Metgagenomic Analysis of the Human Oral Viral Microbiome

描述(申请人提供)：鉴于噬菌体已被证明在众多生态系统的微生物生态中扮演的角色，以及人类病毒在导致口腔疾病中所扮演的角色，我们正在测试一种假设，即存在大量不同的病毒(包括噬菌体和人类病毒)存在于人类口腔中，对其基因组序列的研究将揭示病毒在口腔微生物生态(如生物膜形成和疾病)中的作用的新线索。这个由发现驱动的R21项目的目标是使用Sanger/焦磷酸测序混合采样来确定口腔病毒的遗传多样性，作为更全面地评估人类口腔病毒成分基因组能力的先导。对口腔内的病毒基因库进行测序将使我们深入了解噬菌体和人类病毒在这种环境中所扮演的生物学角色。有一个特定的目标：使用元基因组学方法从丝裂霉素C诱导和非诱导的人唾液和牙龈上菌斑样本中了解病毒(包括人类病毒和噬菌体)的多样性。这种方法与以前通过传统的基于培养的程序鉴定口腔噬菌体的尝试有很大的不同。有许多标准的、可靠的方法来估计环境样本中的病毒总数，以及用于分析病毒元基因组数据的新工具，这将使估计多样性和群落结构成为可能。在我们的方案中，我们将使用透射电子显微镜、荧光定量技术和Sanger/焦磷酸测序混合方法来获得病毒DNA序列。对这些数据的分析将为未来彻底检查存在于人类口腔中的病毒的基因组内容及其在人类疾病中的作用提供框架。公共卫生：这项基于发现的R21提案的目标是确定口腔病毒的遗传多样性，作为更全面地检查人类口腔中的病毒基因库的第一步。虽然这项研究并不全面，但我们仍将对口腔病毒的基因内容有价值的洞察，从而更好地理解这些被忽视的载体如何影响体内平衡和疾病。这些数据的未来应用是通过识别和生产噬菌体编码产品来控制细菌病原体。

DESCRIPTION (provided by applicant): Given the roles that bacteriophages have been shown to play in the microbial ecology of numerous ecosystems and the roles that human viruses play in causing oral disease, we are testing the hypothesis that there are a significant number of diverse viruses (both bacteriophage and human viruses) that inhabit the human oral cavity and that examination of their genome sequences will unveil new clues to the role of viruses in the microbial ecology of the mouth (e.g. biofilm formation and disease). The goal of this discovery-driven R21 project is to determine the genetic diversity of oral viruses using Sanger/pyrosequencing hybrid sampling as a precursor to a more comprehensive evaluation of the genomic capacity of the virus component of the human mouth. Sequencing the viral gene pool within the mouth will give insight into the biological role that phage and human viruses play in this environment. There is one specific aim: To use a metagenomic approach to understand viral (both human viruses and bacteriophages) diversity from Mitomycin C-induced and non-induced human saliva and supra- gingival plaque samples. This approach differs significantly from previous attempts to identify oral bacteriophages through traditional culture-based procedures. There are a number of standard, robust methodologies for the estimation of the total numbers of viruses in environmental samples as well as new tools for the analysis of virus metagenomics data that will enable estimation of diversity and community structure. In our proposal, we will use TEM, epifluorescence quantitation techniques and a Sanger/pyrosequencing hybrid approach to obtain virus DNA sequences. Analysis of these data will provide the framework for future thorough examination of the genomic content of the viruses that exist in the human mouth and their role in human disease. Public Health: The goal of this discovery-based R21 proposal is to determine the genetic diversity of oral viruses as the first step to more comprehensive examination of the viral gene pool in the human oral cavity. Though this study is not meant to be comprehensive, we will still gain valuable insight into the gene content of oral viruses, leading to a better understanding of how these overlooked vectors influence homeostasis and disease. A future application of the data is the control of bacterial pathogens through the identification and production of bacteriophage-encoded products.