Detection and Discrimination of Classical and Atypical L-Type Bovine Spongiform Encephalopathy by Real-Time Quaking-Induced Conversion

Abstract

Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. The real-time quaking-induced conversion (RT-QuIC) test, which is based on prion-seeded fibrillization of recombinant prion protein (rPrP Sen ), is known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Here, we tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrP Sen substrates. Specifically, L-BSE was detected using multiple rPrP Sen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms.T ransmissible spongiform encephalopathies (TSEs), or prion diseases, such as Creutzfeldt-Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE) in cattle, are fatal neurodegenerative disorders characterized by spongiosis, neuronal loss, gliosis, and abnormal deposition of host-encoded prion protein (PrP) in the brain. The infectious agent responsible for TSEs appears to be largely composed of a misfolded and multimeric form of prion protein (PrP Sc ), which is able to induce polymerization and conformational conversion of normal proteasesensitive prion protein (PrP Sen ) into PrP Sc and its partially protease-resistant forms (PrP Res ) (1). BSE and its link to variant Creutzfeldt-Jakob disease (vCJD) in humans have raised important food safety issues. The incidence of classical BSE (C-BSE) has decreased as a result of disease-control programs, such as the ruminant feed ban (2). However, atypical forms of bovine prion disease have been identified in several countries (3). These emerging bovine prion strains can be categorized as H-type BSE (H-BSE) or L-type BSE (L-BSE or bovine amyloidotic spongiform encephalopathy [BASE] when amyloid plaques are present in the brain). These atypical BSE strains are differentiated biochemically by the electrophoretic mobility and glycoform pattern of PrP Res after proteinase K (PK) digestion (4-7). Currently, both of these atypical bovine prion strains, which mainly affect older animals (8), are thought to be sporadic forms of bovine prion diseases (9) and are still rare (less than 100 cases identified worldwide to date).It is suspected that one of these atypical L-BSE or H-BSE strains may have instigated the C-BSE epidemic, as suggested, for example, by the fact that the L-BSE strain can convert to C-BSE following serial passage in mice (5,6,10). Further concerns about the atypical BSE strains have arisen from...

Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

“…We show here that mouse rPrP is better suited for amplification of CWD prions than cervid PrP, indicating that amplification in RT-QuIC does not follow the rules of the species barrier concept, as observed previously [43; 44]. We also found a higher spontaneous conversion rate for cervid rPrP, possibly because of the ‘rigid loop’ structure [45; 46] which makes PrP more aggregation-prone [38].…”

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

“…As noted above, our previous studies demonstrated RTQuIC assays capable of sensitive detection and discrimination of C-BSE and L-BSE using various rPrP Sen substrates (40,47). Here, we describe an ultrasensitive RT-QuIC assay that also detects H-BSE prion seeding activity in H-BSE brain homoge- Ϫ8 within 24 h. To our knowledge, the corresponding sensitivities of animal bioassays and PMCA for the atypical BSE strains have not yet been described, but for C-BSE, our RTQuIC tests appear to be at least as sensitive as bioassays and nearly as sensitive as PMCA (37)(38)(39).…”

“…Previous efforts to improve the practicality and performance of testing for bovine prions led to the adaptation of a highly sensitive and specific assay called real-time quaking-induced conversion (RT-QuIC) for the detection and discrimination of C-BSE and L-BSE (40). RT-QuIC is based upon the prion-seeded assembly of protease-sensitive recombinant PrP C (rPrP Sen ) into thioflavin T (ThT)-positive amyloid fibrils (41,42).…”

Infectious Prions in the Pregnancy Microenvironment of Chronic Wasting Disease-Infected Reeves' Muntjac Deer

“…RT-QuIC provides a rapid methodology for analysis of tissues and fluids containing minute quantities of prions (85,86) and may have utility in quantitative and correlative investigations of early and carrier states of prion diseases and potentially other protein misfolding disorders (86)(87)(88)(89). There is little to no evidence that RT-QuIC generates, or represents, the presence of prion infectivity.…”