Abstract
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.
Highlights
In a seminal series of experiments in the 1980s, Paulson and colleagues showed that the hemagglutinins (HA) of human influenza viruses show binding preference for a2–6 linked sialic acid while avian viruses bound the a2–3 linked form, and that a single amino acid change was sufficient to switch the specificity from mammals to birds and vice versa [1]
As we and others screened human H3N2 influenza viruses, or expressed HAs, on the Consortium for Functional Glycomics (CFG) Glycan Array, we noted considerable diversity in the substructures bound by different isolates [4,5] and significant binding of a2–3 sialylglycans has been reported in some human strains [5,6,7,8]
A/Albany/1/70, A/Albany/42/75 from early passages in rhesus monkey kidney (RMK) cells were already in the database; the sequences after 2–3 passages in Madin-Darby canine kidney (MDCK) cells were identical, and we have seen no change in Oklahoma isolates between isolation in RMK cells and after multiple passages in MDCK cells
Summary
In a seminal series of experiments in the 1980s, Paulson and colleagues showed that the hemagglutinins (HA) of human influenza viruses show binding preference for a2–6 linked sialic acid while avian viruses bound the a2–3 linked form, and that a single amino acid change was sufficient to switch the specificity from mammals to birds and vice versa [1]. The red blood cells recommended for hemagglutination-inhibition tests were changed from chicken to turkey, but by the 2000s it was widely recognized that the avidity for turkey red cells was reduced. Some of the sequence and structural changes responsible have been identified [10], but the low avidity of recent viruses has caused considerable problems with inconsistent hemagglutination-inhibition tests when seeking evidence of antigenic drift and determining when a change in the vaccine virus is needed
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