山东省归国劳务人员中HIV-1感染毒株的序列特征和亚型分析

为了解自1992年以来,山东省归国劳务人员HIV-1感染毒株的分子流行病学特征。我们采集了11份确认为HIV-1感染者的全血分离单核细胞(PBMC),提取前病毒DNA,用套式PCR扩增HIV-1膜蛋白(env)基因,并对其env C2-V3区进行序列测定和分析。结果表明10名归国劳工人员及1名归国劳工配偶的样本经序列测定和基因分析发现了HIV-1的4种基因亚型A、B、C、E亚型。其中C亚型7名,B亚型2名,A和E亚型各1名。由此可见;山东省归国劳务人员中HIV-1毒株的感染情况较复杂,通过计算发现这些毒株间的基因离散率相差较大。提示应加强归国劳务人员的教育、检测和控制以防其它国家毒株传入。     

我国中部HIV-1B′亚型分离株基因组结构特征及系统发育分析

为了分析河南省Humanimmunodeficiencyvirus-1(HIV-1)流行株的基因特征和传染来源,我们从一位HIV-1感染的有偿献血员体内分离HIV-1毒株。提取感染细胞的全基因组DNA,扩增病毒全基因组,Walking法测得全长序列。与HIV-1RL42和HIV-1HXB2株比较,分析基因特征。用Anthewin软件比较分析CNHN24株和RL42株env蛋白的二级结构。用Clustal软件对国内HIV-1分离株和国际参考株的全基因组、env基因和V3环基因分别进行了系统发育分析。结果发现,CNHN24株为HIV-1B′亚型,富含嘌呤,含量达到60.26%。与RL42株及HXB2株比较,pol基因和gag基因变异度较小,env基因及非结构基因变异度较大。与RL42株相比,env蛋白二级结构变异不大,但env保守区C4的氨基酸呈高度变异。系统发育分析显示,CNHN24株与RL42株遗传距离最近,HIV-1Lai株和HXB2株次之,与国内的HIV-1B/C及AE/BC重组毒株的遗传距离较远;从V3序列的比较可以发现,CNHN24株与来自云南省的毒株HIV-1CR206及RL42遗传距离最近。认为HIV-1CNHN24株可能由云南传入。     

我国首次发现的HIV-1和HIV-2双重感染样品中病毒部分基因的序列特征分析

上海市卫生检疫局送检了一例HIV - 1和HIV - 2抗体检测均呈阳性的双重感染样品 ,对其感染的HIV前病毒的 gag和env基因区进行了序列分析 ,首次阐明我国发现的HIV双重感染样品的HIV部分基因特征。从HIV感染者淋巴细胞 (peripheralbloodmononuclearcells,PBMC)中提取前病毒DNA ,分别使用HIV 1和HIV 2特异性引物用套式PCR扩增HIV 1和HIV 2的部分基因区。PCR产物不经克隆直接测序 ,经GenBank检索并使用GCG软件包进行序列分析。结果表明 ,其中感染的HIV 2毒株中gag基因区与德国株HI2PEI2KR相似 ,基因离散率仅为8 1% ,env基因C2 -V3区与来自几内亚比绍的HIV 2U0 5 35 8株最近 ,离散率为 13 0 4% ,在其 gp36区发现与HIV 2U0 5 35 8基因离散率为 10 6 3% ,两个毒株均属HIV 2中的A亚型。而其HIV 1型毒株在 gag和env区都与从尼日利亚分离的H92NG0 83株相似 ,属HIV 1的G亚型。本文首次对我国发现的HIV 2型毒株进行了主要基因区的序列分析 ,表明在非洲较常见的HIV 2A亚型和HIV 1G亚型毒株 ,已随援外劳工传入我国。     

