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.     

HIV-1毒株V3环顶端四肽多态性及原发耐药性研究

目的 探索我国河南省新蔡县既往献血人群中HIV-1病毒流行株env基因V3环顶端四肽多态性和pol基因原发耐药性变异特征.方法 套式聚合酶链反应（Nested-PCR）扩增51例确认HIV阳性样本env、pol基因部分区段并测序,PCR产物纯化后克隆,挑选克隆株鉴定为阳性后测序,以MEGA（version 3.0）等软件进行分析.结果 env基因V3环顶端四肽主要为GPGR、GPGQ、GPGK、GQGR;在pol基因PR区未发现有关蛋白酶抑制剂（PIs）的主要耐药性突变位点,在RT区共发现9条序列存在对NRTIs或NNRTIs中的一类药物耐药性突变.结论 GPGR这一典型的欧美B亚型V3环顶端四肽序列比例最高为44.44%;原发性耐药发生的比例较低,药物敏感性资料表明耐药性突变位点多表现为低度耐药性,因此提示我们在对该地区因献血感染HIV的患者在首次治疗时可不必以耐药性检测做指导.     

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.     

淮安市经异性性传播的HIV感染者中HIV-1病毒基因亚型特征分析

目的 了解淮安市经异性性传播HIV感染者中HIV-1病毒基因亚型分布及其流行特征.方法 从60名异性性传播HIV-1感染者血液中提取DNA或RNA,用巢式PCR或RT-PCR方法扩增gag、env基因区片段,测定序列并分析.结果 60份标本中,确定了48份标本的基因亚型,发现有4种HIV-1亚型和重组型.其中CRF01_AE亚型占62.5％,CRF07_BC亚型占22.9％,CRF08_BC亚型占6.3％,B亚型占6.3％.结论淮安市异性性传播感染HIV者中,流行着CRF01_AE、CRF07_BC、CRF08_BC、B亚型这4种亚型病毒株,CRF01_AE为主要流行亚型.     

我国HIV-1感染者耐药突变的流行性研究

目的 掌握我国大规模用药前耐药突变在我国HIV-1感染者中流行情况的本底资料。方法 在第二次全国HIV流行病调查2002年样本中随机选取20％，对于蛋白酶(PR)基因区全长和逆转录酶(RT)20～230氨基酸位点进行PCR扩增、测序并使用HIVdb-Drug Resistance Algorithm软件进行分析，检测耐药相关突变。结果 分别得到164份PR基因区样本和138份RT基因区样本。PR基因区样本中发现1份(0．61％)样本存在蛋白酶抑制剂(PI)主要相关突变，163份(99．39％)样本存在P1次要耐药相关突变。RT基因区样本中发现8份(5．80％)具有核苷类逆转录酶抑制剂(NRTI)耐药相关突变，2份(1．45％)存在非核苷类逆转录酶抑制剂(NNRTI)耐药相关突变。结论 我国未用药HIV感染者中耐药相关突变尚处于低流行状态，应当在用药过程中定期监控以减少耐药突变的产生与传播。     

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。     

北京市2006年新确认HIV-1感染者耐药突变的流行性研究

目的 研究北京市2006年新确认HIV-1感染者毒株的耐药突变本底数据.方法 随机选取北京市2006年新确认HIV-1感染者抗凝全血标本50份,提取血浆病毒RNA,用逆转录聚合酶链反应扩增HIV-1 pol区基因片段,并进行序列测定及耐药基因型分析.结果 成功扩增出34份标本的pol区基因;在1例样本的蛋白酶编码区检测出1个主要耐药突变,7例样本检测出7个次要耐药突变,主要耐药突变为M46L,毒株是CRF01_AE亚型,次要耐药突变有4种,出现的频率分别为A71T(2个)、A71V(3个)、Q58E(1个)、V11IV(1个).在14例样本逆转录酶编码区检测出一种或多种核苷类和(或)非核苷类逆转录酶抑制剂耐药突变,9例标本检出核苷类逆转录酶抑制剂耐药突变,出现频率分别为:V118I(42.9%)、M184V(7.1%)、A62V(7.1%)、K70T(7.1%)、K65R(7.1%)、K219N(7.1%)、T69d(7.1%)、V75LV(7.1%)、K219R(7.1%);10例标本检出核苷类逆转录酶抑制剂耐药突变,出现的频率分别为V1061(35.5%)、Y181C(15.4%)、K103KR(7.7%)、K103R(7.7%)、L100LV(7.7%)、V1081(7.7%)、V179D(7.7%)、V179DV(7.7%).结论 北京市2006年新确认HIV-1感染者毒株中已经存在一定比例耐药突变,有必要定期进行耐药性监测研究.     

