Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification.
ABSTRACT Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1x10(-12) dilution of scrapie hamster brain, which contains the equivalent of approximately 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.
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ABSTRACT: The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.Biochemical and Biophysical Research Communications 06/2012; 423(4):770-4. · 2.28 Impact Factor
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ABSTRACT: Prions, infectious agents associated with transmissible spongiform encephalopathy, are primarily composed of the misfolded and pathogenic form (PrPSc) of the host-encoded prion protein. Because PrPSc retains infectivity after undergoing routine sterilizing processes, the cause of bovine spongiform encephalopathy (BSE) outbreaks are suspected to be feeding cattle meat and bone meals (MBMs) contaminated with the prion. To assess the validity of prion inactivation by heat treatment in yellow grease, which is produced in the industrial manufacturing process of MBMs, we pooled, homogenized, and heat treated the spinal cords of BSE-infected cows under various experimental conditions. Prion inactivation was analyzed quantitatively in terms of the infectivity and PrPSc of the treated samples. Following treatment at 140[degree sign]C for 1 h, infectivity was reduced to 1/35 of that of the untreated samples. Treatment at 180[degree sign]C for 3 h was required to reduce infectivity. However, PrPSc was detected in all heat-treated samples by using the protein misfolding cyclic amplification (PMCA) technique, which amplifies PrPSc in vitro. Quantitative analysis of the inactivation efficiency of BSE PrPSc was possible with the introduction of the PMCA50, which is the dilution ratio of 10% homogenate needed to yield 50% positivity for PrPSc in amplified samples. Log PMCA50 exhibited a strong linear correlation with the transmission rate in the bioassay; infectivity was no longer detected when the log PMCA50 of the inoculated sample was reduced to 1.75. The quantitative PMCA assay may be useful for safety evaluation for recycling and effective utilization of MBMs as an organic resource.BMC Veterinary Research 07/2013; 9(1):134. · 1.86 Impact Factor
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ABSTRACT: Transmissible spongiform encephalopathies (TSEs), or prion diseases, represent a unique form of infectious disease based on misfolding of a self-protein (PrP(C)) into a pathological, infectious conformation (PrP(Sc)). Prion diseases of food animals gained notoriety during the bovine spongiform encephalopathy (BSE) outbreak of the 1980s. In particular, disease transmission to humans, to the generation of a fatal, untreatable disease, elevated the perspective on livestock prion diseases from food production to food safety. While the immediate threat posed by BSE has been successfully addressed through surveillance and improved management practices, another prion disease is rapidly spreading. Chronic wasting disease (CWD), a prion disease of cervids, has been confirmed in wild and captive populations with devastating impact on the farmed cervid industries. Furthermore, the unabated spread of this disease through wild populations threatens a natural resource that is a source of considerable economic benefit and national pride. In a worst-case scenario, CWD may represent a zoonotic threat either through direct transmission via consumption of infected cervids or through a secondary food animal, such as cattle. This has energized efforts to understand prion diseases as well as to develop tools for disease detection, prevention, and management. Progress in each of these areas is discussed.ISRN veterinary science. 01/2012; 2012:254739.