应考虑到膜区的高变性,gp120糖蛋白的体外表达有利于进一步开展针对我国主要HIV流行毒株膜蛋白致病机制和疫苗学的研究.     

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     

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.     

河北省HIV-1流行株的env基因序列测定和亚型分析

目的了解河北省HIV-1亚型的分布特点和传播方式,推测流行时间,预测流行趋势。方法采集HIV感染者的全血样品,分离外周血单核细胞(PBMC),提取前病毒DNA,使用套式聚合酶链反应(nested-PCR),扩增HIV-1的env基因的C2-V3区并进行序列测定和亚型分析。结果对22份HIV-1感染者的样品,扩增得到了18份HIV-1envC2-V3基因片段,经序列测定和基因分析鉴定出3种HIV-1M亚群基因亚型,即:B′、CRF-BC和C亚型。B′亚型的组内基因离散率为7.84%±3.14%(n=14),基因序列与云南瑞丽株rl42(泰国B亚型)相近;2株CRF-BC亚型与广西毒株的基因离散率为4.60%。与血液途径感染有关的人员均为B′(泰国B)亚型。结论目前,在河北省发现了3种HIV-1亚型。输供血途径中B′亚型仍是主要的流行亚型,可能来源于云南吸毒人群。HIV-1B′亚型在河北省的流行时间大约为7~9年。     

北京市性传播HIV-1感染者流行毒株gag基因序列测定和亚型分析

目的 了解北京市性传播HIV-1感染者流行毒株亚型特点和流行规律.方法 随机采集北京市2008年新确证性传播HIV感染者的抗凝全血标本100份,分离血浆,提取病毒RNA,用套式聚合酶链反应扩增病毒gag基因,并进行序列测定和亚型分析.结果 系统进化分析确定北京市性传播HIV-1感染者流行毒株存在8个亚型或流行重组型,分别为B亚型22份,B'亚型8份,C亚型1份,CRF01_AE 38份,CRF02_AG 2份,CRF07 BC 9份,CRF08_BC 3份,疑似C/CRF01_AE重组型1份.结论 CRF01_AE和B亚型分别占45.2%和26.2%,为性传播感染者主要的亚型,应该加强我市HIV-1亚型流行情况的监测.

Abstract：

Objective To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent among sexual infectors in Beijing. Methods We collected the blood samples from 100HIV sexual infectors in Beijing during 2008 and separated plasma specimens. RNA was extracted from the plasma and the gag gene was amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag gene was performed using the MEGA4 software. Results Among 100 HIV-1 plasma samples,84 gag gene fragments were amplified and analyzed. Eight HIV subtypes including B(22 strains), B'(8 strains),C( 1 strain) ,CRF01_AE (38 strains) ,CRF02_AG (2 strains) ,CRF07_BC(9 strains) ,CRF08_BC(3 strains) and C/CRF01_AE recombinant like strain( 1 strain) were identified circulating in Beijing. Conclusion CRF01 _AE and subtype B were predominant in Beijing account for 45.2% and 26.2% and the surveillance of HIV gene variation should be paid more attention.     

National survey of prevalent HIV strains: limited genetic variation of Korean HIV-1 clade B within the population of Korean men who have sex with men

The evolution of HIV is the result of an explosive combination of factors-a high rate of mutation, replication dynamics, frequent recombination, and natural selection. To understand the evolution of the distinctive Korean HIV-1 B clade, we investigated the characteristics of the genetic variation of the HIV-1 subtype B env gene within the group of Korean men who have sex with men (MSM). From 1985 to 2005, 700 HIV-1-infected Koreans were sequenced at the V1 to V5 region of the HIV-1 env gene. In the phylogenetic analysis, 560 isolates were identified as HIV-1 subtype B, and 489 of the 560 isolates were HIV-1 Korean clade B. Based on epidemiologic investigation, 249 of 700 HIV-1-infected patients were HIV-1 subtype B-infected MSM. Interestingly, the proportion of the GPGS motif in MSM infected by Koreans was 1.6 times higher than in MSM infected by foreigners, and the genetic expansions of diversity and divergence for HIV-1 subtype B in Korean MSM were 2.1% and 2.5%, respectively. This was much lower than those observed in other countries. Therefore, our findings imply that the HIV strains in this group were closely related. This result may be helpful for understanding the evolution of the distinct HIV-1 Korean B clade.     

