The D225G change in 2009 H1N1 influenza virus

sialic-acid-2Last year a mutation in the HA gene of the 2009 H1N1 influenza virus was identified in isolates from patients with severe disease. At the time I concluded that the emergence of this change was not a concern. Recently the Norwegian Institute of Public Health reported that the mutation, which causes a change from the amino acid aspartic acid to glycine at position 225 of the viral HA protein (D225G), has been identified in 11 of 61 cases (18%) of severe or fatal influenza, but not in any of 205 mild cases. Have these observations changed my view of the importance of this mutation?

The cell receptor for influenza A virus strains is sialic acid. Human influenza A strains bind preferentially to sialic acids linked to galactose by an alpha(2,6) bond, while avian and equine strains prefer alpha(2,3) linked sialic acids (pictured). Alpha(2,6) linked sialic acids are dominant on epithelial cells in the human nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi. Alpha(2,3) linked sialic acids are found on nonciliated bronchiolar cells at the junction between the respiratory bronchiole and alveolus, and on type II cells lining the alveolar wall.

The 2009 swine-origin H1N1 influenza virus is known to bind both alpha(2,3) and alpha(2,6) linked sialic acids. This is consistent with the ability of the virus to cause lower respiratory tract disease. The D225G change might be expected to increase affinity for alpha(2,3) linked sialic acids. However, it is not known if increased binding affinity correlates with higher infectivity and pathogenicity. It’s equally likely that high affinity binding might restrict the movement of the virus in lung tissues by causing retention of the virus on nonsusceptible cells.

One view of the D225G mutation is that it is spreading globally and causing more severe disease. However there is no evidence in support of this hypothesis. According to WHO, viruses with the D225G change have been found in 20 countries since April 2009, but there has been no temporal or geographic clustering. As of January, the HA change has been identified in 52 sequences out of more than 2700. Furthermore, the authors of the Norwegian study write, “Our observations are consistent with an epidemiological pattern where the D225G substitution is absent or infrequent in circulating viruses, with the mutation arising sporadically in single cases where it may have contributed to severity of infection”.

One explanation for the sporadic emergence of influenza viruses with the D225G change is that they are selected for in the lower respiratory tract where alpha(2,3) sialic acids are more abundant than in the upper tract. Such selection might be facilitated in individuals with compromised lung function (e.g. asthmatics, smokers) or suboptimal immune responses, in whom the virus more readily reaches the lung. One way to address this hypothesis would be to compare the HA at amino acid 225 of viral isolates obtained early in infection, from the upper tract, with isolates obtained from the lower tract late in disease. However such paired isolates have not yet been obtained. But whether the presence of viruses with D225G increases viral virulence is unknown. Many H1N1 isolates from cases of fatal or severe disease do not contain this amino acid change.

There is an alternative explanation for the isolation of at least some influenza viruses with the D225G change: it is selected by propagation in embryonated chicken eggs. This selection occurs because cells of the allantoic cavity of chicken eggs have only alpha(2,3) linked sialic acids. A change in receptor specificity does not occur when viruses are propagated in MDCK (canine kidney) cells, which possess sialic acids with both alpha(2,3) and alpha(2,6) linkages. Consistent with this hypothesis, WHO reports (pdf) that the D225G substitution in 14 virus isolates occurred after growth in the laboratory.

Studies on the binding of influenza viruses to glycan arrays have shown that attachment is influenced not only by the linkage to the next sugar, but the type of sialic acid as well as the rest of the carbohydrate chain. The distribution of all the possible sialic acid containing sugars in the respiratory tract is unknown, as is the specific molecules that can support productive viral infection. The view that HA preferentially binds to either alpha(2,3) or alpha(2,6) linked sialic acids is likely to be overly simplistic: another casualty of reductionism.

