Serial Protein Misfolding Cyclic Amplification Research Articles

Detection of Chronic Wasting Disease Prions in Prairie Soils from Endemic Regions.

Chronic wasting disease (CWD) is a contagious prion disease that affects cervids in North America, Northern Europe, and South Korea. CWD is spread through direct and indirect horizontal transmission, with both clinical and preclinical animals shedding CWD prions in saliva, urine, and feces. CWD particles can persist in the environment for years, and soils may pose a risk for transmission to susceptible animals. Our study presents a sensitive method for detecting prions in the environmental samples of prairie soils. Soils were collected from CWD-endemic regions with high (Saskatchewan, Canada) and low (North Dakota, USA) CWD prevalence. Heat extraction with SDS-buffer, a serial protein misfolding cyclic amplification assay coupled with a real-time quaking-induced conversion assay was used to detect the presence of CWD prions in soils. In the prairie area of South Saskatchewan where the CWD prevalence rate in male mule deer is greater than 70%, 75% of the soil samples tested were positive, while in the low-prevalence prairie region of North Dakota (11% prevalence in male mule deer), none of the soils contained prion seeding activity. Soil-bound CWD prion detection has the potential to improve our understanding of the environmental spread of CWD, benefiting both surveillance and mitigation approaches.

Inter- and intra-species conversion efficacies of Norwegian prion isolates estimated by serial protein misfolding cyclic amplification

Prion diseases, including chronic wasting disease (CWD) in cervids, are fatal neurodegenerative disorders caused by the misfolding of cellular prion proteins. CWD is known to spread among captive and free-ranging deer in North America. In 2016, an outbreak of contagious CWD was detected among wild reindeer in Norway, marking the first occurrence of the disease in Europe. Additionally, new sporadic forms of CWD have been discovered in red deer in Norway and moose in Fennoscandia. We used serial protein misfolding cyclic amplification to study the ability of Norwegian prion isolates from reindeer, red deer, and moose (two isolates), as well as experimental classical scrapie from sheep, to convert a panel of 16 brain homogenates (substrates) from six different species with various prion protein genotypes. The reindeer CWD isolate successfully converted substrates from all species except goats. The red deer isolate failed to convert sheep and goat substrates but exhibited amplification in all cervid substrates. The two moose isolates demonstrated lower conversion efficacies. The wild type isolate propagated in all moose substrates and in the wild type red deer substrate, while the other isolate only converted two of the moose substrates. The experimental classical scrapie isolate was successfully propagated in substrates from all species tested. Thus, reindeer CWD and classical sheep scrapie isolates were similarly propagated in substrates from different species, suggesting the potential for spillover of these contagious diseases. Furthermore, the roe deer substrate supported conversion of three isolates suggesting that this species may be vulnerable to prion disease.

Phospholipid cofactor solubilization inhibits formation of native prions.

Cofactor molecules are required to generate infectious mammalian prions in vitro. Mouse and hamster prions appear to have different cofactor preferences: Whereas both mouse and hamster prions can use phosphatidylethanolamine (PE) as a prion cofactor, only hamster prions can also use single-stranded RNA as an alternative cofactor. Here, we investigated the effect of detergent solubilization on rodent prion formation in vitro. We discovered that detergents that can solubilize PE (n-octylglucoside, n-octylgalactoside, and CHAPS) inhibit mouse prion formation in serial protein misfolding cyclic amplification (sPMCA) reactions using bank vole brain homogenate substrate, whereas detergents that are unable to solubilize PE (Triton X-100 and IPEGAL) have no effect. For all three PE-solubilizing detergents, inhibition of RML mouse prion formation was only observed above the critical micellar concentration (CMC). Two other mouse prion strains, Me7 and 301C, were also inhibited by the three PE-solubilizing detergents but not by Triton X-100 or IPEGAL. In contrast, none of the detergents inhibited hamster prion formation in parallel sPMCA reactions using the same bank vole brain homogenate substrate. In reconstituted sPMCA reactions using purified substrates, n-octylglucoside inhibited hamster prion formation when immunopurified bank vole PrPC substrate was supplemented with brain phospholipid but not with RNA. Interestingly, phospholipid cofactor solubilization had no effect in sPMCA reactions using bacterially expressed recombinant PrP substrate, indicating that the inhibitory effect of solubilization requires PrPC post-translational modifications. Overall, these in vitro results show that the ability of PE to facilitate the formation of native but not recombinant prions requires phospholipid bilayer integrity, suggesting that membrane structure may play an important role in prion formation in vivo.

