云南省HIV-1 CRF01_AE毒株基因型耐药研究

目的 了解云南省HIV-1 CRF01_AE毒株的流行分布及基因型耐药情况。 方法 收集2018—2021年云南省抗病毒治疗失败的HIV/AIDS患者的人口学资料,采集患者血浆,逆转录聚合酶链反应扩增HIV-1 pol基因蛋白酶和逆转录酶区并进行序列测定,对基因型为CRF01_AE的序列进行耐药情况分析。结果 通过基因型耐药检测共获得967例亚型为CRF01_AE序列,其中男性682例（占70.5%）,耐药509例,耐药率为52.6%,异性性传播813例（占84.1%）,同性性传播95例（占9.8%）。同性性传播的耐药率最高,为65.3%;治疗时长<12个月的耐药率最低,为35.9%,36~<48个月耐药率最高,为58.1%。核苷类反转录酶抑制剂（NRTIs）耐药率为33.9%,非核苷类反转录酶抑制剂（NNRTIs）的耐药率为48.5%,蛋白酶抑制剂（PIs）的耐药率较低为1.3%。NRTIs类药物中阿巴卡韦（ABC）、恩曲他滨（FTC）和拉米夫定（3TC）耐药率较高,分别为为32.9%、32.4%和32.4%;NNRTIs类药物中奈韦拉平（NVP）和依非韦仑（EFV）耐药率较高,分别为47.6%和47.5%,均以高度耐药为主。一线治疗方案耐药率56.2%高于二线治疗方案耐药率29.5%,差异有统计学意义（P<0.001）。NRTIs耐药突变位点中,以M184V/I突变位点为主,突变率为30.0%;NNRTIs耐药突变位点中,以K103N/Q/S突变位点为主,突变率为28.1%;PIs耐药位点突变率相对较低,主要的耐药突变位点是L33F,突变率为3.7%。结论 云南省抗病毒治疗失败亚型CRF01_AE的患者耐药率有所降低,但部分地区及人群的耐药率仍较高,应加强对重点地区和人群的管理,加强耐药监测,对治疗失败的患者及时掌握耐药情况,合理调整治疗方案。     

广西防城港市HIV-1流行株env基因C2V3区序列特征及亚型分析

目的了解近期防城港市HIV-1流行毒株env基因C2V3区亚型及分布特征,为防城港市防治艾滋病工作提供科学依据。方法于2017年10月～2018年4月在防城港市人民医院收集199例定点随访的接受抗病毒治疗的HIV感染者或艾滋病病人(human immunodeficiency virus/acquired immunodeficiency syndrome,HIV/AIDS)的流行病学调查信息和10 ml外周血。从外周血白细胞中提取前病毒DNA,用巢式聚合酶链反应对获得的HIV-1前病毒DNA env C2V3区基因片段进行扩增,并进行DNA测序,获得的DNA序列信息使用Sequencher 5.1、MEGA 5.0软件进行清理和分析。结果本研究共收集199例HIV/AIDS,其人口学特征主要以年龄18～49岁为主,占53.70%;职业以农民和待业为主,分别占42.59%、45.37%;学历以初中及以下为主,占89.81%;民族以汉族为主,占75.93%;传播途径以异性传播为主,占93.52%。成功扩增HIV-1env C2V3区基因108例,其中CRF01_AE(Circulating Recombinant Forms,CRF)亚型103例,CRF BC亚型3例,B、CRF59_01B亚型各1例。发现8种gp120 V3环顶端四肽,其中以GPGQ为主,占50.00%;其次为GPGR和GPGK,分别占22.22%、12.96%。预测辅助受体为CXCR4(chemo-kine receptor 4) 59例,占54.63%;其次是CCR5辅助受体(chemo-kine receptor 5)30例,占27.78%;CXCR4/CCR5辅助受体19例,占17.59%。结论防城港市主要流行HIV-1 CRF01_AE亚型,其流行毒株亚型存在多态性。防城港市HIV-1型CRF01_AE毒株膜区V3环氨基酸顶端四肽存在较高的多态性,使用CXCR4辅助受体的HIV-1毒株比例高于用CCR5辅助受体的毒株。     

