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.

A deletion of ϳ20 amino acids in the stalk of neuraminidase is frequently observed upon transmission of influenza A viruses from waterfowl to domestic poultry. A pair of recombinant H7N1 viruses bearing either a short-or long-stalk neuraminidase was genetically engineered. Inoculation of the long-stalk-neuraminidase virus resulted in a higher cloacal excretion in ducks and led conversely to lower-level oropharyngeal excretion in chickens, associated with a higher-level local immune response and better survival. Therefore, a short-stalk neuraminidase is a determinant of viral adaptation and virulence in chickens but is detrimental to virus replication and shedding in ducks.

For decades, French guinea fowl have been affected by fulminating enteritis of unclear origin. By using metagenomics, we identified a novel avian gammacoronavirus associated with this disease that is distantly related to turkey coronaviruses. Fatal respiratory diseases in humans have recently been caused by coronaviruses of animal origin.

Highly pathogenic avian influenza (HPAI) H7N1 viruses caused a series of epizootics in Italy between 1999 and 2001. The emergence of these HPAI viruses coincided with the deletion of the six amino acids R 225 VESEV 230 at the C terminus of NS1. In order to assess how the truncation of NS1 affected virus replication, we used reverse genetics to generate a wild-type low-pathogenic avian influenza (LPAI) H7N1 virus with a 230 aa NS1 (H7N1 230 ) and a mutant virus with a truncated NS1 (H7N1 224 ). The 6 aa truncation had no impact on virus replication in duck or chicken cells in vitro. The H7N1 230 and H7N1 224 viruses also replicated to similar levels and induced similar immune responses in ducks or chickens. No significant histological lesions were detected in infected ducks, regardless of the virus inoculated. However, in chickens, the H7N1 230 virus induced a more severe interstitial pneumonia than did the H7N1 224 virus. These findings indicate that the C-terminal extremity of NS1, including the PDZ-binding motif ESEV, is dispensable for efficient replication of an LPAI virus in ducks and chickens, even though it may increase virulence in chickens, as revealed by the intensity of the histological lesions.