Virologists plan influenza H7N9 gain of function experiments

A group of virologists lead by Yoshihiro Kawaoka and Ron Fouchier have sent a letter to Nature and Science outlining the experiments they propose to carry out with influenza H7N9 virus.

Avian influenza H7N9 virus has caused over 130 human infections in China with 43 fatalities. The source of the virus is not known but is suspected to be wet market poultry. No human to human transmission have been detected, and the outbreak seems to be under control. According to the authors of the letter, the virus could re-emerge this winter, and therefore additional work is needed to assess the risk of human infection.

The research that the virologists propose involve gain-of-function experiments which provide the H7N9 virus with new properties. The isolation of avian influenza H5N1 viruses that can transmit by aerosol among ferrets is an example of a gain-of-function experiment.

The proposed gain-of-function experiments fall into five general categories:

  • Determine whether viruses with altered virulence, host range, or transmissibility have changes in antigenicity, or the ability of the virus to react with antibodies. The results of these studies would suggest whether, for example, acquisition of human to human transmissibility would have an impact on protection conferred by a vaccine produced with the current H7N9 virus strain.
  • Determine if the H7N9 virus could be adapted to mammals and whether it could produce reassortants with other influenza viruses. The results of this work would provide information on how likely it is that the H7N9 virus would become better adapted to infect humans.
  • Isolate mutants of H7N9 virus that are resistant to antiviral drugs. The purpose of these experiments is to identify how drug resistance arises (the mutations can then be monitored in clinical isolates), determine the stability of drug resistant mutants, and whether they confer other properties to the virus.
  • Determine the genetic changes that accompany selection of H7N9 viruses that can transmit by aerosol among mammals such as guinea pigs and ferrets. As I have written before, the point of these experiments, in my view, is not to simply identify specific changes that lead to aerosol transmission. Such work provides information on the mechanisms by which viruses can become adapted to aerosol transmission, still an elusive goal.
  • Identify changes in H7N9 virus that allow it to become more pathogenic. The results of these experiments provide information on the mechanism of increased pathogenicity and whether it is accompanied by other changes in properties of the virus.

I believe that the proposed gain-of-function experiments are all worth doing. I do not share the concerns of others about the potential dangers associated with gain-of-function experiments: for example the possibility that a virus selected for higher virulence could escape the laboratory and cause a lethal pandemic. Gain-of-function is almost always accompanied by a loss-of-function. For example, the H5N1 viruses that gained the ability to transmit by aerosol among ferrets lost their virulence by this route of infection. When these experiments are done under the proper containment, the likelihood that accidents will happen is extremely small.

All the proposed experiments that would use US funds will have to be reviewed and approved by the Department of Health and Human Services:

The HHS review will consider the acceptability of these experiments in light of potential scientific and public-health benefits as well as biosafety and biosecurity risks, and will identify any additional risk-mitigation measures needed.

While I understand that the authors wish to promote a dialogue on laboratory safety and dual-use research, I question the ultimate value of the communication. Because the letter has been published in two scientific journals, I assume that the target audience of the letter is the scientific community. However, the letter will clearly have coverage in the popular press and I am certain that it will be misunderstood by the general public. I can see the headlines now: “Scientists inform the public that they will continue to make deadly flu viruses”. The controversy about the H5N1 influenza virus transmission studies in ferrets all began with a discussion of the results before the scientific papers had been published. I wonder if the publication of these letters will spark another controversy about gain-of-function research.

In my view, science is best served by the traditional process known to be highly productive: a grant is written to secure funding for proposes experiments, the grant proposal is subject to scientific review by peers, and based on the review the work may or may not be supported. The experiments are done and the results are published. I do not understand why it is necessary to trigger outrage and debate by announcing the intent to do certain types of experiments.

I am curious to know what the many readers of virology blog – scientists and non-scientists – feel about the publication of this letter. Please use the comment field below to express your views on this topic.

TWiV 241: The ferret looks ill

On episode #241 of the science show This Week in Virology, Vincent, Alan, Rich and Kathy review how human placental trophoblasts confer viral resistance via exosome-mediated delivery of microRNAs, and isolation of the first human influenza virus in 1933.

You can find TWiV #241 at www.microbe.tv/twiv.

TWiV 236: Flu gets the VIP treatment

On epside #236 of the science show This Week in Virology, Vincent, Alan and Kathy review novel approaches to preventing influenza virus infection.

