Diversity and ecology of marine RNA viruses

病毒是海洋食物网的一个组成部分，造成几乎每一种海洋生物的疾病和死亡，但海洋病毒组合的多样性仍然很难确定。这对于含有RNA的病毒尤其如此。有几个报告分离的RNA病毒感染海洋原生生物，但培养独立的调查RNA病毒的多样性仍然非常有限。先前在沿海不列颠哥伦比亚省和沿海俄勒冈州进行的研究表明，RNA病毒在温带和亚热带沃茨中具有多样性和持久性。这些分子调查清楚地表明，少数可用的海洋RNA病毒分离株只是冰山一角;随着每一个新样本的分析，新的RNA病毒仍在被发现。许多这些新的基因序列似乎来自海洋原生生物的病毒，它们的高度多样性表明，病毒感染是一个持久的力量塑造原生生物群落组成的海洋。量化这些病毒的丰度、多样性和动态是将它们纳入海洋生态的重要第一步。因此，这项提议的直接目标集中在回答一些重要的一级生态学问题上，即：1）RNA病毒对总病毒库有显著的数量贡献吗？2)在什么样的空间和时间尺度上，海洋RNA病毒群落的多样性和具体组成在这个生态系统中的变化？3)RNA病毒的衰变率和产生率是多少？它们与含有DNA的病毒相比如何？研究人员将估计绝对和相对丰度的RNA病毒，使用落射荧光和电子显微镜在平行的纯化和分馏的病毒组合从海水中收获的RNA和DNA检测，我们将记录的基因组多样性的RNA病毒在亚热带环境中使用宏基因组学方法。专门设计用于处理宏基因组数据的新生物信息学工具和方法也将在该项目中进行测试和实施。研究人员将采用DGGE指纹图谱和RdRP基因测序来确定群落组成在什么尺度上（米到公里，小时到年）变化，以及这种变化如何与测量的环境梯度相关。他们还将通过qRT PCR量化单个RNA病毒的空间分布和时间动态。抽样方案将解决有关规模的变化，在社区组成。这是在微生物多样性调查中经常被忽视的一个基本问题。最后，研究人员将尝试对不同浮游生物大小组分中RNA病毒相对于含DNA病毒的衰变和产生速率进行初步估计。这些估计将为估计RNA病毒对浮游生物死亡率的贡献提供基础。预计该项目的成果将对包括微生物海洋学家、进化生物学家、病毒学家和原生生物生态学家在内的一系列学科的研究人员产生兴趣和价值。 这项研究将提供关于一组研究不足的病原体动态的第一个信息，这些病原体可能对浮游食物网的结构和功能产生重大影响。该项目几乎肯定会极大地扩大RNA病毒基因组多样性的已知范围，将为它们的进化，生态学和生物学提供新的见解，并将有助于开发新的生物信息学工具，广泛适用于宏基因组学。PI实验室定期参加夏威夷大学海洋与科学技术学院举办的两年一度的开放日，两天内吸引了数千名游客，其中包括来自50多所奥陶纪学校的K12学校团体。这个机会是用来教育学生和教师的存在和海洋微生物的重要性，并激发学生对科学研究的兴趣。该项目的PI和博士后也隶属于最近成立的科学和技术中心，称为微生物海洋学研究和教育中心（CMORE）。作为CMORE的成员，我们正在与一个教育和外展团队合作，帮助开发和实施各种外展和教育活动。一个关键的教育目标是与CMORE的教育办公室合作，增加代表性不足的群体，特别是夏威夷土著人和太平洋岛民（NHPI）的多样性。C MORE已经与（1）Ka Imi Ike（“求知者”）建立了强大的合作伙伴关系，旨在招募和留住NHPI的本科生进入海洋和地球科学，以及（2）夏威夷实习计划（HIP），这是一个为期10周的暑期计划，通过密切的指导，让NHPI的本科生和应届毕业生参与动手科学研究。PI和博士后已经通过HIP计划在2007年夏天成功地在他们的实验室接待了一名NHPI学生。这个学生计划在今年秋天继续她在病毒多样性方面的工作，我们预计她的暑期项目的结果将在同行评审的期刊上发表。我们计划在未来几年参与HIP和Ka Imi Ike计划，本提案中预算用于本科研究的资金将用于帮助支持通过这些计划确定的学生的研究。

Viruses are an integral component of the marine food web contributing to the disease and mortality of essentially every type of marine life, yet the diversity of marine viral assemblages remains very poorly characterized. This is especially true of the RNA containing viruses. There are several reports of isolations of RNA containing viruses that infect marine protists, but cultivation-independent surveys of RNA viral diversity are still very limited. Previous studies in coastal British Columbia and in coastal Oahu have shown RNA viruses are diverse and persistent in both temperate and subtropical waters. These molecular surveys have made it clear that the few available isolates of marine RNA viruses are just the tip of the iceberg; novel RNA viruses are still being discovered with each new sample analyzed. Many of these novel gene sequences appear to derive from viruses of marine protists and their high diversity suggests that viral infections are a persistent force shaping protistan community composition in the sea. Quantifying the abundance, diversity, and dynamics of these viruses is an important first step toward incorporating them into the ecology of the sea. The immediate goals with this proposal, therefore, are focused on answering some important first order ecological questions, namely, 1) Do RNA viruses make a significant numerical contribution to the total virus pool?, 2) On what spatial and temporal scales does the diversity and specific composition of the marine RNA virus community in this ecosystem vary?, 3) What are the decay and production rates of RNA viruses and how do they compare to those of DNA containing viruses?The investigators will estimate the absolute and relative abundance of RNA viruses using epifluorescence and electron microscopy in parallel with RNA and DNA assays of purified and fractionated viral assemblages harvested from seawater and we will document the genomic diversity of RNA viruses in a subtropical environment