Empirical cumulative distribution of birth year for patients with H1N1 (blue) and H3N2 (red), from the NCBI dataset, in the United States, between 1995 and 2008.

<p>The significant statistical dissimilarity between the distribution for the year of birth (P(MW) = 2.04E-24 and P(KS) = 1.13E-35) hints to an existing immunity against one of the subtypes in different age groups, possibly carried over from a previous pandemic.</p

The studied datasets from New York State and the NCBI.

<p>a: the median age.</p><p>b: the birth year of the oldest person.</p><p>c: number of counts.</p>*<p>Probabilities computed for Mann-Whitney (P(MN)) and Kolmogorov-Smirnov (P(KS)) tests.</p

Empirical cumulative distribution of ages for patients with H1N1 (blue) and H3N2 (red), from the NCBI dataset, in United States during the influenza seasons of 2006–2007 (left) and 2007–2008 (right).

<p>The significantly low probabilities computed via Mann-Whitney (P(MN)) and Kolmogorov-Smirnov (P(KS)) tests during separate influenza seasons show the consistency in our results among sub-portions of the data and refute the possibility that the previous statistical results are due to a unique season.</p

Empirical cumulative distribution of ages for patietns with H1N1 (blue) and H3N2 (red) in New York State during the 2006–2007 and 2007–2008 influenza seasons.

<p>The significantly low probabilities computed via Mann-Whitney (P(MN)) and Kolmogorov-Smirnov (P(KS)) tests indicate a remarkable dissimilarity between the distributions.</p

Empirical cumulative distribution of ages for patients with H1N1 (blue) and H3N2 (red), from the NCBI dataset, in the United States, between 1995 and 2008 (left) and Oceania, between 2000 and 2007 (right).

<p>Complementary to the results from New York State (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006832#pone-0006832-g001" target="_blank">Fig. 1</a>), the low probabilities computed via Mann-Whitney (P(MN)) and Kolmogorov-Smirnov (P(KS)) tests show a significant difference between the distributions, which is spatially and temporally consistent.</p

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

<div><p>It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)₃ in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population.</p></div

Effect of oseltamivir on virus entry.

<p>Viruses were adsorbed to cells in the presence or absence of oseltamivir for 1 hr, then the inoculum aspirated off, infection medium without drug was added and the plates incubated for 3 days to allow virus growth. Both virus and oseltamivir were titrated at 10-fold dilutions so the errors are ±1 log.</p

Binding profile of the top six glycans (ranked by the sum of the percentile signals, top to bottom) to viruses from 1968 to 2012 (left to right).

<p>The percentile binding of each glycan to each virus is shown, color-coded from 100 (red) to 10 (violet). White cells indicate binding less than 10% of the maximum. The colors show a shift from short branched sialylated structures in early viruses to long linear or long branched glycans in later isolates.</p

Binding of four viruses to the glycan array in the absence or presence of oseltamivir.

<p>BCM/2/92 (A) and OK/483/2008 (C) bind only NeuAcα2–6 glycans and there is no change in the presence of oseltamivir. OK/309/2006 (B) and OK/5342/2010 (D) show binding to many NeuAcα2–3 glycans but this binding is lost in the presence of oseltamivir, indicating it is NA, not HA, that binds to NeuAcα2–3.</p

Relationship between agglutination of turkey red blood cells and the highest binding signal on the Glycan Array.

<p>A. Hemagglutinating units (HAU, red) or fluorescent signal (RFU, blue) are shown per µg or per 100 ng viral protein, respectively, to approximately equalize the magnitude. B. HAU plotted against RFU with the trendline shown in black. Note that both axes are on a log scale. C. Plot of isoelectric point (pI) against binding avidity (the average of HAU and RFU).</p