Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains

Orrú, Christina D.; Groveman, Bradley R.; Raymond, Lynne D.; Hughson, Andrew G.; Nonno, Romolo; Zou, Wen-Quan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron

doi:10.1371/journal.ppat.1004983

Cited by 147 publications

(184 citation statements)

References 47 publications

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“…Briefly, to compensate for differences between the fluorescence plate readers, data sets from replicate wells were averaged and normalized to a percentage of the maximal fluorescence response of the instrument, and the obtained values were plotted against the reaction times. Samples were judged to be RT-QuIC positive using previously described criteria (47). The data are displayed as the averages and standard deviations of the results from four technical replicates, except where indicated.…”

Section: Methodsmentioning

confidence: 99%

“…Sen conversion products and immunoblotting were performed as previously described (47). RT-QuIC reaction products were collected from the bottom of plates by extensive scraping and pipetting using the same tip for all replicate wells and treated with 10 g/ml PK for 1 h at 37°C with orbital shaking at 400 rpm to leave PKresistant recombinant PrP (rPrP Res ) products.…”

Section: Pk Digestion Of Rt-quic Products and Western Blot Analysis mentioning

confidence: 99%

“…2). In contrast, reaction mixtures seeded with the same dilution of brain samples from C-BSE-infected cattle gave much longer lag phases of between 24 and 40 h, while L-BSE reactions consistently displayed intermediate lag phases of 8 to 13 h. For one C-BSE sample, when reaction mixtures were seeded with 10 Ϫ5 dilutions of brain tissue, RT-QuIC kinetics were only ϳ20% faster than in an uninfected negative-control brain homogenate, which started to give spontaneous and prion-independent positive fluorescence after 50 h. The 50-h reaction time therefore marked the point at which it became difficult to clearly discriminate prion-seeded reactions from spontaneously positive reactions; this point is known to vary with substrate, reaction conditions, and sample matrix effects (41,47,51,52). These results indicated that with RT-QuIC using the BV rPrP Sen substrate, H-BSE was consistently more rapidly detectable than with either C-BSE or L-BSE at the same concentration of brain homogenate from clinically affected animals.…”

Section: Pk Digestion Of Rt-quic Products and Western Blot Analysis mentioning

confidence: 99%

“…Another RT-QuIC-based approach to discriminating prion strains is the comparison of the profile of PK-resistant products of RT-QuIC reactions using the BV rPrP Sen 23-230, M 109 substrate by Western blotting (47). We tested this approach by comparing the products of reaction mixtures seeded with brain samples from H-, L-, and C-BSE-affected cattle (Fig.…”

Section: -230 and Sh Rprpmentioning

confidence: 99%

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Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

et al. 2016

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Many mammalian species can be afflicted with prion diseases or transmissible spongiform encephalopathies. Prion diseases are neurodegenerative, transmissible, untreatable, and fatal (reviewed in references 1 and 2). The underlying pathogenesis of prion diseases involves the conversion of the host's normal prion protein, PrP C , to abnormal forms that are usually more protease resistant, multimeric, and insoluble. The multimeric and insoluble forms have been generically called PrP Sc (PrP-scrapie), or more functionally PrP Res (protease resistant) and PrP D (disease associated). At least some of these forms comprise the transmissible agent or prion (3-10). Multiple prion strains can be propagated within a given host species, giving consistently different clinical, pathological, and molecular phenotypes (11-16).The first prion disease to be recognized in cattle was classical bovine spongiform encephalopathy (C-BSE). C-BSE was likely caused primarily by widespread prion contamination of cattle feed (17). After peaking in the early 1990s, the incidence of C-BSE has now been greatly reduced by regulatory measures that limit its horizontal spread. C-BSE is the only known zoonotic prion disease, having caused variant Creutzfeldt-Jakob disease (vCJD) in humans who presumably consumed contaminated beef. Although new clinical cases of vCJD are rare (http://www.cjd.ed.ac .uk/documents/figs.pdf), a recent survey of appendices in the United Kingdom suggests a high incidence of subclinical vCJD infections, at ϳ1:2,000 of the population born between 1941 and 1985 (18).Since the C-BSE epidemic, two atypical strains of BSE, H-type BSE (H-BSE) and L-type BSE (L-BSE), have also been identified in cattle. PrPRes is usually composed of a mixture of glycosylated and unglycosylated molecules, and the various BSE strains can be differentiated biochemically using Western blotting of postmortem brain tissue samples by comparing the proteinase K (PK)-treated PrPRes banding patterns (19)(20)(21)(22). The H and L types of BSE are classified by their respective high and low apparent molecular masses of the unglycosylated PrP Res band. These atypical BSE strains, which are rare (Ͻ100 cases identified worldwide), tend to affect older animals (23) and appear to represent sporadic forms of bovine prion diseases (24). Despite the apparent rarity of the various types of BSE, the facts that H-and L-BSE appear to arise spontaneously and have distinct transmissibilities (20, 25-32) make it important to be able to detect and differentiate them to reduce the risk of transmission to cattle or other species, such as humans.Several in vitro methods have been developed to detect C-, L-, and H-BSE. Commercially available rapid immunochemical tests for PrP D can, in the best cases, give positive responses from 10 Ϫ3 to 10 Ϫ4 dilutions of postmortem brain tissues with high levels of PrP D (33, 34). Immunoblotting for PrP Res can detect BSE-infected tissues with similar sensitivity and also discriminate between the bovine strains based on the relative electro...

