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 232: Gophers go viral

On episode #232 of the science show This Week in Virology, Vincent meets up with Roberto, Reuben, Lou, and Leslie at the University of Minnesota to talk about their work on HIV-1, APOBEC proteins, measles virus, and teaching virology to undergraduates.

You can find TWiV #232 at www.microbe.tv/twiv.

Nature just is

What better way to start 2013 than with a meaningful quote from Jon Yewdell:

We might think we know how Nature should work, and we certainly gain insight into Nature by using our logical powers (endowed by Nature) to predict how Nature might work, but ultimately, we have to understand the way Nature does work. Nature, in all its glorious complexity, is completely impassive. It cares not a whit what we may or may not believe. Nature just is.

It’s astounding how many scientists don’t really get this.

Jon’s statement includes the subject of this blog – viruses – which have no intentions or desires. Viruses are the product of mutation and selection, the goal of which is simply existence. Evolution does not move viruses along a trajectory aimed at perfection. Change comes about by eliminating those viruses that are not well adapted for the current conditions, not by building something that will fare better tomorrow. Viruses just are.

Vaccine-associated poliomyelitis in Pakistan

Poliovirus by Jason RobertsAn outbreak of ten cases of poliomyelitis caused by circulating vaccine-derivied poliovirus type 2 (cVDPV2) is ongoing in Pakistan, centered in the Kila Abdulla/Pishin area of Baluchistan. The same virus strain has spread to the neighboring Kandahar province in Afghanistan, where two paralytic cases have been reported. Vaccine-derived poliomyelitis is a well-known consequence of immunization with the Sabin poliovirus vaccine.

There are three serotypes of poliovirus, each of which causes poliomyelitis. The three vaccine strains developed by Albert Sabin (OPV, oral poliovirus vaccine) contain mutations which prevent them from causing paralytic disease. When the vaccine is taken orally, the viruses replicate in the intestine, and immunity to infection develops. While replicating in the intestinal tract, the vaccine viruses undergo genetic changes. As a consequence, the OPV recipients excrete neurovirulent polioviruses. These so-called vaccine-derived polioviruses (VDPV) can cause poliomyelitis in the recipient of the vaccine or in a contact. During the years that the Sabin poliovirus vaccines were used in the US, cases of poliomyelitis caused by VDPV occurred at a rate of about 1 per 1.4 million vaccine doses, or 7-8 per year. Once the disease was eradicated from the US in 1979, the only cases of polio were caused by VDPVs. For this reason the US switched to the Salk (inactivated) poliovirus vaccine in 2000.

Because VDPVs are excreted in the feces, they can spread in communities. These circulating VDPVs, or cVDPVs, can cause outbreaks of poliomyelitis in under-immunized populations. Examples include outbreaks of poliomyelitis in an Amish community and in Nigeria in 2009 caused by cVDPV2. Nigeria employed trivalent OPV before 2003, the year that this country began a boycott of polio immunization. Because type 2 poliovirus had been eradicated from the globe in 1999, when immunization in Nigeria resumed in 2004, monovalent types 1 and 3 vaccine were used. The source of the VDPV type 2 in Nigeria was the trivalent vaccine used before 2003.

For many years the vaccine used by WHO in the global eradication effort was a trivalent preparation comprising all three serotypes. When type 2 poliovirus was eliminated, many countries began immunizing only against types 1 and 3 poliovirus. As a consequence of this immunization strategy, population immunity to type 2 poliovirus declined. This has likely lead to the emergence of cVDPV2 in Pakistan, together with poor routine immunization coverage.

The resurrection of poliovirus type 2 highlights the difficulties in eradicating a pathogen using a vaccine that can readily mutate to cause the disease that it is designed to prevent. As wild type polioviruses are eliminated, the only remaining polio will be caused by the vaccine. If immunization is then stopped, as planned by WHO, there will likely be outbreaks of polio caused by cVDPV of all three serotypes. The solution to this conundrum is to switch to the inactivated vaccine until cVDPVs disappear from the planet.

Exacerbating the polio situation in Pakistan was the murder in the past week of nine immunization workers in several provinces. The Taliban, which carried out the executions, accused them of being spies. This accusation originates from the CIA operation in 2011 in which a Pakistani doctor ran an immunization program in Abbottabad in an attempt to obtain DNA samples from the Bin Laden family. As a result of this violence, immunization campaigns in Balochistan have been suspended. Coupled with the previous refusal of many parents to have their children immunized, this action makes it likely that poliovirus will spread more extensively in the country, making eradication even more difficult.

