Characterization of Primate Retroviruses

灵长类逆转录病毒的表征

• 批准号: 6433024
• 负责人: RAOUL BENVENISTE
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433024
• 关键词: HIV infections; Macaca; Retroviridae; disease /disorder model; human tissue; nucleic acid sequence; protein sequence; simian AIDSs; simian immunodeficiency virus; virulence; virus antigen; virus genetics; virus infection mechanism; virus protein

Molecular changes in Nef with progression to AIDS . We have compared nef gene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically (E11S) or molecularly cloned SIV/Mne (clone 8). Two samples from each animal obtained early (wk 2-8) or late (wk 21-137) after infection were analyzed. Four substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and a fifth in all but one. Two of the common exchanges are located about 40 residues apart in the Nef core sequence, but are in proximity on the tertiary structure as judged by computer modeling using the structure of the HIV Nef core protein as a guide. Most recurring in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIV/Sm clade. Animals inoculated with virus already containing the "late version" nef gene developed a more aggressive disease. Recombinant virus containing a macaque adapted nef (MA-nef) on the clone 8 backbone was threefold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone8MA-nef contained a large proportion of cells carrying proviruses with defective nef genes. These findings suggest that the nef gene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture which were "corrected" in vivo . Chimeric viruses containing the late nef gene sequence associated with AIDS will be tested for pathogenicity in macaques in vivo . Screening macaques for the presence of type D retroviruses. The SIV macaque model is the preferred nonhuman primate model for AIDS related vaccine, antiviral, and pathogenesis research. A necessary component of this animal model is the availability of macaques that are free of retrovirus infections that might confound pathogenesis and vaccine studies with SIV. Macaques are known to be infected with various retroviruses, including type D viruses (SRV), STLV-I, and foamy (spumaretroviruses). SRV infection is prevalent in wild-caught macaques as well as in colony born animals. Five neutralization types have been identified (SRV1 - SRV5). These infectious type D retroviruses cause an often fatal immunosuppressive disease in macaques that is manifested by progressive weight loss, persistent diarrhea, anemia, opportunistic infections, and unusual neoplasms. Since the experimental infection of macaques with SIV results in similar clinical outcomes, animals used in AIDS experiments must be free of other retrovirus infections. Two species of macaques, rhesus and cynomolgus, have been screened for the presence of type D retroviruses (SRV). A combination of serological, virus isolation and DNA polymerase chain reaction (PCR) assays were used to identify SRV infected macaques. Serological screening for SRV alone is not sufficient because not all infected animals seroconvert. Virus isolation by coculture of peripheral blood mononuclear cells (PBMCs) with various indicator cell lines is time consuming (4 to 8 weeks) and variable due to the presence of antibodies and differing virus titers in infected animals. Several PCR primers were compared for their sensitivity and ability to detect SRV subtypes 1-5 present in PBMC DNA. One primer set has been identified as being the most sensitive for the detection of SRV-1, SRV-2, and Mason Pfizer monkey virus (MPMV, SRV-3). This primer set detected one DNA proviral copy per 50,000 cells and identified the greatest number of SRV positive animals.

NEF的分子变化，进展为AIDS。我们已经比较了通过猕猴的外周血淋巴细胞DNA分离的NEF基因序列，这些NEF链反应已被生物学上（E11S）或分子克隆的SIV/MNE接种（克隆8）。 分析感染后早期获得的每只动物（WK 2-8）或晚期（WK 21-137）的两个样品。 在晚期的所有动物中，在预测的NEF氨基酸序列中进行了四个取代，除一个动物外，所有动物均观察到五分之一。 在NEF核心序列中，有两个常见的交换位于大约40个残基，但使用HIV NEF NEF NEF核心蛋白的结构作为指导来判断的第三纪结构上的距离。 大多数反复出现的体内变化代替了克隆的NEF序列中发现的残基，其中一个存在通过对齐SIV/SM进化片的NEF序列来得出的共识中。接种已接种已经包含“晚期” NEF基因的病毒的动物会出现更具侵略性的疾病。 在克隆8主链上含有猕猴适应性NEF（MA-NEF）的重组病毒比原始病毒更感染性Smagi细胞。 感染SIV-CLONE8MA-NEF的淋巴细胞系包含大部分带有有缺陷NEF基因的原病毒的细胞。 这些发现表明，克隆的SIV/MNE的NEF基因在组织培养过程中的传播过程中经历了衰减突变，这些突变在体内“校正”。 含有与艾滋病相关的晚期NEF基因序列的嵌合病毒将在体内猕猴中的致病性测试。筛查猕猴的存在D型逆转录病毒。 SIV猕猴模型是与艾滋病相关疫苗，抗病毒和发病机理研究的首选非人类灵长类动物模型。该动物模型的必要组成部分是猕猴的可用性，这些猕猴没有逆转录病毒感染，这些感染可能将发病机理和疫苗研究与SIV混淆。 猕猴已被各种逆转录病毒感染，包括D型病毒（SRV），STLV-I和泡沫病（spumaretroviruses）。 SRV感染在野生猕猴以及殖民地诞生的动物中普遍存在。 已经确定了五种中和类型（SRV1 -SRV5）。这些感染性的D型逆转录病毒在猕猴中常常导致致命的免疫抑制疾病，这表现为进行性体重减轻，持续性腹泻，贫血，机会性感染和不寻常的肿瘤。 由于具有SIV猕猴的实验感染会导致相似的临床结果，因此在AIDS实验中使用的动物必须没有其他逆转录病毒感染。 已经对D型逆转录病毒（SRV）进行了筛查，两种猕猴，恒河猴和cynomolgus。血清学，病毒分离和DNA聚合酶链反应（PCR）测定的组合用于鉴定被SRV感染的猕猴。 单独使用SRV的血清学筛查是不够的，因为并非所有感染动物血清复合体。 通过各种指标细胞系的外周血单核细胞（PBMC）共培养的病毒分离为耗时（4至8周），并且由于受感染动物的抗体和病毒滴度不同而导致可变。 比较了几种PCR引物的敏感性和检测PBMC DNA中存在的SRV亚型1-5的能力。一个引物集已被确定为检测SRV-1，SRV-2和Mason Pfizer猴子病毒（MPMV，SRV-3）最敏感的。该底漆集检测到每50,000个细胞的一个DNA病毒拷贝，并确定了最大数量的SRV阳性动物。