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 www.microbe.tv/twiv.

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 www.microbe.tv/twiv.

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 www.microbe.tv/twiv.

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 www.microbe.tv/twiv.

NSABB reverses decision on publication of H5N1 results

The National Science Advisory Board for Biosecurity (NSABB) has re-examined two manuscripts on the transmissibility of influenza H5N1 virus in ferrets:

After careful deliberation, the NSABB unanimously recommended that this revised Kawaoka manuscript should be communicated in full. The NSABB also recommended, in a 12 to 6 decision, the communication of the data, methods, and conclusions presented in this revised Fouchier manuscript.

The NSABB reached this decision using ‘analytical tools that it previously developed for considering the risks and benefits associated with the communication of dual use research of concern.

Apparently information communicated in revised versions of the Fouchier and Kawaoka manuscripts changed the Board’s risk/benefit calculation:

The data described in the revised manuscripts do not appear to provide information that would immediately enable misuse of the research in ways that would endanger public health or national security.

New evidence has emerged that underscores the fact that understanding specific mutations may improve international surveillance and public health and safety.

This decision (full text here) is welcome, although I wonder how the manuscripts have been ‘revised’ – were data added or removed? Furthermore, why does the NSABB now feel that the results do not endanger public health, and can be used to improve international surveillance? These arguments have been made previously but the NSABB discounted them.

I look forward to publication of the Fouchier and Kawaoka findings and a comprehensive discussion of how they influence influenza H5N1 transmission in ferrets.

Update: According to the New York Times, the chair of the NSABB said “the new decision was not a reversal, because the revised manuscripts were so different from the originals. Had these versions been presented originally, the board would not have recommended withholding any details”.

Did the authors remove data from the manuscripts, or just clarify them?

Update 2. According to Kawaoka, quoted in the The Chronicle of Higher Education, the revisions of his manuscript “provided a more in-depth explanation of the significance of the findings to public health and a description of the laboratory biosafety and biosecurity.” His paper, he added, would contain descriptions of all the mutations that enhanced transmission of the virus, the very data that initially concerned the board.” Furthermore, Ron Fouchier wrote to me in an email that “the manuscripts will indeed be published in full.” All this is very good news.

TWiV 173: Going to bat for flu research

On episode #173 of the podcast This Week in Virology, the TWiVites discuss seroevidence for human infection with avian influenza H5N1, and the discovery of a new influenza virus in Guatemalan bats.

You can find TWiV #173 at www.microbe.tv/twiv.

Influenza H5N1 is not lethal in ferrets after airborne transmission

Ron Fouchier has discussed his influenza H5N1 transmission experiments in ferrets at an ASM Biodefense Conference, clarifying several assumptions about the transmissibility of the virus in this animal model.

Two different influenza H5N1 strains were used for Fouchier’s experiments: a wild type virus, and a mutated virus (we’ll call it mutH5N1). He did not reveal the nature of the mutations in this virus but from previous reports they consist of changes introduced into the viral HA protein to allow binding to sialic acid receptors in the avian respiratory tract, and other changes selected during passage in ferrets.

Ferrets are housed in neighboring cages separated by steel grids to allow free air flow between cages. The cages are placed in a class 3 biosafety hood within a BSL3+ facility. A ferret in one cage is inoculated intranasally with virus, and then ferrets in neighboring cages are assayed for presence of virus in the respiratory tract. When ferrets are inoculated with wt H5N1 virus, viral replication ensues in the respiratory tract, but the virus is not transmitted to animals in neighboring cages. When ferrets are inoculated with mutH5N1, the virus is transmitted to 3/4 ferrets in neighboring cages. If the mutH5N1 virus is recovered from these animals and used to infect new ferrets, it is then transmitted to 2/2 ferrets in neighboring cages. The results are summarized in the following figure:

fouchier slide 1

Fouchier concluded that this work identified the mutations that are needed for H5 transmission between ferrets.

Next Fouchier indicated that because the work has not yet been published, and the press has ‘picked up on it’, there are many misconceptions about what can or cannot be concluded. For example, it has been suggested that this virus would spread ‘like wildfire’ if it were to get out of his facility. He presented data indicating that this would not be the case. Although his results demonstrate aerosol transmission of H5N1 among ferrets, the assay is not quantitative, and therefore the efficiency of transmission cannot be deduced. He showed results of ferret transmission studies using the 2009 H1N1 pandemic influenza virus strain. This virus spreads to all ferrets by aerosol and replicates to high titers in the respiratory tract. In comparison, the mutH5N1 virus does not transmit to all ferrets, virus titers are lower, and shedding does not begin until later in infection. He concluded that the mutH5N1 virus does not transmit among ferrets as does a pandemic or seasonal influenza virus.

The second misconception that he addressed is that the mutH5N1 virus would be highly lethal. He showed the results of experiments demonstrating that when ferrets are inoculated intranasally with high doses of mutH5N1 virus, only 1/8 animals show signs of disease. In contrast, 2 of 2 ferrets developed disease when inoculated in the same way with wild type H5N1 virus. When the mutH5N1 virus is transmitted to ferrets via aerosol, none of the recipient animals develop disease. Only when the mutH5N1 virus is delivered to the lower respiratory tract of ferrets by intratracheal intubation does the virus cause disease in 6 of 6 animals.

Finally, Fouchier showed that pre-exposure of ferrets to seasonal influenza virus protects them from disease caused by H5N1 viruses. These findings are summarized on the following figure.

fouchier slide 2

After this presentation Dr. Anthony Fauci, head of the National Institute of Allergy and Infectious Disease, said that “There is a gross, pervasive misunderstanding out there,” and recommended that the data be re-examined by the NSABB.

The data presented by Fouchier appear to be at odds with the conclusions of the NSABB to redact publication. They are also not consistent with statements made by Fouchier and others to Science magazine in November 2011. For example, Fouchier called mutH5N1 “probably one of the most dangerous viruses you can make”, and Paul Keim, head of the NSABB, said “I can’t think of another pathogenic organism that is as scary as this one.”

Update: Here is what Fouchier said about his work at the Malta meeting in September 2011, as reported by New Scientist. The article begins with the statement:

…five mutations in just two genes have allowed the virus to spread between mammals in the lab. What’s more, the virus is just as lethal despite the mutations.

Fouchier is quoted as saying “The virus is transmitted as efficiently as seasonal flu.” This is in direct contrast to what he reported at the ASMBiodefense meeting.

In describing the passage of H5N1 in ferrets, the writer concludes:

The tenth round of ferrets shed an H5N1 strain that spread to ferrets in separate cages – and killed them.

Again this is in direct contrast to what Fouchier reported this past week.

I do not understand the difference between what Fouchier said in Malta in 2011 and in Washington, DC in February 2012. However, there is one way to explain the apparent paradox, which derives from the following statment from the New Scientist article:

The process yielded viruses with many new mutations, but two were in all of them. Those plus the three added deliberately “suggest that as few as five are required to make the virus airborne”, says Fouchier. He will now test H5N1 made with only those five.

Perhaps the results that Fouchier reported in Washington, DC are from experiments using H5N1 virus with only those five mutations.