Mammalian adaptation of an avian influenza A virus involves stepwise changes in NS1

Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral non-structural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host-species barrier. We characterised the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionary distinct NS1s counteract the IFN response using different and mutually exclusive mechanisms: while the NS1s of early EIVs block general gene expression by binding to the cellular polyadenylation specific factor 30 (CPSF30), NS1s from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and became rapidly selected during EIV evolution, highlighting the importance of evolutionary processes on immune evasion mechanisms during IAV adaptation