An alternative AUG codon in segment 5 of the 2009 pandemic influenza A virus is a swine-derived virulence motif

The 2009 influenza A virus (IAV) pandemic (pdm2009) was caused by a swine H1N1 virus with several atypical genetic features. Here, we investigate the origin and significance of an upstream AUG (uAUG) codon in the 5’-untranslated region of the NP gene. Phylogeny indicated that the uAUG codon arose in the classical swine IAV lineage in the mid 20th Century, and has become fixed in the current triple reassortant, variant pdm2009 swine IAV and human pdm2009 lineages. Functionally, it supports leaky ribosomal initiation in vitro and in vivo to produce two isoforms of NP: canonical, and a longer “eNP”. The uAUG codon had little effect on viral gene expression or replication in vitro. However, in both murine and porcine models of IAV infection, removing the uAUG codon gene attenuated pdm2009 virus pathogenicity. Thus, the NP uAUG codon is a virulence factor for swine IAVs with proven zoonotic ability.