HIV-1 B'/C亚型毒株辅助受体的利用

目的分析疾病进展不同阶段分离的B’肥亚型毒株辅助受体的利用情况。方法从长期不进展者和AIDS期患者体内分离病毒株,将获得的B’／C毒株进行体外培养,用病毒株感染U87．CD4、GHOST细胞系并进行培养,通过检测、比较培养基上清液中核衣壳蛋白p24量的差异来确定某一毒株利用的辅助受体。再利用辅助受体抑制剂TAK～799、AMD-3100验证从细胞系实验中得到的结论。结果从AIDS期患者体内分离的毒株大部分利用C—C家族趋化因子受体（C—Cchemokinereceptor,CCR）5辅助受体,部分利用C—X～C家族趋化因子受体（C—X—C chemokine receptor,CXCR）4和CCR5辅助受体。从长期不进展者体内分离的毒株利用CCR5辅助受体。结论从长期不进展者体内分离的病毒主要利用CCR5辅助受体。从AIDS期患者体内分离的病毒主要利用CCR5辅助受体,也有同时利用CCR5和CXCR4辅助受体的现象。     

HIV-1临床毒株的分离培养及生物学表型和基因型鉴定

目的从晚期艾滋病（AIDS）病人中分离培养艾滋病病毒Ⅰ型（HIV-1）毒株,对其生物学表型和基因型进行鉴定,了解中国临床HIV-1毒株的生物学特征,以期探讨HIV-1生物学特征与疾病进展的关系。方法收集6例AIDS晚期病人的静脉全血,分离血浆和外周血单个核淋巴细胞（PBMCs）;流式细胞仪检测CD4细胞,bDNA法检测病毒载量;PBMCs共培养法分离HIV-1毒株;终点稀释法滴定分离株的感染力;逆转录-聚合酶链反应（RT-PCR）扩增分离株的env区;以MT-4细胞检测毒株的融合诱导性。结果6例病例中分离出3株能在PBMCs中连续稳定传代的HIV-1毒株,50%组织培养感染剂量（TCID50/ml）分别为6.3×10^3、10.2×10^3、2.51×10^3;3株毒株均为B亚型,均不能引起MT-4细胞病变。结论共分离出3株感染力相对较高、呈慢/高型复制动力学、M-嗜性、非融合诱导性的HIV-1 B亚型临床分离株。     

我国男男性行为者HIV-1感染早期病毒分离株复制与疾病进展的关系

目的研究中国男男性行为者1型艾滋病病毒(HIV-1)早期感染者病毒分离株的复制能力,分析感染早期复制能力与疾病进展的关系。方法应用外周血单个核细胞(PBMC)共培养法,从HIV-1早期感染者的样本中获得病毒分离株,检测培养上清中的p24滴度,分析病毒分离株的复制能力与疾病进展的关系。结果从28例HIV-1早期感染者的PBMC中分离获得28株病毒。HIV-1感染早期病毒分离株的复制能力分为复制高型和低型(高型16株,占57.1%;低型12株,占42.9%)。复制高型早期感染者的病毒调定点高于复制低型早期感染者(P=0.01);复制高型早期感染者感染1年后的CD4+T淋巴细胞(简称CD4细胞)低于复制低型早期感染者(P=0.02)。感染120天内的早期感染者分离株的复制p24峰值和曲线下面积与病毒调定点呈正相关(R=0.86,P=0.000 6;R=0.81,P=0.002 2);感染1年内的早期感染者分离株的复制p24峰值和曲线下面积与感染1年的CD4细胞计数呈负相关(R=-0.48,P=0.01;R=-0.45,P=0.02)。结论中国HIV-1感染早期病毒分离株的复制能力与疾病进展相关。     

