Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses.
Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses.
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DOI：
10.1111/1462-2920.12100
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发表时间：
2013-08
影响因子：
5.1
通讯作者：
Sullivan MB
中科院分区：
文献类型：
--
作者：
Allers E;Moraru C;Duhaime MB;Beneze E;Solonenko N;Barrero-Canosa J;Amann R;Sullivan MB
微生物驱动生物地球化学循环，为地球提供燃料，它们的病毒（噬菌体）改变微生物种群结构、基因组库和代谢能力。然而，我们理解和量化噬菌体-宿主相互作用的能力受到技术限制。在这里，我们介绍了phageFISH -一种显着改进的geneFISH协议，将基因检测效率从40%提高到bb0.92%，并优化了细胞内和细胞外噬菌体DNA的检测和可视化。应用噬菌体fish表征海洋足病毒-伽马变形菌宿主模型系统的感染动力学，证实了经典的指标（qPCR、斑块测定、FVIC、DAPI），并优于大多数方法，揭示了新的生物学特性。PhageFISH检测复制和封装（细胞内和细胞外）噬菌体DNA，同时在感染的所有阶段识别和定量宿主细胞。此外，phageFISH允许对每个细胞的噬菌体DNA进行相对测量，从而实现单细胞感染状态的记录（例如早期和晚期感染）。此外，它区分了两波感染，这是其他测量方法无法根据人口平均信号进行的。总之，这些发现充分表征了一种新型模型噬菌体-宿主系统的感染动力学，并首次将噬菌体fish作为实验室中研究噬菌体-宿主相互作用的急需工具，在环境调查和自由噬菌体的谱系特异性种群生态学方面具有很大的前景。
Microbes drive the biogeochemical cycles that fuel planet Earth, and their viruses (phages) alter microbial population structure, genome repertoire, and metabolic capacity. However, our ability to understand and quantify phage–host interactions is technique-limited. Here, we introduce phageFISH – a markedly improved geneFISH protocol that increases gene detection efficiency from 40% to > 92% and is optimized for detection and visualization of intra- and extracellular phage DNA. The application of phageFISH to characterize infection dynamics in a marine podovirus–gammaproteobacterial host model system corroborated classical metrics (qPCR, plaque assay, FVIC, DAPI) and outperformed most of them to reveal new biology. PhageFISH detected both replicating and encapsidated (intracellular and extracellular) phage DNA, while simultaneously identifying and quantifying host cells during all stages of infection. Additionally, phageFISH allowed per-cell relative measurements of phage DNA, enabling single-cell documentation of infection status (e.g. early vs late stage infections). Further, it discriminated between two waves of infection, which no other measurement could due to population-averaged signals. Together, these findings richly characterize the infection dynamics of a novel model phage–host system, and debut phageFISH as a much-needed tool for studying phage–host interactions in the laboratory, with great promise for environmental surveys and lineage-specific population ecology of free phages.
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影响因子：
4.2
作者：
BORSHEIM, KY
通讯作者：
BORSHEIM, KY
影响因子：
11
作者：
Kenzaka, Takehiko;Tani, Katsuji;Nasu, Masao
通讯作者：
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影响因子：
5.1
作者：
Hellweger, Ferdi L.
通讯作者：
Hellweger, Ferdi L.
影响因子：
4.4
作者：
Holmfeldt, Karin;Middelboe, Mathias;Riemann, Lasse
通讯作者：
Riemann, Lasse
影响因子：
1.4
作者：
Brum, JR;Steward, GF;Jellison, R
通讯作者：
Jellison, R