Virus isolation and molecular characterization.
Among the 38 cloacal swabs from captured free-living tree sparrows, 25 tested avian influenza virus antigen positive by enzyme-linked immunosorbent assay. These 25 samples were inoculated into embryonated eggs, and four avian influenza viruses were isolated. They were named A/Tree sparrow/Henan/1/04 (H5N1) (Ts/HN/1/04) to A/Tree sparrow/Henan/4/04 (H5N1) (Ts/HN/4/04). Each of eight gene segments of the four viruses was sequenced. The four isolates shared a high homology with each other (96% to 99%), except in their NA and PB2 genes. For the NA gene, only Ts/HN/4/04 did not contain a 20-amino-acid (aa) deletion in the stalk of the NA molecule, so the homology between Ts/HN/4/04 and other sparrow viruses was low (91% to 92%). The homology between the PB2 genes of Ts/HN/2/04 and Ts/HN/3/04 was 99%, but the homology between Ts/HN/1/04 and Ts/HN/2/04 (or Ts/HN/3/04) was 91%, while that between Ts/HN/4/04 and Ts/HN/2/04 (or Ts/HN/3/04) was 95%. Nucleotide sequence analysis revealed that they originated from different sources (Table ).
Percentages of sequence similarity between the tree sparrow virus isolates and other influenza viruses
The HA genes from the four tree sparrows all have the same multiple basic amino acids (RRKKR) at the connecting peptide between HA1 and HA2, which was considered a characteristic of influenza viruses that are highly pathogenic for chickens (11
). All amino acids relevant to receptor binding (aa 91, 130 to 134, 149, 151, 179, 186, 190 to 191, and 220 to 225) were identical to those of Gs/Gd/1/96 (3
). Therefore, it is likely that these viruses bind to cellular receptors with 2,3-NeuAcGal linkages, as has been shown for Gs/Gd/1/96 (7
The HA protein of Ts/HN/1/04, Ts/HN/2/04, and Ts/HN/3/04 had an additional glycosylation site (aa 170 to 172) at the head of the HA molecule and contained a 20-aa deletion in the stalk of the NA molecule, while Ts/HN/4/04 did not. The additional glycosylation site in the HA protein and the deletion of 20 aa in the NA protein were also dominant in other 2004 viruses isolated from poultry and human, which was suggested to be connected with the adaptation of viruses for spreading more efficiently in terrestrial domestic poultry (15
Four tree sparrow viruses had a 5-aa deletion (aa 80 to 84) in the middle of the NS molecule but did not have a mutation of Glu92 in the NS1 protein or Lys627 in the PB2 protein, which had been suggested to be associated with the increased virulence of H5N1 viruses in mice (4
). Moreover, the four viruses did not have mutations at the amino acids Ser31, Leu26, Val27, and Ala30 in the transmembrane region of the M2 protein, which occur in many genotype Z viruses and which have been proved to be associated with amantadine resistance (18
Phylogenetic analysis revealed that the HA genes of tree sparrows formed a branch in the phylogenetic tree with 2000-2004 isolates from Asia (genotypes Z, Z+, Y, A, B, C, D, E, and X). The HK/97 isolates formed another branch. Both branches originated from Gs/Gd/96-like viruses (Fig. ).
FIG. 1. Phylogenetic trees of virus genes isolated from tree sparrows in China. Trees were generated by using neighbor-joining analysis with the Tamura-Nei-gamma model in the MEGA program (version 2.0). Numbers below branches indicate bootstrap value percentages (more ...)
The NA gene tree showed that tree sparrows branched with genotypes A, B, C, D, E, X, Y, Z, and Z+ and with Gs/Gd/96 and formed a branch of avian influenza viruses. Isolates of HK/97 formed a separate branch. Unlike the other three tree sparrow isolates, Ts/HN/02 clustered into the branch of genotype Y, Z, and Z+ viruses of 2002 to 2004 (Fig. ).
Analysis of the internal protein genes shows that four H5N1 sparrow viruses originated from multiple avian influenza viruses. The PB2 genes of Ts/HN/2/04 and Ts/HN/3/04 were almost identical, but they were different from Ts/HN/1/04 and Ts/HN/4/04. Ts/HN/1/04 and Ts/HN/4/04 formed a branch together with genotypes A, B, C, D, E, Y, Z, Z+, and X and with Gs/Gd/96. This branch was distinctly related to the branch of Ts/HN/2/04 and Ts/HN/3/04 and the branch of H5N1/97 (Fig. ). Interestingly, the PB2 gene of DK/Yolohama/aq10/03, which was a genotypically unique H5N1 influenza virus isolated from duck meat in 2003 (14
), was found to be most closely related to that of the tree sparrows (Fig. ).
In the phylogenetic tree of the PB1 gene, the four sparrow viruses formed a separate fork and clustered with the branch of genotypes A, B, C, D, E, Y, Z, and Z+ and the branches of Gs/Gd/96 and HK/97. According to the phylogenetic tree, the PB1 gene of tree sparrows might be the ancestor of genotypes A, B, C, D, E, Y, Z, and Z+ and of Gs/Gd/96 and HK/97. The PB1 gene of genotype X is distinctly related to those of all the above-named genotypes (Fig. ).
In the phylogenetic tree of the PA gene, the four sparrow viruses formed a separate branch. The branch was distinctly related to the branch of genotypes A, B, C, D, E, Y, Z, Z+, and X and to the branch of Gs/Gd/96, the branch of H5N1/97, and that of Gf/HK/38/02 (Fig. ).
In the phylogenetic tree of the NP gene, the four sparrow viruses formed a separate fork and clustered with the branch of genotypes B, C, E, Y, Z, Z+, and X, the branch of HK/97, and that of DK/Yolohama/aq10/03 (14
). The NP genes of the above-named genotypes were similar to those of genotype A and Gs/Gd/96 but distinctly related to that of genotype D (Fig. ).
In the phylogenetic tree of the M gene, the four sparrow viruses formed a separate branch with H3 and H6 subtype viruses. The branch was distinctly related to the branch of genotypes A, B, C, D, E, X, Y, Z, and Z+, that of Gs/Gd/96, and that of HK/97 (Fig. ). The NS genes of four tree sparrows were distinctly related to that of genotypes A, B, C, D, E, X, Y, Z, and Z+, that of H5N1/97, and that of the H3, H4, H6, H9, and H11 subtypes (Fig. ).
According to the above-described analysis of phylogenetic relationships, the four tree sparrow isolates were different from other reported H5N1 genotypes. They were reassortants of Gs/Gd/96-like viruses; the outer genes are derived form Gs/Gd/96-like viruses, and the inner genes are derived from unknown influenza viruses resident in wild birds (Fig. ).