HIV-1 CRF07_BC病毒适应性的体外生长竞争试验的建立

目的:通过突变的方式构建含有鼠 Thy1.1基因或鼠 Thy1.2基因的HIV-1 CRF07_BC感染性克隆,建立CRF07_BC亚型体外竞争试验。 方法:用鼠 Thy1.1基因和鼠 Thy1.2基因替换CRF07_BC亚型感染性克隆pXJDC6291-13 ...     

人类免疫缺陷病毒Ⅰ型包膜糖蛋白gp41的基因重组表达

目的基因重组表达（ＨＩＶ１ｇｐ４１）抗原，并研制一种快速、简便、灵敏性高、特异性强的国产ＨＩＶ１免疫检测试剂。方法选用ＨＩＶ１型ＢＨ１０毒株的包膜糖蛋白ｇｐ４１的部分基因（６９７７７４９７），重组在ＰＢＶ２２１表达载体上。表达产物通过１５％ＳＤＳ聚丙烯酰胺凝胶电泳初步分离纯化，根据ＲＦ值，切下含特异蛋白的胶带，以Ｗｅｓｔｅｒｎｂｌｏｔ法转移在硝酸纤维素膜上；免疫血清法检测合格者制备的抗原检测条带，经国家标准参比血清检测。结果获得１株含ＨＩＶ１ｇｐ４１基因的重组质粒，其蛋白的特异性表达为８％，经ＨＩＶ１阳性血清检测和国家标准参比血清检定，该表达蛋白灵敏性、特异性均为１００％。结论（１）可以选用单一的ｇｐ４１作为ＨＩＶ１感染初筛试剂的抗原。（２）表达载体ＰＢＶ２２１其表达的目的蛋白为非融合蛋白，作为试剂中的抗原，可降低检测中的非特异性。（３）该试剂是一种简便、特异、灵敏性高的试剂。     

我国西南西北地区吸毒人群重组人类免疫缺陷病毒1型毒株的发现

目的寻找人类免疫缺陷病毒１型（ＨＩＶ１型）在中国可能的重组。方法从流行２种以上ＨＩＶ１亚型的地区收集ＨＩＶ感染者的血样。从ＰＭＣｓ中应用套式聚合酶链反应（ＰＣＲ）方法,对ＨＩＶ病毒的ｔａｔ和ｅｎｖ基因进行扩增,ＰＣＲ产物直接测序并进行序列分析。结果对中国Ｂ′亚型和Ｃ亚型流行区域收集的１４个ＨＩＶ１毒株进行序列分析,对ｅｎｖ基因进行测序后没有发现重组毒株的证据。对ｔａｔ基因的第一外显子进行序列分析时,１４个样品中的１０个样品发现了Ｂ′亚型和Ｃ亚型重组的ＨＩＶ１毒株。此外,四川省静脉吸毒者中发现了３个非重组的Ｂ′亚型毒株和１个非重组的Ｃ亚型毒株。结论首次在中国西南部的四川省和西部的新疆维吾尔自治区发现了Ｂ′亚型和Ｃ型的重组ＨＩＶ１毒株。相同的序列和重组方式,表明重组毒株具有相同的起源,同时表明这两个艾滋病流行区域密切相关。由于在新疆只发现了重组毒株,而在四川省则发现了Ｂ亚型、Ｃ亚型和Ｂ′／Ｃ重组毒株,重组很有可能发生在四川而不是新疆。     

我国HIV-1 CRF07_BC流行重组毒株Tat蛋白第一外显子CTL表位预测

目的 通过测定和分析我国HIV-1 CRF07_BC毒株Tat蛋白第一外显子的基因序列预测其CTL表位的分布情况.方法 236份来自第3次全国HIV分子流行病学调查中感染CRF07_BC毒株的患者的血浆样本,应用巢式聚合酶链反应(nested-PeR)扩增tat基因第一外显子基因区并测序,使用BIMAS HLA Peptide Binding Predictions在线软件和统计学软件对CTL表位进行预测分析.结果 236份CRF07_BC毒株来自16个省份,主要为静脉吸毒传播(58.9%),其次为性传播(25.0%).236条CRF07_BC Tat蛋白第一外显子区共分布有12种CTL表位,主要分布于脯氨酸富集区、半胱氨酸富集区和疏水核心区,碱性区和谷氨酰胺富集区没有发现已知的CTL表位分布,这些表位主要受5种HLA基因型(A * 2501、A * 2902、B * 15、B * 5301和Cw * 1203)以及6种HLA血清型(B53、B58、B57、A3、A68和Cw12)限制,其中5种表位的单氨基酸变异频率大于50%.结论 我国HIV-1 CRF07_BC毒株Tat蛋白第一外显子CTL表位分布于不同功能区,并且存在氨基酸变异.     

