Performing Department
(N/A)
Non Technical Summary
Beef and Dairy cattle are important commodities in the United States. During viral growth, BoHV-1 spreads to sensory neurons in the peripheral nervous system and certain cells in the pharyngeal tonsil where the virus establishes and maintains a life-long latent infection. During latency, BoHV-1 does not grow but it lingers in the latently infected cells. Stress, because of dramatic weather changes and shipping cattle long distances can trigger BoHV-1 to grow in latently infected calves (reactivate from latency). Bovine alpha-herpesvirus 1 (BoHV-1) infection of cattle can cause severe nasal congestion, swelling of the upper respiratory tract, temporary immune suppression, and abortions. BoHV-1 is also a cofactor of bovine respiratory disease (BRD), the most important disease in cattle. BRD can lead to life-threatening lung infections, including pneumonia. The goal of this grant is to identify cellular and BoHV-1 genes that induce reactivation from latency in the peripheral nervous system and pharyngeal tonsil. Understanding how reactivation occurs will allow us to develop superior strategies for vaccine viruses that do not respond to stress and reactivate from latency. This will reduce the spread of BoHV-1 and reduce the incidence of BRD.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Goals / Objectives
The goals for this research are summarized in the three Objectives of the grant. These Objectives are summarized below.Objective 1: Analysis of PT cells that harbor viral DNA during latency. Sorting pharyngeal tonsil (PT) single cell suspensions revealed cytokeratin 18+ cells was enriched for bovine alphaherpesvirus 1 (BoHV-1) DNA in latently infected calves. Cytokeratin 18 is expressed on Microfold cells (M-cells), certain epithelial cells, or macrophage like cells. Thus, we hypothesize cytokeratin 18+ PT cells harbor BoHV-1 DNA during latency and support reactivation from latency. Single cell transcriptomic studies of cytokeratin 18+ PT cells will identify cell type(s) harboring viral DNA and differentially expressed cellular genes during reactivation from latency. Objective 2: Analysis of recombinant viruses containing TATA box mutations in the BoHV-1 infected cell protein 0 (bICP0) early promoter or immediate early transcription unit 1 (IEtu1) promoter. The hypothesis of this objective is bICP0 and bICP4 expression trigger reactivation from latency in trigeminal ganglia (TG) and PT. To test this hypothesis, BoHV-1 virus mutants with TATA box mutations in the bICP0 early (E) promoter or IEtu1 promoter will be examined for replication in primary bovine cells and calves.Objective 3. Analysis of cellular transcription factors that cooperate with GR to activate viral gene expression and mediate reactivation from latency. The hypothesis of this objective is glucocorticoid receptor (GR) and the specificity pprotein 1 (Sp1) transcription facotr play crucial roles during viral replication and reactivation from latency in PT and TG. To test this hypothesis, we will examine how GR and Sp1 cooperatively transactivate IEtu1 and bICP0 E promoters, and whether GR stably interacts with Sp1. We will also determine whether certain transcription factors occupy the IEtu1 and bICP0 E promoters during early stages of reactivation of latency.Completing the studies described in these three objectives will provide new insight into the mechanism by which reactivation from latency is triggered in PT and TG. These studies are important because BoHV-1, including all modified live vaccines (MLVs) used in the USA, can reactivate from latency from latency following stressful stimuli, which increases the incidence of bovine respiratory disease(BRD) or abortions.
Project Methods
Objective 1: Analysis of pharyngeal tonsil (PT) cells that harbor viral DNA during latency. To identify cells in PT that are latently infected, we will sort cells and then test each cell type for BoHV-1 DNA. These cells will be explanted and treated with dexamethasone to determine whether BoHv-1 can reactivate from these cells (Objective 1A). For Objective 1B, PT cells that harbor viral DNA, since cell RNA sequencing will be performed to identify viral and cellular genes that are differentially expressed in latently infected cells relative to explant-induced reactivation.Objective 2: Analysis of recombinant viruses containing TATA box mutations in the BoHV-1 infected cell proteins 0 (bICP0) early promoter or IEtu1 promoter. We will test whether mutant viruses replicate as efficiently in cultured cells and in calves. Additional studies will compare the growth properties of the respective mutant viruses to wild-type BoHV-1 and whether they can reactivate from latency in calves.4.3A. Objective 3. Analysis of cellular transcription factors that cooperate with GR to activate viral gene expression and mediate reactivation from latency. Luciferase reporter plasmids containing the bICP0 E promoter and IEtu1 promoter will be utilized to test whether the glucocorticoid receptor and specificity proteins 1 (Sp1) or the Sp3 transcription factor will cooperate with to activate these promoters. Mutagenesis of consensus binding sites for Sp1 and Sp3 will be generated to test how this affects promoter activity. Finally, chromatin immunoprecipitation (ChIP) studies will test whether GR, Sp1, Sp3, and Krüppel like factor 4 (KLF4) or KLF15 occupy the bICP0 E promoter and IEtu1 promoter in transient transfection studies. Finally, ChIP studies be performed using TG from latently infected calves or latently infected calves treated with dexamethasone to trigger reactivation from latency to test whether the cellular transcription factors occupy the bICP0 E promoter or IEtut1 promoter.