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Section: Methodsmentioning

confidence: 99%

Section: Pk Digestion Of Rt-quic Products and Western Blot Analysis mentioning

confidence: 99%

Section: Pk Digestion Of Rt-quic Products and Western Blot Analysis mentioning

confidence: 99%

Section: -230 and Sh Rprpmentioning

confidence: 99%

See 2 more Smart Citations

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

et al. 2016

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“…In the final, recently described application of the RT-QuIC to be presented, recombinant full-length bank vole PrP (rec-bvPrP) was found to be a "universal" substrate, including for previous strains unable to be amplified by RT-QuIC and for differentiating certain animal and human prion strains 22) . Rec-bvPrP allowed RT-QuIC amplification of PrP D seeds from 28 different prion strains derived from humans, cattle, sheep, cervids and rodents, as well as facilitated discrimination of classical and atypical L-type BSE, classical and atypical Nor98 type scrapie and sporadic and variant CJD.…”

Section: Emerging Uses Of Rt-quicmentioning

confidence: 99%

RT-QuIC Assays in Humans … and Animals.

Collins

Sarros

2016

Food Safety

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Prion diseases are neurodegenerative diseases affecting both humans and animal species. The phenotypic spectrum is broad and includes Creutzfeldt-Jakob disease (CJD) and its variant zoonotic form (vCJD) in humans, while in animals, scrapie of sheep and goats, bovine spongiform encephalopathy and chronic wasting disease of deer, elk and moose are naturally occurring forms. Transmission and pathogenesis appear causally linked to the misfolding of the normal form of the prion protein (PrP C ) into disease associated conformers (PrP D ), the latter enriched in β-strand secondary structure. Over the past 10 years two protein amplification techniques, the protein misfolding cyclic amplification (PMCA) assay and real-time quaking induced conversion (RT-QuIC) assay have been developed and successfully deployed in prion biology across a range of scientific and clinical applications, including generation of de novo prions, quantitation of prion infectivity and ultra-sensitive detection of PrP D . While PMCA utilises sonication to facilitate protein amplification, RTQuIC employs vigorous shaking to achieve this outcome, with both techniques sharing the ability to amplify miniscule quantities of PrP D seed present in various tissues and body fluids to levels detectable using routine biochemical methods. The enhanced specificity of the RT-QuIC for detection of PrP D in cerebrospinal fluid (CSF) has spawned international collaborations to rigorously assess and validate the assay for clinical diagnostic purposes. In parallel with collaborative CSF validation studies have been successful efforts to refine the RT-QuIC allowing its use for more accessible body fluids or tissues such as urine and nasal brushings, as well as promote higher sample throughput, shorten assay times and offer accurate quantification of PrP D even at levels below those detectable by animal bioassays. Animal studies support the generic capacity of the RT-QuIC for PrP D detection, underpinning the utility of this assay for studying prion disease and the high likelihood of inter-convertibility of technical refinements for human and animal use.

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Diagnosing Sporadic Creutzfeldt-Jakob Disease by the Detection of Abnormal Prion Protein in Patient Urine

et al. 2016

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Importance CJD is a fatal neurodegenerative disorder associated with the accumulation of infectious abnormal PrP through a mechanism of templated misfolding. A recent report has described the detection of abnormal PrP in vCJD patient urine using protein misfolding by cylic amplification, which was apparently absent in the more common sporadic form of CJD (sCJD). A non-invasive diagnostic test could improve early diagnosis of sCJD, and by screening donations, mitigate the potential risks of prion transmission through human urine-derived pharmaceuticals. Here we describe the adaptation of the direct detection assay, developed originally as a blood test for vCJD, for the detection of disease-associated PrP in urine samples from sCJD patients. Objectives To determine the feasibility of sCJD diagnosis by adaptation of an established vCJD diagnostic blood test to urine. Design Retrospective, cross sectional study. Setting Urine samples obtained during the MRC PRION-1 trial, the National Prion Monitoring Cohort study, and/or referred to the National Prion Clinic or Dementia Research Centre at the National Hospital for Neurology and Neurosurgery in the United Kingdom. Participants Anonymised urine samples from healthy non-neurological control subjects (n=91), patients with non-prion neurodegenerative diseases (n=34) and patients with prion disease (n=37) of which 20 were sCJD. Main Outcome Measure Presence of sCJD infection determined by an assay that captures, enriches, and detects disease-associated prion protein isoforms. Results The assay’s specificity for prion disease was 100% [CI: 97% to 100%] with no false positive reactions from 125 controls, including 34 from a range of neurodegenerative diseases. In contrast to a previous study which used a different methodology, sensitivity to vCJD infection was low with only 1 out of 13 patients testing positive whilst sensitivity to sCJD was unexpectedly high at 40% [CI:19-64%]. Conclusions We determined 40% of sCJD urine samples as positive. This is the first demonstration of an assay that can detect sCJD infection in urine or any target analyte outside of the central nervous system. Urine detection could allow the development of rapid, molecular diagnostics for sporadic CJD and has implications for other neurodegenerative diseases where disease-related assemblies of misfolded proteins might also be present in urine.

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Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains

Cited by 147 publications

References 47 publications

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

RT-QuIC Assays in Humans … and Animals.

Diagnosing Sporadic Creutzfeldt-Jakob Disease by the Detection of Abnormal Prion Protein in Patient Urine

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