Influenza H5N1 is not lethal in ferrets after airborne transmission

1 March 2012

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.

 

  • Deena

    Dear Prof Racceniello,

    Thanks for the update, your summary adds to the CIDRAP statement here:
    http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/news/feb2912forum.html

    Sorry to be pedantic, but I think you might have missed the bracketed word in the sentence quoted below:
    “When the mutH5N1 virus is transmitted to ferrets via aerosol, none of the recipient animals develop [severe] disease.”

    Disease severity measurements in ferrets are not easy to measure and often measured qualitatively by counting sneezes or considering lethargy.  As far as I am aware, the most qualitative measurement of disease in ferrets infected with ‘flu is temperature telemetry, which is achieved by surgically implanting a small thermometer, which continually measures core temperature over time and therefore can catch the short (~8 hour) spike in temperature that is usually caused by ‘flu infection. 

  • http://www.virology.ws profvrr

    I just listened again to Fouchier on video and he mostly says ‘no disease’, only saying ‘severe disease’ once. I didn’t quote him in my article; I summarized his words. I had not read the CIDRAP article previously so I was not quoting that either, or else I would have noted it.

    That’s a spelling of my name that I’ve never seen before. 

  • Fili_bucardo

    Thank you Prof. Racaniello, to keep us updated on H5N1 debate. 

  • EllenHunt

    This whole thing has now attained a squirreliness factor that is much too high for my taste. 
    Things just don’t add up. 

    Why did Fouchier make those statements originally if they weren’t true? Vincent, this isn’t just about not quite true – a little clarification. He has revised his conclusion so that his original statements are complete hogwash.

    Can anyone verify the ferrets didn’t get severe disease – really?

    – I know the answer to the latter is no. Nobody can. We just have to trust him. But he’s changed his tune 180 degrees. That’s just weird. I have never seen a scientist do that before. Think about that.

    Please understand that I am not in the camp that suggests redaction – even if it were 100% fatal to every ferret in the study.  Yes, I know that probably 40+ members of Al Qaeda died in a probable plague manufacturing facility 3 years ago in Algeria. I know they really want to pull off a big event and kill a lot of Americans, the more the better as far as they are concerned. But getting in the way doesn’t help anything.

    We need to keep a healthy level of skepticism and remember that this whole business was raised to a high enough level that it would have been reported as a national security threat to the president.  Get the bunco boys out of Langley on the case and anything could happen. Yes, I could see very strong pressure being exerted to threaten, cajole and appeal to god , country and who knows what,  in order to get Fouchier to change his tune. The rationale would be to throw the bad boys off the scent. The Langley crew would target Fouchier, the source, as the point of control over the story. Without him on board, the show is over.

    I would be extremely careful where I stepped here Vincent. We may have the worst possible outcome of all out of this mess. We just might have a situation where the scientists and physicians who should have the straight story don’t.  Be very careful.

    If I could, I would do a replication study on my own. But I just don’t have the resources.

  • http://twitter.com/Metaviromics Oskar Karlsson

    Dear Prof Racaniello,

    Following the
    discussion closely it strikes me as a possible “tail wagging the dog” situation
    on the rise, where the perspective of biodefense and the public’s fright of human
    induced pandemics lend an inappropriate amount of power to an important but
    still minor function tied to research within infectious pathology.

    I do
    however get a bit more excited over the prospect of actually getting to read
    the article from Dr. Fouchier’s group myself in the future as he now hint’s at
    a more widespread use for the data from a genomics standpoint, not only giving
    insight into air transmissible influenza but also into possible changes in pathogenesis
    due to changed behavior in the virus.

    Being a
    resident in Europe, I’m also fascinated by the modest, in comparison, media
    coverage that we see here compared to the U.S. Even though the reporter have
    little knowledge in the field I believe that this clip, http://bit.ly/ytLSNu , with
    Dr. Fouchier is actually a good indicator of the interest here which seems a
    lot more research oriented and a lot less pandemic doomsday tilted.

    Thanks for
    a great show; it’s one of the weekly highlights for me (understanding
    supervisors even make sure I listen in each week).

  • http://www.virology.ws profvrr

    The difference in the findings reported by Fouchier in 2011 and at this meeting are puzzling, but they could be explained if we assume that since his original work Fouchier then tested an H5n1 virus with only the five mutations that he thought to be important. See the update to the article above for more information.

  • Doc Robb Williams

    A number of well known and not-so-well known influenza scientists have made a career out of scare-mongering about flu pandemics.  Publicity is good for funding.  Sensationalist reports bring the attention of governements that fund research.   Possibly exaggerating the importance of one’s findings is also a common technique which some big-shot or wannabe scientists can’t resist.  It is irresponsible to frighten the public in order to get a bigger share of the research grants.  Looks like the NSBB was fooled too.  The reason Fouchier has back-pedaled on the disease severity is that his lab has effectively been shut down.  He over-egged the pudding and its blown up in his face.  He wants to get the work published and get the next grant. 

