Transcriptomic Characterization of a Chicken Embryo Model Infected With Duck Hepatitis A Virus Type 1

Figure 1. Gross lesions and histopathological lesions in CH- or CH60-infected livers of embryonated eggs. Embryonated eggs were infected with the CH or CH60 strain, and then the livers were collected and soaked in 4% paraformaldehyde solution at 12, 24, 36, and 48 hpi. Microscopic lesions were observed under an optical microscope; magnification, 600×.

Figure 2. Transmission electron microscopy analysis of liver tissue of the mock, CH and CH60 groups at 48 hpi. A: mock group; A2 and A4 are magnifications of the red boxes in A1 and A3, respectively. B: CH group. B2 and B4 are magnifications of the red boxes in B1 and B3, respectively. C and D: CH60 group. C4, D2, and D4 are magnifications of the red boxes in C3, D1, and D3, respectively.

Figure 3. TUNEL assay of the livers of CH- or CH60-inoculated embryonated eggs at 48 hpi. (A) TUNEL staining of the livers of the mock, CH, and CH60 groups at 48 hpi. (B) Number of apoptotic nuclei identified by TUNEL staining. Student’s t-test was used for statistical analysis to compare the mock and treated groups.

Figure 4. Analysis of differentially expressed genes (DEGs) of the mock, CH, and CH60 groups (p-value < 0.01 and fold change > 4). A heatmap was used to classify the gene expression patterns, and a volcano plot displayed the number of DEGs. (A,B) The x-axis represents the experimental conditions. (C) Volcano plot of DEGs between the CH and CH60 groups. (D) Volcano plot of DEGs between the MOCK and CH60 groups. The y-axis indicates the negative logarithm of the p-value; the x-axis indicates the base 2 logarithm of fold change.

Figure 5. Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs). The z-score is assigned to the x-axis, and the negative logarithm of the adjusted p-value is assigned to the y-axis. The areas of the displayed circles are proportional to the number of genes assigned to the term, and the colors correspond to the categories. (A) GO enrichment analysis of DEGs between the mock group and CH60 group (p-value < 0.01 and fold change > 4). (B) GO enrichment analysis of DEGs between the CH group and CH60 group (p-value < 0.01 and fold change > 4).

Figure 6. KEGG enrichment analysis of differentially expressed genes (DEGs) (p-value < 0.01 and fold change > 4). Circles and triangles represent KEGG enrichment analyses of the DEGs between the CH and CH60 groups and the DEGs between the mock and CH60 groups, respectively. The colors of the circles and triangles indicate p-value; the sizes indicate the number of genes assigned to the term.

Figure 7. Confirmation of differentially expressed genes by RT-PCR and methylation status of SOCS3. (A) Gene expression levels were measured by the 2−ΔΔCt method with relative quantification. Differences in the expression levels of the various genes between strains CH and CH60 were analyzed using Student’s t-test and were considered significant as follows: *p < 0.05; **p < 0.01; (B) Methylation status of the SOCS3 gene in liver tissues from Duck hepatitis A virus type 1 (DHAV-1)-infected embryonated eggs. DNA from the liver tissues of embryonated eggs infected with DHAV-1 at 48 hpi was subjected to methylation-specific PCR. M, methylation-specific primers; U, non-methylation-specific primers.