Large-scale sequence analyses of influenza viruses revealed that nonstructural 1 (NS1) proteins from avian influenza viruses have a conserved C-terminal ESEV amino acid motif, while NS1 proteins from typical human influenza viruses have a C-terminal RSKV motif. To test the influence of the C-terminal domains of NS1 on the virulence of an avian influenza virus, we generated a wild-type H7N1 virus with an ESEV motif and a mutant virus with an NS1 protein containing a C-terminal RSKV motif by reverse genetics. We compared the phenotypes of these viruses in vitro in human, mouse, and duck cells as well as in vivo in mice and ducks. In human cells, the human C-terminal RSKV domain increased virus replication. In contrast, the avian Cterminal ESEV motif of NS1 increased virulence in mice. We linked this increase in pathogenicity in mice to an increase in virus replication and to a more severe lung inflammation associated with a higher level of production of type I interferons. Interestingly, the human C-terminal RSKV motif of NS1 increased viral replication in ducks. H7N1 virus with a C-terminal RSKV motif replicated to higher levels in ducks and induced higher levels of Mx, a type I interferon-stimulated gene. Thus, we identify the C-terminal domain of NS1 as a species-specific virulence domain.

In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.

This study analysed the immune response in the intestinal tract of ducks infected with low-pathogenic avian influenza viruses compared with ducks treated orally with R848, a synthetic Toll-like receptor 7 (TLR7) agonist. Influenza virus infection induced a type I interferon (IFN)-dependent immune response characterized by the expression of Mx transcripts in the ileum at levels that were proportional to viral load. Mx transcripts were detected in differentiated enterocytes from influenza virus-infected ducks. By contrast, in R848-treated ducks, Mx transcripts were detected solely in intraepithelial round cells of haematopoietic origin. An increase was detected in the number of intraepithelial TLR7-positive cells and intraepithelial IFN-aproducing cells in influenza virus-infected ducks, albeit to a lower level than in R848-treated ducks. IFN-c expression was also upregulated in the intestine of influenza virus-infected and R848-treated ducks. Finally, interleukin (IL)-1b and IL-8 transcripts were expressed at high levels in R848-treated ducks but were not increased in influenza virus-infected ducks. These findings suggest that a type I IFN-mediated immune response in enterocytes and the activation of IFN-c-secreting cells contribute to the control of influenza virus replication in the duck intestine. INTRODUCTIONDucks are frequently infected with avian influenza viruses and have been shown to shed viruses belonging to multiple subtypes (Olsen et al., 2006). They represent an important reservoir species of avian influenza viruses. These viruses can occasionally be transmitted from ducks to other bird species, such as gallinaceous poultry, or to mammals, including humans (Taubenberger & Kash, 2010). Ducks thus have a central role in the epidemiology of influenza virus infection.The vast majority of viruses isolated from ducks are lowpathogenic avian influenza (LPAI) viruses (Munster et al., 2007). LPAI viruses replicate asymptomatically in epithelial cells from the duck intestine and are excreted in the faeces (Kida et al., 1980;Webster et al., 1978). Highly pathogenic avian influenza (HPAI) viruses have also been isolated from ducks (De Marco et al., 2005;Sturm-Ramirez et al., 2005). Interestingly, whilst abrupt death is the usual outcome of HPAI virus infection in gallinaceous poultry, HPAI virus-infected ducks usually present only mild clinical signs and recover from infection (Perkins & Swayne, 2003;Wood et al., 1995). Asian HPAI H5N1 viruses from the clade 2.2 genotype represent an exception, as they have been described as being lethal to ducks (Kim et al., 2008). However, lethality is mainly observed and reproduced experimentally in young ducks (Löndt et al., 2010;Pantin-Jackwood et al., 2007). By contrast, older ducks are able to survive infection with the Asian HPAI H5N1 viruses from the clade 2.2 genotype, suggesting that ducks have the ability to control the replication of the most virulent HPAI viruses. Importantly, HPAI viruses have a similar tropism in poultry and ducks, and comparative analyses have ...