TWiV 363: Eat flu and dyad

On episode #363 of the science show This Week in Virology, The TWiVers reveal influenza virus replication in the ferret mammary gland and spread to a nursing infant, and selection of transmissible influenza viruses in the soft palate.

You can find TWiV #363 at

The neuraminidase of influenza virus

influenza virusThe influenza virus particle is made up of the viral RNA genome wrapped in a lipid membrane (illustrated). The membrane, or envelope, contains three different kinds of viral proteins. The hemagglutinin molecule (HA, blue) attaches to cell receptors and initiates the process of virus entry into cells. I have written about the HA and its function during infection (article one and two) but not about the neuraminidase (NA, red) or M2 (purple) proteins. Let’s first tackle NA.

An important function of the NA protein is to remove sialic acid from glycoproteins. Sialic acid is present on many cell surface proteins as well as on the viral glycoproteins; it is the cell receptor to which influenza virus attaches via the HA protein. The sialic acids on the HA and NA are removed as the proteins move to the cell surface through the secretory pathway. Newly released virus particles can still potentially aggregate by binding of an HA to sialic acid present on the cell surface. Years ago Peter Palese showed that influenza virus forms aggregates at the cell surface when the viral neuraminidase is inactivated. The NA is therefore an enzyme that is essential for release of progeny virus particles from the surface of an infected cell.

The NA protein also functions during entry of virus into the respiratory tract. The epithelial cells of the respiratory tract are bathed in mucus, a complex protective coating that contains many sialic acid-containing glycoproteins. When influenza virions enter the respiratory tract, they are trapped in mucus where they bind sialic acids. This interaction would prevent the viruses from binding to a susceptible cell were it not for the action of the NA protein which cleaves sialic acids from glycoproteins. When the virus particle encounters a cell, it binds the sialic acid-containing receptor and is rapidly taken into the cell before the NA protein can cleave the carbohydrate from the cell surface.

The essential nature of the NA for virus production has been exploited to develop new drugs designed to inhibit viral release. Both Tamiflu (Oseltamivir) and Relenza (Zanamivir) are structural mimics of sialic acid that bind tightly in the active site of the NA enzyme. When bound to drug, the NA cannot remove sialic acids from the cell surface, and consequently newly synthesized virus remains immobilized. The result is an inhibition of virus infection because virions cannot spread from one cell to another.

This article is part of Influenza 101, a series of posts about influenza virus biology and pathogenesis.

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 230: Gene goes to Washington, flu chickens out

On episode #230 of the science show This Week in Virology, Vincent, Rich, Alan and Kathy review H7N9 infections in China, the debate over patenting genes, and receptor-binding by ferret-transmissible avian H5 influenza virus.

You can find TWiV #230 at

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

Virology lecture #5: Attachment and entry

Download: .wmv (386 MB) | .mp4 (131 MB)

There are some errors in this lecture – I’ll correct them during the next session.

Visit the virology W3310 home page for a complete list of course resources.

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.

The D225G change in 2009 H1N1 influenza virus is not a concern

sialic-acid-2The Norwegian Institute of Public Health recently identified a mutation in 2009 H1N1 influenza virus isolated from two patients who died and one with severe disease. It has been suggested that this mutation, which causes a change from the amino acid aspartic acid to glycine at position 225 of the viral HA protein (D225G), could make the virus more likely to infect deeper in the airways and cause more severe disease. What is the basis for this concern and does it have merit?

Attachment of all influenza A virus strains to cells requires sialic acids. There are a number of chemically different forms of sialic acids, and influenza virus strains vary in their affinity for them. 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.

The type of sialic acid preferred by influenza viruses is controlled by amino acids in the HA protein. Amino acids 190 and 225 are important determinants of receptor binding specificity of the 1918 H1 hemagglutinin. The HA of the 1918 strain A/South Carolina/1/18 prefers alpha(2,3) linked sialic acids; the New York variant, isolated in September 1918, binds both alpha(2,3) and alpha(2,6) sialic acids. These two H1 hemagglutinins differ only by a single amino acid, position 225, which is aspartic acid (D) in the South Carolina strain and glycine (G) in the NY strain. When amino acid 190, which is D in both strains, is changed to E in the NY HA, the virus (AV18) preferentially binds alpha(2,3) sialic acids. These findings are summarized in the table.


Different isolates of the 2009 H1N1 influenza virus have D at HA at amino acid 190 and mostly D at amino acid 225. The virus prefers to bind to alpha(2,6) linked sialic acids. The amino acid change D225G would be expected to produce a virus with preference for both alpha(2,3) and alpha(2,6) linked sialic acids.