HIV-1 CRF01_AE毒株gp41蛋白生物信息学预测

目的：对HIV-1 gp41蛋白的膜外区和跨膜区进行预测,并分析该蛋白的结构和功能。方法：从NCBI GenBank数据库获取gp41基因及其蛋白编码序列,利用ExPASy ProtParam tool、ProtScale、SignalP 4.0 Server、TMHMM Server v.2.0、SWISS-MODEL、PredictProtein等工具对gp41蛋白的生物学特性进行预测。结果：HIV-1 gp41基因ORF长度为666 bp,编码222个氨基酸,蛋白分子式为C1164H1815N317O325S6,等电点（isoelectric point,pI）＝8.60,属于稳定、疏水性蛋白。该蛋白具有跨膜区,信号肽切割位点位于第20和21位氨基酸处。其二级结构由无规则卷曲、β折叠和α螺旋组成,属于全螺旋型蛋白。而且该蛋白具有N-端信号肽,能引导蛋白分泌到胞外。此外gp41蛋白具有4个蛋白质-蛋白质和1个蛋白质-核酸结合位点,这些位点能调控gp41蛋白的多种生物学功能。结论：对gp41蛋白的结构和功能进行了预测,为深入研究其参与疾病发生发展的分子机制奠定了基础。     

基于HIV-1 pNL4-3构建含CRF07_BC V3的嵌合分子克隆

目的在感染性克隆pNL4-3骨架上构建含CRF07_BC V3的嵌合分子克隆。方法通过融合PCR将CRF07_BC pXJDC13的V3环与pNL4-3的env骨架进行融合并克隆至pNL4-3上。经过鉴定后将阳性V3嵌合克隆pNL4-3/XJDC13V3转染到293T细胞进行病毒包装并检测病毒的感染性。V3嵌合病毒的细胞嗜性分别用Ghost细胞系和MT-2细胞进行测定。结果利用融合PCR克隆方法在pNL4-3骨架上成功构建了含CRF07_BC pXJDC13 V3的嵌合分子克隆pNL4-3/XJDC13V3。该嵌合病毒利用的辅助受体为CCR5,不能利用CXCR4,也不能在MT-2细胞上诱发合胞体。结论在pNL4-3骨架上成功构建了含CRF07_BC pXJDC13 V3嵌合分子克隆pNL4-3/XJDC13V3。该嵌合分子克隆为进一步研究CRF07_BC嗜性转变中V3关键位点提供了有利工具。     

沈阳市1份人类免疫缺陷病毒的基因序列分析

目的 :对 2 0 0 4年我院门诊检出的 1例艾滋病感染者体内的HIV 1病毒进行基因序列分析和亚型鉴定。方法 :从HIV 1感染者的全血标本中提取脱氧核糖核酸 (DNA)作为PCR扩增的模板 ,套式引物扩增HIV 1前病毒DNA的p17与p2 4交界部分的基因片断并进行测序。所测定序列与各亚型国际参考株及亚洲流行参考序列进行比较 ,确定被检标本的亚型 ,并进行基因序列分析。结果 :该患HIV 1病毒属于B’亚型毒株 ,经输供血途径感染。该毒株与泰国B’亚型参考株B TH 92 92TH0 14的基因距离最为接近。结论 :在沈阳市有效地控制艾滋病经输供血途径传播应引起我们高度的重视     

