TWiV 190: The second ferret of the Apocalypse

On episode #190 of the science show This Week in Virology, Vincent, Alan, and Kathy review selection of influenza H5N1 viruses that can transmit among ferrets by aerosol.

You can find TWiV #190 at

Influenza H5N1 virus versus ferrets, round two

H5N1 mutationsThe second of two papers on avian influenza H5N1 virus that caused such a furor in the past year was published today in the journal Science. I have carefully read the paper by Fouchier and colleagues, and I assure you that it does not enable the production of a deadly biological weapon. The results describe the requirements for airborne transmission of influenza viruses among ferrets, but it provides no information about human to human transmission. Failure to publish this work would have substantially restricted our understanding of influenza transmission.

The authors modified the HA protein of an Indonesian strain of influenza H5N1 virus so that it could attach to cell receptors in the ferret respiratory tract. They also added a change in one of the subunits of the viral RNA polymerase, called PB2 protein, that improves replication in mammalian cells (E627K). This H5N1 virus, with the amino acid changes HA Q222L, G224S and PB2 E627K, did not transmit through the air among ferrets.

In an attempt to select a virus with airborne transmissibility, the authors passed their modified H5N1 virus in ferrets. They inoculated a ferret intranasally with virus, waited 4 days, harvested virus from the respiratory tract, and infected the next animal. After ten ferret-to-ferret passages, the pool of viruses produced by the last animal contained mutations in all but one of the 8 viral RNA segments. The original alterations were present (HA Q222L and G224S, PB2 E627K) together with a new change in the HA protein, T156A. This amino acid change prevents the addition of a sugar group to the protein near the receptor binding site, thereby increasing virus binding to mammalian cell receptors.

After ten ferret to ferret passages, the modified H5N1 virus could transmit from one animal to another housed in neighboring cages, e.g. by the aerosol route. All viruses acquired by ferrets by this route had five amino acid changes in common:  the original three introduced by mutagenesis (HA Q222L and G224S, PB2 E627K) and two selected in ferrets, HA H103Y and T156A.

The modified H5N1 virus does not transmit with high efficiency among ferrets, and it is not lethal when acquired by aerosol transmission. For this reason, and because we do not know if the virus would transmit among humans, it would not be an effective biological weapon.

A minimum of five amino acid changes in H5N1 virus are required for aerosol transmission among ferrets. This conclusion is based on the observation that the viruses acquired by ferrets through aerosol infection all had five amino acid changes in common. The actual number could be higher. For example, one virus that was studied in more detail differed from the parent H5N1 virus by nine amino acid changes, and other mutations were identified in other isolates. Determining the exact number will require introducing mutations in various combinations into H5N1 virus and testing transmission in ferrets. At present these experiments cannot be because there is a moratorium on H5N1 transmission research.

How do these results compare with those of Kawaoka and colleagues? Those authors found that five amino acid changes in the H5 HA are needed for airborne transmission among ferrets. However, they used a different virus, the 2009 H1N1 pandemic virus with an H5 HA protein. The latter was modified so it could recognize mammalian receptors. They found that amino acid changes that shift the HA from avian to human receptor specificity reduce the stability of the virus. The amino acid changes HA N158D and T318I, which were selected during infection of ferrets, restore stability. The T318I change is near the HA fusion peptide, distant from the receptor binding site. [In the figure, changes identified by Kawaoka and colleagues are in red; black are those identified by Fouchier and colleagues].

It is quite possible that both Kawaoka and Fouchier independently found that virion stability is an important property of viruses that can be transmitted through the air among ferrets. I wonder if Fouchier’s alterations to the HA, Q222L and G224S, destabilized the protein, like those introduced by Kawaoka. This possibility is suggested by the presence of the HA T318I amino acid change that was selected in ferrets. Amino acid 156 is in the HA trimer interface and could confer stability to the protein (the viral HA protein is composed of three copies of one polypeptide; the interface of these  three proteins determines its stability). It is an hypothesis that can be easily tested (even with the moratorium on H5N1 transmission research).

The results demonstrate that 5 to 9 amino acid changes are sufficient to allow influenza H5N1 virus to transmit by the aerosol route among ferrets. The findings provide no information about aerosol transmissibility of H5N1 virus in humans. We cannot conclude from this work that a similar number of changes in H5N1 virus will allow transmission among humans. That information can only come from the study of a pandemic H5N1 strain (should such a virus ever emerge).

There is a great deal of good science in this paper, and I cannot imagine hiding it in a vault, or only providing it to certain individuals. I find the findings intriguing, and I am sure that other virologists will be similarly fascinated. One of them might do a seminal experiment on H5N1 transmission as a consequence. But that would never happen if the paper were not published.

The new manuscript is very different from the version submitted in 2011. That paper, in typical Science article style, contained only one paragraph of background information. The experimental findings are described tersely and with little explanation. In contrast, the first five pages of the revised manuscript read like a review article, with substantial detail on influenza virus biology, host range, and the precautions taken during conduct of the experiments. The experimental results are carefully explained. The style is considered and soothing, in contrast with the stark presentation of the original manuscript. I now understand why the NSABB changed their mind and decided to publish this version.

TWiV 186: From Buda to grinding stumps

On episode #186 of the science show This Week in Virology, the TWiV chiefs tackle reader email about how to pronounce Buda, Texas, grinding tree stumps, and much more.

You can find TWiV #186 at

TWiV 185: Dead parrots and live Wildcats

On episode #185 of the science show This Week in Virology, Vincent visits with members of the Department of Microbiology and Immunology at Northwestern University School of Medicine to discuss their work on herpesviruses and parainfluenzaviruses.

You can find TWiV #185 at

TWiV 183: Bats out of hell

On episode #183 of the science show This Week in Virology, Connor Bamford joins the TWiV team to discuss bats as hosts for major mammalian paramyxoviruses.

You can find TWiV #183 at

TWiV 182: One flu over the ferrets’ nest

On episode #182 of the science show This Week in Virology, Michael Imperiale joins the TWiV crew to discuss the recently published influenza H5N1 transmission paper and how it was viewed by the NSABB.

You can find TWiV #182 at

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

h5 ha changesOne 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.

Too dangerous to publish?

Science magazine will be conducting a live chat on whether some scientific research is too dangerous to publish, and how governments are getting involved in regulating such studies. It will be moderated by Science writer David Malakoff and will include Gregory Viglianti of Boston University School of Medicine.

The live chat begins at 3 PM EST on Thursday, 26 April at this link.

Building the perfect bug

This past February I was interviewed by the Australian Broadcasting Company on the topic of the Fouchier and Kawaoka experiments on avian influenza virus H5N1. The video, Building the Perfect Bug, has been released by Journeyman Pictures and includes interviews with S.T. Lai, Laurie Garrett, Michael Osterholm, and Ron Fouchier (transcript available). It is far too alarmist for my taste, but both sides of the issue are represented.

The video includes sequences of me working in the cell culture laboratory. Note that I did wear a tie for my interview while Michael Osterholm did not.

TWiV 177: Live in Dublin

On episode #177 of the science show This Week in Virology, Vincent, Connor Bamford, Wendy Barclay, and Ron Fouchier discussed avian influenza H5N1 transmission experiments in ferrets and novel bunyaviruses at the 2012 Spring Conference of the Society for General Microbiology in Dublin, Ireland.

You can find TWiV #177 at