Kilander A, Rykkvin R, Dudman SG, & Hungnes O (2010). Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009-2010. Euro surveillance : bulletin europeen sur les maladies transmissibles = European communicable disease bulletin, 15 (9) PMID: 20214869

Takemae N, Ruttanapumma R, Parchariyanon S, Yoneyama S, Hayashi T, Hiramatsu H, Sriwilaijaroen N, Uchida Y, Kondo S, Yagi H, Kato K, Suzuki Y, & Saito T (2010). Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs. The Journal of general virology, 91 (Pt 4), 938-48 PMID: 20007353

Garcia-Sastre, A. (2010). Influenza Virus Receptor Specificity. Disease and Transmission American Journal Of Pathology DOI: 10.2353/ajpath.2010.100066

It’s not easy to make the 2009 H1N1 influenza virus a killer

influenza-rna-2The second RNA segment of some influenza virus strains encodes a protein called PB1-F2 that might contribute to virulence. Speaking about the 2009 pandemic H1N1 strain, Peter Palese noted that “If this virulence marker is necessary for an influenza virus to become highly pathogenic in humans or in chickens, then the current swine virus doesn’t have what it takes to become a major killer.” If the pandemic virus mutated so that the PB1-F2 protein is produced, would it become a killer?

The PB1-F2 protein is not produced in cells infected with the 2009 H1N1 strain because there are three translation stop codons at nucleotide positions 12, 58, and 88.  To determine if this protein plays a role in virulence, the second RNA segment of the A/California/04/2009 H1N1 strain was genetically altered to code for a full-length PB1-F2 protein. When mice or ferrets were infected intranasally with the modified virus, no significant differences in symptoms of infection were observed compared with mice infected with Cal/09 virus. The parameters measured included weight loss, viral replication in the lungs, and lung pathology.

The PB1-F2 protein has been shown to increase the severity of primary viral and secondary bacterial infections in mice. However, no increased mortality was observed in mice co-infected with Streptococcus pneumoniae and Cal/09 virus that can produce PB1-F2 protein.

Some differences were observed that might be attributed to the production of PB1-F2 protein. Synthesis of this protein was associated with enhanced replication in a human respiratory cell line. Furthermore, mice infected with the modified virus produced higher levels of some pro-inflammatory cytokines than mice infected with Cal/09 virus. The significance of these observations is unclear. Higher levels of virus production can influence transmission of infection among hosts, but the effect of PB1-F2 on this property was not examined. While increased proinflammatory cytokines could exacerbate or ameliorate disease, there was no effect on pathogenesis in mice.

The authors conclude that “mutations enabling the production of PB1-F2 in the Cal/09 influenza virus do not have a significant impact on virus virulence in mice or in ferrets.” Whether similar results would be observed in humans is unknown. But not all PB1-F2 proteins are the same: that produced by the genetically altered Cal/09 virus is different from the protein made by the 1918 H1N1 virus. It would be interesting to determine if the nature of the PB1-F2 protein has any effect on the virulence of the 2009 pandemic virus.

Hai, R., Schmolke, M., Varga, Z., Manicassamy, B., Wang, T., Belser, J., Pearce, M., Garcia-Sastre, A., Tumpey, T., & Palese, P. (2010). PB1-F2 expression by the 2009 pandemic H1N1 influenza virus has minimal impact on virulence in animal models Journal of Virology DOI: 10.1128/JVI.02717-09

Influenza PB1-F2 protein and viral fitness

influenza-rna-2The second RNA segment of the influenza virus genome encodes the PB1 protein – part of the viral RNA polymerase – and, in some strains, a second protein called PB1-F2. The latter protein is believed to be an important determinant of influenza virus virulence. The absence of a full-length PB1-F2 protein has been suggested as one possible determinant for the low pathogenicity of the 2009 influenza H1N1 pandemic strain. Analysis of the evolutionary history of PB1-F2 suggests that it does not contribute significantly to viral fitness – the ability of the virus to replicate.

PB1-F2 binds to mitochondria, leading to a release of cytochrome c and induction of apoptosis in CD8 T-cells and alveolar macrophages. The protein increases the severity of primary viral and secondary bacterial infections in mice, and is associated with the high pathogenicity of avian H5N1 and the 1918 H1N1 pandemic virus.

