Duck hepatitis A virus 1 (DHAV-1) is the primary cause of duck viral hepatitis, leading to sudden mortality in ducklings and significant economic losses in the duck industry. However, little is known about how DHAV-1 affects duckling liver at the molecular level. We conducted an analysis comparing the expression patterns of mRNAs and miRNAs in DHAV-1-infected duckling livers to understand the underlying mechanisms and dynamic changes. We identified 6,818 differentially expressed mRNAs (DEGs) and 144 differentially expressed microRNAs (DEMs) during DHAV-1 infection. Functional enrichment analysis of DEGs and miRNA target genes using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed their potential involvement in innate antiviral immunity, mitophagy, and pyroptosis. We constructed coexpression networks of mRNA-miRNA interactions and confirmed key DEMs (novel-mir333, novel-mir288, novel-mir197, and novel-mir71) using RT-qPCR. Further investigation demonstrated that DHAV-1 activates the RLRs signaling pathway, disrupts mitophagy, and induces pyroptosis. In conclusion, DHAV-1-induced antiviral immunity is closely linked to mitophagy, suggesting it could be a promising therapeutic target.

The maintenance of viral protein homeostasis depends on the interaction between host cell proteins and viral proteins. As a molecular chaperone, heat shock protein 70 (HSP70) has been shown to play an important role in viral infection. Our results showed that HSP70 can affect translation, replication, assembly, and release during the life cycle of duck hepatitis A virus type 1 (DHAV-1). We demonstrated that HSP70 can regulate viral translation by interacting with the DHAV-1 internal ribosome entry site (IRES). In addition, HSP70 interacts with the viral capsid proteins VP1 and VP3 and promotes their stability by inhibiting proteasomal degradation, thereby facilitating the assembly of DHAV-1 virions. This study demonstrates the specific role of HSP70 in regulating DHAV-1 replication, which are helpful for understanding the pathogenesis of DHAV-1 infection and provide additional information about the role of HSP70 in infection by different kinds of picornaviruses, as well as the interaction between picornaviruses and host cells.

The cytokine storm induced by duck hepatitis A virus type 1 (DHAV-1) infection significantly contributes to severe, rapid deaths and economic losses in the duck industry in Egypt. This study aimed to investigate the potential inhibitory effect of a nanoemulsion containing turmeric and black pepper oil on the immune response and pathogenesis of DHAV-1 in ducklings. A total of 105 ducklings from nonvaccinated breeders were divided into 5 experimental groups, each comprising 21 birds. The negative control group (G1) remained noninfected with DHAV-1 and nontreated with nanoemulsion, while the positive control group (G2) was infected with DHAV-1 but not treated with nanoemulsion. The other 2 groups (G3, the supplemented group which was noninfected with DHAV-1), and group 4 (the prophylactic group G4) which was infected with DHAV-1, both received nanoemulsion throughout the experiment. Group 5 (G5, the therapeutic group), on the other hand, which was infected with DHAV-1 received nanoemulsion only from the onset of clinical signs. At 5 days old, the ducklings in the positive control (G2), the prophylactic (G4), and the therapeutic group (G5) were infected with DHAV-1. All the ducklings in the infected groups exhibited depression, anorexia, and opisthotonos, and their livers displayed various degrees of ecchymotic hemorrhage, liver enlargement, and microscopic pathological lesions. Notably, the positive control group (G2) experienced the most severe and pronounced effects compared to the other infected groups treated with the nanoemulsion. Meanwhile, the viral RNA loads were lower in the liver tissues of the infected ducklings treated with the nanoemulsion (G4, and G5) compared to the positive control group G2. Additionally, the nanoemulsion effectively modulated proinflammatory cytokine expression, antioxidant enzymes, liver enzymes, and lipid profile of treated ducklings. In conclusion, the turmeric and black pepper oil nanoemulsion has the potential to be a therapeutic agent for regulating and modulating the immune response, decreasing DHAV-1-induced cytokine storms, and minimizing mortality and economic losses in the duck business. More research is needed to understand how turmeric and black pepper oil nanoemulsion alleviates DHVA-1-induced cytokine storms and lowers duckling mortality.

