Influenza A H1N1pdm09 Virus; Chicken embryo; Phylogenetic tree; Egg-adaptation; Antigenic site; Drug-resistance site; Abstract: To investigate egg-adapted mutation in influenza A H1N1pdm09 viruses isolated from the Hubei influenza surveillance network, a comparative analysis was performed of three influenza A H1N1pdm09 viruses isolated in chicken embryo and the corresponding MDCK cell-derived viruses. Analyses included examination of the phylogenetic tree, evolutionary rates, amino acid substitutions, egg-adapted mutation and homology modeling. We found differences between the egg-adapted viruses and MDCK cell-derived viruses based on phylogenetic trees and evolutionary rates; the viruses showed a trend of " NA>HA>MP". Four amino acid substitutions(Q223R,V527 I,M19Iand H275Y)were found in three egg-adapted viruses.Q223 Rand V527Iwere present in the haemagglutinin protein, while M19I and H275Y were detected in neuraminidase.The Q223R mutation changed the structure of antigenic sites between Sb and Ca2. H275Y is a classic neuraminidase resistance mutation. The results suggest that the egg-adapted mutations were introduced when influenza viruses were isolated in chicken embryos from the Hubei influenza surveillance network. These mutations may affect the selection of vaccine candidates and vaccine efficacy; therefore, monitoring of egg-adapted mutations should be strengthened in the influenza surveillance network.