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。     

Analysis of HIV-1 subtype B third variable region peptide motifs for induction of neutralizing antibodies against HIV-1 primary isolates

The HIV-1 gp120 V3 loop is a potent inducer of neutralizing antibodies for T cell line adapted-HIV-1, but less so for primary isolates. We hypothesized that peptides representative of the diversity of natural HIV-1 V3 loop variants might capture elements of conserved higher order structures and so stimulate broadly reactive neutralizing antibodies. We designed a panel of 29 subtype B V3 sequences postulated to reflect the range of V3 diversity. These peptides were used to immunize guinea pigs. The most effective peptide (62.19) clustered around the subtype B consensus sequence and induced antibodies that reproducibly neutralized 31% of the subtype B HIV-1 primary isolates evaluated, but exhibited limited cross-neutralization of non-subtype B HIV-1 strains. Taken together, these data demonstrated that the limited neutralization profile of antibodies induced by optimal subtype B V3 motifs likely represents the maximum breadth of neutralization of subtype B HIV-1 primary isolates attainable by anti-V3 peptide antibodies.     

中国HIV-1型B、C亚型主要流行株外膜蛋白基因V3-V4区序列的变异分析

目的　研究我国人类免疫缺陷病毒 1型 (HIV 1)B、C亚型主要流行株在感染过程中基因变异的特点及其与选择压力的关系。方法　应用巢式聚合酶链反应 (nested PCR)对 2 5 8例HIV 1感染者血样中的HIV 1外膜蛋白 (env)基因进行扩增 ,并使用ABI 377型测序仪对扩增产物测序后 ,选择其中 37份B亚型和 35份C亚型HIV 1毒株env基因包括V3～V4区的序列进行比较分析 ,并计算和分析氨基酸同义替换与非同义替换的比值 (Ks Ka)。结果　B亚型毒株V3～V4区的基因离散率高于C亚型毒株。无论B亚型 ,还是C亚型毒株 ,其V4区基因序列较V3区变异更大。在C亚型毒株中 ,V3区基因序列变异甚至比V3上游区和C3区小。B和C亚型毒株整个V3～V4基因区的Ks Ka比值均 <1,差异有非常显著性 (P <0 0 0 1) ,其中B亚型毒株以V3区的Ks Ka比值最小 ,而C亚型毒株则以V4区的Ks Ka比值最小。结论　B和C亚型毒株env基因的变异主要发生在V4区而不是V3区。C亚型毒株V3区较V3上游区和C3区还要保守 ,是本研究的特殊发现。这两种亚型在我国快速流行中发生的变异是在选择压力下发生的 ,而不是随机进化的结果 ,而且选择压力对这两种亚型毒株V3、V4区的作用程度也不一样。这将为我国艾滋病防治策略的制定和疫苗研究提供科学的依据。     

Genotypic and phenotypic analysis of the env gene from South African HIV-1 subtype B and C isolates

The objective of the study was to assess the genotypic and phenotypic properties of 18 viral strains from human immunodeficiency virus-1 (HIV-1) positive patients and to identify subtype C isolates for vaccine design strategies. All the isolates were non-syncytium-inducing (NSI) in both the primary and MT-2 cell cultures. The amino acid charge of the V3 loop correlated with the NSI phenotype of the strains. The V3 competitive peptide enzyme immunoassay and DNA sequencing of the partial gp120 region gave concordant results on the 15 subtype C strains, whereas the three B genotypes gave a positive to B, a nonreactive to B, and a dual reaction to the B-D peptides, respectively. Sixteen of the isolates used only CCR5 as coreceptor whereas two isolates made use of additional coreceptors including CXCR4. In summary, all our subtype C isolates are NSI phenotypically and almost all of them use CCR5 exclusively as their coreceptor.     

Variability of human immunodeficiency virus type 2 (hiv-2) infecting patients living in france