2015—2019年南京市HIV-1 CRF07BC毒株流行特征及分子进化分析

目的了解2015—2019年南京市HIV-1 CRF07_BC的流行史、进化模式和传播特征, 为精准防控该毒株的传播提供科学依据。方法对南京市319例HIV-1 CRF07_BC患者的pol区基因序列扩增测序并构建最大似然树。使用MCMC(Markov Chain Monte Carlo)算法生成MCC树(Maximum Clade Credibility tree), 使用BSP(Bayesian Skyline Plot)法重塑有效人口规模数变化趋势。采用成对基因距离法构建分子网络探究其传播特征。结果 319名HIV-1 CRF07_BC感染者中, 95.0%为男性, 多性伴(2个及以上)比例达82.8%, 仅4.4%病例自报一直使用安全套, 男男同性性行为(MSM)为最主要的感染途径(77.4%)。进化树结果显示, HIV-1 CRF07_BC呈现两大流行簇:Cluster1和Cluster2, Cluster1以MSM感染病例为主, Cluster2以异性性行为感染病例为主。Cluster1和Clu...     

固有性抗人类免疫缺陷病毒-1因子的研究

人类免疫缺陷病毒(human immunodeficiency vi-rus,HIV)是获得性免疫缺陷综合征(acquired immu-nodeficiency syndrome,AIDS)的病原体,引起人类致病的主要是HIV-1。人体在HIV-1病毒感染后,主要通过固有性免疫和获得性免疫控制和清除病毒。前者主要有(natural killer cell,NK ce     

人类免疫缺陷病毒Ⅰ型和丙型肝炎病毒核酸联合检测试剂的评价

目的 了解人类免疫缺陷病毒Ⅰ型（HIV-1）和丙型肝炎病毒（HCV）核酸联合检测试剂检测的敏感性、特异性以及检测中国不同亚型样品的适应性。方法 用酶联免疫试剂对来自于不同地区的HIV感染者或可疑感染者献血员样品进行HIV抗体和HCV抗体检测。对HIV抗体阳性者进行抗体确证。用HIV-1和HCV核酸联合检测试剂对样品进行检测，对初次检测阳性者，用HIV RNA和HCV RNA鉴别试剂单独检测，区分是HIV RNA阳性或是HCV RNA阳性。结果 74份HIV和HCV抗体均阳性的样品，HIV／HCV RNA检测也为阳性，5份HIV和HCV抗体均阴性的样品，HIV／HCV RNA检测也为阴性；84份HIV抗体确证为阳性的样品，有82份检测为HIVRNA阳性，7份HIV抗体阴性的样品检测均为HIV RNA阴性，12份HIV抗体不确定的样品，有6份检测为HIV RNA阳性；81份HCV抗体阳性样品中有70份检测为HCV RNA阳性，22份HCV抗体阴性样品中有18份检测为HCV RNA阴性，4份检测为HCV RNA阳性。结论 该试剂的灵敏度较高，尤其在HCV抗体阴性样品中检出HCV RNA阳性者。因此，可用于献血员筛查，减少窗口期的传播。     

人类免疫缺陷病毒-1型感染对U937前单核细胞基因表达的影响

目的：研究人类免疫缺陷病毒-1型(HIV-1) 急性感染对U937前单核细胞基因表达的影响，探索AIDS的病理机制。方法:应用基因芯片技术分析HIV-1感染2-3 d后U937前单核细胞的550个RNA序列的表达水平,并用半定量RT-PCR加以验证。结果:感染HIV-1 后2-3 d的U937细胞有38 个基因受到不同的调节：26个基因被下调，12个基因上调。这些基因编码着功能各异的宿主 蛋白，这些蛋白质涉及不同的细胞内反应过程，包括受体介导的信号转换、亚细胞信号转导 、 凋亡、转录调节和化学趋向等。结论:HIV-1感染能引起U937前单核细胞 许多基因表达发生改变。     

重组腺病毒HIV-1 vpr基因的构建和表达

目的 构建携带HIV-1 vpr基因的重组腺病毒,使CD4 T淋巴细胞C8166内源性的高表达Vpr蛋白.方法 利用AdEasy-1系统, 通过将含有目的 基因片段的穿梭载体pAdTrack-CMV-vpr和骨架质粒pAdEasy-1在BJ5183细菌内同源重组的方法构建重组腺病毒质粒Ad-vpr, 用脂质体法将重组质粒转染至HEK293A细胞包装, 获得重组腺病毒Ad-vpr, 荧光显微镜观察Ad-vpr感染C8166细胞GFP的表达 , Western blotting鉴定Vpr在C8166细胞内的特异性表达,流式细胞术检测Ad-vpr感染 C8166细胞的效率.结果 成功构建携带HIV-1 vpr基因的重组腺病毒 ,Western blotting结果表明重组腺病毒Ad-vpr感染的C8166细胞内源性的高表达Vpr 蛋白,流式细胞术检测结果表明Ad-vpr感染C8166细胞效率高(44.07±3.62)%.结论 成功构建出携带HIV-1 vpr基因的重组腺病毒,使C8166细胞内源性的高表达Vpr蛋白.     

