RNA-seq analysis of chlorogenic acid intervention in duck embryo fibroblasts infected with duck plague virus.

INTRODUCTION: Chlorogenic acid, the primary active component in Chinese medicines like honeysuckle, exhibits anti-inflammatory and antiviral effects. It has been demonstrated that chlorogenic acid effectively prevents and treats Duck enteritis virus (DEV) infection. This study aims to further elucidate the mechanism by which chlorogenic acid prevents DEV infection. METHODS: Duck embryo fibroblast (DEF) cells were pre-treated with chlorogenic acid before being infected with DEV. Cell samples were collected at different time points for transcriptomic sequencing, while qPCR was used to detect the proliferation of DEV. Additionally, 30-day-old ducks were treated with chlorogenic acid, and their lymphoid organs were harvested for histopathological sections to observe pathological damage. The proliferation of DEV in the lymphoid organs was also detected using qPCR Based on the transcriptomic sequencing results, NF-κB1 gene was silenced by RNAi technology to analyze the effect of NF-κB1 gene on DEV proliferation. RESULTS: Compared to the viral infection group, DEF cells in the chlorogenic acid intervention group exhibited significantly reduced DEV load (P < 0.05). Transcriptomic sequencing results suggested that chlorogenic acid inhibited DEV proliferation in DEF cells by regulating NF-κB signaling pathway. The results of RNAi silencing suggested that in the three treatment groups, compared with the DEV experimental group, there was no significant difference in the effect of pre-transfection after transfection on DEV proliferation, while both the pre-transfection after transfection and the simultaneous transfection group showed significant inhibition on DEV proliferation Furthermore, compared to the virus infection group, ducks in the chlorogenic acid intervention group showed significantly decreased DEV load in their lymphoid organs (P < 0.05), along with alleviated pathological damage such as nuclear pyretosis and nuclear fragmentation. CONCLUSIONS: Chlorogenic acid effectively inhibits DEV proliferation in DEF and duck lymphatic organs, mitigates viral-induced pathological damage, and provides a theoretical basis for screening targeted drugs against DEV.

Untargeted metabolomics of the intestinal tract of DEV-infected ducks.

INTRODUCTION: Duck enteritis virus (DEV) mainly causes infectious diseases characterized by intestinal haemorrhage, inflammation and parenchymal organ degeneration in ducks and other poultry. However, the mechanism by which it causes intestinal damage in ducks is not well understood. Metabolomics can provide an in-depth understanding of the full complexity of the disease. METHODS: In this study, 24 clinically healthy green-shell ducks (weight 1.5 kg ± 20 g) were randomly divided into 2 groups (experimental group, 18; control group, 6). The experimental group was intramuscularly injected with 0.2 mL of DEV virus in solution (TCID50 3.16 × 108 PFU/mL), and the control group was injected with 0.2 mL of sterile normal saline. Duck duodenum and ileum tissue samples were collected at 66 h, 90 h and 114 h post-injection (12 h of fasting before killing), and metabolomics analysis of duck duodenum and ileum tissues at the three time points (66, 90, 114 h) was performed by liquid chromatography-mass spectrometry (LC-MS) to screen for and analyse the potential differentiated metabolites and related signalling pathways. RESULTS: Screening was performed in the positive/negative mode (Pos: Positive ion mode; the ionization of substances at the ion source with positive ions such as H+, NH4+, Na+ and K+; Neg: Negative ion mode; the ionization of substances at the ion source with negative ions such as Cl-, OAc-), and compound abundance was compared to that in the control group. The total number of differentially abundant compounds in the duodenum at 66 h, 90 h and 114 h of DEV infection gradually increased, and metabolites such as cytidine, 2'-deoxyriboside and 4-guanidinobutyric acid were differentially abundant metabolites common to all three time periods. The metabolic pathways related to inflammatory response and immune response were tryptophan acid metabolism, cysteine-methionine metabolism, histidine metabolism and other amino acid metabolism and fat metabolism. Among them, the metabolic pathways with more differentially abundant metabolites were amino acid biosynthesis, cysteine and methionine metabolism, tryptophan metabolism, unsaturated fatty acid biosynthesis and purine metabolism, and the metabolic pathways with more enrichment factors were the IgA-related intestinal immune network pathway and lysosome pathway. Compared with the control group, there were 16 differentially abundant metabolites in the ileum tissue of DEV-infected ducks at 66 h of infection, 52 at 90 h of infection, and 40 at 14 h of infection with TD114. The metabolic pathways with more enriched differentially abundant metabolites were pyrimidine metabolism, tyrosine metabolism, phenylalanine metabolism and tryptophan biosynthesis. The metabolic pathways with the most enrichment factors were the mTOR signalling pathway, ferroptosis pathway, tryptophan metabolism pathway and caffeine metabolism pathway. CONCLUSION: Comparative analysis showed that the number of differentially abundant metabolites in the duodenum and ileum differed to some extent after DEV infection, with significantly more differentially abundant metabolites in duodenal tissues and fewer in ileal tissues; after DEV infection, the highest number of differentially abundant metabolites was obtained at 114 h of DEV infection, followed by the second highest at 90 h of infection and the lowest at 66 h of infection. The common differentially abundant metabolites in duodenal and ileal tissues were prostaglandins, arachidonic acid, and arachidonic ethanolamine. The main metabolic pathways in the duodenum were the IgA-associated intestinal immune network pathway and the lysosomal pathway, and the metabolic pathways with more enriched factors in the ileum were the mTOR signalling pathway, the ferroptosis pathway, and the tryptophan metabolism pathway.