Apr 25, 2013 (CIDRAP News) – A new study suggests that a lab-derived hybrid H5N1 influenza virus that is capable of airborne transmission among ferrets may well be capable of doing the same thing in humans.
Writing in Nature, an international team of researchers says that the hemagglutinin (HA) protein from the hybrid H5N1 strain described last year by Yoshihiro Kawaoka, DVM, PhD, and colleagues has a strong preference for human airway cell receptors over avian receptors. In flu viruses, HA is the surface protein that grabs onto host cells to start the infection process.
Also, the researchers say the bond formed between the virus's H5 HA and human cell receptors is structurally similar to the bond formed by previous pandemic flu viruses with human receptors.
The study is a close examination of the receptor-binding properties of the HA from the mutant virus described by Kawaoka and colleagues. That study, and a similar one from the Netherlands, sparked a major controversy when they were first described in general terms in late 2011. The other study was led by Ron Fouchier, PhD, of Erasmus Medical Center in Rotterdam.
Out of fear that someone could exploit the findings to create a highly dangerous virus, the US National Science Advisory Board for Biosecurity (NSABB) recommended in December 2011 that both studies be stripped of key details before publication. But after the authors provided some additional information and clarifications, the board in March 2012 endorsed publication of full versions of both studies. Publication of Kawaoka's study followed in May 2012 and Fouchier's in June.
Kawaoka is a coauthor of the new study, along with scientists from China, the United Kingdom, and Switzerland. The lead author is Xiaoli Xiong and the senior author is Steven J. Gamblin; both are with the UK Medical Research Council's National Institute for Medical Research in London.
In Kawaoka's earlier study, his team induced random mutations in the HA of a 2004 H5N1 strain from Vietnam and then identified those that caused increased binding to human-type cell receptors, known as alpha2,6 receptors. The researchers then created reassortant viruses consisting of the genes for the mutant H5 HAs joined to seven other genes from the 2009 pandemic H1N1 virus.
The team subsequently infected ferrets—which are considered the best experimental model for human flu—intranasally with several different reassortant viruses and placed them in cages next to uninfected ferrets. After some of the latter animals became infected, their viruses were analyzed to identify mutations that were associated with respiratory-droplet transmission. In this and further experiments, the scientists found four such changes.
In the new study, the researchers conducted biophysical measurements of the receptor-binding properties of the HA from the ferret-transmissible virus, using two different measurement techniques (microscale thermophoresis and surface biolayer interferometry). In addition, they developed an algorithm for predicting the strength of the virus's binding to surfaces, based on the tightness of single-receptor bonds with HA.
The team found that the affinity of the mutant HA with human receptors is slightly greater than that of HA from the wild-type 2004 H5N1 strain from Vietnam, whereas its affinity for avian receptors is greatly decreased. The net result as predicted by the authors' algorithm is that the mutant HA "has a 200-fold preference for binding human over avian receptors," the report says.
While the mutant HA strongly favors human over avian receptors, its ability to latch onto human cells is well below that of some past flu viruses, the authors report. They found that the capability is about fivefold weaker than that of the H3N2 strain that sparked the 1968 flu pandemic.
The report says the findings suggest that the mutant virus's properties "would favour infection of cells in the human upper respiratory tract that have been shown to display alpha2,6-linked sialic acids." Also, the inability to bind to avian receptors means that airway mucus, which is rich in alpha2,3-linked sialic acids, would not catch and hold the virus.
"Avoidance of mucins is probably important for the ferret transmissibility of the transmissible-mutant virus given that its human-receptor-binding properties are modest compared to pandemic viruses," the report adds.
The researchers also determined the crystal structure of the complex formed by the mutant HA with analogs of human receptors. For comparison, they also examined the structure of the bonds formed by HAs from the 2004 Vietnam H5N1 strain and a 2005 H5N1 strain from Turkey with human receptor analogs.
This analysis revealed that the mutant H5 "has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957, 1968, and 2009 pandemic viruses," the report says. "This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor."
