Positions 239 and 220 are localized within the HA antigenic site called Ca. The amino acid S220, though not exposed to the surface, is localized in the receptor binding domain (RBD), and its change could affect the transmissibility and infectivity of H1N1 in humans. The fixed mutation, S220T, has been found at high percentage (76.7%) in this study. To test whether change 220T could contribute to antigenic drift, it would be interesting to compare its antigenic profile against a wildtype isolate (S220). This mutation, probably, has become fixed in all pandemic strains through optimization of viral fitness, rather than immune selection or adaptation to the host. Substitution S101N has been proposed previously as a reversion to the seasonal H1N1 residue 101N and thus possibly an adaptation to the human host, being found in some studies at high frequencies. Its global impact, however, is controvertible because it was found in only 0.2% of our sequences. Substitution E391K, found at 15.6% in our study, has been identified as part of a highly conserved epitope in the 1918 H1N1 virus with a possible role in membrane fusion [22] . Another proximal substitution found in other studies, N387H, was found in only 1.7% of our sequences.