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ARS Home » Pacific West Area » Pullman, Washington » Animal Disease Research » Research » Research Project #441236

Research Project: Genetic Approaches and Tools to Prevent, Control, and Eradicate Transmissible Spongiform Encephalopathies

Location: Animal Disease Research

2022 Annual Report

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

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.

Progress Report
In support of Objective 1, a collaborative agreement was established with collaborators at the Canadian Food Inspection Agency, Ottawa, Ontario, Canada, to provide ARS researchers at Pullman, Washington, with archival and newly acquired samples from up to 900 Canadian sheep exposed to scrapie and up to 800 Canadian Rocky Mountain elk exposed to chronic wasting disease. The collaborators have readied archival samples for shipment and entered needed animal and herd information as available. In addition, newly acquired case samples from infected populations are being examined by the collaborators and are simultaneously being processed for shipment if suited to the parameters of the planned genomic studies. ARS researchers worked with samples already at the location to further optimize the process of genomic DNA extraction from archival samples and the enzymatic conditioning necessary to produce the high-quality DNA necessary for these studies. Genomic analyses on elk from U.S. wildlife populations have continued using samples provided by collaborators. In support of Objective 2, production of the substrates necessary for use in misfolding assays was begun. Full length bacterial recombinant prion protein genotypes representing the major classical scrapie-susceptible alleles of sheep and goats, and that of the bank vole were produced, characterized, and tested for misfolding activity seeded by classical scrapie prions using the quaking-induced conversion (QuIC) assay. Refinement of bacterial recombinant protein production with and without purification by chromatography continued. The brain of the transgenic (tg) mouse, tg338, serves as a natural substrate suitable for the protein misfolding cyclic amplification (PMCA) assay. Substrate stocks of tg mouse brains were prepared for planned experiments in which the blood in the brain was either removed by transcardial perfusion with saline or left in place. These substrate stocks have been biochemically characterized, including the determination of heme concentration. As expected, perfusion profoundly reduces the presence of heme in the substrate. These substrates will be used to determine if the difference in heme concentration reduces the sensitivity in detecting prion-seeded misfolding activity using PMCA like what has been found to be inhibitory to the QuIC assay. A recombinant form of the bacterial protein, heme acquisition system (Has)A, has been produced, purified, and characterized. Recombinant HasA strongly binds heme and will be used to measure and mitigate the inhibitory effects of heme in misfolding assays. A transgenic mouse line known as tg12 expresses the prion protein of elk and will be used to carry out bioassays for prion infectivity and as a source of brain tissue used as a substrate for the PMCA assay. The newly acquired mice were found to be infected with a highly transmissible bacterial pathogen but were cleared of this nuisance infection through a lengthy process of rederivation. The rederived tg12 mice were bred and have produced offspring from which substrate has been prepared and shown to be sensitive to misfolding activity seeded by chronic wasting disease prions. Experiments to optimize the conditions of PMCA have begun. An important goal of this objective is to produce an array of recombinant prion proteins from eukaryotic cells that include the wide variety of genotypes present in agriculturally important species. The study aims to do this using the commercially scalable baculovirus-insect cell system (BICS). Components for an improved BIC system were obtained and the VRQ allele of sheep was successfully expressed. The recombinant prion protein was characterized and testing of its sensitivity to various prion diseases of sheep and cervids has begun. Efficiencies in the process of incorporating the prion protein are being explored, after which production of the other prion protein sequences of sheep, goats, and cervid species will commence. Work to extract the prion protein alleles of elk has commenced. Transgenic mouse lines infected with tissues from the progeny of Nor98-like scrapie continue to be monitored and processed as end-point incubation times or intercurrent diseases have required. Tissues from goats bearing the GS127 allele, a genotype that prolongs scrapie incubation, have all been processed and biochemically analyzed to confirm transmission but delayed disease kinetics in GS127 goats. In addition, a postmortem examination was conducted on an SS127 goat. Scrapie was observed by immunoassays in the peripheral tissues and brain of this animal, confirming that the S127 allele is not protective against infection but rather only delays the kinetics of infection even in the homozygous state. Tissues from goats bearing the fully susceptible GG127 genotype have been biochemically analyzed and preparation has commenced to using these samples as large sources of brain and lymph node scrapie materials for the planned bioassay and misfolding assay experiments. Goats inoculated with classical scrapie, but which express the NS146 allele of the prion protein, were highly resistant to developing signs of infection even after 12 years post-inoculation. Tissues from these animals have been prepared and inoculated into tg338 mice to determine if any prion infectivity is present. Significant progress was also made in the subordinate project 58-2090-1-001 which endeavors to optimize and validate an RT-QuIC protocol for diagnosing chronic wasting disease in white-tailed deer. A pre-analytical procedure for preparing the rectal mucosal samples was determined and the effect of buffer conditions was established. The optimized procedure and assay were applied to all samples of rectal biopsy tissues provided to us by our collaborator at the Animal Plant Health Inspection Agency, Fort Collins, Colorado. All tissues were tested using two sources of assay substrate, one from our collaborator at the United States Geological Survey, Madison, Wisconsin, and the other from a now commercial source from the University of Minnesota, Minneapolis, Minnesota. The diagnostic performance measures were generally equivalent between these sources of the substrate and were deemed high enough to warrant an inter-laboratory study at six National Animal Health Laboratory Network sites. The ARS researchers at Pullman, Washington, established an online protocol of the optimized RT-QuIC procedure and prepared the uniform aliquots of testing materials. Recently completed, the measures of inter-laboratory agreement and diagnostic accuracies were high but indicate a need for further improvement of implementation at various laboratory sights. These experiments and findings of this subordinate project support our efforts to similarly optimize the RT-QuIC assay for diagnosing classical scrapie by rectal biopsy in small ruminants. In addition, experiments were conducted to optimize the pre-analytical procedure for preparing and testing the retropharyngeal lymph node of white-tailed deer by RT-QuIC. With some surprising differences, the procedures have been optimized and the blinded study for diagnostic accuracy has commenced. The full-length sequence of the coding region of the prion protein in nearly half of these tissues has now been determined. This data will enhance our understanding of factors that might impact the diagnostic performance of this assay. Finally, a statistical analysis of the diagnostic performance of tonsil biopsies from white-tailed deer has been completed. Tonsil biopsy had poor diagnostic sensitivity in cases of early preclinical infection, which was associated with a lower proportion of affected follicles.

