H5Cam (HQ664938, see also ref. 30 ), H5Anh (ABD28180), H5Ind (ABP51969), H5Qin (ABE68923)), mutants AnhM1-M6, CamM1-M3 (Table 1 ) and N1 gene (ABO10176) from A/ Cambodia/JP52a/2005 were synthesized as human codon optimized genes (GENEARTH, Regansburg, Germany) and subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen). Mutants AnhM7 and AnhM8 were generated by sitedirected mutagenesis using QuikChange site-directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) according to the manufacturer's instructions. To generate soluble H5-HA constructs, the transmembrane domain (TMD) of the HA was removed, and the polybasic cleavage site was changed into a monobasic cleavage site RESR by site-directed mutagenesis to avoid the potential influence of H5-HA cleavage in cells on the purification of sHA proteins, which involve multiple steps. The truncated HAs were then subcloned into pcDNA3.1 (Invitrogen). All H5 plasmids were tagged with the FLAG-epitope at the C-terminal and sequenced to confirm that they contain only the expected mutations as indicated in Table 1 .