广西HIV-1重组毒株env区快速基因分型方法的建立

目的建立一种快速简便的基因分型方法，对广西HIV-1重组毒株env基因区进行亚型鉴定。方法从HIV阳性样品中提取核酸，使用HIV-1M组通用引物对env区进行第一轮扩增，第二轮则使用分别检测B′/C或C亚型和CRF01-AE亚型的二套特异性引物放入同一反应管中进行扩增，根据不同亚型扩增的目的带位置不同来判断亚型。将通用引物扩增出的所有样本均进行基因测序和系统树分析以验证结果。结果50份样本中，经基因测序和系统树分析证实CRF08-BC样本3份（6％），CRF01-AE样本43份（86％），4份（8％）样本无法确定亚型。经亚型特异性引物PCR法检测得出B′/C或C亚型样本3份（100％），CRF01-AE样本39份（90．7％），灵敏度为91．3％，特异度为100％。两种方法检测结果经差异性检验显示X^2=2．25，P〉0.05，差异无统计学意义，结果一致者占92％。与基因分析结果吻合。重复实验显示CRF08-BC平均重复性为100％（10／10），CRF01．AE为93．8％（61／65）。结论该方法是一种简便、快速、低成本，具有高度灵敏性和特异性的HIV-1毒株env基因区分型法，能够直接对广西HIV-1 CRF01-AE重组毒株进行鉴定。     

Molecular clock-like evolution of human immunodeficiency virus type 1

The molecular clock hypothesis states that the rate of nucleotide substitution per generation is constant across lineages. If generation times were equal across lineages, samples obtained at the same calendar time would have experienced the same number of generations since their common ancestor. However, if sequences are not derived from contemporaneous samples, differences in the number of generations may be misinterpreted as variation in substitution rates and hence may lead to false rejection of the molecular clock hypothesis. A recent study has called into doubt the validity of clock-like evolution for HIV-1, using molecular sequences derived from noncontemporaneous samples. However, after separating their within-individual data according to sampling time, we found that what appeared to be nonclock-like behavior could be attributed, in most cases, to noncontemporaneous sampling, with contributions also likely to derive from recombination. Natural selection alone did not appear to obscure the clock-like evolution of HIV-1.     

In-utero transmission of quasispecies among human immunodeficiency virus type 1 genotypes

Samples from infants infected in-utero by the human immunodeficiency virus type 1 (HIV-1) subtypes A, C, D, and recombinants from Dar es Salaam, Tanzania, were examined for the presence of viral genetic quasispecies. HIV-1 envelope diversity was measured on peripheral blood mononuclear cells collected within the first 48 h of life from 53 infants. Phylogenetic analysis of C2-C5 envelope nucleotide sequences was used for HIV-1 subtype classification. Forty-two of 53 samples (79%) showed a heteroduplex mobility assay (HMA) suggestive of transmission of a single quasispecies, while 21% showed infection with multiple quasispecies. No differences among HIV-1 subtypes were found in the proportion of single to multiple quasispecies transmitted in-utero (Likelihood ratio test, P = 0.83), nor were differences found among single to multiple quasispecies transmitted and maternal viral load (Mann-Whitney test, P = 0.44). This suggests that differences in perinatal transmission between subtypes we previously observed in this cohort could not be associated with the likelihood for multiple independent infections during in-utero infections.     

Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop

The crystal structure of the human immunodeficiency virus type 1 (HIV-1) neutralizing, murine Fab 83.1 in complex with an HIV-1 gp120 V3 peptide has been determined to 2.57 A resolution. The conformation of the V3 loop peptide in complex with Fab 83.1 is very similar to V3 conformations seen previously with two other neutralizing Fabs, 50.1 and 59.1. The repeated identification of this same V3 conformation in complex with three very different, neutralizing antibodies indicates that it is a highly preferred structure for V3 loops on some strains of the HIV-1 virus.     

