Table 1.
Oligonucleotide primers used in the experiments.
Figure 1.
Duck model establishment and verification of infection with DHV-1.
A: No symptoms were evident in duckling organs without the specific bands of the conserved regions in the DHV-13D gene in the control group. B and C: Some symptoms were observed in duckling organs, including an enlarged liver with yellow or yellow-brown spots on kidneys, hyperemia and swelling, spleen enlargement with the specific bands of the conservative regions in the DHV-13D gene in the experimental ducklings (B: morbid group; C non-morbid group).
Figure 2.
Expression profile of CD8A transcripts in duck tissues.
Tissues analyzed include the thymus (Th), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), cerebrum (Cr), cerebellum (Ce), and muscle (Mu). Expression data for each tissue were analyzed from three randomly selected individuals. Vertical bars represent the mean±standard deviation (S.D.) (n = 3). Significant differences relative to controls are indicated with adjacent letters (P<0.05) and with separate letters (P<0.01).
Figure 3.
CD8A expression in duckling tissue treated with DHV-1.
Tissues analyzed include the thymus (Th), liver (Li), spleen (Sp), lung (Lu). Expression data for each tissue were analyzed from three randomly selected individuals. Vertical bars represent the mean±S.D. (n = 3). Significant differences relative to controls were indicated with * (P<0.05) and ** (P<0.01).
Figure 4.
The differential methylation level of the CD8A promoter in the duck.
(A) The schematic diagram of the CD8A promoter. An arrow indicates the transcription start site (TSS) (+1 bp), and short vertical lines indicate the positions of the methylation sites relative to the TSS. Ten CpG islands were detected in the region. (B) Bisulfite sequencing analysis of the DNA methylation profile of the individual CpG sites in the CD8A promoter in the morbid, non-morbid, and control groups. The solid and open circles indicate methylation and unmethylation status, respectively. Each PCR product was subcloned, and three samples were subjected to sequencing analysis (n = 3, mean ± S.D.).
Figure 5.
Examples of bisulfite genomic sequencing chromatography.
DNA was amplified and sequenced using each primer set on an ABI automated sequencer with dye terminators. After bisulfite treatment, unmethylated cytosine was converted to thymine, while methylated cytosine was invariable in the CpG sites.
Figure 6.
Comparison of global DNA methylation among morbid, non-morbid and control ducklings isolated from peripheral blood treated with DHV-1.
Significant difference in global DNA methylation between morbid ducklings and non-morbid ducklings. * means P<0.05 (n = 3).