Introduction

Duck viral hepatitis (DVH) is a highly fatal contagious disease of ducklings that is characterized by clinical signs of opisthotonos and by hemorrhagic lesions of the liver [1]. This causes mortality rate is approximately 95% in ducklings within a week. The causative agents of DVH include duck hepatitis virus type 1 (DHV-1), DHV-2, and DHV-3. Recently, DHV-1, along with two other types, was classified as a member of the species Avihepatovirus A of the genus Avihepatovirus in the family Picornaviridae and was renamed as duck hepatitis A virus (DHAV) [2]. DHAV was furtherly divided into three genotypes: DHAV-1, DHAV-2, and DHAV-3.

DHAV-1 is distributed worldwide, and DHAV-2 has been isolated only in Taiwan to date [3, 4]. The presence of DHAV-3 was mainly reported in Korea and China [5,6,7,8]. Generally, DHAV-1 causes lesions in the liver that are typical of DVH. However, Guérin et al. reported in 2005 that several DHV infections of young Muscovy ducklings resulted in nervous symptoms and pancreatitis [9]. Several outbreaks of DHAV-1, which were characterized by yellow coloration and hemorrhage in pancreatic tissues, occurred in China in 2011 [10]. The causative agent was subsequently named "pancreatitis-associated DHAV-1". Genome sequencing indicated a variation rate of 3.4-6.5% in the genome of pancreatitis-associated DHAV-1 compared with that of classical-type DHAV-1 [11]. The antigenic association between pancreatitis-associated DHAV-1 and the classical-type DHAV-1 indicated large variation [12]. The comparative pathogenicity of pancreatitis-associated DHAV-1 and the classical type DHAV-1 in the ducklings of different species has been well documented [13, 4C). The BP categories mainly included genes that were involved in the oxidation-reduction process, negative regulation of the apoptotic process, and myeloid leukocyte activation. The CC categories mainly included genes that affect the extracellular space, protein-extracellular matrix, and ribosome. The majority of the corresponding genes in the MF categories are associated with antioxidant activity, G-protein-coupled peptide receptor activity, and structural constituents of the ribosome.

Pathway analysis of DEGs based on KEGG after infection with different DHAV-1 types

The KEGG database was used to analyze specific pathways in order to further define DEG function in duckling pancreatic tissue following infections with different DHAV-1 types. The top 20 enrichment KEGG pathways are listed in Figure 5 according to their Q-value (Q < 0.05) (Table 2).

Fig. 5
figure 5

Top 20 KEGG pathways enriched in the pancreas of ducklings in classical-type DHAV-1 vs. control (A), pancreatitis-associated DHAV-1 vs. control (B), and pancreatitis-associated DHAV-1 vs. classical type DHAV-1 (C)

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Table 2 Signal pathway enrichment evaluated by KEGG in the pancreas of ducklings in classical-type DHAV-1 vs. control, pancreatitis-associated DHAV-1 vs. control, and pancreatitis-associated DHAV-1 vs. classical-type DHAV-1
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A total of six functional categories were identified that play important roles in the classical-type DHAV-1 FZ86 and pancreatitis-associated DHAV-1 GD1206 infections. These categories were mainly associated with the immune system, including the Toll-like receptor signaling pathway. However, significant KEGG enrichment in the pancreatitis-associated DHAV-1 group was also involved in metabolism, including the glycine, serine, and threonine metabolism pathways.

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 involved in immune and metabolism-associated functions. These genes were also involved in host immune defense responses and metabolic function noted in the 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 (P < 0.05), indicating the reliability of the transcriptome sequencing data (Table 3).

Table 3 Verification by real-time RT-PCR for some differentially expressed genes
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Discussion

In the present study, ducklings that were inoculated with the classical-type DHAV-1 strain FZ86 developed massive haemorrhages on the liver surface. The ducklings that were inoculated with pancreatitis-associated DHAV-1 strain GD1206 exhibited loss of appetite, lying prone, diarrhea, and depression. The gross lesions of the yellowed or hemorrhagic pancreatitis were observed in the pancreatitis-associated-DHAV-1-infected ducklings, but no consistent nervous disorders were observed in the present study, which is not in line with the observations of Guérin et al. [9]. Cha et al. reported that a DHAV-3 strain induced only liver discoloration without hemorrhagic mottling, lymphocyte infiltration, or bile duct hyperplasia, as determined by histology of the lesions [21]. These studies reveal the diversity of the pathogenic effects of DHAV infection.

Transcriptome analysis is a promising tool that can provide a comprehensive understanding of the molecular mechanisms involved in specific biological processes and diseases [22]. Previous studies have described the transcriptome profiles of DHAV, reovirus, and DHBV infections in ducks [19, 23,24,25]. In the present study, transcriptome sequencing was employed to explore and compare the gene expression patterns of infection with different DHAV-1 types in duck pancreatic tissues, with the aim of comparing different molecular events during pancreatitis-associated DHAV-1 infection.

