Quantification of virus particles
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| Virus particle abundance |
|---|
| Approach: epifluorescence microscopy, flow cytometry, plaque assay, or MPN |
| Context: in situ, incubation, lab |
| Spatial scale: point sample |
| Temporal scale: hours to days (changes in abundance) |
| Units: particles L-1; particles cell-1 (burst size) |
| Community captured: bulk viral community |
| Co-measurements: host cell abundance |
Method Overview
Virus-like particle (VLP) concentrations in seawater are determined by one of several approaches: (1) Epifluorescence microscopy after staining with SYBR Green I or DAPI: filtered samples on anodisc or polycarbonate membranes are stained with a nucleic-acid-specific fluorescent dye and counted under the microscope. (2) Flow cytometry: samples are stained with SYBR Green I and run on a flow cytometer; VLPs are resolved as a distinct population based on their green fluorescence and side scatter[1]. (3) Plaque assays: serial dilutions of a water sample are applied to a lawn of susceptible host cells and plaques (clearings) are counted. (4) Most probable number (MPN) methods: serial dilution and host infection in multiwell plates.
Scale of measurement
Each measurement gives a snapshot of viral abundance at a single time point. Time-series measurements reveal viral production and decay rates.
Data generated
Virus-like particle concentration (VLPs L-1); virus-to-bacterium ratio (VBR). Changes in VLP concentration in dilution incubations can estimate viral production rates.
Units & currency
Units are particles L-1. The currency is virus particles.
Sample size
Typical samples are < 1 L in volume.
Repositories & databases
Limitations
All particle-counting methods quantify VLPs (virus-like particles) without distinguishing infective from non-infective viruses. Plaque and MPN assays only count viruses infective to the specific host cell line used, missing the majority of marine viral diversity. SYBR Green staining does not distinguish viruses from other small particles with nucleic acid. The assumption that all VLPs are bacteriophage or eukaryotic viruses (vs. gene transfer agents, membrane vesicles) introduces uncertainty.
Example Applications & Protocols
Classic examples
- Marie et al. (1999) Enumeration of marine viruses in culture and natural samples by flow cytometry [1]
- Hennes & Suttle (1995) Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy [2]
Recent applications
- Brussaard (2004) Optimisation of procedures for counting viruses by flow cytometry [3]
Common calculations/conversions
- Virus-to-bacterium ratio (VBR) = VLP concentration / bacterial cell concentration; typically 10–100 in marine environments.
- Viral production rate (VP, VLPs L-1 h-1) from dilution experiments: VP = ΔVLP / Δt corrected for virus decay.
References
- ↑ 1.0 1.1 Marie, D., Brussaard, C. P. D., Thyrhaug, R., Bratbak, G., & Vaulot, D. (1999). Enumeration of marine viruses in culture and natural samples by flow cytometry. Applied and Environmental Microbiology, 65(1), 45–52. https://doi.org/10.1128/aem.65.1.45-52.1999
- ↑ Hennes, K. P., & Suttle, C. A. (1995). Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy. Limnology and Oceanography, 40(6), 1050–1055. https://doi.org/10.4319/lo.1995.40.6.1050
- ↑ Brussaard, C. P. D. (2004). Optimisation of procedures for counting viruses by flow cytometry. Applied and Environmental Microbiology, 70(3), 1506–1513. https://doi.org/10.1128/aem.70.3.1506-1513.2004