广西HIV-1重组毒株env区快速基因分型方法的建立

目的建立一种快速简便的基因分型方法，对广西HIV-1重组毒株env基因区进行亚型鉴定。方法从HIV阳性样品中提取核酸，使用HIV-1M组通用引物对env区进行第一轮扩增，第二轮则使用分别检测B′/C或C亚型和CRF01-AE亚型的二套特异性引物放入同一反应管中进行扩增，根据不同亚型扩增的目的带位置不同来判断亚型。将通用引物扩增出的所有样本均进行基因测序和系统树分析以验证结果。结果50份样本中，经基因测序和系统树分析证实CRF08-BC样本3份（6％），CRF01-AE样本43份（86％），4份（8％）样本无法确定亚型。经亚型特异性引物PCR法检测得出B′/C或C亚型样本3份（100％），CRF01-AE样本39份（90．7％），灵敏度为91．3％，特异度为100％。两种方法检测结果经差异性检验显示X^2=2．25，P〉0.05，差异无统计学意义，结果一致者占92％。与基因分析结果吻合。重复实验显示CRF08-BC平均重复性为100％（10／10），CRF01．AE为93．8％（61／65）。结论该方法是一种简便、快速、低成本，具有高度灵敏性和特异性的HIV-1毒株env基因区分型法，能够直接对广西HIV-1 CRF01-AE重组毒株进行鉴定。     

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。     

Distribution of human immunodeficiency virus type 1 subtypes in the State of Amazonas,Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible “homogenous” subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country. Key words: HIV-1; Subtypes; Phylogenetic analysis; Blood donors; Molecular and epidemiological characterization     

Genetic diversity of HIV type 1 in rural eastern Cameroon

To monitor the presence of genotypic HIV-1 variants circulating in eastern Cameroon, blood samples from 57 HIV-1-infected individuals attending 3 local health centers in the bordering rural villages with Central African Republic (CAR) were collected and analyzed phylogenetically. Out of the 40 HIV-1 strains with positive polymerase chain reaction (PCR) profile for both gag and env-C2V3,12 (30.0%) had discordant subtype or CRF designation: 2 subtype B/A (gag/env), 1 B/CRF01, 2 B/CRF02, 1 CRF01/CRF01.A, 2 CRF11/CRF01, 1 CRF13/A, 1 CRF13/CRF01, 1 CRF13/CRF11, and 1 G/U (unclassified). Twenty-eight strains (70.0%) had concordant subtypes or CRF designation between gag and env: 27 subtype A and 1 F2. Out of the remaining 17HIV-1 strains negative for PCR with the env-C2V3 primers used, 10 (58.8%) had discordant subtype or CRF, and 7 (41.2%) had concordant one based on gag/pol/env-gp41 analysis. Altogether, a high proportion (22/57, 38.6%) of the isolates were found to be recombinant strains. In addition, an emergence of new forms of HIV-1 strains, such as subtype B/A (gag/env), B/CRF01 and B/CRF02, was identified. The epidemiologic pattern of HIV-1 in eastern Cameroon, relatively low and high prevalence of CRF02 and CRF11, respectively, was more closely related to those of CAR and Chad than that of other regions of Cameroon, where CRF02 is the most predominant HIV-1 strain. These findings strongly suggest that this part of Cameroon is a potential hotspot of HIV-1 recombination, with a likelihood of an active generation of new forms of HIV-1 variants, though epidemiologic significance of new HIV-1 forms is unknown.     

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.     

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.     

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。     

Genetic analysis of HIV-1 strains in rural eastern Cameroon indicates the evolution of second-generation recombinants to circulating recombinant forms

The HIV-1 genetic diversity in most parts of Cameroon is well described and shown to be very broad. However, little is known about the composition of the HIV-1 epidemic in the rural parts of eastern Cameroon. Therefore, we investigated 25 specimens from this region for their subtypes in gag, pol, and env gene fragments. Along with genetic material of subtypes A1, C, G, CRF01_AE, CRF02_AG, and CRF11_cpx, we also identified a large number (24%, 6/25) of distinct env sequences within the subtype A radiation. CRF02_AG was the predominant genetic form in all genes studied. Half of the specimens studied were considered "pure" based on concordant subtypes in the genes studied, whereas the other half were unique recombinant forms (URFs). Except for 1 URF, all were second-generation recombinants (SGRs), 90% of which contained genetic material of CRF02_AG in at least 1 gene. Notably, we identified individuals from 3 different villages infected with CRF01_AE(gag)CRF02_AG(pol)A(env) strains, which is indicative of the evolution of this URF to a circulating recombinant form (CRF). In addition, we identified a CRF02_AG(pol)C(env) recombinant infecting a man and a woman living in the same village, suggesting horizontal transmission of this recombinant. The current study emphasizes the power of HIV-1 recombination through the generation of SGRs and the evolution of URFs into CRFs. These findings suggest that, in a region where a predominant HIV-1 strain cocirculates among several subtypes, recombination could eventually decrease the proportion of this strain over time, such as CRF02_AG in Cameroon.     

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.