Detection and Analysis of Mouse Retroviruses in Humans

人类小鼠逆转录病毒的检测和分析

• 批准号: 8349466
• 负责人: Frank Maldarelli
• 金额: $ 25.48万
• 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/8349466
• 关键词: Award; Biological Assay; Blood specimen; CCR; Cells; Characteristics; Chronic; Chronic Fatigue Syndrome; Collaborations; Conflict (Psychology); Controlled Study; DNA; Data; Detection; Development; Disease; Disease Association; Employee; Endogenous Retroviruses; Epidemiology; Fatigue; Gammaretrovirus; Genetic Variation; Genomics; Geographic Distribution; Goals; HIV; HIV Drug Resistance Program; HIV Infections; Health; Human; Immune; Immunology; Individual; Infection; Institutes; Laboratories; Legal patent; Light; Malignant neoplasm of prostate; Measures; Methods; Mitochondrial DNA; Murine leukemia virus; Mus; National Institute of Allergy and Infectious Disease; Nucleic Acids; Pathogenesis; Patients; Performance; Peripheral Blood Mononuclear Cell; Phylogenetic Analysis; Plasma; Population; Population Genetics; Prevalence; Quality Control; RNA; Reporting; Research; Retroviridae; Risk; Role; Safety; Sampling; Sampling Studies; Sequence Analysis; Series; Signal Transduction; Source; System; Techniques; Testing; Time; Tissues; United States; Urologic Oncology; Variant; Vascular blood supply; Viral; Virus; Whole Blood; bench to bedside; design; genome sequencing; human disease; member; mouse genome; novel; nucleic acid detection; particle; population based; prevent; virology

XMRV is a newly discovered member of the gammaretrovirus genus of Retroviruses that has been recently associated with two human disorders, prostate cancer and chronic fatigue syndrome (CFS). Since it was first reported in 2006, XMRV has been intensely investigated, but conflicting data regarding XMRV detection have been reported, and no clear picture of prevalence, geographic distribution, disease association, or role in pathogenesis has emerged. The identification of XMRV in otherwise healthy individuals raises additional concerns regarding the safety of the blood supply. Several serious limitations prevent in-depth understanding of the role of XMRV in human infection and disease, including systems to detect and sequence XMRV nucleic acid. Currently, there is a lack of adequate XMRV nucleic acid detection systems with robust single-copy detection characteristics. In addition, understanding the origin, spread, and pathogenesis of XMRV is hampered by inability to obtain extensive phylogenetic information using standard population-based amplification and sequencing. We have initiated studies to detect and quantify XMRV by using real-time PCR techniques and have developed single-copy assays for analysis of XMRV RNA in plasma and whole blood and for analysis of XMRV DNA in PBMC and tissue-derived material. As a result of the expertise in our Virology Core, we are one of four laboratories in the United States testing panels of XMRV standards and samples. In the first analyses studying a panel of spiked blood samples, the Core assays demonstrated excellent detection characteristics with single-copy sensitivity. The development of XMRV real-time PCR assays incorporated an important design feature that permits distinguishing XMRV from typical endogenous retroviruses. This novel aspect in XMRV detection design affords particular advantages in XMRV detection in humans, as there are new data suggesting that other endogenous viruses may be present in humans as well as the described XMRV. As a consequence of this novel aspect of the assay, we are nowpreparing an Employee Invention Report for possible patent application. In the course of developing these quantification assays, it became clear that control studies are essential to rule out the possibility of contaminating substances generating artifactual signal. In addition to typical adventitious amplification, XMRV detection is particularly sensitive to murine contamination; the mouse genome contains hundreds if not thousands of copies of endogenous retroviruses, representing a substantial load of retrovirus that may be amplified in XMRV assays. To identify potential sources of contamination, we have developed sensitive assays to detect low-level mouse contamination, including single-copy assays for mouse genomic and mitochondrial DNA. In addition, we have also developed a quantitative assay for DNA of intracisternal A type particle (IAP), a virus amplified to a high degree in mouse cells. Taken together, this suite of amplification assays represents a comprehensive approach to detect XMRV in human-derived samples. The Core has also established new single-genome sequencing (SGS) approaches to analyze genetic diversity in the XMRV population. As we have demonstrated previously with HIV, SGS permits detailed analysis of viral population genetics. As XMRV may have substantial geographic distribution, SGS will be particularly useful in analysis of XMRV epidemiology. SGS will also provide a substantial measure of quality control and determine whether identified sequences are the result of laboratory contamination. These new assays will be essential in determining the role of XMRV in disease pathogenesis. We have established useful collaborations with Drs. W. Marston Linehan and Peter Pinto (Urologic Oncology Branch, CCR) to study samples from patients with prostate cancer, and with Drs. Frank Ruscetti and Kathryn Jones (Laboratory of Experimental Immunology, CCR) and Dr. Judy Mikovits (Whittemore Peterson Institute) to study patients with severe CFS. As immune deficiency may contribute to infection by XMRV, we have also established collaborations with NIAID to obtain samples from study individuals with immune deficiency, including HIV-infected patients and patients with both HIV infection and prostate cancer. In addition, we are collaborating with Dr. Vinay Pathak (HIV Drug Resistance Program, CCR) in a study of XMRV pathogenesis and prostate cancer as part of a Bench to Bedside Award to Dr. Pathak. Reports of XMRV infection in individuals with chronic fatigue and in otherwise healthy individuals raised concerns regarding new health risks. Within a year of these reports, we have optimized a series of detection and analytical assays with excellent performance characteristics. In the next year, we will apply these approaches to shed new light on the potential role of mouse-related viruses in human disease.

