Upstream start codon in segment 4 of North American H2 avian influenza A viruses.

H2N2 influenza A virus was the cause of the 1957 pandemic. Due to its constant presence in birds, the H2 subtype remains a topic of interest. In this work, comparison of H2 leader sequences of influenza A segment 4 revealed the presence of an upstream in-frame start codon in a majority of North American avian strains. This AUG is located seven codons upstream of the conventional start codon and is in a good Kozak context. In vivo experiments, using a luciferase reporter gene fused to leader sequences derived from North American avian H2 strains, support the efficient use of the upstream start codon. These results were corroborated by in vitro translation data using full-length segment 4 mRNA. Phylogenic analyses indicate that the upstream AUG, first detected in 1976, is stably nested in the North American avian lineage of H2 strains nowadays. The possible consequences of the upstream AUG are discussed.