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

Trivalent influenza vaccine for the 2010-2011 season

influenza-vaccineThe World Health Organization and the US Food & Drug Administration have decided on the composition of the influenza virus vaccine that will be used during the 2010-2011 season in the northern hemisphere. The trivalent preparation will contain the following influenza virus strains: A/California/7/2009 (H1N1); A/Perth/16/2009 (H3N2); and B/Brisbane/60/2008. The same trivalent vaccine is also being used to prepare for the upcoming winter in the southern hemisphere.

The A/California/7/2009 (H1N1) virus is the pandemic strain that was used in the 2009 H1N1 monovalent vaccine. That virus has not yet undergone sufficient antigenic drift to warrant selection of a new strain for the vaccine. Note that a seasonal H1N1 strain from previous years will not be included in the vaccine. This change has been made because epidemiological evidence suggests that these viruses will probably not circulate at significant levels during the 2010-2011 northern hemisphere season. Although the vast majority of circulating influenza viruses in humans are related to the 2009 H1N1 pandemic strain, sporadic influenza A(H3N2) activity continues to be reported in several countries. This is the reason why an H3N2 component is part of the vaccine.

The selection of viruses for seasonal flu vaccines is based on which influenza viruses circulate during the previous season. Sample viruses are collected by 130 national influenza centers in 101 countries and data on disease trends are analyzed by the four World Health Organization (WHO) Collaborating Centers for Reference and Research on Influenza. Vaccine viruses are selected which will most likely protect against the main circulating viruses during the next influenza season. WHO makes recommendations about which specific virus strains should be included in the vaccine. Individual countries then decide which viruses will be included in the influenza vaccine.

Even though the 2009 H1N1 strain has not undergone significant antigenic changes, it’s important to be immunized again in anticipation of the next influenza season. That’s because immunity conferred by the vaccine isn’t particularly long lasting. As Adolfo Garcia-Sastre told me today*, even if influenza didn’t change, you would still have to be immunized every year to protect against infection.

*I recorded our conversation. Look for it at TWiV within the next few weeks.

Protection against 2009 influenza H1N1 by immunization with 1918-like and classical swine viruses

LetterheadInfluenza A viruses typically cause severe respiratory disease mainly in the very young or the elderly. The 2009 swine-origin H1N1 virus is unusual because it preferentially infects individuals under 35 years of age. We’ve previously noted that being older is a good defense against 2009 H1N1 influenza virus, in part because older people have antibodies that block infection. Experiments done in mice show that immunization with 1918-like or classical H1N1 swine influenza viruses protects against infection with 2009 H1N1 virus.

When mice are inoculated intranasally with a high dose of the 2009 H1N1 influenza virus, the virus replicates in the lungs and leads to significant weight loss and lethality. After a sublethal virus dose the mice develop protective antibodies that protect against severe disease. These observations establish the mouse as a valid model for studying immunization against the 2009 H1N1 strain.

One explanation for why older individuals are protected against infection with the 2009 H1N1 virus is that they were infected years ago with a related virus. To examine this possibility, mice were immunized with different inactivated H1N1 vaccines made from viruses that circulated from 1918–2009. Immunization with two different classical swine H1N1 viruses (Sw/30 or NJ/76), 1918 virus-like particles, and a human H1N1 virus isolated in 1943 (Wei/43) protected against death from 2009 pandemic H1N1 challenge. However, only partial protection against challenge was afforded by immunization with seasonal H1N1 viruses that circulated from 1977–2007.

These results suggest that the 1918, SW/30, and NJ/76 viruses induce antibodies that cross-react with the 2009 H1N1 strain. This possibility was confirmed by examining mouse sera by hemagglutinin inhibition assay. Sera from animals that were immunized with 1918 virus-like particles, SW/30 or NJ/76 had hemagglutination-inhibition activity (titer ≥ 40) against 2009 H1N1 viruses.

The 1918, SW/30 and NJ/76 viruses have a similar protein sequence on the viral HA molecule that gives rise to protective antibodies. This protein sequence, known as an epitope, is shown in light blue in the image of the viral HA molecule (click for a larger version). This epitope, called Sa, is located on the top of the HA molecule and is conserved in the 1918, SW/30, ad NJ/76 viruses.

In other words, if you lived before 1943, or received the 1976 swine flu vaccine, you may be protected against infection with 2009 H1N1 virus. After the 1976 swine H1N1 outbreak at Fort Dix, NJ, approximately 40 million people in the United States were immunized with an NJ/76 vaccine. The NJ/76 swine virus never spread in the general population, but the vaccine against it has finally proven useful.

