Gross lesions of the classical type DHAV-1 and the pancreatitis-type DHAV-1-infected ducklings
The infection experiments were conducted with the pancreatitis-type DHAV-1 GD1206 and the classical type DHAV-1 FZ86 strains at the dose of 105.0 ELD50 per ducks. The ducks infected with the classical type 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 the pancreatitis-type DHAV-1 GD1206 strain demonstrated no typical signs during the course of infection. The major affected organ in the pancreatitis-type DHAV-1 group was the pancreas, which exhibited lesions of yellowing or hemorrhage. No hemorrhagic change was noted in the liver of the pancreatitis-type DHAV-1-infected ducklings. No gross lesions were observed in the mock-infected control group. The pancreatic samples were the classical type DHAV-1- or the pancreatitis-type DHAV-1-positive in the classical type DHAV-1- or the pancreatitis-type DHAV-1-group as determined by reverse transcription polymerase chain reaction (RT-PCR) analysis.
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 (Figs. 1). In order to obtain a global view of the change in duck gene expression among different experimental groups, three paired comparisons (classical type DHAV-1 vs. control, pancreatitis-type DHAV-1 vs. control, pancreatitis-type vs. classical type DHAV-1) were performed. RNA-seq analysis detected 3,340 and 5,919 genes, which were expressed at significantly different levels in classical type DHAV-1- and pancreatitis-type DHAV-1-infected animals, respectively compared with those noted in the control group (P < 0.05). Classical type DHAV-1 infection contributed to the differential expression of 2,031 genes that were upregulated and 1,309 genes that were downregulated in the pancreatic tissues of the infected animals compared with those noted in the control ducklings. Moreover, the expression levels of 3,308 genes were upregulated and those of 2,611 genes were downregulated in pancreatitis-type DHAV-1-infected animals. A total of 1,913 genes were differentially expressed in pancreatitis-type DHAV-1-infected animals compared with classical type DHAV-1-group. Specifically, the expression levels of 834 genes were downregulated and those of 1,079 genes were upregulated (Fig. 2).
Functional analysis of the transcriptome data derived from the pancreatic tissues of different subtype 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 the classical type DHAV-infected ducklings were analyzed using GO. The analysis of the GO categories of the classical type DHAV-1-group indicated that the differentially expressed genes were mapped to 61 categories of BP, CC and MF (Fig. 3A). The categories of BP included the genes mainly involved in the positive regulation of interleukin-12 production, myeloid leukocyte activation and T cell proliferation. The majority of the corresponding genes in the CC categories were involved in the external side of plasma membrane, the oligosaccharyltransferase complex and the integral component of the membrane. The majority of the corresponding genes in the MF categories 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 pancreatitis-type DHAV-1 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 (Fig. 3B). The majority of the corresponding genes in the BP categories were involved in cholesterol efflux, positive regulation of cell cycle and translation. The categories of the CC included genes that were mainly involved in the cytosolic large ribosomal subunit, extracellular space and ribosome. The majority of the genes of the MF categories included genes involved in the structural constituents of the ribosome, cytokine activity and NADH dehydrogenase (ubiquinone) activity.
The putative functions of the unigenes in the pancreatic libraries of the pancreatitis-type DHAV-1-infected ducklings were compared with those of the classical type 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 (Fig. 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. The majority of the corresponding genes in the MF category were involved in the antioxidant activity, G-protein coupled peptide receptor activity and structural constituents of the 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 subtype DHAV-1-infections. The top 20 enrichment KEGG pathways were listed in Fig. 4 according to their Q-value (Q < 0.05) (Table 1).
A total of 6 functional categories were identified that played important roles in the classical type DHAV-1 FZ86- and the pancreatitis-type DHAV-1 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 pancreatitis-type DHAV-1 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 that were involved in immune and metabolism-associated functions. These genes were also involved in host immune defense responses and metabolic function noted in the two subtype DHAV-1 infection groups. The genes examined were as follows: GNMT-like, GCAT, CBS, PHGDH, SERCA, PLCγ, TLR2, TLR4, TLR7 and IFNα. They were differentially expressed compared with the control ones (P < 0.05), indicating the reliability of the transcriptome sequencing data (Table 2).