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.     

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.     

应用多重巢式PCR法快速鉴定广西HIV-1主要亚型

目的 建立快速鉴定广西HIV-1主要流行亚型CRF01_AE、CRF07_BC、CRF08_BC、B和C的多重巢式PCR方法.方法 针对HIV-1 gag基因设计CRF01_AE、CRF07_BC、CRF08_BC、B和C亚型特异性引物,建立多重巢式PCR法,用该法鉴定来自广西的HIV-1毒株的亚型,并与基因测序法的鉴定结果比较.结果 多重巢式PCR法能正确鉴别5种已知亚型的样本,10份HIV阴性样本均无扩增,在72份未知亚型样本中,正确鉴定出66份,分别属于CRF01_AE、CRF07_BC、CRF08_BC、B亚型,多重巢式PCR法的灵敏度为91.7%,特异度达100%.结论 多重巢式PCR法能准确鉴定广西CRF01_AE、CRF07_BC、CRF08_BC和B亚型毒株,是一种简便、快速、低成本的亚型鉴定方法.     

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.     

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.     

广州市吸毒强戒人员HIV-1基因亚型分布的初步研究

目的分析吸毒人群HIV感染者中HIV-1的基因序列特征,初步确定其感染的基因亚型和各亚型的流行趋势。方法在Genbank中查找HIVgag基因的序列,设计巢式PCR引物。从全血中提取样本的基因组DNA,进行巢式PCR,对PCR产物进行琼脂糖凝胶电泳和测序,所获序列与国际参考株序列进行比对,确定基因型或亚型。结果对36例HIV-1感染者样本进行扩增,PCR共检测出29例阳性样本,27例测序成功,其中16例为CRF08-BC重组亚型、7例为CRF07-BC重组亚型、4例为CRF01-AE重组亚型,15例阴性对照均无目的条带。结论在广州吸毒强戒人员HIV-1型中以CRFBC亚型为主,其次为CRF01-AE亚型。应加强对HIV-1毒株亚型的监测,以制定更好的防治策略。     

广东省珠江三角洲地区吸毒者中流行的HIV-1 ENV基因序列分析

目的 了解广东省珠江三角洲地区吸毒者HIV-1亚型的流行规律及其与国际参考株的同源性.方法 应用套式聚合酶链反应(PCR)对43例采自广东省珠江三角洲HIV-1抗体阳性的吸毒者淋巴细胞富集液,并对其核酸样品进行扩增,使用ABI377型测序仪对扩增产物测序后,对其ENV基因C2-V3段的核酸序列进行比较分析.结果 43份血样中,29份为07-BC重组亚型,与国际参考株CN.97.C54A最近,基因离散率为(2.291±1.055)%,组内离散率为(3.534±1.306)%;9份为AE重组亚型,与国际参考株01AE.TH.90.CM240最近,基因离散率为(8.068±0.534)%,组内离散率为(2.823±1.235)%;3份为08-BC重组亚型,与国际参考株97CNGX-9F最近,基因离散率为(1.403±0.681)%,组内离散率为(1.507±0.681)%;2份为泰国B(B')亚型,与国际参考株RL42最近,基因离散率为(7.335±3.330)%,组内距离为8.52%.结论 广东省珠江三角洲地区吸毒者感染的HIV-1以曾经主要在西北地区吸毒人群中流行的07-BC重组亚型为主,也存在主要以性传播途径感染的人群中流行的AE重组亚型,提示珠江三角洲地区吸毒者中流行的HIV-1亚型趋于多样化.     

使用多重巢式PCR对我国HIV-1主要流行株亚型鉴定方法的建立

目的 建立一套新的亚型鉴定方法，仅仅使用巢式PCR，一次扩增，即可对我国HIV-1主要流行株B、C和CRF01-AE进行亚型鉴定。方法 从HIV阳性样本中提取核酸，使用能覆盖HIV-1型M组gag区的引物进行第一轮扩增，第二轮扩增则使用分别检测B、C、CRF01-AE亚型的三套特异性引物进行扩增，三套引物放在同一个反应管中。反应产物经琼脂糖电泳后观察，不同亚型的位置不同，以此来判断亚型。另外设计一套引物，专门检测我国重组株CRF07-BC和CRF08-BC。所有样品均经过基因测序、系统进化树分析，以进行结果验证。结果 在检测的119份样品中，经基因测序和系统进化树分析证实B亚型样品43份(欧美B11份，泰国B32份)，C、CRF01-AE、A和D亚型样品分别为54份、17份、3份和2份。其中C亚型的样品，有52份属于CRF07-BC和CRF08-BC。而经过上述多重巢式PCR方法检测到的B亚型样品为35份(81．4％)，C亚型46份(85．2％)和CRF01-AE13份(76．5％)。另外，检测CRF07-BC和CRF08-BC重组株的引物特异性地检测到43份(82．7％)样品。上述结果与基因分析结果吻合，各个亚型之间无交叉，一种亚型的特异性引物只对该亚型有反应，而对其他亚型无反应，特异性达到100％。虽然有时会有非特异扩增带，但一般不影响结果判断。结论 我们建立了一套简单快速的H1V-1亚型鉴定方法，不需基因测序，即可检测我国主要流行株B、C、CRF01-AE、CRF07-BC和CRF08-BC。该方法具有高度特异性和敏感性，可以作为初筛方法在我国及其他国家HIV-1实验室推广使用。     