Duck hepatitis A virus type 1 (DHAV-1) can cause severe liver damage in infected ducklings and is a fatal and contagious pathogen that endangers the Chinese duck industry. The objective of this study was to explore the correlation mechanism of liver metabolism-gut microbiota in DHAV-1 infection. Briefly, liquid chromatography-mass spectrometry and 16S rDNA sequencing combined with multivariate statistical analysis were used to evaluate the effects of DHAV-1 infection on liver metabolism, gut microbiota regulation, and other potential mechanisms in ducklings. In DHAV-1-infected ducklings at 72 h postinfection, changes were found in metabolites associated with key metabolic pathways such as lipid metabolism, sugar metabolism, and nucleotide metabolism, which participated in signaling networks and ultimately affecting the function of the liver. The abundance and composition of gut microbiota were also changed, and gut microbiota is significantly involved in lipid metabolism in the liver. The evident correlation between gut microbiota and liver metabolites indicates that DHAV-host gut microbiome interactions play important roles in the development of duck viral hepatitis (DVH).

Duck hepatitis A virus 1 (DHAV-1) is one of the main contagious pathogens that causes rapid death of ducklings. To illuminate the potential of DHAV-1-infected underlying mechanisms, we analyzed the mRNA and microRNA (miRNA) expression profiles of duck embryonic hepatocytes (DEHs) in response to DHAV-1. We found 3410 differentially expressed genes (DEGs) and 142 differentially expressed miRNAs (DEMs) at 36 h after DHAV-1 infection. Additionally, DEGs and the target genes of miRNA expression were analyzed and enriched utilizing GO and KEGG, which may be crucial for immune responses, viral resistance, and mitophagy. For instance, the dysregulation of DDX58, DHX58, IRF7, IFIH1, STING1, TRAF3, CALCOCO2, OPTN, PINK1, and MFN2 in DHAV-1-infected DEHs was verified by RT-qPCR. Then, the association analysis of mRNAs and miRNAs was constructed utilizing the protein-protein interaction (PPI) networks, and the expressions of main miRNAs were confirmed, including miR-132c-3p, miR-6542-3p, and novel-mir163. These findings reveal a synthetic characterization of the mRNA and miRNA in DHAV-1-infected DEHs and advance the understanding of molecular mechanism in DHAV-1 infection, which may provide a hint for the interactions of virus and host.

Type I interferon (IFN-I) is essential for the regulation of host-virus interactions, and viruses have evolved strategies to escape the host immune response. Duck hepatitis A virus type 1 (DHAV-1) causes severe liver necrosis and hemorrhage, neurological symptoms, and high mortality in ducklings. However, how DHAV-1 interacts with the duck innate immune system remains unclear. In this study, DHAV-1-encoded proteins were cloned, and DHAV-1 2A2 was shown to strongly suppress IFN-β-luciferase activity, triggered by Sendai virus and polyriboinosinic polyribocytidylic acid [poly(I:C)], along with the transcription of IFN-β and downstream antiviral genes, including OASL, PKR, and TNF-a. In addition, 2A2 interacts with the central adaptor proteins mitochondrial antiviral signaling (MAVS) and TANK-binding kinase 1 (TBK1) by its N-terminal 1-100 amino acids (aa), thus leading to the inhibition of IFN-β production. Importantly, the deletion of the N-terminal 1-100 aa region of 2A2 abolished inhibition of IFN-I production. Moreover, the transmembrane domain of the MAVS protein and the ubiquitin domain of TBK1 were demonstrated to be required for interaction with DHAV-1 2A2. These findings revealed a novel strategy by which DHAV-1 hijacks cellular immunosurveillance and provided new insights into controlling the disease.

Duck hepatitis A virus type 1 (DHAV-1) infection causes an acute and highly fatal disease in young ducklings. Exosomes are nano-sized small extracellular vesicles secreted by various cells, which participate in intercellular communication and play a key role in the physiological and pathological processes. However, the role of exosomes in DHAV-1 transmission remains unknown. In this study, through RT-PCR, WB analysis and TEM observation, the complete DHAV-1 genomic RNA, partial viral proteins, and virions were respectively identified in the exosomes derived from DHAV-1-infected duck embryo fibroblasts (DEFs). The productive DHAV-1 infection was transmitted by exosomes in DEFs, duck embryos, and ducklings, and high titers of neutralizing antibodies completely blocked DHAV-1 infection but did not significantly neutralize exosome-mediated DHAV-1 infection. To the best of our knowledge, this is the first report that exosome-mediated DHAV-1 infection was resistant to antibody neutralization in vivo and in vitro, which might be an immune evasion mechanism of DHAV-1.

