The new virophages were originally isolated from water from Lake Ontario.Brian Lasenby / Shutterstock

In a single drop of water from Lake Ontario, you can find an abundance of algae. In these algae, scientists in 2015 found a new virus belonging to an enigmatic group called giant viruses. And nested inside these giant viruses, scientists have now found yet more novel viruses—three tiny ones that they have named CpV-PLV Larry, Curly, and Moe.

“I originally named them to see if I can get away with it,” says Joshua Stough, now a postdoctoral research fellow at the University of Michigan. He’s a co-author of a new paper describing and naming the Three Stooges, so, in fact, he has gotten away with it.

All three of these viruses are what are known as virophages, viruses that specialize in infecting other viruses. Virophages were first discovered infecting giant viruses from a water-cooling tower in 2008. Since then, scientists have isolated only a handful more—all from giant viruses that infect microscopic organisms such as algae or amoebas. It’s a virus inside a virus inside a cell. “They’re like the Russian doll,” says Curtis Suttle, a virologist at the University of British Columbia who was not involved with the new study.

Giant viruses themselves are strange and poorly understood. Viruses are traditionally considered not to be alive, because they are unable to reproduce on their own like bacteria or multicellular organisms. But giant viruses seem to blur the line between alive and dead—some are almost as big as bacteria, and their genomes sometimes have many of the genes necessary for replication. What’s more, bacteria can be infected by specialized viruses called bacteriophages. Giant viruses can also be infected by specialized viruses, which are of course called virophages. In some ways, giant viruses seem to have more in common with living bacteria than with a simple influenza virus.

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Stough’s colleagues managed to sequence DNA from the giant virus found in Lake Ontario, which was given the the far less colorful name CpV-BQ2. As far as Stough knew, they were sequencing pure DNA from CpV-BQ2 viruses. But an oddity in the data made him realize that he was also looking at three completely new virophages. In particular, they had similarities to a virophage that parasitizes another giant virus, called CroV.

Suttle had unknowingly isolated CroV at least as far back as 1995. It would be another decade before other scientists realized that giant viruses even existed, and his lab spent years trying to study CroV. Over the years, he would start a student on the project. They would defrost a sample from storage. The CroV would grow and then mysteriously stop growing. “A couple years later, someone would come along to try again,” Suttle says. It wasn’t until DNA-sequencing technology improved and Matthias Fischer joined the lab that they finally figured out what was going on: Their samples also contained virophages that were “killing” the giant viruses. Freezing destroyed most of the virophages, but each time someone took the giant viruses out of the freezer and grew them in the lab, the virophages would start replicating and attacking the giant viruses. Suttle and Fischer published a paper describing all this in 2011.

The virophages that Suttle found could also explain the origin of another strange genetic phenomenon. Transposons, or “jumping genes,” are DNA sequences that can move around within the genomes of living organisms. The DNA sequences of the virophages that Suttle and Fischer found were similar to those of a certain type of transposon called Polintons. Virophages are now known to infect giant viruses, which in turn infect host cells such as algae or amoebas. Suttle and Fischer hypothesized that this might have gone one step further in the past: Ancient virophages might have become part of the host cell over time as part of a mutually beneficial arrangement—the virophages killed attacking giant viruses for the host and got a safe place to hang out. Eventually, those virophages became transposons.

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The Three Stooges virophages that Stough described from Lake Ontario are genetically similar to these Polinton transposons, too. He didn’t physically observe any virus particles. But it’s possible, he says, that the three virophages are somehow hiding out inside the giant virus CpV-BQ2.

Stough did also trawl through more sequencing data from Lake Erie and Lake Tai in China. He found evidence of virophages in Lake Tai halfway across the world from North America. What’s more, he looked at not just DNA but also RNA, which is present only if the virophages are replicating, which it appears they are. Bernard La Scola, a virologist at Aix-Marseille Université, says that giant viruses and virophages are not uncommon in sequencing data from different environments. “Probably giant viruses and virophages are very common and present in all environments, everywhere,” he says. Until recently, we just didn’t know to look for them.

Fischer, who is now a virologist at the Max Planck Institute for Medical Research, points out that to find giant viruses and virophages, you first need to look for protists, a catchall group of poorly studied organisms that are not bacteria or archaea or animals or plants or fungi. They include single-celled algae, amoebas, slime molds, plankton, and many even more obscure organisms. Scientists have only just started to explore the world of protists. Inside protists, there is a whole world of giant viruses. And inside giant viruses, apparently, a whole world of virophages.

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