H5N1 highly pathogenic avian influenza virus (HPAIV) is not only a catastrophic pathogen for poultry, but it poses a severe threat to the public health and may cause a future influenza pandemic. In 1997, highly pathogenic H5N1 avian influenza virus caused outbreaks in chickens in Hong Kong and was transmitted to humans, causing the deaths of 6 of 18 people infected (4
). The H5N1 outbreaks in poultry, which became widespread in late 2003, affected at least 10 Asian countries initially, but since then, H5N1 viruses have been isolated from wild birds (3
) and poultry in multiple countries in Asia, Europe, and Africa (http://www.oie.int
). H5N1 influenza virus infections have occurred in several mammalian species, such as pigs, domestic cats, tigers, and leopards (http://www.oie.int
). More importantly, human cases of H5N1 infections have been reported in many countries (http://www.who.int
), with greater than 50% mortality caused by H5N1 viruses among infected humans. Such findings have sparked great interest in pandemic preparedness as well as in understanding the genetic determinants of influenza virus pathogenicity and the ability of the virus to cross species barriers to mammalian hosts.
The pathogenicity of influenza viruses is determined by many factors, including virus-specific determinants encoded within the virus genome. In the H5 and H7 subtypes of influenza viruses, the multiple basic amino acids adjacent to the cleavage site of the hemagglutinin (HA) glycoprotein are a prerequisite for lethality in chickens and mice (12
). For H5N1 influenza viruses, a reverse genetics study demonstrated that a single-amino-acid substitution at position 627 of the PB2 protein from glutamic acid to lysine is responsible for virulence in mammalian species (12
). Moreover, the amino acid at position 701 in PB2 plays a crucial role in the ability of H5N1 viruses of duck origin to replicate and be lethal in mice (16
). This same PB2 amino acid residue contributes to the increased lethality of an H7N1 avian influenza virus in a mouse model (9
Several studies have reported that the NS1 protein is also associated with the virulence and host range of influenza viruses in different animal models (17
). Influenza viruses in which the NS1 gene was deleted exhibited an attenuated phenotype in mice and pigs (23
). The glutamic acid at position 92 of the NS1 protein of the H5N1 influenza virus that transmitted to humans in 1997 was shown to be critical in conferring virulence and resistance to antiviral cytokines in pigs (27
). However, H5N1 virus with this amino acid residue is no longer circulating in nature and glutamic acid is not found in the NS1 proteins of other influenza viruses. Another amino acid substitution at position 149 of the NS1 protein from valine to alanine was shown to be responsible for the replication of a goose H5N1 influenza virus in chickens (17
); however, this mutation did not affect virus virulence in mammals (H. Chen, unpublished data). Thus, the specific amino acid residues in avian NS1 that are responsible for conferring high virulence in mammals remain unclear.
Host factors, such as the immune responses, also play a role in determining influenza virus pathogenicity (14
). The interferon (IFN) response represents an early host defense mechanism against viral infections and is an important component of innate immunity (33
). The presence of double-stranded RNA (dsRNA) is a signal to the host cell that virus infection and replication are occurring and triggers a plethora of antiviral host defense mechanisms (5
). The presence of dsRNA induces the synthesis of alpha/beta IFN (IFN-α/β) proteins through the activation of several transcription factors, including IRF-3, IRF-7, NF-κB, and c-Jun/ATF2. Influenza viruses have dsRNA species of replication intermediates that elicit the host IFN response. The secreted IFN-α/β induces an antiviral state in influenza virus-infected and uninfected neighboring cells by stimulating the transcription of IFN-stimulated response element promoter-containing genes via the JAK/STAT pathway (29
). However, influenza and other viruses have developed strategies to counteract host IFN-α/β production, through inhibiting the activation of transcription factors involved in IFN activation (10
) and by attenuating host gene expression (20
). Antagonism of the innate response by influenza virus is a property of the NS1 protein (7
). Although data in this area of research are growing, there remain facets of host range and virulence determination that need further examination.
In this study, we characterized two H5N1 avian influenza viruses that were isolated from ducks, A/Duck/Guangxi/12/2003 (DK/12) and A/Duck/Guangxi/27/2003 (DK/27), in the Guangxi province of China in 2003. These two viruses are highly pathogenic for chickens but differ in their virulences in mice. We used reverse genetics to determine the molecular basis for the difference in virulence in mice and to explore the possible underlying mechanisms. We found a specific amino acid in NS1 that confers lethality in mice to an avian H5N1 virus.