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BALB/c mice were immunized with formalin-treated influenza A/CK/Hubei/327/2004 virus. Six monoclonal antibodies specific to HA were selected, designed 1H8, 1D11, 2B7, 2C9, 2H4 and 4C9, respectively. The six Mabs probed linear epitopes by western blot assays. In ELISA additivity assays, the low additivity indexes (≤28.3) of each pair Mabs indicated that the epitopes recognized by the six Mabs were located on the globular head of HA1. The neutralization activity of anti-HA1 Mabs and chicken polyclonal sera to various AIV H5N1 strains from different hosts was followed by virus neutralization with MDCK cells. All Mabs except 2C9 and chicken polyclonal serum showed highest neutralizing activity to lowly virulent A/Duck/XF/XFY/2004 from different phylogenetic lineage, and lowest neutralization efficiency to highly virulent A/CK/XF/XFJ/2004. For the other two highly virulent viruses, 1D11, 2H4, 4C9 and chicken polyclonal sera had higher neutralization to A/Goose/ZF/ZFE/2004 than A/CK/Hubei/327/2004, and 1H8 and 2B7 had considerable level of neutralizing efficiency to them. These findings suggested that the neutralizing antibodies showed lower neutralization efficiency to highly virulent virus strains than lowly virulent virus strains and strong cross-neutralizing reaction between virus strains located in different phylogenetic lineages. Moreover, the neutralizing Mabs could more efficiently neutralize AIV H5N1 strains from the natural hosts generally, such as waterfowl.