Characterization of human T-cell lines harboring defective human immunodeficiency virus type 1

Defective HIV-producing T-cell lines were subcloned from MT-4/HIV_HTLV-IIIB MOLT-4/HIV_HTLV-IIIB, and H9/HIV_HTLV-IIIB cell lines chronically infected with HIV. The NY-M10 cell line derived from MOLT-4/HIV_HTLV-IIIB and the NY-H6 cell line derived from H9/HIV_HTLV-IIIB produce defective HIV, which lacks the ability to infect human T-cell lines. NY-M10 cells retain the capacity to form multinucleated giant cells in cocultivation with HIV-uninfected CD4-positive cells. However, NY-H6 cells failed to fuse with CD4-positive cells. Electron microscopic analysis indicated that the defective HIV produced from NY-M10, like those reported previously, lacked the structure of the nucleocapsid, and the virion released from NY-H6 was indistinguishable from those of authentic HIV particles. Southern and Northern blotting analyses of NY-M10 and NY-H6 cleared that the genome of those defective viruses was not significantly deleted, suggesting minor mutation(s) should take place on the viral genome.     

Productive infection of human immunodeficiency virus type 1 in dendritic cells requires fusion-mediated viral entry

Human immunodeficiency virus type 1 (HIV-1) enters dendritic cells (DCs) through endocytosis and viral receptor-mediated fusion. Although endocytosis-mediated HIV-1 entry can generate productive infection in certain cell types, including human monocyte-derived macrophages, productive HIV-1 infection in DCs appears to be dependent on fusion-mediated viral entry. It remains to be defined whether endocytosed HIV-1 in DCs can initiate productive infection. Using HIV-1 infection and cellular fractionation assays to measure productive viral infection and entry, here we show that HIV-1 enters monocyte-derived DCs predominately through endocytosis; however, endocytosed HIV-1 cannot initiate productive HIV-1 infection in DCs. In contrast, productive HIV-1 infection in DCs requires fusion-mediated viral entry. Together, these results provide functional evidence in understanding HIV-1 cis-infection of DCs, suggesting that different pathways of HIV-1 entry into DCs determine the outcome of viral infection.     

Insulin-like growth factor II mRNA binding protein 1 modulates Rev-dependent human immunodeficiency virus type 1 RNA expression

Human immunodeficiency virus type 1 (HIV-1) needs to overcome cellular counter mechanisms such as to successfully propagate itself. Results of our recent studies show that overexpression of insulin-like growth factor II mRNA binding protein 1 (IMP1) inhibits production of infectious HIV-1 particles through adversely affecting virus maturation. Here, we report that IMP1 interacts with HIV-1 Rev protein and its ectopic expression causes relocation of Rev from the nucleus to the cytoplasm. In accordance with this observation, ectopic expression of IMP1 severely diminishes Rev-dependent expression of CAT enzyme and disturbs HIV-1 RNA expression by causing accumulation of the multiple spliced viral RNA. Results of mutagenesis analysis further reveal that the KH4 domain represents the key element of IMP1 in modulating HIV-1 RNA expression. Taken together, these data suggest, in addition to hampering virus assembly, that IMP1 also has an effect on Rev-dependent viral RNA expression.     

An ultrastructural observation of negatively-stained human immunodeficiency virus type 1 (HIV-1) cores

Cores of human immunodeficiency virus type 1 (HIV-1) were observed using negative staining for electron microscopy. On the surfaces of cores isolated from viral particles with a Nonidet P40 and glutaraldehyde mixture, mortar-like units were detected in the wide and narrow margins. In some of the cores which were burst with distilled water, the units were linked together revealing a beaded structure.     

HIV-1包膜V3区在病毒侵入靶细胞中的增强作用

目的　明确Ⅰ型人免疫缺陷病毒 (humanimmunodeficiencyvirustype 1,HIV 1)包膜糖蛋白gp12 0第三可变区 (variableregion ,V3)在病毒进入靶细胞的早期阶段中的作用。方法　合成了 3个环状V3多肽 ,包括V3 BH10、V3 ADA和V3 89.6以及直链和短链多肽V3 BH10 /CA、V3 NNT2 4和V3 IRI12。构建了 3株分别带HIVHXB2株、ADA株和 89.6株包膜的伪病毒并观察了多肽对不同嗜性HIV侵入靶细胞能力的影响。结果　 (1)HIV 1V3区多肽以毒株特异性模式增强HXB2、ADA和 89.6进入靶细胞 ;(2 )直链V3多肽与其环状多肽具有相似作用 ;带有C末端的短肽V3 NNT2 4也有与长链V3 BH10 /CA相近的作用 ,只有中央保守区的短链V3 IRI12没有类似功能。结论　本研究首次发现HIV 1V3区在病毒进入靶细胞早期阶段中具有毒株特异性增强病毒侵入的作用。该发现有助于进一步明确HIV 1进入靶细胞的机制