You can find TWiV #236 at www.microbe.tv/twiv.

Inefficient influenza H7N9 virus aerosol transmission among ferrets

ferretThere have been 131 confirmed human infections with avian influenza H7N9 virus in China, but so far there is little evidence for human to human transmission. Three out of four patients report exposure to animals, ‘mostly chickens‘, suggesting that most of the infections are zoonoses. Whether or not the virus will evolve to transmit among humans is anyone’s guess. Meanwhile it has been found that one of the H7N9 virus isolates from Shanghai can transmit by aerosol among ferrets, albeit inefficiently.

Ferrets were inoculated intranasally with influenza A/Shanghai/02/2013 virus or A/California/07/2009, the 2009 pandemic H1N1 virus. One to two days later the ferrets developed fever, sneezing, coughing, and nasal discharge; both viruses induced similar clinical signs. Virus was shed in nasal secretions for 7 days. Six infected ferrets were then divided among three separate cages, and each group was housed with a naive ferret, and a second uninfected animal was placed in an adjacent cage. Airflow was controlled so that air flowed from the cage of infected animals towards the cage of naive animals. Transmission of infection was measured by observing clinical signs, and measuring virus shedding in nasal secretions and hemagglutination-inhibition antibodies in serum.

Of the three ferrets housed in the same cage with H7N9 virius-infected animals, all three had signs of infection (sneeze, cough, nasal discharge), shed virus in nasal secretions, and developed anti-HA antibodies. All three ferrets in neighboring cages developed signs of infection, but only one shed virus in nasal secretions, and two of three seroconverted. From these data the authors conclude that H7N9 virus is ‘efficiently transmitted between ferrets by direct contact, but less efficiently by airborne exposure’. In contrast, transmission of H1N1 virus to naive ferrets by contact or aerosol was efficient (3/3 animals in both cases).

The authors also found that pigs could be infected intranasally with A/Shanghai/02/2013 virus: the animals shed virus in nasal secretions and developed clinical symptoms. However the infected pigs transmitted infection inefficiently to other pigs by contact or aerosol, or to ferrets by aerosol.

The  authors’ equivocal conclusion that “Under appropriate conditions human to human transmission of the H7N9 virus may be possible” could have been reached even before these experiments were done. Their results provide no information on whether the virus can undergo human to human transmission because animal models are not definitive predictors of what might occur in humans. I disagree with the authors’ statement on page 5, “Efficient transmission of influenza viruses in ferrets is considered as a predictor of human to human transmissibility’. While many influenza virus strains that transmit among humans by aerosol also do so in ferrets, this does not mean that human transmission of a novel virus can be predicted by animal experiments.

Infection of ferrets with A/Shanghai/02/2013 or or A/California/07/2009 virus results in mild disease with no mortality. In contrast, 32 humans infected with H7N9 virus have died, and many humans have died after H1N1 infection. These findings further emphasize the differences in influenza virus pathogenesis in ferrets and humans.

Further defense of the Chinese H1N1 – H5N1 study

Robert Herriman of The Global Dispatch interviewed me this week on the H1N1 – H5N1 reassortant study that has been in the headlines:

There was much written concerning the research published earlier this month in Science, where researchers from China’s Harbin Veterinary Research Institute reported creating an  avian H5N1 (highly pathogenic) and pandemic 2009 H1N1 (easily transmissible) hybrid, that according to them, achieved airborne spread between guinea pigs.

Read the rest of the article at The Global Dispatch.

Ferreting out the truth on Science Sunday Hangout on Air

I joined Buddhini SamarasingheScott Lewis, Tommy Leung, and William McEwan for a discussion of the avian influenza H5N1 virus transmission experiments done in ferrets.

 

Influenza H5N1 x H1N1 reassortants: ignore the headlines, it’s good science

Those of you with an interest in virology, or perhaps simply sensationalism, have probably seen the recent headlines proclaiming another laboratory-made killer influenza virus. From The Independent: ‘Appalling irresponsibility: Senior scientists attack Chinese researchers for creating new strains of influenza virus’; and from InSing.com: ‘Made-in-China killer flu virus’. It’s unfortunate that the comments of several scientists have tainted what is a very well done set of experiments. Let’s deconstruct the situation with an analysis of the science that was done.

