Gross lesions of DHAV-1 and DHAV-1a-infected ducklings
The infection experiments were conducted with DHAV-1a GD1206 and DHAV-1 FZ86 strains at the dose of 105.0 ELD50 per ducks. The ducks infected with DHAV-1 FZ86 strain indicated clinical symptoms of depression, lethargy, and opisthotonos. The major lesions that were typically enlarged with petechial and ecchymotic hemorrhages in the liver and kidney were observed during necropsy. No visible lesions were noted in the pancreas. In contrast to these observations, the ducklings infected with DHAV-1a GD1206 strain demonstrated no typical signs during the course of infection. The major affected organ in the DHAV-1a group was the pancreas which exhibited lesions of yellowing or hemorrhage. No hemorrhagic change was noted in the liver of the DHAV-1a-infected ducklings. No gross lesions were observed in the mock-infected control group. The pancreatic samples were DHAV-1- or DHAV-1a-positive in the DHAV-1- or DHAV-1a-group as determined by RT-PCR.
RNA-seq and assembly of transcriptome data from the pancreas of the ducklings
Following Illumina-Solexa deep sequencing, a total of 183 M raw reads were obtained in the cDNA library derived from pancreatic tissues. The removal of low-quality reads (i.e., reads containing only adaptors and empty reads) resulted in the identification of clean reads with total residues of 53.9 Gb clean data. Following de novo sequence assembly, a total of 29,597 unigenes with an average length of 993.43 bp were generated. BLAST and ORF analyses indicated that only 9,387 targets matched the known genes among the total number of unigenes. The sequencing data from the GD1206- and FZ86-infected groups were submitted to the NCBI database (accession number: SRR7239978, SRR7239979, SRR7239984, SRR7239985, SRR7239988, and SRR7239989).
The heatmaps indicated the top differentially expressed genes (DEGs) and the classification of gene expression profiles in different subtype DHAV-1-infection (Figures 1). In order to obtain a global view of the change in duck gene expression among different experimental groups, three paired comparisons (DHAV-1 vs. control, DHAV-1a vs. control, DHAV-1a vs. DHAV-1) were performed. RNA-seq analysis detected 3,340 and 5,919 genes, which were expressed at significantly different levels in DHAV-1- and DHAV-1a-infected animals, respectively compared with those noted in the control group (P <0.05). DHAV-1 infection contributed to the differential expression of 2,031 genes that were up-regulated and 1,309 genes that were down-regulated in the pancreatic tissues of the infected animals compared with those noted in the control ducklings. Moreover, 3,308 genes were up-regulated and 2,611 genes were down-regulated in the DHAV-1a-infection animals. A total of 1,913 genes were differentially expressed in DHAV-1a-infected animals compared with those of the DHAV-1-group. Specifically, 834 genes were down-regulated and 1,079 genes were up-regulated (Figure 2).
Function analysis of the transcriptome data derived from the pancreatic tissues of different type DHAV-infected animals
We performed GO enrichment analysis by functional annotation clustering of DEGs and annotated DEGs into three groups, including biological processes (BP), cellular components (CC), and molecular function (MF).
The putative functions of the unigenes in the libraries derived from DHAV-infected ducklings were analyzed using GO. The analysis of the GO categories of the DHAV-1-group indicated that the differentially expressed genes were mapped to 61 categories of BP, CC and MF (Figure 3A). The category of BP included mainly the genes involved in the positive regulation of interleukin-12 production, myeloid leukocyte activation and T cell proliferation. The majority of the corresponding genes in CC category were involved in the external side of plasma membrane, the oligosaccharyltransferase complex and the integral component of the membrane. The category of MF included genes that were involved in the non-membrane spanning protein tyrosine kinase activity, antioxidant activity and calcium-dependent phospholipid binding.
The putative functions of the unigenes in the pancreatic libraries of the DHAV-1a-infection were analyzed using GO. The analysis of the GO categories indicated that the differentially expressed genes were mapped to 61 categories including BP, CC and MF (Figure 3B). The majority of the corresponding genes in the BP category were involved in cholesterol efflux, positive regulation of cell cycle and translation. The category of the CC included genes mainly involved in the cytosolic large ribosomal subunit, extracellular space and ribosome. The majority of the genes of the MF category included genes involved in the structural constituent of ribosome, cytokine activity and NADH dehydrogenase (ubiquinone) activity.
The putative functions of the unigenes in the pancreatic libraries of DHAV-1a-infected ducklings were compared with those of the DHAV-1-infected ducklings and were analyzed using GO. The analysis of the GO categories indicated mapping of differentially expressed genes to 61 categories of BP, CC and MF (Figure 3C). The BP category included mainly genes that were involved in the oxidation-reduction process, the negative regulation of apoptotic process and the myeloid leukocyte activation. The CC category included mainly genes that were involved in the extracellular space, protein-extracellular matrix and ribosome. Most of the corresponding genes in the MF category were involved in the antioxidant activity, G-protein coupled peptide receptor activity and structural constituent of ribosome.
Pathway analysis of DEGs based on KEGG following different DHAV-1-infection
The KEGG database was used to analyze specific pathways in order to further define DEG function in the duckling pancreatic tissue following different DHAV-1-infections. The top 20 enrichment KEGG pathways were listed in Figure 4 according to their Q-value < 0.05 (Table 1).
A total of 6 functional categories were identified that played important roles in the DHAV-1 FZ86- and DHAV-1a GD1206-mediated infections. These categories were mainly classified into immune system categories, including the Toll-like receptor signaling pathway. However, significant KEGG enrichment in the DHAV-1a group was also involved in the metabolism function, including the glycine, serine and threonine metabolism pathway.
Verification of DEG identification by real-time RT-PCR
In order to verify the differential gene expression levels obtained from the transcriptome sequencing data, we analyzed the expression levels of 10 genes of immune and metabolism-associated genes that were mainly involved in host immune defense responses and metabolism in the two phenotype DHAV-1 infection groups. The genes examined as following: GNMT-like, GCAT, CBS, PHGDH, SERCA, PLCγ, TLR2, TLR4, TLR7 and IFNα, were significantly differently expressed compared with the control ones (P <0.05), indicating the reliability of the transcriptome sequencing data (Table 2).