湖北地区人类免疫缺陷病毒-1主要流行株外膜蛋白基因V3-V4区序列变异分析

目的 分析湖北地区HIV-1主要流行株外膜蛋白V3-V4区序列特征,了解其流行特点和变异规律.方法 对湖北地区HIV-1主要流行区域进行流行病学调查,应用套式PCR对102例HIv-1感染者的env基因V3-V4区进行扩增,对阳性扩增样本进行基因测序和序列分析.比较基因距离的差异用卡方检验;基因距离的变异性分析使用描述性分析法.结果 湖北地区共发现4种HIV亚型和重组亚型,其中B'亚型占82.69%,B'/C重组毒株、CRF01_AE重组毒株各占7.69%,C亚型占1.92%.湖北地区HIV-1B'亚型与来源于云南和河南等地的HIV-1B'亚型代表株之间的基因距离分别为7.08±2.19和7.88±2.28.其流行时间约为10年;氨基酸序列变异分析显示,HIV-1B'亚型毒株env基因V3、C3、V4区域均发生不同程度变异,其中以V4区的变异程度最大,V3环顶端四肽表现为GPGR、GPGK、GPGQ和GQGR,分别占46.5%、30.2%、13.6%和9.3%;V3环辅助受体预测显示,其中16.28%为CCR5型,13.95%为CXCR4型,69.77%无法预测;糖基化位点分析显示.湖北地区HIV-1主要流行株env V3-V4区9个糖基化位点有8个存在不同程度丢失.结论 B'亚型仍是湖北地区HIV优势流行株,与来源于云南和河南等地的毒株有较高同源性.     

1例输入性HIV-1G亚型毒株的检出及基因组序列分析

目的对1例输入性HIV-1G亚型感染者病毒基因组进行序列测定,并进行系统进化分析。方法采集在本地区艾滋病监测中发现的1例HIV-1G亚型感染者的血清,提取病毒RNA,利用反转录巢式-PCR技术扩增并测定病毒近似全长基因序列。结果获得此毒株近似全长基因组序列,长度为8695 bp。重组分析结果显示,此毒株序列中无其他亚型的基因型片段插入,为HIV-1G亚型毒株;系统进化树显示,此毒株与尼日利亚流行株G.NG.1995.NG1937.AF069937亲缘关系最近,基因离散率为8.95%。此G亚型毒株以趋化性细胞因子受体5作为辅助受体,V3区有1个氨基酸缺失,糖蛋白GP120中有8个位点因多糖空洞而导致糖基化位点缺失。结论本研究从分子流行病学角度证实该毒株为境外输入性毒株,此毒株序列的测定和分析对于本地区HIV流行趋势和HIV毒株多样性研究均具有重要意义,提示我们需加强艾滋病监测,防止境外毒株与本地毒株发生重组和流行。     

长春市HIV感染者病毒基因亚型分布状况及CRF01_AE毒株基因特征

目的了解长春市艾滋病病毒(HIV)感染者病毒基因亚型分布状况及当前主要流行株CRF01_AE毒株的基因变异特征。方法采集HIV感染者血液标本,提取病毒核糖核酸(RNA),应用巢式聚合酶链反应对HIV的gag区(HXB2:863~1486)基因进行扩增并测序。PhyML3.0软件构建系统进化树,采用HIV database数据库中Entropy、N-Glycosite、VESPA对CRF01_AE毒株两个独立进化簇的氨基酸多态性、糖基化位点、特征性氨基酸进行分析。结果成功测得gag序列的97例感染者中,经男男同性传播感染者71例(73.2%);经异性传播感染者13例(13.4%),双向传播感染者1例,12例感染途径未知。基因亚型分析结果显示,CRF01_AE 73例(75.3%),CRF07-BC 18例(18.6%),B亚型5例(5.2%)。URFs1例(0.1%)。系统进化树显示CRF01_AE在感染者中形成两个独立的进化簇,其中簇Ⅰ44株(61.1%),簇Ⅱ28株(38.9%)。进一步对CRF01_AE两个进化簇分析显示:这两个独立进化簇有10个特征性氨基酸存在差异,簇Ⅰ糖基化位点多态性大于簇Ⅱ,但氨基酸位点多态性小于簇Ⅱ。结论长春市HIV感染者主要以男男同性接触感染为主,CRF01_AE已成为本地最主要流行株。CRF01_AE毒株流行簇Ⅰ和Ⅱ存在特征性氨基酸,糖基化位点和氨基酸位点存在多态性差异,提示应密切关注本地区HIV的基因变异特征。     