我国中部HIV-1 B''''亚型分离株基因组结构特征及系统发育分析

为了分析河南省Human immunodeficiency virus-1(HIV-1)流行株的基因特征和传染来源,我们从一位HIV-1感染的有偿献血员体内分离HIV-1毒株.提取感染细胞的全基因组DNA,扩增病毒全基因组,Walking法测得全长序列.与HIV-1 RL42和HIV-1 HXB2株比较,分析基因特征.用Anthewin软件比较分析CNHN24株和RL42株env蛋白的二级结构.用Clustal软件对国内HIV-1分离株和国际参考株的全基因组、env基因和V3环基因分别进行了系统发育分析.结果发现,CNHN24株为HIV-1 B'亚型,富含嘌呤,含量达到60.26%.与RL42株及HXB2株比较,pol基因和gag基因变异度较小,env基因及非结构基因变异度较大.与RL42株相比,env蛋白二级结构变异不大,但env保守区C4的氨基酸呈高度变异.系统发育分析显示,CNHN24株与RL42株遗传距离最近,HIV-1Lai株和HXB2株次之,与国内的HIV-1 B/C及AE/BC重组毒株的遗传距离较远;从V3序列的比较可以发现,CNHN24株与来自云南省的毒株HIV-1 CR206及RL42遗传距离最近.认为HIV-1 CNHN24株可能由云南传入.     

我国中部HIV-1B’亚型分离株基因组结构特征及系统发育分析

为了分析河南省Human immunodeficiency virus—I(HIV-1)流行株的基因特征和传染来源,我们从一位HIV-1感染的有偿献血员体内分离HIV-1毒株。提取感染细胞的全基因组DNA,扩增病毒全基因组,Walking法测得全长序列。与HIV-1RL42和HIV-1HXB2株比较,分析基因特征。用Anthewin软件比较分析CNHN24株和RL42株env蛋白的二级结构。用Clustal软件对国内HIV-1分离株和国际参考株的全基因组、env基因和订环基因分别进行了系统发育分析。结果发现,CNHN24株为HIV-1B’亚型,富含嘌呤,含量达到60．26％。与RL42株及HXB2株比较,pol基因和gag基因变异度较小,env基因及非结构基因变异度较大。与RL42株相比,env蛋白二级结构变异不大,但env保守区C4的氨基酸呈高度变异。系统发育分析显示,CNHN24株与RL42株遗传距离最近,HIV-1Lai株和HXB2株次之,与国内的H1V-1B／C及AE／BC重组毒株的遗传距离较远;从份序列的比较可以发现,CNHN24株与来自云南省的毒株HIV-1CR206及RL42遗传距离最近。认为HIV-1CNHN24株可能由云南传入。     

HIV-1 B亚型gag基因密码子优化及其免疫原性的研究

从河南HIV-1流行区感染者中克隆HIV-1 B亚型gag基因,通过序列比对获得其一致性共有序列,对该共有序列按照哺乳动物优势密码子的使用原则进行优化,以Western blot方法比较优化前后gag基因体外表达量.发现对gag基因进行密码子优化可显著提高其表达水平.将优化后的mod.gag基因插入重组腺病毒载体,构建了重组病毒rAdV-mod.gag.在BALB/c小鼠体内分别以108PFIJ及108PFU rAdV-mod.gag疫苗单独免疫两次均可产生较高水平的gag特异性细胞免疫反应.由此得出结论,对gag基因的密码子优化是成功的;表达优化后gag基因的重组腺病毒疫苗,可以在小鼠体内诱导较强的gag基因特异性CTL应答.     

HIV-1 C／B’亚型多价重组MVA病毒疫苗免疫原性的研究

目的:检测人类免疫缺陷病毒1型(HIV-1) C/B'亚型与痘苗病毒安卡拉株(MVA)的多价重组疫苗的免疫原性,此疫苗中包含HIV-1 C/B'CRF 株的5个基因,分别是env,gag,pol,nef,tat.方法:设105pfu/ml,106 pfu/ml,107 pfu/ml ADMVA 3个剂量组,用ELISPOT方法检测细胞免疫反应.同时,以Gag蛋白作为包被抗原,使用间接ELISA的方法检测体液免疫反应.结果:免疫后的小鼠对插入基因env,gag,pol和nef所表达蛋白均产生了特异性细胞免疫应答,且免疫效果与免疫剂量呈正相关.ELISA结果表明, MVA重组疫苗免疫诱导产生了特异性体液免疫,抗HIV-1Gag的抗体滴度与免疫次数和免疫剂量都存在正相关性.结论:多价MVA重组疫苗能有效地诱导小鼠产生特异的细胞免疫和体液免疫反应.     