Poliovirus image courtesy of Jason Roberts.

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.

TWiV 83: An hour with Dr. Kiki

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Kirsten Sanford

On episode #83 of the podcast This Week in Virology, Vincent, Alan, Rich, and special guest Dr. Kirsten Sanford talk about her career in science media, then consider whether smallpox eradication led to the AIDS pandemic, high fidelity RNA synthesis, and a new Ebola virus vaccine.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #83 (66 MB .mp3, 91 minutes)

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Poliovirus vaccine safety

Albert SabinThe contamination of the rotavirus vaccine Rotarix with porcine circovirus 1 DNA was revealed by deep sequencing. The same technique was also used to demonstrate that oral poliovirus vaccine does not contain viruses that can cause poliomyelitis.

The oral poliovirus vaccine strains developed by Albert Sabin (pictured) were licensed in the United States in 1962, and over the next 37 years immunization with these vaccines lead to the eradication of poliomyelitis in this country. During that period, the vaccine was responsible for 5-10 cases of poliomyelitis each year, either in recipients of the vaccine or in their contacts. Some of these individuals have sued the manufacturers of the vaccine, claiming that they made a defective product.

OPV contains three different poliovirus strains which were selected by Sabin because they do not cause poliomyelitis. We call such vaccine strains avirulent or attenuated. The mutations in the genetic information of the virus that prevent the development of paralysis have been identified. Unfortunately, these mutations are unstable. After oral administration, OPV replicates in the intestinal tract. During this phase the vaccine viruses undergo genetic change and eventually lose the mutations that made them avirulent. As a consequence, nearly every infant who receives OPV sheds in the feces polioviruses that are significantly more neurovirulent than those that were ingested.

Vaccine-associated poliomyelitis is caused by vaccine revertants that accumulate in the alimentary tract of immunized individuals. These neurovirulent viruses arise not because the vaccine is improperly prepared, but as a consequence of mutation during replication in the intestine. Proving this point to lay juries has been difficult. Now deep sequencing of poliovirus vaccine can show whether or not vaccine preparations are contaminated with neurovirulent viruses.

Deep sequence analysis of OPV manufactured by Bharat Biotech was done to detect mutations associated with neurovirulence. There are four mutations in the genome of type 1, two in the genome of type 2, and three in the genome of type 3 that are important for the attenuated property of the vaccine. The base present at each of these positions in the neurovirulent wild type viruses, and in the vaccine strains, is shown in the table.

Determinants of attenuation

The results of sequence analysis show that the Bharat vaccine does not contain any of the ‘wild type’ bases at these nine positions. Any vaccine-associated poliomyelitis associated with this vaccine is not a consequence of faulty production, but the fact that vaccine strains mutate during replication in the human gut.

There have been many lawsuits involving vaccine-associated poliomyelitis in which plaintiffs claim that the OPV was incorrectly manufactured, leading to a product of unacceptably high neurovirulence. Deep sequencing analysis of these lots of vaccine could have resolved this claim in a way that a lay jury could understand.

Reassortment of the influenza virus genome

Mutation is an important source of RNA virus diversity that is made possible by the error-prone nature of RNA synthesis. Viruses with segmented genomes, such as influenza virus, have another mechanism for generating diversity: reassortment.

When an influenza virus infects a cell, the individual RNA segments enter the nucleus. There they are copied many times to form RNA genomes for new infectious virions. The new RNA segments are exported to the cytoplasm, and then are incorporated into new virus particles which bud from the cell.

If a cell is infected with two different influenza viruses, the RNAs of both viruses are copied in the nucleus. When new virus particles are assembled at the plasma membrane, each of the 8 RNA segments may originate from either infecting virus. The progeny that inherit RNAs from both parents are called reassortants. This process is illustrated in the diagram below, which shows a cell that is co-infected with two influenza viruses L and M. The infected cell produces both parental viruses as well as a reassortant R3 which inherits one RNA segment from strain L and the remainder from strain M.

influenza-reassortment

One example of the evolutionary importance of reassortment is the exchange of RNA segments between mammalian and avian influenza viruses that give rise to pandemic influenza. For example, the 2009 H1N1 pandemic strain is a reassortant of avian, human, and swine influenza viruses, as illustrated.

influenza-h1n1-reassortment

Reassortment can only occur between influenza viruses of the same type. Why influenza A viruses never exchange RNA segments with influenza B or C viruses is not understood. However, the reason is probably linked to the packaging mechanism that ensures that each influenza virion contains at least one copy of each RNA segment.

