Figure 4

PB1-F2 alters virus shedding. A, B Oropharyngeal (A) and cloacal swabs (B) of wt (blue) and ΔF2 (red) animals were titrated by plaque assay. Statistical analysis was done by Two-Way ANOVA. C RNA was extracted from tissue samples of brain, jejunum, lung and spleen. Subsequently, real-time reverse-transcription PCR (RT-qPCR) was performed. Standard curves of H7N7 were included in the run, allowing the calculation of virus content in PFU/ml by plotting the Ct-values of the standard curves. Data is depicted as log (10) of PFU per gram of tissue (PFU/g) equivalents. For statistical analysis Two-Way ANOVA was performed. Virus antigen score for intestinal mucosa proves viral replication after wt but not after ΔF2 infection. Influenza virus matrix (M1) protein detection by IHC, blind scoring, dots represent individual scores, bar represents median group score. Scores given as no antigen = 0, rare/focal = 1, multifocal = 2, coalescing = 3, diffuse = 4 (D). Representative images for IHC-based detection of M1 protein in enterocytes after wt (E) but not after ΔF2 (F) infection. IHC, using avidin and biotinylated enzyme method, 3-Amino-9-Ethylcarbazole chromogen (red), and haematoxylin (blue) counterstain. Bar 50 µm, inlay with a cross section of the small intestine and rectangle showing the selected representative image section.