Determination of drug concentration

In order to eliminate the toxic effect of chlorogenic acid on DEF and control the effect of chlorogenic acid on DEF within the safe range, the optimal safe concentration of the drug was determined. As shown in Table 1, with the increase of concentration, the cell activity showed the trend of increasing and then decreasing, but all of them were lower than the cell activity of 0 mg/mL, and the degree of decrease of the cell activity was not significant, which indicated that the low concentration of chlorogenic acid had a small toxicity on DEF, and the cell activity was the highest when the concentration was up to 0.250 mg/mL, which was selected as the safe concentration of the test drug.

Group N, to which only 3% maintenance medium + CCK-8 solution was added, is the solution control group, and the concentration of 0 mg/mL without chlorogenic acid was added, and is the cell control group.

Cell activity assay

In order to understand whether chlorogenic acid will alleviate the effect of DPV on DEF cells when acting on infected cells, the CCK-8 method was used to detect the cell activity of each group of cells after different treatments at 24 h, 36 h and 48 h of incubation, as shown in Table 2, the cell activity of group C did not change significantly compared with group A (P > 0.05), and the cell activity of group F increased extremely significantly compared with group D (P < 0.01). It indicates that DPV infection leads to a decrease in cell activity, and the cell activity of DEF cells after chlorogenic acid action is significantly increased compared to no treatment in DPV infection.

Table 2 Results of DEF cell viability assay in each groupInfected cell virus qRT-PCR detection results

In order to understand the viral load of cells in each group at each time point after DPV infection, qRT-PCR was used. As shown in Fig. 2, compared with the cell control group, the DPV load of the virus-infected group increased significantly at all three time points (P < 0.05); compared with the virus-infected group, the DPV load of the cells in the chlorogenic acid-intervention group showed a significant decrease at all three time points (P < 0.05), which indicated that chlorogenic acid could inhibit the proliferation of DPV in DEF cells.

Fig. 2

Detection of viral load at various periods after the action of chlorogenic acid on infected cells the same lowercase letters represent non-significant differences between the groups compared at the same time, and different lowercase letters represent significant differences between the groups compared at the same time, followed by the same

Transcriptomics analysis resultsGlobal changes in gene expression

In order to study the gene expression of chlorogenic acid during DPV infection of DEF, total cellular RNA was extracted, and sequencing library was constructed and used for RNA-seq. To reduce the interference of subsequent sequencing, the data were filtered and clean reads were obtained, and the percentage of clean reads of high-quality data obtained after filtration was higher than 90%, and the overall quality of the data was high, which lays the foundation of subsequent analyses (Additional file 2: Table S2). The results of PCA showed that the distribution of samples in the same group was more concentrated, and the distribution between samples in different groups was more dispersed, suggesting that the samples were biologically well duplicated (Fig. 4a); the heat map of sample correlation showed that the samples in the same group were similar in color, and the correlation was high (Fig. 4b). Overall statistical analysis of differential genes and visualization by volcano diagram (Additional file 3: Fig. S1) showed that the number of down-regulated differential genes was significantly higher than the number of up-regulated differential genes in group A compared to group D, suggesting that DPV infection with DEF mainly caused down-regulation of gene transcript levels; the number of up-regulated differential genes was significantly higher than the number of down-regulated genes in group C compared to group F, and the number of up-regulated differential genes was significantly higher than the number of down-regulated genes in group D compared to group F, suggesting that chlorogenic acid mainly causes up-regulation of genes to inhibit DPV proliferation in DEF (Fig. 3).

Fig. 3

Sample relationship analysis. a sample principal component analysis plot, PCA1 is the first principal component, PCA2 is the second principal component, and percentage refers to the contribution of the comparison sample; b sample correlation heat map

Results of overall statistical analysis of differential genes

Comparison between groups at the same time point, the results are shown in Fig. 4: when chlorogenic acid and DPV acted DEF for 24 h, there were a total of 308 differentially expressed genes in group A and D, of which 125 were up-regulated genes and 183 were down-regulated expressed genes; a total of 292 differentially expressed genes in group C and D, of which 176 were up-regulated genes and 116 were down-regulated genes; a total of 246 differentially expressed genes in group C and F, of 193 up-regulated genes and 53 down-regulated genes; group D and group F differentially expressed genes totaled 362, including 258 up-regulated genes and 104 down-regulated genes.when chlorogenic acid and DPV acted DEF for 36 h, there were a total of 898 differentially expressed genes in group A and D, including 231 up-regulated genes and 667 down-regulated genes; there were a total of 931 differentially expressed genes in group C and D, including 696 up-regulated genes and 235 down-regulated genes; there were a total of 1719 differentially expressed genes in group C and F, including 1225 up-regulated genes and 494 down-regulated genes; and there were a total of 362 differentially expressed genes in group D and F. 494 down-regulated genes; Group D and Group F differentially expressed genes totaled 348, including 206 up-regulated genes and 142 down-regulated genes.when chlorogenic acid and DPV acted DEF for 48 h, the total number of differentially expressed genes in group A and D was 660, including 84 up-regulated genes and 576 down-regulated genes; the total number of differentially expressed genes in group C and D was 731, including 147 up-regulated genes and 584 down-regulated genes; the total number of differentially expressed genes in group C and F was 223, including 154 up-regulated genes and 69 down-regulated genes; the total number of differentially expressed genes in group D and F was 1425, including 206 up-regulated genes and 142 down-regulated genes. The total number of differentially expressed genes in group C and group F was 223, 154 up-regulated genes and 69 down-regulated genes; the total number of differentially expressed genes in group D and group F was 199, 138 up-regulated genes and 61 down-regulated genes.