深圳市HIV-1毒株的流行状况

目的了解深圳地区人类免疫缺陷病毒1型(HIV-1)毒株亚型及流行情况,分析其传染来源和传播规律。方法应用巢式聚合酶链反应技术,对122例在深圳市发现的HIV-1感染者外周血单个核细胞中的env基因和gag基因进行扩增,并对各基因区核苷酸序列进行测定和分析。结果122份样本中共存在CRF01_AE、CRF08_BC、CRF07_BC3种重组毒株以及B'、B、C3种亚型,其在所有分析样本中的比例分别为45.1%(55/122)、31.1%(38/122)、6.6%(8/122)、14.8%(18/122)、1.6%(2/122)和0.8%(1/122)。CRF01_AE、CRF08_BC、CRF07_BC、B和B'亚型间的组内离散率分别为(4.455±1.478)%、(2.997±1.345)%、(4.380±2.024)%、(5.186±2.487)%和(4.869±2.638)%,与部分国内和国际参考株01AE.TH.90.CM240、97CNGX-9F、CN.97.C54A、B.US.83.JRFL、RL42核苷酸序列间的离散率分别为(5.228±0.823)%、(3.634±1.073)%、(4.233±1.119)%、(4.950±2.564)%、(5.795±2.198)%。CRF07_BC和CRF08_BC亚型以吸毒人群为主,CRF01_AE重组亚型以性途径和吸毒人群为主,B和B'亚型主要分布在静脉吸毒、性传播和献/输血人群。结论深圳地区有3种亚型和3种重组亚型HIV-1毒株流行,CRF01_AE、CRF08_BC重组亚型和B'亚型为主要流行毒株,CRF01_AE是性传播人群中的主要流行毒株,CRF08_BC和CRF01_AE是静脉吸毒人群中的主要流行毒株。     

我国人类免疫缺陷病毒-1主要流行株外膜蛋白基因V3-V4区及其临近区域的特征性氨基酸分析

目的鉴定我国HIV-1主要流行毒株亚型的env,V3-V4区及其临近区域的特征性氨基酸,并阐明其在追踪传染源和研究疫苗中的作用。方法应用nested—PCR对157份来自我国12个省份的HIV-1毒株env区序列进行扩增,并使用ABI377型测序仪测序,然后应用BLAST、GCG、MEGA和VESPA等生物学软件或程序对env基因V3-V4区及其临近区域序列进行基因型鉴定、系统树分析及特征性氨基酸鉴定。结果157份样本包括54份B′(34．40％)、61份B′／C(38．85％)和42份CRF01-AE(26．75％)毒株。系统树分析结果显示,B′亚型毒株序列均与B．CN．RL42十分接近,B′／C毒株主要与97CN54A和97CNGX6F聚成一簇。而CRF01—AE序列与THCM240和97CNGX2F聚在一起．而且分别聚成明显不同的两个亚组。特征性氨基酸分析发现,我国B′和B′／C毒株分别具有8个保守的特征性氨基酸,而且与代表株的相同位点氨基酸基本一致。而CRF01-AE重组毒株具有11个保守的特征性氨基酸,其中有9个位点与97CNGX2F和TH.CM240不一致。包括这9个特征性氨基酸的样本主要来自除云南省以外的其他省份。结论目前流行于我国的B′和B′／C毒株具有单一的共同传染源,而CRF01-AE毒株可能是通过不同输入源或不同传播途径先后从泰国传入我国的。这将对我国艾滋病防治策略的制定和正在进行的疫苗研究具有重要的指导意义。     

1型人类免疫缺陷病毒tat基因重组分泌型真核表达载体的构建

目的构建人类免疫缺陷病毒1型(HIV-1)tat基因分泌型真核表达载体。方法聚合酶链反应(PCR)法从缺失pol基因的重组HIV-1基因组pNL4-3中扩增tat基因;将PCR获得的tat基因片段和pSecTag2B分泌型真核表达载体分别用Hind III酶切;pSecTag2B载体片段经小牛碱性磷酸酶去磷酸化后,用T4 DNA连接酶将tat基因与pSecTag2B载体片段连接过夜并转化感受态大肠杆菌DH5α,提取阳性克隆质粒进行Hind III酶切鉴定,并利用Nco I酶切鉴定克隆的tat基因反向。最后选取正向tat基因克隆质粒送基因测序。结果 PCR扩增产物在1%琼脂糖凝胶上约320bp位置出现条带,与预期的324bp大小一致;经Hind III和Nco I分别酶切鉴定,获得2个正向克隆;正向克隆经测序,克隆的tat基因序列与Genbank中登记的HIV-1 tat基因100%同源。结论本研究方法可以成功地构建HIV-1 tat基因分泌型真核表达载体。     

