广东两株人免疫缺陷病毒的分离和鉴定

从我省两名经性途径感染艾滋病的病人中采血,分离外周血单核细胞（ＰＢＭＣｓ）,将其与正常人ＰＢＭＣｓ共培养分离人免疫缺陷病毒（ＨＩＶ）,３周后检测上清ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ法）超过阈值。将共培养第四周细胞和上清分别感染Ｈ９细胞（Ｔ细胞淋巴瘤传代细胞）,一周后检测ＨＩＶ－１ ｐ２４怕,证明有病毒生长。用ＨＩＶ－１ ＥＮＶ基因引物的套式聚合酶链反应（Ｎｅｓｔｅｄ ＰＣＲ）证实,两株新分离的病毒     

抗原捕获聚合酶链式反应（AC－PCR）直接分型检测单纯疱疹病毒

用抗单纯疱疹病毒（ＨＳＶ）型共同性ｇＣ和ｇＤ羊克隆抗体（ＭｃＡｂ）,包被即Ｅｐｐｅｎｄｏｒｆ管,捕捉ＨＳＶ,同时加入３个引物：一个是ＨＳＶ─１／ＨＳＶ─２型共同性上游引物,另两个分别是ＨＳＶ─１和ＨＳＶ─２型特异性下游引物。借此建立了能直接分型检测ＨＳＶ的抗原捕获聚合酶链式反应（ＡＣ─ＰＣＲ）。ＨＳＶ─１的扩增产物为４７７ｂｐ,ＨＳＶ─２的为３９９ｂｐ两型病毒经ＡＣ─ＰＣＲ扩增后产生分子量不同的ＤＮＡ片段,致使ＡＣ─ＰＣＲ能直接分型检测ＨＳＶ。ＨＳＶ─１和ＨＳＶ─２扩增产物的克隆和序列分析表明,本方法特异性好。用本法检测Ｂａｌｂ／ｃ幼鼠中枢神经系统ＨＳＶ感染的脑标本,进一步证实本方法不仅敏感、特异,而且分型准确。     

从一名国内感染的艾滋病人分离人免疫缺陷病毒（HIV）

从一名国内感染的艾滋病人采血,分离其外周血单核细胞（ＰＭＣｓ）。首先与正常的ＰＭＣｓ共培养,４周后检测其ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ）达到峰值。用此时的细胞及其上清分别感染Ｊｕｒｋａｔ－ｔａｔ、ＣＥＭ、ＭＴ４细胞,可很快地在这三株细胞中检测到ＨＩＶ生长,ＨＩＶ在Ｊｕｒｋａｔ－ｔａｔ细胞中生长情况最好,同时用病人血清直接感染Ｊｕｒｋａｔ－ｔａｔ和ＭＴ４细胞,４周后检测其细胞上清ＨＩＶ－１ｐ２４抗原（ＥＬＩＳＡ法）为阳性,但ＯＤ值很低（约为ＰＭＣ共培养组的一半）。用病人的少量全血与正常ＰＭＣｓ共培养,得到的结果与分离病人ＰＭＣｓ法相近。应用间接免疫荧光法（ＩＦＡ）、免疫酶法（ＩＥＡ）、蛋白印迹法及ＨＩＶ－１ＰＯｌ基因和Ｅｎｖ基因特异引物的聚合酶链反应（ＰＣＲ）等证实为ＨＩＶ－１病毒。分离的ＨＩＶ在Ｊｕｒｋａｔ－ｔａｔ细胞中连续传代,细胞被感染后２－３天即出现以大量融合细胞为主的细胞病变,感染后７－１０天细胞几乎全部死亡。病毒在连续传代过程中的生长特征及致细胞病变特征不变。此病毒命名为ＣＡ－２毒株。     

缺氧诱导因子1和红细胞生成素3‘—增强子调节内皮细胞？…

目的和方法：将红细胞生成素（ＥＰＯ）３＇－增强子野生片断及点突变片断借脂质体主人脐静脉内皮细胞株ＥＣＶ－３０４,用半定量ＲＴ－ＰＣＲ测定正常秘缺氧诱导因子－１（ＨＩＦ－１）诱导剂氧化钴（ＣｏＣｌ２）作用下培养６ｈ的细胞环氧合酶２（ＣＯＸ－２）和血栓素合酶（ＴＸＳ）的ｍＲＮＡ。结果：ＨＩＦ－１诱导剂ＣｏＣｌ２可放ＣＯＸ－２和ＴＸＳ基因转明显增强２,向细胞导入野生ＥＰＯ３＇增强子片断可阻断ＣｏＣｌ２诱     

