virology blog
About viruses and viral disease
On episode #321 of the science show This Week in Virology, Paul Duprex joins the TWiV team to discuss the current moratorium on viral research to alter transmission, range and resistance, infectivity and immunity, and pathogenesis.
You can find TWiV #321 at www.microbe.tv/twiv.
The gain of function experiments in which avian influenza H5N1 virus was provided the ability to transmit by aerosol among ferrets were met with substantial outrage from both the press and even some scientists; scenarios of lethal viruses escaping from the laboratory and killing millions proliferated (see examples here and here). The recent publication of new influenza virus gain of function studies from the laboratories of Kawaoka and Perez have unleashed another barrage of criticism. What exactly was done and what does it mean?
According to critics, virologists should not be entrusted to carry out gain of function studies with influenza virus; they are dangerous and of no scientific value. The headline of a Guardian article is “Scientists condemn ‘crazy, dangerous’ creation of deadly airborne flu virus†(the headline is at best misleading because the influenza virus that was reconstructed by Kawaoka and colleagues is not deadly when transmitted by aerosol). The main opponents of the work appear to be Lord May*, former President of the Royal Society; Harvard epidemiologist Mark Lipsitch; and virologist Simon Wain Hobson. They all have nasty things to say about the work and the people doing it. To his credit, author of the Guardian article Ian Sample (who likely did not write the headline) does present both sides of the study, and attempts to explain what was done. He even quotes Kawaoka on the value of the work. But much is left unsaid, and without a detailed analysis of the study, its importance is not readily apparent.
The work by Kawaoka and colleagues attempts to answer the question of whether an influenza virus similar to that which killed 50 million people in 1918 could emerge today. First they identified in the avian influenza virus sequence database individual RNA segments that encode proteins that are very similar to the 1918 viral proteins.
Next, an infectious influenza virus was produced with 8 RNA segments that encode proteins highly related to those of the 1918 virus. Each RNA segment originates from a different avian influenza virus, and differs by 8 (PB2), 6 (PB1), 20 (PB1-F2), 9 (PA), 7 (NP), 33 (HA), 31 (NA), 1 (M1), 5 (M2), 4 (NS1), and 0 (NS2) amino acids from the 1918 virus.
The 1918-like avian influenza virus was less pathogenic in mice and ferrets compared with the 1918 virus, and more pathogenic than a duck influenza virus isolated in 1976. Virulence in ferrets increased when the HA or PB2 genes of the 1918-like avian influenza virus were substituted with those from the 1918 virus.
Aerosol transmission among ferrets was determined for the 1918-like avian influenza virus, and reassortants containing 1918 viral genes (these experiments are done by housing infected and uninfected ferrets in neighboring cages). The 1918 influenza virus was transmitted to 2 of 3 ferrets. Neither the 1918-like avian influenza virus, nor the 1976 duck influenza virus transmitted among ferrets. Aerosol transmission among ferrets was observed after infection with two different reassortant viruses of the 1918-avian like influenza virus: one which possesses the 1918 virus PB2, HA, and NA RNAs (1918 PB2:HA:NA/Avian), and one which possesses the 1918 virus PA, PB1, PB2, NP, and HA genes (1918(3P+NP):HA/Avian).
It is known from previous work that amino acid changes in the viral HA and PB2 proteins are important in allowing avian influenza viruses to infect humans. Changes in the viral HA glycoprotein (HA190D/225D) shift receptor specificity from avian to human sialic acids, while a change at amino acid 627 of the PB2 protein to a lysine (627K) allows avian influenza viruses to efficiently replicate in mammalian cells, and at the lower temperatures of the human upper respiratory tract.
These changes were introduced into the genome of the 1918-like avian influenza virus. One of three contact ferrets was infected with 1918-like avian PB2-627K:HA-89ED/190D/225D virus (a mixture of glutamic acid and aspartic acid at amino acid 89 was introduced during propagation of the virus in cell culture). Virus recovered from this animal had three additional mutations: its genotype is 1918-like avian PB2-627K/684D:HA-89ED/113SN/190D/225D/265DV:PA-253M (there are mixtures of amino acids at HA89, 113, and 265). This virus was more virulent in ferrets and transmitted by aerosol more efficiently than the 1918-like avian influenza virus. The virus recovered from contact ferrets contained yet another amino acid change, a T-to-I mutation at position 232 of NP. Therefore ten amino acid changes are associated with allowing the 1918-like avian influenza virus to transmit by aerosol among ferrets. Aerosol transmission of these viruses is not associated with lethal disease in ferrets.
Previous studies have shown that changes in the HA needed for binding to human sialic acid receptors reduced the stability of the HA protein. Adaptation of these viruses to aerosol transmission among ferrets required amino acid changes in the HA that restore its stability. Similar results were obtained in this study of the 1918-like avian influenza virus, namely, that changes that allow binding to human receptors (HA-190D/225D) destabilize the HA protein, and changes associated with aerosol transmission (HA-89D and HA- 89D/113N) restore stability.