HIV-1中国流行株CRF01_AE env基因改造及其重组DNA疫苗的构建

目的 构建表达含有密码子优化的HIV-1 CRF01_AE亚型env基因的DNA疫苗,为多载体艾滋病治疗性疫苗的应用提供候选疫苗.方法 对HIV-1感染者血液样本进行型别分析,分型得到的AE亚型标本采用亚型特异性引物克隆env基因,通过序列比对获得其共有序列,对该序列按照哺乳动物密码子使用的偏嗜性进行优化,将人工合成的优化基因克隆至pVR载体,构建DNA疫苗.通过Western Blot方法比较优化前后env基因的表达.结果 成功获得32条具有完整的开放性阅读框的env克隆,型别分析确认均为CRF01_AE亚型.成功对其共有序列进行了优化、合成并构建了DNA疫苗pVR-mod.AE env,该疫苗与野生型的env基因（wt.AE env）相比可以高水平表达env基因,且不依赖Rev的存在.结论 对HIV-1中国流行株CRF01_AE env基因的优化改造及重组DNA疫苗的构建是成功的.     

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。     

Rapid identification of human immunodeficiency virus type 1 CRF01_AE and BC recombinants by subtype-specific PCR

A subtype-specific PCR approach is described for the identification of HIV-1 intersubtype CRF01_AE and BC recombinants, the two predominant subtypes in Southern China. Primers were designed based on the env and gag regions of the HIV-1 genome. Nested PCRs with primers targeting the env region were performed to amplify subtype C, CRF01_AE, or BC recombinants. To differentiate BC recombinants from subtype C virus, a BC recombinant specific gag PCR was then performed. In order to identify the CRF07_BC and CRF08_BC recombinant forms, an additional PCR step was included. Four HIV-1 samples of known subtype, 77 samples with unknown-subtype, and 30 HIV-negative control samples were tested by the new assay. The results of this PCR-based subtyping approach were compared with that of a sequence-based phylogenetic analysis. In total, 73 (94.8%) samples were amplified by the subtype-specific PCR reactions, of which 39 were identified as CRF01_AE, 14 as CRF07_BC, and 20 as CRF08_BC. The sensitivity of this assay was 90.7% for the CRF01_AE recombinant and 100% for BC recombinants. The specificity was 100% when used to identify 30 HIV-negative samples. The reproducibility was 93.8% for CRF01_AE, and 100% for BC recombinants. This subtype-specific PCR technique represents a simple, rapid, and low-cost assay for the identification of HIV-1 CRF01_AE and BC recombinants in Southern China.     

Gut barrier structure,mucosal immunity and intestinal microbiota in the pathogenesis and treatment of HIV infection

Background

Since the first HIV-1 case in 1989, Hebei province has presented a clearly rising trend of HIV-1 prevalence, and HIV-1 genetic diversity has become the vital barrier to HIV prevention and control in this area. To obtain detailed information of HIV-1 spread in different populations and in different areas of Hebei, a cross-sectional HIV-1 molecular epidemiological investigation was performed across the province.

Methods

Blood samples of 154 newly diagnosed HIV-1 individuals were collected from ten prefectures in Hebei using stratified sampling. Partial gag and env genes were amplified and sequenced. HIV-1 genotypes were identified by phylogenetic tree analyses.

Results

Among the 139 subjects genotyped, six HIV-1 subtypes were identified successfully, including subtype B (41.0 %), CRF01_AE (40.3 %), CRF07_BC (11.5 %), CRF08_BC (4.3 %), unique recombinant forms (URFs) (1.4 %) and subtype C (1.4 %). Subtype B was identified as the most frequent subtype. Two URF recombination patterns were the same as CRF01_AE/B. HIV-1 genotype distribution showed a significant statistical difference in different demographic characteristics, such as source (P < 0.05), occupation (P < 0.05) and ethnicity (P < 0.05). The distributions of subtype B (P < 0.05), CRF01_AE (P < 0.05), CRF07_BC (P < 0.05) and subtype C (P < 0.05) showed significant differences in all ten prefectures, and the distributions of all six subtypes were significantly different in Shijiazhuang (P < 0.05) and Xingtai (P < 0.05), but not in other prefectures (P > 0.05). The differences in HIV-1 genotype distribution were closely associated with transmission routes. Particularly, all six subtype strains were found in heterosexuals, showing that HIV-1 has spread from the high-risk populations to the general populations in Hebei, China. In addition, CRF01_AE instead of subtype B has become the major strain of HIV-1 infection among homosexuals.

