According to the Influenza A Virus Genotype Tool 12 the studied genes of the investigated Swedish isolates belonged to the following lineages: PB2 (K), PB1 (G), PA (D), HA (5J), NP (F), NA (1J), MP (F), NS (1E).

All twelve Swedish H5N1 isolates in this study belonged to the 2.2. clade and the phylogenetic trees of all eight genes had similar topologies. Representative trees of the HA and PB2 genes are shown (Figures 1 and 2). These data along with those generated from the other genes confirmed the close genetic relationship of H5N1 HPAIVs isolated in the northern region of Germany, Denmark and Sweden in early 2006. Two isolates out of the Swedish ones (A/tufted duck/Sweden/599/06 and A/herring gull/Sweden/1116/06) grouped together with sub-clade 2.2.1. viruses while the other ten belonged to sub-clade 2.2.2. No viruses of sub-clade 2.2.3. were identified among the studied ones.

HA amino acid residue 403 was observed to characterize 2.2.1. (isolates mainly from Southern parts of Germany) and 2.2.2. (German isolates from the North) sub-clade German H5N1 viruses because the former group contained mainly D while the latter N at this position. All sub-clade 2.2.2. Swedish H5N1 viruses possessed N at HA 403 position together with A/tufted duck/Sweden/599/06 sub-clade 2.2.1. isolate, and only 2.2.1. isolate A/herring gull/Sweden/1116/06 had D at this site.

As far as the NA gene concerned residues 34 I/V, 44 C/R, 305 S/N appeared to be discriminative of sub-clade 2.2.1./2.2.2. isolates, respectively, consistently in case of Swedish viruses and predominantly in the analyzed additional 100 sequences. Also, at NA amino acid position 305 sub-clade 2.2.1. Swedish isolates uniquely had an S while all other viruses that were analyzed (sub-clade 2.2.2. and 2.2.3. viruses) possessed N at this position due to a AAT→AGT transition. Sub-clade 2.2.2. Swedish viruses, and A/great crested grebe/Denmark/7498/06, A/grey lag goose/Denmark/6692/06, A/buzzard/Denmark/6370/06, A/tufted duck/Denmark/6540/06, and A/swan/Germany/R65/06 isolates possessed D at NA position 316 while all other analyzed viruses had G at this site.

The separation of the Swedish H5N1 HPAIVs into two subgroups was already demonstrated on the basis of NS gene sequences 10 and this finding was consistent for all eight genes of the isolates (herein summarized in Additional file 1). No reassortant variant was found among the sequenced twelve Swedish isolates.

Characteristic findings regarding the preservation/substitutions at particular amino acid positions along with potential distinctive molecular markers for the Swedish H5N1 viruses are summarized in Additional file 1.

A single amino acid substitution, from glutamic acid (E) to Lysine (K) in position 627 in PB2 is a determinant of mammalian host range 1314. Most avian isolates have E in this position. The substitution to K in this position converts a nonlethal H5N1 influenza A virus isolated from a human to a lethal virus in mice 13. Among the H5N1 HPAIV sequences we investigated a larger proportion of those originating from 1998–2005 had PB2-E627 than more recent isolates. The 2.2.2.-like Swedish viruses along with the most closely related Danish and German isolates encoded K at this site while the two sub-clade 2.2.1.-like Swedish isolates (A/tufted duck/Sweden/599/06 and A/herring gull/Sweden/1116/06) possessed E at position 627. The mutations D701N and S714R in PB2 contribute to virulence by enhancing polymerase activity 15. All Swedish isolates had D and S at position 701 and 714, respectively.

PB1-F2 has been identified as a proapoptotic mitochondrial protein expressed from a second open reading frame of the PB1 gene 16 and it has been shown to contribute to viral pathogenesis in mice 17. Aspargine in position 66 in PB1-F2 has been demonstrated to play a key role in the pathogenicity of H5N1 viruses 18 and its presence was determined in all Swedish viruses. Furthermore, Swedish 2.2.1. subclade viruses had a K26Q substitution compared to 2.2.2. subclade viruses. Isolate A/tufted duck/Sweden/599/06 solely contained a T323I and a H562P, while A/herring gull/Sweden/1116/06 a V719M substitution, respectively. The H5N1 viral polymerase activity is enhanced by the presence of PB2 701N and 714R, PB1 13P, PA 615N, further, NP 319K and 678N 15. Among the Swedish isolates the presence of PB1 13P was determined.

The HA sequences of isolates A/Mute swan/Sweden/827/06, A/Canada goose/Sweden/978/06, and A/peregrine/Denmark/6632/06 proved to be identical. The amino acid sequence flanking the cleavage site of the HA gene was PQGERRRKKRGLF alike all other 2.2. viruses with the exception of some French isolates that had the PQGERKRKKR/G sequence 11. The identified amino acid markers of H5N1 influenza viruses isolated at Qinghai and Poyang Lakes from migratory birds (HA-I99, HA-N268, and NA-R110) were present in all Swedish isolates as well 11. No "sub-clade"-specific amino acid changes were identified in the HA among the two subgroups of Swedish isolates. All the Swedish isolates had the 238Q and 240G (numbered from the H5 start codon) which indicates preferred receptor specificity for the avian α-(2,3) linkage to galactose 1920. All HA sequences contained 6 N-linked potential glycosylation sites, as analysed with NetNglyc server (threshold: 0.5) at the following positions: 27, 39, 181, 302, 500, 559; none of them is adjacent to the cleavage site. Furthermore, the substitutions S145L and A172T, which are associated with viral adaptation to poultry 21 were not determined in association with the Swedish H5N1 viruses.