Fig. 4

Overall statistical results of differential genes. The X axis represents the difference comparison scheme for each group, and the Y axis represents the corresponding number of differential genes (DEG). Red represents the number of up-regulated DEGs and blue represents the number of down-regulated DEGs

The above results showed that the number of down-regulated genes was significantly higher than the number of up-regulated genes in group A compared with group D, suggesting that the infection of DEF by DPV mainly caused the down-regulation of gene transcription level; the number of up-regulated genes was significantly higher than the number of down-regulated genes in group C compared with group F, and the number of up-regulated genes was significantly higher than the number of down-regulated genes in group D compared with group F, suggesting that chlorogenic acid mainly caused gene up-regulation to inhibit the proliferation of DPV in DEF.

Differential gene GO enrichment analysis

The differentially expressed genes appeared in DPV-infected DEF cells after chlorogenic acid intervention were analyzed and categorized by comparing with GO database in order to observe which biological processes the differentially expressed genes were involved in. As shown in Fig. 5, the differential genes were mainly involved in biological processes such as immune response, cell adhesion, signal transduction, cell chemotaxis, and viral defense response at 24 h post-intervention; at 36 h post-intervention, the differential genes were mainly involved in biological processes such as immune response, inflammatory response, neutrophil chemotaxis, and activation of neutrophils; and at 48 h post-intervention, the differential genes were mainly involved in immune response, cell adhesion, activation of complement, cell chemotaxis, acute phase response, and cytokine-mediated signaling pathway.

Fig. 5

GO enrichment analysis of differentially expressed genes involved in biological processes. The X-axis is -lg (Qvalue), the Y-axis is the name of the participating function, and the broken line is the number of differential genes

KEGG enrichment analysis of differentially expressed genes

The differential metabolites were compared with KEGG large database to analyze the enrichment of differential genes in relevant pathways after chlorogenic acid intervention in DPV-infected DEF cells. As shown in Fig. 6, the differentially expressed genes at 24 h of intervention were mainly involved in signaling pathways such as toll-like receptor signaling pathway, chemokine signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway and IL-17 signaling pathway. The differentially expressed genes at 36 h mainly participated in TNF signaling pathway, NF-kappa B signaling pathway, calcium signaling pathway, toll-like receptor signaling pathway and DNA sensing pathway. The differentially expressed genes at 48 h were mainly involved in DNA sensing pathway, NF-kappa B signaling pathway, TNF signaling pathway and IL-17 signaling pathway.

Fig. 6

Bubble enrichment of KEGG pathway of differentially expressed genes. X axis is enrichment ratio, Y axis is KEGG Pathway, bubble size represents the number of genes annotated on a KEGG Pathway, color represents enrichment significance value, the redder the color, the smaller the significance value

Correlation factor analysis and validation resultsData preprocessing and quality control

As shown in Fig. 7, the overlap in the VENN plot comparing the normal control group with the viral control group at each time point indicates 37 differentially expressed genes (Fig. 7a), the overlap in the VENN plot comparing the chlorogenic acid intervention group with each other at each time point indicates 71 differentially expressed genes (Fig. 7b), the overlap in the VENN plot comparing the chlorogenic acid intervention group with the chlorogenic acid control group at each time point indicates 29 differentially expressed genes (Fig. 7c), and the overlap in the VENN plot comparing the chlorogenic acid intervention group with the viral control group at each time point indicated 17 differentially expressed genes (Fig. 7d). Pooling these genes, the screening enumerated 8 meaningful differentially expressed genes, of which TNFSF15, TNFAIP2, IFNAR1 and CCL26 were pathway-related genes. RT-qPCR was used to analyze the above 8 DEGs to verify the accuracy of the RNA-seq data, and the results showed that the overall trend of the selected genes was consistent, proving the reliability of our RNA sequencing (Additional file 4: Fig. S2). In addition, the differentially expressed genes were annotated to the KEGG enrichment signaling pathway to analyze the expression changes of genes in the NF-κB dominated signaling pathway (Table 3).

