For years, public health officials have been watching the H5N1 bird flu virus warily. When it hops from birds to people, it has a disturbing tendency to kill them. So far, however, it has been unable to spread from person to person, which has kept the world safe from a lethal pandemic. That posed a rather significant question: could the bird flu ever evolve the ability to spread among mammals, and if it did, would it remain lethal?

Two teams of researchers, one in the US and the other in the Netherlands, set out to answer that question by selecting for a virus that could spread among ferrets, an animal commonly used for flu research. But the publications describing their work have been held up, as the US National Science Advisory Board for Biosecurity debated whether the possibility of weaponizing the virus posed too large a risk. Now, finally, after months of debate, the first of the papers will appear in Nature. It provides a partial answer to the big question—it should be possible for the virus to spread among mammals—but doesn't address whether this would pose a threat.

The rough outline of the results has been known for months, as the researchers presented them at meetings and submitted them to Science and Nature. The NSABB got involved, and asked the journals to hold off on publishing. After a series of debates both public and private, the papers were given the go-ahead (although not everyone involved in that decision agrees).

The Dutch group, which submitted their paper to Science, has ended up bogged down by their nation's export control laws, allowing Nature to go first. And the latter journal is taking its role seriously, penning an editorial entitled "Publishing risky research" and commissioning an external group to do a risk/benefit analysis of releasing the publication. The benefits—better informed efforts to monitor and develop vaccines against the bird flu—were considered to outweigh the risks of weaponization.

The analysis, however, warns that, "This information could be misrepresented by a willful media, in the absence of a knowledgeable public." We'll see if we can avoid that.

The basic science

Flu viruses contain about a dozen genes, spread across eight pieces of RNA (we've got a flu biology primer with more details). One of these genes—the H of the HxNx terminology—latches on to molecules on the surface of cells, providing the virus with the opportunity to infect them. The form in the avian flu, H5, is specific for a molecular structure that is found on the cells of birds, but not those of mammals. As a result, it plays a large role in keeping the bird flu restricted to the birds.

But it's not the only thing. Other proteins produced by the virus are sensitive to the conditions inside infected cells, or must interact with proteins made by their hosts. There are subtle differences between birds and mammals, like pH, body temperature, salt conditions, and the shape of the host's proteins. These mean that, once inside a mammalian cell, the bird flu virus may not work well—or at all. So many changes may be needed before H5N1 can pose a threat to mammals.

The new work, led by Wisconsin's Yoshihiro Kawaoka, focuses only on the H5 protein. To make sure that the virus would work if it could attach to a mammalian cell's surface, Kawaoka's team inserted the H5 gene into the backbone of the 2009 pandemic flu virus. So, the research only addresses a somewhat limited question: what changes are needed in the H5 protein before it can efficiently latch on to mammalian cells and allow the virus to enter them?

(This approach—sticking the H5 protein into a virus that already targeted humans, may seem a bit like cheating. But it's actually not far off from something that happens naturally. More on that below.)

Past research had identified areas on the H5 protein that were essential in attaching to host cells, so the authors decided it would be easiest to just do some accelerated evolution. They introduced random mutations to the DNA that encoded these regions, and then selected for proteins that could latch on to the human versions of their molecular targets. A number of different mutations all conferred this ability, which was confirmed by testing whether they could attach to pieces of human tracheal tissue.

When these tests indicated the mutant viruses could work in mammals, the researchers turned to ferrets, a common model for flu research. Sets of six of the animals were inoculated with different mutated viruses, and the course of infection followed for several days. Several showed infections peaking on day three, and one had an infection that remained high six days after the initial infection. These viruses had actually picked up an additional mutation after infection, giving them a total of three.

The researchers then placed uninfected ferrets in adjacent cages. These animals also ended up infected, although an additional mutation was required, bringing the total to four.

Biochemically, the authors were able to determine that some of the mutations that allowed the virus to bind to a different target actually destabilized the protein, making it more prone to falling apart at elevated temperatures. One of the mutations that was acquired later restored some stability, specifically by adapting it to the pH conditions found in the mammalian respiratory tract.

All of this sounds a bit dry. In fact, it was a bit dry; a typical sentence from the results of the paper reads like this: "The parental control virus (designated VN1203/ PR8) with avian-type receptor-binding specificity agglutinated untreated TRBCs (which possess both human- and avian-type receptors on their surface), but not TRBCs possessing predominantly human-type receptors (Siaa2,6-TRBCs; Supplementary Table 1)." But there are a number of significant findings buried in the details.

What it means

One of the key results of the paper was reported almost incidentally: none of the infected animals died over the course of the study. Most saw flu-like symptoms and experienced some weight loss, but they didn't do any worse than those infected with the unmodified 2009 pandemic virus. So, based on these results, there's no indication that the modified virus is a killer.

That said, the virus carrying these four mutations is very infective, showing a profile similar to that of the 1918 pandemic virus, which probably killed over 100 million people.

The problem with extrapolating from these results is that they are starting with a virus that is mostly derived from the 2009 pandemic, which wasn't especially lethal. The fact is that we don't know what a modified avian flu virus might look like. This is a result of the fact that the virus's genes are distributed among eight different pieces of RNA. If two different viruses infect the same cell (something known to happen in pigs), it's possible for these segments to mix randomly. So, the avian flu might end up jumbled up with pieces of the 2009 pandemic virus, just as was done in this paper. But it's also entirely possible that it could reassort with something else entirely.

That makes these experiments more relevant than they might otherwise appear. But it also means that we have no way of knowing what a human-transmissible form of H5N1 might actually end up looking like—just that it's possible that one could evolve.

That's the bad news. There is some good news in all of this, though. There's already a prototype vaccine for H5N1, and tests with the serum from vaccinated individuals indicate that they contain plenty of antibodies that react to the viruses generated in these experiments. In addition, oseltamivir (better known as Tamiflu) was able to interfere with the virus' normal activity, indicating that current therapies could be effective.

And, as the cost/benefit analysis conditioned by Nature suggested, the results provide valuable information for those on the front lines who are monitoring H5N1 around the globe. The general outlines of the changes caused by the four mutations (loss of a specific site where the H5 protein is chemically modified, tolerance for a different pH) have been hinted at by previous work, and provide clear guidelines for identifying cases where the virus is looking like a more serious threat.

Unfortunately, the relevance of these results would be strengthened a great deal if it turns out that the Dutch team saw similar things. We can only hope that the government of the Netherlands decides to give the publication its approval before too long.

Finally, there's the bioterrorism issue. Overall, I'd say this doesn't raise that risk. Anyone wishing to kill a lot of people would probably do better by sticking with the 1918 pandemic virus, which has a proven track record in this regard. The biggest threat posed by this research is that a commercial or public research group would try to replicate the work (possibly with a beneficial goal in mind) without having the appropriate containment procedures in place.

Nature, 2012. DOI: 10.1038/nature10831 (About DOIs).