The PB1-F2 protein is not produced in cells infected with the 2009 H1N1 strain because there are three stop codons at nucleotide positions 12, 58, and 88.  The PB1 segment of the 2009 H1N1 strain is related to PB1 of H1N2 and H3N2 swine viruses from 1998 and human H3N2 viruses. Curiously, all the relatives of the 2009 H1N1 strain in swine and in humans encode a complete PB1-F2 protein. A truncated PB1-F2 is encoded by the genome of classical swine H1N1 viruses and human H1N1 viruses since 1947. But 96% of the avian influenza virus sequences deposited in NCBI as of 2007 encode the full length version of the protein.

Because the full-length PB1-F2 protein is not encoded in the genome of many influenza viruses, its evolutionary role and contribution to the fitness of the virus is unclear. To answer these questions, the evolution of PB1-F2 was compared with PB1 and two other open reading frames of similar size within the same RNA segment that are not translated into protein.

PB1-F2 is complete in all H1N1 human isolates before 1947, when a stop codon appeared which leads to production of a shorter version of the protein – 57 amino acids. If the complete protein conferred a functional advantage to the virus, a change in the evolutionary rates of the human H1N1 PB1-F2 proteins should have occurred in 1947. No such change is observed.

Results of sequence analysis reveal that the PB1-F2 open reading frame is as conserved, and maintained as a full-length protein, as other non-coding regions of the same RNA segment and of a randomly generated PB1 segment. These observations, and the fact that PB1-F2 is truncated in many virus isolates, suggest that the evolutionary role of PB1-F2 in animal hosts is minimal. Why the full length protein is produced by some viruses – and unfortunately leads to higher virulence – remains a puzzle.

Trifonov, V., Racaniello, V., & Rabadan, R. (2009). The Contribution of the PB1-F2 Protein to the Fitness of Influenza A Viruses and its Recent Evolution in the 2009 Influenza A (H1N1) Pandemic Virus PLoS Currents: Influenza

The problems with Barry’s “The Great Influenza”

great-influenzaWhen the 2009 H1N1 pandemic influenza virus emerged earlier this year, I began re-reading John Barry’s The Great Influenza. I came across the sentences that I had underlined during my first read identifying errors in basic virology. Because this is a very popular book, it’s important to identify the mistakes and correct them.

Barry is not a virologist, or any type of scientist. He’s a historian who happens to have written on influenza. This does not excuse the virological errors in his book; he  should have had a virologist fact-check the manuscript before publication.

Page citations refer to the Penguin Books paperbound version.

When a virus successfully invades a cell, it inserts its own genes into the cell’s genome, and the viral genes seize control from the cell’s own genes. [page 100]

This sentence implies that the reproductive cycle of every virus includes integration of the genome into that of the host. Barry’s statement is incorrect; only genomes of certain viruses (e.g. retroviruses) are introduced into the host DNA.

Soon a pit forms in the cell membrane beneath the virus, and the virus slips through the pit to enter entirely within the cell… [page 103]

Only some viruses enter the cell from the ‘pit’ formed at the plasma membrane. In many cases the ‘pit’ eventually becomes a vesicle known as an endosome which moves deep into the cytoplasm. Influenza viruses enter cells from  endosomes.

If for some reason the influenza virus cannot penetrate the cell membrane, it can detach itself and then bind to another cell that it can penetrate. Few other viruses can do this. [page 104]

I presume Barry is referring to the ability of influenza NA to remove sialic acids from the cell surface, thereby liberating surface-bound virions. Other viruses have this ability. Viruses that do not possess a neuraminidase probably have other ways to leave the cell surface, such as a weak virus-receptor interaction.

The following description concerns the entry of influenza virus into cells:

Inside this vesicle, this bubble, shape and form shift and create new possibilities as the hemagglutinin faces a more acidic environment. This acidity makes it cleave in two and refold itself into an entirely different shape. [page 104]

Cleavage of the HA does not occur during endosomal entry. Whether or not the viral HA is cleaved (which is required for infectivity) is determined during assembly of the virus particle.

In the following sentence, Barry seems intent on making a retrovirus out of influenza virus:

Soon the genes of the virus spill into the cell, then penetrate to the cell nucleus, insert themselves into the cell’s genome, displace some of the cell’s own genes, and begin issuing orders. [page 104]

The influenza virus genome does not integrate into the DNA of the host cell, as noted above.

