Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity

Abstract

Adaptation of avian influenza viruses for replication and transmission in the human host is believed to require mutations in the hemagglutinin glycoprotein (HA) which enable binding to human alpha2-6 sialosides and concomitant reduction in affinity for avian alpha2-3 linked sialosides. Here, we show by glycan microarray analyses that the two mutations responsible for such specificity changes in 1957 H2N2 and 1968 H3N2 pandemic viruses, when inserted into recombinant HAs or intact viruses of some recent avian H5N1 isolates (clade 2.2), impart such attributes. This propensity to adapt to human receptors is primarily dependent on arginine at position 193 within the receptor-binding site, as well as loss of a vicinal glycosylation site. Widespread occurrence of these susceptible H5N1 clade 2.2 influenza strains has already occurred in Europe, the Middle East, and Africa. Thus, these avian strains should be considered high-risk, because of their significantly lower threshold for acquiring human receptor specificity and, therefore, warrant increased surveillance and further study.

Fig. 1

Phylogenetic tree of selected hemagglutinin genes from H5N1 viruses. Phylogenetic trees were inferred from nucleotide sequences by the neighbor-joining method. The lengths of the horizontal lines are proportional to the numbers of nucleotide differences, as indicated by the scale bars. Bootstrap analysis values >90% are shown. The numerical fractions denote the frequency of the specified amino acids in each clade among all H5 HAs of the Goose/Guangdong/96 (H5N1) lineage available in public databases. Strains used in this study are highlighted in red text, and all but the Egret06 virus were isolates from humans infected by H5N1 avian viruses. Only clades whose viruses have infected humans are shown. For a full tree, readers should consult reference 1 or the WHO website.

Fig. 2

Assessment of viral dilutions on detection of sialoside ligands on the glycan microarray. Examples of the dilution series performed on the glycan microarray for virus suspensions. Viet04 wild type was analyzed in 2-fold serial dilutions for hemagglutination (HA) titers ranging from 512 to 64.

Fig. 3

Comparison of receptor specificity of representative H5N1 viruses (column 1) and corresponding recombinant HAs (column 2) using a glycan microarray. A number of HAs from 1997 to 2005 isolated from human infections were analyzed and compared. Viruses were analyzed at a HA titer of 128 per 50μl while 15 μg of recombinant HA was used for the complementary analysis. Binding to different categories of glycans on the array are highlighted as follows: red, α2-3 sulfated; green, α2-3 di-sialylated, yellow, linear α2-3 sialosides; orange, fucosylated α2-3 sialosides; blue, α2-3 internal sialosides; and pink α2-6 ligands in same order as listed for the α2-3 sialosides (see Table 2 for name list and structures). Because additional glycans were added to the microarrays during the course of this study, glycans # 1, 21, 22, 29, 31, 36 and 53 are absent on certain graphs.

Fig. 4

Impact of LS mutations propensities of recent H5N1 isolates to bind human-type receptors. Mutations responsible for adaptation of the 1968 H3 pandemic strain (Gln226Leu, Gly228Ser) to human-type receptor specificity were introduced into H5N1 viruses by reverse genetics (column 1) and the corresponding recombinant HAs (column 2). Mutations in the clade 1 strain, A/Vietnam/1203/2004 (Row 2) and a clade 2.1 strain; A/Indonesia/05/2005 (Row 3) both show reduced avian α2-3 specificity, and acquire weak binding to α2-6 glycans, in particular to α2-6 biantennary glycans (#40, 41). Most striking is the effect of the same mutations on the clade 2.2 avian virus, A/Egret/Egypt/1162/NAMRU-3/2006 (row 4), which shows substantially reduced avian α2-3 specificity, and significant binding to a wide spectrum of α2-6 glycans. Virus and recombinant HAs were analyzed as in Figure 3.

Fig. 5

Relationship of clade-specific mutations to the receptor binding domain of H5 HA. A) Structural model showing amino acid differences in and around the RBD of A/Indonesia/05/2005 compared to A/Vietnam/1203/2004. Seven changes are highlighted (with the Viet04 sequence labeled first), but only two, at positions 133 and 193, are within the RBD and likely to directly affect receptor binding. B) Structural model of the Viet04 HA trimer illustrating the direction that α2-6 sialosides exit the HA RBD and the role that a vicinal glycosylation site may play in steric hindrance. One monomer of the trimer was modeled with the lactoseries pentasaccharide, LSTc from the swine H9 HA co-crystal structure (pdb:1JSI) in the RBD. A biantennary glycan, commonly found on influenza viruses ; , was manually appended to the glycosylation site at Asn158 to illustrate the size of such a structure and how it may influence access to the RBD.

Fig. 6

Analysis of clade-specific mutations that increase propensity for binding to human-type receptors. Mutants were generated to assess the effect of Arg193 on the clade 1 strain, A/Vietnam/1203/2004 (Viet04-RLS, Row 1) and glycosylation at position 158 in HA on clade 1 (Viet04-ALS, Row 2) and clade 2.1 strain, A/Indonesia/05/2005 (Indo05-ALS, row 3), in addition to human H3 and H2 serotype mutations (Gln226Leu/Gly228Ser). The equivalent viruses shown were generated by reverse genetics and revealed a similar binding profile compared to the recombinant HA. Viruses were analyzed at an HA titer of 128. Because additional glycans were added to the microarrays during the course of this study, glycans #1, 21, 22, 29, 31, 36 and 53 are absent from some of the graphs.

Fig. 7

Structural models for binding of sialosaccharides in the RBD of the Viet04 HA. Truncated fragments of human lactoseries tetrasaccharides a and c (LSTa and LSTc) receptor analogs were modeled into the RBD of the Viet04 strain (PDB:2FK0 11) by superimposing the structures of H5 A/Duck/Singapore/3/97 and H9 A/Swine/Hong Kong/9/98 HAs (PDB:1JSN and 1JSI 31) with α2-3 and α2-6 analogs bound. Residues were substituted from the Viet04 model (A) to yield a model for (B) the mutant RLS. Putative hydrogen bonds between the sialoside and the RBD are shown as hashed green lines. Figures were generated with MacPymol .