    Of course, there are lots of excellent scientists that just get along with their work without having to hype it all the time, and if others find the results useful or interesting, they are glad.

  • A.T.

     Statements like “I would do a replication study on my own” are EXACTLY why the NSABB has tried to keep the full methods section from being published.  To do them safely requires a lot of resources, but repeating them at a lower BSL/BCL level does not; this is the worry, that others will attempt to repeat the studies unsafely leading to accidental release of the virus. 

    You should not lend credence to their arguments.

  • Drosha

    What I don’t understand is that how can NSABB make a decision like that without requesting to see the full data. They have been making claims about working on this for a long time, apparently they were working on the wrong things. And I thought the idea was that these results were submitted to Science or Nature. How can they submit the hyped ideas when their experimental results clearly show the reality?

  • Dorian McILROY

    Looks like it’s difficult to say which virus caused what level of pathology. Only publication will actually help us understand what’s going on here, otherwise we’re going to be left with a lot of ambiguity between results presented last year, and this more recent communication.

    On a biosaftey note, does anyone know whether the research staff working with these viruses get prophylactic tamiflu? If not, this is perhaps something that could be done to reduce risks of an accidental escape of the viruses.

    DMc

  • Wollclark

    What do you mean, “We may have the *worst possible outcome*…”?

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  • http://www.facebook.com/people/Carol-Smith/1385517334 Carol Smith

    Dorian asked: “does anyone know whether the research staff working with these viruses get prophylactic tamiflu?” 
    The Erasmus MC website has an FAQ about Fouchier’s research that has been up for a couple months. (At: http://bit.ly/t7ojp7 last accessed March 4 2012) Under the question, “Can researchers become infected with the virus and then spread it?”, the answer includes this statement: “The staff members have been vaccinated.”  Vaccinated against what strain of influenza? Repeated queries about this from reporters and outside experts have gone unanswered. What is the vaccine efficacy of the vaccine used to vaccinate the staff? That would be impossible to know, since the virus passed in the ferrets has apparently not infected any human beings to date. And yet, the statement “The staff members have been vaccinated” is intended to be part of the reassuring answer to the question: “Can researchers become infected with the virus and then spread it?”

    Note also that the very first sentence of the Erasmus MC bird flu FAQ states: “Erasmus MC researchers have discovered that the avian influenza virus spreads more easily among humans than previously thought.”

    In HUMANS, it says!

    Dr. R., what can you infer about a research group that for months refuses to correct an obvious error on its own website (“spreads more easily in humans”) ,and refuses to clarify a curious assertion (“the staff members have been vaccinated”), despite numerous requests and queries? 

    It certainly raises questions in my mind about the researchers’ interest in being responsive, transparent and candid with the public. And yet the researchers are upset that the public “misunderstands” them!

  • josh

    What is even more worrisome as I’ve preached many times is how the authors of the study glorify what they have done with the mass media.  They hype the importance, scare up interest by repeatedly being quoted by mass media and then ultimately we allocate more funds to H5N1 research.  Also I agree with A.T. statements, the disconnect some virologists have with research and potential outbreak or more “rolling of the eyes” at safety measures. There is so much pressure by universities and labs to “hype” research, take chances and publish before having a good handle on things. Many universities hire their own PR firms, have flashy web-pages and churn out data to obtain grants and students due to the pressure to perform.  I don’t care so much if the H5N1 studies are published in full…Nature and Science sure love them and want to publish…quality science is going in the crapper.  Take a deep look virologists….have a good handle on what your doing..be ready for the fall-out and back it up.  There is an old saying…”You should be ready to publish the follow-up publication even before the initial studies are in print”

  • http://twitter.com/coolvirus Xenobio

    The table in the 2nd slide is horrible. The text in the 2 left-hand columns appears to be running off the bottom of the slide. Maybe I haven’t had enough coffee today, but I can’t figure out which viruses, doses, and routes on the left go with which results in the morbidity/mortality and days post-infection columns on the right. I should go and watch the video later.

  • http://www.psandman.com/ Jody Lanard M.D.

    But as Fouchier said on camera in your March 26 interview in Dublin (TWiV 177): 

    “What was also in the original manuscript, and what I’ve also presented in Malta, is that if the ferrets receive the virus by aerosol they only got sick, they don’t drop dead at all.” 

    So he was claiming that the “original work” submitted to Science last August 11 contained the data that ferrets infected via the aerosol route did not die. That particular information was not dependent on subsequent research.

    (Sorry I can’t put a time-stamp on that quote. In the original broadcast, it came about 32:50 minutes into the discussion. But I don’t know if you are going to edit out the first 20 or so minutes of that broadcast, before the interviews really started.)

  • http://www.virology.ws profvrr

    So it would seem, and I believe him, having heard it in person. Hopefully we will soon see the manuscript and all will be revealed. In this case I don’t understand why NSABB was so scared of a virus that transmits from one ferret to another but doesn’t cause serious disease. It’s not even clear that the virus can pass serially (from ferret to ferret) by the airborne route, as does human influenza.

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