(A) HA activation pH measured by syncytia assay. Viruses were inoculated into Vero cells at an MOI of 3 PFU/cell. Recombinant viruses with HA and NA segments from G15 and P4 were rescued in the background of the six internal genes from A/TN/09 using reverse genetics. Representative images of three independent experiment results are shown. (B) HA inactivation pH measured by loss of infectivity as a function of acid exposure. Virus aliquots were treated with pH-adjusted PBS, re-neutralized, and subjected to measurement of residual virus infectivity by TCID50. (C) Virus growth capacity in swine testicular (ST) and MDCK cells. Viruses were inoculated into ST and MDCK cells at an MOI of 0.01 PFU/cell. Cell culture supernatants were harvested at the indicated time points and quantified by TCID50. (D) Binding specificities toward α(2, 6)- or α(2, 3)-linked sialic acid receptors. Viruses were inoculated onto fetuin-coated plates. Virus binding affinities toward α2,6- or α2,3-linked sialylglycopolymers were measured by solid-phase receptor binding assay. Recombinant A/Puerto Rico/9/1934 (H1N1) with the HA segment from A/Mallard/Alberta/383/2009 (H5N1) was used as a control. All data were means ± SD of at least two independent experiments.