Based on the PN-SIA49-C179 competition assay results, a large panel of HA mutants carrying an alanine substitution were generated [29, 30, 31, 32] . The binding of PN-SIA49 to these mutants was then evaluated by FACS analysis. Data obtained revealed that the binding of PN-SIA49 was decreased by His25Ala, Asn336Ala, Pro338Ala mutants on HA1 and Me-t360Ala, Asp362Ala, Gly363Ala, Trp364Ala, Thr384Ala, Va-l395Ala, Asn396Ala, Glu400Ala mutants located on HA2 (sequence numbering refers to A/PR/8/34, GenBank accession number ABO21709). Importantly, these residues are extremely conserved among viruses belonging to H1N1 subtype spanning from 1918 and 2009 and also highly conserved in H2N2 and H5N1 subtypes ( Figure 3 ). All the other mutations did not have any effect on the antibody PN-SIA49 binding to the HA ( Figure 3 ). To exclude the possiblity that the reduced PN-SIA49 binding to HA was due to reduced expression of HA on the cell surface, we performed a FACS analysis in which wild-type HA and mutants HA were stained with a mouse anti-influenza A HA (H1 subtype) monoclonal antibody. This antibody was directed against a linear epitope in order to evaluate the expression level for each HA on cell surface. As shown in figure S4 , the HA mutants are expressed on the cell surface at a similar levels to that of wild type HA.