河南和新疆地区HIV-1毒株gp120和gag基因的变异分析

目的分析中国河南和新疆地区艾滋病病毒Ⅰ型(HIV-1)毒株gp120和gag基因全长序列的变异特征。方法PCR扩增河南和新疆HIV-1确认阳性样本前病毒DNA gp120和gag全长基因,测序得到40份gp120和41份gag基因序列,用GCG软件包等软件进行分析。结果河南省HIV-1毒株为B′亚型,新疆维吾尔自治区HIV-1毒株为CRF07 BC亚型。B′亚型毒株gp120基因中变异程度最大的区段是V5区段,最小的是C1区段;CRF07BC亚型毒株中变异最大的是V1区段,最小的是C4区段。两种亚型毒株gp120和gag基因的Ks/Ka值<1(P<0.05)。结论对于病毒基因全长序列的分析可以更多地反映病毒的特性。gp120基因各个区段在B′和CRF07BC亚型毒株中的变异程度不一致。B′和CRF07 BC亚型毒株的gp120基因和gag基因都受到正向选择压力的作用。     

河南省艾滋病病毒感染者HIV毒株膜蛋白基因序列研究

对河南省部分地区HIV-1感染者提取外周血单核细胞(PBMC)DNA,经套式PCR扩增env基因片断,对C2—V3区350—450核苷酸序列进行测定和分析.结果表明所采样品均属HIV—1B亚型,与其它国际亚型的基因离散率在20%以上;与流行于云南省的B亚型毒株基因离散率为2.8%左右,样品间的基因离散率很小.结果提示,流行于河南省的HIV属B亚型,并与云南省的流行株密切相关;其在河南省的流行时间在3年左右.     

一例HIV-1急性感染者奠基病毒的gp160基因序列特征及其假病毒构建

目的观察急性感染期艾滋病病毒I型（HIV-1）gp160的全长基因序列及感染特征。方法从一例处于Feibig I期HIV-1感染者血浆中提取RNA,扩增gp160全长基因并测序,分析其生物学信息;将gp160全长基因与pcDNA3.1His/V5真核表达载体连接,构建Env-pcDNA3.1真核表达质粒,与骨架质粒pNL4-3.Luc.R-E-共转染293细胞,包装出假病毒。用包装的假病毒感染ghost细胞,测定感染细胞的荧光素酶活性（RLU）,鉴定假病毒的感染活性。结果成功扩增出gp160全长基因,嗜性预测为CCR5,N-糖基化位点数与标准株HXB2相同,但gp120糖基化程度更高,氨基酸变异主要集中在V1-V5区。假病毒感染试验显示,RLU值达到7log。结论获得了处于急性感染期的HIV-1gp160基因序列和高感染活性的假病毒。     

1996～1998年中国流行的E亚型艾滋病病毒1型毒株的分子流行病学研究

目的通过对1996～1998年采集的艾滋病病毒1型(HIV-1)毒株样本的env基因的序列分析,阐明在中国流行的E亚型HIV-1毒株的特点、来源和传播方式。为中国E亚型HIV-1疫苗的研制和应用提供基础资料。方法 从HIV感染者淋巴细胞(PBMC)中提取前病毒DNA.使用嵌套式聚合酶链反应(PCR)方法扩增HIV-1的env基因的C2V5区。PCR产物不经克隆直接测序并使用GCG软件包进行序列分析。结果样品采自1996～1998年中国29个省(自治区,直辖市),总共发现37个E亚型HIV-1感染者。他们中大部分是通过性途径感染(23人,占62.2％);部分在静脉吸毒人群中发现(10人,占27.0％);少数是在职业献血员中发现(4人,占10.8％)。经C2-V3区序列分析发现,大部分中国E亚型HIV-1毒株与泰国株很相近,而与非洲株相差很大。而来自广西壮族自治区的毒株与越南吸毒人群中的流行株U48720相一致;系统树分析结果发现,中国的E亚型HIV-1株与泰国(CM240X、H93TH966)、越南(U48720)的代表株聚在一起。结论 中国E亚型HIV-1毒株目前仅在东南沿海地区流行,涉及静脉吸毒、输供血和性乱等各种人群,通过env区的序列分析发现其主要来源于泰国,部分来源于与中国接壤的越南。     

