Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR

Abstract

Although A/Hong Kong/156/97 (H5N1/97)-like viruses associated with the "bird flu" incident in Hong Kong SAR have not been detected since the slaughter of poultry in 1997, its putative precursors continue to persist in the region. One of these, Goose/Guangdong/1/96 (H5N1 Gs/Gd)-like viruses, reassorted with other avian viruses to generate multiple genotypes of H5N1 viruses that crossed to chickens and other terrestrial poultry from its reservoir in geese. Whereas none of these recent reassortants had acquired the gene constellation of H5N1/97, these events provide insight into how such a virus may have been generated. The recent H5N1 reassortants readily infect and kill chicken and quail after experimental infection, and some were associated with significant mortality of chickens within the poultry retail markets in Hong Kong. Some genotypes are lethal for mice after intra-nasal inoculation and spread to the brain. On this occasion, the early detection of H5N1 viruses in the retail, live poultry markets led to preemptive intervention before the occurrence of human disease, but these newly emerging, highly pathogenic H5N1 viruses provide cause for pandemic concern.

Figure 1

Phylogenetic trees for the H5 HA1 (a), NP (b), and NS (c) genes of influenza A viruses. The nucleotide sequences were analyzed with PAUP by using a maximum-parsimony algorithm. Nucleotides 43 to 1,023 (981 bp) of the HA gene, 42 to 1,002 (961 bp) of the NP gene, and 41 to 824 (784 bp) of the NS gene were used for the phylogenetic analysis. The H5 HA1 phylogenetic tree is rooted to A/Japan/305+/57 (H2N2). The nucleotide trees of NP and NS gene are rooted to A/Equine/Prague/1/56 (H7N7). 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. Names and abbreviations of viruses studied are listed in Table 1, and the abbreviations are denoted in the footnote. Other sequences can be found in GenBank.

Figure 2

Generalized phylogenies in diagrammatic form of PB2, PB1, PA, NP, M, and NS genes of H5N1 influenza viruses. The nucleotide sequences of PB2 (992 bp), PB1 (979 bp), PA (645 bp), NP (961 bp), and NS (784 bp) were used in the phylogenetic analysis. Only the major terminal branches of the Eurasian avian lineages of the trees are shown. The genes of H5N1 viruses isolated in 2001 are shown in blue, and those of the precursor Gs/Gd/1/96 are in orange.

Figure 3

The genotypes of H5N1 reassortants and their derivation from 1997 to 2001. The eight gene segments in each schematic virus particle are represented in the order (top to bottom) PB2, PB1, polymerase (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and nonstructural (NS) genes. A different color is used to represent each distinct virus lineage. The virus abbreviations used are: H9N2 (G1), Quail/Hong Kong/G1/97; H6N1 (W312), Teal/Hong Kong/W312/97; H5N1 Gs/Gd, Goose/Guangdong/1/96; H9N2/Y280, Chicken/Hong Kong/Y280/97; and H5N1/97, A/Hong Kong/156/97-like viruses. Abbreviations used for poultry are explained in the footnote. The reassortment events that most likely led to the genesis of the pathogenic viruses of 1997 (H5N1/97) are included for reference. ? denotes that genotype A viruses have not so far been isolated from aquatic poultry in Hong Kong.