Analysis of the clean reads with BLAST and ORF resulted in a total of 9,387 matched known genes. The DEGs were annotated and categorized by GO and KEGG signaling pathway analyses, which demonstrated that the majority of these genes in the classical-type DHAV-1and pancreatitis-associated DHAV-1 infection groups were classified in the immune system and metabolism categories. Comparisons of the transcriptomes of classical-type-DHAV-1-infected and the control ducklings indicated that classical-type DHAV-1 infection caused downregulation of genes associated with metabolic pathways and inhibition of the metabolism of the host cell. In addition, upregulation of immune-associated genes was associated with inhibition of viral replication and progression of viral infection [26]. Similarly, in the present study, upregulation of immune genes and downregulation of certain metabolism-related genes were observed in the pancreatitis-associated DHAV-1 infection group compared with the control group. However, significant KEGG enrichment was observed in the pancreatitis-associated DHAV-1 infection group compared with the classical-type DHAV-1 infection group that was mainly involved metabolism, including the glycine, serine, and threonine metabolism pathways. These results suggest that differences in metabolism functions in the DHAV infection group may contribute to the different DHAV-1 phenotypes.

D-3-phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP) are involved in three steps of serine biosynthesis [27]. Initially, 3-phosphoglycerate is converted to 3-phosphohydroxypyruvate by the enzyme PGDH. Subsequently, PSAT converts 3-phosphohydroxypyruvate to 3-phosphoserine. Finally, 3-phosphoserine is converted to L-serine by PSP [28]. L-serine is an important precursor involved in various processes, such as synthesis of proteins and phospholipids as well as the synthesis of tetrahydrofolate metabolites and specific amino acids, namely glycine, cysteine, and D-serine [29, 30]. Previous studies have shown that L-serine supplementation can inhibit alcoholic fatty liver formation in mice and rats [31]. In the present study, the expression levels of the PHGDH, PSAT, and PSP genes were significantly lower in pancreatitis-associated-DHAV-1-infected ducklings than in the classical-type DHAV-1 group, suggesting that serine metabolism disorders are involved in the pancreatitis-associated DHAV-1 infection.

L-serine dehydratase (SDH) catalyzes the deamination of L-serine to yield ammonia and pyruvate. This enzyme uses L-threonine as a substrate to yield 2-oxobutanoate, which is a part of the valine, leucine, and isoleucine biosynthetic pathways. The decrease in serine dehydratase levels suggests that the pathway of conversion of serine to pyruvate was impaired, further affecting the valine, leucine, and isoleucine biosynthetic pathways [32,33,34].

TLRs play a critical role in innate immune responses. In recent years, the role of innate immunity and its interaction with adaptive immunity have been extensively investigated. In the TLR pathway, TLR2 plays a critical role in the induction of innate and inflammatory responses [35, 36]. Although TLR2 recognizes various bacterial components, recent studies have indicated that TLR2 is triggered by the hepatitis C virus core protein and NS3, leading to the activation of inflammatory cells [37, 38]. In the present study, the expression levels of the TLR2 gene were 10.63- and 7.62-fold higher in the pancreatitis-associated DHAV-1 and classical-type DHAV-1 infection groups, respectively, than in the control group, indicating that TLR2 may be involved in the host response to DHAV infection. TLR4 and CD14 have recently been shown to be major lipopolysaccharide (LPS) receptors. Mutations in mouse and human TLR4 were found to be associated with hyporesponsiveness to LPS and to confer an increased risk of infection with Gram-negative bacteria [39,40,41]. In addition to its interaction with LPS, the TLR4/CD14 complex interacts with viruses and proteins, such as the respiratory syncytial virus and fibrinogen [42,43,44]. In the present study, the TLR4/CD14 expression levels in the pancreatitis-associated DHAV-1 and classical-type DHAV-1 infection groups were much higher than in uninfected ducklings, indicating that TLR4/CD14 may be involved in the host response to pancreatitis-associated DHAV-1 and classical-type DHAV-1 infection. Surprisingly, TLR4 expression was significantly higher in the pancreatitis-associated DHAV-1 and classical-type DHAV-1 groups than in the mock-infected group, with an increase of 51.63- and 38.59-fold, respectively. TLR7 can detect single-stranded RNA (ssRNA) molecules and induce pro-inflammatory factors, such as the type I interferon, to stimulate the body's nonspecific immune response. Activation of TLR7 initiates downstream signaling cascades via induction of transcription factors such as IRF7. This induces the production of pro-inflammatory cytokines and chemokines that are involved in various viral infection outcomes, including spontaneous clearance and viral persistence [45]. In the present study, upregulation of TLR7, IRF7, and IFN-β expression was observed in the pancreatitis-associated DHAV-1 and classical-type DHAV-1 groups, which is consistent with the findings of previous studies [46, 47].

In summary, transcriptome analysis of pancreatic tissues derived from classical-type DHAV-1- and/or pancreatitis-associated-DHAV-1-infected ducks was performed. Infection with pancreatitis-associated DHAV-1 caused yellowing and hemorrhagic lesions in the pancreatic tissues of ducklings and was associated with differences in the expression levels of D-3-phosphoglyceratedehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which are involved in the glycine, serine, and threonine metabolism pathways. These genes were significantly downregulated in the pancreatitis-associated-DHAV-1-infected group compared with the classical-type-DHAV-1-infected group, indicating that intensive metabolism disorders may contribute to the different phenotypes of DHAV-1 infection.