Molecular characterization of H9N2 influenza viruses: were they the donors of the "internal" genes of H5N1 viruses in Hong Kong?

Abstract

The origin of the H5N1 influenza viruses that killed six of eighteen infected humans in 1997 and were highly pathogenic in chickens has not been resolved. These H5N1 viruses transmitted directly to humans from infected poultry. In the poultry markets in Hong Kong, both H5N1 and H9N2 influenza viruses were cocirculating, raising the possibility of genetic reassortment. Here we analyze the antigenic and genetic features of H9N2 influenza viruses with different epidemiological backgrounds. The results suggest that the H9N2 influenza viruses of domestic ducks have become established in the domestic poultry of Asia. Phylogenetic and antigenic analyses of the H9N2 viruses isolated from Hong Kong markets suggest three distinct sublineages. Among the chicken H9N2 viruses, six of the gene segments were apparently derived from an earlier chicken H9N2 virus isolated in China, whereas the PB1 and PB2 genes are closely related to those of the H5N1 viruses and a quail H9N2 virus-A/quail/Hong Kong/G1/97 (Qa/HK/G1/97)-suggesting that many of the 1997 chicken H9 isolates in the markets were reassortants. The similarity of the internal genes of Qa/HK/G1/97 virus to those of the H5N1 influenza viruses suggests that the quail virus may have been the internal gene donor. Our findings indicate that the human and poultry H5N1 influenza viruses in Hong Kong in 1997 were reassortants that obtained internal gene segments from Qa/HK/G1/97. However, we cannot be certain whether the replicate complex of H5N1 originated from Qa/HK/G1/97 or whether the reverse transfer occurred; the available evidence supports the former proposal.

Figure 1

Phylogenetic trees for the H9 HA1 and N2 NA genes of influenza A viruses. The nucleotide sequences of the HA1 and NA genes were analyzed with paup by using a maximum-parsimony algorithm. Nucleotides 43 to 1,047 (1,005 bp) of H9 HAs and nucleotides 21 to 1,393 (1,373 bp) of N2 NA were used for the phylogenetic analysis. The HA1 phylogenetic tree is rooted to duck/Alberta/60/76, DALB (H12N5). The N2 NA phylogenetic tree is rooted to A/ruddy turnstone/New Jersey/60/85, RTNJ60–85 (H4N9). The lengths of the horizontal lines are proportional to the minimum number of nucleotide differences required to join nodes. Vertical lines are for spacing branches and labels. Virus names and abbreviations are listed in Table 1, and the remaining sequences can be found in GenBank by using the following accession numbers: D90305, M12051, M11925, U42630, J02173, U42776, U43421, D00713, D29659, L37330, and M11205. All viruses underlined in the NA tree are H9N2 influenza viruses.

Figure 2

Phylogenetic trees for the NS and PB1 of avian influenza viruses from Eurasia. Phylogenetic relationships were determined with the paup program. The nucleotide tree of the NS gene is rooted to A/Equine/Prague/1/56 (H7N7). The PB1 tree is rooted to B/Lee/40. Nucleotides 39 to 842 (804 bp) of NS gene and nucleotides 25 to 2,237 (2,213 bp) of PB1 gene were used for the phylogenetic analysis. Only the terminal branches of the Eurasian avian lineage of the trees are shown. Virus names and abbreviations are listed in Table 1 or can be found in ref. or GenBank by using the following accession numbers: AF036360, U49492, U49493, M17070, M60800, M55484, Z46440, Z26865, and AF036362. All viruses underlined are H9N2 influenza viruses.

Figure 3

Generalized phylogenies in diagrammatic form of NP, M, PB2, and PA genes of H9N2 influenza viruses. The nucleotide sequences of NP (1,381 bp), M (935 bp), PB2 (2,177 bp), and PA (2,125 bp) were used in the phylogenetic analysis. Only the major terminal branches of the Eurasian avian lineages of the trees are shown. Abbreviations are listed in Table 1.