抗人类免疫缺陷病毒-1的治疗研究

获得性免疫缺陷综合征(AIDS)是由人类免疫缺陷病毒(HIV)感染引起的一种严重的疾病，当HIV侵入人体后借细胞表面的CD4分子及某些趋化因子受体侵犯宿主细胞，造成机体免疫功能破坏，进而引起严重的机会感染及肿瘤。现就该病治疗的中心环节——抗病毒治疗，尤其是高效逆转录病毒疗法(HAART)的治疗现状及研究进展简介如下。     

人类免疫缺陷病毒1型AE重组亚型毒株全长gp120基因序列分析

目的研究人类免疫缺陷病毒1型(HIV-1)AE重组型(CRF01-AE)毒株全长gp120基因的变异特征,为针对CRF01-AE重组型的免疫学研究和疫苗的研制提供帮助。方法应用巢式PCR对福建省2004至2005年期间100例HIV-1CRF01-AE感染者随机抽取21份血样的外膜蛋白全长gp120进行扩增、回收、克隆至T载体后测序,应用MEGA、DNASIS、CLUSTAL1·83、N-GLYCOSITE等软件对HIV-1全长gp120序列进行分析。结果基因系统树显示这21份样本属于CRF01-AE重组型HIV-1株,样本组内基因距离为9·5%±2·5%。V3环顶端四肽存在4种类型:GPGQ(71·43%),GPGR(19·05%),GPGH(4·76%),GQGQ(4·76%)。根据V3环关键氨基酸推测辅助受体使用情况的结果显示,16份(76·19%)可能使用CCR5作为辅助受体,1份(4·76%)样本可能使用CCR5/CXCR4序列,4份样本(19·05%)不能做出预测。21条HIV-1全长gp120氨基酸序列中,C1-C5比较保守,而V1-V5变化较大,其中V3相对保守。N糖基化位点分析发现大多数位点较为保守,少数发生糖基化位点丢失和增加。结论福建省大部分CRF01-AE重组型毒株与东南亚代表株关系密切,来源较为复杂,大部分CRF01-AE重组毒株为NSI型,gp120基因变异大,该研究为以后的病毒免疫学和疫苗的研究提供丰富的材料和基础数据。     

Genetic characterization of three CRF01_AE full-length HIV type 1 sequences from Fujian Province, China

Background One of the major characteristics of the human immunodeficiency virus type 1 （HIV-1） is its unusually high degree of genetic variability, which involves in genetic diagnosis, subtyping, vaccine design, and epidemiology. HIV-1 CRF01_AE is a main prevalent HIV-1 recombinant strain in China. In this study, three full-length CRF01_AE genomes from Fujian Province, China were cloned, sequenced, and analyzed; and the further genetic diversity defining and epidemiologic analysis were carried out. Methods Proviral DNA was extracted from non-cultured peripheral blood mononuclear cells, the near full-length HIV-1 genome was amplified and the PCR products were cloned into pCR-XL-TOPO vector and sequenced. 5＇-long terminal repeat （LTR） and 3＇-LTRs were amplified by additional independent PCR and cloned into pMD18T vector. Gene-based phylogenic tree was constructed and genetic distances were calculated by MEGA 3.1. Simplot was used for Bootscan analysis. Results The phylogeny and genetic distance analysis of the three near full-length sequences confirmed that these three samples clustered with CRF01_AE isolates, more close to Thailand CRF01_AE strain CM240, and were distantly related to African CRF01_AE strain 90CF402. Analysis of their genomic organization revealed the presence of nine potential open reading frames. There were no major deletions, rearrangements, or insertions in the three sequences, but an in-frame stop codon was found in tat gene of Fj051. LTRs of the three sequences contained a few nucleotides mutation. We did not find new mosaic recombinant in the three sequences. The V3 motif was GPGQ in all the three sequences, and there were only few amino acids differences in all three V3 loop sequences. Conclusion This report reveals the background of the three full-length CRF01_AE genomes, the most dominantly circulating HIV-1 strain in Fujian Province, China. The work is essential for the design and development of an effective AIDS vaccine for the region.     

