After months of anticipation, the second of two controversial experiments to increase the virulence of H5N1 avian influenza has been published.

Many scientific questions are raised by the findings, and the same safety concerns remain that provoked massive public outcry and a temporary halt to the research. But if there were any doubts about H5N1's ability to become airborne, they're gone.

In the experiment, researchers made H5N1 strains that passed through the air between ferrets, which are often used as a model for human flu infection. Catching the highly lethal virus typically requires close contact with an infected bird or person.

The resulting virus didn't kill the animals, and it's uncertain if a ferret-infecting strain could also infect people. But the fact that just five genetic mutations were needed to produce the airborne strain is troubling.

"One of the next key questions is, 'How likely is it that such a virus could evolve in nature?'" said virologist Derek Smith of Cambridge University at a 20 June press conference. The research was led by virologists Sander Herfst and Ron Fouchier of Erasmus University in the Netherlands and published 21 June in Science.

"It's now clear that we're living on a fault line," continued Smith. "It could really do something. What we need to now is how likely that is."

The researchers started by genetically modifying H5N1 to contain three mutations identified in previous pandemic flu strains as increasing viral transmissibility.

Thus modified, the strain was manually introduced into the noses of ferrets. Once infection was established, the researchers swabbed the ferrets and infected another group. They repeated the cycle multiple times, mimicking a chain of infection between people.

At each stage, some strains became more adapted to surviving and replicating in the ferrets' upper respiratory tracts, a precursor to making flu spread by cough or sneeze. By the 10th stage's end, the mutant H5N1 was airborne, passing between ferrets in separate cages.

Ferrets infected by the airborne strains didn't die, raising the possibility that becoming highly infectious makes H5N1 less lethal. This, however, is far from certain, and perhaps more time would have produced a more-lethal variant.

Making the engineered H5N1 go airborne required a total of five mutations. "Within the first three or four passages, we already see strong adaptations of the virus," said Fouchier during the press conference. "Only a limited chain of transmission is sufficient in ferrets. We assume that in humans it would take a low number of events for these or similar mutations to accumulate."

In a study accompanying the findings, researchers led by Smith and microbiologist Colin Russell, also of Cambridge University, looked for the five mutations in naturally circulating strains of H5N1. They found them, though the mutations occurred individually or in pairs, not united simultaneously in single strains.

Whether they could gather in one strain is now a pressing question, as is a more fundamental scientific question: Do the mutations represent a mechanism by which H5N1 could become contagious in humans, or just in ferrets? And if they are directly relevant to human infections, do they represent a primary route, or one of many possible paths?

That's unknown, but simply being able to see how mutations alter influenza should help researchers, said biomedical informaticist Raul Rabadan of Columbia University, who specializes in analyzing flu genomes.

"Beyond the particular mutations in each paper, these studies point out key necessary elements for mammalian adaptation and transmission," Rabadan said. "The mutations are important, but more important is what they are telling us about the mechanisms for host adaptation and airborne transmission."

Smith and Fouchier's teams hope the findings will guide surveillance efforts, providing researchers who track flu's natural evolution in humans and other animals. But in a commentary also published in Science, epidemiologists Marc Lipsitch and Barry Bloom of Harvard University said the findings were less useful than advertised.

According to Lipsitch and Bloom, too little is known about flu genetics or its natural evolution to make lab-generated changes a reliable guide for real-world surveillance. "Predictions about how particular influenza strains will behave in humans or, even more important, how they will evolve, remain highly speculative," they wrote.

Calculating the risks and benefits of these experiments, along with experiments that made H5N1 more virulent by mixing it with the pandemic 2009 flu strain, is a complicated and controversial matter.

Supporters of the research argue that potential benefits outweigh the risks, which are enormous. Beyond giving recipes to would-be bioterrorists, accidental disease exposures at high-security research laboratories are not uncommon, and flu is extraordinarily difficult to control.

Some researchers put H5N1's human mortality rate at 60 percent, a number likely inflated by epidemiologists overlooking cases that lack severe symptoms. But a mortality rate of just 2.5 percent killed 40 million people in the 1918 pandemic.

Should the findings prove less useful than expected, justification for H5N1 engineering would evaporate. If the findings do prove useful, there's still powerful disagreement over how the research should be conducted. Many critics say H5N1 experiments should be restricted to a few highly skilled people in the world's most secure laboratories.

The National Institutes of Health is currently drafting formal policies to handle this and other potentially dangerous research, a task that should have been completed years ago.

Asked about the study published today, epidemiologist Stephen Morse of Columbia University said, "At least it's forced us to confront this issue directly, which was never really resolved and badly needs some good policy."

Correction 6/21: The original wording of the opening paragraphs implied that the new H5N1 strain could infect humans. That is unknown.

Citations: "Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets." By Sander Herfst, Eefje J. A. Schrauwen, Martin Linster, Salin Chutinimitkul, Emmie de Wit, Vincent J. Munster, Erin M. Sorrell, Theo M. Bestebroer, David F. Burke, Derek J. Smith, Guus F. Rimmelzwaan, Albert D. M. E. Osterhaus, Ron A. M. Fouchier. Science, Vol. 336 Issue 6088, June 22, 2012.

"The Potential for Respiratory Droplet-Transmissible A/H5N1 Influenza Virus to Evolve in a Mammalian Host." By Colin A. Russell, Judith M. Fonville, André E. X. Brown, David F. Burke, David L. Smith, Sarah L. James, Sander Herfst, Sander van Boheemen, Martin Linster, Eefje J. Schrauwen, Leah Katzelnick, Ana Mosterín, Thijs Kuiken, Eileen Maher, Gabriele Neumann, Albert D. M. E. Osterhaus, Yoshihiro Kawaoka, Ron A. M. Fouchier, Derek J. Smith. Science, Vol. 336 Issue 6088, June 22, 2012.

"Evolution, Safety, and Highly Pathogenic Influenza Viruses." By Marc Lipsitch, Joshua B. Plotkin, Lone Simonsen, Barry Bloom. Science, Vol. 336 Issue 6088, June 22, 2012.