How Canadian researchers reconstituted an extinct poxvirus for $100,000 using mail-order DNA

Eradicating smallpox, one of the deadliest diseases in history, took humanity decades and cost billions of dollars. Bringing the scourge back would probably take a small scientific team with little specialized knowledge half a year and cost about $100,000.

That’s one conclusion from an unusual and as-yet unpublished experiment performed last year by Canadian researchers. A group led by virologist David Evans of the University of Alberta in Edmonton, Canada, says it has synthesized the horsepox virus, a relative of smallpox, from genetic pieces ordered in the mail. Horsepox is not known to harm humans—and like smallpox, researchers believe it no longer exists in nature; nor is it seen as a major agricultural threat. But the technique Evans used could be used to recreate smallpox, a horrific disease that was declared eradicated in 1980. "No question. If it’s possible with horsepox, it’s possible with smallpox,” says virologist Gerd Sutter of Ludwig Maximilians University in Munich, Germany.

Evans hopes the research—most of which was done by research associate Ryan Noyce—will help unravel the origins of a centuries-old smallpox vaccine and lead to new, better vaccines or even cancer therapeutics. Scientifically, the achievement isn't a big surprise. Researchers had assumed it would one day be possible to synthesize poxviruses since virologists assembled the much smaller poliovirus from scratch in 2002. But the new work—like the poliovirus reconstitutions before it—is raising troubling questions about how terrorists or rogue states could use modern biotechnology. Given that backdrop, the study marks "an important milestone, a proof of concept of what can be done with viral synthesis,“ says bioethicist Nicholas Evans—who's not related to David Evans—of the University of Massachusetts in Lowell.

Bringing back an extinct virus that is related to smallpox, that's a pretty inflammatory situation Paul Keim, Northern Arizona University

The study seems bound to reignite a long-running debate about how such science should be regulated, says Paul Keim, who has spent most of his career studying another potential bioweapon, anthrax, at Northern Arizona University in Flagstaff. "Bringing back an extinct virus that is related to smallpox, that's a pretty inflammatory situation,” Keim says. "There is always an experiment or event that triggers closer scrutiny, and this sounds like it should be one of those events where the authorities start thinking about what should be regulated.”

Little-noticed discussion

David Evans acknowledges that the research falls in the category of dual-use research, which could be used for good or bad. "Have I increased the risk by showing how to do this? I don't know," he says. "Maybe yes. But the reality is that the risk was always there."

Evans discussed the unpublished work in November 2016 at a meeting of the Advisory Committee on Variola Virus Research at the World Health Organization (WHO) in Geneva, Switzerland. (Variola is the official name of the virus that causes smallpox.) A report from that meeting, posted on WHO's website in May, noted that Evans's effort "did not require exceptional biochemical knowledge or skills, significant funds or significant time.” But it did not draw much attention from biosecurity experts or the press.

Also little noticed was a press release issued by Tonix, a pharmaceutical company headquartered in New York City with which Evans has collaborated, which also mentioned the feat. Tonix says it hopes to develop the horsepox virus into a human smallpox vaccine that is safer than existing vaccines, which cause severe side effects in a small minority of people. Evans says it could also serve as a platform for the development of vaccines against other diseases, and he says poxvirus synthesis could also aid in the development of viruses that can kill tumors, his other area of research. "I think we need to be aware of the dual-use issues," Evans says. "But we should be taking advantage of the incredible power of this approach.”

The double-stranded variola genome is 30 times bigger than the poliovirus genome, which Eckard Wimmer of State University of New York at Stony Brook assembled from mail-ordered fragments in 2002. Its ends are also linked by structures called terminal hairpins, which are a challenge to recreate. And though simply putting the poliovirus genome into a suitable cell will lead to the production of new virus particles, that trick does not work for poxviruses. That made building variola "far more challenging," says Geoffrey Smith of the University of Cambridge in the United Kingdom, who chairs WHO's variola advisory panel.

