Flow cytometry is a powerful tool for the study of cells and their components. Virus particles were, until recently, believed to be too small to be detectable with flow cytometry, but recent developments have made it possible to detect and characterize viruses with higher success.
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The problem of detecting viruses with flow cytometry
Most flow cytometers, the machines used for flow cytometry, have resolution limits of 300 to 500 nm. Virus particles range in sizes from around 17 nm at the smallest to 350 nm at the larger, enveloped vaccinia viruses.
The issue with detecting viruses with flow cytometers has not been that the viruses are undetectable, but rather that the signals from the viruses get lost in the background noise. The range of most viruses is the same as that of the background noise of optical, electrical, and filtered sheath buffer.
The problems associated with flow cytometry on virus particles, which is also referred to as flow virometry, has been overcome by developments in labeling techniques. New fluorescent technology allows for the labeling of viral particles, and newly available generations of dyes and instrumentation make the analyses clearer.
How flow virometry works
Initial flow virometry focused on larger viruses and viral parts, such as poxvirus and T4 phages. Recently, the repertoire of viruses exposed to flow cytometry has come to include lambda phage, herpes simplex virus 1 (HSV-1), mouse hepatitis virus (MHV), human immunodeficiency virus (HIV), dengue virus, and vaccinia virus, among others.
In normal flow cytometers, detectors of forward light scatter are used to determine sample size and differentiate between several small and one large particles. Whereas traditional flow cytometers capture light that is emitted at an 0.5° to 15°, virus detection by flow cytometry uses reduced wide-angle forward light scatter to detect light in the 15° to 70° range. This enables them to block out the light in the 0° to 15° to reduce noise.
Similarly, the power of the lasers and the sensitivity of the detectors in traditional flow cytometry is more optimized towards larger, full cells rather than viruses and viral envelopes. Flow cytometers for the detection of viruses overcome this problem by using lasers with more than 10 times the wattage levels as that of standard instruments (300 mW compared to 10-20 mW).
As for detectors, photomultiplier tubes are generally more sensitive and better equipped for virus detection than photodiode detectors (except for avalanche photodiodes).
Photodetectors can also be a factor in the effectiveness of flow virometry. More modern flow cytometers tend to use high-performance photomultiplier tubes rather than photodiodes due to higher sensitivity. As a more virus-specific optimization, many flow virometers have reduced filter pore size.
The standard pore size for filtering the sheath buffer is 0.22 μm, whereas smaller pore sizes of 0.1 μm can reduce background signals from impurities and make it easier to detect viruses.
Image Credit: https://jvi.asm.org/content/92/3/e01765-17
The uses of flow virometry
As the methods have improved, the number of applications of flow virometry has also grown. One of the earliest applications, and still a dominant one, of flow virometry, is single virus genomics. This has proven to be an incredible advantage because it removes the need for cultivation before the characterization of novel viruses.
Understanding virus replication is a key part of understanding their infectiousness. Flow viromics has proven useful here, too. Studies on herpes simplex virus 1 (HSV-1) nuclear capsids, which are produced during infections, have shown that flow viromics can achieve greater purity of C-capsids than standard biochemical approaches. This is particularly important because it is the C-capsids, and not other capsids, that incorporate the viral genome and mature into virions.
Other applications of flow cytometry on viruses include discriminating different viruses, developing neutralizing antibodies, nuclear capsid sorting, surface protein analysis, protein heterogeneity, size markers for liposomes, enumeration, and isolation. From a more clinical perspective, flow virometry has been used for vaccine quality control, viral fitness, virion maturation, envelope conformation, and the impact of hosts on viruses.
One of the more recent hallmark studies on flow virometry was able to use flow cytometry on viruses for several simultaneous purposes. Looking at Junin virus particles, the authors were able to analyze infectivity, virus size, and RNA content.
Furthermore, they could compare those secreted by Vero cells with those secreted by human physiologically relevant macrophages. This meant that not only did they successfully analyze the small lipid vesicles with flow cytometry, but they also managed to do so while keeping the vesicles biologically active.
- Lippé, R., 2017. Flow Virometry: a Powerful Tool To Functionally Characterize Viruses. Journal of Virology, 92(3).
- Brussaard, C., Marie, D., and Bratbak, G., 2000. Flow cytometric detection of viruses. Journal of Virological Methods, 85(1-2), pp.175-182.
- Gaudin, R., and Barteneva, N., 2015. Sorting of small infectious virus particles by flow virometry reveals distinct infectivity profiles. Nature Communications, 6(1).