The neuraminidase guarantees that new viruses can escape to invade other cells. Again, few viruses do anything similar. [page 104]

Members of other virus families do have neuraminidases which probably serve similar functions during infection as the influenza NA. It’s not correct to write that ‘few’ viruses do anything similar.

Antibodies, for example, carry thousands of receptors on their surface to recognize and bind to a target antigen. [page 108]

A single antibody does not have the ability to bind thousands of antigens; only one. Collectively, antibodies can recognize thousands of epitopes.

Dendritic cells attack bacteria and viruses indiscriminately, engulf them, then “process” their antigens and “present” those antigens – in effect they chop up an invading microorganism into pieces and display the antigens like a trophy flag. [page 108]

Dendritic cells don’t engulf viruses and bacteria – they take up extracellular proteins by endocytosis, then display them to lymphocytes. Perhaps Barry is thinking of phagocytic cells such as macrophages.

But of all parts of the influenza virus that mutate, the hemagglutinin and neuraminidase mutate the fastest. [page 109]

The mutation rate of all influenza virus RNA segments is similar. What Barry means is that the HA and NA proteins vary more than do other viral proteins. This is because the HA and NA are structurally plastic and can accommodate amino acid substitutions. Changes in the protein are not mutations; this term refers specifically to nucleic acid.

When an organism of weak pathogenicity passes from living animal to living animal, it reproduces more proficiently, growing and spreading more efficiently. This often increases virulence. [page 177]

These conclusions simply are not correct. I discussed this issue previously.

Initially Ebola has extremely high mortality rates, but after it goes through several generations of human passages, it becomes far milder and not particularly threatening. [page 177]

One of the problems with The Great Influenza is that statements such as this one are not supported by literature references. There has been so little person to person spread of ebolavirus that this conclusion cannot be made.

The following statements that implies that there were multiple waves of influenza in 1918 accompanied by mutation to higher and then lower virulence:

All over the world, the virus was adapting to humans, achieving maximum efficiency. And all over the world, the virus was turning lethal. [page 193]

Even when the virus mutated toward mildness, it still killed efficiently. [page 363]

At first those processes had made the virus more lethal. Whether it first jumped from an animal host to man in Kansas or in some other place, as it passed from person to person it adapted to its new host, became increasingly efficient in its ability to infect, and changed from the virus that caused a generally mild first wave of the disease in the spring of 1918 to the lethal and explosive killer of the second wave in the fall. [page 370]

As time went on, it became less lethal. [page 371]

But it mutated enough, its antigens drifted enough, to rekindle the epidemic. [page 373]

It continued to attack, but with far less virulence, partly because the virus mutated further toward its mean, toward the behavior of most influenza viruses. [391]

As I’ve written before, we have no evidence for an increase or decrease of the 1918 virus with time, because there are no virus isolates other than one reconstructed from November 1918. All these statements are therefore without any proof and remain highly speculative.

It’s not my intent to severely criticize the book – it’s a compelling description of a very serious pandemic. I simply want to ensure that everyone understands the scientific underpinnings of the outbreak. When authors write about science for a general audience, they have an obligation to get the science right.

Is the 2009 H1N1 influenza virus more dangerous than we think?

Mustela_putorius_furoThe results of experiments comparing the virulence in animals of the 2009 H1N1 influenza virus with seasonal strains have spawned the headline Study Suggests H1N1 Virus More Dangerous Than Suspected. In my view, the best experiment is now being done in humans: infection of millions with the pandemic virus. The results show that the virus is no more virulent than last season’s H1N1 strain.

In mice, ferrets, and non-human primates, the 2009 H1N1 swine-origin influenza virus (S-OIV) replicated more efficiently, and caused more severe lesions in the lungs than a seasonal H1N1 virus. These findings lead the lead author of the study to comment:

There is a misunderstanding about this virus. People think this pathogen may be similar to seasonal influenza. This study shows that is not the case. There is clear evidence the virus is different than seasonal influenza.