Trifonov, V., Khiabanian, H., & Rabadan, R. (2009). Geographic Dependence, Surveillance, and Origins of the 2009 Influenza A (H1N1) Virus New England Journal of Medicine DOI: 10.1056/NEJMp0904572

The trajectory of evolution

quasispecies-selectionScientists and philosophers have long debated the trajectory of evolution. Some of the questions they consider include: is there a predictable direction for evolution, and if there is, what is the pathway? Are there evolutionary dead ends?

Viruses are excellent subjects for the study of evolution: they have short generation times, high yields of offspring, and prodigious levels of mutation, recombination, and reassortment. Furthermore, selection pressures can be readily applied in the laboratory, and may be often be identified in nature.

When studying evolution of viruses, it is important to avoid judging outcomes as ‘good’ or ‘bad’. Anthropormorphic assessments of virus evolution come naturally to humans, but concluding that viruses become ‘better adapted’ to their hosts, for example, fails to recognize the main goal of evolution: survival. Or, in the case of the non-living viruses, existence.

Evolution does not move a viral genome from simple to complex, or along a trajectory aimed at perfection. Change comes about by eliminating those viruses that are not well adapted for the current conditions, not by building something that will fare better tomorrow.

Virulence: A positive or negative trait for evolution?

1856663523_cffa76bfbc_mWith just 141 confirmed deaths so far, an interesting question is whether the 2009 H1N1 influenza virus could mutate into something more lethal (“How a Mild Virus Might Turn Vicious“). Of course it could – but is it beneficial for the virus?

A fundamental principle of viral evolution is that viruses must spread from host to host to maintain the viral population. A virus spreads only if an infected individual passes the virus on to more than one new host. Furthermore, infection can spread only if population density exceeds a minimal value.

Some scientists believe that increased viral virulence reduces transmissibility. When infected hosts die faster, exposure to uninfected hosts is reduced. According to Ian Lipkin:

“A really aggressive flu that quickly kills its host” – like SARS and H5N1 avian flu – “gives itself a problem”.

According to this hypothesis, virulence is selected against as the virus spreads in humans. This idea leads to statements like this one:

In the last year, dozens of H5N1 cases have been confirmed in toddlers, almost all of whom have survived – which led some experts to speculate that those are cases of a less lethal version of H5N1 that is better adapted to humans.

Why is reduced lethality equated with being better adapted to humans? And how could the virus become better adapted to humans when human to human transmission has been minimal?

There is insufficient evidence to conclude that increased viral virulence leads to reduced transmission. For example, the 1918 influenza virus strain was extremely virulent, yet spread very efficiently among humans.  SARS and H5N1 influenza aren’t good examples – SARS transmission was probably stopped by containment efforts, and H5N1 influenza virus hasn’t transmitted well among humans, if at all.

In today’s highly crowded and mobile society, even a very lethal virus can be transmitted well. Acute viral infections are preceded by an incubation period, during which virus is shed but symptoms are not yet severe enough to lead to hospitalization. And even a highly pathogenic virus will cause mild or no disease in some individuals – further increasing the chances of spreading infection.

It seems more likely that increased viral virulence could lead to better transmission. For example, a more virulent influenza virus might cause more coughing and sneezing, which would be more effective in transmitting infection. Perhaps we should focus on transmissibility, not virulence, as the property that drives viral evolution. Viruses evolve so they can be efficiently transmitted to other hosts. According to this hypothesis, any other properties that accompany transmissibility, such as virulence, are secondary effects. If this idea were true, then all viruses would evolve to be maximally infectious and avirulent. But this is not the case. Perhaps, as Peter Palese said, viral virulence has unknown benefits:

“Look, I believe in Darwin. Yes, the fittest virus survives. But it’s not clear what the ultimate selection parameter is.” A mutation that confers lethality, he explained, may confer another advantage scientists have not pinned down.