To define the underlying mechanism of the beneficial role of Chrysanthemum indicum polysaccharides (CIPS) and phosphorylated Chrysanthemum indicum polysaccharides (pCIPS) in duck viral hepatitis (DVH), we evaluated the protective effects of the CIPS and pCIPS against DVH in terms of antioxidation and mitochondrial function. Fluorescence probes and several assay kits were used to determine the oxidative stress and mitochondrial dysfunction in vitro and vivo. Additionally, transmission electron microscopy was applied to observe the changes of mitochondrial ultrastructure in the liver tissue. Our results indicate that the CIPS and pCIPS significantly enhanced the survival of duck hepatitis A virus type 1 (DHAV-1) infected ducklings. Moreover, the CIPS and pCIPS suppressed oxidative stress and preserved mitochondrial function, such as enhanced antioxidant enzyme activity, increased ATP production, and stabilized mitochondrial membrane potential (MMP). Meanwhile, the results of hematoxylin-eosin (HE) staining and serum biochemical examination demonstrated that treatment with the CIPS and pCIPS could decrease focal necrosis and infiltration of inflammatory cells, which in turn reducing liver injury. Furthermore, the CIPS and pCIPS were able to preserve liver mitochondrial membrane integrity in DHAV-1 challenged ducklings. Notably, the pCIPS was significantly outperformed the CIPS on all measures of liver and mitochondrial function. These results suggested that mitochondrial homeostasis plays an important role in alleviating oxidative damage in the livers, and the hepatocyte protective effects of the CIPS were enhanced after phosphorylation modification.

Duck hepatitis A virus 1 (DHAV-1) is a highly contagious etiological agent that causes acute hepatitis in young ducklings. MicroRNAs (miRNAs) play important regulatory roles in response to pathogens. However, the interplay between DHAV-1 infection and miRNAs remains ambiguous. We characterized and compared miRNA and mRNA expression profiles in duck embryo fibroblasts cells (DEFs) infected with DHAV-1. In total, 36 and 96 differentially expressed (DE) miRNAs, and 4110 and 2595 DE mRNAs, were identified at 12 and 24 h after infection. In particular, 126 and 275 miRNA-mRNA pairs with a negative correlation were chosen to construct an interaction network. Subsequently, we identified the functional annotation of DE mRNAs and target genes of DE miRNAs enriched in diverse Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which may be important for virus resistance, cell proliferation, and metabolism. Moreover, upregulated miR-222a could negatively regulate DHAV-1 replication in DEFs and downregulate integrin subunit beta 3 (ITGB3) expression by targeting the 3\' untranslated region (3\'UTR), indicating that miR-222a may modulate DHAV-1 replication via interaction with ITGB3. In conclusion, the results reveal changes of mRNAs and miRNAs during DHAV-1 infection and suggest miR-222a as an antiviral factor against DHAV-1.

The duck hepatitis A virus 1 (DHAV-1) 2C protein was predicted to be a superfamily III helicase member and includes nucleotide binding (NTB) and putative RNA helicase activity motifs. To study whether DHAV-1 2C protein has NTB activity, we expressed DHAV-1 2C protein with maltose binding protein (MBP) to solve its poor solubility in a prokaryotic expression system. We showed that the DHAV-1 2C protein has nucleoside triphosphatase (NTPase) activity by measuring the released phosphate. The NTPase of the DHAV-1 2C protein is Mg2+ indispensable and affected by other biochemical characteristics such as Mn2+, Ca2+, Zn2+, Na+ and pH. Guanidine hydrochloride (GdnHCl), a potent inhibitor of viral RNA replication, inhibited ATPase activity of the DHAV-1 2C protein in a dose-dependent manner. Finally, we constructed three mutants to identify the key site for the ATPase activity of the DHAV-1 2C protein. These results indicate that lysine at position 151 of the DHAV-1 2C protein is very important for NTPase activity. Here, we demonstrated and partially characterized that the DHAV-1 2C protein has NTPase activity and showed that mutation of the lysine in the conserved Walker A impairs that activity. The results serve to confirm what is readily predicted from previous work on picornavirus 2C proteins. It also provides a basis for further study of the 2C protein and the function of NTPase activity on the viral life cycle.