树Ju免疫细胞体外感染Ⅰ型人免疫缺陷病毒的实验研究

至今可以感染Ⅰ型人免疫缺陷病毒（ＨＩＶ－１）的动物只有黑猩猩和长臂猿,这严重阻碍了ＨＩＶ－１的疫苗研究和治疗研究。因此,寻找新的可以感染ＨＩＶ－１的动物模型成为十分迫切的课题。已知树Ｊu对许多重要的医学病毒易感,为了探讨树Ｊu是否可以感染ＨＩＶ－１,利用不同辅助受体的５种ＨＩＶ－１病毒株。体外感染云南野生成年树Ju的淋巴细胞和单核／巨噬细胞;同时还用这些病毒感染人外周血淋巴细胞或单核细胞。然后用ＲＴ－ＰＣＲ、ＰＣＲ和流式细胞术分别进行了检测,用ＲＴ－ＰＣＲ方法未检测到感染上清中有病毒粒子的存在,用ＰＣＲ法未能发现树Ｊu的这些免疫细胞中有前病毒ＤＮＡ,用流式细胞术也未能在这些感染ＨＩＶ－１的树Ｊu细胞的表面检测到特异抗原;而感染ＨＩＶ－１的人免疫细胞均为阳性结果。实验结果表明树Ju的这些免疫细胞在体外未能感染上ＨＩＶ－１,可能的原因是树Ｊu的这些免疫细胞的ＨＩＶ－１受体（ＣＤ４）和辅助受体（ＣＣＲ５或ＣＸＣＲ４）与人的免疫细胞胞差别较大。     

转译优化对EPO基因在COS7细胞中表达的影响

利用ＣＯＳ７细胞暂时表达系统,研究转译起始序列对ＥＰＯ－ｃＤＮＡ表达的影响。通过ＤＮＡ重组技术,构建了原ＥＰＯ－ｃＤＮＡ表达载体ｐＣＳＶ－ＥＰＯ（１）,其转译起始序列为５＇ＡＡＴＴＣＡＴＧＧ３＇。同时通过定点突变技术,将起始序列改变成５＇ＣＣＡＣＣＡＴＧＧ３＇,而构建了另一表达载体ＰＣＳＶ－ＥＰＯ（２）。后经序列分析证明无误后和前均通过ＤＥＡＥ－ｄｅｘｔｒａｎ法转染ＣＯＳ７细胞上清,测定结果为     

6株A型口蹄疫病毒结构蛋白VP1（1D）基因的核苷酸序列分析

早期收集保藏的６株Ａ型口蹄疫病毒（ＦＤＭＶ）（ＡＬ１－ＡＬ６）,适应ＢＨＫ２１单层细胞后,提取６株细胞增殖病毒的ＲＮＡ。用一对ＦＭＤＶ通用引物经反转录（ＲＴ）－ＰＣＲ法扩增了约５００ｂｐ的期望的ＤＮＡ片段。克隆目的的基因后,采用双脱氧ＤＮＡ链末端终止法测得了６株病毒的ＶＰ１基因２１０－６３９核苷酸序列。分析表明,３株病毒缺失３个核苷酸,从推导的氨基酸序列比较,确定缺失的３个核苷酸正好是一个密码子,     

HIV—1抗体阳性血浆中HIV—1病毒基因分型研究

为了解ＨＩＶ抗体阳性血浆中的ＨＩＶ１病毒基因亚型的情况,应用逆转录ＰＣＲ和ＤＮＡ序列测定技术,对６份获自高危人群的抗ＨＩＶ１阳性血浆进行序列分析和基因亚型分型的研究,结果表明均属ＨＩＶ１Ｂ亚型。Ｖ３环氨基酸序列分析指出这些ＨＩＶ１Ｂ亚型病毒株与泰国ＨＩＶ１Ｂ亚型病毒株核苷酸和氨基酸序列相似;同时发现ＨＩＶ１ｃＤＮＡ和氨基酸序列均相同,推测这６份标本可能来自同时感染同一株ＨＩＶ病毒的感染者。本研究对了解高危人群中ＨＩＶ１流行的遗传变异和ＨＩＶ１亚型病毒株的分子流行病分析具有一定的意义。     

