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

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.

学界普遍认为，人类流感病毒结合带有α2-6型连接唾液酸（sialic acid）的聚糖（glycans），禽流感病毒则结合带有α2-3型连接唾液酸的聚糖，而改变结合特异性的突变可能会改变病毒的宿主嗜性（host tropism）。我们注意到人类H3N2亚型流感病毒的结合特异性存在显著差异，因此开展了一项针对代表性人类H3N2流感病毒的研究：这些病毒分离自1968年至2012年，且仅在哺乳动物细胞（mammalian cells）中进行了最低限度的传代，从未使用鸡胚（eggs）培养。
45株病毒均在MDCK细胞（Madin-Darby Canine Kidney cells）中培养、纯化，经荧光标记（fluorescently labeled）后，于功能糖组学联盟聚糖芯片（Consortium for Functional Glycomics Glycan Array）上进行筛选。同一季节分离的病毒具有相似的结合特异性谱，但该谱存在显著的年度间差异。
芯片上的610种聚糖（其中166种为唾液酸化聚糖（sialylated glycans））均未与所有45株病毒结合；结合能力最强的是线性或双天线型的Neu5Acα2–6(Galβ1–4GlcNAc)3，可结合42株病毒。
早期人类H3N2病毒优先结合短链分支唾液酸化聚糖，而近年病毒则更倾向于结合以唾液酸结尾的长链聚乳糖胺链（polylactosamine chains）。1996、2006、2010及2012年分离的病毒可结合带有α2-3型连接唾液酸的聚糖；针对2006、2010及2012年的病毒，该结合可被奥司他韦（oseltamivir）抑制，表明神经氨酸酶（neuraminidase）介导了对α2-3型唾液酸化聚糖的结合。更值得注意的是，奥司他韦可抑制2010和2012年病毒对MDCK细胞的入侵。
上述所有病毒均为在全球传播的流行毒株代表，均可高效感染人类并在人间传播。我们的研究结论为：受体结合特异性的年度间差异是抗原漂移（antigenic drift）驱动的氨基酸序列改变所导致的结果，而结合特异性和亲和力（avidity）差异显著的病毒，同样具备在人群中感染和传播的适配能力。