Statistical analysis
All statistical analyzes were performed using GraphPad Prism for Windows, version 3.0 (GraphPad Software, San Diego California USA). Due to the limited number of cats integrated in the study, we refrained from using a parametric approach in the statistical tests. As such tests require a larger sample size than n = 4, it was not possible to calculate p-values for the induction in expression of each gene shown in Figure2. Differences between treatment groups in proliferation of PBMCs were analyzed with a Mann–Whitney test and expression of co-stimulatory molecules on the surface of these cells were analyzed with a Wilcoxon signed rank test, where values for each cat were paired. Relative Mx mRNA expression, OD values from viral inhibition assays, ratios for FeLV provirus and Ct values for FeLV viral RNA loads were analyzed with a Wilcoxon signed rank test with pairing of values for each cat when treatment with different supernatants were compared, or a Mann–Whitney test when incubation with supernatant was opposed to treatment with medium alone or rfeIFNα. Normalized and relative TLR9 mRNA expression ratios between different animal age groups were also compared with a Mann–Whitney test. Longitudinal effects on FeLV viral and proviral loads were compared with each other using a Mann–Whitney test carried out with Area Under the Curve (AUC) values. Correlations were assessed using the Spearman test. p-values < 0.05 were considered statistically significant.
Figure 2 ODN 2216 induces an antiviral gene expression profile in PBMCs of adult cats. (A-D) mRNA expression factors of the indicated genes were measured in PBMCs isolated from fourteen cats belonging to the indicated age groups and stimulated with ODN 2216 for 24 h. (E) mRNA expression factors of the indicated IFNα subtype genes were measured in PBMCs isolated from four adult cats (group 2) and stimulated with ODN 2216 for 24 h. (F) mRNA levels of TLR9 were measured by real-time qPCR in unstimulated PBMCs of all four groups of cats and normalized to the expression of a feline housekeeping gene (GAPDH). (G) Relative TLR9 mRNA expression factors were measured in ODN 2216 stimulated PBMCs from cats of all four groups. *p < 0.05, Perf = perforin, GranB = granzyme B.