In recent years, concerns about low-pathogenic influenza has been increased, since it may be able to replicate well and possibly cause disease in humans, as observed during the 2009 H1N1 pandemic4,49,50. Mutations in the polymerase complex are critical for influenza viruses to infect and adapt mammals; notable examples of such mutations are 271A, 627K, 701N, and 591R36–39,51,52. Our prototypical EA H1N1 IAVs-S have 701N. For H1N1/2009 pandemic PB2, it is reported that 271T and 591R confer efficient replication and transmission in mammals38,39. All of our strains with H1N1/2009 pandemic PB2 bear 271A and 591R. These indicate that 14 IAVs-S in our study have mammalian-adapting mutations in their PB2 genes (Table 3). Importantly, IAVs-S containing these particular marker combinations are transmissible in ferrets, as previously described25.