The ability of current influenza virus vaccines and antivirals to block replication with ferret transmissible versions of the 1918-like avian influenza virus was determined. Sera from humans immunized with the 2009 pandemic H1N1 strain poorly neutralized the virus, indicating that this vaccine would likely not be protective if a similar virus were to emerge. However replication of the ferret transmissible 1918-like avian influenza virus is inhibited by the antiviral drug oseltamivir.
Examination of influenza virus sequence databases reveals that avian viruses encoding PB2, PB1, NP, M, and NS genes of closest similarity to those of the 1918-like avian virus have circulated largely in North America and Europe. The PB2-627K change is present in 168 of 4,293 avian PB2 genes (4%), and the HA-190D change is in 9 of 266 avian H1 HA sequences (3%), and one also had HA-225D.
Most of the viral sequences used in this work were obtained quite recently, indicating that influenza viruses encoding 1918-like proteins continue to circulate 95 years after the pandemic. Â Now that we have discussed the work, we can summarize why it is important:
Do these experiments constitute an unacceptable risk to humans? Whether or not the 1918-like avian influenza virus, or its transmissible derivatives, would replicate, transmit, and cause disease in humans is unknown. While ferrets are a good model for influenza virus pathogenesis, they cannot be used to predict what will occur in humans. Nevertheless it is prudent to work with these avian influenza viruses under appropriate containment, and that is how this work was done. The risk is worth taking, not only because understanding transmission is fundamentally important, but also because of unanticipated results which often substantially advance the field.
The Guardian quotes Lipsitch as saying that “Scientists should not take such risks without strong evidence that the work could save lives, which this paper does not provideâ€. The value of science cannot only be judged in terms of helping human health, no matter what the risk. If we only did work to improve human health, we would not have most of the advances in science that we have today. One example is the biotechnology industry, and the recombinant DNA revolution, which emerged from the crucial discovery of restriction enzymes in bacteria – work that was not propelled by an interest in saving lives.
The results obtained from the study of the reconstruction of a 1918-like avian influenza virus are important experiments whose value is clear. They are not without risk, but the risk can be mitigated. It serves no useful purpose to rail against influenza virus gain of function experiments, especially without discussing the work and its significance. I urge detractors of this type of work to carefully review the experiments and what they mean in the larger context of influenza virus pathogenesis. I understand that the papers are complex and might not be easily understood by those without scientific training, and that is why I have tried to explain these experiments as they are published (examples here and here).
In the next post, I’ll explain the gain of function experiments recently published by the Perez laboratory.
*May’s objection is that the scientists carrying out the work are ‘grossly ambitious people’. All scientists are ambitious, but that is not what drives Kawaoka and Perez to do this work. I suggest that Lord May read the papers and base his criticism on the science.
To the collection of avian influenza viruses known to sporadically infect humans – H5N1, H7N9, H7N2, H7N3, H7N7, H9N2, and H10N7 – we can now add H10N8, recently found in two individuals in China.
Avian influenza virus H10N8 was first detected in tracheal aspirates from a 73 year old woman who was hospitalized in November 2013 for severe respiratory illness. The patient, who died, had previously visited a live poultry market. A second infection with this virus was detected in January 2014.
Virus isolated from tracheal aspirates on day 7 of illness was named A/Jiangxi-Donghu/346/2013(H10N8). Nucleotide sequence analysis of the viral genome reveals that it is a reassortant. The HA gene most closely resembles that of a virus isolated from a duck in Hunan in 2012, while the NA gene resembles that of a virus isolated from a mallard in Korea in 2010. All six other RNA segments resemble those from circulating H9N2 viruses in China. These viruses have also provided genes for H7N9 and H5N1 viruses.
Examination of the viral protein sequences provides some clues about virulence of the virus. The HA protein sequence reveals a single basic amino acid at the cleavage site, indicating that the virus is of low pathogenicity in poultry, like H7N9 virus. The sequence in the sialic acid binding pocket of the HA protein indicates a preference for alpha-2,3 linked sialic acids, typical  for avian influenza viruses (human influenza viruses prefer alpha-2,6 linked sialic acids). A lysine at amino acid 627 in the PB2 protein is known to enhance the ability of the virus to replicate at mammalian temperatures; the H10N8 virus has a mixture of lysine and glutamic acid, the residue associated with less efficient replication. The sequence of the M2 protein indicates that the virus is resistant to the antiviral adamantanes. In vitro testing indicated sensitivity to NA inhibitors Tamiflu and Relenza.
It is not known if this novel H10N8 virus will spread further in the human population. A novel influenza H7N9 virus was first detected in humans in early 2013 and has since caused 250 human infections with 70 deaths. Similar incursions of avian influenza viruses into humans have probably taken place for as long as humans have had contact with poultry. We are now adept at detecting viruses and therefore we are noticing these infections more frequently.
Live poultry markets are clearly a risk factor for humans to acquire infections with avian influenza viruses, as noted by Perez and Garcia-Sastre:
Live bird markets in Asia are undoubtedly the major contributor in the evolution of avian influenza viruses with zoonotic potential, a fact for which we seem to remain oblivious.
