We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice.

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.

In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/ 27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-B pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.

In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.