Conformational diversity in purified prions produced in vitro.

Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level.

Chronic wasting disease prions in mule deer interdigital glands.

Chronic wasting disease (CWD) is a geographically expanding, fatal neurodegenerative disease in cervids. The disease can be transmitted directly (animal-animal) or indirectly via infectious prions shed into the environment. The precise mechanisms of indirect CWD transmission are unclear but known sources of the infectious prions that contaminate the environment include saliva, urine and feces. We have previously identified PrPC expression in deer interdigital glands, sac-like exocrine structures located between the digits of the hooves. In this study, we assayed for CWD prions within the interdigital glands of CWD infected deer to determine if they could serve as a source of prion shedding and potentially contribute to CWD transmission. Immunohistochemical analysis of interdigital glands from a CWD-infected female mule deer identified disease-associated PrPCWD within clusters of infiltrating leukocytes adjacent to sudoriferous and sebaceous glands, and within the acrosyringeal epidermis of a sudoriferous gland tubule. Proteinase K-resistant PrPCWD material was amplified by serial protein misfolding cyclic amplification (sPMCA) from soil retrieved from between the hoof digits of a clinically affected mule deer. Blinded testing of interdigital glands from 11 mule deer by real-time quake-induced conversion (RT-QuIC) accurately identified CWD-infected animals. The data described suggests that interdigital glands may play a role in the dissemination of CWD prions into the environment, warranting future investigation.

BSE can propagate in sheep co-infected or pre-infected with scrapie

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.

Decrease in Skin Prion-Seeding Activity of Prion-Infected Mice Treated with a Compound Against Human and Animal Prions: a First Possible Biomarker for Prion Therapeutics

Previous studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

Sensitive protein misfolding cyclic amplification of sporadic Creutzfeldt\u2013Jakob disease prions is strongly seed and substrate dependent

Unlike variant Creutzfeldt–Jakob disease prions, sporadic Creutzfeldt–Jakob disease prions have been shown to be difficult to amplify in vitro by protein misfolding cyclic amplification (PMCA). We assessed PMCA of pathological prion protein (PrPTSE) from 14 human sCJD brain samples in 3 substrates: 2 from transgenic mice expressing human prion protein (PrP) with either methionine (M) or valine (V) at position 129, and 1 from bank voles. Brain extracts representing the 5 major clinicopathological sCJD subtypes (MM1/MV1, MM2, MV2, VV1, and VV2) all triggered seeded PrPTSE amplification during serial PMCA with strong seed- and substrate-dependence. Remarkably, bank vole PrP substrate allowed the propagation of all sCJD subtypes with preservation of the initial molecular PrPTSE type. In contrast, PMCA in human PrP substrates was accompanied by a PrPTSE molecular shift during heterologous (M/V129) PMCA reactions, with increased permissiveness of V129 PrP substrate to in vitro sCJD prion amplification compared to M129 PrP substrate. Combining PMCA amplification sensitivities with PrPTSE electrophoretic profiles obtained in the different substrates confirmed the classification of 4 distinct major sCJD prion strains (M1, M2, V1, and V2). Finally, the level of sensitivity required to detect VV2 sCJD prions in cerebrospinal fluid was achieved.

Early preclinical detection of prions in the skin of prion-infected animals

A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To determine the feasibility of using skin for preclinical diagnosis, here we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detects skin PrPSc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay reveals earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263K-inoculated animals, mock-inoculated animals show detectable skin/brain PrPSc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease.

