5, 60–80% of genes exhibited induction in the 4 contrasts that include: NV–C severe day 5 vs. NV–C mild day 5, NV–C severe day 1 vs. NV–NC day 1, NV–C severe day 5 vs. NV–NC day 5, NV–C severe day 1 vs. NV–C severe day 5. Many differentially expressed genes showed large fold changes. In the 4 contrasts described, Akt inhibitor 25–31% of differentially expressed genes had a fold change of 3 or greater. Heatmaps were generated to characterize

patterns of gene expression between similar contrasts by including genes with a minimum q value of 0.05 in any contrast ( Fig. 3 and Fig. 4). The NV–C mild group on day 5 showed more similarities to the V–NC groups than to the challenged groups ( Fig. 3). The remaining challenged groups, both vaccinated and non-vaccinated, exhibited similar expression patterns. The only group with notable expression changes over time was the NV–C severe group ( Fig. 4). Gene ontology analysis focused on biological process terms among significant genes. Larger numbers of significantly enriched GO terms were found in contrasts with a higher number of genes with differential expression. Three GO terms related to response (response to stimulus, response to stress, and defense response) were discovered among significantly differentially expressed genes in the contrast of NV–C

severe day 1 vs. NV–C severe 5. Among the other 3 contrasts described (NV–C severe day 5 vs. NV–C mild day 5, NV–C severe day 1 vs. NV–NC control day 1, NV–C severe day 5 vs. NV–NC control day 5), a variety of metabolic and biosynthetic processes were common. Prominent within NV–C severe day 5 vs. NV–C mild day 5, were

GO terms for signal transduction, immune system processes, Fulvestrant mouse ion homeostasis and, surprisingly, several GO terms centered on reproduction. Within NV–C severe vs. NV–NC control, response terms pheromone were prominent on day 1, and ion homeostasis and DNA structural terms were prominent on day 5. GO analysis of unique and shared differentially expressed genes for 3 contrasts (Fig. 2) was carried out; NV–C severe day 5 vs. NV–C mild day 5, NV–C severe day 1 vs. NV–NC control day 1, NV–C severe day 5 vs. NV–NC control day 5. Many of the genes shared among contrasts were related to immune response. These included (a) CD4, tumor necrosis factor receptor, and Rab11a shared by all 3 contrasts; (b) ATPase, CD5, interferon gamma receptor, and toll-like receptor 15 shared by NV–C severe day 1 vs. NV–NC control day 1 and NV–C severe day 5 vs. NV–NC control day 5; (c) ATPase, CD3ε, CD200R1, toll-like receptor 7 shared by NV–C severe day 5 vs. NV–NC control day 5 and NV–C severe day 5 vs. NV–NC control day 5. Avian beta-defensins, CD74 and interleukin-8 were unique to NV–C severe vs. NV–NC control on day 1 (e). Unique to NV–C severe vs. NV–C mild on day 5 (f) were genes related to ion transport and energy (ATPases and ATP synthases), immune response (CD28, CD79b, interleukin 4 receptor, interleukin 10 receptor beta, toll-like receptor 21), and reproduction.