山东省HIV-1流行毒株的基因特征和系统树分析

从13名山东省HIV感染者的淋巴细胞(PBMC)中提取前病毒DNA,使用套式PCR方法扩增HIV-lenv基因的C2-V3区。经序列分析发现13份样品均为B′,亚型(泰国B亚型),其与国际参考序列Bcon、泰国代表株93th067A、中国云南省代表株RL42、云南省共享序列96Yn-Bcon的基因离散率分别为7.31％、5.72％、4.25％、3.76％,而远离其它亚型。由系统树可以更清楚看到13个毒株全部与93th067A、RL42等B,亚型流行株聚在一起。通过GCG软件包计算这些毒株彼此间的基因离散率的变异系数为2.71％。以上数据提示HIV-1在山东省的流行时间不长。且与云南省相同亚型的HIV-1毒株密切相关。     

北京市同性恋HIV-1感染者的包膜基因C2-V3区序列测定和亚型分析

目的 了解北京市同性恋HIV-1感染者HIV-1的亚型类型及传播来源和流行时间。方法 应用套式聚合酶链式反应（PCR）对12份1993～2001年北京市HIV-1阳性同性恋者外周血单个核细胞（PBMC）的核酸样品进行扩增,并对其包膜区的C2-V3段的306个核酸序列进行测定和分析。结果12份样品全部是B亚型的HIV-1毒株序列,其亚型内的基因离散率为 10.35± 2.06,与国际 A-E亚型共享序列比较后发现其与 A、C、D、E亚型的共享序列的基因离散率均大于 25％,而与国际 B亚型共享序列的基因离散率仅为11.25±3.60。系统树分析显示,12个毒株与B亚型共享序列聚在一起并远离其它国际亚型,并且12个毒株与SF162紧密相连,而与国际B亚型共享序列和泰国B亚型代表株TH14可以分开。对gp120中最重要的中和抗体决定簇V3环序列进行对比分析发现,12毒株在V3环中变化较大,其中4毒株带有GPGR这一欧美B亚型V3环顶端四肽序列特征,占33.33％,1个毒株带有GLGR,占8.33％,而其它7个毒株为GWGR,占58.34％。结论HIV-1在北京市同性恋人群中流行的为B亚型,流行来源为欧美,流行时间10年左右,V3环顶端四肽序列特征以GWGR为主。     

四川省≥50岁HIV-1感染者的分子流行病学调查

目的了解四川省≥50岁艾滋病病毒Ⅰ型(HIV-1)感染者的流行毒株亚型、人群和地域分布及原发性耐药情况。方法通过抽样调查的方法,在成都、泸州、达州、内江、广元5个市州采集2017年3—5月新报告、未经抗病毒治疗的≥50岁HIV-1感染者的血样235份,扩增HIV-1 pol基因区,对获得的190份核酸序列进行系统进化分析分型。比较不同亚型毒株感染者的人口学特征和地域分布特征。序列提交斯坦福大学HIV耐药数据库进行耐药位点分析。结果四川省≥50岁HIV-1感染者的流行毒株亚型为CRF01_AE、CRF07_BC、CRF08_BC、B亚型和CRF85_BC。毒株分布显示出明显的地域特征。男性感染者的毒株亚型在非婚非商业和非婚商业两种不同感染途径中的分布差异有统计学意义(χ~2=12.519,P=0.028)。190例中,原发性耐药突变率为5.8%;泸州地区的原发性耐药比例最高,为9.7%。尚未发现具有双重耐药或多重原发性耐药突变的患者。结论四川省≥50岁年龄组感染的HIV-1主要毒株有独特的流行态势,应加强对HIV毒株的监测,掌握主要流行亚型和耐药毒株的流行特点和规律,针对性地进行艾滋病防治。     

Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-1 gp120.

