Postnatal epigenetic differences in calves following transient fetal infection with bovine viral diarrhea virus

Abstract

Background: Bovine viral diarrhea virus (BVDV) is the most detrimental pestivirus within the cattle industry. Infection with vertically transmissible BVDV prior to 125 days of gestation results in the generation of a persistently infected (PI) calf. These PI calves are unable to clear the virus in utero, due to an incomplete immune response. However, when infection with BVDV occurs after 150 days of gestation, the fetus clears the transient infection (TI) in utero and is born with antibodies specific to the infecting strain of BVDV. Variations in DNA methylation have been identified in white blood cells (WBC) from TI heifers at birth. It was hypothesized that epigenomic alterations persist into the postnatal period and contribute to previously undocumented pathologies. To study these possible effects, DNA was isolated from the WBCs of 5 TI heifers and 5 control heifers at 4 months of age and subjected to reduced representation bisulfite sequencing (RRBS).

Results: Differential analysis of the methylome revealed a total of 3,047 differentially methylated CpG sites (DMSs), 1,349 of which were hypermethylated and the other 1,698 were hypomethylated. Genes containing differential methylation were associated with inflammation, reactive oxygen species (ROS) production, and metabolism. Complete blood count (CBC) data identified a higher lymphocyte percentage in TI heifers. When compared in the context of the CD45⁺ parent population, spectral flow cytometry revealed increased intermediate monocytes, B cells, and CD25⁺/CD127^- T cells, and decreased CD4⁺/CD8b⁺ T cells. Comparative analysis revealed differential methylation of CpG sites contained in 205 genes, 5 promoters, and 10 CpG islands at birth that were also present at 4 months of age. Comparison of differential methylation in TI heifers and PI heifers at 4 months of age showed 465 genes, 18 promoters, and 34 CpG islands in common.

Conclusion: Differential methylation of WBC DNA persists to 4 months of age in TI heifers and is associated with dysregulation of inflammation, metabolism, and growth. Analysis of differential methylation in TI heifers contributes to the understanding of how fetal infection with BVDV induces postnatal detriments related to profit loss.

Keywords: BVDV; Epigenetics; Fetal infection; Methylation; Transient infection.

Fig. 1

Body weight is decreased in TI heifers at 4 months of age. Quantification of body weight in kilograms of control and TI heifers at 4 months of age. A single outlier (Control—127.27 kg) was excluded from significance testing but is denoted within the figure as a hollow circle. Following removal of the outlier, the data passed a Shapiro–Wilk normality test. Data are displayed as mean ± standard error of the mean (SEM). Asterisks denote the significance between groups, ** P < 0.01

Fig. 2

Differential methylation of CpG sites in TI heifers. The CpG sites are defined as sites containing a ‘CCGG’ motif within the genome. A Hierarchical clustering of sample methylation profiles based using 1—Pearson’s correlation distance. Control heifers are denoted in red and heifer ID numbers are followed by ‘.C’. Transiently infected TI heifers are denoted in blue and animal ID numbers are followed by ‘.TI’. B Visualization of differentially methylated CpG sites identified in TI heifers compared to controls by chromosome. Each data point represents a differentially methylated CpG site in respect to its location on the chromosome. Hypermethylated CpG sites are denoted in green and are positioned outside of the solid chromosome bars. Hypomethylated CpG sites are denoted in red and are positioned within the solid chromosome bars. C Visualization of the hypermethylated and hypomethylated CpG sites identified in TI cattle compared to controls. The –log10(q-value) Y axis threshold was set at 2 and denoted as a horizontal, grey dashed line. The % differential methylation X axis thresholds were set at − 25 and 25 and are denoted as vertical, grey dashed lines. Hypermethylated CpG sites are denoted in green and hypomethylated CpG sites are denoted in red

Fig. 3

IPA predicted impacts on TI cattle through utilization of differentially methylated CpG sites. Top 5 canonical pathways and machine learning (ML) disease pathways as determined by IPA. Green denotes hypermethylation while red denotes hypomethylation. The total number of genes included in the IPA pathway is denoted to the right of each stacked bar. The number of hypo- or hyper- methylated genes identified in the dataset are denoted within each stacked bar. A Canonical pathways were sorted according to the absolute value of the predictive z-score determined by IPA. B Disease pathways were sorted according to the -log(p-value) determined by IPA

Fig. 4

IPA predicts increased risk of arrhythmia and bleeding, decreased growth of muscle and lymphoid tissue. Modified IPA figures depict genes containing differential methylation that contribute to central diseases or altered functions. A Genes associated with arrhythmia. B Genes associated with increased bleeding. C Genes associated with decreased growth of muscle tissue. D Genes associated with decreased growth of lymphoid tissue. Green indicates hypermethylation, red indicates hypomethylation. Dashed lines ending with a flat line end indicate inhibition. Dashed lines ending with an arrow indicate contribution to the central disease or altered state. Asterisks following a gene name indicate that multiple identifiers in the dataset map to a single gene or chemical in the Global Molecular Network

Fig. 5

Markers utilized to identify immune cell populations. A A schematic depicting the fluorescent markers used to identify various immune cell populations through flow cytometry. Colors used here correspond to those utilized in Table 4. Boxes shaded in light grey were not identified in populations, while boxes shaded in various colors (including white) indicate positive fluorescence in a cellular population. B Schematic representation of the gating strategy used to identify various immune cell populations. Colors here correspond to colors representing various populations in Table 6

Fig. 6

Transient fetal infection with BVDV results in epigenetic alterations and differences in intermediate monocytes, B cells, and body weight. A graphical summary of the findings identified in postnatal heifers following transient fetal infection with BVDV. The findings include decreased body weight as well as elevated intermediate monocytes and B cells