Hot spots. Three mutations in or near hemagglutinin's binding site (yellow) and one on its stalk increased transmissibility.

H.-L. Yen and J. S. M. Peiros, Nature, Adavanced Online Edition, (2012)

One of Two Hotly Debated H5N1 Papers Finally Published

One of two influenza papers at the center of an intense, 6-month international debate has finally seen the light of day. Today, Nature published a controversial study in ferrets that shows how scientists can engineer an avian influenza strain to transmit between mammals through respiratory droplets such as those created by coughing or sneezing.

The 11-page study, led by Yoshihiro Kawaoka of the University of Wisconsin, Madison, and the University of Tokyo, describes how the research group stitched a mutated version of a key viral protein called hemagglutinin from the bird flu virus known as H5N1 onto the human H1N1 virus that caused a relatively mild pandemic in 2009. A mere four mutations in the avian hemagglutinin—the H5—allowed this hybrid virus to bind more strongly to mammalian cells and copy itself at high enough levels to readily transmit via respiratory droplets. If the same holds true in humans, that means the virus might be able to trigger a pandemic.

The paper is one of two studies that the U.S. National Science Advisory Board for Biosecurity (NSABB) in December said should not be published in full. 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. That cleared the way for Nature on 2 May to publish the first of the two experiments. (The other of the two papers, by Ron Fouchier of Erasmus MC in Rotterdam, the Netherlands, has been held up because the Dutch government insisted that Fouchier obtain an export license to submit it, which he has done. It is now under review by Science.)

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 globe-shaped head that contains the binding site. Several groups had previously reported that mutations to the binding site could make the virus switch its preference from bird to human cells. 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 identified two such mutations. But the mutations alone still didn't make the virus transmissible between ferrets through respiratory droplets.

Kawaoka's group carried out a series of experiments that coaxed out additional mutations with that effect. The effort included screening 2 million randomly created mutants and infecting ferrets to let strains further adapt to them. Eventually, they found a virus that transmitted from infected animals to four of six healthy ferrets in neighboring cages. It did not kill any of them.

That virus had three mutations in the receptor-binding domain but also one in the stalk that appears to be crucial. Hemaggluttinin's second job—after latching onto the host receptor—is to fuse viral and host cells' membranes once 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 who studies influenza binding at the Scripps Research Institute in San Diego, California.

Nancy Cox, a flu researcher at the U.S. Centers for Disease Control and Prevention in Atlanta and another co-author of the Virology study, 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.

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."

For more on the Kawaoka paper and the debate over H5N1 studies, check out Friday's print edition of Science.

Also check out our full coverage of the flu controversy.