The structural analysis helps explain why a mutation known as Gln227Leu plays a key role in changing the mutant HA's binding preference from avian to human receptors, the scientists said.
In some ways, they said, the properties of the mutant HA "suggest that H5 viruses could take a similar evolutionary pathway in humans to that followed in 1957 and 1968 by avian H2 and H3 viruses. Thus, the preference of the transmissible mutant for binding to human versus avian receptors, and the structural manner by which it binds them, are highly characteristic of pandemic viruses."
However, the fact that the mutant virus sacrifices its avian binding ability to gain its human affinity "distinguishes it from known pandemic viruses," the authors add.
The study drew sharply different reactions from two other experts.
Vincent Racaniello, PhD, Higgins Professor of microbiology and immunology at Columbia University in New York City and author of Virology Blog, said the study offers important contributions but cautioned that receptor binding is not the only factor that determines a flu virus's ability to infect and spread among ferrets and humans.
In e-mailed comments, Racaniello observed that the authors "find that there is a limit on how many HA–sialic acid interactions can occur with one virus particle, and that this explains how modest decreases in the dissociation constant [the strength of single HA-receptor interactions] (~15-fold) can lead to a complete loss of binding and the resulting effects on receptor specificity. I think this is an important contribution which unifies the physical measurements with the ability of viruses to infect cells."
"To summarize the important contributions of this work," Raniello said, "the ferret transmissible HA has a small increase in affinity for human receptors and a large decrease for avian receptors; alpha-2,6 sialic acid binds this HA in a way typical of human pandemic strains, and different from how wild-type H5 binds avian sialic acids."
"They [the authors] suggest that the transmissible H5 would not arise in waterfowl, given its lack of binding to avian receptors," he continued. "These data help guide analysis of viral sequences for pandemic potential, and make important links between HA sequences, structure, and receptor binding.
"However, it is important to remember that aerosol transmission is far more complex than just being able to bind a receptor, and involves other parameters such as efficient shed from cells, the ability to replicate, stability, and probably others," he said. For humans, he added, the nature of those other changes is unknown.
'A novel human virus'
Simon Wain-Hobson, PhD, a veteran HIV researcher and outspoken opponent of "gain of function" experiments like Kawaoka's and Fouchier's, expressed alarm over the findings.
"What I conclude here is that this virus does bind the sugar receptor as would a mammalian virus, in which case, as we know it goes from ferret to ferret, it's very reasonable to assume this is a novel human virus," he told CIDRAP News in an interview. "The world is a more dangerous place."
Wain-Hobson is a professor at the Pasteur Institute in Paris and chairs the board of the Foundation for Vaccine Research, a privately funded group that seeks increased funding for vaccine studies. In March the foundation formally asked President Obama's Commission for the Study of Bioethical Issues to review the ethics of experiments designed to increase the transmissibility of H5N1 viruses.
Commenting further by e-mail, Wain-Hobson said the report "says that the virus will replicate in the upper respiratory tract due to affinity for the alpha2,6-linked sialic acids—just like any good mammalian influenza virus. It also says that as the virus has lost its binding for alpha2,3-linked sialic acids it won't get stuck on the mucin surfaces in the airways.
"This means that airborne virus will get down to the upper respiratory tract almost unhindered. So it will get into the right place and it will replicate. And because it won't get stuck on the mucin, it will get out."
Wain-Hobson said further that if Kawaoka's (or Fouchier's) virus escaped from a lab, it might not become a pandemic strain immediately, but it could begin spreading in humans and quickly evolve into one. "So even if this Kawaoka configuration isn't the BIG ONE, it could rapidly lead to it," he asserted.
Xiong X, Coombs PJ, Martin SR, et al. Receptor binding my a ferret-transmissible H5 avian influenza virus. (Letter) Nature 2013 (published online Apr 24) [Preview]
May 3, 2012, CIDRAP News story "Report details changes that may boost H5N1 spread in mammals"
Mar 29 CIDRAP News story "Scientists seek ethics review of H5N1 gain-of-function research"