Epigenetic Modifications of White Blood Cell DNA Caused by Transient Fetal Infection with Bovine Viral Diarrhea 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.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1ß. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1ß production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1ß production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

Epigenomic and Proteomic Changes in Fetal Spleens Persistently Infected with Bovine Viral Diarrhea Virus: Repercussions for the Developing Immune System, Bone, Brain, and Heart.

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.

Fetal Lymphoid Organ Immune Responses to Transient and Persistent Infection with Bovine Viral Diarrhea Virus.

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.

Attenuated lymphocyte activation leads to the development of immunotolerance in bovine fetuses persistently infected with bovine viral diarrhea virus†.

Bovine viral diarrhea virus continues to cost the cattle industry millions of dollars each year despite control measures. The primary reservoirs for bovine viral diarrhea virus are persistently infected 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 bovine viral diarrhea virus naïve heifers were inoculated with bovine viral diarrhea virus 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 persistently infected fetuses compared to controls. Reverse transcription quantitative polymerase chain reaction on select targets validated the microarray revealing a downregulation of type I interferons and lymphocyte markers in day 190 persistently infected fetuses compared to controls. Protein was visualized using western blot and tissue sections were analyzed with hematoxylin and eosin staining and immunohistochemistry. Data collected indicate that fetal immunotolerance to bovine viral diarrhea virus 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.