Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Clinical Science
Non Technical Summary
These studies will provide: (i) improved understanding of the role interferon plays in BTV disease severity in the natural host, (ii) establish novel methodologies to identify host cellular and transcriptional targets as well as methods of detection of virally infected cells, and (iii) establish a resource for collaboration with laboratories whose research involves non-traditional models.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Aim 1. Employ RNA flow cytometry to identify immune cell populations and IFN-γ induction in naïve and BTV-infected sheep. Probes designed for IFN-γ transcripts will be used to determine expression in circulating immune cell populations of BTV-infected sheep. Data validation will be corroborated by ELISA and RT-qPCR . Aim 2. Identify the temporal BTV viral genome burden in longitudinal sheep infections. RNA probes designed against BTV serotype 10 will be used to determine the viral burden within circulating blood cell populations in longitudinal sheep infections.
Project Methods
Animal inoculation and tissue harvest: ?Using a cohort of n=10 sheep, 2 cohorts of n=5/each. One cohort of 5 will be inoculated subcutaneously at the nape of the neck with BTV17. We have previously shown efficacy of infecting a similar cohort of sheep using the same inoculation methods using BTV17 serotype (Figure 1). The remaining 5 animals will be maintained as naïve and will serve as non-infected controls. All animals will be monitored daily for clinical signs of disease including, mouth ulcers, facial edema, salivation, nasal discharge, malaise, and cough. Prior to inoculation, each day after inoculation until day 5, and every 3 days after that, we will bleed the sheep (3 ml/draw) via jugular vein. At the conclusion of the study, the animals will be sacrificed and tissues known to play critical roles in BTV infections will be collected (draining lymph nodes, spleen, lungs, kidney, and oral tissues). All of these procedures have been approved by CSU IACUC protocol #1402.Collection and Evaluation of tissues: The peripheral blood taken from the animals at each time point will be processed that day for several analyses. A portion of the whole blood will be set aside and banked for traditional viremia detection by RT-qPCR. From the remaining blood plasma and peripheral mononuclear blood cells (PMBCs) will be isolated. Plasma will be aliquoted and frozen for later analysis. PBMCs will be processed for analysis by traditional and RNA flow cytometry. The samples will be assayed for cellular markers denoting major classes of cells (CD4, CD8, B, and macrophages). The antibodies used to identify these cell types are the result of the aforementioned costly trial and error, and limit us to fairly superficial grouping. Once more fully established, we aim to utilize RNA flow to gain an understanding of subclasses of interest within each of these groups. The cells will then be processed for RNA Flow using probes targeted to BTV viral RNA genomes, sheep IFN-γ transcript, and sheep actin transcript (housekeeping gene control) following the manufacturer's instructions (ThermoFisher Primeflow kit). Briefly, the cells will be surface stained with the antibodies to determine cell classes. These cells will then be fixed and permeabilized, followed by staining with probes and fluorescent markers for the transcripts of interest (ß-actin, IFN-γ, and BTV-17 genomes). The cells will be washed extensively and analyzed on the CSU flow cytometry core facility Cytek Aurora spectral analyzer. After acquisition, the .fcs files will be analyzed using FlowJo software v10 and compared internally at each time point, and for changes over the duration of the study. The banked whole blood, PBMCs, and serum will be used for virus detection (by RTqPCR), and bulk IFN-γ protein production to compare to previous findings as well as act as a confirmatory assay to out RNA flow assays.