1) RT-QuIC is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie had not yet been tested. Therefore, in collaboration with Dr. Rohana Dassanayake and colleagues at the USDA, we aimed to (a) evaluate whether prion seeds in brain tissues of goats with scrapie could be detected using RT-QuIC, (b) further optimize RT-QuIC conditions to improve scrapie detection, and (c) compare the performance of the RT-QuIC for the detection of PrPSc to the more commonly used western blot and enzyme-linked immunosorbent assays (ELISA). We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie infected and normal goat brain samples within 15 h. Our findings indicated that the RT-QuIC is at least 10,000-fold more sensitive than the ELISA and western blot assays for the detection of scrapie seeding activity in goat brain samples. Taken together, these findings suggested that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples. 2) Prion diseases of cattle include the classical (C) bovine spongiform encephalopathy (BSE), as well as the atypical H-BSE and L-BSE strains. Although the C- and L-BSE strains can be detected and discriminated by ultrasensitive real time quaking-induced conversion (RT-QuIC) assays, no such test had been described for the detection of H-BSE or the discrimination of each of the major bovine prion strains. We demonstrated an RT-QuIC assay for H-BSE that can detect as little as 10-9 dilutions of brain tissue and neat cerebrospinal fluid from clinically affected cattle. Moreover, comparisons of reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K-treated RT-QuIC reaction products indicated that H-, L- and C -BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis. 3) Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, was first documented nearly fifty years ago in Colorado and Wyoming, and has since spread across North America and to the Republic of Korea. The expansion of this disease makes the development of sensitive diagnostic assays and antemortem sampling techniques crucial for the mitigation of spread; this is especially true in cases of relocation/reintroduction, or prevalence studies in large or protected herds where depopulation may be contraindicated. We collaborated with Dr. Nicholas Haley of the University of Kansas, and other colleagues at Colorado State University, the U.CS. Geological Survey and the National Park Service in their evaluation of the sensitivity of the real-time quaking-induced conversion (RT-QuIC) assay in rectal mucosal associated lymphoid tissue (RAMALT) biopsies and nasal brushings collected antemortem. Antemortem findings were then compared to results from ante- and postmortem samples evaluated using immunohistochemistry (IHC). RAMALT samples were collected from populations of both farmed and free-ranging Rocky Mountain elk (n=316), with nasal brushes collected from a subpopulation of these animals (n=205). We hypothesized the sensitivity of RT-QuIC would be comparable to IHC in RAMALT, and would correspond to postmortem results. We found a high sensitivity through RAMALT testing (77.3%), which was highly correlative between RT-QuIC and IHC. Sensitivity was lower in nasal brushings (34%), though both RAMALT and nasal brush sensitivities were highly dependent on both genotype and obex score. These data suggested that RT-QuIC, like IHC, is a fairly sensitive assay for detection of CWD prions in RAMALT biopsies and other antemortem samples, and with further investigation has potential for large scale and rapid automated testing for CWD diagnosis. 4) The real-time quaking-induced conversion (RT-QuIC) assay for prion seeding activity has been applied to many prion diseases and provides for specific antemortem diagnostic testing. We evaluated RT-QuIC's long-term consistency and varied multiple reaction parameters. Repeated assays of a single scrapie sample using multiple plate readers and recombinant prion protein (rPrP(Sen)) substrates gave comparable results. N-terminal truncated hamster rPrP(Sen) (residues 90-231) hastened both prion-seeded and prion-independent reactions but maintained a clear kinetic distinction between the two. Raising temperatures or shaking speeds accelerated RT-QuIC reactions without compromising specificity. When applied to nasal brushings from Creutzfeldt-Jakob disease patients, higher temperatures accelerated RT-QuIC kinetics, and the use of hamster rPrP(Sen) (90-231) strengthened RT-QuIC responses. Elongation of shaking periods reduced scrapie-seeded reaction times, but continuous shaking promoted false-positive reactions. Furthermore, pH 7.4 provided for more rapid RT-QuIC reactions than more acidic pHs. Additionally, we showed that small variations in the amount of sodium dodecyl sulfate (SDS) significantly impacted the assay. Finally, RT-QuIC performed in multiplate thermoshakers followed by fluorescence readings in separate plate readers enhanced assay throughput economically. Collectively, these results demonstrated improved speed, efficacy and practicality of RT-QuIC assays and highlight variables to be optimized for future applications. 5) We also provided assistance in a project spear-headed by Bruce Chesebro and coworkers to assess early accumulation of PrPSc and prion seeding activity after local stereotactic inoculation of scrapie into the brains of mice. We performed RT-QuIC assays to help them assess the loss and replication, respectively in mice that either lacked or expressed PrPC. 6) We also participated in the writing of two major reviews of the applications of RT-QuIC to the diagnosis of human prions diseases.
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