I’m puzzled by this statement. As far as I know, the 2009 H1N1 strain has so far likely infected millions of people, and most have concluded that the disease is no more severe than seasonal influenza. Are mice, ferrets, and non-human primates more reliable indicators of influenza virus virulence than humans?

I agree that the 2009 H1N1 influenza virus does seem to multiply more extensively in the respiratory tract than a seasonal H1N1 strain, as does the 1918 virus. But how many influenza virus strains have been studied in such animals? There are probably others that can replicate in the lower tract of experimental animals but are not very pathogenic in humans.

Two other research groups published the results of similar experiments in ferrets. They both found that the 2009 H1N1 virus replicated to higher titers, and more extensively in the lower respiratory tract, than seasonal H1N1 influenza virus. One of the groups concluded:

Our results indicated that the 2009 A(H1N1) influenza virus replicates efficiently in the upper and lower respiratory tract of ferrets, is associated with mild or moderate clinical signs and pathological changes and is transmitted efficiently between ferrets via aerosols or respiratory droplets. These results are in agreement with observations in humans, where generally mild disease but relatively efficient human-to human transmission has been observed

In other words, although the 2009 H1N1 replicated more efficiently, and in different parts of the lungs than seasonal H1N1 virus, these authors felt the observations were consistent with the low virulence of the virus humans.

It’s worth noting that when the authors of the Nature paper infected miniature pigs with the 2009 H1N1 influenza virus, the virus replicated without clinical symptoms. This result emphasizes the importance of remembering that animals are models for studying virus infections; they rarely duplicate the effects of a virus infection in humans. Furthermore, the virulence of a virus strain may vary dramatically depending on the dose and the route of infection, as well as on the species, age, gender, and susceptibility of the host. Virulence is a relative property. Consequently, when the degree of virulence of two very similar viruses are compared, the assays must be identical.

Do we really want to conclude that the new H1N1 strains is ‘more dangerous’ than we think based on tests in animal models which may or may not accurately reflect what occurs in humans? The ongoing infection of millions of humans with the new H1N1 virus seems a better source of data – and so far the results indicate that the new pandemic strain is no more dangerous than seasonal influenza.

Of course, it is possible that a more virulent version of the 2009 H1N1 virus could emerge in the coming months – what will happen as this virus evolves in humans is anyone’s guess – but who knows if it will retain the ability to multiply in the lower tract? The only thing that is certain is that it will be a different virus from the one that has been studied in mice, ferrets, and small pigs.

Y. Itoh1 et al. (2009). In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses Nature adv. online pub 13 July 2009.

Maines, T., Jayaraman, A., Belser, J., Wadford, D., Pappas, C., Zeng, H., Gustin, K., Pearce, M., Viswanathan, K., Shriver, Z., Raman, R., Cox, N., Sasisekharan, R., Katz, J., & Tumpey, T. (2009). Transmission and Pathogenesis of Swine-Origin 2009 A(H1N1) Influenza Viruses in Ferrets and Mice Science DOI: 10.1126/science.1177238

Munster, V., de Wit, E., van den Brand, J., Herfst, S., Schrauwen, E., Bestebroer, T., van de Vijver, D., Boucher, C., Koopmans, M., Rimmelzwaan, G., Kuiken, T., Osterhaus, A., & Fouchier, R. (2009). Pathogenesis and Transmission of Swine-Origin 2009 A(H1N1) Influenza Virus in Ferrets Science DOI: 10.1126/science.1177127

Riding the influenza pandemic wave

1973927918_ce00011ef5_mOne notable characteristic of the four previous influenza pandemics is that they occurred in multiple waves. The 1918 pandemic began with outbreaks of low mortality in the spring and summer, followed by a more lethal wave in the winter. This pattern has fueled speculation that the current H1N1 pandemic strain will undergo mutation that leads to the emergence of a more lethal virus. What is the evidence that pandemic waves of increasing virulence are a consequence of viral mutation?