Subtype and sequence analysis of HIV-1 strains in Heilongjiang Province

Background Human immunodeficiency virus (HIV-1) is divided into two types, HIV-1 (groups M, N and O) and HIV-2. Heilongjiang Province located in the northeast of China, and the feature of the subtype distribution and sequence characteristics of HIV-1 strains prevalent in Heilongjiang Province is still uncertain. The aim of this study was to investigate the subtype distribution and genetic characteristics of HIV-1 strains in one hospital in Heilongjiang Province. Methods HIV-1 env gene was amplified by nested-PCR from peripheral blood mononuclear cells (PBMCs) obtained from 19 HIV-1 seropositive individuals in Heilongjiang Province. The C2-V3 region was sequenced. Aligned the nucleotide sequence of 19 samples with CLUSTAL W (BioEdit) software, results were acquired and used for phylogenetic tree analysis after artificial adjustment. Reference sequence, downloaded from Los Alamos HIV Sequence Database, was used to identify the subtype of obtained sequence. Genetic distance between sequences was assessed using the software MEGA 3.1 Kimura 2-parameter, and the Phylogenetic tree was reestablished with Neighbor-Joining method.Results Phylogenetic tree analysis showed that 19 Heilongjiang strains clustered closely to subtype B strain from Thailand and were far from other international subtype reference strains. Statistical test showed no significant discrepancy between the genetic distance of interclass and intra-class (P&gt;0.05). The analysis of V3 loop amino sequence of 19 Heilongjiang B strains revealed that V3 tip motif of 10 samples (52.63%) was GPGQ, and of 4 samples (21.53%) was GPGR.Conclusions The subtype of 19 HIV-1 seropositive individuals in Heilongjiang Province is B’, and it is introduced from He’nan Province. V3 tip motifs of the HIV-1 isolates are mainly GPGQ and GPGR.     

湖南省20株狂犬病毒株与疫苗株N基因序列分析

目的 分析湖南省狂犬病毒株与疫苗株的N基因序列及遗传进化关系,从基因角度解决疫苗的选择问题,为狂犬病的防治提供科学依据.方法 采用KT-PCK法从市售家犬脑组织标本内获得N基因并进行测序,采用DNAStar软件包中的MegAlign软件,将所得结果与国内外发表的代表性疫苗株相应基因进行核苷酸、氨基酸序列同源性比对分析,并以Neighbor-Joining法构建系统发生树.结果 湖南省20株狂犬病毒与国内外13株疫苗代表的株核苷酸同源性在85.3%～94.2%(中位数88.6%).相应地,N基因编码的氨基酸序列同源性为95.4%～99.6%(中位数99.2%).其中,与中国疫苗株CTN、SRV-9,国外疫苗株CVS、RBE3-15、SADB19-1st及HEP-Fllury株的氨基酸序列同源性范围在99.2%～99.6%,并以与CTN疫苗株同源性中位数最高(90%).系统发生分为两大群.湖南省20株研究毒株与中国人用CTN株具有很高的亲缘性,构成第一群.其余的疫苗毒株构成第二基因群.结论 湖南省狂犬病毒株与中国人用疫苗株CTN同源性最高、亲缘关系最近,因此在湖南省使用CTN疫苗株效果可能较好.     

HIV-1gag基因p24编码区的变异性分析

目的　了解我国HIV感染者p2 4蛋白编码区的基因变异情况。 方法　收集我国河南及上海地区 2 8份已经证实的HIV - 1感染患者的血浆标本并抽提出RNA ;采用逆转录和Nested PCR的方法扩增所需的目的基因 ,使用DNA测序仪直接测序 ;应用CLUSTALW、PHYLIP等软件包对序列进行比对及进化树分析。结果　2 8份gag样本中 2 5份为B亚型 ,3份为A亚型。与共享序列相比 ,2 5例B亚型的 p2 4编码区中发生核苷酸改变的位点比例为 0 .4 %～ 4 .8% ,平均为 1.4 % ,其中A→G占 2 0 .5 % ,G→A占 17.3% ;3例A亚型发生核苷酸改变的位点比例平均为 0 .2 4 %。在所有变异位点中均未发现G→A的高度突变。B亚型和A亚型内的基因离散率平均为 2 .9%和 0 .5 8% ,A亚型与B亚型之间的基因离散率平均为 11.1%。 2 5例B亚型根据基因序列所推测的p2 4蛋白发生氨基酸改变的比例为 0 .4 %～ 5 .2 % ,平均为 2 .2 %。进化树分析表明在我国河南地区HIV感染B亚型多为与泰国株相近的B’亚型。结论　HIV 1p2 4编码区仍相对保守 ,选择无变异发生的区域有助于免疫治疗及疫苗研制等方面的研究     

