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.

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.

Influenza A/Mexico/2009 (H1N1) – Questions and answers

Here are answers to questions send to virology blog about the new strain of influenza H1N1 that is spreading globally.

Q: Regarding this marker (PB1-F2) – is it something that was present as well in the early wave of the 1918 virus, which was also considered mild up until August, or was it something that was acquired during its passage through humans? Do the steps being currently taken reduce the likelihood of sufficient human-to-human transmission to adapt and become more virulent?

A: It’s a good question. The 1918 sequences were obtained from autopsy material collected in September and November of 1918. Therefore we
can’t address the question of whether any changes occurred during propagation in humans. We can reduce but not eliminate transmission; therefore selection for viruses of greater virulence is still possible.

Q: Thank you very much for sharing with us the interview done to Ruben Donis. It is very provocative so I have several questions.

1. Has he uploaded to NCBI the sequence of the ninth virus that was not identical to those isolated in US? If not, Do you have any explanation for keeping secret this information.

A: Most of the sequences from Mexican isolates have been uploaded to GISAID.

2. He explains how it can be created new viruses by taking advantage of viral modules to make reassortments and the use of ferrets to check for pathogenicity. If by accident release infected ferrets into the wild, wouldn´t this a way to generate a new epidemic? If not, please explain.

A: Not necessarily. The particular combination of genes might not survive in the wild – for example they might not transmit well. But this is why these experiments are done under high containment.

3. Please explain Donis saying, and quote “the hemagglutinins we are seeing in this strain are a lonely branch” end of quote. Wouldn´t be this the reason for the higher pathogenicity of the viruses straines found in Mexico?

A: I assume he means that there aren’t many viruses with HA related to these strains. This observation makes no implications about pathogenicity. In any case all the strains appear to be on the same lonely branch.

Q: It would be very informative to know the origin of the specific H1 (and other) genes- avian or swine; it is unlikely that the H1 gene is from a current H1N1 human virus because it would be too similar to previous human H1N1 influenza viruses to presumably cause a pandemic.

A: Eurosurveillance has an article on bioinformatic analysis of the H1N1 sequences. They report: “Six segments of the virus are related to swine viruses from North America and the other two (NA and M) from swine viruses isolated in Europe/Asia. The closest clusters (for the HA segment) in the NCBI data base are North America swine influenza A(H1N2) and H3N2s.  The closest relatives of the neuraminidase (NA) gene of the new virus, are influenza A isolates from 1992. The North American ancestors are related to the multiple reassortants, H1N2 and H3N2 swine viruses isolated in North America since 1998. In particular, the swine H3N2 isolates from 1998 were a triple reassortment of human, swine and avian origin.”

Q: After reading these news that a man seemingly infected pigs with flu on a farm in Canada, don’t you think that we should reconsider following pigs with more caution, I think that if this was true that pigs could catch the virus from humans, they could act as “Transit” station for re-emergence of the virus again and again, I don’t know if I am experiencing my mind well here, I mean given that pigs show mild or no symptoms at all, and that now they could acquire the virus from man, a farmer could give the pigs the virus and then leave the rest for the pigs to re-transmit the virus for other people, which in turn could transmit to other people and so on. This was a thought that I want to here what do you think.

A: The infection of Canadian pigs by a human with influenza returning from Mexico is disturbing, but there are many questions, elucidated in a previous post here. In short, it is incredible that such contamination was allowed to occur. Clearly pig-human interactions need to be carefully monitored. Culling herds should only be done when there is a known risk for spread of infection.

Q: I’m a bit confused: is it H1N1 or H3N2 from Eurasian pig flu virus? And does it directly copy it’s own RNA genome with the viral RNA polymerase?

A: NA and M genes are from Eurasian pig influenza virus. And yes, the viral RNAs are copied directly by the viral RNA polymerase.

Three related questions:

Q: How long would you expect human immunity to exist within an individual infected with h1n1 virus? I had the swine flu in the mid 70’s. Am I immune? Does getting H1N1 confer immunity in the future, and if so, for how long?

A: Natural infection confers lifelong immunity, but only to the infecting strain, not one that has drifted antigenically. As far as being infected by swine flu in the mid 1970s – this probably will not confer protection as the HA and NA of the two viruses are quite different.

Q: Are you aware of research out of the Kobe University Center for Infectious Diseases that more than 10% of pigs in Indonesia carry H5N1? Does this change your level of concern regarding the possibility of a recombination?

