Non Technical Summary
Our research project focuses on a disease in cattle called Bovine Viral Diarrhea Virus (BVDV),which isa big problem for farmers because it affects the health and immune system of young cows, known as calves. This virus comes in two forms: one that causes persistent infections called non-cytopathic (NCP) and another called cytopathic (CP), which cannot cause persistent infections and is therefore used in vaccines to prevent the disease. However, both types of the virus can damagean importantorgan in the calves called the thymus.The thymus is crucial because it helps develop specialcells called T-cells, which areakeypart of the immune system that fights off infections.When the thymus is damaged, it doesn't workproperly, and asa result, the calves may not respond well to infections or even vaccines. Our preliminary studies have shown that after being infected with BVDV, the thymus might become smaller andpermanently damaged, affecting the calf's ability to fight other diseases.This damage is seen in howcertainimmune cells, called CD8 cells, don'trespondas well as they should. Another problem is with something called the T-cell receptor (TCR) diversity. TCRs are on the surface of T-cells and help them recognize and fight different infections. If there isn't enough diversity in these receptors, the calves mightnot be ableto fight offinfectionseffectively. The goal of our project isto study these effects in more detail to understand how BVDV affects the thymus and T-cell receptors so we canbetterprotect calves from this virus and improve vaccines.This research could lead to better health management in cattle and ensure that vaccines are effective, helping farmers maintain healthy animals.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3440	1090	50%
311	3440	1101	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3440 - Meat, dairy cattle;

Field Of Science
1090 - Immunology; 1101 - Virology;

Goals / Objectives
Assess T-cell receptor (TCR) Repertoire Diversity Post-BVDV Infection in calves.Thisinvolves detailed profiling and comparison of the T-cell receptor (TCR) repertoire in calves infected with BVDV. The focus will be on analyzing how the virus affects the diversity of TCRs, which are crucial for recognizing and responding to pathogens. This will include sequencing the TCRs from different T-cell populations (naive, effector, and memory T-cells) to assess changes in diversity and clonality post-infection. The longitudinal study will track changes in the TCR repertoire over time from the acute phase through the recovery phase. This will provide insights into the dynamics of TCR diversity following viral infections and its potential impact on long-term immunity.

Project Methods
The study aims to characterize the α/β TCR repertoire of CD4 and CD8 T-cells in naïve and antigen-experienced (effector and memory) cells in calves exposed to BVDV and after a subsequent IDV infection. We will use the peripheral blood mononuclear cells (PBMCs) samples previously collected and cryopreserved toward this goal. PBMC collected from calves on days post BVDV infection 0, 21 (IDV day 0), and 42 (IDV day 21) will be used. Cells will be sorted at our core facility to obtain pure populations of the CD4 and CD8 naïve and effector plus memory cells. Our panel will include: live/dead reagent (Invitrogen), anti-bovine CD4 (ILA11), CD8 (BAQ111A), and CD45RO (ILA16) (all from the Washington State University MAb Center) and anti-human CCR7 (3D12, Invitrogen). Using this strategy, we will be able to sort CD4 T-cells (naïve, and not naïve-i.e., effector plus memory cells) as well as CD8 T-cells (same phenotypes). All antibodies required for the flow cytometry assays are commercially available, and our group has previously optimized protocols. The RNA from T-cell subsets was purified using PureLink RNA Mini Kit. RNA will be extracted using an automated benchtop extraction method (Promega). Specific custom probes for each of the TCR genes (α, β) will be designed based on the annotated TCR sequences from the ImMunoGeneTics information system.Prepared cDNA will be used for the library preparation using SMARTerTCR Kit (Takara). This preparation includes rapid amplification of cDNA ends, enabling complete sequencing of the transcript's V(D)J variable regions. The expected output is over 1M total reads/sample (300bp, paired reads). Sequence quality will be accessed with Fastq, and low-quality sequences, including Phred-type Q-score < 20 (<99% base call accuracy), will be removed. Sequence analysis will be conducted using the MixCR pipeline in MedGenomes's proprietary MAnGO bioinformatics platform to retrieve the full-length TCR. The homogeneity test of variances will initially analyze data within groups. A P-value < 0.05 will be considered statistically significant. Analyses will be performed using GraphPad Prism software.