Genomic Diversity of Human Immunodeficiency Viruses

Background

Globally circulating strains of human immunodeficiency virus type one (HIV-1) exhibit an extraordinary degree of genetic diversity. Sequences derived from HIV-1 strains have historically been classified on the basis of their phylogenetic relationships. The viruses have been classified into groups, subtypes or clades and circulating recombinant forms (CRFs). Groups were originally named M for main, O for outlier and N for Non-M-Non-O. The identification of subtypes and CRFs provides a means of tracking the dissemination of the pandemic.

Methods

Various methods to study the molecular epidemiology of HIV-1 are virus isolation, cloning, DNA sequencing, restricted fragment length polymorphism of the molecularly cloned and amplified genome (PCR-RFLP), RNase mismatch cleavage analyses of RNA, RNA heteroduplexes derived from culture amplified virus, primer mismatch sensitive PCR to identify specific mutations, single strand conformational polymorphism (SSCP) to localize mutations arising over short regions of env gene, denaturing gradient gel electrophoresis and serological assays based on V3 peptide. Except for PCR-RFLP and denaturing gradient gel electrophoresis, these techniques do not easily allow simultaneous analyses of multiple sequence variants and include the laborious and selective process of virus co-cultivation or molecular cloning prior to analyses. The extensive DNA sequence analyses remain the gold standard for epidemiological investigations.

Conclusions

Both HIV-1 and HIV-2 are present in India. The Indian strains of HIV1 also show diverse subtypes with HIV1 subtype C predominance. Tracking the genetic diversity has implications towards understanding the evolution of the epidemic, immunopathogenesis, natural course of infection, response to therapy and most importantly vaccine design.Key Words: Genetic diversity, Human immunodeficiency virus type one (HIV-1), Subtypes     

Detection of two amino acid deletions in HIV-1 Nef protein from Chinese former paid blood donors

HIV-1 Nef protein plays an important role in HIV-1 pathogenesis, including downregulation of the cell surface CD4 and class I major histocompatibility complex （MHC）, enhancement of virion infectivity and alteration of a variety of cellular signal transduction pathways.1.2 A recent report demonstrated that Nef down-regulates dendritic cell surface expression of MHC class I and up-regulates molecules known to be critical for apoptosis induction, such as TNF-α and FasL^3 It is also very interesting that Nef expression alone may be sufficient to cause an AIDS-like disease in transgenic mice.2 In many human cohort studies it has been reported that long-term nonprogressors （LTNPs）, who remain asymptomatic for more than one decade without antiretroviral therapy, harbored nef-deleted viruses.4-7 Intense studies on nef genes from LTNPs have been done in Europe and United States but none of such studies have been done in China.[第一段]     

Full-length clone and characterization of a human immunodeficiency virus type 1 subtype B’ isolated from Hubei Province, China

There are two types of Human Immunodeficiency Virus （HIV）： HIV-1 and HIV-2. HIV-1 dominates epidemics in many different parts of the world, and HIV-2 is principally responsible for the epidemic in westem Africa. In China, Zeng et al have reported the first individual infected with HIV-1 in 1985. And in the 1990s, there was a severe epidemic involving the HIV-1 B＇ strain among people who sold blood and plasma in Henan, Hubei and adjacent provinces. To further study in HIV/AIDS vaccines and HIV-1 drug resistance for people in these regions, we need to construct an infectious HIV-1 B＇ molecular clone which is representative of the virus in these areas. To this end, we have isolated a HIV-1 B＇ virus from a child who was infected with HIV-1 from his mother in Hubei province, and have constructed a full-length clone from this genome.