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Single virus sorting: a revolution in flow cytometry

“Sorting individual viral particles makes it possible to identify and sequence the genomes of viruses one by one,” states Òscar Fornàs, head of the Flow Cytometry Unit of Pompeu Fabra University and the Centre for Genomic Regulation. The goal behind this new application is to be able to know any type of virus present on Earth. In fact, as Fornàs tells Biocores, “we estimate that we only know 1% of the viruses that exist on the planet.”

At present, viruses are considered to be the most abundant biological entity on the face of the Earth. Without a doubt, they present the greatest biodiversity, and at the same time, a public health hazard in the cases of pathogenic viruses that threaten human health, like Zika, currently, or Ebola in the recent past. 

Figure 1.- Ebola virus. Source: CDC Global (Wikimedia)


While amplification and sequencing of microorganisms’ genomes has completely changed our understanding of microbial ecology or medicine, the same cannot be said for virology. According to the team of Shannon J. Williamson at the Craig J. Venter Institute, in an article published in the journal PLOS One, “viral genomics continues to face many difficulties related with isolation and characterization of viruses that we have not yet been able to culture.”

To overcome this challenge, Williamson’s group presented in that study a new method based on flow cytometry. With it, they intended to isolate and sequence the complete genome of individual viral particles. Thanks to the technique, researchers were able to characterize two known viruses: the lambda and T4 bacteriophages of Escherichia coli, isolated using flow cytometry and immobilized in an agarose matrix. Their results, as they stated in 2011, could open new pathways in the study of virus biodiversity, as well as their evolution, adaptation and ecology. 

Figure 2.- CRG/UPF flow cytometry team. Source: Centre for Genomic Regulation.

Five years on, how has flow cytometry changed our knowledge of viruses? In the words of Fornàs, the technique “is an extremely powerful tool for individual particle analysis. The main advantage is that we are down to the level of nanoparticles, smaller in size than cells”. Using the flow cytometer makes it possible to deploy a key asset: the capability to identify and separate viral particles one by one, to later study and characterize them.

Over the last five years we’ve made significant advances in this field. One of the most interesting results of flow cytometry is virus sorting, described just a few months ago in Nature Communications. The study, by Raphaël Gaudin and Natasha S. Barteneva, at the Harvard Medical School, managed to classify viruses based on their infectivity profile. “The nature and concentration of lipids and proteins on the viral surface are essential parameters to determine their infective capacity,” according to the scientists. This type of analysis is usually done by examining clusters of viral particles, mainly because individual viruses could not be isolated and characterized individually.


Figure 3.- Arenavirus. Source: CDC / C. S. Goldsmith (Wikimedia)

The work published in Nature Communications showed that flow cytometry is a worthy tool with which to analyze the properties of the Junin virus, a kind of arenavirus involved in Argentine hemorrhagic fever. This was done by isolating and identifying the viral particles individually, to later characterize the relationship between infectivity, viral size and their RNA content, discover their differences and establish different viral profiles.

“We are stretching the technique to levels we never imagined when it was introduced,” says Fornás. Flow cytometry applied to virus sorting has reached resolution well below the theoretical limits of sensitivity. “This opens new doors”, says the UPF-CRG specialist, “that will change our knowledge on viral particles and their impact on ecology or medicine, to mention just two of the most immediate applications.”