EAGER: Viral tagging: Combining flow cytometry and genomics to explore virus-host interactions

EAGER：病毒标签：结合流式细胞术和基因组学探索病毒与宿主的相互作用

• 批准号: 0940390
• 负责人: Matthew Sullivan
• 金额: $ 20.68万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0940390&HistoricalAwards=false
• 关键词: EAGER; Viral; tagging; Combining; flow

The investigator will develop a high-throughput viral-tagging method to investigate virus-host interactions in natural marine communities. In this approach, cultured or wild viruses are tagged by fluorescent labeling of their nucleic acid and allowed to adsorb to cells, thereby labeling the cells for differential sorting of viral-tagged cells by flow cytometry. Preliminary experiments using this method to detect, sort, and identify ocean microbes with attached viruses (i.e., viral-tagged cells) have been accurate and specific, showing distinct fluorescent shifts for viral-tagged target host cell populations and greatly reduced tagging of non-host cells. This project will develop and rigorously evaluate this viral-tagging method for use with wild populations of viruses and microbes. First, additional systematic characterization of viral-tagging methodology with cultured virus-host pairs will be needed before application to wild populations. The investigator will use representatives of the three major types of marine DNA viruses with ecologically important host cell lineages to evaluate the effects of variable cell physiological states, experimental conditions, and host specificity on viral-tagging efficiency and signal. In addition, cryopreservation is critical for use of this method at remote sites. The investigator will test various cryopreservation techniques for impact on viral-tagging signatures and on downstream molecular biology. Parallel challenges face single-cell genomic researchers, making intellectual cross-fertilization desirable and likely. Finally, in experiments using cultured hosts as "hooks" to capture wild viral diversity, one must enrich for viral DNA after sorting viral-tagged cells. Based on stable isotope probing protocols, the investigator will test using cultured "bait" microbes grown on a 13C-labeled substrate to create heavy host DNA, thus allowing density-gradient separation of host from viral DNA. Light viral DNA is then linker-mediated amplified and pyrosequenced to reveal the host-specific viral metagenome. Broader impacts: An experimentally validated viral-tagging method offers to alleviate three bottlenecks in understanding virus-microbe interactions. Specifically, the investigator anticipates that the method would allow a researcher to perform high-throughput experiments that could (1) map the in situ host range of a virus, (2) define the natural range of viruses that infect a given host, and (3) enable targeted metagenomic sequencing to further uncover the myriad ways that viruses impact microbial metabolism in the wild. Such advances would be useful across fields of viral and microbial ecology, as well as to biogeochemists and ecological modelers seeking population-scale datasets. A post-doctoral researcher will receive significant methodological training in advanced flow cytometry and molecular biological methods, as well as the opportunity to gain experience in grant and paper writing, teaching and cross-discipline and cross-cultural collaboration through collaboration with a Fulbright scholar and a systems administrator to enable high throughput data analysis pipelines to be established.

研究人员将开发一种高通量病毒标记方法，以研究天然海洋社区中的病毒宿主相互作用。在这种方法中，培养或野生病毒被其核酸的荧光标记标记，并允许吸附到细胞上，从而通过流式细胞仪标记细胞以差异为病毒标记的细胞分类。使用此方法进行初步实验来检测，分类和识别具有附着病毒的海洋微生物（即病毒标记的细胞）是准确且具有特异性的，显示了病毒标记的靶宿主细胞群体的明显荧光移动，并大大降低了非宿主细胞的标记。该项目将开发并严格评估这种病毒式标记方法，用于与病毒和微生物的野生种群一起使用。首先，在应用野生种群之前，需要将病毒式方法与培养病毒宿主对的额外系统表征。研究者将使用具有生态重要的宿主细胞谱系的三种主要类型的海洋DNA病毒的代表来评估可变细胞生理状态，实验条件以及宿主特异性对病毒tag效效率和信号的影响。此外，冷冻保存对于在远程站点使用此方法至关重要。研究者将测试各种冷冻保存技术，以影响病毒标记和下游分子生物学。平行挑战面对单细胞基因组研究人员，使智力交叉剥夺是理想的和可能的。最后，在使用培养的宿主作为捕获野生病毒多样性的“钩子”的实验中，在对病毒标记的细胞进行分类后，必须富集病毒DNA。基于稳定的同位素探测方案，研究者将使用在13C标记的底物上生长的培养的“诱饵”微生物来测试，从而产生重宿主DNA，从而允许宿主与病毒DNA的密度分离分离。然后将接头介导的放大并进行焦磷酸化以揭示宿主特异性病毒元基因组。更广泛的影响：一种经过实验验证的病毒标记方法提供了减轻三种瓶颈了解病毒 - 微生物相互作用的瓶颈。 Specifically, the investigator anticipates that the method would allow a researcher to perform high-throughput experiments that could (1) map the in situ host range of a virus, (2) define the natural range of viruses that infect a given host, and (3) enable targeted metagenomic sequencing to further uncover the myriad ways that viruses impact microbial metabolism in the wild.这种进步将在病毒和微生物生态学领域以及寻求人口规模数据集的生物地球运主义者和生态建模者方面有用。博士后研究人员将接受高级流式细胞仪和分子生物学方法的重大方法学培训，以及通过与富布赖特学者和系统管理员的合作来建立高通量数据分析管道，从而获得赠款和纸质写作，教学和跨文化合作的机会。