Location: Animal Disease Research
Project Number: 2090-32000-042-000-D
Project Type: In-House Appropriated
Start Date: Oct 18, 2021
End Date: Oct 17, 2026
Objective:
Objective 1: Identify genes outside of PRNP that are associated with prion disease.
Objective 1A: Identify and characterize genes outside of PRNP that are associated with Scrapie in North American small ruminants.
Objective 1B: Identify and characterize genes outside of PRNP that are associated with CWD in North American cervids.
Objective 2: Determine the influence of prion genotypes and tissue constituents on the detection of TSE prions.
Objective 2A: Develop improved methods for antemortem detection of Scrapie in small ruminants.
Objective 2B: Determine the influence of genetic variations in the prion protein on the detection and susceptibility of prion infection in small ruminants
Approach:
The goal of Objective 1 is to identify genetic markers outside the prion protein gene (PRNP) associated with prion disease. Variation in PRNP can substantially impact the development of TSEs. Still, only a few PRNP genotypes in small ruminants, and none in cervids, are known to confer strong resistance to prion infection. Studies in several species, including sheep and deer, indicate that genetic factors other than PRNP also impact prion diseases. Therefore, genome-wide association and prediction studies will be performed to identify regions outside of PRNP associated with Scrapie in sheep (Objective 1A) and Chronic Wasting Disease in elk (Objective 1B).
The goal of Objective 2 is to determine the influence of PRNP genotypes and tissue constituents on the detection of prions. The sensitivity of current diagnostic methods is poor compared to bioassay. In contrast, modern protein-misfolding assays known as RT-QuIC and sPMCA can be equivalently sensitive as bioassay but are variably inhibited by tissue factors, including omnipresent blood. To improve the applied assay performance, Objective 2A will test the hypothesis that heme, a significant blood component, is present in various types of diagnostic samples at concentrations that reduce the sensitivity of protein misfolding assays. The inhibitory mechanisms of heme will be investigated using the RT-QuIC assay since its reagents are fully defined. A novel strategy to mitigate assay inhibition through heme-sequestration will be examined in RT-QuIC and sPMCA assays. In addition, the utility of these misfolding assays to detect prions in different species relies on the genotype of the prion protein used as substrate. The RT-QuIC assay uses bacterial recombinant prion protein as substrate. In contrast, sPMCA assay performance depends on prion protein produced in a eukaryotic system, usually as brain homogenate of transgenic mice. Objective 2B aims to overcome the limited genetic representation and animal use of current substrate sources by producing recombinant protein substrate using a scalable baculovirus-insect cell system (BICS). An array of PRNP genotypes will be screened for sensitivity as a substrate for the sPMCA assay in detecting multiple forms of prions from sheep, goats, and cervid species. The BICS recombinant substrates will be used to test the hypotheses (1) that the seeded-conversion profiles of sPMCA using different substrate genotypes can differentiate interspecies transmission of CWD to sheep from naturally occurring forms of scrapie in sheep, and (2) that the PRNP S146 and K222 genotypes found in goats both confer strong resistance to oral infection by classical scrapie prions.