The amino acid substitutions R178I and I248V in HA that were found in the domestic birds of the Danish isolates 22 were not present in any of the Swedish viruses, nor the V73I substitution that was found in the Danish swan isolates. However, the D387N substitution found in the German and most of the Danish isolates was also present in the Swedish isolates.

The H5N1 virus isolated from a mink (A/Sweden/mink/2006/V907) was examined in order to reveal any possible adaptation towards mammals. As a result, a unique E513G substitution was found in the HA gene but no substitutions that could be regarded as host-related were found, which is consistent with previous findings, i.e. that a single passage in mammals is not necessarily associated with changes in receptor-binding sites 9.

As in the other 2.2. viruses, NA-R110 was present in the Swedish isolates, and a 20 amino acid deletion was also found at positions 49–68 similarly to the majority of the recent H5N1 strains 22. The N228S substitution was present only in A/Herring gull/1116/06 Swedish 2.2.1. virus (alike with several other member of the sub-clade) and not in A/Tufted duck/Sweden/599/06 isolate. These two isolates differed further in amino acid residues 414 and 434 by bearing N/K and S/G corresponding to A/Herring gull/1116/06 and A/Tufted duck/Sweden/599/06 viruses, respectively. Interestingly, while the Danish and German isolates shared unique amino acids in the NA (G336D), PB1 (K531R) and NS2 (G63E) proteins the Swedish isolates were not homogenous in this regard: although NA-G336D was a characteristic of the Swedish viruses too, two isolates retained the PB1-531K, and NS2-63G. Reported substitutions in NA, inducing oseltamivir resistance 9, were not found in the Swedish isolates.

The NP-10Y amino acid residue, which may affect the pathogenicity of AIVs 15, was present in all of the Swedish isolates. Concerning the M2 gene, all Swedish viruses contained the L26-V27-A30-S31-G34 amino acid pattern, thus, no adamantan drug resistant variant was revealed 9. Substitutions S64A and E66A that were present in the M2 genes of H5N1 AIV isolates from Hong Kong 11 did not appear in Swedish viruses.

The complete characterization of the NS genes from these isolates was described by Zohari et al., 10, and is not further discussed here.

The effect of substitutions on the predicted antigenicity was investigated among the Swedish isolates for the surface glycoproteins (HA and NA) and for those primarily targeted by the host's cellular immune response (PB2, PA, and NP 23) (Table 1). The observed amino acid alterations affected the predicted antigenic epitopes in few cases. Regarding the HA in all but one cases 22 epitopes were predicted by the Kolaskar-Tongaonkar approach 24, the exception was strain A/herring gull/Sweden/1116/06, bearing a V201M substitution, which resulted in splitting the corresponding GKLCDLDGVKPLILRDCSVAGW predicted epitope (between amino acid residues 55–76) into two smaller ones: GKLCDLD (aa residues 55–61) and PLILRDCSVAGW (aa residues 65–76). The predicted numbers of epitopes in NA were higher for Swedish 2.2.1. viruses than for 2.2.2. viruses (19–20 compared to 17–18). However, in this case no splitting of epitope(s) was predicted due to a change in the amino acid sequence, but rather, the substitutions could be associated with the appearance of newer epitopes (data not shown). No changes in the number of predicted epitopes was found in for PB2 and PA. In general, the Swedish viruses coded for 15 epitopes on the NP with the only exception of sublineage 2.2.2. virus A/eagle owl/Sweden/V618/06, which had an additional epitope of seven amino acids between residues 22–28. In summary, the detected amino acid changes among the Swedish viruses appeared to have greater effect on the composition of proteins targeted by the humoral than those targeted by the cellular immune responses, in particular, on the NA gene.

The isolates involved in this study are shown in Table 2. They were collected during the HPAI outbreak in Northern Europe in spring 2006 10.

The collection of specimens, RNA extraction, and RT-PCR amplification of the NS1 sequences was described earlier and the same RNA batches were used for this study that served as targets in the previous investigation 10. In order to obtain possibly the full length nucleotide sequences of the coding regions of the influenza virus isolates several approaches were combined that comprised of either published protocols/primers 222627 or those developed and used by the Influenza Genome Sequencing Project [28; the primer sequences were kindly provided by David Spiro, The J. Craig Venter Institute, Rockville, Maryland, USA), or designed by ourselves. The primer and PCR protocols for sequencing are available from the authors upon request.

For the phylogenetic analyses, a set of H5N1 AIVs that were isolated in Europe, Asia and Africa in 2005 – 2006 was selected and used for all genes. These were collected from the Influenza Virus Resource at NCBI 29 and these were included in the phylogenetic analyses.

Sequence assembly, multiple alignment and alignment trimming were performed with the CLC Combined Workbench 3.0.2. bioinformatics software (CLC bio A/S, Aarhus, Denmark). Distance based neighbor joining and character based maximum parsimony phylogenetic trees were generated using the Molecular Evolutionary Genetics Analysis (MEGA) software v.4.0. 30 with 1000 bootstrap replicates. For the neighbor-joining trees, the Kimura-2 substitution model was used. Other models were also tested which showed similar topologies. The evolutionary divergence between the sub-clades was investigated by pairwise analyses over all sequence pairs between the groups by using the MEGA software also. The occurrence and distribution of synonymous and nonsynonymus substitutions was investigated by the DNA Sequence Polymorphism software (Version 4.50.3) software 31. Computational analysis of the antigenic sites was carried out by using the Kolaskar-Tongaonkar method 24.

Nucleotide sequences from Swedish H5N1 virus isolates included in this study have been submitted to GenBank with the following accession numbers: PB2: EU889035–EU889046, PB1: EU889047–EU889058, PA: EU889059–EU889070, HA: EU889071–EU889082, NP: EU889083–EU889094, NA: EU889095–EU889106, M: EU889107–EU889118.