If you are less than 35 years old, you are more likely to be infected with the 2009 H1N1 virus because you did not receive the NJ/76 vaccine, nor were you infected with viruses that circulated from 1918-1943.

Perhaps most interesting is the idea that the H1 hemagglutinin has remained ‘antigenically frozen’ in domestic pigs. In 1918, the H1N1 virus entered humans and pigs at roughly the same time. In humans the H1N1 virus underwent significant antigenic drift; as a consequence H1N1 viruses that circulated after 1943 do not protect against infection with 2009 H1N1. During the same period swine H1N1 virus underwent little antigenic drift in pigs, as shown by the ability of the NJ/76 strain to induce protective immunity in mice against the 2009 H1N1 virus. Why the virus does not evolve rapidly in domestic pigs is unknown, but could be related to the short lives of these food animals – there is little pre-existing immunity in pigs which could apply selective pressure for antigenic drift.

The fact that the H1 HA has remained antigenically stable in pigs since 1918 suggests that this animal could be a source of future pandemic strains. In one scenario, while the H1N1 virus remains in pigs for several generations, population immunity to the H1 HA declines until it is low enough to allow another pandemic outbreak. The human H3 HA has also become established in domestic pigs. With the likely disappearance of the H3N2 seasonal strain from humans this year, population immunity to this HA will wane. Perhaps in 60-70 years a virus with an H3 HA will emerge from domestic pigs as a new pandemic strain.

Manicassamy B, Medina RA, Hai R, Tsibane T, Stertz S, Nistal-Villán E, Palese P, Basler CF, & García-Sastre A (2010). Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines. PLoS pathogens, 6 (1) PMID: 20126449

TWiV 69: They’re all safecrackers

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich review recent outbreaks of mumps in the UK, US, and Israel, protection of mice against 2009 H1N1 influenza A virus by 1918-like and classical swine H1N1 vaccines, and a virus-like particle vaccine for chikungunya virus.

This episode is sponsored by Data Robotics Inc. Use the promotion code VINCENT to receive $50 off a Drobo or $100 off a Drobo S.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #69 (59 MB .mp3, 82 minutes)

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Links for this episode:

Weekly Science Picks

Rich John Moran Florida Nature Photography
Alan Periodic Table of Videos
Vincent The Protein Databank Educational Resources

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Radio Sandy Springs interview

radio_sandy_springsI was recently interviewed on Radio Sandy Springs by Sharon Sanders of FluTrackers. We talked about pandemic influenza H1N1 virus. Listen to the show below.

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Download Infectious Disease Hour January 4 2010 (20 MB .mp3, 57 minutes)

Radio Sandy Springs 1620 AM is a low-powered Atlanta-based talk radio station that simulcasts on the Internet.  They broadcast a weekly ‘Infectious Disease Update’ with interviews with clinicians, scientists, researchers, and even historians. You can find an archive of recent Infectious Disease Hour shows here.

TWiV 64: Ten virology stories of 2009

3D_InfluenzaHosts: Vincent Racaniello, Alan Dove, and Rich Condit

Vincent, Alan, and Rich discuss ten compelling virology stories of 2009.

Click the arrow above to play, or right-click to download TWiV #64 (68 MB .mp3, 94 minutes)

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Ten virology stories of 2009:

  1. Pandemic influenza: Swine-origin H1N1 virus (TWiV 36)
  2. XMRV, prostate cancer, and chronic fatigue syndrome (TWiV 50, 55)
  3. AIDS vaccine ‘success’ (TWiV 51)
  4. Colony collapse disorder (TWiV 46, 49)
  5. AIDS-like disease in wild chimps (TWiV 45)
  6. Diverse viral community in Antarctic lake (TWiV 58)
  7. Polyomavirus seroepidemiology in humans (TWiV 26)
  8. Poxvirus threatens UK red squirrels (TWiV 63)
  9. Polio spreads from Nigeria (TWiV 29)
  10. How mosquitoes survive Dengue virus infection (TWiV 21)

Picture book on viruses for kids (Thanks Soraia!)

Weekly Science Picks
Rich Surely You’re Joking, Mr. Feynman! by Richard P. Feynman, Ralph Leighton, Edward Hutchings, and Albert R. Hibbs
Vincent The Art and Politics of Science by Harold Varmus

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Reinfection with 2009 influenza H1N1

immune-memoryIn healthy individuals, the first encounter with a virus leads to a primary antibody response. When an infection occurs with the same or a similar virus, a rapid antibody response occurs that is called the secondary antibody response. Antibodies are critical for preventing many viral infections, including influenza. But reinfection may occur if we encounter the same virus before the primary response is complete.

