We celebrated the 200th episode of TWiV by visiting the National Emerging Infectious Diseases Laboratories at Boston University Medical Center, where we met with Elke, Paul, and Ron to talk about building and working in a BSL4 facility. It was an amazing visit that will be fully documented in an upcoming video. Here are some behind-the-scenes photographs of two memorable days.
On episode #200 of the science show This Week in Virology, Vincent, Alan, and Rich visit the National Emerging Infectious Diseases Laboratories at Boston University Medical Center, where they meet with Elke, Paul, and Ron to talk about building and working in a BSL4 facility.
You can find TWiV #200 at www.microbe.tv/twiv.
On episode #192 of the science show This Week in Virology, Vincent, Alan, and Rich answer listener email about bioinformatics, insects, influenza, laboratory classes, commensalism, reproducibility of data, and more.
You can find TWiV #192 at www.microbe.tv/twiv.
I still wonder why the influenza virus H5N1 ferret transmission studies generated such fear and misunderstanding among the public, the press, and even some scientists. I still cannot fully explain what transpired, but now that the papers have been published some new clues have emerged.
In my opinion, the main catalyst of the storm was the article Scientists brace for media storm around controversial flu studies by Martin Enserink. It began with the inflammatory statement ‘Locked up in the bowels of the medical faculty building here and accessible to only a handful of scientists lies a man-made flu virus that could change world history if it were ever set free’. Fouchier said that he created ‘probably one of the most dangerous viruses you can make’. Members of the NSABB were quoted as saying ‘I can’t think of another pathogenic organism that is as scary as this one’, and ‘This work should never have been done’.
This article presented a one-sided view because only Fouchier or NSABB members were quoted. I don’t understand why Fouchier made some of the statements that he did; perhaps he was quoted out of context. The NSABB members were on the way to restricting publication of the paper, so their views were clear. What Enserink did not do – what he should have done – was to speak with other virologists. This he could not do because the manuscript describing the work had not been made public. He violated a main tenet of journalism, to present both sides of the story.
With the publication of the Fouchier and Kawaoka papers, it became immediately apparent that all of the inflammatory statements in the Enserink article are wrong. For example, after 10 passages in ferrets, an altered H5N1 virus does transmit in the air among ferrets, but inefficiently and without killing the animals. Hardly one of the most dangerous viruses you can make.
To be fair, Enserink was not the first to report these findings. Fouchier presented the results of his H5N1 ferret transmission studies at a meeting in Malta in September 2011. A week later, New Scientist published an article on the findings entitled Five easy mutations to make bird flu a lethal pandemic. In the first paragraph, the author writes ‘…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.’ A few paragraphs later: ‘The tenth round of ferrets shed an H5N1 strain that spread to ferrets in separate cages – and killed them.’ Both the title and these statements are all wrong. Fouchier’s published paper does not prove that five mutations are sufficient for aerosol transmission among ferrets, and the virus does not kill ferrets when it is transmitted through the air. Also problematic is Fouchier quoted as saying that ‘The virus is transmitted as efficiently as seasonal flu’. Given the published data, and his comments at an ASM Biodefense Meeting in February 2012, I do not understand this statement.
It is easy to see how the misinformation in these two articles ignited the fear. Their stories were repeated by countless other publications without verifying whether or not they were correct, amplifying the false conclusions and spreading misinformation even further. Those of us who pointed out inconsistencies were dismissed as risk-takers. Even the New York Times – without ever having seen the data – declared that the experiments should not have been done and the virus stocks should be destroyed.
A comparison of the original Fouchier manuscript with the version recently published in Science provides additional insight (I do not have the original version of the Kawaoka paper). The first version of the manuscript was submitted to Science and reviewed by the NSABB, whose members recommended that it should be published in redacted form. Fouchier and colleagues then submitted a revised version which was reviewed by the NSABB, who then decided that the entire paper could be published.
Curiously, the titles of the paper are different. The original: Aerosol transmission of avian influenza H5N1 virus. The published version: Airborne transmission of influenza A/H5N1 virus between ferrets. The first is clearly ‘scarier’.
Another big difference between the two manuscripts is the length. A research article in Science is typically brief: it begins with a paragraph or two of background information, then delves right into the results. This is how the original Fouchier manuscript was constructed. In contrast, it is not until page five of the published version do we reach the data: the previous pages are filled with background material reminiscent of a review article. Included is information on the basic biology of influenza viruses, the functions of individual proteins, virulence, why the authors decided to do these studies, what is known to control host range and transmission, and the containment procedures that were undertaken. It is an impressive amount of background information, none of which was present in the original version. The additional material does help to put the experiments in their proper context.
I found two examples of changes in the wording that I feel could make substantive changes in reader perceptions of the results.
In the abstract of the original manuscript, the authors wrote:
The virus acquired the ability to transmit via aerosols or respiratory droplets while remaining highly pathogenic to ferrets.
In the revised manuscript, this sentence has been changed:
None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses.
The second version better represents the data. The first version is incorrect as stated because the virus is virulent only when inoculated intratracheally into ferrets, not after transmission by aerosols.
A second example occurs during discussion of the response of ferrets to infection with mutant H5N1 virus. In the original manuscript the authors note that after intransal inoculation, the animals have signs of disease but did not die. However, after intratracheal inoculation with the virus, all six ferrets died. Their conclusion:
These data are similar as described previously for A/H5N1wildtype and thus do not point to reduced virulence (italics mine).
In the revised manuscript this sentence has been modified:
These data are similar to those described previously for A/H5N1wildtype in ferrets. Thus, although the airborne-transmissible virus is lethal to ferrets upon intratracheal inoculation at high doses, the virus was not lethal after airborne transmission.
The first version is misleading because it does not clearly state that virulence was assessed by intratracheal inoculation.
For the most part the same data are presented in the two versions of the manuscripts. A virologist would not draw different conclusions from the two manuscripts despite the longer introduction and the two modifications noted above. I remain puzzled as to why the first manuscript raised such a furor. I cannot believe that it was simply a consequence of overzealous writers and a few scientific overstatements.
Although the Kawaoka and Fouchier papers have been published, the effect of the H5N1 storm will linger for a long time. The moratorium on H5N1 transmission research continues, meaning that important questions cannot be answered. On 29 March 2012 the United States government issued its Policy for Oversight of Life Sciences Dual Use Research of Concern (pdf). According to this new policy, seven different types of ongoing or proposed research (including transmission studies) on 15 different pathogens (including highly pathogenic avian influenza viruses) must now be reviewed by a committee for risk assessment and if the development of mitigation plans. Once the work is in progress, no deviations from proposed experiments are permitted without further review. I understand from a number of virologists that this policy has had a chilling effect on avian influenza virus research. As a consequence, this area of investigation is likely to substantially contract, depriving us of potentially important findings that could be useful in limiting influenza and other viral diseases.
We are in this position because access to the Fouchier and Kawaoka papers was restricted, and few could actually read them to understand exactly what was done. I can’t think of a better reason for unrestricted publication of scientific findings.
The 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.
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.
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.
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.
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 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.
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.”
…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.
Several panelists from the recent influenza H5N1 dual-use forum at the New York Academy of Sciences spoke with Brendan Maher of Nature News to discuss their position. Participants in this video include Laurie Garrett, Michael Osterholm, Ian Lipkin, Vincent Racaniello, and Veronique Kiermer.
Update: The New York Academy of Sciences has posted video of the full two hour panel discussion.