Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3SL-PAA and 6SLN-PAA, which contained, respectively, 3-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q3L, increased binding to 6SLN-PAA, while among H1 swine viruses, the 190E3D and 225G3E mutations in the HA appeared important for the increased affinity of the viruses for 6SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.Influenza A viruses contain eight segments of negativesense, single-stranded RNA (reviewed in reference 16). Each RNA segment encodes at least one protein, and two of these proteins, hemagglutinin (HA) and neuraminidase (NA), project through the viral envelope and are available for interactions with cellular molecules. The abundance of each protein varies among virus subtypes, with the HA-NA ratio of influenza virus A/WSN/33 (H1N1) being approximately 10 to 1 (21). Since HA and NA recognize the same molecule (sialic acid) with conflicting activities, it can be assumed that drastic changes in either activity would affect viral replication.The HA, a type I integral membrane glycoprotein, is cleaved into two disulfide-linked chains, HA1 and HA2, by host proteases. Such cleavage is critical for viral infectivity, because it exposes the membrane fusion peptide located at the amino terminus of the HA2 subunit (reviewed in reference 14). The HA functions as a homotrimer of noncovalently linked monomers and plays two major roles during the replication of influenza A virus in host cells. First, it attaches the virus to the cell surface by binding to sialic-acid-containing receptors and promotes viral penetration by mediating fusion of the endosomal and viral membranes. The conserved sialic acid receptor-binding pocket, located on the HA1 subunit at the distal end of the molecule, binds to monovalent sialic acid receptor analogs with relatively low affinity (dissociation constant, approximately 0.1 to 1 mM [11]); however, the high abundance of HA molecules on the virion surface permits a sufficient number of low-affinity interactions to allow virus attachment and entry into host cells.The NA molecule, a type II integral membrane glycoprotein (7, 28), consists of a box-like catalytic head, a centrally attached stalk with a hydrophobic transmembrane-spanning region that attaches the molecule to the plasma ...

No abstract

Synthetic sialylglycoconjugates bearing 3'-sialyllactose, 6'-sialyllactose, or 6'-sialyl(N-acetyllactosamine) moieties attached to the polyacrylic acid carrier (P-3-SL, P-6-SL, and P-6-SLN, respectively) were prepared and tested for their ability to bind to influenza virus isolates from different hosts in a competitive solid phase assay. The virus panel included egg-grown avian and porcine strains, as well as human viruses isolated and propagated solely in mammalian (MDCK) cells and their egg-adapted variants. A clear correlation was observed between the pattern of virus binding of two glycopolymers, P-3-SL and P-6-SLN, and the host species from which the virus was derived. Avian isolates displayed a high binding affinity for P-3-SL and a two to three orders of magnitude lower affinity for P-6-SLN. By contrast, all non-egg-adapted human A and B viruses bound P-6-SLN strongly but did not bind P-3-SL. Unlike the "authentic" human strains, their egg-adapted counterparts acquired an ability to bind P-3-SL, indicative of a shift in the receptor-binding phenotype toward the recognition of Neu5Ac2-3Gal-terminated sugar sequences. Among the porcine viruses and human isolates with porcine hemagglutinin, few displayed an avian-like binding phenotype, while others differed from both avian and human strains by a reduced ability to discriminate between P-3-SL and P-6-SLN. Our data show that sialylglycopolymers may become a useful tool in studies on molecular mechanisms of interspecies transfer, tissue specificity, and other structure-function relationships of the influenza virus hemagglutinin.

Receptor specificity of influenza A/H5 viruses including human 2003-04 isolates was studied. All but two isolates preserved high affinity to Sia2-3Gal (avian-like) receptors. However, two isolates (February, 2003, Hong Kong) demonstrated decreased affinity to Sia2-3Gal and moderate affinity to a Sia2-6Gal (human-like) receptors. These two viruses had a unique Ser227-Asn change in the hemagglutinin molecule. Thus, a single amino acid substitution can significantly alter receptor specificity of avian H5N1 viruses, providing them with an ability to bind to receptors optimal for human influenza viruses. Asian 2003-04 H5 isolates from chickens and humans demonstrated highest affinity to the sulfated trisaccharide Neu5Acalpha2-3Galbeta1-4(6-HSO3)GlcNAcbeta (Su-3'SLN) receptor but, in contrast to 1997 isolates, had increased affinity to fucosylated Su-3'SLN. American poultry H5 viruses also had increased affinity to Su-3'SLN. These data demonstrate that the genetic evolution of avian influenza A(H5N1) viruses is accompanied during adaptation to poultry by the evolution of their receptor specificity.