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One H5N1 Paper Finally Goes to Press; Second Greenlighted

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Science  04 May 2012:
Vol. 336, Issue 6081, pp. 529-530
DOI: 10.1126/science.336.6081.529

They have been called the most famous papers that were never published. But now, one of two controversial studies that shows how to make H5N1 avian influenza more transmissible in mammals is up on Nature's Web site for all the world to scrutinize—including, some worry, would-be bioterrorists who might use the information to set off a pandemic. The other paper received a crucial go-ahead from the Dutch government last week and will likely be published by Science soon.

In December, the U.S. National Science Advisory Board for Biosecurity (NSABB) recommended that the two studies—both of which use ferrets, a popular model for human influenza—not be published in full. But after an expert panel convened by the World Health Organization (WHO) disagreed with the decision, NSABB reviewed revised versions of the manuscripts and changed its position (Science, 6 April, p. 19). That cleared the way for Nature on 2 May to publish the first of the two experiments, led by Yoshihiro Kawaoka of the University of Wisconsin, Madison, and the University of Tokyo.

Kawaoka's unusually long, 11-page article describes how his group stitched a mutated version of the hemagglutinin protein of the bird flu virus—the H5—onto the H1N1 virus that caused a relatively mild pandemic in 2009. The team explains that introducing into H5 a mere four mutations, which are delicately balanced with each other, allowed this hybrid virus to bind more strongly to mammalian cells and copy itself at high enough levels to readily transmit via respiratory droplets. Although the researchers don't know whether these mutations would “support sustained human-to-human transmission,” many virologists worry mightily about this scenario. To date, H5N1 has not spread easily between humans, but in 355 of 602 confirmed cases, the patient has died.

First to finish.

Yoshihiro Kawaoka (above) had his paper published this week.

CREDIT: M. ENSERINK/SCIENCE

The second paper has been held up in an extra bureaucratic tangle. After NSABB's initial recommendation, the Dutch government insisted that Ron Fouchier of Erasmus MC in Rotterdam apply for an export license before submitting his revised manuscript (Science, 20 April, p. 285). He called the decision “pure censorship,” but on 24 April he filed the application, still disputing his obligation to do so and stressing that he did not want to set a precedent. Fouchier sent the paper to Science soon after the Dutch government issued the permit on 27 April.

Early this week, the paper was still under review and being edited. Both Fouchier and Kawaoka had hoped the two papers, whose fates had been closely linked for 6 months, would appear simultaneously, which Science Executive Editor Monica Bradford says would have been “ideal” as well. “I regret that both papers will not appear online together, but our priority must be to serve our authors and readers,” Nature Editor-in-Chief Philip Campbell said in a statement. “You can't blame them,” Fouchier says.

Despite the massive debate about these two papers, the Kawaoka study adds only a piece to a puzzle that his and other groups have been putting together for years. Influenza researchers have long attempted to understand the mutations and mechanisms that make a strain spread readily in humans. Kawaoka's study is an “important additional step along the way,” says Malik Peiris, a flu researcher at the University of Hong Kong, who co-wrote an article in Nature about the Kawaoka paper.

Influenza infection begins when hemagglutinin binds to receptors on the host cell. The protein is shaped like a mushroom, with a long stalk and a globular head that contains the binding site. Three of the mutations Kawaoka's group describes are in or near the binding site. They make the virus prefer receptors on human cells to avian ones.

Hot spots.

Mutations near hemagglutinin's binding site (yellow) and stalk increased transmissibility.

CREDIT: H.-L. YEN AND J. S. M. PEIRIS, NATURE (ADVANCED ONLINE EDITION) ©2012 MACMILLAN PUBLISHERS LIMITED

Several groups revealed similar binding site mutations earlier. Indeed, on 5 November 2011, while NSABB was debating the wisdom of publishing the Kawaoka and Fouchier papers in full, a report appeared online in Virology that showed respiratory transmission in one of two ferrets with a lab-made H5 virus that had two of the binding-site mutations also reported by Kawaoka's group. But these researchers stressed that respiratory transmission required additional mutations.

Nancy Cox, a flu researcher at the U.S. Centers for Disease Control and Prevention in Atlanta who co-authored that study—which an internal biosecurity committee said could be published—applauds Kawaoka and his colleagues for their “absolutely fantastic work” and says their mutant “definitely moved the transmission bar to the right towards being fully transmissible.” But she notes that even the new mutant does not spread as readily as common, seasonal flu strains.

Cox and others say the most novel finding in the Kawaoka study involves a mutation in the stalk. This fourth mutation surfaced after a series of experiments that coaxed out mutations to make the virus spread more easily in ferrets. The effort included screening 2 million randomly created mutants and infecting ferrets to let strains further adapt to them. The best transmitter spread from infected animals to four of six healthy ferrets in neighboring cages. It did not kill any of the animals.

The role of the stalk mutation became clear in a set of additional experiments. Hemagluttinin's second job—after latching onto the host receptor—is to fuse viral and host cells' membranes as the virus enters the cell. The mutations at the binding site make it difficult for the protein to do that in the slightly acidic environment of human mucosa, the researchers say, but the mutation on the stalk compensates by enabling the protein to operate in a more acidic environment. “It's the major discovery in the study,” says James Paulson, a co-author of the Virology paper with Cox who studies influenza binding at the Scripps Research Institute in San Diego, California.

Keiji Fukuda, a flu expert at WHO, says the paper will help guide surveillance for viruses that may cause great harm in humans. That's not just because they highlight specific mutations, Cox adds. “What we're really looking for is generalizable patterns of changes that occur when viruses become more transmissible in a mammalian model. … You can't be focused on a set of four specific mutations.”

Now, influenza scientists are eagerly awaiting publication of Fouchier's study, still under wraps, which Kawaoka recently said has “striking similarities” with his own. It's unclear how these publications will affect a self-imposed moratorium on studies that involve modifying the transmissibility or lethality of H5N1. Announced by researchers from the world's most active H5N1 labs in January, the moratorium was extended indefinitely at a February meeting organized by WHO.

A related, vigorous debate continues about whether H5N1 modification studies should be confined to the most secure laboratories—known in the United States as biosafety level 4 facilities—or “enhanced” BSL-3 laboratories, where both Kawaoka and Fouchier performed their experiments. WHO will not take sides, nor will it advise on the moratorium, Fukuda says. Others say the moratorium should remain in force until the safety issues are resolved. “We should not rush forward when the stakes are so high,” Thomas Inglesby, director of the Center for Biosecurity at the University of Pittsburgh Medical Center in Pennsylvania, told a U.S. Senate committee at a hearing on 26 April.

Anthony Fauci, head of the U.S. National Institute of Allergy and Infectious Diseases, which funded the two studies, agrees. Before U.S. officials support lifting the ban, he said at last week's hearing, they will have to “feel comfortable” that H5N1 labs understand how to evaluate the dual-use potential of their work and take steps to mitigate safety and security risks.

  • * With reporting by David Malakoff.

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