Fig. 7

VENN analysis results of differentially expressed genes in each group. a Comparison at various time points between the virus group and the normal control group; b Each time point in the intervention group was compared respectively; c Comparison between intervention group and chlorogenic acid control group at each time point; d Comparison between intervention group and virus group at various time points

Table 3 Analysis results of NF-κB, toll like receptor and TNF signal pathwayResults of critical path analysis

As shown in Table 4, chlorogenic acid inhibits DPV proliferation in vitro, and up- and down-regulates genes in the NF-κB signaling pathway, toll-like receptor signaling pathway, TNF signaling pathway, and IL-17 pathway at different time intervals. IL-6 and IL-8 are inflammatory factors, and IL-8 transmits signals to activate the toll-like receptor signaling pathway, and TLR4 and NFKBIA are NF-κB signaling inhibitors. pathway inhibitors, chlorogenic acid can regulate IL-8, TLR4 and NFKBIA, down-regulate the inflammatory-related factors in the toll-like receptor signaling pathway, coordinate IL-18R and NFKBIA in the TNF signaling pathway, and cause a large amount of secretion of CCL20 in the IL-17 signaling pathway, alleviate inflammatory injury to cells by DPV, inhibit the activity of NF-κB signaling pathway activated by DPV and inhibit the activity of NF-κB signaling pathway activated by DPV, to resist cellular damage and interfere with DPV proliferation in cells.

Table 4 Effect of RNA interference NF-κB1 on DEV proliferationEffect of NF-κB1 RNAi on DEV proliferation in DEF cells

pGPU6/GFP/Neo-NF-κB-2 and DEV were infected and transfected using three different methods, respectively. Cells were collected at 24, 36, and 48 h, followed by qPCR detection of the DEV-NP gene. The results demonstrated that the group with interference for 24 h followed by treatment with poison achieved an inhibition efficiency of up to 83% at 48 h. Additionally, the group treated with both poison and interference exhibited an inhibition efficiency of up to 77% at 72 h. Notably, the group with interference for 24 h followed by poison treatment showed the highest inhibition efficiency (refer to Table 4, Fig. 8).

Fig. 8

Transfection of pGPU6/GFP/Neo-NF-κB1-2 (100 x)

Observations on tissue lesions after DPV infection in ducks

In order to study the effect of DPV-infected ducks on various lymphoid organs, pathological sections were prepared. There were no obvious histopathological changes in various lymphoid organs in group N. In group N, there were no obvious histopathological changes in various lymphoid organs. Splenic organs: Group V showed massive congestion of the splenic sinus at 24 h, nuclear consolidation and nucleolysis at 36 h, and a significant decrease in the number of splenic lymphocytes at 48 h, accompanied by a large number of inflammatory cell infiltration and tissue disintegration (Additional file 5: Fig. S3a, Group V, the arrowheads pointing to the above histopathological lesions); Group M showed an increase in the number of splenic lymphocytes at 24 h, a reduction in the nuclear consolidation and nucleolysis phenomenon at 36 h, and a significant reduction in the hemorrhage or bruising phenomenon of the splenic sinus at 48 h (Additional file 5: Fig. S3a Group M. The arrows point to slight lesions in the above tissues. Thymus: Group V showed eosinophilic staining in both the cortex and medulla at 24 h, with a large number of vacuoles, slight hemorrhage and local inflammatory cell infiltration in the cortex at 36 h, and tissue disintegration and nuclear consolidation in the thymus at 48 h (Additional file 5: Fig. S3b, Group V, the arrowheads pointing to the above histopathology); in Group M, lymphocytosis at 24 h increased in comparison with that of Group V, with a small number of vacuoles at 36 h, and slight inflammatory cell infiltration and lymphocytosis in the cortex and medulla at 48 h. The lymphocytes were increased at 24 h, with a small number of vacuoles at 36 h. In group M, a small number of vacuoles appeared at 48 h compared with group V. At 48 h, the cortex and medulla showed a slight inflammatory cellular infiltration with lymphocytosis, and the histologic lesions did not worsen with the prolongation of the time of infection (Additional file 5: Fig. S3b, group M, the arrowheads pointing to the abovementioned slight histologic lesions). Bursa fascicularis: 24 h markedly reduced basophilic granulocytes in the medulla,36 h nuclear consolidation, nuclear fragmentation, reduced lymphocytes, 48 h tissue disintegration and a small number of hemorrhagic spots (Additional file 5: Fig. S3c Group V, the arrows pointing to the above histopathology); Group M ducks with bursa fascicularis infection 24 h markedly increased basophilic granulocytes in the medulla, 36 h localized irregular fissures and a small amount of hemorrhage, 48 h a slight tissue disintegration (Additional file 5: Fig. S3c Group M, the arrow pointing to the above histopathology). In group M, there was a significant increase in basophils in the medulla of ducks at 24 h after bursa infection. The above results further indicated that honeysuckle could reduce the damage of lymphoid organs caused by DPV infection in ducklings.

Results of qRT-PCR detection of duck immune organ virus in infected ducks

In order to study the actual intervention effect of chlorogenic acid, qPCR was used to detect the viral load in each immune organ of ducks in each group at each time point after DPV infection. As shown in Fig. 9, compared with group N, the DPV load in all lymphoid organs of ducks in group V increased significantly (P < 0.05) at all three time points, and compared with group V, the DPV load in all lymphoid organs of ducks in group M decreased. The DPV load of group N was zero in all lymphoid organs. The results showed that chlorogenic acid inhibited the proliferation of DPV in all lymphoid organs of ducklings.

Fig. 9

The viral content of each immune organ after DPV infection in ducks