Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Cattle are essential food animals and are also raised to produce milk in the United States. Bovine alpha-herpesvirus 1 (BoHV-1) continues to be a signficant problem in dairy and beef cattle. BoHV-1 infects cattle via the ocular, nasal, and/or oral cavity. Sexual transmission of the virus can also occur. Viral growth leads to cell destruction and inflammation in the ocular and nasal cavity, which can trigger secondary bacterial infections and life-threatening pneumonia. Infection also causes fever and short-term immune suppression. As a result of these clinical symptoms, infected animals do not eat or drink very much, which costs the producer money and complicates recovery from infection. BoHV-1 is also an important cofactor of bovine respiratory disease (BRD), the most important disease in cattle. Finally, it is well established that BoHV-1 can induce abortions, in part because the virus gains access to the fetus and replicates to high levels. The focus of this grant is to compare the ability of BoHV-1 to establish, maintain, and reactivate from latency in pharyngeal tonsil relative to sensory neurons in trigeminal ganglia. We will also examine the mechanism by which stress interrupts latency and leads to virus growth and viral transmission; this is referred to as reactivation from latency. A recent publication found that cellular transcription factors, including glucocorticoid receptor and specificity protein 1 (Sp1) or Sp3 cooperatively activates the infected protein 0 (ICP0) early promoter that is a key viral promoter that mediates reactivation from latency. Ongoing studies will identify cell types in pharyngeal tonsil that harbor BoHV-1 during latency and whether these cell populations support viral reactivation from latency. Compelling preliminary studies revealed dendritic cells, micro-fold cells, and natural killer cells contain viral DNA whenthese cells are in the pharyngeal tonsil. Completion of these studies will provide new insight into how reactivation from latency occurs in the pharyngeal tonsil. Understanding these complex viral-host interactions are important because they will provide new strategies designed to developnew therapeutic strategies designed to reduce the incidence of BoHV-1 reactivation from latency. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student,Sankha Hewawasam, was first author of the Virus paper published in 2025. These studies are a crucial part of his dissertation. Dr. El-Mayet, a coauthor on the Virus paper, also contributed to these studies. How have the results been disseminated to communities of interest?I talked aobut these studies at CRWAD in January 2025. Furthermore, a paper published in Viruses (https://doi.org/10.3390/v17020229) was part of the studies proposed in this grant. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Analysis of PT cells that harbor viralDNA during latency.Transcriptomic studies focused on identifying cellular signaling pathways that are differentially expressed during reactivation from latency will be identified. Furthermore, we will test whether viral gene exression during reactivation inDC, Micro-fold cells andNKcells is the same or if their are cell-type specific changes in viral gene expression during reactivation from latency. A manuscript describing these studies will be submitted to an appropriate peer-reviewed journal, J of Virology or PLOS Pathogens. Objective 2: Analysis of recombinant viruses containing TATA box mutations in the bICP0 early promoter or IEtu1 promoter. This bICP0 E TATA box mutant virus wil be repeated and if the results are consistent a manuscript will be submitted. Objective 3. Analysis of cellular transcription factors that cooperate with GR to activate viral gene expression and mediate reactivation from latency.We will complete studies demonstrating GR occupies the IEtu1 and bICP0 E promoters when latently infected calves are treated with dexamethasone for 3 hours. Additional studies will confirm that the histone 3 marker associated with activate chromatin, H3K9 acetylation, also occupiesthese crucial viral promoters during reactivation but not during latency. Finally, we will confirm histone 3 trimethylated at lysine 9, a heterochromatin marker, occupied these viral promoters in TG of latently ifnected calves. A manuscript will be submitted to J Virology describing these studies.

Impacts
What was accomplished under these goals? Objective 1: Analysis of PT cells that harbor viral DNA during latency. Tigeminal ganglia and pahryngeal tonsils latently infected calves and latently calves treated with dexamethasone (DEX) were recently collected. Cell sorting of pharyngeal single cell suspensions were prepared. BoHV-1 DNA was readily detected indendrtitic cells (DC), Micro-fold cells and Natural Killer (NK) cells.Conversely, B and T-cells did not contain detectable levels of viral DNA. Single cell transcriptomic analysis will be performed on DC, Micro-fold cells, and NK cells is in the process to compareviral and cellular gene expression during reactivation from latency. Objective 2: Analysis of recombinant viruses containing TATA box mutations in the bICP0 early promoter or IEtu1 promoter. The hypothesis of this objective is bICP0 and bICP4 expression trigger reactivation from latency in TG and PT. To test this hypothesis, BoHV-1 virus mutants with TATA box mutations in the bICP0 E promoter or IEtu1 promoter will be examined for replication in primary bovine cells and calves. An intial study demonstrated that the bICP0?TATA box mutant grows less efficently than wild-type BoHV-1 and calves infected with the mutant did not exhibit clinical disease. Furthermore, low levels of reactivation occurred in thebICP0?TATA box mutant versus wild-type virus. 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 GR and Sp1 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. A manuscript was recently published that demonstrated GR and Sp1 or Sp3 cooperatively transactivates the BoHV-1 bICP0 and immediate early transcirption unit 1 (IEtu1) promtoers. As denoted above, this paper was published in 2025 in Viruses. Additional studies revealed that the glucocorticoid receptor (GR) occupies thebICP0 and immediate early transcirption unit 1 (IEtu1) promtoers during early stages or reactrivation from latency. Notably, it was also clear that during latency these promoters are silent becuase they are occupied by heterochromatin. When DEX was added to calves to initiate reactivation from latency, the silent heterochromatin was remodeld by GR so transcription could occur. These two viral promtoers drive expression of the two most important viral transcriptional regulators, bICP0 and bICP4.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Hewawasam SG, F.S. El-Mayet, and C. Jones. 2025. Glucocorticoid receptor (GR) and specificity protein 1 (Sp1) or Sp3 transactivate the bovine herpesvirus 1 (BoHV-1) infected cell protein 0 early promoter. Viruses 17(2), 229.