福建艾滋病病毒的分离和微量全血分离法的建立

目的从HIV/AIDS病例血液中分离HIV并进行微量全血分离方法的研究.方法采集20份在福建发现的HIV1感染者肝素抗凝血,分离外周血单核细胞(PBMC),与健康人PBMC共培养,使用含神经氨酸酶(NA)的细胞培养液进行HIV-1的分离,建立微量全血分离法,通过检测p24抗原、间接免疫荧光试验(IFA)等确定病原分离结果.用MT4细胞感染试验分析病毒表型,并通过测定病毒分离株DNA的C2-V3区的核苷酸序列鉴定亚型.结果从20例HIV/AIDS病例PBMC标本中分离到18株HIV-1,分离率达90%,其中对HIV感染者的分离率为84.6%(11/13),对AIDS患者的分离率为100%(7/7).预刺激和同时刺激PBMC共培养对分离结果没有明显影响,可从10～125μl感染者全血中分离出病毒,与大量法分离比较,结果没有明显差异.18株病毒分离株只有1株可在MT4细胞中稳定传代,其它为M嗜性株;HIV-1A亚型1株,B亚型3株,E亚型13株,E亚型基因离散率为13.724±3.595.结论添加NA可能有助于提高HIV的分离率,微量全血分离法可用于病毒的分离,福建病毒分离株主要为HIV-1E亚型的M嗜性慢低复制株.     

HIV-1感染、致病机理和人体CCR5、CCR2等基因多态性之间的相互关系研究进展

1 概述 HIV-1感染人体后引起进行性的AIDS病变,通常经历三个典型的时期,第一为急性感染期,表现为广泛的病毒血症;接着是无症状期,此时在淋巴器官中大多可检测出病毒的复制;最后是免疫系统破坏,并伴随病毒血症的再次出现,并产生继发性感染等而死亡。引起AIDS的病毒株有以下几种:(1)嗜巨噬细胞HIV-1株:它主要通过性传播,从初期感染者体内分离到的原代病毒,它感染细胞后不诱生合胞体(syncytia);(2)嗜T细胞HIV-1病毒株:是从发病的AIDS病人身上来源的或实验室培养的毒株。它的毒性很强,感染后,病情呈进行性发展;(3)双嗜性HIV-1病毒株,即感染巨嗜细胞,又感染T细胞。     

R5嗜性的人类免疫缺陷病毒-1在疾病不同阶段的生物学特性

目的 研究HIV-1感染者不同时期分离的R5毒株的生物学特性.方法 采用传统的共培养方法分离并培养HIV-1,用表达CD4和CC趋化因子受体5(CCR5)或CXC趋化因子受体4(CXCR4)的GHOST细胞系,通过流式细胞仪测定病毒辅助受体的利用和感染性,从而判断所分离毒株的CCR5嗜型(R5型毒株);使用2 ng P24病毒量感染正常人分离的外周血单个核细胞(PBMC),ELISA法检测第1、3、5、7、10、15天的HIV-1 P24抗原,反映病毒复制能力;采用HIV-1核酸荧光定量检测试剂盒测定血浆病毒载量.数据分析采用t检验.结果 HIV-1B'亚型感染者22例,其中CD4+细胞>0.2×109/L和CD4+细胞≤0.2×109/L各11例;所分离的病毒仅利用CCR5辅助受体,均为R5型毒株,感染性的结果显示,来自CD4+细胞≤0.2×109/L的11株R5毒株的感染性为(7.392 7±4.584 2)%,而CD4+细胞>0.2×109/L的为(2.613 6±1.610 5)%,差异有统计学意义(t=3.262,P<0.05);两组病毒复制滴度在第7天开始明显上升,培养第7、10、15天,两组病毒复制动力学差异有统计学意义(t值分别为3.771、2.509和2.260;P<0.05),CD4+细胞≤0.2×109/L的R5毒株的复制能力较CD4+细胞>0.2×109/L的明显增强;CD4+细胞≤0.2 × 109/L R5型毒株的病毒载量的对数值为(5.606 8±0.815 1)拷贝/mL,CD4+细胞>0.2 × 109/L的为(4.729 8±0.431 6)拷贝/mL,两组差异有统计学意义(t=3.771,P<0.05).结论 疾病进展过程中,即使病毒的辅助受体利用未从CCR5转变为CXCR4,但病毒的感染性和复制能力已有明显改变.     

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.     