Amino acid sequences inducing neutralizing antibodies to HIV-1 were sought. Murine monoclonal antibodies (MAbs) were characterized by their reactivity with the envelope precursor gp160 or the Escherichia coli recombinant DNA products pB1 and pE3 representing the carboxy- and amino-terminal halves of mature envelope gp120. Fine mapping of the MAb determinants was performed using defined 15-mer synthetic peptides spanning the entire envelope gp120 region of HIV-1. One group of MAbs recognizes epitopes (amino acids 304-323) occurring in a small region with variable and conserved amino acid sequences of gp120. These MAbs mediate neutralization of the HIV-1 strain HTLV-IIIB (HIV-1IIIB) which was used for immunization. Nine out of 11 primary HIV-1 isolates were neutralized well or moderately well. In addition, prominent serological reactivity was noted with peptide sequences of strains of various European or American origins, but not with two HIV-1 strains of African origin. The cross-reactivity contrasts with previously described type-specific reactions to other sequences of this region. The reactivity to the short conserved site GPGR with its flanking amino acids may explain the broad sequence cross-reactivity seen with our neutralizing MAbs. Two other MAbs recognize conserved epitopes (amino acids 79-103) situated in the amino-terminal region of gp120. These MAbs did not neutralize HIV-1IIIB.     

Unique V3 loop sequence derived from the R2 strain of HIV-type 1 elicits broad neutralizing antibodies

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies. In this study, DNA vaccines were constructed to express the gp120 subunit of Env from the isolate HIV-1(R2) using both wild-type and codon-optimized gene sequences. Three copies of the murine C3d were added to the carboxyl terminus to enhance the immunogenicity of the expressed fusion protein. Mice (BALB/c) vaccinated with DNA plasmid expressing the gp120(R2) using codon-optimized Env sequences elicited high-titer anti-Env antibodies regardless of conjugation to C3d. In contrast, only mice vaccinated with DNA using wild-type gp120(R2) sequences fused to mC3d(3), had detectable anti-Env antibodies. Interestingly, mice vaccinated with DNA expressing gp120(R2) from codon-optimized sequences elicited antibodies that neutralized both homologous and heterologous HIV-1 isolates. To determine if the unique sequence found in the crown of the V3 loop of the Env(R2) was responsible for the elicitation of the cross-clade neutralizing antibodies, the codons encoding for the Pro-Met (amino acids 313-314) were introduced into the sequences encoding the gp120(ADA) (R5) or gp120(89.6) (R5X4). Mice vaccinated with gp120(ADA)-mC3d(3)-DNA with the Pro-Met mutation had antibodies that neutralized HIV-1 infection, but not the gp120(89.6)-mC3d(3)-DNA. Therefore, the use of the unique sequences in the Env(R2) introduced into an R5 tropic envelope, in conjunction with C3d fusion, was effective at broadening the number of viruses that could be neutralized. However, the introduction of this same sequence into an R5X4-tropic envelope was ineffective in eliciting improved cross-clade neutralizing antibodies.     

gp120 sequences from HIV type 1 subtype C early seroconverters in India

A limited number of full-length gp120 sequences are currently available for subtype C HIV-1 from India. Sequence data from HIV-1 subtype C in early seroconverter stage virus are also very limited. With the objective of identifying the sequence variation in early seroconverters, we compared Indian subtype C gp120 sequences obtained from six early seroconverters presented in this study with non-Indian subtype C sequences from other parts of the world obtained from the Los Alamos database and subtype C potential vaccine candidate sequences. All these samples were collected within a few weeks of seroconversion and hence they represent gp120 sequences of currently circulating viral strains in India. The phylogenetic tree indicated that the Indian sequences compared here clustered together within the C clade. The seroconverter sequences presented in the study would surely help in identifying the immunogenic epitopes and could be utilized further for developing effective prophylactic strategies against HIV-1 subtype C for India.     

An inducible HIV type 1 gp41 HR-2 peptide-binding site on HIV type 1 envelope gp120

Synthetic peptides of sequences within the HIV-1 gp41 heptad repeat-regions (HR-1 and HR-2) can effectively inhibit cell fusion and viral entry. DP178 (T-20), an HR-2 peptide, acts by inhibiting the association between HR-1 and HR-2, thereby interfering with HIV-1 fusion and viral entry. HR-2 peptide binding is predicted to be an important indicator of the presence of Env gp41 fusion intermediate conformation. A stabilized HR-2/Env conjugate might be an HIV-1 vaccine candidate and have the potential for inducing antibodies against transiently exposed epitopes on HIV-1 Env. To explore the possibility of design of HR-2 stabilized-HIV-1 immunogens, we studied the ability of HIV-1 Env to bind to HR-2 peptides. Using surface plasmon resonance (SPR)-binding assays and precipitation of soluble Env gp120 proteins with HR-2 peptide DP178, we have found that there is an HR-2 peptide-binding site on soluble HIV-1 recombinant gp120. Binding of DP178 was induced by sCD4 and by the anti-gp120 human mAb A32. The induction of DP178 binding was inhibited > 80% by the HIV-1 coreceptor-binding site mAb 17b. Binding of DP178 to gp120 was also inhibited by gp120 C4 peptides with sequences that are centrally located within the HIV-1 coreceptor-binding site. Thus, in addition to interactions with the gp41 HR-1 region, the fusion inhibitor peptide DP178 binds to triggered soluble HIV-1 recombinant gp120 following its interaction with sCD4 or CD4 mimic mAb A32. This may prove to be an important consideration when designing an HIV vaccine that utilizes constrained HIV Env proteins.     

山西某县农村外来嫁入女性及配偶中HIV感染者病毒序列特征分析

目的 分析山西省某县农村外来嫁人女性及配偶中感染艾滋病病毒（HIV）的毒株分布和传染源，为制定有效的艾滋病防治措施提供科学依据。方法 采集17名感染HIV的农村外来妇女及这些外来妇女的4例阳性配偶的外周血，提取前病毒DNA，应用套式PCR进行艾滋病病毒gag基因检测，使用GCG软件包对扩增序列进行分析，并对这些HIV感染者进行相关流行病学调查。结果 在送检的21份HIV确认阳性的样本中，共获得18份gag基因序列。基于gag基因区的序列分析结果表明：其中13份为CRF01_AE亚型（占72．2％），1份为B’亚型（占5．6％），2份为B’／C重组型（占11．1％），1份为C亚型（占5．6％），另有1例样本不确定。B’／C重组与新报告的云南瑞丽发现的B／C重组亚型相似性最大。18份样本中有3对为HIV双阳性的夫妻，系统树分析表明，每对夫妻感染毒株在系统树上聚在一起，处于一个分支，彼此间的基因距离均很小（0．007～0．009），推测为近期的家庭内传染。结论 山西以往的HIV感染者的主要来源为采供血途径，近年外来高危人口的大量输入，已是山西HIV感染人群的重要毒株来源，使该地区的流行毒株从单一型向多样化发展，并已有家庭内传播的发生。这种形式使该省艾滋病的防控工作更加复杂，今后外来高危人口应纳入当地省艾滋病疫情的重点监测对象，降低传播情况的发生。     

The V3 loops of the HIV-1 and HIV-2 surface glycoproteins contain proteolytic cleavage sites: a possible function in viral fusion?

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.     

Antigenicity and immunogenicity of the V3 domain of HIV type 1 glycoprotein 120 expressed on the surface of Streptococcus gordonii

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.     

Peptides selected from a phage display library with an HIV-neutralizing antibody elicit antibodies to HIV gp120 in rabbits, but not to the same epitope

Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide-protein fusions were expressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal antibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically inhibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide-protein fusions produced antisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120.     

HIV-1 in genital tract and plasma of women: compartmentalization of viral sequences, coreceptor usage, and glycosylation

Worldwide, 90% of HIV-1 infections are transmitted heterosexually. Because the genital mucosa are the sites of initial contact with HIV-1 for most exposed individuals, study of the virus from the genital tract is critical for the development of vaccines and therapeutics. Previous analyses of HIV-1 in various tissues have documented compartmentalization of viral genomes. Whether compartmentalization was associated with viral phenotypic differences or immune status, however, was not well understood. We compared HIV-1 gp120 env sequences from the genital tract and plasma of 12 women. Eight women displayed compartmentalized HIV-1 RNA genomes, with viral sequences from each site that were clearly discrete, yet phylogenetically related. The remaining four exhibited env sequences that were intermingled between the two sites. Women with compartmentalized HIV-1 genomes had higher CD4+ cell counts than those displaying intermingled strains (P = 0.02). Intrapatient HIV-1 recombinants comprising sequences that were characteristic of both sites were identified. We next compared viral phenotypes in each compartment. HIV-1 coreceptor usage was often compartmentalized (P 0.01). The number of N-linked glycosylation sites, associated with neutralization resistance, also differed between compartments (P < 0.01). Furthermore, disparities between the density of gp120 glycosylations in each compartment correlated with higher CD4+ counts (P = 0.03). These data demonstrate that the genital tract and plasma can harbor populations of replicating HIV-1 with different phenotypes. The association of higher CD4+ cell counts with compartmentalization of viral genomes and density of gp120 glycosylations suggests that the immune response influences the development of viral genotypes in each compartment. These findings are relevant to the prevention and control of HIV-1 infection.