It is known that avian influenza H5N1 viruses can occasionally infect but not transmit among humans, while the 2009 pandemic H1N1 virus (which continues to circulate) readily transmits from person to person. The investigators asked whether reassortants of the two viruses – which could arise in nature – might confer transmissibility to H5N1 virus. To answer this question they produced 127 different reassortants of the two viruses, and tested their ability to transmit by aerosol among guinea pigs. The latter have been used for transmission studies on influenza, notably to understand the seasonality of infection. Ferrets have been more famously used for influenza virus transmission studies.

Rather than describe the results, I’ve made an illustration that shows what I believe to be the most important conclusions of the study (click for a larger version):

h1n1 h5n1 reassortants

The H5N1 virus (red RNAs) is not transmissible among guinea pigs, while the H1N1 virus (green RNAs) has highly efficient transmission. Exchange of the H5N1 RNA coding for PA or NS from H1N1 produces a highly transmissible virus. Exchange of the H5N1 RNA coding for NA or M produces a less efficiently transmitted virus. These are interesting and novel findings. It will be of great interest to determine how the PA, NS, NA, or M genes mechanistically enhance aerosol transmission. This is important information because our understanding of the determinants of transmission is very poor.

All the reassortant viruses shown in the figure have the H5 HA; when only the H1 of the H1N1 virus was substituted with the H5 HA, the reassortant virus transmitted efficiently among guinea pigs. In ferrets the H5 HA is not compatible with aerosol transmission. Therefore guinea pigs are clearly different from ferrets with respect to the determinants of transmissibility.

I cannot understand why some scientists have called these experiments ‘appallingly irresponsible’ and of no scientific use. I can only assume that they are not familiar with the literature on viral transmission and do not appreciate how the results advance our understanding of the field. It also seems irresponsible to predict that these viruses, should they escape from the laboratory, could kill millions of people. If you accept guinea pigs as a predictor of human pathogenicity – which I do not – then there is no reason for fear because none of the reassortants were lethal. I do not believe that any animal model predicts what will occur in humans, and so I am even less concerned about the safety of these experiments. I firmly believe that laboratory-constructed viruses do not have what it takes to be a human pathogen: only viral evolution in nature can produce the right combination of RNA segments and mutations. I also believe that scientists are quite responsible when it comes to safe handling of pathogens. If we worry about every type of transmission experiment involving influenza H5N1 virus, we will never make progress in understanding why this virus does not transmit among humans. The moratorium on H5N1 transmission research is over; let’s move beyond the sensational headlines and get back to the science.

In summary, I believe that these are well designed experiments which show that single RNA exchanges with H1N1 virus can produce an H5N1 virus that transmits via aerosol among guinea pigs. The relevance of these findings to humans is not known; nevertheless understanding how the individual viral proteins identified in this study enhance transmission may be mechanistically informative. I believe that the news headlines depicting these experiments as irresponsible and dangerous are based on uninformed statements made by scientists who are not familiar with the literature on influenza virus transmission. I wonder if they even read the paper in its entirety before making their comments.

TWiV 230: Gene goes to Washington, flu chickens out

On episode #230 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review H7N9 infections in China, the debate over patenting genes, and receptor-binding by ferret-transmissible avian H5 influenza virus.

You can find TWiV #230 at www.microbe.tv/twiv.

Harvard University: Great virology, bad science writing

Harvard virologyHarvard University is home to some of the world’s finest virologists. But apparently they do not communicate with the writers at Harvard Magazine, where a botched story on the avian H5N1 influenza virus has just been published.

The problems begin with the first paragraph:

But when Dutch researchers recently created an even more deadly strain of the virus in a laboratory for research purposes, they stirred grave concerns about what would happen if it escaped into the outside world.

Readers of virology blog will know by now that the Dutch researchers did not make an ‘even more deadly strain of the virus’ – they made one that could be transmitted by aerosol, but which had lost its lethality.

The title of the article, ‘The Deadliest Virus’, presumably refers to the H5N1 virus that transmits by aerosol among ferrets. This title is simply wrong, because the virus is not deadly to ferrets.

The first paragraph also contains an equally egregious statement by epidemiologist Marc Lipsitch:

If you make a strain that’s highly transmissible between humans, as the Dutch team did, it could be disastrous if it ever escaped the lab.

Dr. Lipsitch seems to be saying that the Dutch group created an H5N1 virus that transmits among humans. As far as I know, ferrets are not humans.