Recently three cases of confirmed infection with 2009 influenza H1N1 were reported in Chile. The first patient had laboratory confirmed infection; treatment with oseltamivir resolved symptoms after 48 hours. Twenty days later the patient developed a second bout of laboratory confirmed influenza which was treated with amantadine. The second patient acquired laboratory confirmed influenza in hospital, was treated with oseltamivir and recovered. Two weeks later, while still in hospital, the patient had a new episode of laboratory confirmed influenza infection. Treatment with oseltamivir again resolved the infection. The third patient also acquired laboratory infection in hospital, was successfully treated with oseltamivir, and was discharged. He was readmitted 18 days later with confirmed pandemic H1N1 2009, and again successfully treated with oseltamivir.

These individuals were likely resusceptible to reinfection with the same strain of influenza virus due to a confluence of unusual events. First, all three were reinfected within three weeks, before their primary adaptive response had sufficiently matured. Another contributing factor was the high level of circulation of the pandemic strain. This issue was compounded for patients two and three who probably acquired both infections while in the hospital (called nosocomial transmission).

Could reinfection also occur after immunization with influenza vaccine? Yes, if the immunized individual encounters the virus before the primary antibody response matures, which occurs in 3-4 weeks. This is more likely to occur during pandemic influenza when circulation of the virus is more extensive than in non-pandemic years.

Perez CM, Ferres M, & Labarca JA (2010). Pandemic (H1N1) 2009 Reinfection, Chile. Emerging infectious diseases, 16 (1), 156-7 PMID: 20031070

TWiV 62: Persistence of West Nile virus

The_Persistence_of_MemoryHosts: Vincent Racaniello, Dickson Despommier, and Alan Dove

On episode #62 of the podcast This Week in Virology, Vincent, Dickson, and Alan discuss STEP HIV-1 vaccine failure caused by the adenovirus vector, presence of West Nile virus in kidneys for years after initial infection, adaptation of the influenza viral RNA polymerase for replication in human cells, and the significance of the D225G change in the influenza HA protein.

Click the arrow above to play, or right-click to download TWiV #62 (47 MB .mp3, 66 minutes)

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Links for this episode:

Weekly Science Picks
Dick Smallpox – The Death of a Disease by DA Henderson
Alan Olympus Bioscapes Digital Imaging Competition
Vincent Microbe Magazine

Send your virology questions and comments (email or mp3 file) to or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at and tag them with twiv.

Influenza virus growth in eggs

candle-eggsBefore the development of cell culture, many viruses were propagated in embryonated chicken eggs. Today this method is most commonly used for growth of influenza virus. The excellent yield of virus from chicken eggs has led to their widespread use in research laboratories and for vaccine production. In fact the vast majority of influenza vaccines – both inactivated and infectious – are produced in chicken eggs. How is influenza virus propagated in eggs?

The illustration below shows a cutaway view of an embryonated chicken egg. The different routes of inoculation into the egg are shown, as well as the different compartments in which viruses replicate.


For propagation of influenza virus, pathogen-free eggs are used 11-12 days after fertilization. The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac. This is marked with a pencil. After all the eggs have been ‘candled’ in this way, a small nick is made in the shell at this position using a jeweler’s scribe. Next, a hole is drilled at the top of the egg with a Dremel motorized tool. If this is not done, when virus is injected, the pressure in the air sac will simply force out the inoculum.

After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe – a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle. The needle passes through the hole in the shell, through the chorioallantoic membrane, and the virus is placed in the allantoic cavity, which is filled with allantoic fluid. The two holes in the shell are sealed with melted paraffin, and the eggs are placed at 37 degrees C for 48 hours.

During the incubation period, the virus replicates in the cells that make up the chorioallantoic membrane. As new virus particles are produced by budding, they are released into the allantoic fluid. To harvest the virus, the top of the egg shell – the part covering the air sac – is removed. We used to have a special tool to do this, which was placed over the egg. When the handle of this tool is squeezed, it makes a neat crack around the top of the egg. It was then easy to remove the flap of shell with tweezers. The shell membrane and chorioallantoic membrane are pierced with a pipette which is then used to remove the allantoic fluid – about 10 ml per egg. Sufficient virus may be produced in one or two eggs (depending on the viral strain) to produce one 15 microgram dose of vaccine.

We used to grow so much influenza virus that a large walk-in warm room was used as an egg incubator. When you opened the door of the incubator and heard peeping, it meant that someone had left unused eggs too long and they had hatched. Then you were left with the task of catching the evasive chicks.