Among influenza virus proteins, PB2 is considered a major viral determinant of influenza viruses in mammals. The 627K and 701N amino acid residues in PB2 are known to play an important role in mammalian fitness for influenza viruses34–38. Most of IAVs-S with EA H1N1-origin PB2 genes have 701N. For H1N1/2009 pandemic virus, it was reported that 271A in PB2 enhances the transmissibility of influenza A in ferrets and 591R is important for H1N1/2009 pandemic in mammalian adaptation38–40. In our study, the IAVs-S with H1N1/2009 pandemic PB2 contain 271A and 591R.