XMRV是逆转录病毒γ型病毒属的新成员，最近与两种人类疾病有关，即前列腺癌和慢性疲劳综合征（CFS）。 自2006年首次报道以来，已经对XMRV进行了深入研究，但已经报道了有关XMRV检测的数据，并且没有出现清晰的患病率，地理分布，疾病关联或在发病机理中的作用。在其他健康个体中，XMRV的识别引起了有关血液供应安全性的其他问题。几个严重的局限性阻止了对XMRV在人类感染和疾病中的作用的深入了解，包括检测和序列XMRV核酸的系统。当前，缺乏具有鲁棒单拷贝检测特征的足够的XMRV核酸检测系统。此外，了解XMRV的起源，扩散和发病机制受到无法使用基于标准人群的放大和测序获得广泛的系统发育信息的阻碍。 我们已经开始使用实时PCR技术来检测和量化XMRV的研究，并开发了单拷贝测定，用于分析血浆和全血中XMRV RNA，并分析PBMC和组织衍生材料中XMRV DNA。由于我们的病毒学核心方面的专业知识，我们是美国测试XMRV标准和样品的四个实验室之一。在研究一组尖刺的血液样本的第一个分析中，核心测定表现出具有单拷贝灵敏度的出色检测特征。 XMRV实时PCR分析的开发融合了一个重要的设计特征，该特征允许将XMRV与典型的内源性逆转录病毒区分开。 XMRV检测设计中的这一新方面在人类的XMRV检测方面具有特殊的优势，因为有新的数据表明，人类以及所述的XMRV可能存在其他内源性病毒。由于该测定法的这一新颖方面，我们现在正在为可能的专利申请提供一份员工发明报告。 在开发这些定量测定过程的过程中，很明显，控制研究对于排除污染产生人为信号的物质的可能性至关重要。除了典型的不定放大外，XMRV检测对鼠污染特别敏感。小鼠基因组包含数百份甚至数千份的内源性逆转录病毒，代表大量逆转录病毒，可以在XMRV测定中扩增。 为了确定潜在的污染源，我们开发了敏感测定方法以检测低级小鼠污染，包括用于小鼠基因组和线粒体DNA的单拷贝测定。此外，我们还开发了针对肠内A型颗粒（IAP）的DNA的定量测定，该病毒在小鼠细胞中被扩增至高度。综上所述，这套放大测定套件代表了一种检测人类衍生样品中XMRV的综合方法。 该核心还建立了新的单基因组测序（SGS）方法来分析XMRV人群中的遗传多样性。 正如我们先前使用HIV所证明的那样，SGS允许对病毒种群遗传学的详细分析。由于XMRV可能具有大量的地理分布，因此SGS将在XMRV流行病学分析中特别有用。 SGS还将提供对质量控制的实质性衡量，并确定确定的序列是否是实验室污染的结果。 这些新测定对于确定XMRV在疾病发病机理中的作用至关重要。我们已经与Drs建立了有用的合作。 W. Marston Linehan和Peter Pinto（泌尿外科肿瘤科，CCR）研究了来自前列腺癌患者和DRS的样本。 Frank Ruscetti和Kathryn Jones（CCR实验免疫学实验室）和Judy Mikovits博士（Whittemore Peterson Institute）研究了严重CFS患者。由于免疫缺陷可能导致XMRV感染，因此我们还与NIAID建立了合作，以从具有免疫缺乏症的研究个体中获取样本，包括感染HIV的患者和HIV感染和前列腺癌患者。此外，我们正在与Vinay Pathak博士（CCR HIV耐药计划，CCR）合作，作为XMRV发病机理和前列腺癌的研究，这是授予Pathak博士的卧床奖的一部分。 患有慢性疲劳和其他健康个体患者XMRV感染的报告引起了人们对新健康风险的担忧。在这些报告的一年内，我们优化了一系列具有出色性能特征的检测和分析测定。在第二年，我们将采用这些方法来新阐明与小鼠相关病毒在人类疾病中的潜在作用。