Yet another avian influenza virus, H10N8, infects humans

chicken market

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.

TWiV 270: Homeland virology

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.

Influenza H7N9 gain of function experiments on Dispatch Radio

I spoke with Robert Herriman, executive editor of The Global Dispatch, about the proposed avian influenza H7N9 virus gain of function experiments on Dispatch Radio.

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.

A single amino acid change switches avian influenza H5N1 and H7N9 viruses to human receptors

HA receptor binding siteTwo 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 –  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) 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.

TWiV 233: We’re surrounded

On episode #233 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review aerosol transmission studies of influenza H1N1 x H5N1 reassortants, H7N9 infections in China, and the MERS coronavirus.

You can find TWiV #233 at www.microbe.tv/twiv.

Avian influenza H7N9 viruses isolated from humans: What do the gene sequences mean?

Influenza A virionThere have been over 60 human infections with avian influenza virus H7N9 in China, and cases have been detected outside of Shanghai, including Beijing, Zhejiang, Henan, and Anhui Provinces. Information on the first three cases has now been published, allowing a more detailed consideration of the properties of the viral isolates.

The first genome sequences reported were from the initial three H7N9 isolates: A/Shanghai/1/2013, A/Shanghai/2/2013, and A/Anhui/1/2013. These were followed by genome sequences from A/Hongzhou/1/2013 (from a male patient), A/pigeon/Shanghai/S1069/2013), A/chicken/Shanghai/S1053/2013), and A/environment/Shanghai/S1088/2013, the latter three from a Shanghai market.

Analysis of the viral genome sequences reveals that all 8 RNA segments of influenza A/Shanghai/1/2013 virus are phylogenetically distinct from A/Anhui/1/2013 and A/Shanghai/2/2013, suggesting that the virus passed from an animal into humans at least twice. Similar viruses have been isolated from pigeons and chickens, but the source of the human infections is not known. There is as yet no evidence for human to human transmission of the H7N9 viruses, and it seems likely that all of the human infections are zoonotic – transmission of animal viruses to humans. Since the H7N9 viruses are of low pathogenicity in poultry, infected animals may not display disease symptoms, further facilitating transmission to humans.

The RNA sequences reveal that the H7N9 viruses isolated from humans are all triple reassortants, which means that they contain RNA segments derived from three parental viruses. The gene encoding the hemagglutinin protein (HA) is most closely related to the HA from A/duck/Zhejiang/12/2011 (H7N3), while the NA gene is most similar to the NA gene from A/wild bird/Korea/A14/2011 (H7N9). The remaining 6 RNA segments are most related to genes from A/brambling/Beijing/16/2012-like viruses (H9N2). The type of animal(s) in which the mixed infections took place is unknown.

Some observations on the relatedness of these sequences:

  • A/Shanghai/2/2013, A/Anhui/1/2013, and A/Hangzhou/1/2013 were isolated in distant cities yet have over 99% identity. The pigeon, chicken, and environmental isolates are also very similar except for one gene of A/pigeon/Shanghai/S1069/2013. Long-range shipping of infected poultry might explain these similarities.
  • There are 53 nucleotide differences between A/Shanghai/1/2013 and A/Shanghai/2/2013. Perhaps A/Shanghai/1/2013 and the remaining viruses originated from different sources.

When the gene sequences of these human viral isolates are compared with closely related avian strains, numerous differences are revealed. The locations of the proteins in the influenza virion are shown on the diagram; click for a larger version (figure credit: ViralZone).

  • All seven H7N9 viruses do not have multiple basic amino acids at the HA cleavage site. The presence of a basic peptide in this location allows the viral HA to be cleaved by proteases that are present in most cells, enabling the virus to replicate in many organs. Without this basic peptide, the HA is cleaved only by proteases present in the respiratory tract, limiting replication to that site. This is one reason why the H7N9 viruses  have low pathogenicity in poultry.
  • All seven viruses have a change at HA amino acid 226 (Q226L) which could improve binding of the viruses to alpha-2,6 sialic receptors, which are found throughout the human respiratory tract. Avian influenza viruses prefer to bind to alpha-2,3 sialic acid receptors. This observation suggests that the H7N9 isolates should be able to infect the human upper respiratory tract (alpha-2,3 sialic acid receptors are mainly located in the lower tract of humans). However, viruses which bind better to alpha-2,3 sialic acids still bind to alpha-2,6 receptors and can infect humans.
  • All seven viruses have a change at HA amino acid 160 from threonine to alanine (T160A). This change, which has been identified in other circulating H7N9 viruses, prevents attachment of a sugar to the HA protein and could lead to better recognition of human (alpha-2,6 sialic acid) receptors.
  • Five amino acids are deleted from the neuraminidase (NA), the second viral glycoprotein, in all seven viruses. In avian H5N1 influenza virus this change may influence tropism for the respiratory tract and enhance viral replication, and might regulate transmission in domestic poultry. This change is believed to be selected upon viral replication in terrestrial birds.
  • One of the viruses (A/Shanghai/1/2013) has an amino acid change in the NA glycoprotein associated with oseltamivir resistance (R294K).
  • An amino acid change in the PB1 gene, I368V, is known to confer aerosol transmission to H5N1 virus in ferrets.
  • An amino acid change in the PB2 gene, E627K, is associated with increased virulence in mice, higher replication of avian influenza viruses in mammals, and respiratory droplet transmission in ferrets.
  • Changes of P42S in NS1 protein, and N30D and T215A in M1 are associated with increased virulence in mice, but these changes are also observed in circulating avian viruses.
  • All seven viruses have an amino acid change in the M2 protein known to confer resistance to the antiviral drug amantadine.
  • All seven viruses lack a C-terminal PDZ domain-binding motif which may reduce the virulence of these viruses in mammals.

