Many More Viruses Revealed Below the Seafloor

Researchers at JAMSTEC have developed a new method to extract viruses from deep-sea sediments, yielding up to hundreds of times more virus particles than former techniques. The improved method suggests that there are many more viruses below the seafloor than previously detected.

Despite their bad reputation for being agents of disease, viruses play an important ecological role. By bursting open cells and killing off microorganisms, they help cycle nutrients through the environment. Viruses occur wherever there are cells, but it can be tricky to study them, especially in soil and marine sediments, where they stick to the surrounding grains of minerals. 

Now there is a better way to extract viruses from sediments, thanks to research by DCO Deep Life Community members Donald Pan, Yuki Morono, Fumio Inagaki, and Ken Takai (all at Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Japan). Using the new protocol, the researchers extracted hundreds of times more virus particles from a range of subseafloor sediments, compared to existing methods. The findings suggest that we have vastly underestimated the number of viruses in deep sediments and their contribution to subseafloor carbon. The researchers describe the improved method in a new paper in Frontiers in Microbiology [1]. 

fluorescent viruses
This image from under the microscope shows that the new “density separation method” extracts more viruses from subseafloor sediments (right) than conventional techniques (left). Each green dot is a virus tagged with a fluorescent dye. Credit: Pan et al.

When Pan arrived at JAMSTEC for his postdoc in 2016, he was interested in viruses in deep, low-biomass environments where there are few microbial cells to infect. There has been little research in this area because of the difficulty of working with sediment viruses. “Progress in this field has been slow compared to the study of viruses in water, where viruses are plentiful and free floating,” said Pan. 

Previously, Morono had developed a protocol for extracting microbial cells from subseafloor sediments. His method can detect cells from extraordinarily low-biomass sediments, equivalent to finding a few grains of sand in an Olympic-sized swimming pool, so Pan decided to adapt the method for extracting viruses. The protocol involves separating the viruses from sediment through a dense solution of a compound called Nycodenz by spinning in a centrifuge, causing the sediment particles to move through the Nycodenz layer to the bottom of the tube, while the lighter viruses stay suspended up top. Pan could then remove the top layer, stain the viruses with a fluorescent dye, and count them under the microscope.

When Pan extracted and counted viruses from a variety of sediments stored at JAMSTEC and the Kochi Core Center (Japan), he was able to count as much as 350 times more virus particles than reported in previous studies using those sediments. “When I first looked at the results under the microscope, I was shocked,” said Pan. “It was such an enormous improvement over previous results that I thought maybe something was wrong.” 

“We found so many more viruses with the new method compared to former methods,” said Pan. “This suggests that everything we knew about how many viruses there are in the subsurface may have to be reconsidered.” As a result, viruses likely make up a much larger component of the organic carbon in subseafloor environments than scientists had previously realized.

It is still not yet clear what function viruses serve in subseafloor sediments. If they are actively infecting cells, they may free up nutrients for the surviving cells in deep sediments where food is scarce. However with better separation methods, advanced genomic techniques that require substantial amounts of genetic material might become feasible. With traditional virus extraction methods, scientists may need enormous amounts of sediment to yield enough viruses to effectively sequence their genetic material. The new method may make it possible to use smaller amounts of deep sediment to sequence the viruses present, called a viral metagenome, which would enable them to study the viruses that make up the subseafloor “virosphere.” 

Currently, Pan is using the new extraction technique to study viruses from cores collected during International Ocean Discovery Program Expedition 370, T-Limit of the Deep Biosphere off Muroto. During this expedition, the team drilled into the hot Nankai Trough subduction zone off Cape Muroto, Japan, with the goal of finding the upper temperature limit that microbes can withstand. 

“Our newly developed method makes it possible to probe the limits of the deep virosphere and to study viruses in low-biomass sediments that had previously been beyond the limits of detectability,” said Pan. “However, more studies will be needed to elucidate what function they may have in the deep subsurface.”

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