Molecular evolution of H6 influenza viruses from poultry in Southeastern China: prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry

Abstract

The A/teal/Hong Kong/W312/97 (H6N1) influenza virus and the human H5N1 and H9N2 influenza viruses possess similar genes encoding internal proteins, suggesting that H6N1 viruses could become novel human pathogens. The molecular epidemiology and evolution of H6 influenza viruses were characterized by antigenic and genetic analyses of 29 H6 influenza viruses isolated from 1975 to 1981 and 1997 to 2000. Two distinct groups were identified on the basis of their antigenic characteristics. Phylogenetic analysis revealed that all H6N1 viruses isolated from terrestrial poultry in 1999 and 2000 are closely related to A/teal/Hong Kong/W312/97 (H6N1), and the nucleotide sequences of these viruses and of A/Hong Kong/156/97 (H5N1) were more than 96% homologous. The hemagglutinin (HA) of the 1999 and 2000 terrestrial viruses does not have multiple basic amino acids at the site of cleavage of HA1 to HA2; however, a unique insertion of aspartic acid in HA1 between positions 144 and 145 (H3 numbering) was found. The neuraminidase of these terrestrial H6N1 viruses has a deletion of 19 amino acids characteristic of A/Hong Kong/156/97 (H5N1). Evolutionary analysis suggested that these H6N1 viruses coevolved with A/quail/Hong Kong/G1/97-like H9N2 viruses and became more adapted to terrestrial poultry. These terrestrial 1999 and 2000 A/teal/Hong Kong/W312/97 (H6N1)-like viruses, along with the H9N2 viruses, could have been involved in the genesis of the pathogenic H5N1 influenza viruses of 1997. The presence of H6N1 viruses in poultry markets in Hong Kong that possess seven of the eight genes of the A/Hong Kong/156/97 (H5N1) virus raises the following fundamental questions relevant to influenza pandemic preparedness: could the pathogenic H5N1 virus reemerge and could the H6N1 viruses directly cross the species barrier to mammals?

FIG. 1.

Phylogenetic trees based on the sequences of the H6 HA and N1 NA genes of influenza viruses. Nucleotides 29 to 1730 (1,701 bp) of the H6 HA gene were used to construct the HA phylogenetic tree, which was rooted to A/mallard/Potsdam/178-4/83 (H2N2). Nucleotides 66 to 1373 (1,439 bp) of the N1 NA gene were used to construct the NA phylogenetic tree, which was rooted to A/WSN/33 (H1N1). H6 influenza viruses characterized in this study are underlined (NA phylogenetic tree). *, viruses with characteristic deletion of codons for 19 amino acids from the NA gene. 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. Gene sequences used in this study are listed under the following EMBL Nucleotide Sequence Database accession numbers: D90303, X5226, K02252, AF036357, AF057292, AF098548, AF144304, AF098551, and AF208598. Abbreviations: Sh, shearwater; Pa, parrot; Ab, aquatic bird.

FIG. 2.

Phylogenetic trees based on the sequences of the NS1 and PB2 genes of influenza viruses. H6 influenza viruses characterized in this study are underlined. The ranges of nucleotide sequences used to construct the phylogenetic trees are shown in Table 4. The NS1 phylogenetic tree is rooted to A/equine/Prague/1/56 (H7N7), and the PB2 phylogenetic tree is rooted to B/Lee/40. 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. Gene sequences used in this study are listed under the following EMBL Nucleotide Sequence Database accession numbers: AF156479, M17070, M60800, M55484, Z26865, U49492, L25830, AF156476, AF262212, U49493, AF250502, AF156482, AF156481, AF036360, AF098569, AF098571, AF250483, AF156477, AJ278649, AJ404735, AF156480, AF156472, AF156475, AF156440, AF156439, M55471, AF255624, AF156434, AF156437, AF156438, AF156436, AF156433, AF156430, AJ404630, AJ404630, AF036363, AF098577, AF098579, AF250476, and AF156435. Sw, swine.

FIG. 3.

Generalized phylogeny of M1, NP, PA, and PB1 genes of influenza viruses. H6 influenza viruses characterized in this study are underlined. Nucleotide ranges used to construct the phylogenetic trees are shown in Table 4. Gene sequences used in this study are listed under the following EMBL Nucleotide Sequence Database accession numbers: AF098560, AF036358, AF156463, AF250482, AJ278646, AF057293, AF036359, AF156470, AF250480, AJ289871, AF098604, AF046095, AF156449, AF250478, AJ404637, AF098590, AF036362, AF156421, AF250477, and AJ404634.