In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJDV180I) have been found to share a unique pathological prion protein (PrPSc) that lacks the protease-resistant PrPSc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrPSc from their cellular PrP (PrPC). To investigate the seeding activity of these unique PrPSc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrPC substrates. We observed that the seeding of PrPSc from VPSPr or fCJDV180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrPSc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrPSc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrPC from the mixture of normal TgMM and Tg mouse brain expressing PrPV180I mutation (Tg180) but not TgV180I alone was converted into PrPSc by seeding with the VPSPr or fCJDV180I. The RT-QuIC seeding activity of PrPSc from VPSPr and fCJDV180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrPSc does not affect the glycoforms of in vitro newly amplified PrPSc.

Preserving prion strain identity upon replication of prions in vitro using recombinant prion protein

Last decade witnessed an enormous progress in generating authentic infectious prions or PrPSc in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrPSc with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrPSc gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate. Up to now, it remains unclear whether prion strain identity can be preserved upon replication in rPrP. The current study reports that faithful replication of hamster strain SSLOW could be achieved in vitro using rPrP as a substrate. We found that a mixture of phosphatidylethanolamine (PE) and synthetic nucleic acid polyA was sufficient for stable replication of hamster brain-derived SSLOW PrPSc in serial Protein Misfolding Cyclic Amplification (sPMCA) that uses hamster rPrP as a substrate. The disease phenotype generated in hamsters upon transmission of recombinant PrPSc produced in vitro was strikingly similar to the original SSLOW diseases phenotype with respect to the incubation time to disease, as well as clinical, neuropathological and biochemical features. Infrared microspectroscopy (IR-MSP) indicated that PrPSc produced in animals upon transmission of recombinant PrPSc is structurally similar if not identical to the original SSLOW PrPSc. The current study is the first to demonstrate that rPrP can support replication of brain-derived PrPSc while preserving its strain identity. In addition, the current work is the first to document that successful propagation of a hamster strain could be achieved in vitro using hamster rPrP.

Prion replication environment defines the fate of prion strain adaptation

The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrPC and donor PrPSc as well as strain-specific structure of PrPSc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrPSc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.

Cross-seeding of prions by aggregated α-synuclein leads to transmissible spongiform encephalopathy.

Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer’s, Parkinson’s, Huntington’s, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK)-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.

An in vitro model for assessing effective scrapie decontamination

Scrapie infectivity enters the environment via a multiplicity of routes from infected animals. Environmentally associated scrapie persists on farms when infected animals have been removed and is particularly resistant to disinfection. Infectivity within the farm is not adequately removed by current recommended guidelines for farm decontamination. We describe an in vitro method for modelling decontamination, specifically the removal of scrapie prions from the surface of concrete fomites within buildings that have housed scrapie infected animals. Concrete that had been spiked with low amounts of a diluted scrapie positive brain homogenate was sampled before and after decontamination. Extracts were used to seed a semi-quantitative serial protein misfolding cyclic amplification assay (sPMCA). We demonstrate that methods currently recommended for prion decontamination result in inadequate reduction of prion seeding activity within this in vitro assay. Effective treatment was achieved using repeat dosing of surfaces with 20,000ppm available chlorine for 4h.

In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

ABSTRACTThe in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrPSc to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrPSc from 3 distinct experimental isolates of H-BSE, could detect PrPSc in as little as 1×10−12 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrPSc with distinct protease K-resistance and protease K-resistant PrPSc molecular weights.

Evidence of scrapie transmission to sheep via goat milk.