1. The placenta of sheep with atypical scrapie is not infectious. Selective breeding has successfully increased the number of sheep having the ARR/ARR prion protein genotype—a genotype highly resistant to the classical forms of naturally transmissible scrapie. Nor98-like scrapie is a sporadic atypical form of scrapie thought to spontaneously arise in the central nervous system of aging sheep, including ARR/ARR sheep. A concern for natural transmission was raised when infectious atypical prions were demonstrated in peripheral nerves, lymphoid tissues, and muscles of infected sheep. ARS researchers at Pullman, Washington, discovered a progressive accumulation of an atypical prion-like protein in the placenta of ARR/ARR sheep years after experimental infection; however, these placental proteins were not found to be infectious. This knowledge supports the epidemiologic evidence that Nor98-like scrapie is a spontaneous disease of sheep with low to no risk of natural transmission. Furthermore, this knowledge supports the continued use of selective breeding for the ARR/ARR genotype to mitigate the occurrence of classical forms of scrapie in sheep.

Review Publications
Piel III, R., McElliott, V.R., Stanton, J.B., Zhuang, D., Madsen-Bouterse, S.A., Hamburg, L.K., Harrington, R.D., Schneider, D.A. 2022. PrP-res in placental tissue following experimental transmission of atypical scrapie in ARR/ARR sheep is not infectious by Tg338 mouse bioassay. PLoS ONE. 17(1). Article e0262766.