Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls

Abstract

In wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses circulating in black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H16 and the previously described subtypes H1 to H15. Genetically, H16 HA was found to be distantly related to H13 HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H13 and H16 HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H16 viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N3 genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. We propose that sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes.

FIG. 1.

Phylogeny of HA genes from Swedish and Dutch black-headed gull influenza A viruses. DNA maximum-likelihood trees were generated by using the HA₀ ORFs of prototype H13 influenza A viruses and all isolates described in this study (A) or representative sequences for all HA subtypes, when available from Eurasian avian origin (B). DNA maximum-likelihood trees were built by using 100 bootstraps and 3 jumbles, and branch lengths were recalculated for the resulting consensus tree. The scale bars roughly represent 10% of nucleotide changes between close relatives. MD, A/Gull/Maryland; ME, A/Pilot whale/Maine; AS, A/Gull/Astrakhan; NL, A/Black-headed Gull/The Netherlands; SE, A/Black-headed Gull/Sweden. Small numbers in trees represent bootstrap values.

FIG. 2.

Amino acid sequence identity between pairs of HA sequences. The average percent amino acid (aa) sequence identities and standard deviations are shown for all pairs of HA sequences within a subtype (A) or for all pairs of HA sequences from two closely related subtypes (B). Black, white, and gray bars represent HA₀, HA₁, and HA₂ sequences, respectively. HA subtype 13′ represents the combined group of H13 and H16 HA sequences. The percentages and standard deviations were calculated by using all HA sequences available from public databases upon sequence alignment per subtype (A) or per two subtypes (B) and subsequent calculation of pairwise Hamming distances (13).

FIG. 3.

Double radial immunodiffusion assay with hyperimmune rabbit antisera raised against HA of gull influenza A viruses. Concentrated hyperimmune antisera raised against H13 HA of A/Gull/Maryland/704/77 (A) or HA of A/Black-headed Gull/Sweden/5/99 (B) were loaded in wells S1 and S2, respectively. Lysates prepared from concentrated virus stocks of A/Gull/Maryland/704/77 (H13), A/Shoveler/The Netherlands/18/99 (H11), and A/Black-headed Gull/Sweden/5/99 were loaded in antigen wells A1, A2, and A3, respectively. Precipitates were allowed to form for 48 h, after which the gels were dried and stained with Coomassie brilliant blue. Specific precipitates were observed only between wells A1 (H13, A/Gull/Maryland/704/77) and S1 (anti-H13, A/Gull/Maryland/704/77) and wells A3 (H16, A/Black-headed Gull/Sweden/5/99) and S2 (anti-H16, A/Black-headed Gull/Sweden/5/99), as indicated by arrowheads.

FIG. 4.

Alignment of predicted amino acid sequences of HA subtype H16. An alignment of representative H16 amino acid sequences (2/99, A/Black-headed Gull/Sweden/2/99; 5/99, A/Black-headed Gull Sweden/5/99) is shown with periods representing identical amino acid residues and dashes representing gaps. Potential N-linked glycosylation sites (N-X-T/S) are boxed and numbered. Asterisks above the sequences indicate amino acid positions at which differences were observed between HA genes from Laridae and Anatidae (positions 191, 215, 222, 227, 228, 229, and 231 in H3 HA) (36). The predicted signal peptide and the HA₁ and HA₂ domains are indicated.

FIG. 5.

Phylogeny of NA genes from Swedish and Dutch black-headed gull influenza A viruses. A tree was generated with all 649 full-length NA amino acid sequences available from public databases (A). Aligned sequences were bootstrapped 100 times, and the Kimura amino acid distance matrices were converted to trees by using a UPGMA clustering algorithm. Very similar trees were obtained when distance matrices were generated by using Hamming distances or the Jones-Taylor-Thornton model (5, 13). The consensus tree was used for recalculation of the branch lengths with the Fitch program of Phylip. Representative nucleotide sequences, when available from Eurasian avian origin, were used to generate a DNA maximum-likelihood tree (B). This tree was built by using 100 bootstraps and 3 jumbles, after which branch lengths were recalculated for the consensus tree. NL/1/00, SE/1/99, SE/2/99, and SE/5/99 represent the NA sequences of black-headed gull viruses of subtypes H13N8, H13N6, H16N3, and H16N3 from The Netherlands and Sweden, respectively. Scale bars roughly represent 10% of changes between close relatives. Small numbers in trees represent bootstrap values.

FIG. 6.

Phylogenetic trees representing the internal genes of influenza A viruses. Trees were constructed based on a 2,262-nucleotide fragment of gene segment 1 (PB2) (A), 2,267 nucleotides of gene segment 2 (PB1) (B), 2,142 nucleotides of gene segment 3 (PA) (C), 1,486 nucleotides of gene segment 5 (NP) (D), 947 nucleotides of gene segment 7 (MA) (E), and 794 nucleotides of gene segment 8 (NS) (F). Sequences obtained from influenza viruses A/Black-headed Gull/The Netherlands/1/00 (H13N8) (NL 1), A/Black-headed Gull/Sweden/1/99 (H13N6) (SE 1), A/Black-headed Gull/Sweden/2/99 (H16N3) (SE 2), and A/Black-headed Gull/Sweden/5/99 (H16N3) (SE 5) were compared with those of reference strains available from GenBank, representing the known genetic lineages of influenza A virus (8). The DNA maximum-likelihood trees were built by using 100 bootstraps and 3 jumbles, and branch lengths were recalculated for the resulting consensus tree. Scale bars roughly represent 10% of nucleotide changes between close relatives. Small numbers in trees represent bootstrap values.