The world just needs to accept the fact that you can do this and now we have to figure out what is the best strategy for dealing with that David Evans, University of Alberta

In 2015, a special group convened by WHO to discuss the implications of synthetic biology for smallpox concluded that the technical hurdles had been overcome. "Henceforth there will always be the potential to recreate variola virus and therefore the risk of smallpox happening again can never be eradicated,” the group's report said. But Evans felt like the matter was never really put to rest. "The first response was, ‘Well let's have another committee to review it,’ and then there was another committee, and then there was another committee that reviewed that committee, and they brought people like me back to interview us and see whether we thought it was real,” he says. "It became a little bit ludicrous.”

Evans says he did the experiment in part to end the debate about whether recreating a poxvirus was feasible, he says. "The world just needs to accept the fact that you can do this and now we have to figure out what is the best strategy for dealing with that,” he says.

Two rejections

Evans declines to discuss details of his work because, after two rejections, he is about to resubmit a paper about it for publication. But the WHO report says the team purchased overlapping DNA fragments, each about 30,000 base pairs in length, from a company that synthesizes DNA commercially. (The company was Geneart, in Regensburg, Germany, Evans says.) That allowed them to stitch together the 212,000-base-pair horsepox virus genome. Introducing the genome into cells infected with a different type of poxvirus led these cells to start producing infectious horsepox virus particles, a technique first shown to work in a 2002 paper in the Proceedings of the National Academy of Sciences. The virus was then “grown, sequenced and characterized,” the report notes, and had the predicted genome sequence.

Evans says Science and Nature Communications both rejected the paper. Caroline Ash, an editor at Science, says the paper wasn't formally submitted to the journal, but that Evans inquired about publication and provided the Tonix press release. “While recognizing the technical achievement, ultimately we have decided that your paper would not offer Science readers a sufficient gain of novel biological knowledge to offset the significant administrative burden the manuscript represents in terms of dual-use research of concern," Ash says she replied to Evans.

Evans says he has run his draft papers by Canadian government officials involved in export and trade as well as the Public Health Agency of Canada and the Canadian Food Inspection Agency, which were "very helpful and provided timely and sensible guidance," he says. “These things potentially fall under export legislation, because technically it could be viewed as instructions for manufacturing a pathogen,” he says. To avoid running afoul of international conventions, Evans says he “provided sufficient details so that someone knowledgeable could follow what we did, but not a detailed recipe.”

Peter Jahrling, a virologist at the National Institutes of Allergy and Infectious Diseases in Bethesda, Maryland, says the paper should definitely be published. "Not only is it novel,“ he says. "It is also extremely important.“

Regulatory questions

Producing the variola virus in the same fashion would be prohibited under WHO regulations and rules in place in many nations. Labs are not allowed to make more than 20% of the variola genome, and the companies that make and sell DNA fragments have voluntary checks in place to prevent their customers from ordering ingredients for certain pathogens unless they have a valid reason. But controlling every company in the world that produces nucleic acids is impossible, Keim says. "We’ve recognized for quite a few years that regulating this type of activity is essentially impossible,“ he says.

Instead, Keim says, there should be an international permit system for researchers who want to recreate a virus no longer found in nature. Current U.S. rules already require federally-funded researchers who plan to do an experiment that “generates or reconstitutes an eradicated or extinct agent” that is on a 15-agent list of dual-use agents to undertake a special review and risk assessment. That U.S. list of regulated agents includes variola, but not horsepox, because it's not considered a dangerous virus itself.

The system in Canada is different, says Gregory Koblentz, a biodefense expert at George Mason University in Fairfax, Virginia, who has been looking into the experiment since noticing the Tonix press release in March. There, the rules say even research that does not involve certain dangerous pathogens, but that could nonetheless generate knowledge that poses a dual-use risk, should be reviewed. “That should have captured the horsepox synthesis,” he says. Evans talked to federal agencies in Canada, which was not even required of him, and his university did look at the safety aspect of bringing back an animal pathogen. “But as far as I understand, they did not engage in a systematic review of the broader dual-use implications of synthesizing an orthopox virus,” says Koblentz. "I don't think this experiment should have been done."