To determine the prevalence of human immunodeficiency virus type 2 (HIV-2) subtypes circulating in France and to identify possible relationships between these subtypes and pathogenesis, we studied 33 HIV-2-infected patients living in France. HIV-2 DNA was directly amplified from peripheral blood mononuclear cells by nested PCR with specific HIV-2 env primers, and the env gene was sequenced. The serological consequences of antigenic variability were studied by using a panel of peptides and by Western blotting. Phylogenetic analysis classified the 33 HIV-2 strains as subtype A (n = 23) or B (n = 10). There were no significant clinical or epidemiological differences between patients infected with either of these two subtypes. There was some evidence for geographical clustering. Subtype A strains from patients originating from the Cape Verde Islands and Guinea Bissau clustered together. The majority of patients infected with subtype B strains originated from the Ivory Coast or Mali. Strains from patients originating in Mali also clustered in subtype A but distinctly from the Cape Verde or Guinea Bissau strains. The subtype B strains showed greater diversity and included some highly divergent strains relative to those previously characterized. The V3 loop of HIV-2 subtypes A and B was found to be quite conserved in comparison with HIV-1. A strong HIV-2 subtype B serological cross-reactivity was found on HIV-1 env antigen by Western blot mostly in the gp41 transmembrane glycoprotein. This could partly explain the double HIV-1 and HIV-2 reactive profiles found in countries where HIV-2 subtype B is prevalent.     

Molecular subtyping of the HIV-1 V3 loop sequences detected in HIV-1-positive patients in southern Taiwan

Polymerase chain reaction and nucleotide sequence analysis were performed to amplify and determine the V3 loop sequences of human immunodeficiency virus type 1 (HIV-1) from ten seropositive patients at National Cheng Kung University Hospital, Tainan. The nucleotide sequences and the deduced amino acid (a. a.) sequences of these V3 regions were compared with those of known HIV-1 prototypes. The V3 loop a. a. sequences detected in eight individuals belong to subtype B which predominates in North America and Europe, whereas two individuals were infected with HIV-1 subtype E which is mainly found in the heterosexual populations of Thailand. Sequence analysis of these variant HIV-1 strains revealed a number of interesting features and a phylogenetic tree was also constructed according to the V3 loop nucleotide sequences of these variant strains and HIV-1 isolates from other parts of the world. Furthermore, our results suggest that the north vs south geographical separation in terms of HIV-1 epidemiology in Taiwan is insignificant.     

Genetic analysis of HIV-1 Circulating Recombinant Form 02_AG, B and C subtype-specific envelope sequences from Northern India and their predicted co-receptor usage

HIV-1 epidemic in India is largely driven by subtype C but other subtypes or recombinants have also been reported from several states of India. This is mainly due to the co-circulation of other genetic subtypes that potentially can recombine to generate recombinant/mosaic genomes. In this study, we report detail genetic characterization of HIV-1 envelope sequences from North India (Delhi and neighboring regions). Six of 13 were related to subtype C, one B and the rest six showed relatedness with CRF02_AG strain. The subtype C possessed the highly conserved GPGQ motif but subtype B possessed the GPGR motif in the V3 loop as observed earlier. While most of the sequences suggested CCR5 co-receptor usage, one subtype C sample clearly indicated CXCR4 usage. A successful mother to child transmission was established in two pairs. Thus, co-circulation of multiple subtypes (B and C) and the recombinant CRF02_AG strains in North India suggests a rapidly evolving scenario of HIV-1 epidemic in this region with impact on vaccine formulation. Since this is the first report of CRF02_AG envelope from India, it will be important to monitor the spread of this strain and its impact on HIV-1 transmission in India.     

Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

The HIV epidemic in the Amazon Basin is driven by prototypic and recombinant HIV-1 subtypes B and F

This paper describes genetic subtypes of HIV-1 found in blood samples from 31 HIV-1-infected people who visited the Counseling and Testing AIDS Center of Instituto de Medicina Tropical in Manaus, Brazil. Manaus, the main city in Brazil's Amazon Basin, is also the closest urban connection for more than 100,000 Indians living in the rain forests of this region. Although to date there is no evidence of increased incidence of HIV-1 infection among the indigenous population, our understanding of both the prevalence and nature of the epidemic in the region as a whole is limited. From the 31 samples analyzed by C2V3 sequencing, we found almost equal proportions of HIV-1 strains belonging to subtype B (n = 16; 51.6%) and subtype F (n = 15; 48.4%), a finding that differs from results from previous studies conducted in urban areas of southeastern Brazil. We also observed the presence of the GWGR amino-acid sequence in the critical tetra-peptide crown of the env V3 loop in the HIV-1 subtype B samples analyzed. Among these samples, we also found 14 mosaic genomes (45.16%) in which different combinations of subtypes B, C, and F were identified between the p24 gag, pro, and env regions. Our data support the hypothesis that the Amazonian HIV-1 infections linked to the urban epidemic in southeastern Brazil. The genetic diversity and the prevalence of mosaic genomes among the isolates in our study confirm an integral role of recombination in the complex Brazilian epidemic.