FIG 6.

The distribution of VFs in the cytoplasm is dependent on an intact actin cytoskeleton and microtubule network, but alterations in VF distribution do not alter viral replication. DF-1 cells were infected with the PBG98-VP1-TC virus at an MOI of 1. Cells were fixed at 20 hpi and stained with DAPI and either an anti-tubulin mouse monoclonal antibody, followed by a goat anti-mouse secondary antibody conjugated to Alexa Flour 488 to visualize the microtubule network (A) or phalloidin conjugated to Alexa Flour 488 to visualize the actin cytoskeleton (B). Scale bars, 20 μm. Images of the boxed regions are shown enlarged, and the locations where the cytoskeleton colocalized with the VFs are indicated by white arrows. (C) The number of VFs per cell was determined for 30 infected cells and plotted. (D) The average area of the VFs in an infected cell was determined using the surface tool in Imaris 9 software (bitplane) and plotted for 30 infected cells. The line represents the mean, and the error bars represent the SD of the mean. (E) Cell supernatants obtained 24 hpi were titrated, and the TCID₅₀ values were determined. Virus titers were compared by one-way ANOVA and a Tukey’s multiple-comparison test following a Shapiro-Wilk normality test to confirm whether the data followed a normal distribution for parametric or nonparametric testing. The mean area and number of VFs were compared using Kruskal-Wallis one-way ANOVA. ns, not significant; ***, P < 0.001; ****, P < 0.0001.