Bovine Viral Diarrhea Virus (BVDV) fetal infections occur in two forms; persistent infection (PI) or transient infection (TI), depending on what stage of gestation the fetus is infected. Examination of lymphoid organs from both PI and TI fetuses reveals drastically different fetal responses, dependent upon the developmental stage of the fetal immune system. Total RNA was extracted from the thymuses and spleens of uninfected control, PI, and TI fetuses collected on day 190 of gestation to test the hypothesis that BVDV infection impairs the innate and adaptive immune response in the fetal thymus and spleen of both infection types. Transcripts of genes representing the innate immune response and adaptive immune response genes were assayed by Reverse Transcription quatitative PCR (RT-qPCR) (2^−ΔΔCq; fold change). Genes of the innate immune response, interferon (IFN) inducible genes, antigen presentation to lymphocytes, and activation of B cells were downregulated in day 190 fetal PI thymuses compared to controls. In contrast, innate immune response genes were upregulated in TI fetal thymuses compared to controls and tended to be upregulated in TI fetal spleens. Genes associated with the innate immune system were not different in PI fetal spleens; however, adaptive immune system genes were downregulated, indicating that PI fetal BVDV infection has profound inhibitory effects on the expression of genes involved in the innate and adaptive immune response. The downregulation of these genes in lymphocytes and antigen-presenting cells in the developing thymus and spleen may explain the incomplete clearance of BVDV and the persistence of the virus in PI animals while the upregulation of the TI innate immune response indicates a more mature immune system, able to clear the virus.

Bovine viral diarrhea virus (BVDV) infections cause USD 1.5–2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle.

Bovine viral diarrhea virus (BVDV) continues to cost the cattle industry millions of dollars each year despite control measures. The primary reservoirs for BVDV are persistently infected (PI) animals, which are infected in utero and shed the virus throughout their lifetime. The difficulty in controlling the virus stems from a limited understanding of transplacental transmission and fetal development of immunotolerance. In this study, pregnant BVDV naïve heifers were inoculated with BVDV on day 75 of gestation and fetal spleens were collected on gestational days 82, 97, 190, and 245. Microarray analysis on splenic RNA from days 82 and 97 revealed an increase in signaling for the innate immune system and antigen presentation to T cells in day 97 PI fetuses compared to controls. RT-qPCR on select targets validated the microarray revealing a downregulation of type I interferons and lymphocyte markers in day 190 PI fetuses compared to controls. Protein was visualized using western blot and tissue sections were analyzed with H&E staining and immunohistochemistry. Data collected indicates that fetal immunotolerance to BVDV developed between days 97 and 190, with mass attenuation of the immune system on day 190 of gestation. Furthermore, lymphocyte transcripts were initially unchanged then downregulated, suggesting that immunotolerance to the virus stems from a blockage in lymphocyte activation and hence an inability to clear the virus. The identification of lymphocyte derived immunotolerance will aid in the development of preventative and viral control measures to implement before or during pregnancy. BVDV is an infectious RNA virus which is a major cause of production losses in cattle due to its contribution to bovine respiratory disease in feedlot animals and reproductive losses in cow-calf (breeding) herds. Persistently infected (PI), immunotolerant animals are the main source of BVDV infections in cattle. The generation of PI animals by vertical transmission of BVDV during early pregnancy has been known for over 50 years; however, the mechanisms responsible for the immunotolerance to and persistence of BVDV in PI animals have not been elucidated [1-3]. This in vivo study provides not only a unique perspective on the development of immunotolerance to BVDV in PI fetuses, but contributes to our understanding the development of the bovine fetal immune system.

Bovine viral diarrhea virus (BVDV) infection during early gestation results in persistently infected (PI) immunotolerant calves that are the primary reservoirs of the virus. Pathologies observed in PI cattle include congenital defects of the brain, heart, and bone as well as marked functional defects in their immune system. It was hypothesized that fetal BVDV infection alters T cell activation and signaling genes by epigenetic mechanisms. To test this, PI and control fetal splenic tissues were collected on day 245 of gestation, 170 days post maternal infection. DNA was isolated for reduced representation bisulfite sequencing, protein was isolated for proteomics, both were analyzed with appropriate bioinformatic methods. Within set parameters, 1951 hypermethylated and 691 hypomethylated DNA regions were identified in PI compared to control fetuses. Pathways associated with immune system, neural, cardiac, and bone development were associated with heavily methylated DNA. The proteomic analysis revealed 12 differentially expressed proteins in PI vs. control animals. Upregulated proteins were associated with protein processing, whereas downregulated proteins were associated with lymphocyte migration and development in PI compared to control fetal spleens. The epigenetic changes in DNA may explain the immune dysfunctions, abnormal bone formation, and brain and heart defects observed in PI animals.

Current technology to identify blastocysts with the greatest chance of pregnancy success relies on visual inspection of the embryos before transfer, which contributes to variation in embryo transfer pregnancy rates. It was hypothesized that ribonucleic acid (RNA)-sequencing (RNA-Seq) could be employed to discover differentially expressed transcripts between Grade 1 (G1, excellent/good) and Grade 3 (G3, poor) quality day 7 in vitro produced (IVP) embryos. Bovine oocytes were collected, fertilized, cultured, and then graded on day 7. Using RNA-Seq of pooled early and expanded blastocysts by quality grade, three messenger RNA transcripts were found to be upregulated in G3 vs G1 embryos. These transcripts were studied in G3 vs G1 whole or biopsied morula-stage embryos in three independent trials. A pre-amplified complementary deoxyribonucleic acid method using primers for all three transcripts and housekeeping genes was developed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). These transcripts were either lowly expressed or not expressed at all in G1 quality morulae and biopsies. Expression of these transcripts in G3 embryos and biopsies had significantly greater variation (Levene’s test). Some of the G3 embryos had transcript levels that were consistent with G1 embryos, potentially indicating a higher quality compared to subjective visual methods of evaluation. In conclusion, an RNA extraction and RT-qPCR method for studying marker transcript expression in a single IVP embryo and biopsy sample was validated. Three transcript markers were identified that differ in expression based on embryo grade in both blastocysts and morulae. These transcripts in addition to other embryo evaluations may identify the best candidates for embryo transfer.