A: I haven’t seen that study published yet. But if it’s correct then of course there will be more concern about recombination. The more H5N1 replicates in pigs, the greater the chance.

Q: This may not be the best place to post this, but I find no other mention of it. There was an interesting ProMed-mail post today. Basically, the author, from the British Columbia Centre for Disease Control, says that while testing for H1N1, a lot of new cases of H3N2 were found, and that the H3N2 strain had changed.  The author speculated that some of the late season Mexico flu reports could have actually been due to the new H3N2 variant.

A: It’s not surprising that H3N2 viruses are continuing to change as they circulate. What remains to be seen if they disappear with continued circulation of the new H1N1 strains. We won’t know the answer to this question for some time, and continued surveillance will be needed. A very interesting time for influenza virology.

Keep your questions coming to virology@virology.ws – I enjoy answering them.

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.

h1n1-pb2-f2

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

New swine influenza viruses in humans

swineA new strain of swine influenza virus has been recently isolated from seven persons in the US. Is it time to break out the swine flu vaccine of 1976?

Last week the CDC reported that swine influenza virus had been isolated from two children with respiratory illness in California. The cases were not linked and the children recovered from the illness. The virus was identified as a swine influenza H1N1 strain, similar to viruses that have circulated in American pigs for the past ten years. However some of the viral genes are derived from Eurasian swine influenza viruses. The isolates are new because this particular combination of swine influenza virus RNAs has not been observed before among swine or human viruses.

A similar virus was subsequently identified in five additional individuals in Texas. It’s curious that one of the California children had traveled to Texas before becoming ill, but whether or not the cases are related has not been revealed.

What is the origin of these new swine viruses? None of the people who were infected had known contact with pigs. Others must have acquired the virus from pigs, who then passed it on – demonstrating that the virus can be transmitted among humans.

At the moment these infections don’t seem to be cause for alarm. Because influenza virus surveillance is more intense than ever before, it is likely that new viruses will always be detected. Furthermore, respiratory disease caused by these new viruses has not been very severe. Another mitigating factor is that the influenza season is nearly over – viral transmission wanes when the weather becomes warmer and more humid.

It is believed that swine influenza originated in 1918-19, when pigs became infected with the pandemic influenza virus strain. Since that time, the H1N1 swine virus has been transmitted back to humans. The hypothesis for the origin of swine influenza is supported by the finding that pigs can be experimentally infected with the human 1918 pandemic influenza virus strain. Furthermore, other human influenza virus strains are known to infect pigs. For example, in the early 1970s, a human H3N2 subtype entered the European swine population.

Pigs can be infected with both human and avian influenza virus strains because the cells of their respiratory tract bear receptors for both kinds of viruses. Based on this observation, it has been suggested that influenza viruses pass from birds through pigs on their way to infecting people. For example, if a pig is infected with avian and human influenza A viruses, reassortment of the viral RNAs occurs, leading to new virus strains to which humans are not immune. The 1957 and 1968 human pandemic viruses were reassortants of human and bird strains, although there is no evidence that these viruses arose in pigs. The role of pigs as a ‘mixing vessel’ for influenza virus has been questioned in view of the recent transmission of avian influenza viruses directly to humans.

Swine influenza viruses probably routinely pass among humans and swine; in this case they were detected as a consequence of heightened surveillance. Gerald Ford won’t be rolling over in his grave over this incident.

Weingartl, H., Albrecht, R., Lager, K., Babiuk, S., Marszal, P., Neufeld, J., Embury-Hyatt, C., Lekcharoensuk, P., Tumpey, T., Garcia-Sastre, A., & Richt, J. (2009). Experimental Infection of Pigs with the Human 1918 Pandemic Influenza Virus Journal of Virology, 83 (9), 4287-4296 DOI: 10.1128/JVI.02399-08

de Jong, J., Smith, D., Lapedes, A., Donatelli, I., Campitelli, L., Barigazzi, G., Van Reeth, K., Jones, T., Rimmelzwaan, G., Osterhaus, A., & Fouchier, R. (2007). Antigenic and Genetic Evolution of Swine Influenza A (H3N2) Viruses in Europe Journal of Virology, 81 (8), 4315-4322 DOI: 10.1128/JVI.02458-06

Van Reeth, K. (2007). Avian and swine influenza viruses: our current understanding of the zoonotic risk Veterinary Research, 38 (2), 243-260 DOI: 10.1051/vetres:2006062