Fig. 7: D113A decreases Ca²⁺ requirements for viral replication and enhances aerosol infection under Ca²⁺ -depleted conditions.

a MDCK cells were infected with 10-fold dilutions of hVIC/11 NA mutants and the control sOH/04 virus in media containing 2 (orange), 0.5 (cyan), or 0 mM Ca²⁺ (purple). At 72 hpi plaque size diameter was measured for each condition. p values were obtained by ordinary one-way ANOVA. b Effect of Ca²⁺ concentration on plaque size represented as the percentage of the plaque diameter compared to the plaque size at 2 mM Ca²⁺ that was arbitrarily set to 100%. p values were obtained by ordinary one-way ANOVA. c Viral particle release assay showing the HA units in 50 μL of supernatant of MDCK cells infected with hVIC/11, sOH/04, hVIC/11-A138S, hVIC/11-D113A, and hVIC/11-A138S/D113A. Cells were infected at an MOI of 0.1 and at 5 hpi the media was replaced with fresh media containing the mentioned Ca²⁺ concentrations. Values represent the mean ± SD of three independent experiments. d Viral growth kinetics of hVIC/11, sOH/04, hVIC/11-A138S, hVIC/11-D113A, and hVIC/11-A138S/D113A in aerosol infected MDCK cells. Viruses were aerosolized with (orange) or without (purple) Ca²⁺ and viral titers were recorded at 0, 12, 24, 48, and 72 hpi. Values represent the mean ± SD of three independent experiments. e Viral growth kinetics of hVIC/11, sOH/04, hVIC/11-A138S, hVIC/11-D113A, and hVIC/11-A138S/D113A in aerosol infected PK15 cells. Viruses were aerosolized with (orange) or without (purple) Ca²⁺ and viral titers were recorded at 0, 12, 24, 48, and 72 hpi. Values represent the mean ± SD of three independent experiments.