Emerging Technologies Applied to the Discovery of Human Tumor Viruses

描述(申请人提供)：全世界约20%的人类癌症是由感染引起的。然而，已知致癌感染剂的名单出人意料地短，而且在过去十年中没有发现新的人类肿瘤病毒。目前的肿瘤病毒发现方法并不全面，很可能会错过新的药物家族的发现。使用这些技术的阴性研究不排除肿瘤病毒的存在，可能会错过以前未知的病原体。我们建议修改长序列基因表达分析(SAGE)作为一种无偏见的病毒发现方法，将其用于硅胶数字转录本消减(DTS)。我们在这里展示了一种实用的方法，在电子消减短转录标签的整个转录组中执行，允许区分人类和非人类序列。一旦发现一个候选的肿瘤病毒序列，它就可以作为病毒基因组行走和表征的起点。为了证明这种方法的可行性，我们在感染潜伏的KSHV病毒的肿瘤细胞系上进行了DTS的初步研究。DTS快速、唯一地鉴定了5个KSHV转录本，占细胞转录组总数的0.44%。这项技术是通过将KSHV感染的细胞系RNA添加到未感染的人类肿瘤组织RNA中来定量复制的。我们还确定了使用DTS将大多数人类多态与病毒SAGE标签区分开来的实用截止水平。最后，我们对3个鳞状细胞结膜癌(SCCC)肿瘤进行了试验性DTS，这是一种与免疫缺陷相关的恶性肿瘤。在108,000个SAGE标签的电子消减中，产生了46个候选序列，其中包括12个被评估为可能的SCCC试剂序列的高概率标签。我们表明，这项技术出人意料地对RNA降解免疫，因此它可以用于发生部分RNA降解的稀有或档案材料。我们寻求第二阶段R33资金，对4个SCCC样本进行DTS，将我们的初步研究扩展到5-10个转录/百万水平的SCCC全面分析。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性能，以应用于其他可疑的感染性肿瘤 评估：

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: