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Single virus genomics reveals missed marine viral communities

An international team of scientists has discovered 44 new types of marine virus. The sorting of individual viral particles, and the sequencing of their genomes, facilitated the find, published in the journal Nature Communications. Through the use of these techniques, the researchers, among whom are members of the Centre for Genomic Regulation (CRG), Pompeu Fabra University (UPF), the University of Alicante, the Institute of Marine Sciences (ICM-CSIC) or Miguel Hernández University, claim that it will be possible in the future to find emergent pathogenic viruses, or study the entirety of viruses that populate the human body.

The goal of the study was to identify and characterize the marine viruses naturally found on the surface of the Mediterranean sea, and the depths of the Atlantic Ocean, with the support of the Malaspina expedition. Scientists used virus sorting, a method based on flow cytometry, fluorescence and confocal microscopy to individually separate 2,234 viral particles. Next, the complete amplification of the genome managed to isolate 392 virus genomes, from which researchers randomly selected 44 genomes to be characterized using massive sequencing technology.

Source: Pixabay.

According to the results, for a majority of the viruses, (32 of 44), a single large genome contig was obtained (from ≈10–78 kb, mean ≈20 kb). The later annotation of the genome and the protein analysis revealed that 22 of the 44 viral genomes analyzed belonged to the Caudovirales order and that 37 belonged to species not previously described. The article published in Nature Communications sheds light on new evidence of these techniques’ potential “to uncover the genomics of some of the most abundant marine viruses”, according to the researchers.

Unknown viral diversity

The methods for culturing viral particles in the laboratory are inefficient when it comes to showing all of the diversity existing on the planet. According to a study published in the journal Nature Reviews in Mircobiology there exist more than 1030 viruses in the oceans, responsible for approximately 1023 viral infections per second in marine communities. Nonetheless, many of these particles go unnoticed or are completely unknown to the scientific community, despite advancements in metagenomic techniques.

According to Òscar Fornàs, head of the Flow Cytometry Unit of the Centre for Genomic Regulation and Pompeu Fabra University, “we estimate that we only know 1% of the viruses that exist on the planet,” In recent years, individual genomes of very abundant prokaryotic organisms, of key ecological importance in marine and other ecosystems, have been sequenced thanks to the use of single-cell genomics (SCG). These powerful tools are expanding horizons to overcome the limitations of conventional metagenomic and culture techniques, helping to better understand the biology, ecology and evolution of microbial communities.

One of the first studies to demonstrate the potential of single-cell genomics was published in Science in 2014. The study showed that these technologies were capable of separately assembling individual genomes and offered a more complete analysis of natural genetic variability. SCG had been applied to isolate and study individual particles from a culture of lambda and T4 Escherichia coli bacteriophages, as was shown in a study in PLOS One, and to separate and characterize over 5,000 viruses never cultured until then from a sample taken from the sea, as was published by another team in the ISME Journal.

The study recently published in Nature Communications proves the possibilities of the flow cytometry method to individually identify and separate viral particles, at the nanoparticle level, and far below cell size. Researchers have subsequently been able to study and characterize them through other technologies such as massive sequencing. “It is not only useful to discover new viruses, or see the ecology of large virus groups in the samples studied, it also lays the groundwork to study the different viruses present in a specific ecosystem. Along these lines, the human body is a specific ecosystem, and this is where the future of this project, and possible emerging projects, lies,” states Fornàs, whose group is now working to apply these techniques in the analysis of human saliva.

Among the limitations of this technique, scientists mention the need to have optimal fluorescent staining of viral nucleic acids and highly sensitive equipment. “For instance, the detection of viral particles with very small genomes, in particular those with ssDNA and RNA genomes, is difficult due to the low levels of fluorescence signal per viral particle achieved with commercially available fluorescence dyes”, state the researchers. The possible contamination of the DNA or the difficulties in relating the viruses found with their hosts are two other challenges of this incipient technology. Nevertheless, the conclusions of the article presented in Nature Communications reveal a “huge potential” that could in the future reveal the true extent of viruses’ genetic diversity.