Role of Intrauterine Bovine Viral Diarrhea Virus in Modulating Host Gene Expression at the Maternal-Fetal Interface

Abstract

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. Infections with BVDV have a substantial economic impact on beef and dairy producers in the United States and worldwide. This virus affects multiple organs including the respiratory, reproductive, and gastrointestinal systems, and immunosuppression is the primary underlying cause of clinical disease. Viral maintenance in cattle herds is due in large part to the capability of BVDV to cross the placenta and infect the fetus. Reproductive consequences of BVDV infection include stillbirths, abortions, calves with congenital malformations, or calves born persistently infected (PI) with the virus (Baker 1995). Once established on a farm, the virus can remain undetected in cattle herds because persistently infected animals continuously shed large amounts of virus and animals may show no clinical signs. These undetected persistently infected animals provide a continual source of the virus to the herd and maintain this cycle of BVDV exposure to serologically naïve heifers and cows. BVDV control programs have been established to increase protective serologic titers and to reduce the incidence of PI fetuses. Despite attempts to improve maternal immunity through vaccination programs, vaccines do not provide 100% fetal protection. To further understand why vaccination provides high levels of humoral immunity but does not adequately protect the fetus, we examined T cell associated responses during transplacental BVDV infections. We hypothesized efficient transplacental transmission of the virus is associated with a predominant Th-2 microenvironment of the pregnant uterus. We identified evidence of immune recognition of the virus in placentomes with consequences of dysregulation of normal local immunoregulatory processes at the maternal-fetal interface. The primary objectives were to examine the gene expression profile of T cell- associated cytokines, including some T regulatory cell associated molecules in the normal bovine placenta, cytokeratin enriched primary placental cells, and peripheral blood mononuclear cells collected during normal, early to mid-pregnancy. The goal for this research was to determine if immune recognition by the local maternal cellular immune response occurs following BVDV infection and to characterize this response. We hypothesized the virus is more likely to avoid elimination by the host T cell response in the uterus than the systemic circulation due to the presence of negative regulators or inhibitory factors necessary for maintenance of pregnancy following implantation to gestational day 150. The overall goals detailed in this dissertation were to establish normal immune regulatory parameters in bovine placentomes and the peripheral circulation during early to mid-gestation. Some of the experiments examined the role of bovine viral diarrhea virus on these immune parameters at the placental level. These investigations were performed with placental tissue and leukocytes obtained from peripheral blood samples collected from pregnant and non-pregnant heifers between 89-150 days of gestation, permitting three investigations. The first study was an in vivo experimental trial in which placental samples representing the maternal-fetal interface were analyzed to evaluate immune regulatory molecules potentially exerting an immunosuppressive function during mid-gestation. The literature provides minimal background regarding the anticipated cytokine and chemokine expression patterns during mid-gestation bovine pregnancy. This trial was carried out by experimental inoculation of pregnant heifers with BVDV. This virus is capable of infecting fetuses by vertical transmission, and causing multiple adverse reproductive outcomes. Our hypothesis was that BVDV downregulates immune responses in the pregnant bovine uterus from gestational ages 75-150 to allow for persistence of the virus and escape from the maternal immune response. The second investigation centered on primary cell cultures propagated from maternal epithelial cells obtained from 75-day-gestation placentas from serologically negative heifers, and provided identification of immune regulatory genes in these primary cells. The hypothesis for the in vitro experimental study was that trophoblasts provide a substantial role in intercellular communication at the maternal-fetal interface, primarily functioning as an immunologic sensor involved with immune recognition and trafficking. Bovine trophoblasts were found to be susceptible to BVDV and appeared immunologically responsive to BVDV infection. For the third study, peripheral blood leukocytes from non-pregnant and pregnant heifers were treated as samples from control and treated groups, respectively, to evaluate expression patterns of potential immune regulatory molecules during normal bovine pregnancy during the 89 to 150-day period. The sampling time point was within the gestational period determined to be optimal for vertical transmission of BVDV and establishment of persistent fetal infection. Immunological signaling mechanisms in the bovine placenta during this phase of gestation are largely unknown. Our goal was to develop increased baseline knowledge about immune signaling in cattle at this specialized interface between the mother and fetus. This work provides a detailed comparison of immune regulatory networks between humans and cattle and expands on how infectious agents such as BVDV alter the normal mechanisms of immune homeostasis, disrupting the symbiotic relationship between the mother and fetus during pregnancy.