Location: Animal Disease Research
2023 Annual Report
Objectives
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.
Progress Report
This report documents progress in fiscal year (FY) 2023 for project 2090-32000-042-000D, titled, “Genetic Approaches and Tools to Prevent, Control, and Eradicate Transmissible Spongiform Encephalopathies”.
In support of Objective 1, archival and new samples appropriate for genomic analyses were received from collaborators at the Canadian Food Inspection Agency. The samples received include approximately 760 Canadian sheep exposed to scrapie and 160 Canadian Rocky Mountain elk exposed to chronic wasting disease (CWD). Animal and herd data for these samples were also received. Additional newly acquired case samples from infected populations are being examined by the collaborators and processed for shipment if suited to the parameters of the planned genomic studies. The enzymatic process for genomic DNA extraction suitable for analyses was optimized and applied to 165 sheep and 160 elk samples. Most sheep samples, 595, were received as DNA and have been checked for quality and quantity. Genomic analyses of about 900 elk from U.S. wildlife populations have continued using samples provided by collaborators at the National Park Service.
In support of Objective 2A, ARS researchers in Pullman, Washington determined the naturally occurring concentration of heme—a known inhibitor of ultrasensitive prion detection assays, using a panel of tissues from sheep. The heme concentrations ranged from several to hundreds of micromolar (µM). The protein misfolding cyclic amplification (PMCA) assay is a method that relies on brain homogenate as the source of cellular prion protein. Given the omnipresent blood in tissues, ARS researchers tested the performance of PMCA in detecting scrapie prions using brains cleared and not cleared of blood before homogenization. Both homogenates produced equivalent ultrasensitive detection of sheep scrapie despite brain perfusion reducing heme in homogenates from 20 to 2 µM. The assay's sensitivity was dramatically reduced when the heme content of the homogenate was artificially increased to 100 µM, indicating a narrow window before significant PMCA assay inhibition by heme. The inhibitory mechanisms of heme were further investigated using the quaking-induced conversion (QuIC) assay. This essentially bloodless assay does not rely on tissue-derived factors and instead uses bacterial recombinant prion protein as the essential substrate. Blood inhibition was greater than that which could be accounted for by heme bound to hemoglobin and unbound heme, indicating components other than heme also contribute to assay inhibition. There was no evidence that extreme exposure to heme alone directly impacts prions in the tissue. In contrast, heme appeared to interact with two critical components of the QuIC assay reaction mixture: the prion protein substrate and thioflavin T (ThT). Heme dose-dependently precipitated the recombinant prion protein substrate, and a direct interaction of approximately six molecules of recombinant prion protein to each heme molecule was detected by spectrometry. The ThT molecule functions in the reaction mixture as a reporter of fibrillar aggregation of the prion protein. Heme dose-dependently reduced the baseline fluorescence of ThT and its maximum fibril-mediated gain in fluorescence. Experimentation to elucidate these inhibitory mechanisms continues, but simply increasing these two components in the reaction mixture was insufficient to fully restore the assay's sensitivity. As a novel method to mitigate the inhibitory effects of heme, the recombinant form of a bacterial “heme acquisition system” (HasA) protein was produced and characterized. Recombinant HasA sequestered heme from its interaction with cellular prion protein but also precipitated the recombinant substrate. This suggests that using HasA to mitigate the inhibitory effects of heme may be best achieved during tissue preparation rather than in the assay reaction mixture.
In support of Sun-objective 2B, several species and genotypes of cellular prion protein were created to represent the spectrum of sheep, goat, elk, and deer genotypes known or suspected to influence the pathophysiology of scrapie and CWD. Novel modifications to the prion protein were also used in experimentation. Production of recombinant prion protein in bacterial expression systems, the standard substrate source in the QuIC assay, was refined by adapting an on-column refolding and purification protocol. The ability to finely control the refolding process and monitor eluting protein over time helped recover the highest quality fractions. Bacterially produced recombinant prion protein works poorly as a substrate in PMCA. Thus, recombinant prion protein was produced using a eukaryotic cell culture system to supplant the use of animal brains in substrate production for PMCA. The baculovirus-insect cell system (BICS) production system was optimized to produce cultures of insect cells expressing recombinant sheep prion protein. Lysates of the cultured cells were then prepared for use in PMCA. As expected, scrapie prions were detected by PMCA when using brain homogenate substrate made from prion protein-expressing mice but not when made from “null” mice, that is, mice not expressing prion protein. However, the BICS recombinant prion protein failed to substitute as a PMCA substrate, whether used as the cell lysate alone or when added to the null mouse brain homogenate. The reasons for this failure to replicate earlier work have not been determined. In other work, PMCA and QuIC assays are being developed as possible alternatives to a two-mouse line infection model (dual bioassay) previously shown to discriminate scrapie from CWD in small ruminants. PMCA substrates were created from prion-naïve mice using the same two transgenic lines, one expressing a sheep prion protein, the other elk. Scrapie prions derived from small ruminants were not detected by PMCA when using the ‘elk’ substrate. In contrast, CWD prions were detected in samples from elk and deer. The same experiment using the ‘sheep’ substrate is underway. For QuIC, bacterial recombinant substrates were prepared using sheep and mutated hamster prion protein genotypes. Small ruminant scrapie isolates were detected in the QuIC assay using either substrate. In contrast, cervid isolates of CWD were only detected when using the ‘elk’ substrate. These results collectively are consistent with the hypothesis that the metabolic pathways that undoubtedly play a role in the pathogenesis of prion disease in transgenic mice are not required to differentiate small ruminant-origin scrapie prions and cervid-origin CWD prions using benchtop assays. In other support of Objective 2B, tissues of the last second-generation descendants of ewes experimentally infected with Nor98-like scrapie were examined after seven years of living symptom-free. Immunohistochemical examination of brain and peripheral lymphoid tissues did not detect infection, nor has transgenic mouse bioassay detected symptoms of infection. Similarly, symptoms of prion infection in transgenic mice have not been observed after inoculation with tissues from goats bearing the prion genotype NS146 and orally exposed to classical scrapie.
Significant progress was also made in the subordinate project 58-2090-1-001, which endeavors to optimize and validate a QuIC protocol for diagnosing CWD in white-tailed deer. A blind study measured the diagnostic performance of QuIC and a commercial substrate source for testing samples of the medial retropharyngeal lymph node, routinely the first tissue in which early infection can be detected by immunohistochemistry. Compared to the gold standard diagnosis based on immunohistochemical evaluation, the overall diagnostic sensitivity of QuIC on homogenates of this lymph node was very good but somewhat less in cases of early preclinical infection. To understand the role of infected white-tailed deer genotypes and the impact of using an imperfect diagnostic gold standard, the prion protein genotypes of these deer were determined, quantification of immunohistochemically stained tissues by whole-slide image analysis was begun, and diagnostically ‘suspect’ samples are being assessed for prion infectivity by transgenic mouse bioassay.
Accomplishments
1. Tonsil biopsy has limited ability to detect chronic wasting disease during early infection. Strategies to limit the geographic spread of chronic wasting disease (CWD) in North American elk and deer crucially depend on detecting affected stock early in infection. ARS scientists in Pullman, Washington, collaborated with the APHIS-Cervid Health Program to determine the immunohistochemical diagnostic sensitivity of a tonsil biopsy procedure in naturally infected white-tailed deer. Though the biopsy procedure produced samples of sufficient quality for diagnostic evaluation, it failed to reliably detect infected deer during early preclinical infection. The results inform diagnosticians and disease managers about the limitations of this biopsy procedure as a live animal test to identify early cases of CWD infection.