The only virus available from the 1918 pandemic was rescued from an Alaskan influenza victim who was buried in permafrost in November of that year, when higher mortality was already evident. This makes it impossible to correlate any genetic changes in the virus with increased virulence. Furthermore, as discussed on ProMedMail,

…there are many different ways of interpreting these differences other than more virulent virus. Some of these are differences in populations affected, more circulation of pneumococci and staphylococci during cold weather, more circulation of other viral pathogens, more virulence and larger inocula with the crowding and cold air inhaled.

The November 1918 influenza virus certainly has genetic and phenotypic properties expected of a virulent virus. These include the ability to multiply in the absence of trypsin*, lethality in mice and embryonated chicken eggs, and efficient replication in human bronchial epithelial cells. But we don’t know if these properties were absent from the virus that circulated in the spring of 1918.

Do the pandemics of 1957 and 1968, which also occurred in waves of increasing lethality, provide any information? Viruses are available from different stages of these pandemics, but to my knowledge the virulence studies have not been done.

This uncertainty makes it impossible to conclude that the 2009 H1N1 pandemic strain will become more virulent. Nevertheless, speculation is rampant, and accompanied the recent release of the Brazilian isolate. Another example is an amino acid change in the viral PB2 protein observed in some 2009 H1N1 isolates. According to Recombinomics,

Acquisition of E627K is a concern because it allows for optimal replication at 33 C, the temperature of a human nose in the winter, in contrast to E627, which is in the avian version of PB2 and allows for optimal replication at 41 C, the body temperature of birds. The appearance of E627K raises concerns that the level of swine flu with E627K will markedly increase in colder months. In 1918, the flu in the spring was mild, but the fall version of the virus, which had E627K, was much more virulent and targeted young, previously healthy adults…

If the amino acid at 627 is an important determinant of virulence, we would expect to find E627 in viruses isolated early in the 1918 pandemic – but such viruses are not available. Therefore the role of this amino acid change in virulence in humans cannot be tested. Further complicating the situation is that other amino acids in the viral PB2 protein can influence viral replication at low temperatures.

Fortunately, new H1N1 isolates are obtained every week, which provide a very accurate sampling of the entire pandemic. Should the new H1N1 strain become more virulent, it will be a relatively straightforward task to determine the genetic changes that accompany this property. Finally we will be able to determine if  pandemic waves of increasing virulence are a consequence of specific changes in the viral RNA.

*We’ll discuss the requirement of proteases for influenza virus replication next week.

Miller, M., Viboud, C., Balinska, M., & Simonsen, L. (2009). The Signature Features of Influenza Pandemics — Implications for Policy New England Journal of Medicine, 360 (25), 2595-2598 DOI: 10.1056/NEJMp0903906

Tumpey, T. (2005). Characterization of the Reconstructed 1918 Spanish Influenza Pandemic Virus Science, 310 (5745), 77-80 DOI: 10.1126/science.1119392.

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.

WHO will redefine pandemic

pandemic-influenzaThe World Health Organization, whose duties include directing and coordinating authority for health within the United Nations system, will soon be writing science textbooks.

That statement isn’t true, of course. But it was my reaction to reading the latest announcement from Geneva:

Bowing to pressure, the World Health Organization announced Friday that it would rewrite its rules for alerting the world to new diseases, meaning the swine flu circling the globe will probably never be declared a full-fledged pandemic. Dr. Keiji Fukuda, the deputy director general making the W.H.O. announcement, said that he could not predict exactly what the new rules would be but that criteria would include a “substantial risk of harm to people,” not just the geographic spread of a relatively benign virus.

Apparently members of the United Nations don’t like the fact that WHO has been using ‘pandemic’ to describe the global spread of the new H1N1 influenza strains. They feel that the word pandemic implies that the virus is lethal and capable of causing many deaths – like the 1918 strain of influenza. Problem is, the new H1N1 strain isn’t any more lethal than seasonal strains of the virus. Apparently using the p-word gets everyone frightened as pandemic preparedness plans shift into gear.

According to the virology textbooks (one of which I wrote), the word pandemic means ‘global epidemic’. Even wikipedia has a benign definition: “A pandemic (from Greek παν pan all + δήμος demos people) is an epidemic of infectious disease that spreads through populations across a large region; for instance a continent, or even worldwide.”

