Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses - PubMed (original) (raw)

. 2013 Aug;15(8):2306-18.

doi: 10.1111/1462-2920.12100. Epub 2013 Mar 14.

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Free PMC article

Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses

Elke Allers et al. Environ Microbiol. 2013 Aug.

Free PMC article

Abstract

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.

© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

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Figures

Figure 1

Figure 1

One-step growth infection dynamics of a marine virus–host system.A. Host abundance as total cell counts and extracellular virus abundance as virus abundance per cell by gene presence (qPCR) and plaque assays (PFU).B. Relative abundance of phage containing cells as FVIC by TEM count (black squares) and phageFISH count (coloured circles); pie charts indicate area size distribution among phage signals as determined by phageFISH.C. One-step growth model for phage PSA-HP1 and its host H100.Error bars indicate standard deviation except for FVIC data in which error bars indicate 95% confidence intervals. All data are based on measurements from two biological replicates. All data at T-19 are corrected by a 1/100 factor to be comparable to values measured after dilution of cultures.

Figure 2

Figure 2

Progression of infection over time: epifluorescence micrographs of virus-infected and uninfected host cells after phageFISH.A: T0–T51, B: T66–T96, C: T111–T46.Left: host only. Centre: virus only. Right column: overlay of host cells in green (Alexa488) and virus in red (Alexa594).Colour designation for arrows: white = cell lysis and release of free phages, cyan = new infection, blue = rRNA and phage localization, yellow = advanced infection, violet = infection by two phage. The scale bar indicates 5 μm.

Figure 3

Figure 3

Epifluorescence micrograph of free virus after phageFISH (red, Alexa594) and SYBR staining (green).On the left: probe targeting a viral gene, on the right: negative control gene probe.Top: overlay of SYBR Green and phageFISH, centre: phageFISH only, bottom: SYBR Green only. Scale bar indicates 2 μm.

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