Summary Avian influenza A viruses rarely infect humans, but if they do and transmit among them, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern due to the appreciable case fatality rate associated with these infections (>25%), potential instances of human-to-human transmission1, and the lack of pre-existing immunity among humans to viruses of this subtype. Here, we therefore characterized two early human A(H7N9) isolates, A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9; hereafter referred to as Anhui/1 and Shanghai/1, respectively). In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011; H7N9; Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/04/2009; H1N1; CA04). Anhui/1, Shanghai/1, and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates (NHPs), Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs upon intranasal inoculation. Most critically, Anhui/1 transmitted via respiratory droplets in one of three pairs of ferrets. Glycan arrays demonstrated that Anhui/1, Shanghai/1, and A/Hangzhou/1/2013 (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was less sensitive than a pandemic 2009 H1N1 virus to neuraminidase inhibitors, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets, and NHPs and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

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The hemagglutinin (HA) of influenza A virus is a homotrimeric glycoprotein with an ectodomain composed of a globular head and stem region (26). Both regions carry N-linked oligosaccharide chains. The appearance or disappearance of oligosaccharides on the globular head has been reported to occur naturally during antigenic drift of influenza A/H3N2 viruses from 1968 to 1975 (15, 24, 25). The acquisition of new oligosaccharides is an important mechanism underlying the antigenic drift of HA. Antigenic drift occurs by accumulation of a series of point mutations resulting in amino acid substitutions in antigenic sites on the surface of the HA (1, 25). These substitutions prevent neutralization by antibodies directed against previous epidemic strains.Based on analysis of the HA sequences of A/H3N2 viruses isolated from 1968 to 2002, the oligosaccharide chains on the globular head show large variations in number among different A/H3N2 isolates, although five glycosylation sites at Asn residues 8, 22, 38, 285, and 483 on the stem region are strictly conserved. Most of the A/H3N2 viruses that circulated between 1968 and 1974 (represented by A/Aichi/2/68) had only two oligosaccharides at residues 81 and 165 on the globular head of the HA (Fig. 1A). However, viruses isolated in 1975 (represented by A/Victoria/3/75) had lost a glycosylation site at residue 81 and gained two new sites at residues 63 and 126. The 1986 isolates (represented by A/Memphis/6/86) had acquired a new carbohydrate attachment site at residue 246, and the 1997 isolates (represented by A/Sydney/5/97) had obtained two additional sites at residues 122 and 133. Some recent isolates (represented by A/Panama/2007/99) had often obtained a novel site at residue 144. Thus, the A/H3N2 viruses recently circulating have six or seven glycosylation sites on the globular head of the HA, although whether these are glycosylated is not known. Moreover, the HAs of influenza A/H1N1 viruses and influenza B viruses isolated recently also possess several oligosaccharide chains on their globular head. These observations suggest that the addition of new oligosaccharides to the globular head of the HA may provide influenza viruses with an increased ability to prevail among humans (14). Interestingly, however, examination of the available HA sequences of influenza A/H2N2 viruses showed that none of the HAs had obtained a new glycosylation site on the globular head, and they had only one carbohydrate chain at position 169 (21).We previously studied the antigenic structure of the HA of A/H2N2 virus and revealed that most of the escape mutants selected by monoclonal antibodies had acquired a new glycosylation site at position 131, 160, or 187 on the tip of the HA (21,22,23). The results indicated that A/H2N2 viruses have the potential to gain at least one additional oligosaccharide on the globular head of the HA, although this has never occurred during 11 years of its circulation in humans. We constructed HA glycosylation site mutants containing one to three oligosaccharides at posi...