For the most part we do not know the significance of any of the amino acid changes for viral replication and virulence in humans.

I believe that these H7N9 viruses might take one of two pathways. If they are widespread in birds, they could spread globally and cause sporadic zoonotic infections, as does avian influenza H5N1 virus. Alternatively, the H7N9 viruses could cause a pandemic. Influenza H7N9 virus infections have not occurred before in humans, so nearly everyone on the planet is likely susceptible to infection. Global spread of the virus would require human to human transmission, which has not been observed so far. Some human to human transmission of avian H7N7 influenza viruses was observed during an outbreak in 2003 in the Netherlands, but those viruses were different from the ones isolated recently in China. Whether or not these viruses will acquire the ability to transmit among humans by aerosol is unknown and cannot be predicted. If a variant of H7N9 virus that can spread among humans arises during replication in birds or humans, it might not have a chance encounter with a human, or if it did, it might not have the fitness to spread extensively.

What also tempers my concern about these H7N9 viruses is the fact that the last influenza pandemic (H1N1 virus) took place in 2009.  No influenza pandemics in modern history are known to have taken place 4 years apart, although only 11 years separated the 1957 (H2N2) and 1968 (H3N2) pandemics. I suppose that is not much consolation, as there are always exceptions, especially when it comes to viruses.

Meanwhile a vaccine against this H7N9 strain is being prepared (it will be months before it is ready), surveillance for the virus continues in China and elsewhere, and health agencies ready for a more extensive outbreak. These are not objectionable courses of action. But should this be our response to every zoonotic influenza virus infection of less than 100 cases?

Sources

Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus.

Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013.

TWiV 227: Lacks security and bad poultry

On episode #227 of the science show This Week in Virology, the complete TWiV team reviews the controversial publication of the HeLa cell genome, a missing vial of Guanarito virus in a BSL-4 facility, and human infections with avian influenza H7N9 virus.

You can find TWiV #227 at www.microbe.tv/twiv.

First human infections with avian influenza H7N9 virus

comingled birdsFourteen people in China have been infected with avian influenza H7N9 virus, leading to five deaths. This avian influenza virus has never been isolated from humans.

Influenza A viruses with the H7 hemagglutinin protein circulate among birds, and some, such as H7N2, H7N3, and H7N7, have been previously found to infect humans. It is not known how the individuals in China acquired the H7N9 virus. Some of the infections have occurred in Shanghai, where a similar virus was found in pigeon samples collected at a marketplace in that city. It is not clear what types of pigeon samples tested positive for the virus, nor is it known whether the virus spread from poultry to pigeons or vice versa. In response the city has begun mass slaughter of poultry to stem further spread of the virus.

Influenza H7N9 virus is typically a low-pathogenicity virus, which means that infection of chickens causes mild respiratory disease, depression, and decrease in egg production. The virus does not have a basic peptide between HA1 and HA2. The presence of a basic peptide in this location allows the viral hemagglutinin glycoprotein to be cleaved by proteases that are present in most cells, enabling the virus to replicate in many organs. Without this basic peptide, the HA is cleaved only by proteases present in the respiratory tract, limiting replication to that site.

According to Brian Kimble on Google+, the nucleotide sequence reveals that the H7N9 human isolate is a reassortant* with 6 RNA segments encoding the internal proteins PB1, PB2, PA, NP, M, and NS derived from H9N2 virus, and the HA and NA from H7N9 virus. The significance of this observation is not clear, because I do not know if H7N9 viruses isolated from birds are also reassortants. One possibility is that reassortment produced a virus that can infect humans. It is known that reassortants of H9N2 viruses with the 2009 pandemic H1N1 strain can transmit via aerosols in ferrets.

An important question is whether this H7N9 virus isolated from humans has pandemic potential. So far there is no evidence for human to human transmission of the virus. There is no vaccine for this subtype of influenza virus, but the virus is susceptible to neuraminidase inhibitors oseltamivir and zanamivir. WHO has released the following statement:

Any animal influenza virus that develops the ability to infect people is a theoretical risk to cause a pandemic. However, whether the influenza A(H7N9) virus could actually cause a pandemic is unknown. Other animal influenza viruses that have been found to occasionally infect people have not gone on to cause a pandemic.

*Because the influenza virus genome occurs as 8 segments of RNA, when multiple viruses infect a single cell, new viruses can be produced with combinations of the parental segments, a process known as reassortment.

Update: Peter Palese notes that the human H7N9 isolates do not have a serine in position 61 (as does the 1918 virus). This change is a human virulence marker for some animal influenza viruses. Brian Kimble notes that the H7N9 isolates possess a L226 equivalent in the HA, which confers human-like receptor binding in other viruses. Human influenza viruses prefer to bind to alpha-2,6 sialic acid receptors, while avian strains bind alpha-2,3 sialic acids. If the human H7N9 viruses can bind alpha-2,6 sialic acid receptors then they are adapted to infect the human upper respiratory tract.