Alanine scanning mutagenesis study. On the basis of these results, HA mutants carrying an alanine substitution in position 361 (Ile361Ala) or in position 362 (Asp362Ala) were generated and the PN-SIA28 binding to the mutants was evaluated by FACS analysis evidencing that PN-SIA28 was not longer able to bind to the mutated HAs. In order to identify other amino acidic residues involved in the binding of PN-SIA28 to HA, a large panel of HAalanine mutants was generated (Table S1 ). FACS analysis showed that binding of PN-SIA28 to HA was decreased by His25Ala, His45Ala mutants on HA1 and Thr358Ala, Met360Ala, Ile361Ala, Asp362Ala, Gly363Ala, Trp364Ala, Thr384Ala and Val395Ala mutants on HA2. (Figure 2 ). All other mutations listed in the Table S1 did not have any effect on PN-SIA28 binding. An extra HA-mutant in position 361 (Ile361Val) was generated. Indeed, in position 361 either an Isoleucine or a Valine residue can be present in different isolates belonging to either to group 1 either to group 2 ( Figure 2 ). No binding reduction of PN-SIA28 to this mutant was observed.