Conclusions

Our study revealed HIV-1 evolution and genotype distribution by investigating newly diagnosed HIV-1 individuals in Hebei, China. This study provides important information to enhance the strategic plan for HIV prevention and control in China.

     

Human Immunodeficiency Virus Type 1 Subtypes Prevalence in Central China

Purpose

To study the epidemic characteristics, transmission sources and routes of various subtypes of human immunodeficiency virus type 1 (HIV-1) and sequence variations in Henan, central China. To provide theoretical foundation for Acquired Immune Deficiency Syndrome (AIDS) prevention strategy in this region where the primary HIV transmission route was through former paid blood donation.

Materials and Methods

HIV-1 gene env and gag were amplified by nested polymerase chain reaction (PCR) from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 1,287 HIV-1 confirmed samples in Henan.

Results

Among 1,287 samples, 5 HIV-1 strains were found including subtypes B'' (95.9%), C (0.47%) and recombinant subtypes CRF 07_BC (1.09%), CRF 08_BC (1.79%) and CRF 01_AE (0.78%). Phylogenetic tree analysis found that 1,234 Henan subtype B'' were closely related to those commonly found in Thailand, and were distantly related to other international subtypes. The dominant strain in former blood plasma donors (FPDs) was subtype B'', and the dominant strains in sexual transmission were subtype B'' and BC. Among HIV patients who were most likely infected through routes other than paid blood donation, the percentage of non-B'' subtypes was much higher than those of FPD.

Conclusion

These findings suggest that the prevailing strain of HIV-1 in Henan is subtype B'', similar to the B'' subtype found in Thailand. In addition, for the first time we found subtypes C and recombinant subtypes CRF07_BC, CRF08_BC and CRF01_AE in this region. Indicating that the subtype feature of HIV-1 became more complicated than before in central China.     

Distinguishing molecular forms of HIV-1 in Asia with a high-throughput, fluorescent genotyping assay, MHAbce v.2

High-resolution HIV-1 genotyping of large sample sets is crucial to define the evolving and dynamic epidemics in Asia. Here we present MHAbce v.2, a multi-region hybridization assay that individually discriminates subtypes B, C, CRF01_AE, and virtually all of their described recombinants, based on real-time PCR using subtype-specific TaqMan probes in 8 regions throughout the viral genome. In a validation panel (n=70), the assay performed with a sensitivity of 95.7% and specificity of 99.8%. The assay was field-tested on samples from a retrospective MTCT cohort (n=180; Lampang Province, Northern Thailand; 1996-1998). 177/180 of the samples were typeable, and 94.4% were typed as CRF01_AE. The remaining strains represented even proportions of subtype B and B/CRF01_AE recombinants and were confirmed by sequencing, revealing early links between the heterosexual and IDU HIV-1 epidemics in Thailand. MHAbce v.2, with an area of application including China, India, Southeast Asia, and the Pacific Rim, can be used to develop a comprehensive and detailed picture of this important component of the HIV/AIDS pandemic.     

Temporal trends and molecular epidemiology of HIV-1 infection in Taiwan from 1988 to 1998

Eight hundred and seventy-nine HIV-1-infected patients (comprising 46% of reported HIV-1/AIDS cases in Taiwan) were recruited for this study of the molecular epidemiology of HIV-1 in Taiwan from 1988 to 1998. HIV-1 subtypes were determined using a modified peptide-enzyme immunoassay complemented with DNA sequencing and phylogenetic analysis. Of the 807 HIV-1 infected men, 68.2% were infected with HIV-1B, 29.5% with HIV-1 circulating recombinant form (CRF)01_AE and 2.3% with other subtypes. Of the 72 HIV-1-infected women, 72.2% were infected with HIV-1 CRF01_AE, 13.9% with HIV-1B, and 13.9% with other subtypes. All of 8 foreign-born, Southeast Asian women and 6 of 7 (85.7%) Taiwan-native female commercial sex workers were infected with HIV-1 CRF01_AE. Fourteen of the 33 (42.4%) heterosexual married men with CRF01_AE had transmitted HIV-1 to their wives, whereas only 1 of 17 (5.9%) men with HIV-1 B had transmitted HIV-1 to their spouses (p < .01). Of 18 heterosexual male injecting drug users, 1 of 12 (8.5%) with HIV-1B and 5 of 6 (83.3%) with HIV-1 CRF01_AE had had sexual contact with female commercial sex workers (p < .01). Therefore, in this population, CRF01_AE was preferentially associated with heterosexual risk groups, a finding compatible with differences in transmission capability between B and non-B subtypes.