武汉重症手足口病EV-A71分离株全基因组特征分析北大核心CSCD

目的分析武汉重症肠道病毒A71型(Enterovirus A71,EV-A71)病毒株全基因组特征。方法自湖北武汉2例手足口病Ⅱ期患儿咽拭子中分离EV-A71,利用overlap-PCR扩增EV-A71全基因组的6个首尾重叠的基因片段,利用生物信息学软件DNAStar5.0、MEGA-Ⅹ和SimPlot3.5.1分析基因组特征。结果获得2株EV-A71分离株wh064和wh170的全基因组序列,二者核苷酸和氨基酸序列同源性分别为99.95%和99.86%。2株EV-A71病毒株属于C4a2亚型。系统进化分析和遗传距离计算发现,在P1区域,2株病毒株与C基因型EV-A71位于同一进化分支,遗传距离最小;在P2区域,2株病毒株与B基因型EV-A71和CVA16位于同一分支,与B基因型EV-A71之间遗传距离最小(C4亚型除外);在P3区域,2株病毒株与B3亚型EV-A71和CVA16位于同一分支,与B3亚型EV-A71和CVA16之间遗传距离较小。基因重组分析发现,2株分离株P1区域与C基因型EV-A71相似度最高,P2区域与B基因型EV-A71、CVA4、CVA5、CVA14和CVA16原型株的相似度高于C基因型EV-A71,P3区域与B3亚型EV-A71、CVA4、CVA14和CVA16原型株相似度高于其他病毒株,重组断点位于2A-2B连接处和3C处。此外,2株病毒株基因组中突变位点5’UTR-61T、VP1-27S(L)、VP1-98K和3D-396S亦存在于武汉其他重症和死亡病例EV-A71分离株。结论武汉2株EV-A71是重组病毒株,可能由C基因型和B3亚型EV-A71病毒株重组而来,亦或由C基因型EV-A71和CVA4、CVA14、CVA16重组而来。某些位点突变可能影响EV-A71毒力。     

一起儿童病毒性脑炎暴发肠道病毒ECHO6型分离株VP1基因特征分析

目的 对埃可病毒6型(ECHO6)福建龙岩分离株进行分子生物学分析,阐明ECHO6病毒福建龙岩分离株的分子生学特征及其与世界其它分离株的基因关系。方法 采集暴发病例脑脊液标本,用RD细胞进行分离病毒,阳性分离物采用中和试验及PCR序列分析确定其血清型别;RT-PCR扩增VP1基因全长,测定序列进行同源性及亲缘进化树分析。结果 13例细胞培养阳性分离物经中和试验及序列分析结果均为ECHO6。随机选取4株病毒株进行VP1全基因序列分析显示: 4个病毒株之间同源性＞99%;与山东病毒株E6/SD/sewage/081226/2-2同源性高于其他参考株。在遗传进化树上,4株分离病毒株同属一个分支, 为C2亚型。结论 ECHO6是引起本次福建省龙岩市长汀县儿童病毒性脑炎暴发的病原体,其基因型为C2亚型。     

福建省1株登革Ⅱ型病毒的鉴定

目的对2005年福建省分离的1株登革病毒(DV)进行鉴定,并从分子水平追踪其可能的传染源。方法采用酶联免疫吸附试验(ELISA)检测疑似登革热患者血清中DV(IgM、IgG抗体;同时应用C6/36细胞、单克隆抗体间接免疫荧光(McAb-IFA)、逆转录(套式PCR法分别进行病毒的分离和鉴定,并对分离株的部分基因进行核苷酸序列分析。结果患者血清登革病毒特异性IgM抗体阳性、IgG抗体可疑,表明该患者在近期感染过登革病毒。患者血清接种C6/36细胞,观察到登革病毒特有的CPE。受感染的C6/36细胞能与登革病毒Ⅱ型单克隆抗体反应,表明分离的病毒株为登革Ⅱ型病毒。分离株的核酸提取物经RT(PCR扩增,登革病毒通用引物可扩增出511bp的特异性条带,型特异性引物扩增出119bp的特异性条带,进一步证实分离的病毒株为登革Ⅱ型病毒。分离株RT(PCR扩增产物的核苷酸序列与30株不同地域来源的登革Ⅱ型病毒相应序列构建的系统发生树表明,此毒株与东南亚地区的毒株比较接近。此次分离株的序列与1999年登革Ⅱ型病毒福建株的对应序列在亲缘关系上有一定程度距离。结合流行病学调查资料,进一步确定此病例为输入性感染病例。结论福建省首次从输入性登革热患者血清中分离出登革Ⅱ型病毒,该病毒来源于东南亚地区。     

Analysis of insertions and deletions in the gag p6 region of diverse HIV type 1 strains

