Kawaoka paper published on aerosol transmission of H5 influenza virus in ferrets
2 MAY 2012
One of two papers on avian influenza H5N1 virus that caused such a furor in the past six months was published today in the journal Nature. I have read it, and I can assure you that the results do not enable the construction of a deadly biological weapon. Instead, they illuminate important requirements for the airborne transmission of influenza viruses among ferrets. Failure to publish this work would have compromised our understanding of influenza viral transmission.

The paper from Kawaoka’s group focuses on the viral hemagglutinin (HA) protein, an important determinant of whether influenza viruses can infect birds or mammals. In the image, the HA is shown as blue ‘spikes’ on the virion surface; a single HA molecule is shown at right. Avian influenza viruses prefer to attach to cells via a specific form of sialic acid that differs from the form bound by mammalian influenza viruses. This difference in receptor preference is one reason why avian influenza viruses do not transmit among mammals.

Kawaoka’s group used a random mutagenesis and selection approach to identify amino acid changes in the avian H5 HA protein that allow it to bind human receptors. These changes are located around the sialic acid binding pocket in the HA head (figure). Some of the amino acid changes were previously known, but there are also some new ones reported, expanding our understanding of how the HA binds sialic acids. Some of the HA amino acid changes allow virus binding to ciliated epithelial cells of the human respiratory tract (wild type H5 HA cannot). All of this is important new information.

The H5 HA genes with these amino acid changes were then substituted for the HA gene in a 2009 H1N1 pandemic virus, and this reassortant virus was inoculated intranasally into ferrets. The viruses did not replicate well in the ferret trachea, but viruses recovered from the animals contained a new change in the HA protein that improves replication. This change (asparagine to aspartic acid at amino acid 158) is known to prevent attachment of a sugar group to the HA and enhance binding to human receptors. Viruses with this change probably have a replicative advantage in ferrets.

A reassortant virus with HA amino acid changes N158D/N224K/Q226L transmitted through the air to 2 of 6 ferrets. Viruses recovered from one of the animals contained a new change in the HA protein, T318I. A virus with four amino acid changes in the H5 HA (N158D/N224K/Q226L/T318I) replicates well in ferrets and transmits efficiently, although the infection is not lethal.

Even more interesting are the results of experiments to understand how these HA amino acid changes affect viral transmission. The N224K/Q226L amino acid changes that shift the HA from avian to human receptor specificity reduce the stability of the HA protein. The N158D and T318I changes, which were selected in ferrets, restore stability of the HA.

There are three key questions concerning this work that must be answered.

Would an H5N1 virus with the changes N158D/N224K/Q226L/T318I transmit among humans? Probably not. The virus tested by the authors derived 7 of 8 RNA segments from a human H1N1 strain, which is well adapted for human transmission. It is likely that changes in other avian influenza viral proteins would be needed for human transmission. It might also be that entirely different changes in the H5 HA are required for transmission in humans compared with ferrets.

Is this information useful for the surveillance of circulating H5N1 strains; specifically, would the emergence of these HA changes signify a virus with pandemic potential? I don’t believe so. These are mutations that enhance the transmission of H5 viruses in ferrets, and their effect in humans is unknown. Ferret transmission experiments are not meant to be predictive of what might occur in humans.

If these results are not predictive of what might happen in humans, why were these experiments done? (to paraphrase Laurie Garret at the New York Academy of Sciences Meeting on Dual Use Research). A substantial portion of this work goes far beyond surveillance of H5N1 strains: it provides a mechanistic framework for understanding what regulates airborne transmission of avian H5 influenza viruses. In the Kawaoka study, amino acid changes that improve the stability of the HA protein were selected for during replication and transmission of the H5 viruses in ferrets. In other words, stability of the HA protein is an important property that allows efficient airborne transmission among ferrets. Additional experiments can now be designed to extend this idea. If such stabilizing changes can be shown to be important for transmission of human strains, then they might be a valuable marker of influenza transmission.

The Kawaoka paper is a significant piece of work that substantially advances our understanding of what viral properties control airborne transmission of influenza viruses. To view it as enabling construction of a bioweapon is highly speculative and fundamentally incorrect.

