Fig 2. Conformational stability and membrane fusion properties of HK, R5, R2 and R7.

(a) pH of acid-induced conformational transition of HA. Solid-phase adsorbed viruses were incubated in acidic buffers and treated with proteinase K. Viral binding of fet-HRP was assayed, and pH values that corresponded to 50% reduction of HA binding activity (pH₅₀) were determined from binding-versus-pH curves. (b) pH threshold of polykaryon formation. Inoculated MDCK cells were cultured for 16 h, treated with trypsin and exposed to different pH buffers. After returning to neutral medium and incubation for 3 h, the cells were fixed, stained and analysed under the microscope. The data show highest pH values at which polykaryon formation was detected. (c) Inhibition of viral infection by ammonium chloride. MDCK cells were inoculated in the presence of various concentrations of NH₄Cl, incubated overnight, fixed, and immunostained for NP. Concentrations of NH₄Cl that reduced numbers of infected cells by 50% (IC₅₀) were determined from dose-response curves. (d) HA stability at elevated temperature. Solid-phase adsorbed viruses were incubated in PBS at 65°C for different time periods and assayed for their binding to fet-HRP to determine incubation time required for 50% reduction of the binding activity (t₅₀). (e) HA stability in chaotropic buffer. Solid-phase adsorbed viruses were incubated in buffers containing GnHCl for 60 min at 4°C washed with PBS and assayed for binding to fet-HRP. Data show concentrations of GnHCl that reduced viral binding activity by 50%. (f) Reduction of infectivity after incubation of the viruses for 2 h at 45°C determined by focus assay in MDCK cells. All panels show data points, mean values and SDs from 1 to 4 independent experiments performed with 2 to 7 replicates. P values for the differences between the viruses were determined with Tukey’s multiple comparison procedure.