The article is accompanied by a photograph of two scientists working in BSL4 suits. The legend reads:

The modified H5N1 virus could infect a billion people if it escaped a biocontainment lab like the Canadian facility shown above.

And later Lipsitch is quoted as saying:

It could infect millions of people in the United States, and very likely more than a billion people globally, like most successful flu strains do. This might be one of the worst viruses—perhaps the worst virus—in existence right now because it has both transmissibility and high virulence.

For Lipsitch to say that the virus is both transmissible and of high virulence in humans is a misrepresentation of the Dutch group’s findings. He seems to be making up numbers and scenarios.

Perhaps Dr. Lipsitch does not know that ferret studies are not predictive of how viruses will behave in humans. With so many virologists at Harvard, the writer could have checked Dr. Lipsitch’s statements. But he did not, and the result looks as foolish as the New York Times.

The risks and benefits of influenza H5N1 research

Both Nature and the New York Times have weighed in on the resumption of influenza H5N1 research. In an editorial from 23 January 2013, Nature opines that “Experiments that make deadly pathogens more dangerous demand the utmost scrutiny”:

As several critics point out, the assessments of the relative risks and benefits of such research remain restricted to largely qualitative arguments. The formal, quantitative risk assessment common in the nuclear power and other industries could have helped to nail down and quantify risks, and would have informed the debate better. One year on, an irreproachable, independent risk–benefit analysis of such research, perhaps convened by a body such as the World Health Organization (WHO), is still lacking.

The Times editors, who looked foolish in January 2012 after remarking that the H5N1 ferret transmission research should not have been done*, simply tow Nature’s line.

To clarify a point, the Fouchier and Kawaoka experiments on influenza H5N1 transmission did not make the virus more dangerous – they made it less dangerous for ferrets. How they affect the virus in humans is unknown.

I suspect that no one, not even WHO, has done a quantitative risk-benefit analysis of H5N1 research because it cannot be done. What basic research will reveal is frequently unknown – if the outcome could be predicted, then it would not be research. Scientists ask questions, and design experiments to answer them, but the results remain elusive until the experiments are done. How can the benefits be quantified if the outcome isn’t certain?

For example, one of the benefits of influenza H5N1 research is to understand what regulates aerosol transmission of the virus. It is without doubt an important question, but whether or not research will provide an answer is unknown. At best, we might identify the determinants of aerosol transmission in ferrets – but not in humans. I don’t know the solution to this problem –  should we simply assume that we will get answers to all the questions we ask? Should we conclude that H5N1 research will allow us to understand H5N1 transmission and pathogenesis, thereby leading to vaccines and antiviral drugs or novel therapies? In this case there is no doubt that the benefits of H5N1 research are very high, but I can’t put a number on it. Nature calls this a ‘qualitative’ argument. But if someone tried to make a quantitative risk-benefit analysis of H5N1 research it would be fiction.

What is the risk of influenza H5N1 virus research? Many influenza researchers feel that it is low, if work with infectious virus is carried out under the right containment conditions. Perhaps the more relevant question is what is the risk of releasing experimental results that could be used for nefarious purposes. Because H5N1 transmission experiments utilize animal models, the results cannot be directly extrapolated to humans. If a virus is isolated that transmits by aerosol among ferrets, it cannot be concluded that the same virus will transmit among humans. Also remember that gain of aerosol transmission among ferrets was accompanied by a loss of fitness – the altered virus did not cause lethal disease when transmitted by aerosol. It seems unlikely that these research findings could be used to successfully produce a biological weapon.

It seems unlikely that someone intent on producing an H5N1 biological weapon would base it on work done in ferrets, or any other animal model. Their solution would be to passage the virus in humans – an unethical experiment, but which one could imagine being done by unethical individuals. Even the outcome of this experiment would not be assured – no one knows if an H5N1 virus selected for aerosol transmission among humans would have high lethality.

I understand why the Times would ask for a cost-benefit analysis of basic scientific research – the editors are not scientists and do not understand the unpredictable nature of research. But I expected more from the science journal Nature. Have the editors who wrote this opinion forgotten how scientific research is done?

*Without having read the papers, the Times editors decided that the H5N1 ferret experiments should not have been done. When the papers were published we all learned that the modified H5N1 viruses were not lethal to ferrets.