M. Imai, T. Watanabe, M. Hatta, S.C. Das, M. Ozawa, K. Shinya, G. Zhone, A. Hanson, H. Katsura, S. Watanabe, C. Li, E. Kawakami, S. Yamada, M. Kiso, Y. Suzuki, E.A. Maher, G. Neumann, Y. Kawaoka. 2012. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.   doi: 10.1038/nature10831.

http://www.ims.u-tokyo.ac.jp/imsut/jp/lab/microbiologyimmunology/

WEDNESDAY, MAY 02, 2012
Nature Publishes The Kawaoka H5N1 Study

BSL-4 Lab Worker - Photo Credit –USAMRIID

# 6312

Although it was Ron Fouchier’s announcement last September (see New Scientist: Five Easy Mutations) of his successful manipulation of the H5N1 virus to make it transmissible among ferrets that set off biosecurity alarm bells (see NPR: Bio-Terrorism Concerns Over Bird Flu Research) and instigated an international debate, in a laboratory halfway around the world Dr. Yoshiro Kawaoka was quietly duplicating the feat.

Today (the embargo having lifted at 1pm) the journal Nature publishes Kawaoka’s study, which details how he created a modified H5N1 – H1N1 hybrid that transmits via respiratory droplets among ferrets.

While its virulence in ferrets was reduced over that of its parental H5N1 virus (exactly why is unknown), the big news is that it only took the introduction of 4 mutations into the H5N1’s hemagglutinin (HA) to allow it to bind efficiently to the alpha (2-6) sialic acid receptors found in ferret (and human) upper respiratory tracts.

Three of these mutations were directly involved in the binding to the a2,6 receptor cell, while the 4th stabilized the virus to fuse with receptor cells at pH levels typically found in the upper respiratory tract.

An affinity for a2,6 receptor cells has long been thought vital for the virus to adapt to humans, but this 4th mutation - that facilitates virus-cell fusion - may prove equally important.

Given its highly public and bumpy road to publication, Kawaoka’s study (see below) is certain to receive an inordinate amount of scrutiny and review over the next couple of days.

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to areassortant H5 HA/H1N1 virus in ferrets

doi:10.1038/nature10831
Masaki Imai, Tokiko Watanabe, Masato Hatta, Subash C. Das, Makoto Ozawa, Kyoko Shinya, Gongxun Zhong, Anthony Hanson, Hiroaki Katsura, Shinji Watanabe, Chengjun Li , Eiryo Kawakami , Shinya Yamada, Maki Kiso5,Yasuo Suzuki , Eileen A. Maher, Gabriele Neumann & Yoshihiro Kawaoka

Kawaoka’s 11-page study is, as you might imagine, highly technical and may prove tough sledding for those without a solid background in virology.

Nature published a 2-page synopsis by Hui-Ling Yen & Malik Peiris that helps to explain some of the details, but this is a pay-to-view article.

Bird flu in mammals

An engineered influenza virus based on a haemagglutinin protein from H5N1 avian influenza, with just four mutations, can be transmitted between ferrets, emphasizing the potential for a human pandemic to emerge from birds.

According to Malik Peiris, this study reaffirms the pandemic potential of the H5N1 virus, and reinforces the need for better surveillance of the virus in the wild.

While Kawaoka identified several mutations in the H5 HA that allowed the virus to bind to a2,6 receptors there are likely other combinations that could lead to greater transmissibility.

For the best all-around coverage of this story, we can always rely on Helen Branswell of The Canadian Press. Helen has just posted the following article on this research’s publication.

Bird flu study: Three mutations might give virus ability to transmit to humans
By: Helen Branswell, The Canadian Press

There will, undoubtedly, be a large number of stories and opinion pieces regarding the publication today of Kawaoka’s research.

Already I note that Vincent Racaniello has blogged on this publication:

Kawaoka paper published on aerosol transmission of H5 influenza virus in ferrets
2 May 2012

Meanwhile Laurie Garrett takes a more cautionary route in:

Manmade H5N1 Paper Finally Published, And Worrying

I’ll update this blog with more links to articles and opinion pieces on this research later today or tomorrow.

Posted by Michael Coston at 1:51 PM
Labels: H5N1, Kawaoka, Nature, Research