In the human respiratory tract, alpha(2,6) linked sialic acids are dominant on epithelial cells in the 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.

Based on these considerations, it could be hypothesized that the D225G change would allow the 2009 H1N1 virus to replicate deeper in the respiratory tract. But 2009 H1N1 virus without this amino acid change can already replicate deep in the respiratory tract of ferrets, and probably also in humans. Cells with alpha(2,6) linked sialic acids are present in the lower respiratory tract of humans. So it’s not clear if any effect on virulence would be conferred by the ability of the 2009 H1N1 strain to bind alpha(2,3) linked sialic acids.

An important consideration is that the D225G amino acid change has a negative impact on transmission. The change from D to G at amino acid 225 of the 1918 HA significantly impairs transmission among ferrets. When both D225G and D190E are present, transmission is abolished. These changes do not impair viral replication or virulence in the respiratory tract of inoculated animals.

Transmissibility is clearly a positive selection factor for viral evolution. There may be selection for increased virulence only if there is no negative impact on viral transmission. Given these considerations, the choice between an H1 HA amino acid at position 225 that allows efficient transmission (D225) or one that impairs transmission and might or might not allow multiplication deeper in the lung (D225G) seems obvious.

Tumpey, T., Maines, T., Van Hoeven, N., Glaser, L., Solorzano, A., Pappas, C., Cox, N., Swayne, D., Palese, P., Katz, J., & Garcia-Sastre, A. (2007). A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission Science, 315 (5812), 655-659 DOI: 10.1126/science.1136212

Shen J, Ma J, & Wang Q (2009). Evolutionary Trends of A(H1N1) Influenza Virus Hemagglutinin Since 1918. PloS one, 4 (11) PMID: 19924230

Influenza virus attachment to cells: role of different sialic acids

As we discussed previously, attachment of all influenza A virus strains to cells requires sialic acids. However, there are a number of chemically different forms of sialic acids, and influenza virus strains vary in their affinity for them. These differences may determine which animal species can be infected.

In the example shown below, sialic acid is linked to the sugar galactose by what is called an alpha(2,3) linkage. This means that the carbon atom at position number 2 of the sialic acid hexose is joined, via an oxygen atom, to the carbon at position 3 of the hexose of galactose.


Avian influenza virus strains preferentially bind to sialic acids attached to galactose via an alpha(2,3) linkage. This is the major sialic acid on epithelial cells of the duck gut. In contrast, human influenza virus strains preferentially attach to sialic acids attached to galactose by an alpha(2,6) linkage. This is the major type of sialic acid present on human respiratory epithelial cells. Alpha(2,3) linked sialic acids are found on ciliated epithelial cells, which are a minor population within the human respiratory tract, and also on some epithelial cells in the lower tract.

This receptor specificity has implications for human infection with avian influenza virus strains. For example, highly pathogenic avian H5N1 influenza viruses undergo limited replication in the human respiratory tract due to the presence of some cells with alpha(2,3) linked sialic acids. However, efficient human to human transmission requires that the avian viruses recognize sialic acids with alpha(2,6) linkages. Consistent with this hypothesis, the results of studies of early influenza virus isolates from the 1918, 1957, and 1968 pandemics suggest that these viruses preferentially recognized alpha(2,6) linked sialic acids.

Epithelial cells of the pig trachea produce both alpha(2,3) and alpha(2,6) linked sialic acids. This is believed to be the reason why pigs can be infected with both avian and human influenza virus strains and serve as a ‘mixing vessel’ for the emergence of new viruses. However, Peter Palese has his doubts, as recorded in The Great Influenza by John Barry:

Dr. Peter Palese….considers the mixing-bowl theory unnecessary to explain antigen shift: “It’s equally likely that co-infection of avian and human virus in a human in one cell in the lung [gives] rise to the virus…There’s no reason why mixing couldn’t occur in the lung, whether in pig or man. It’s not absolute that there are no sialic acid receptors of those types in other species. It’s not absolute that that avian receptor is really that different from the human, and, with one single amino acid change, the virus can go much better in another host”.

Ito T, Couceiro JN, Kelm S, Baum LG, Krauss S, Castrucci MR, Donatelli I, Kida H, Paulson JC, Webster RG, & Kawaoka Y (1998). Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. Journal of Virology, 72, 7367-7373

Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, Castrucci MR, Donatelli I, & Kawaoka Y. (2000). Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. Journal of Virology, 74, 8502-8512