Figure 7
Comparison of biological properties of the virulence factor PB1-F2 of A/equine/Ohio/1/2003 and A/equine/Paris/1/2018. A Disruption of mitochondrial membrane potential (ΔΨm) in A549 cells expressing HA-tagged PB1-F2 variants from A/equine/Paris/1/2018 (HA-PB1-F2PARIS2018) and A/equine/Ohio/1/2003 (HA-PB1-F2OHIO2003) viruses. Cells were fixed 48 h post transfection and processed for indirect immunofluorescence staining with an anti-HA-tag rat antibody and an anti-rat secondary antibody coupled with Alexa Fluor 488 (green). Mitochondria were revealed using the ΔΨm-sensitive mitochondrial dye MitoTracker CMX Ros (magenta), and nuclei were revealed with Hoechst (blue). Scale bars, 10 μm. B Membrane permeabilization assay using recombinant forms of PB1-F2 encoded by A/equine/Paris/1/2018 (PB1-F2PARIS2018) (blue dots) and A/equine/Ohio/1/2003 (PB1-F2OHIO2003) (red dots) viruses. LUVs mimicking mitochondrial outer-membrane composition containing the fluorophore probe (ANTS) and quencher (DPX) were incubated with serial dilutions of PB1-F2 forms. The experiment was carried out 4 times in triplicate. Statistical analysis was carried out with REML F(1,99) = 55.01, P < 0.0001, and Å Ãdák’s multiple comparison. P values are indicated in the figure.