An international research project published in Nature Communications some months ago heralded the discovery of 44 new viruses existing on the surface of the Mediterranean Sea, and in the depths of the Atlantic Ocean. This landmark finding was made possible by the use of a groundbreaking technique called single virus genomics (SVG) the application of which—according to the authors at that time—could help detect emergent pathogenic viruses or identify the viruses present in the human body.
That prediction is now coming true. A team of scientists from the University of Alicante, the Centre for Genomic Regulation and Pompeu Fabra University have used single virus genomics to analyze the saliva samples of 15 volunteers. Their results, published in the journal Viruses have enabled the isolation of 1,328 viruses and the sequencing of the genome of 200. A total of eight viruses were completely unknown up to now, a demonstration of the viral diversity naturally found in saliva.
Isolating and individually sequencing viruses
One of the major challenges facing metagenomics is to accurately read complete viral genomes. Normally, researchers analyze samples from widely diverse viral populations, and with unequal abundance, achieving coverages below 2–5×. Single virus genomics is a tool that is complementary to this methodology, that untangles the genetic complexity of microbial communities, individually sequencing the genome of the viruses once each viral particle has been isolated.
Scientists use a technique known as single virus sorting to perform the separation. It is based on flow cytometry, and gives them the ability to isolate and later sequence the genome of individual particles. This strategy, already successfully tested to analyze the viruses present in marine communities, has been applied to discover the diversity of the human saliva virome. “The oral cavity represents an excellent model to apply SVGs, since it is one of the most densely-populated habitats of the human body, with ≈108 virus-like particles (VLPs) per mL of saliva,” state the authors.
The team, led by microbial ecology specialist Manuel Martínez García and Oscar Fornas of the CRG/UPF FACS Unit, took five milliliters of saliva from 15 volunteers with good oral hygiene, which were later filtered to eliminate any remnants of bacteria and eukaryotic cells. The group used epi-fluorescence microscopy to confirm the presence of viral particles. The samples were processed using viromics methodology; three of them were also analyzed using single virus genomics. After sequencing and analyzing their genome with Illumina technology, they discovered eight new, previously unidentified, viruses.
According to the authors, “our study also highlights that SVGs pipeline optimization is likely necessary for particular environments. Saliva, unlike other types of samples such as seawater, is chemically and biologically more complex, with a higher amount of extracellular genetic material.” Single virus genomics methodology can be applied to the study of any liquid sample, which will help—as already indicated by other researchers in PLoS One—to analyze viral biodiversity and other evolutionary, adaptive and ecological aspects that were once difficult to examine.