中国HIV-1 B’亚型毒株不同基因区与全长基因组系统进化比较

本研究旨在探索不同基因区的使用对HIV-1 B’亚型毒株系统进化分析结果的影响。首先利用既往研究中已发表的共计47条来自泰国,缅甸和中国多个地区不同传播途径的B’毒株近全长基因组序列,将其按基因区分为不同的数据集 (gag, pol, vif, vpr, vpu, env, nef),并分别进行系统进化分析研究。比较不同基因区系统进化分析的结果发现,B’亚型毒株 pol基因在分析的基因区中,具有最低的复杂度和进化速率,可以较好的区分B’TH和B’YN毒株,重复近全长基因组序列的分析结果;尽管env基因则具有最高的复杂度和进化速率,但无法获得类似结果。本研究比较了不同基因区对HIV-1 B’亚型毒株系统进化分析结果的影响,对进一步开展HIV分子流行病学调查,分析我国B’毒株在我国的传播奠定了基础。     

静脉注射吸毒感染者体内HIV-1CRF07_BC的准种变异特征

为了研究静脉注射吸毒者(IDU)体内HIV-1CRF07_BC病毒准种变异和遗传多样性特征,本文采用单基因组扩增(SGA)技术分别从6份CRF07_BC感染者血浆获得HIV-1病毒准种env基因gp120片段序列11～28条,采用构建系统进化树的方法进行聚类分析,描述感染者血浆中CRF07_BC病毒准种的变异特征;运用Simplot软件、分段系统进化树和对平均两两比对基因距离进行遗传多样性作图(Diversity plot)的方法分析病毒准种间的重组。系统进化树分析结果显示仅1个病例具有较高的遗传同质性,而其他5个病例的遗传异质性较高,主要表现为系统进化树上形成2～4个次级进化簇。此外,有3个病例存在病毒准种间的重组。本研究表明SGA技术能有效地分析感染者体内病毒准种复杂的变异特征。     

Human immunodeficiency virus type 1 subtypes defined by env show high frequency of recombinant gag genes. The UNAIDS Network for HIV Isolation and Characterization.

Genetic subtypes of human immunodeficiency virus type 1 can be distinguished on the basis of phylogenetic analysis of their envelope (env) gene. A significant proportion of human immunodeficiency virus type 1 strains was retrospectively shown to result from recombination events between viruses belonging genetically to distinct subtypes (D. L. Robertson, P. M. Sharp, F. E. McCutchan, and B. H. Hahn, Nature [London] 374:124-126, 1995). To establish the frequency of natural infections with recombinant viruses and to exclude tissue culture artifacts, we analyzed plasma samples from the UNAIDS sample collection. The collection includes samples from 53 individuals infected with subtype A (n = 9), subtype B (n = 15), subtype C (n = 1), subtype D (n = 13), and subtype E (n = 15) on the basis of V3 region analysis. Phylogenetic analysis of the gag gene fragment showed intersubtype recombinant genomes in 23 cases: 3 of 9 (33%) of subtype A, 2 of 15 (13%) of subtype B, 3 of 13 (23%) of subtype D, and all of subtype E. Of the 23 recombinant viruses, 19 had a gag gene from one subtype and env from another (B(env)/C(gag), A(env)/C(gag), D(env)/A(gag), and E(env)/A(gag)). Phylogenetic analysis clustered the A(gag) of subtype E viruses as an outgroup of subtype A, suggesting that these viruses may belong to a distinct A' cluster. The remaining four recombinant viruses (B(env)/B(p17)F(p24), A(env)/A(p17)D(p24), A(env)/A(p17)C(p24), and D(env)/ D(p17)A(p24)) had breakpoint crossover sites in the proximity of the p17-p24 protein processing site. We conclude that recombination in the gag gene is highly frequent among the major env subtypes and that selection of recombinants is apparently based on particularly beneficial combinations of gag and env gene products.     

Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in Central Africa

The purpose of this study was to document the genetic diversity of human immunodeficiency virus type 1 (HIV-1) in the Democratic Republic of Congo (DRC; formerly Zaire). A total of 247 HIV-1-positive samples, collected during an epidemiologic survey conducted in 1997 in three regions (Kinshasa [the capital], Bwamanda [in the north], and Mbuyi-Maya [in the south]), were genetically characterized in the env V3-V5 region. All known subtypes were found to cocirculate, and for 6% of the samples the subtype could not be identified. Subtype A is predominant, with prevalences decreasing from north to south (69% in the north, 53% in the capital city, and 46% in the south). Subtype C, D, G, and H prevalences range from 7 to 9%, whereas subtype F, J, K, and CRF01-AE strains represent 2 to 4% of the samples; only one subtype B strain was identified. The highest prevalence (25%) of subtype C was in the south, and CRF01-AE was seen mainly in the north. The high intersubtype variability among the V3-V5 sequences is the most probable reason for the low (45%) efficiency of subtype A-specific PCR and HMA (heteroduplex mobility assay). Eighteen (29%) of 62 samples had discordant subtype designations between env and gag. Sequence analysis of the entire envelope from 13 samples confirmed the high degree of diversity and complexity of HIV-1 strains in the DRC; 9 had a complex recombinant structure in gp160, involving fragments of known and unknown subtypes. Interestingly, the unknown fragments from the different strains did not cluster together. Overall, the high number of HIV-1 subtypes cocirculating, the high intrasubtype diversity, and the high numbers of possible recombinant viruses as well as different unclassified strains are all in agreement with an old and mature epidemic in the DRC, suggesting that this region is the epicenter of HIV-1 group M.     

HIV-1 subtypes in Russia in 1987-1988

The subtyping of 350 isolates of HIV-1, isolated on the territories of 38 subjects of the Russian Federation, was carried out. The analysis was made by the method of the comparative heteroduplex mobility assay, as well as by the determination of the sequence of genes env [correction of ens] (gp 120) and gag (p17-p24). The study revealed that more than 50% of all cases of HIV-1 infection were caused by closely related variants of subtype A virus. The number of cases of HIV-1 infection caused by recombinant virus A/B was not less than 25%. The total number of cases caused by viruses of subtypes C, D, E, F and H was not more than 5%.     

Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate

Immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the na?ve animals. Further, one of six immunized animals was completely protected whereas all six na?ve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L).     

Antigenicity and immunogenicity of a synthetic human immunodeficiency virus type 1 group m consensus envelope glycoprotein

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 subtypes. When used as a DNA vaccine followed by a recombinant vaccinia virus boost in BALB/c mice, CON6 env gp120 and gp140CF elicited gamma interferon-producing T-cell responses that recognized epitopes within overlapping peptide pools from three HIV-1 Env proteins, CON6, MN (subtype B), and Chn19 (subtype C). Sera from guinea pigs immunized with recombinant CON6 Env gp120 and gp140CF glycoproteins weakly neutralized selected HIV-1 primary isolates. Thus, the computer-generated "consensus" env genes are capable of expressing envelope glycoproteins that retain the structural, functional, and immunogenic properties of wild-type HIV-1 envelopes.     

Re-mapping the molecular features of the human immunodeficiency virus type 1 and human T-cell lymphotropic virus type 1 Brazilian sequences using a bioinformatics unit established in Salvador, Bahia, Brazil, to give support to the viral epidemiology studies

The analysis of genetic data for human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type 1 (HTLV-1) is essential to improve treatment and public health strategies as well as to select strains for vaccine programs. However, the analysis of large quantities of genetic data requires collaborative efforts in bioinformatics, computer biology, molecular biology, evolution, and medical science. The objective of this study was to review and improve the molecular epidemiology of HIV-1 and HTLV-1 viruses isolated in Brazil using bioinformatic tools available in the Laboratório Avan?ado de Sáude Pública (Lasp) bioinformatics unit. The analysis of HIV-1 isolates confirmed a heterogeneous distribution of the viral genotypes circulating in the country. The Brazilian HIV-1 epidemic is characterized by the presence of multiple subtypes (B, F1, C) and B/F1 recombinant virus while, on the other hand, most of the HTLV-1 sequences were classified as Transcontinental subgroup of the Cosmopolitan subtype. Despite the high variation among HIV-1 subtypes, protein glycosylation and phosphorylation domains were conserved in the pol, gag, and env genes of the Brazilian HIV-1 strains suggesting constraints in the HIV-1 evolution process. As expected, the functional protein sites were highly conservative in the HTLV-1 env gene sequences. Furthermore, the presence of these functional sites in HIV-1 and HTLV-1 strains could help in the development of vaccines that pre-empt the viral escape process.     

Quantitative analysis of serum neutralization of human immunodeficiency virus type 1 from subtypes A, B, C, D, E, F, and I: lack of direct correlation between neutralization serotypes and genetic subtypes and evidence for prevalent serum-dependent infectivity enhancement.

