The evolution of H5N1 influenza viruses in ducks in southern China

Abstract

The pathogenicity of avian H5N1 influenza viruses to mammals has been evolving since the mid-1980s. Here, we demonstrate that H5N1 influenza viruses, isolated from apparently healthy domestic ducks in mainland China from 1999 through 2002, were becoming progressively more pathogenic for mammals, and we present a hypothesis explaining the mechanism of this evolutionary direction. Twenty-one viruses isolated from apparently healthy ducks in southern China from 1999 through 2002 were confirmed to be H5N1 subtype influenza A viruses. These isolates are antigenically similar to A/Goose/Guangdong/1/96 (H5N1) virus, which was the source of the 1997 Hong Kong "bird flu" hemagglutinin gene, and all are highly pathogenic in chickens. The viruses form four pathotypes on the basis of their replication and lethality in mice. There is a clear temporal pattern in the progressively increasing pathogenicity of these isolates in the mammalian model. Five of six H5N1 isolates tested replicated in inoculated ducks and were shed from trachea or cloaca, but none caused disease signs or death. Phylogenetic analysis of the full genome indicated that most of the viruses are reassortants containing the A/Goose/Guangdong/1/96-like hemagglutinin gene and the other genes from unknown Eurasian avian influenza viruses. This study is a characterization of the H5N1 avian influenza viruses recently circulating in ducks in mainland China. Our findings suggest that immediate action is needed to prevent the transmission of highly pathogenic avian influenza viruses from the apparently healthy ducks into chickens or mammalian hosts.

Fig. 1.

Weight change of mice inoculated with H5N1 duck influenza viruses representative of the four pathotypes. Groups of five mice were inoculated i.n. with 10⁶ eID₅₀ (in 50 μl) of DKGX/22/01 (⋄) (nonpathogenic), DKGD/07/00 (○) (low pathogenicity), DKSH/13/01 (▵) (medium pathogenicity), or DKFJ/01/02 (*) (high pathogenicity) virus or with PBS as control (□) and weighed daily for 14 days.

Fig. 2.

Phylogenetic trees for the HA (a), NA (b), PB2 (c), PB1 (d), PA (e), NP (f), M(g), and NS (h) genes of the H5N1 influenza A viruses analyzed. The trees were generated by using megalign software (DNASTAR) on the basis of the following gene sequences: nucleotides 29–1,732 (1,704 bp) of HA, 21–1,367 or 1,427 (1,347 or 1,407 bp) of NA, 28–2,304 (2,276 bp) of PB2, 25–2,295 (2,271 bp) of PB1, 25–2,172 (2,148 bp) of PA, 45–1,539 (1,495 bp) of NP, 26–781 (756 bp) of M, and 27–708 or 725 (682 or 699 bp) of NS. The length of each pair of branches represents the distance between sequence pairs, and the units at the bottom of the tree indicate the number of substitution events. The colors indicate the pathotypes identified in mice: red, high pathogenicity; pink, medium pathogenicity; green, low pathogenicity; black, nonpathogenic. Table 1 lists the name of the viruses; sequences of the underlined viruses were obtained from GenBank.

Fig. 3.

The genotypic evolution of the H5N1 viruses isolated from ducks in southern China from 1999 through 2002. The eight gene segments in each schematic virus particle are (top to bottom) the PB2, PB1, polymerase (PA), HA, NP, NA, matrix (M), and NS genes. Genes of the same lineage are shown in the same color. The capital letters indicate the genotypes. Table 2 lists the viruses of each genotype.