Antigenic diversity of H 5 highly pathogenic avian influenza viruses of clade 2 . 3 . 4 . 4 isolated 2 in Asia 3 4

30 H5 highly pathogenic avian influenza viruses (HPAIVs) have spread in both poultry and 31 wild birds since the late 2003. The continued circulation of HPAIVs in poultry in several regions 32 of the world has led to antigenic drift. In this study, we analyzed the antigenic properties of H5 33 HPAIVs isolated in Asia using four neutralizing monoclonal antibodies (MAbs) recognizing the 34 hemagglutinin, which were established using A/chicken/Kumamoto/1-7/2014 (H5N8), belonging 35 to clade 2.3.4.4 and also using polyclonal antibodies. Viruses of clades 1.1, 2.3.2.1, 2.3.4, and 36 2.3.4.4 had different reactivity patterns to the panel of MAbs, thereby indicating that the 37 antigenicity of the viruses of clade 2.3.4.4 were similar but differed from other clades. In 38 particular, the antigenicity of the viruses of clade 2.3.4.4 differed from those of the viruses of 39 clades 2.3.4 and 2.3.2.1, which suggests that the recent H5 HPAIVs have further evolved 40 antigenically divergent. In addition, reactivity of antiserum suggest that the antigenicity of 41 viruses of clade 2.3.4.4 differed slightly among group A, B, and C. Vaccines are still used in 42 poultry in the endemic countries, so the antigenicity of H5 HPAIVs should be monitored 43 continually to facilitate the control of avian influenza. The panel of MAbs established in the 44 present study will be useful for detecting antigenic drift in the H5 viruses that emerge from the 45 current strains. 46 47