Emerging Technologies Applied to the Discovery of Human Tumor Viruses

应用于发现人类肿瘤病毒的新兴技术

• 批准号: 7627315
• 负责人: PATRICK S. MOORE
• 金额: $ 15.59万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7627315
• 关键词: Biological Assay; Case-Control Studies; Cell Line; Cells; Computer Simulation; Conjunctival Squamous Cell Carcinoma; Development; Discrimination; Drug or chemical Tissue Distribution; Emerging Technologies; Expression Library; Family; Funding; Gene Expression Profile; Generations; Genetic Polymorphism; Human; Human Herpesvirus 8; Immune; Immunologic Deficiency Syndromes; Infection; Infectious Agent; Joints; Malignant Neoplasms; Methods; Oncogenic Viruses; Pathologic; Performance; Phase; Pilot Projects; Probability; RNA; RNA Degradation; Sampling; Sequence Determination; Silicon Dioxide; Specimen; Techniques; Technology; Testing; Tissues; Transcript; Tumor Cell Line; Tumor Tissue; Uganda; Validation; Viral; Viral Genome; Virus; Walking; base; case control; digital; pathogen; quality assurance; serial analysis of gene expression; tumor

DESCRIPTION (provided by applicant): Infection contributes to ~20% of human cancers worldwide. The list of known carcinogenic infectious agents, however, is surprisingly short and no new human tumor viruses have been discovered over the past decade. Current tumor virus discovery methods are not comprehensive and are likely to miss discovery of new families of agents. Negative studies using these techniques do not rule out the presence of a tumor virus and may miss a previously unknown agent. We propose modifying long Serial Analysis of Gene Expression (SAGE) as an unbiased means for virus discovery using in silica digital transcript subtraction (DTS). We show here a practical method to perform transcriptome-wide in silico subtraction of short transcript tags, allowing discrimination between human and nonhuman sequences. Once a candidate tumor virus sequence is found, it can be used as a start point for viral genome walking and characterization. To demonstrate the feasibility of this method, we performed pilot studies of DTS on a tumor cell line infected with latent KSHV virus. DTS rapidly and uniquely identified 5 KSHV transcripts de novo comprising 0.44% of the total cell transcriptome. The technique was quantitatively reproduced by spiking KSHV-infected cell line RNA into uninfected human tumor tissue RNA. We also identified practical cut-off levels that distinguish most human polymorphisms from viral SAGE tags using DTS. Finally, we performed pilot DTS on 3 squamous cell conjunctival carcinoma (SCCC) tumors, an immunodeficiency-related malignancy. In silico subtraction of 108,000 SAGE tags generated 46 candidate sequences, including 12 high probability tags that are being evaluated as possible SCCC agent sequences. We show that this technique is surprisingly immune to RNA degradation so that it can be used on rare or archival materials in which partial RNA degradation has occurred. We seek phase II R33 funding to perform DTS on 4 SCCC samples, extending our pilot studies into a full analysis of SCCC at the 5-10 transcripts per million level. This will allow us to either identify or exclude a likely tumor virus causing this immunodeficiency-related cancer. This also completes development of DTS technology, allowing us to fully optimize its performance for application to other suspected infectious tumors ASSESSMENT:

描述（由申请人提供）：感染在全球范围内约有20％的人类癌症。然而，已知的致癌传染剂的清单令人惊讶地短，并且在过去十年中没有发现新的人类肿瘤病毒。当前的肿瘤病毒发现方法并不全面，很可能会错过新的特工家族的发现。使用这些技术的负研究不排除肿瘤病毒的存在，可能会错过以前未知的药物。我们提出了对基因表达（SAGE）的长期序列分析，作为在二氧化硅数字转录本中使用（DTS）中发现病毒发现的一种无偏见的手段。我们在这里展示了一种实用方法，可以在短转录标签的硅基减法中执行整个转录组，从而歧视人类和非人类序列。一旦发现了候选肿瘤病毒序列，就可以用作病毒基因组步行和表征的起点。为了证明这种方法的可行性，我们对感染潜在KSHV病毒的肿瘤细胞系进行了DTS的试点研究。 DT迅速且独特地鉴定出5 kSHV转录本，占总细胞转录组的0.44％。该技术通过将KSHV感染的细胞系RNA刺激到未感染的人肿瘤组织RNA中进行了定量再现。我们还确定了使用DTS将大多数人类多态性与病毒鼠尾草标签区分开的实际截止水平。最后，我们在3个鳞状细胞结膜癌（SCCC）肿瘤上进行了试点DTS，这是一种与免疫缺陷相关的恶性肿瘤。在108,000个鼠尾草标签的计算机减法中，产生了46个候选序列，其中包括12个高概率标签，这些标签正在评估为可能的SCCC代理序列。我们表明，该技术令人惊讶地免疫RNA降解，因此可以在发生部分RNA降解的稀有或档案材料上使用。我们寻求II期R33资金以对4个SCCC样品进行DTS，将我们的试点研究扩展到对SCCC的完整分析，以每百万级的5-10个成绩单。这将使我们能够识别或排除可能导致这种免疫缺陷相关癌症的肿瘤病毒。这也完成了DTS技术的开发，使我们能够充分优化其在其他涉嫌传染性肿瘤中的应用 评估：