Given their role in transmitting new viruses from animals to humans, I wonder why live poultry markets are not permanently closed.
Update: George Gao agrees that the live poultry markets in China should be closed.
On episode #270 of the science show This Week in Virology, Vincent and Rich discuss avian influenza virus and an antiviral drug against smallpox with Dennis and Yoshi at the ASM Biodefense and Emerging Diseases Research Meeting in Washington, DC.
You can find TWiV #270 at www.microbe.tv/twiv.
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:
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.
Two back-to-back papers were published last week that provide a detailed analysis of what it would take for avian influenza H5N1 and H7N9 viruses to switch to human receptors.
Influenza virus initiates infection by attaching to the cell surface, a process mediated by binding of the viral hemagglutinin protein (HA) to sialic acid. This sugar is found on glycoproteins, which are polypeptide chains decorated with chains of sugars. The way that sialic acid is linked to the next sugar molecule determines what kind of influenza viruses will bind. Human influenza viruses prefer to attach to sialic acids linked to the second sugar molecule via alpha-2,6 linkages, while avian influenza viruses prefer to bind to alpha-2,3 linked sialic acids. (In the image, influenza HA is shown in blue on the virion (left) and as a single polypeptide at right. Alpha-2,3 linked sialic acid is shown at top).
Adaptation of avian influenza viruses to efficiently infect humans requires that the viral HA quantitatively switches to human receptor binding – Â defined as high relative binding affinity to human versus avian receptors. Such a switch is caused by amino acid changes in the receptor binding site of the HA protein. The HA of the H1N1, H2N2, and H3N2 pandemic viruses are all derived from avian influenza viruses that underwent such a quantitative switch in binding from avian to human sialic acid receptors.
Avian H5N1 influenza viruses have not undergone a quantitative switch to human receptor binding, which is one of the reasons why these viruses do not undergo sustained human-to-human transmission. It has been possible to introduce specific amino acid changes in the H5 HA protein that enable these viruses to recognize human sialic acid receptors. Such changes were required to select variants of influenza H5N1 virus that transmit via aerosol among ferrets. However none of these viruses have quantitatively switched to human receptor specificity.
In the H5N1 paper, the authors compared the structure of an H5 HA bound to alpha-2,3 linked sialic acid with the structure of an H2 HA (its closest phylogenetic neighbor) bound to alpha-2,6 linked sialic acid, revealing substantial differences in the receptor binding site. To predict what residues could be changed in the H5 HA to overcome these differences, the authors developed a metric to identify amino acids within the receptor binding site that either contact the receptor or might influence the interaction. They examined these amino acids in different H5 HAs, and identified residues which might change the H5 HA to human receptor specificity. As a starting point they picked two H5 viruses that have already undergone amino acid changes believed to be important for human receptor binding. The changes were introduced into the HA of a currently circulating H5 HA by mutagenesis and then binding of the HAs to purified sialic acids and human tracheal and alveolar tissues was determined.
The HA receptor binding site amino acid changes required for aerosol transmission of H5N1 viruses in ferrets did not quantitatively switch receptor binding of a currently circulating H5 HA from avian to human (the ferret studies were done using H5N1 viruses that circulated in 2004/05). The authors note that “These residues alone cannot be used as reference points to analyze the switch in receptor specificity of currently circulating and evolving H5N1 strains”.
However introducing other amino acid changes which the authors predicted would be important did switch the H5 HA completely to human receptor binding. Only one or two amino acids changes are required for this switch in recently circulating H5 HAs.
This work is important because it defines structural features in the receptor binding site of H5 HA that are critical for quantitative switching from avian to human receptor binding, a necessary step in the acquisition of human to human transmissibility. These specific residues can be monitored in circulating H5N1 strains as indicators of a quantitative switch to human receptor specificity.
Remember that switching of H5 HA to human receptor specificity is not sufficient to gain human to human transmissibility; what other changes are needed, in which genes and how many, is anyone’s guess.
These authors have also published (in the same issue of Cell) a similar analysis of the recent avian influenza H7N9 virus which has emerged in China to infect humans for the first time. They model the binding of sialic acid in the H7 HA receptor binding site, and predict that the HA would have lower binding to human receptors compared with human-adapted H3 HAs (its closest phylogenetic neighbor). This prediction was validated by studies of the binding of the H7N9 virus to sections of human trachea: they find that staining of these tissues is less intense and extensive than of viruses with human-adapted HAs. They predict and demonstrate that a single amino acid change in the H7 HA (G228S)Â increases binding to human sialic acid receptors. This virus stains tracheal sections better than the H7 parental virus.
These results mean that the H7N9 virus circulating in China might be one amino acid change away from acquiring higher binding to human alpha-2,6 sialic acid receptors. I wonder why a virus with this mutation has not yet been isolated. Perhaps the one amino acid change in the viral HA exerts a fitness cost that prevents it from infecting birds or humans. Of course, as discussed above, a switch in receptor specificity is likely not sufficient for human to human transmission; changes in other genes are certainly needed. In other words, the failure of influenza H7N9 virus to transmit among humans can be partly, but not completely, explained by its binding properties to human receptors.