BackgroundPrevious studies confirmed that classical scrapie can be transmitted via milk in sheep. The current study aimed to investigate whether scrapie can also be transmitted via goat milk using in vivo (new-born lambs fed milk from scrapie-affected goats due to the unavailability of goat kids from guaranteed scrapie-free herds) and in vitro methods (serial protein misfolding cyclic amplification [sPMCA] on milk samples).ResultsIn an initial pilot study, new-born lambs of two different prion protein gene (PRNP) genotypes (six VRQ/VRQ and five ARQ/ARQ) were orally challenged with 5 g brain homogenate from two scrapie-affected goats to determine susceptibility of sheep to goat scrapie. All sheep challenged with goat scrapie brain became infected based on the immunohistochemical detection of disease-associated PrP (PrPsc) in lymphoid tissue, with an ARQ/ARQ sheep being the first to succumb. Subsequent feeding of milk to eight pairs of new-born ARQ/ARQ lambs, with each pair receiving milk from a different scrapie-affected goat, resulted in scrapie in the six pairs that received the largest volume of milk (38–87 litres per lamb), whereas two pairs fed 8–9 litres per lamb, and an environmental control group raised on sheep milk from healthy ewes, did not show evidence of infection when culled at up to 1882 days of age. Infection in those 12 milk recipients occurred regardless of the clinical status, PrPsc distribution, caprine arthritis-encephalitis virus infection status and PRNP polymorphisms at codon 142 (II or IM) of the donor goats, but survival time was influenced by PRNP polymorphisms at codon 141. Serial PMCA applied to a total of 32 milk samples (four each from the eight donor goats collected throughout lactation) detected PrPsc in one sample each from two goats.ConclusionsThe scrapie agent was present in the milk from infected goats and was able to transmit to susceptible species even at early preclinical stage of infection, when PrPsc was undetectable in the brain of the donor goats. Serial PMCA as a PrPsc detection method to assess the risk of scrapie transmission via milk in goats proved inefficient compared to the bioassay.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0807-4) contains supplementary material, which is available to authorized users.

Reversible off and on switching of prion infectivity via removing and reinstalling prion sialylation.

The innate immune system provides the first line of defense against pathogens. To recognize pathogens, this system detects a number of molecular features that discriminate pathogens from host cells, including terminal sialylation of cell surface glycans. Mammalian cell surfaces, but generally not microbial cell surfaces, have sialylated glycans. Prions or PrPSc are proteinaceous pathogens that lack coding nucleic acids but do possess sialylated glycans. We proposed that sialylation of PrPSc is essential for evading innate immunity and infecting a host. In this study, the sialylation status of PrPSc was reduced by replicating PrPSc in serial Protein Misfolding Cyclic Amplification using sialidase-treated PrPC substrate and then restored to original levels by replication using non-treated substrate. Upon intracerebral administration, all animals that received PrPSc with original or restored sialylation levels were infected, whereas none of the animals that received PrPSc with reduced sialylation were infected. Moreover, brains and spleens of animals from the latter group were completely cleared of prions. The current work established that the ability of prions to infect the host via intracerebral administration depends on PrPSc sialylation status. Remarkably, PrPSc infectivity could be switched off and on in a reversible manner by first removing and then restoring PrPSc sialylation.

Protein Misfolding Cyclic Amplification Cross-Species Products of Mouse-Adapted Scrapie Strain 139A and Hamster-Adapted Scrapie Strain 263K with Brain and Muscle Tissues of Opposite Animals Generate Infectious Prions

Transmission of prions between mammalian species is limited by a "species barrier," a biological effect involving an increase in incubation period to decrease the percentage of animals succumbing to disease. In this study, we used protein misfolding cyclic amplification (PMCA) technique, which accelerates the conversion of prion proteins in vitro. Direct interspecies PMCA involving 144 cycles confirmed that both mouse-adapted scrapie strain 139A and hamster-adapted 263K could use brain homogenates of opposite species to form proteinase K (PK)-resistant PrP proteins (PrPres). Newly formed interspecies prions could stably propagate themselves in subsequent serial PMCA passages. The two types of PMCA-generated cross-species PrPres changed their glycosylation profiles, which was similar to that observed during interspecies infection by the mouse agent 139A in vivo. These profiles were distinct from individual seeded PrPSc and possessed properties of new hosts. Comparative analysis with respect to PK resistance showed no significant diversity between PMCA-PrPres and native PrPSc or between brain and muscle PrPres. However, PrPres from the relatively early cycles of serial PMCA showed lower PK resistance than those from later cycles. Inoculation of these PMCA products amplified with homogeneous or heterogeneous brain tissues (cross-species products) induced experimental transmissible spongiform encephalopathies. These results suggested that PMCA can help prion strains to overcome species barrier and to propagate efficiently both in vitro and in vivo.

Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep

BackgroundClassical scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Our previous bioassay studies in lambs revealed that scrapie prions could be detected in association with peripheral blood monocular cells (PBMC), B lymphocytes and platelet-rich plasma fractions. In the present study, bioassay in lambs was again used to determine if scrapie prions are associated with the other two subsets of PBMC, monocytes and T lymphocytes.ResultsPBMC, monocytes and T lymphocytes were isolated from two preclinically affected VRQ/VRQ sheep naturally infected with classical ovine scrapie and intravenously transfused into VRQ/VRQ lambs post-weaning. As determined using standard immunohistochemistry for scrapie, abnormal isoforms of prion protein were detected in lymphoid tissues of lambs inoculated with PBMC (4/4 recipient lambs), monocytes (2/5) and T lymphocytes (1/4). Prion protein misfolding activity was detected by serial protein misfolding cyclic amplification (sPMCA) in PBMC from monocyte and T lymphocyte recipient sheep in agreement with antemortem rectal biopsy results, but such prion protein misfolding activity was not detected from other recipients.ConclusionsThese findings show that scrapie prions are associated with monocytes and T lymphocytes circulating in the peripheral blood of sheep naturally infected with classical scrapie. Combined with our previous findings, we can now conclude that all three major subsets of PBMC can harbor prions during preclinical disease and thus, present logical targets for development of a sensitive assay to detect scrapie prions. In this regard, we have also demonstrated that sPMCA can be used to detect scrapie prions associated with PBMC.

Subcritical Water Hydrolysis Effectively Reduces the In Vitro Seeding Activity of PrPSc but Fails to Inactivate the Infectivity of Bovine Spongiform Encephalopathy Prions

The global outbreak of bovine spongiform encephalopathy (BSE) has been attributed to the recycling of contaminated meat and bone meals (MBMs) as feed supplements. The use of MBMs has been prohibited in many countries; however, the development of a method for inactivating BSE prions could enable the efficient and safe use of these products as an organic resource. Subcritical water (SCW), which is water heated under pressure to maintain a liquid state at temperatures below the critical temperature (374°C), exhibits strong hydrolytic activity against organic compounds. In this study, we examined the residual in vitro seeding activity of protease-resistant prion protein (PrPSc) and the infectivity of BSE prions after SCW treatments. Spinal cord homogenates prepared from BSE-infected cows were treated with SCW at 230–280°C for 5–7.5 min and used to intracerebrally inoculate transgenic mice overexpressing bovine prion protein. Serial protein misfolding cyclic amplification (sPMCA) analysis detected no PrPSc in the SCW-treated homogenates, and the mice treated with these samples survived for more than 700 days without any signs of disease. However, sPMCA analyses detected PrPSc accumulation in the brains of all inoculated mice. Furthermore, secondary passage mice, which inoculated with brain homogenates derived from a western blotting (WB)-positive primary passage mouse, died after an average of 240 days, similar to mice inoculated with untreated BSE-infected spinal cord homogenates. The PrPSc accumulation and vacuolation typically observed in the brains of BSE-infected mice were confirmed in these secondary passage mice, suggesting that the BSE prions maintained their infectivity after SCW treatment. One late-onset case, as well as asymptomatic but sPMCA-positive cases, were also recognized in secondary passage mice inoculated with brain homogenates from WB-negative but sPMCA-positive primary passage mice. These results indicated that SCW-mediated hydrolysis was insufficient to eliminate the infectivity of BSE prions under the conditions tested.