Postnatal epigenetic alterations of cattle following fetal infection with bovine viral diarrhea virus

Abstract

Bovine viral diarrhea virus (BVDV) remains a costly disease despite extensive research, vaccine usage, and the implementation of control strategies. While it is difficult to approximate the overall profit loss due to BVDV infections, the global estimation is in the billions annually. As the main reservoir of the virus, persistently infected (PI) cattle that are infected early in gestation are the main targets for management of the disease and cost analysis. In contrast, infections that occur late in gestation and generate a transiently infected (TI) calf have generally been regarded as inconsequential even though research suggests an increased susceptibility to disease and decreased weight gain. Per the developmental origins of health and disease theory (DOHaD), environmental cues such as nutrition, stress, or pathogen exposure have the potential to alter epigenetic regulation of gene expression and lead to postnatal disease. In fact, alterations of the methylome have been demonstrated in fetal spleen of PIs at gestational day 245. It was hypothesized that these epigenetic changes in PI fetuses persist into the postnatal period and continue to correspond to observed pathologies. To this end, whole blood was collected from 4-month-old PI heifers at a local, cooperating ranch and age-matched control heifers housed at Colorado State University (CSU). Zoetis Inc. utilized whole blood to determine complete blood counts (CBCs). Peripheral WBCs were isolated from whole blood and subjected to spectral cytometry analysis in collaboration with Zoetis Inc. The DNA was extracted from peripheral WBCs for reduced representation bisulfite sequencing (RRBS) to assess alterations of the methylome. The CBC analysis identified elevated monocytes, microcytic anemia, and elevated platelets with decreased mean platelet volume in PI heifers. Spectral cytometry revealed increased intermediate monocytes, B cells, CD4+/CD8b+ T cells, and CD25+/CD127- T cells; the analysis also identified decreased γδ+, CD4+, and CD4-/CD8b- T cells in PI heifers. Comparison of the methylome in PI heifers to that of control heifers revealed 8,367 differentially methylated CpG sites (DMSs) contained within genes associated with postnatal pathologies of the immune system, cardiac system, and skeletal system, similar to the methylome of PI fetal spleen. The identified list of genes containing differential methylation in PI fetal spleen was compared to those containing differential methylation in 4-month-old PI peripheral white blood cells. A total of 797 genes contained differential methylation in both the PI fetal spleen and 4-month-old peripheral WBCs. It was concluded that epigenetic alterations in PI fetuses persist into the postnatal period and continue to correspond to observed pathologies. It was subsequently hypothesized that epigenetic alterations occur in calves due to transient infection with BVDV in late gestation. It was further postulated that these alterations would be present in the postnatal period and correspond to altered expression of transcripts within peripheral WBCs. To study this effect, 12 control and 11 TI calves were generated through maternal inoculation with non-cytopathic BVDV2 type 96B2222 on gestational day 175. Whole blood was collected from generated calves at 4 months of age and 17 months of age; these samples were used to isolate peripheral WBCs, extract DNA, and perform RRBS to quantify and compare the methylation of CpG sites throughout the genome. Concurrently, peripheral WBCs were isolated for CBC and flow cytometry analysis in collaboration with Zoetis Inc. Peripheral WBCs from whole blood were also collected for single cell RNA sequencing at 17 months of age in collaboration with Zoetis Inc. The CBCs at 4 months of age demonstrated elevated lymphocyte percentage in TI heifers. Spectral cytometry analysis at 4 months of age revealed increased intermediate monocytes, B cells, and CD25+/CD127- T cells; TI heifers also had decreased CD4+/CD8b+ T cells. Comparison of 4-month-old TI methylomes to that of age-matched controls identified a total of 3,047 DMSs contained in genes related to inflammation, metabolism, and growth. Identified DMSs were compared to those identified in TI heifers at birth. A total of 205 genes, 5 promoters, and 10 CpG islands were found to contain differential methylation at birth and 4 months of age. Moreover, genes containing differential methylation in TI heifers at 4 months of age were compared to those containing differential methylation in PI heifers at 4 months of age and revealed 1,098 genes, 34 CpG islands, and 18 promoters in common. At 17 months of age, no differences were identified in CBCs. Flow cytometry once again demonstrated a decrease in CD4+/CD8b+ T cells in addition to a decrease in natural killer T cells in TI heifers. A total of 5,508 DMSs were identified when comparing the epigenome of TIs to that of controls and Ingenuity Pathway Analysis (IPA) predicted inhibition of pathways related to the immune system. Single cell RNA sequencing identified 25 unique cell clusters; TIs had greater proportions of B cell population 5 and smaller proportions of myeloid population 5. Four genes were identified as differentially expressed. The TI heifers demonstrated increased expression of PDE4D in a B cell population, increased expression of SFMBT2 in a second B cell population, increased expression of ARHGEF18 in a myeloid cell population, and decreased expression of HNRNPLL in a population of natural killer T cells. While SFMBT2 was found to contain hypomethylation at 17 months and corresponds to the observed increase in expression, neither ARHGEF18 nor HNRNPLL contained differential methylation. Interestingly, PDE4D, as well as other phosphodiesterase genes, contained hypermethylation at 17 months. Comparison of differential methylation at birth, 4 months of age, and 17 months of age in TI heifers revealed that 347 genes that contained differential methylation compared to controls at all 3 timepoints. One gene contained differential methylation within a promoter region and 6 contained differential methylation within a CpG island. Of those 347 genes, only 64 maintained the same differential methylation status at all three timepoints; for example, leukemia inhibitory factor (LIF) contained at least one hypermethylated CpG site in TI heifers at birth, 4 months, and 17 months of age. Together, this data demonstrates that epigenetic alterations due to fetal infection with BVDV persist in the postnatal period and at least partially correspond to differences in gene expression.