Nicholas Evans, the bioethicist, thinks that new rules need to be put in place given the state of the science. “Soon with synthetic biology ... we're going to talk about viruses that never existed in nature in the first place,“ he says. "Someone could create something as lethal as smallpox and as infectious as smallpox without ever creating smallpox.“ WHO should create an information sharing mechanism obliging any member state to inform the organization when researchers plan to synthesize viruses related to smallpox, he argues.

The genie is out of the lamp Peter Jahrling, National Institute of Allergy and Infectious Diseases

Evans’s experiment may also render moot a long-running debate on whether to destroy the two last known caches of variola. After smallpox was eradicated in 1980, labs around the world agreed to destroy their remaining smallpox samples or ship them to the Centers for Disease Control and Prevention (CDC) in Atlanta or to the Russian Research Institute of Viral Preparations in Moscow. (The Russian samples were later moved to the State Research Centre of Virology and Biotechnology in Novosibirsk.) Since then, the fate of those remaining stocks has been the focus of intense debate. "Destructionists" have argued that wiping out the last strains would make the world a safer place, whereas "retentionists" say keeping the virus—and studying it—could help the world prepare for future outbreaks.

Now that variola can be synthesized, the decision hardly matters, Jahrling says. “You think it’s all tucked away nicely in freezers, but it’s not,“ he says. "The genie is out of the lamp." Evans's work is "a gamechanger for the discussion," confirms Andreas Nitsche of the Robert Koch Institute in Berlin, who attended the WHO meeting where Evans presented his work last fall.

Fears of a return of smallpox—which kills up to one-third of its victims—ran high in the United States after 9/11 and the anthrax letters mailed to U.S. politicians and media figures a few weeks later. The events led the U.S. government to amass big new stockpiles of smallpox vaccine and start a vaccination campaign for so-called first responders. But though a smallpox outbreak would almost certainly create panic and pose an unprecedented test for public health systems, scientists familiar with the disease say an outbreak could probably be contained quite easily because smallpox is not highly infectious and spreads slowly—qualities that made it possible to eradicate it in the first place.

Mysterious origins

Much less is known about horsepox. Pox viruses are known to infect many animals, and horsepox is frequently mentioned in historic accounts, but it seems to have disappeared from nature, possibly because of modern husbandry practices. Scientists at the Plum Island Animal Disease Center in New York published a genome sequence for horsepox in 2006, based on a virus isolated from sick horses in Mongolia 40 years earlier. That virus is still held at CDC; Evans says one reason he decided to synthesize a new virus was that he could not get permission to use the CDC samples for commercial purposes.

Evans says his project has academic value as well: It could help elucidate the early history of smallpox immunization. The vaccine used to eradicate smallpox—the world's oldest vaccine—is itself a living virus named vaccinia; it was first used in 1796 by Edward Jenner, a U.K. doctor. Popular accounts usually have Jenner using cowpox to inoculate people after he noticed that dairymaids appeared to be immune to smallpox. But there are also stories implicating horsepox, and the published horsepox genome looks very similar to some old vaccinia strains, bolstering the hypothesis that the vaccine was derived from horses. (To add another layer of confusion, both horsepox and cowpox may originally have been rodent poxviruses that only occasionally infected livestock.)

Evans hopes to study the function of some horsepox genes by making specific deletions, which could shed light on how the vaccine strain arose. “This is the most successful vaccine in human history, the foundation of modern immunology and microbiology, and yet we don't know where it came from," he says. "There is a huge, interesting academic question here."

Updated, 7/7/2017 at 2:30 p.m.: This story has been updated to includes comments from Gregory Koblentz on Canada's system of regulating and reviewing dual use research.