Currently circulating H5N1 influenza viruses have undergone a complex evolution since the appearance of their progenitor A/Goose/Guangdong/1/96 in 1996. After the eradication of the H5N1 viruses that emerged in Hong Kong in 1997 (HK/97 viruses), new genotypes of H5N1 viruses emerged in the same region in year 2000, that were more pathogenic for both chickens and mice than HK/97 viruses. These, as well as virtually all highly pathogenic H5N1 viruses since 2000, harbor a deletion of amino-acids 80-84 in the unstructured region of NS1 linking its RNA-binding domain to its effector domain. Segments NS harboring this mutation have since been found in non-H5N1 viruses, and we asked whether this 5-aa deletion could have a general effect not limited to the NS1 of H5N1 viruses. We genetically engineered this deletion in the NS segment of a duck-origin avian H1N1 virus, and compared the in vivo and in vitro properties of the wild-type and NSdel8084 viruses. In experimentally infected chickens, the NSdel8084 virus showed both an increased replication potential and an increased pathogenicity. This in vivo phenotype was correlated with a higher replicative efficiency in vitro, both in embryonated eggs and in a chicken lung epithelial cell line. Our data demonstrate that the increased replicative potential conferred by this small deletion is a general feature not restricted to NS1 from H5N1 viruses, and suggest that viruses acquiring this mutation may be positively selected in the future.