Transcriptomic and Translatomic Analyses Reveal Insights into the Signaling Pathways of the Innate Immune Response in the Spleens of SPF Chickens Infected with Avian Reovirus

Figures

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  Figure 1. Characteristics of ribosome profiling data in the CON and ARV groups. (A) Length distribution of RPFs. (B) The percentage of RPFs located in CDS, 5′ UTR and 3′ UTR. (C) The total number of RPFs along CDS start and stop codon regions in ARV group. A codon contains three bases, which are represented by red, blue and green.

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  Figure 2. Translation efficiency analysis. (A) Violin plot of TE in the CON and ARV groups. Asterisk indicates significant difference (**** p < 0.0001). (B) Relative TE ratio. Genes were classified based on their rounded log2 TE values. (C) Volcano map of DTEGs. The gray dot represents the non-differentially expressed translation efficiency genes. (D) Gene ontology (GO) analysis of DTEGs. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DTEGs.

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  Figure 3. Avian reovirus altered gene expression at both the transcriptional and translational levels. (A) Scatter plot of the fold change in the ARV/CON group at the transcriptional and translational levels. Nine squares in different colors indicate nine response groups (|log2 fold change| ≥ 1 and FDR < 0.01). (B) GO enrichment analysis of genes in quadrants C and G.

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  Figure 4. Functional gene screening. The circle maps of the top 30 DEGs in the transcriptome (A) and translatome (B). (C) The expression abundance of IL4I1 at the transcriptional and translational levels. (D) The relative level of IL4I1 mRNA expression. Asterisks indicate significant differences (** p < 0.01, **** p < 0.0001).

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  Figure 5. IL4I1 inhibits the replication of ARV in DF1 cells. DF-1 cells were transfected with pEF1α-Myc-IL4I1 or pEF1α-Myc plasmids, and both real-time PCR (A) and Western blot (B) confirmed high levels of IL4I1 expression in DF-1 cells. DF-1 cells were transfected with pEF1α-Myc-IL4I1 or pEF1α-Myc plasmids and infected with the ARV S1133 strain at an MOI of 1. Viral replication was detected by real-time PCR (C) and viral titer (D) 24 h postinfection. Comparison of the inhibition efficiency of three siRNAs of IL4I1 by real-time PCR (E). DF1 cells were transfected with si1357 or siNC and infected with the ARV S1133 strain of virus at an MOI of 1. Viral replication was detected by real-time PCR (F) and viral titer (G) 24 h postinfection. Data are represented as the mean ± SD of three independent experiments. Asterisks indicate significant differences (** p < 0.01).

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  Figure 6. Effect of IL4I1 on the innate immune response during ARV infection. DF-1 cells were transfected with pEF1α-Myc-IL4I1 or pEF1α-Myc plasmids and then infected with the ARV S1133 strain at an MOI of 1. Expression changes in genes associated with the innate immune signaling pathway were detected by real-time PCR (A) 24 h postinfection. DF1 cells were transfected with si1357 or siNC and infected with the ARV S1133 strain of virus at an MOI of 1. Expression changes in genes associated with the innate immune signaling pathway were detected by real-time PCR (B) 24 h postinfection. Data are represented as the mean ± SD of three independent experiments. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01).

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  Figure 7. IL4I1 in DF1 cells interacts with ARV σA and σC proteins. PEF1α-HA-σA or pEF1α-HA-σC were transfected into DF1 cells to overexpress ARV σA or σC proteins, respectively. Determination of IL4I1 expression by real-time PCR (A). We cotransfected cells with pEF1α-Myc-IL4I1 and pEF1α-HA-σA/σC in the experimental group and pEF1α-Myc-IL4I1 and pEF1α-HA or pEF1α-Myc and pEF1α-HA-σA/σC in the control group. After 24 h of transfection, cells were lysed to collect protein samples, and the interaction of IL4I1 with ARV σA and σC proteins in DF-1 cells was verified by Co-IP. Verification of IL4I1 interaction with ARV σA by Co-IP (B). Verification of IL4I1 interaction with ARV σC by Co-IP (C). Asterisks indicate significant differences (* p < 0.05).