药物对病毒感染培养心肌细胞Ca^2＋内流及CVB3—RNA复制的影响

应用放射性同位素＾４５Ｃａ＾２＋示踪技术及光敏生物素标记ｃＤＮＡ探针杂交方法,观察了维拉帕米、黄芪、地塞米松等药物对感染柯萨奇Ｂ３病毒（ＣＶＢ３）的大鼠培养心肌细胞Ｃａ＾２＋内流及细胞中ＣＶＢ３－ＲＮＡ复制的作用。结果发现：在病毒感染４８ｈ,上述三药均可显著减少感染细胞及正常对照的心肌Ｃａ＾２＋内流;若在病毒感染后即加入上药物,经４８ｈ培养后,黄芪组细胞中的ＣＶＢ３－ＲＮＡ含量显著少于病毒对照组,     

鸡传染性支气管炎病毒S1基因在昆虫细胞中表达水平初探

将含有鸡传染性支气管炎病毒 Ｓ１ 基因ｃ Ｄ Ｎ Ａ 的重组转移质粒ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３ Ｓ１ ． Ｈｏｌｔｅ 和ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４ Ｓ１ ． Ｈｏｌｔｅ 分别与粉纹夜蛾核型多角体病毒 Ｔｎ Ｎ Ｐ Ｖ Ｓ Ｖ Ｉ－ Ｇ Ｄ Ｎ Ａ（ Ｏ Ｃ Ｃ－ ,ｇａｌ＋ ） 共转染草地夜蛾（ Ｓｆ９） 细胞,经空斑纯化得到重组病毒 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 和 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 。将重组毒株分别感染 Ｔｎ５ Ｂ１ 细胞,并进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 与 Ｗｅｓｔｅｒｎｂｌｏｔ 检测。结果表明, Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 在感染的细胞中高效表达了 Ｓ１ 蛋白, Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 凝胶薄层色谱分析结果显示,感染病毒后７２ ｈ Ｓ１ 蛋白的表达量占细胞内总蛋白量的３５ ．８ ％ ,而 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 感染的细胞内检测不出 Ｓ１ 蛋白。经分析认为这一差异主要来自 Ｓ１ 基因翻译起始位点及其附近的周围环境。     

Alpha interferon inhibits human T-cell leukemia virus type 1 assembly by preventing Gag interaction with rafts

Alpha-2a interferon (IFN-alpha2a) has beneficial clinical effects on human T-cell leukemia virus type 1 (HTLV-1) infection, but its antiviral mechanism of action is unknown. Antiviral effects of IFN-alpha2a were studied in 293T cells expressing HTLV-1 proviral DNA and in HTLV-1-infected cells (HOS/PL, MT2, and HUT102). In 293T cells, an 50% inhibitory concentration of 10 U of IFN-alpha2a/ml was determined by p19 antigen ELISA. Analysis of IFN-treated cells demonstrated no defect in viral protein synthesis but did show a decrease in the level of released virus, as determined by immunoblot assays. Electron microscopy studies of IFN-treated cells revealed neither a defect in the site of virus budding nor tethering of virus particles at the plasma membrane, thus arguing against an effect on virus release. Cell fractionation studies and confocal microscopy showed no effect of IFN on Gag association with membranes. However, the level of Gag association with lipid rafts was decreased, suggesting a role of IFN in inhibiting HTLV-1 assembly.     

In vivo cellular tropism of human T-cell leukemia virus type 1.

To establish the phenotype of human T-cell leukemia virus type 1 (HTLV-1)-infected cells in peripheral blood, the polymerase chain reaction was used to detect and quantitate viral DNA in subpopulations of leukocytes obtained from patients with tropical spastic paraparesis and asymptomatic carriers. HTLV-1 could not be detected in peripheral blood mononuclear cells thoroughly depleted of T lymphocytes (E- CD3-), nor could it be detected in highly enriched populations of B lymphocytes (E- CD19+), monocytes (E- CD14+), or natural killer cells (E- CD16+). T lymphocytes were strongly positive for HTLV-1, and fractionation of this population revealed that 90 to 99% of the HTLV-1 DNA segregated with the CD4+ CD8- and CD45RO+ subsets. No difference between the cell type distribution of HTLV-1 in the asymptomatic carrier and the subjects with tropical spastic paraparesis was evident. Southern hybridization of genomic DNA prepared from the peripheral blood of HTLV-1 carriers indicated that up to 10% of circulating leukocytes may carry the HTLV-1 provirus.     