using a metagenomics approach. New bioinformatic tools and approaches specifically designed to handle metagenomic data will also be tested and implemented in this project. The investigators will employ DGGE fingerprinting and sequencing of the RdRP gene to determine on what scales (meters to kilometers, hours to years) the community composition varies and how this variability relates to measured environmental gradients. They will also quantify the spatial distribution and temporal dynamics of individual phylotypes of RNA viruses by qRT PCR. The sampling program will resolve the relevant scales of variability in community composition. This is a fundamental issue that is frequently ignored in investigations of microbial diversity. Finally, the investigators will attempt to make the first estimates of decay and production rates of RNA viruses relative to DNA containing viruses in different plankton size fractions. These estimates will provide a basis for estimating the contribution of RNA viruses to mortality among the plankton.It is expected that the output from this project will be of interest and value to researchers in a range of disciplines including microbial oceanographers, evolutionary biologists, virologists, and protistan ecologists. This study will provide the first information on the dynamics of a poorly studied group of pathogens, which are likely to have significant influence on structure and function of the planktonic food web. The project is almost certain to dramatically expand the known range of genomic diversity among RNA viruses, will provide new insights into their evolution, ecology, and biology, and will contribute to the development of new bioinformatics tools of broad applicability for metagenomics.The PIs lab participates routinely in the bi-annual open house held by the School of Ocean and Science and Technology at the University of Hawaii, which draws thousands of visitors over two days including K12 school groups from over 50 Oahu schools. This opportunity is used to educate students and teachers about the presence and importance of microbes in the sea and to motivate student interest in scientific research. The PI and post-doc on this project are also affiliated with a recently established Science and Technology Center known as the Center for Microbial Oceanography - Research and Education (CMORE). As members of CMORE, we are working with an education and outreach team to help develop and implement a variety of outreach and education activities. A key educational goal is to work with CMORE's education office to increase diversity among under represented groups, particularly Native Hawaiians and Pacific Islanders (NHPI). C MORE has already established a strong, working partnership with (1) Ka Imi Ike ("Seekers of Knowledge"), aimed at recruiting and retaining NHPI undergraduates into the ocean and earth sciences, and (2) the Hawaiian Internship Program (HIP), a 10-week summer program that engages NHPI undergraduates and recent graduates in hands-on scientific research through close mentoring. The PI and post-doc have already hosted one NHPI student in their lab through the HIP program for the summer of 2007 with great success. This student plans to continue her work on viral diversity this fall and we anticipate that the results from her summer project will lead to a publication in a peer reviewed journal. We plan to participate in the HIP and Ka Imi Ike programs in future years and funds budgeted for undergraduate research in this proposal will be used to help support research by students identified through these programs.