Human immunodeficiency virus type 1 (HIV-1) M group strains have been assigned to date to nine distinct genetic subtypes, designated A through I, according to phylogenetic analyses of nucleotide sequences of their env or gag genes. Whether there is any relationship between phylogenetic subtypes and the neutralization serotypes is not clear, yet defining the nature of any such relationship by mathematical means would be of major importance for the development of globally effective HIV-1 vaccines. We have therefore developed a quantitative method to analyze serum neutralization of HIV-1 isolates and to identify HIV-1 neutralization serotypes. This method involves calculations of the neutralization index, N(i), a newly defined parameter derived from plots generated from in vitro neutralization assays, calculations of pairwise serum-virus vector distances, and cluster analyses. We have applied this approach to analyze three independent neutralization matrices involving primary HIV-1 strains and sera from genetic subtypes A, B, C, D, E, F, and I. Detailed serum and HIV-1 isolate cluster analyses have shown that in general, the identified neutralization serotypes do not directly correlate with HIV-1 genetic subtypes. These results suggest that neutralization serotypes do not during natural HIV-1 infection are not governed by antibodies directed against simple epitopes within gp120 monomers. A significant proportion (28%) of 1,213 combinations of sera and HIV-1 isolates caused serum-dependent infectivity enhancement [negative N(i) values] rather than neutralization. We also noted that negative N(i) values tended to correlate better with certain HIV-1 isolates rather than with HIV-1-positive sera. Syncytium-inducing variants of HIV-1 were slightly more likely than non-syncytium-inducing variants to undergo serum-dependent infectivity enhancement, although the latter variants could clearly be susceptible to enhancement.     

Genetic characterization of new West African simian immunodeficiency virus SIVsm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop.

It has been proposed that human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that are natural infections of sooty mangabeys (Cercocebus torquatus atys). To test this hypothesis, SIVs from eight sooty mangabeys, including six new viruses from West Africa, were genetically characterized. gag and env sequences showed that while the viruses of all eight sooty mangabeys belonged to the SIVsm/HIV-2 family, each was widely divergent from SIVs found earlier in captive monkeys at American primate centers. In two SIVs from sooty mangabeys discovered about 100 miles (ca. 161 Km) from each other in rural West Africa, the amino acids of a conserved gag p17-p26 region differed by 19.3%, a divergence greater than that in four of five clades of HIV-2 and in SIVs found in other African monkey species. Analysis of gag region sequences showed that feral mangabeys in one small troop harbored four distinct SIVs. Three of the newly found viruses were genetically divergent, showing as much genetic distance from each other as from the entire SIVsm/HIV-2 family. Sequencing and heteroduplex analysis of one feral animal-derived SIV showed a mosaic genome containing an env gene that was homologous with other feral SIVsm env genes in the troop but having a gag gene from another, distinct SIV. Surprisingly a gag phylogenetic tree based on nucleotide sequences showed that the African relatives closest to all three household-derived SIVs were HIV-2 subtypes D and E from humans in the same West African areas. In one case, the SIV/HIV-2 cluster was from the same village. The findings support the hypothesis that each HIV-2 subtype in West Africans originated from widely divergent SIVsm strains, transmitted by independent cross-species events in the same geographic locations.     

Polymorphism of the Human Immunodeficiency Virus Type 1 in Brazil: genetic characterization of the nef gene and implications for vaccine design

Most of the Brazilian HIV-1 samples have been characterized based on the structural genes (env, gag and pol) and no data concerning the variability of the accessory genes such as nef have been available so far. Considering the role of the nef on virus biology and the inclusion of this region in some HIV/AIDS vaccine products under testing, the purpose of this study was to document the genetic diversity of the nef gene in third-four HIV-1 Brazilian samples previously subtyped based on the env C2-V3 region. Although only few non-subtype B samples have already been analyzed so far, the cytotoxic Tlymphocyte epitopes encoded in this region were relatively conserved among the subtypes, with some amino acid signatures mainly in the subtype C samples. Considering the increasing of the non-B HIV-1 subtypes worldwide, in special the subtype C, more data should be generated concerning the genetic and antigenic variability of these subtypes, as well as the study of the impact of such polymorphism in HIV/AIDS vaccine design and testing.