I can already see how the WHO edict will influenza future versions of textbooks. For example, the current edition of  Clinical Virology states “Over the past 300 years, at least six pandemics of influenza have probably occurred, including three well-characterized ones in the 20th century”. In the next edition, this will have to be rewritten: “Until recently, at least six pandemics of influenza have probably occurred, including three well-characterized ones in the 20th century. In 2009, a new strain of H1N1 influenza emerged and spread globally, but it was not considered a pandemic by the new WHO rules”.

WHO redefining pandemic is absurd. Pandemic is an epidemiological definition that has nothing to do with virulence. A pandemic of influenza occurs when a new viral strain emerges to which the population has little or no immunity. Although pandemic is most frequently associated with influenza virus, other infectious agents may cause worldwide epidemics. The world is currently in the midst of an AIDS pandemic, one of the worst in history.

WHO should leave textbook writing to others. To paraphrase Andre Lwoff, a pandemic is a pandemic. The word implies nothing about virulence – and has little to do with politics.

Influenza A/Mexico/2009 (H1N1): Absence of crucial virulence marker

influenza-rna-2The second RNA segment of the influenza virus genome encodes two proteins, PB1 and PB1-F2.  The latter protein is believed to be an important determinant of virulence of influenza virus. Can we learn anything about the virulence of the new influenza virus H1N1 strains from a study of this protein?

During influenza virus infection, PB1-F2 is targeted to the mitochondria, where it induces a form of cell death known as apoptosis. Experiments in a mouse model of influenza virus infection have shown that PB1-F2 regulates lethality of the virus. By comparing the infection of mice with two strains of influenza virus, one of which produces much lower levels of the PB1-F2 protein, it was found that the protein enhances inflammation and increases frequency and severity of secondary bacterial pneumonia. A specific amino acid at position 66 of this protein appears to be an important determinant of viral virulence. This amino acid is a serine in the 1918 H1N1 influenza virus, in a 1997 avian H5N1 isolate from the Hong Kong outbreak, and in the H2N2 (1957) and H3N2 (1968) pandemic strains. Other less pathogenic influenza virus isolates have an asparagine at this position. Two viruses were constructed which differ at amino acid 66 of the PB1-F2 protein, and the virulence of these viruses was determined in mice. The influenza virus with a serine at amino acid 66 was pathogenic in mice, while the virus with an asparagine was significantly less virulent. Increased pathogenicity of the virulent virus was associated with higher levels of virus replication in the lungs. The results of these studies show that the PB1-F2 protein affects pathogenicity in a mouse model, and that position 66 plays an important role.


Truncated PB1-F2

Because the amino acid change N66S of PB1-F2 is present in the three previous pandemic influenza virus strains – 1918 H1N1, 1957 H2N2, and 1968 H3N2 – it would be of interest to determine which amino acid, N or S, is present in the new H1N1 influenza virus strain that is spreading globally. However, examination of the nucleotide sequence of RNA from the current H1N1 isolates shows that these viruses do not even produce a PB1-F2 protein – a stop codon is present after amino acid 11 (see figure). In fact, many other influenza virus strains do not produce the protein. While the PB1-F2 protein is not the only determinant of influenza virus virulence, we can at least eliminate any contribution of this viral protein to increased lethality. As Peter Palese has written in today’s Wall Street Journal, “If this virulence marker is necessary for an influenza virus to become highly pathogenic in humans or in chickens, then the current swine virus doesn’t have what it takes to become a major killer.”

Conenello, G., Zamarin, D., Perrone, L., Tumpey, T., & Palese, P. (2007). A Single Mutation in the PB1-F2 of H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence PLoS Pathogens, 3 (10) DOI: 10.1371/journal.ppat.0030141

MCAULEY, J., HORNUNG, F., BOYD, K., SMITH, A., MCKEON, R., BENNINK, J., YEWDELL, J., & MCCULLERS, J. (2007). Expression of the 1918 Influenza A Virus PB1-F2 Enhances the Pathogenesis of Viral and Secondary Bacterial Pneumonia Cell Host & Microbe, 2 (4), 240-249 DOI: 10.1016/j.chom.2007.09.001