Sequence variation in the gag p6 region in subtype B HIV-1 has been associated with changes in viral replication capacity and antiretroviral drug susceptibility. We examined sequence variation in the HIV-1 gag p6 region using plasma samples from 22 individuals with non-subtype B HIV-1 infection [subtypes A, C, D, F, and G, and circulating recombinant forms (CRFs) CRF01-AE and CRF02_AG]. An additional 105 gag sequences from the Los Alamos National Laboratory database were also analyzed. Extensive length variation was observed in the p6 gag region. Specific patterns of insertions and deletions were observed in different subtypes and CRFs, and no two subtypes or CRFs had the same general pattern. PTAP duplications were more common in subtype C than other strains (3 of 14 in subtype C vs. 2 of 113 in other strains, p = 0.004), and KQE duplications were seen only in subtype B. Further studies are needed to determine whether such genotypic differences influence viral replication capacity, antiretroviral drug susceptibility, or other phenotypic properties of these strains.     

Conserved neutralizing epitopes of HIV type 1 CRF01_AE against primary isolates in long-term nonprogressors

Linear conserved B cell epitopes in envelope glycoprotein of long-term nonprogressors (LTNPs) HIV-1 CRF01_AE were determined. The envelope sequences of HIV-1 subtype E from Thailand were aligned to define consensus sequences. Then the peptides corresponding to these predicted regions were synthesized as peptides represent C1, C2, C3, C5, V2, V3, and gp41 regions. After that, the neutralizing B cell epitopes were determined by neutralized competitive assay with pool sera of typical progressor and LTNP HIV-1 CRF01_AE patients against HIV-1 CRF01_AE 24 primary isolates (PI) and laboratory strains (TCLA). We found that the strength and breadth of neutralization were greater for sera from LTNPs compared with sera from typical progressors. Peptides C1E and C2E could inhibit primary isolates but not the TCLA strain in LTNP sera. The new B cell epitopes, which were located in the C1 and C2 regions of CRF01_AE against primary HIV-1 isolates, were identified in HIV-1 CRF01_AE LTNPs. This may be important in HIV-1 vaccine development and trial.     

Genetic variability of gag and env regions of HIV type 1 strains circulating in Slovenia

The aim of this study was to investigate the genetic diversity of HIV-1 strains circulating in Slovenia. Proviral DNA isolated from peripheral blood mononuclear cells (PBMCs) of 20 randomly selected HIV-1-infected individuals was classified into subtypes by sequence-based phylogenetic analysis of the env (C2V3) and gag (p24) regions of the viral genome. The phylogenetic tree based on env C2V3 sequences showed that 15 of the 20 samples were subtype B, two A1, one F1, one CRF01_AE, and one CRF02_AG. The phylogenetic analysis of the gag gene yielded identical results expect for one sample that had a discordant subtype; it was identified as subtype A1 in the env and AE in the gag region. Our study confirmed that although subtype B predominates, other subtypes and circulating recombinant forms (CRFs) are also present in Slovenia. The high intrasubtype genetic diversity of subtype B sequences suggests a multiple introduction of subtype B strains into Slovenia.     

Temporal relationship between V1V2 variation,macrophage replication,and coreceptor adaptation during HIV-1 disease progression

BACKGROUND: Specific mutations in VPR and V2 potentially restrict HIV-1 replication in macrophages. Such restriction could potentially limit HIV replication in long-term non-progressors (LTNP), thus accounting for low viral load and delayed progression to AIDS. OBJECTIVE: To examine whether a specific VPR phenotype (truncated versus non-truncated) correlates with disease progression and whether elongated V2 restricts viral replication in macrophages or alters viral tropism. METHODS: Sequence analysis was carried for VPR and V1-V3 env from four rapid progressors (RPs), six late progressors (LPs), and three LTNPs in cohort of HIV-1-infected homosexual men. The replication kinetics of sequential isolates was examined in primary CD4 cells and macrophages and coreceptor usage was determined by GHOST infection assays. RESULTS: No differences were found in the VPR protein from RP and LTNP isolates. Analysis of the V2 region revealed that all RPs maintained similar V2 lengths (40 aa), whereas LPs and LTNPs acquired additional amino acids (2-13 aa) in the V2 region. Coreceptor specificity revealed that RP switch from CCR5 to multiple coreceptor usage, whereas LTNPs maintained R5 viruses. Sequential isolates from each group revealed comparable replication efficiencies in both T-cells and macrophages, regardless of the V2 length or coreceptor utilization. In addition, cross-section analysis of six LTNPs from Australia revealed extended V2 with consistent usage of CCR5 coreceptor. CONCLUSION: The present results suggest that acquisition of a V2 extension over time in HIV-1-infected LPs/LTNPs appears to correlate with maintenance of CCR5 usage among LTNPs. These findings may be important for a better understanding of the host interactions and disease progression.     