Sho-Saiko-To,a Traditional Kampo Medicine,Enhances the Anti-HIV-1 Activity of Lamivudine (3TC) In Vitro

Sho-saiko-to (SST), a traditional Kampo medicine, has been examined for its inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in peripheral blood mononuclear cells (PBMCs). SST alone moderately inhibited HIV-1 replication at a concentration of 25 μg/ml. When SST was combined with zidovudine (AZT), lamivudine (3TC) or AZT plus 3TC, SST enhanced the anti-HIV-1 activity of 3TC. In contrast, SST slightly enhanced the anti-HIV-1 activity of AZT plus 3TC but did not enhance the activity of AZT alone. These results suggest that the combination of SST and 3TC has potential as a chemother-apeutic modality of HIV-1 infection.     

Herpes simplex virus resistance and sensitivity to phosphonoacetic acid.

Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.     

Human T-cell leukemia virus type 1 (HTLV-1) infection of mice: proliferation of cell clones with integrated HTLV-1 provirus in lymphoid organs

Human T-cell leukemia virus type 1 (HTLV-1) is suggested to cause adult T-cell leukemia after 40 to 50 years of latency in a small percentage of carriers. However, little is known about the pathophysiology of the latent period and the reservoir organs where polyclonal proliferation of cells harboring integrated provirus occurs. The availability of animal models would be useful to analyze the latent period of HTLV-1 infection. At 18 months after HTLV-1 infection of C3H/HeJ mice inoculated with the MT-2 cell line, which is an HTLV-1-producing human T-cell line, HTLV-1 provirus was detected in spleen DNA from eight of nine mice. No more than around 100 proviruses were found per 10(5) spleen cells. Cellular sequences flanking the 3' long terminal repeat (LTR) and the clonalities of the cells which harbor integrated HTLV-1 provirus were analyzed by linker-mediated PCR. The results showed that the flanking sequences are of mouse genome origin and that polyclonal proliferation of the spleen cells harboring integrated HTLV-1 provirus had occurred in three mice. A sequence flanking the 5' LTR was isolated from one of the mice and revealed the presence of a 6-nucleotide duplication of cellular sequences, consistent with typical retroviral integration. Moreover, PCR was performed on DNA from infected tissues, with LTR primers and primers derived from seven novel flanking sequences of the three mice. Data revealed that the expected PCR products were found from lymphatic tissues of the same mouse, suggesting that the lymphatic tissues were the reservoir organs for the infected and proliferating cell clones. The mouse model described here should be useful for analysis of the carrier state of HTLV-1 infection in humans.     

Human immunodeficiency virus type 1 DNA synthesis, integration, and efficient viral replication in growth-arrested T cells.

Human immunodeficiency virus type 1 (HIV-1) replicates efficiently in nonproliferating monocytes and macrophages but not in resting primary T lymphocytes. To determine the contribution of cell division to the HIV-1 replicative cycle in T cells, we evaluated HIV-1 expression, integration of proviral DNA, and production of infectious progeny virus in C8166 T-lymphoid cells blocked in cell division by treatment with either mitomycin, a DNA cross-linker, or aphidicolin, a DNA polymerase alpha inhibitor. The arrest of cell division was confirmed by assay of [3H]thymidine uptake; the nondividing cells remained viable for at least 3 days after treatment. HIV-1 was expressed and replicated equally well in nondividing and dividing C8166 cells, as judged by the comparison of the levels of p24 core antigens in culture supernatants, the proportion of cells expressing HIV-1 specific antigens, the pattern and quantity of HIV-1 DNA present in the extrachromosomal and total cellular DNA fractions, and the biological activity of progeny viruses. A polymerase chain reaction-based viral DNA integration assay indicated that HIV-1 provirus was integrated in C8166 cells treated with either of the two inhibitors of cell division. Similar results were obtained by using growth-arrested Jurkat T-lymphoid cells. We conclude that cell division and cellular DNA synthesis are not required for efficient HIV-1 expression in T cells.