TWiV 336: Brought to you by the letters H, N, P, and Eye

On episode #336 of the science show This Week in Virology, the TWiVsters explore mutations in the interferon pathway associated with severe influenza in a child, outbreaks of avian influenza in North American poultry farms, Ebolavirus infection of the eye weeks after recovery, and Ebolavirus stability on surfaces and in fluids.

You can find TWiV #336 at

Reconstruction of 1918-like avian influenza virus stirs concern over gain of function experiments

ferretThe 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.  Now that we have discussed the work, we can summarize why it is important:

  • An infectious 1918-like avian virus can be assembled from RNA segments from circulating viruses that is of intermediate virulence in ferrets. Ten amino acid changes are sufficient to allow this virus to transmit by aerosol among ferrets.
  • Confirmation that transmissibility of influenza virus among ferrets depends on a stable HA glycoprotein. This result was a surprising outcome of the initial studies on aerosol transmission of H5N1 avian influenza viruses among ferrets, and provided mechanistic information about what is important for transmission. Experiments can now be designed to determine if HA stability is also important for influenza virus transmission in humans.
  • We understand little about why some viruses transmit well by aerosol while others do not. Transmission should be a selectable trait – the virus with a random mutation can reach another host by aerosol, where it replicates and can transmit further. Why types of mutation allow better transmission? Why don’t avian influenza viruses become transmissible among humans more frequently? Are there fitness tradeoffs to becoming transmissible? These and similar questions about transmission can be answered with sutdies of the types discussed here. The list is not confined to influenza virus: aerosol transmission of measles virus, rhinovirus, adenovirus, and many others, is poorly understood. This work shows what can be done and will surely inspire similar work with other viruses.

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 provide”. 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.

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

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

Avian influenza H7N7 virus outbreak: Lessons for H7N9

Influenza H7 diversityAn outbreak of high-pathogenicity avian influenza H7N7 virus that took place on 255 poultry farms in the Netherlands during 2003 has been used to provide clues about the current avian influenza H7N9 viruses in China. During the Dutch outbreak 453 humans showed symptoms of illness and 89 were confirmed to have infection with the virus. Some interesting observations from that outbreak:

  • Conjunctivitis (inflammation of the membranes surrounding the eyelids) was observed in many of the human cases, as well as in later human infections with H7 influenza viruses. Apparently these viruses replicate well in the eye, which bears alpha-2,3 sialic acid receptors. From there the viruses could reach the nasal cavity via the nasolacrimal duct. 
  • There was one fatal infection during the Dutch outbreak, and virus isolated from this individual contained the amino acid change E627K in viral protein PB2, which is associated with higher replication of avian influenza viruses in mammals. This change likely arose during replication of virus in the patient as it was not observed in other isolates. The recent H7N9 isolates from China all have the PB2 E627K mutation.
  • In the Dutch outbreak there was no evidence for human to human transmission of H7N7 viruses. This conclusion is in part supported by phylogenetic analysis of viral sequences, which showed that during the outbreak the viruses diversified into multiple lineages with human strains at the ends of the trees (Figure; click to enlarge. Credit: Eurosurveillance).

So far the H7N9 virus does not appear to be spreading from human to human. This observation suggests that the virus is widespread in poultry in China, and that there have been multiple introductions into humans. It seems likely that these novel viruses arose relatively recently in China and some time thereafter had to opportunity to infect humans.

The question on everyone’s mind is whether the avian influenza H7N9 viruses will acquire the ability to transmit among humans. On this subject the authors have the following comment:

Although human infections with H7 influenza viruses have occurred repeatedly over the last decades without evidence of sustained human-to-human transmission, the absence of sustained human-to-human transmission of A(H7N9) viruses does not come with any guarantee.

It is possible that during replication in birds or humans, the H7N9 viruses might randomly acquire a mutation that allows for transmission. In the right place at the right time, such a virus could spread through the human population. Alternatively, such a transmission-facilitating mutation might interfere with the overall fitness of the virus, thereby preventing it from spreading. I favor the latter hypothesis because the H7N9 viruses have been transmitting since at least February 2013; they have undergone many replication cycles without such a mutation arising. If the virus has not entered wild birds, culling poultry could eradicate it from China – assuming that it has not gone elsewhere.

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

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.

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.