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 Kawaokaâ€™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 acidÂ 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.
Kawaokaâ€™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 donâ€™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. Â doi: 10.1038/nature10831.
It seems to me as if there is a better reason that the information in the paper will not be very useful for surveillance (in birds). Â
While there appear to be a reasons for selection of these mutations in ferrets, it’s far from obvious to me that there should be any selection for these mutations in birds, where the mode of Â virus transmission is probably different.
Â maybe in swine ?
Thanks for the tip
Thanks for the tip
Henry Niman says
There are several related issues that address the ability of the Kawaioka H5 to transmit in humans and most of these additional considerations are in play in Egypt.Â The first acquisition, N158D is common in clade 2.2, which is endemic in Egypt (humans and birds).Â Only one human isolate (from 2008) does not have the glycosylation site abolished.Â Moreover, N224K has alos been identified in Egypt.Â Â In addition,Â 2010 clade 2.2.1 F sequences were released in association with the 2011 PLOS paper Webby (St Judes) and colleagues, which had H1N1pdm09 and seasonal H1N1 sequnces in two of the polymerase genes (PB1 and PB2), which was recently re-confirmed by re-sequencing.
Henry Niman says
Examples of key mammalian transmission polymorphisms appearing in birds are well known.Â The most dramatic example is PB2 E627K, which was one of the three polymorphisms introduced in the Fouchier (Science) paper.Â It was also in the clade 1 internal genes used in the Donis (Virology) study.
E627K was noted in a subset of cases in the 1997 H5N1 Hong Kong outbreak.Â Kawaoka noted that it increased H5N1 virulence in mice, and also noted that polymerase activity was higher at lower temperatures (33 C), similar to the temperature in a human nose in the cold.Â Moreover E627K was in seasonal influenza A, again linking to mammalian influenza.Â The E at position 627 was linked to avian H5N1, consistent with a higher body temperature in birds.
However, E627K was found in clade 2.2 wild birds at Qinghai Lake in 2005.Â The isolates also had increased virulence in mice, but was fixed in clade 2.2 in birds (and was associated with detection in the upper respiratory tract of birds).Â E627K is still fixed in clade 2.2 in Egypt, 7 years after it was identified in bar headed geese at Qinghai Lake in central China.
Similarly, the abolishishment of the glycosylation site at position 158 (either due to N158D and/or T160A).Â N158D was associated witth ferret transmission in the Kawaoka study (and is probably one of the two acquired changes in the Fouchier study).Â However, N158D was also in the egret isolate used in the Donis study, and is present in most clade 2.2.1 G avian isolates in Egypt (and those that have N at position 158 have T160A).
Moreover, the additional change linked to ferret transmission in the Kawaoka study, creation of a glycosylation site at position 240 via A242S or A242T in ferrets infected with N158D or N158D with T318I, is also in birds.Â A242T is widepead in clade 2.2.1 F birds in Egypt.Â These birds also have Q196K, which is closely related to the Q196R used in the Donis study.
A242T is described here
Why did Fouchier have to obtain an export license when Kawaoka was doing similar experiments with the same virus?
The export license covered the manuscript, not the virus. Fouchier and Kawaoka worked on differen viruses: Fouchier was modified H5N1, Kawaoka was 2009 H1N1 with the H1 substituted with the H5.
but that was not the argument that was given, afaik.
I thought it was just because of the different legislation systems.