RNA sequencing-based analysis of the spleen transcriptome following infectious bronchitis virus infection of chickens selected for different mannose-binding lectin serum concentrations

Abstract

Background: Avian infectious bronchitis is a highly contagious disease of the upper-respiratory tract caused by infectious bronchitis virus (IBV). Understanding the molecular mechanisms involved in the interaction between innate and adaptive immune responses to IBV infection is a crucial element for further improvements in strategies to control IB. To this end, two chicken lines, selected for high (L10H line) and low (L10L line) serum concentration of mannose-binding lectin (MBL) were studied. In total, 32 birds from each line were used. Sixteen birds from each line were infected with IBV and sixteen were left uninfected. Eight uninfected and infected birds from each line were euthanized at 1 and 3 weeks post infection. RNA sequencing was performed on spleen samples from all 64 birds and differential gene expression analysis was performed for four comparisons: L10L line versus L10H line for uninfected birds at weeks 1 and 3, respectively, and in the same way for infected birds. Functional analysis was performed using Gene Ontology (GO) Immune System Process terms specific for Gallus gallus.

Results: Comparing uninfected L10H and L10L birds, we identified 1698 and 1424 differentially expressed (DE) genes at weeks 1 and 3, respectively. For the IBV-infected birds, 1934 and 866 DE genes were identified between the two lines at weeks 1 and 3, respectively. The two most enriched GO terms emerging from the comparison of uninfected birds between the two lines were "Lymphocyte activation involved in immune response" and "Somatic recombination of immunoglobulin genes involved in immune response" at weeks 1 and 3, respectively. When comparing IBV-infected birds between the two lines, the most enriched GO terms were "Alpha-beta T cell activation" and "Positive regulation of leukocyte activation" at weeks 1 and 3, respectively.

Conclusions: Healthy birds from the two lines showed significant differences in expression profiles for subsets of adaptive and innate immunity-related genes, whereas comparison of the IBV-infected birds from the two lines showed differences in expression of immunity-related genes involved in T cell activation and proliferation. The observed transcriptome differences between the two lines indicate that selection for MBL had influenced innate as well as adaptive immunity.

Fig. 1

Structure and timeline of the experiment together with comparisons of gene differential expressions. The figure illustrates the experimental timeline together with the structure of the experiment. In total, 64 birds, 32 from each experimental line, L10H and L10L, were used. In addition, differential expression comparisons are shown

Fig. 2

Venn diagram of differentially expressed (DE) genes between comparisons of uninfected and infected birds at different time points. a Differentially expressed (DE) genes between lines when the lines are uninfected (left circle) and infected (right circle). Numbers in the intersection correspond to DE genes that are in common between lines for uninfected and IBV infected birds at week 1. b DE genes between lines when the lines are uninfected (left circle) and infected (right circle). Numbers in the intersection correspond to DE genes that are in common between lines for uninfected and IBV infected birds at week 3. Numbers outside of circles represent sum of DE genes presented in circles

Fig. 3

Functional map of differentially expressed genes enriched for GO Immune System terms. The top categories of the GO Immune System terms associated with differentially expressed (DE) genes. All categories were statistically significant (adjusted p-value < 0.001). The chart fragments represent the number of genes associated with the terms as a proportion of the total number of genes within the respective GO term. Terms which have not been grouped are shown in grey

Fig. 4

Differentially expressed genes associated with the GO Immune System term. Heatmap representation of the differentially expressed (DE) genes associated with the GO Immune System terms for the four comparisons between the two lines, L10H and L10L, uninfected and infected groups at two time points, weeks 1 and 3. The heat map is constructed using the average values of counts per millions for each group