May 25, 2007

Reovirus genetics system opening doors for discovery

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Working to manipulate the reovirus genome are, from left, Jim Chappell, M.D.,Ph.D., Takeshi Kobayashi, Ph.D., and Terence Dermody, M.D. (photo by Anne Rayner)

Reovirus genetics system opening doors for discovery

It's an exciting time to be a reovirus researcher.

Vanderbilt University Medical Center investigators have reported the first system for manipulating the reovirus genome, providing a platform for discovery and potential vaccine development.

“A way to alter genomes of viruses has been possible for every major class of animal virus — from the very smallest to the very largest — except for the reovirus family,” said Terence Dermody, M.D., professor of Pediatrics and Microbiology & Immunology, a senior author of the Cell Host & Microbe paper describing the new “reverse genetics” system.

“This is really a breakthrough technology for our field,” Dermody added.

Reoviruses are members of a large family of viruses that cause disease in humans and animals. The reoviruses themselves are relatively harmless, except in newborn animals, where they can cause serious disease. But their “siblings,” the rotaviruses, are the most common cause of viral gastroenteritis — inflammation of the stomach and intestines — in human infants. Rotavirus infections kill about half a million infants and young children each year, mostly in Africa and India.

“We're working hard with our colleagues now to extend the system that we've developed for reovirus to studies of rotavirus,” Dermody said. “Our hope is that a rotavirus reverse genetics system will allow generation of improved rotavirus vaccines.”

Reverse genetics refers to the ability of investigators to make specific, targeted changes to the viral genome and study the outcome of those changes. Without such a system, researchers must rely on “forward genetics,” a strategy that involves identifying viruses with different behaviors that arise through natural selection and attempting to find the genetic reasons for the difference.

Developing reverse genetics technology for reovirus was a particularly vexing problem, Dermody said. Reoviruses have a double-stranded RNA genome that is segmented into 10 parts. To create a reverse genetics system, each of these segments had to be precisely “copied” into a DNA plasmid, a vector useful for manipulating pieces of DNA, and all 10 had to function correctly in cultured cells to produce reovirus particles.

Dermody attributes the team's accomplishment to the creativity and perseverance of postdoctoral fellow Takeshi Kobayashi, Ph.D.

“It was the kind of project that required a tremendous amount of imagination and commitment,” Dermody said. “Takeshi simply would not give up. He always had another idea, no matter how many roadblocks he encountered along the way.”

With the reverse genetics system working, the entire laboratory came on board to make mutations in specific genes, recover mutant reoviruses from cells, and perform biochemical or animal experiments with the mutant reoviruses.

“One of the most exciting endeavors in my whole career was the involvement of my entire laboratory in this project,” Dermody said.

The group has now generated more than 100 different mutant viruses, he said. In the Cell Host & Microbe report, the investigators described a single amino acid mutation in the reovirus attachment protein that stabilized it against attack in the intestine. The mutant reoviruses grew well in the intestine and even disseminated in the mouse and reached the brain, something the wild-type reovirus cannot do.

“This system allows us to identify with a high level of precision which regions of a viral protein mediate a particular function,” Dermody said. “As a platform for discovery for reovirus, this system is established.

“The next application is: can we manipulate the genome for vaccine development?”

Toward that end, the investigators swapped out an entire gene in the reovirus genome and replaced it with a gene encoding a fluorescent marker protein. They are now starting to engineer HIV antigens into the reovirus genome and will test in animals whether such recombinant reoviruses produce an immune response to the HIV antigens.

Adenoviruses engineered to express HIV antigens have shown promise in the development of AIDS vaccines, Dermody said.

“Now with the advent of our reovirus work, we have another candidate that might lead to the development of vaccines to prevent AIDS, tuberculosis, malaria, and a number of other infectious diseases.”

In a commentary about the work in the same issue of Cell Host & Microbe, Ralph Baric, Ph.D. and Amy Sims, Ph.D., from the University of North Carolina wrote that “it is not possible to overstate the importance of the development of a dsRNA (double-stranded RNA) reverse genetics strategy.

“The history of reoviruses reaffirms the critical importance of basic science and model organisms as catalysts for key breakthroughs in the scientific process,” they wrote.

Authors contributing to the work included co-senior author James Chappell, M.D., Ph.D., Annukka Antar, Karl Boehme, Ph.D., Pranav Danthi, Ph.D., Elizabeth Eby, Kristen Guglielmi, Geoffrey Holm, Ph.D., Elizabeth Johnson, Melissa Maginnis, Ph.D., Sam Naik, Wesley Skelton, Denise Wetzel, and Gregory Wilson, M.D.

The research was supported by a fellowship from the Naito Foundation, the National Institutes of Health, and the Elizabeth B. Lamb Center for Pediatric Research.