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?


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 217: I just flu in and my arms are shot

On episode #217 of the science show This Week in Virology, Vincent, Alan, Rich, and Dickson review influenza vaccines.

You can find TWiV #217 at www.microbe.tv/twiv.

End of moratorium on influenza H5N1 research

In early 2012 influenza virus researchers around the world decided to stop working on highly pathogenic avian influenza H5N1 virus. This decision came after work from the Fouchier and Kawaoka laboratories revealed the isolation of influenza H5N1 strains that can be passed among ferrets by aerosol. The moratorium on influenza H5N1 virus research has now been lifted, as described in a letter from influenza virologists to Science and Nature.

Lifting the embargo on H5N1 research is an important step forward for understanding what regulates influenza transmission. In my view it was an ill-conceived move, done to quell the growing concern over the adaptation of influenza H5N1 virus to aerosol transmission in ferrets. We now know that these viruses are not lethal for ferrets, and much of the outrage expressed about this work was misguided. In my view the moratorium has accomplished little other than delaying the conduct of important virology research.

According to the influenza virus researchers who signed on to the moratorium, its purpose was to:

…provide time to explain the public-health benefits of this work, to describe the measures in place to minimize pos- sible risks, and to enable organizations and governments around the world to review their policies (for example on biosafety, biosecurity, oversight, and communication) regarding these experiments.

An important consideration is the level of containment that will be required for studying influenza H5N1 transmission. WHO has released recommendations on risk control measures for H5N1 research, and individual countries will decided how to proceed. The US has not yet made a decision on the level of containment needed for H5N1 virus transmission research. Influenza virologists who participated in the moratorium have their own view:

We consider biosafety level 3 conditions with the considerable enhancements (BSL-3+) outlined in the referenced publications (11–13) as appropriate for this type of work, but recognize that some countries may require BSL-4 conditions in ac- cordance with applicable standards (such as Canada).

Their last statement forms the crux of the issue on H5N1 transmission research:

We fully acknowledge that this research—as with any work on infectious agents—is not without risks. However, because the risk exists in nature that an H5N1 virus capable of transmission in mammals may emerge, the benefits of this work outweigh the risks.

Viruses on Time

Poliovirus recently made the cover of Time magazine. Prompted by a reader question, I searched the Time archive to find out if there have been other virology-themed covers. I found fifteen in all, depicting poliovirus (3), herpesvirus (1), HIV/AIDS (4), influenza (5), and SARS coronavirus (2) (I did not distinguish between US and international editions).

The earliest virus-themed cover that I found has Jonas Salk on the cover of the 29 March 1954 issue. Behind Salk is an image of poliovirus particles, probably drawn from an electron micrograph. Salk’s field trial of inactivated poliovirus vaccine had begun in 1954, and in April of the next year the results would be announced:

Jonas Salk

John Enders made the cover of the 17 November 1961 issue, with poliovirions in the background. Enders had been awarded the Nobel Prize in 1954, along with Weller and Robbins, for being the first to propagate the virus in cell culture. This finding paved the way for Salk’s vaccine work.

John Enders

Viruses were not on the cover of Time for 23 years. The 2 August 1982 cover did not have a virus image, but touted herpes simplex virus as ‘Today’s scarlet letter”:


The 4 July 1983 cover featured disease detectives and AIDS:


AIDS returned on 12 August 1985, this time with an image of HIV:

AIDS threat

The 3 November 1986 cover featured the giant headline VIRUSES with a colorized scanning electron micrograph in the background. AIDS was also mentioned:


The Man of the Year for 1996 was virologist David Ho, who graced the cover of the 30 December 1996 issue, with virions reflected in his glasses. This is one of the coolest of the Time virus covers, in my opinion. Naming Ho Man of the Year was fully deserved and helped propel the virology field into the spotlight it deserved.

David Ho

The cover of the 23 February 1998 issue features the flu hunters and a background electron micrograph of influenza virus. This story followed the 1997 outbreak of influenza H5N1 in Hong Kong:

Flu hunters

The Hong Kong outbreak was also featured on the 9 March 1998 cover, with influenza virions in the green lettering:

Flu hunters 2

The SARS outbreak made the 5 May 2003 cover. There were two versions distributed in different countries:


SARS Nation

Avian influenza was later featured on two more covers, 9 February 2004 and 26 September 2005:

Bird flu

Death threat

The 2009 influenza H1N1 pandemic was on the cover of the 24 August 2009 issue:


And the latest virus on the cover is poliovirus, 14 January 2013:

Killing polio

Did I miss any?

The following covers did not feature viruses but were certainly relevant to virology. The antiviral interferon was featured on the 31 March 1980 issue:


Herbert Boyer, one of the pioneers of recombinant DNA technology, was on the 9 March 1981 issue:

Herbert Boyer

The 23 May 1998 cover featured a story on how the immune system fights off disease:

Immune system

Science under siege (sound familiar?) was the story on the 12 September 1994 issue:

Science under siege

The 12 September 1994 Time cover asked if we are losing the war against infectious diseases:

Killer microbes

 There have been 6,169 Time covers, and viruses have been featured on only fifteen. I understand that Time is not a science magazine, but I think it could do more for virology, and science in general (there were other science themed covers that I found, but not that many more).

I wonder how many viruses have been on the cover of Newsweek? Life Magazine? Scientific American?

TWiV 213: Not bad for a hobby

On the final episode of the year of the science show This Week in Virology, the TWiV team reviews twelve cool virology stories from 2012.

You can find TWiV #213 at www.microbe.tv/twiv.

TWiV 192: Viral tertulia

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.

Origin of the H5N1 storm

ferretI 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.

TWiV 190: The second ferret of the Apocalypse

On episode #190 of the science show This Week in Virology, Vincent, Alan, and Kathy review selection of influenza H5N1 viruses that can transmit among ferrets by aerosol.

You can find TWiV #190 at www.microbe.tv/twiv.

Influenza H5N1 virus versus ferrets, round two

H5N1 mutationsThe 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.

TWiV 186: From Buda to grinding stumps

On episode #186 of the science show This Week in Virology, the TWiV chiefs tackle reader email about how to pronounce Buda, Texas, grinding tree stumps, and much more.

You can find TWiV #186 at www.microbe.tv/twiv.