Substitution of hiv type-1 non-B env genes in C2, a subtype B cassette system, results in functional chimeric viruses

Env gene glycoprotein products are essential to viral infectivity and important targets for a host's humoral and cellular immune responses. We have reported the construction of C2, an effective env gene cassetting system for assessing biological properties of HIV-1 subtype B env gene glycoprotein products within a constant genetic background (Zheng NN and Daniels RS: AIDS Res Hum Retroviruses 2001;17:1501-7506). Here we report the ability of C2 to produce chimeric subtype A, C, D, A/E, F, and J HIV-1 and studies of the viruses' biological properties. Virus RNAs were extracted and full-length env genes rescued by RT-PCR. Expression-competent env genes were cloned into the C2 cassette and chimeric recombinant viruses produced by transfecting 293T cells. For each subtype, X4 viruses yielded higher TCID(50) than R5 viruses and the TCID(50) of chimeric viruses were either the same as or lower than their parental viruses. The limited coreceptor usage of R5-tropic parent viruses was retained in the chimeric viruses. Generally, with the exception of the subtype C virus (SE12808), the X4-tropic parental viruses utilized CXCR4 and a wide range of additional coreceptors, while their respective chimeric viruses retained CXCR4 usage but showed a more limited range in respect of other coreceptors. The replication rates of non-B subtype chimeric viruses were generally lower (1.5- to 13.6-fold) than their respective parental viruses with the exception of C2-92UG029, an X4-tropic subtype A chimeric virus. This study demonstrates that C2 is a functional cassette capable of producing infectious chimeric viruses to allow study of the biological phenotypes and functions of HIV-1 subtype B and non-B subtype glycoproteins.     

Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon,Portugal

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.     

HIV-1 viral diversity and its implications for viral load testing: review of current platforms

The 2008 Recommendations for care of the International AIDS Society reaffirmed the importance of both accurate and sensitive viral load assessment, and by necessity, access to viral load assays. HIV-1 viral load testing is considered essential when initiating antiretroviral therapy (ART), when monitoring ART response, and when considering switching ART regimens. The demand for accurate, reproducible, and cost-effective viral load assays is therefore a global issue. Although the North American and Western European experience has historically been with HIV-1 group M subtype B virus, this paradigm is changing rapidly as migrants and refugees from developing countries with non-B subtype infections often now present for care in the developed world, and travelers to developing countries acquire non-B subtype infection abroad and present for care at home. Awareness of any clinical or laboratory differences between the common HIV-1 group M subtype B and the newer HIV-1 strains being seen in practice is therefore increasingly important. This review of current HIV-1 viral load testing is focused on the potential value of a standardized genotype assignment for HIV-1 viral subtypes, regular monitoring of the performance of available commercial HIV viral load assays on emerging non-B HIV subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs), and a discussion of the implications for resource-limited settings.     

Identification and characterization of HIV type 1 subtypes present in the Kingdom of Saudi Arabia: high level of genetic diversity found

Saudi Arabia has a very low prevalence of HIV infections and nothing is known about HIV strains present in the population. Here specimens were collected from 62 HIV-1-infected patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Viral sequences were PCR amplified using primers for HIV-1 group M in gag p24, pol integrase, and env gp41 and genetic subtype was determined by phylogenetic analysis. HIV-1 viral sequences were amplified from 56 of the 62 specimens. Based on phylogenetic analysis of viral sequences, subtype C was the most common subtype present and accounted for 39.3% of the infections followed by subtype G (25%), subtype B (17.9%), subtype D (3.6%), and subtypes A and CRF02_AG (1.8% each). In addition, for six specimens subtype classifications were discordant between gag, pol, and/or env; these intersubtype recombinant viruses account for 10.7% of the infections and consisted of recombinants of subtypes A/CRF01, A/CRF02, A/G, B/G, and D/CRF02. The high HIV-1 strain diversity suggests that there have been multiple introductions of HIV-1 into Saudi Arabia from several sources. Within the study population, there were five husband/wife pairs. For each pair, the viral sequences obtained were closely related to each other showing that heterosexual transmission occurred.