Friday, August 17, 2012

Public at Last, H5N1 Study Offers Insight Into Virus's Possible Path to Pandemic

[This is an excerpt, dated back in June 2012 It is an excerpt describing the mutations used in the two studies by Kawaoka & Foucher, to become a Pandemic]

Vol. 336 no. 6088 pp. 1494-1497
DOI: 10.1126/science.336.6088.1494

There's a key difference between the studies, however. Kawaoka created a hybrid virus: He took the gene for a viral protein called hemagglutinin from an avian H5N1 strain and stitched it together with seven other gene segments from the pandemic H1N1 virus that swept the world in 2009 and 2010, and which is already well-adapted to humans. From this starting point, it took just four mutations in the hemagglutinin gene to create a virus that could travel through the air from one infected ferret—a popular animal model for human infection—and infect another. But Kawaoka's hybrid has not yet been found in nature.

In contrast, “the strong point” of Fouchier's study, Cox says, is that it started out with an actual H5N1 virus isolated from a human victim in Indonesia. In an e-mail to Science, Kawaoka agreed that Fouchier's study addresses the most urgent question more directly. “Ron's data are very important,” he said.

Fouchier's team first inserted several mutations they knew might help the virus adapt for mammalian spread. One key target was the virus's receptor binding site, the area within the hemagglutinin molecule that makes first contact with the host cell; scientists already knew that two mutations there can make the virus prefer mammalian cells over bird cells. Another mutation, in the polymerase protein complex, allows the virus to replicate in the cool environment of the human upper respiratory tract rather than in bird intestines, the much warmer environment where it usually resides.

These initial mutations alone didn't do the trick, however, so Fouchier's team decided to try a time-honored method to encourage a pathogen to adapt to a new host: They passed the virus from ferret to ferret by directly inoculating uninfected animals with nasal samples from infected ones and repeated the procedure a total of 10 times. (In his Malta talk, Fouchier called this a “really stupid” approach, a phrase widely interpreted to mean he regretted it. In fact, he says, he just meant that the technique, called passaging, is a simple one compared to the sophistication of creating targeted mutations. The confusion may have stemmed in part from the fact that the Dutch word for “stupid” can also mean “simple.”)

The end result was a virus that could move through the air from one caged ferret to another right next to it; in a first experiment, the virus transmitted from cage to cage in three out of four instances.

Prior to publication, media reports suggested that airborne transmission required five mutations. The reality is more complex. Each of Fouchier's transmissible viruses had at least nine mutations, five of which were shared by all. This core quintet may be sufficient, the team writes, but the big question is whether one or more of the other changes also plays an important role, Peiris says.

Fouchier already knows part of the answer. Once his team achieved transmission in the summer of 2011, the researchers began additional experiments to identify the minimum set of mutations needed to make the virus airborne. But before those experiments were finished, they submitted their manuscript to Science, worrying that Kawaoka or other scientists might beat them to the punch. “Usually when you discover something important, somebody else is discovering it, too,” Fouchier says. (He was right: Kawaoka, who says he didn't know about Fouchier's work, had submitted his manuscript less than 2 weeks earlier.) Now, Fouchier declines to discuss the results from the additional experiments, which are on hold as a result of the moratorium.

The published paper shows that the core set of five mutations includes the three that the team introduced themselves and two more that arose during passaging. And the resemblance to what Kawaoka found is “quite remarkable,” says James Paulson, a glycobiologist at the Scripps Research Institute in San Diego, California. Both teams found that two mutations at the receptor binding site—one of them identical in the two studies—are important; both discovered an additional mutation that makes hemagglutinin lose a sugar group, which apparently helps make room for the mammalian host cell receptor. Kawaoka also found a mutation in the hemagglutinin's stalk that improves the virus's stability and compensates for the other mutations, Paulson says. “It's tempting to think” that one of Fouchier's mutations plays a similar role—although it's not in the stalk but in another area where three hemagglutinin molecules align to form a so-called trimer.

Cox says she's “surprised” that it didn't take more mutations. In a study with Paulson published online by Virology in November, her team also mutated the receptor-binding site, but they had to make several other changes—including slotting in a human-adapted version of another viral gene called neuraminidase—to get airborne transmission. That led the authors to conclude that the virus might require “extensive evolution” to become pandemic. Fouchier's paper upsets that reassuring notion.

Both papers will aid surveillance efforts because they suggest which genetic changes to look out for in H5N1, Peiris says. But they also point to a limitation: Several mutations can have the same effect on the virus. “It wouldn't be appropriate to focus just on these mutations,” Peiris says. “The virus has different ways to go from A to B.” More research is needed to discover how many ways, he says.

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