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Researchers discover large dormant virus can be reactivated in model green alga

Researchers had been studying the green alga Chlamydomonas reinhardtii for decades without seeing evidence of an active virus within it—until a pair of Virginia Tech researchers waded into the conversation.

Maria Paula Erazo-Garcia and Frank Aylward not only found a virus in the alga but discovered the largest one ever recorded with a latent infection cycle, meaning it goes dormant in the host before being reactivated to cause disease.

"We've known about latent infections for a long time," said Aylward, associate professor in the Department of Biological Sciences.

"A lot of viruses are temperate in the sense that they can integrate their genomes in that of the host and sometimes lay dormant for long periods of time, but what's remarkable about this virus is that it's so large. This is the largest temperate virus discovered to date with this particular infection cycle."

Erazo-Garcia, a Ph. D. candidate and member of the Aylward Lab, was the lead author of the paper detailing the findings, which was in Science.

The researchers leveraged international collaborations and campus resources in both the Fralin Life Science Institute's Genomics Sequencing Center and the Virginia Tech's Nanoscale Characterization and Fabrication Laboratory to unearth the virus, which they named Punuivirus.

They demonstrated that when integrated into a host's genome, it can reactivate and spontaneously produce viral particles that are released in healthy cell cultures.

Along with the novel findings, the researchers believe the study will also shed light on potential biotechnological applications, especially those involved in the delivery and integration of DNA. This could potentially be exploited for gene-editing applications.

"It's fascinating to think that all these things we're trying to engineer in other systems are already happening naturally here," Aylward said. "It's a wonderful opportunity to learn how viral enzymes initiate DNA integration because they can be co-opted for biotechnological purposes."

According to Aylward, Chlamydomonas reinhardtii is one of the most well-studied green algae. Scientists have information dating back to the 1970s that indicates viral production in otherwise healthy cultures of some green algae. They suspected it could be from the activity of latent viruses, but this was difficult to prove because of technological limitations and the possibility of environmental contamination.

Typically, if a healthy alga with a full green, dense culture is infected, the cells will die rapidly and there will be visual evidence. The cells will often group together, and the green flask will quickly turn clear.

"[Originally,] we weren't seeing this at all," Aylward said. "The culture looked completely healthy. You wouldn't know that there was any viral infection unless you did these sophisticated molecular analyses."

To check for viral elements in the alga, researchers conducted long-read sequencing, a process that analyzes long strands of DNA, to test a particular alga strain known for containing viral elements. This work was conducted in the Genomics Sequencing Center with additional guidance from the Max Planck Institute for Biology Tübingen.

They then assessed whether the viral element was active and could produce viral particles by monitoring the parent virus's production in cultures from inoculation to stationary phase and found a distinct population of viral particles.

"We needed evidence that it was there," said Erazo-Garcia, who is studying biological sciences. "And getting images is one of the most convincing ways to convince the community that this is an active virus."

The cultures showed that parent viruses were produced at late exponential and early stationary phases of host growth and that only a small number of cells contained active viruses, which is why the infection was difficult to detect.

Further investigation at Virginia Tech's Nanoscale Characterization and Fabrication Laboratory showed that viral particles were present in the cultures, again demonstrating latent infection.

Collaboration was key, as Aylward and Garcia also worked with researchers at the University of Lund in Sweden and the Royal Institute of Sea Research in the Netherlands, comparing findings that indicated similar infection dynamics, and at the University of Miami, where a collaborator helped lay the groundwork that led to the discovery.