Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification

Universidad Autónoma de Madrid, Madrid, Madrid, Spain
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2006; 281(46):35245-52. DOI: 10.1074/jbc.M603964200
Source: PubMed


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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Available from: Paula Saá, Dec 09, 2014
    • "In our efforts to detect the minute amounts of amplifiable prions present in fetal and maternal tissues, we used sPMCA, a highly sensitive and specific method of PrP res detection (Saá et al., 2006), superior to both IHC and Western blotting of unamplified material. We proceeded to evaluate the probability of contamination in our technique , as well as the optimal number of rounds of amplification necessary and sufficient to demonstrate the presence of amplification-competent material in each sample. "
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    ABSTRACT: The presence of disease-associated prions in tissues and bodily fluids of chronic wasting disease (CWD)-infected cervids has received much investigation, yet little is known about mother to offspring transmission of CWD. Our previous work demonstrated that mother to offspring transmission is efficient in an experimental setting. To address the question of relevance in a naturally-exposed free-ranging population, we have assessed maternal and fetal tissues derived from 19 elk dam-calf pairs collected from free-ranging Rocky Mountain elk from Northcentral Colorado, a known CWD endemic region. Conventional immunohistochemistry (IHC) identified 3/19 CWD positive dams, whereas a more sensitive assay - the serial protein misfolding cyclic amplification (sPMCA) - detected CWD prion seeding activity (PrPCWD) in 15/19 dams. PrPCWD distribution in tissues was widespread and included the central nervous system (CNS), lymphoreticular system (LRS), reproductive, secretory, excretory and adipose tissues. Interestingly, five of fifteen sPMCA positive dams showed no evidence of PrPCWD in either CNS or LRS, sites typically assessed in diagnosing CWD. Analysis of fetal tissues harvested from the fifteen sPMCA positive dams revealed PrPCWD in 80% of fetuses (12/15), regardless of gestational stage. These findings demonstrate that PrPCWD is more abundant in peripheral tissues of CWD exposed elk than current diagnostic methods suggest, and that transmission of prions from mother to offspring may contribute to the efficient transmission of the CWD in naturally exposed cervid populations.
    No preview · Article · Sep 2015 · Journal of General Virology
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    • "The lack of detection in this article is most likely due to the low sensitive techniques (western blots or ELISA) employed to analyze the presence of PrP Sc . Indeed, as we reported previously, PMCA has a power of detection, which is several millions times higher than western blots or ELISA (Saá et al., 2006). In order to estimate the amount of PrP Sc present in stem and leaves coming from contaminated soil, we performed a quantitative PMCA study, as previously described (Chen et al., 2010). "
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    ABSTRACT: Prions are the protein-based infectious agents responsible for prion diseases. Environmental prion contamination has been implicated in disease transmission. Here, we analyzed the binding and retention of infectious prion protein (PrP(Sc)) to plants. Small quantities of PrP(Sc) contained in diluted brain homogenate or in excretory materials (urine and feces) can bind to wheat grass roots and leaves. Wild-type hamsters were efficiently infected by ingestion of prion-contaminated plants. The prion-plant interaction occurs with prions from diverse origins, including chronic wasting disease. Furthermore, leaves contaminated by spraying with a prion-containing preparation retained PrP(Sc) for several weeks in the living plant. Finally, plants can uptake prions from contaminated soil and transport them to aerial parts of the plant (stem and leaves). These findings demonstrate that plants can efficiently bind infectious prions and act as carriers of infectivity, suggesting a possible role of environmental prion contamination in the horizontal transmission of the disease. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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    • "Many similarities can be found between DNA amplification by PCR and PrP TSE amplification by saPMCA, one of them being the possibility of cross-contamination, which is most likely to take place during sample manipulation for next round preparation in the latter . Due to the ultrasensitive nature of saPMCA, capable of detecting one single infectious unit of PrP TSE (Saá et al., 2006b), stringent precautionary measures need to be put in place to minimize such risk and appropriate negative controls need to be included in each experiment. Interestingly, real time quantitative PCR (RT-qPCR) is widely used in molecular disease diagnosis and it is the method of choice in some cases despite associated specificity issues being reported (Phillips et al., 2009a,b). "
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    ABSTRACT: Transmissible spongiform encephalopathies (TSEs) most commonly known as prion diseases are invariably fatal neurological disorders that affect humans and animals. These disorders differ from other neurodegenerative conformational diseases caused by the accumulation in the brain of misfolded proteins, sometimes with amyloid properties, in their ability to infect susceptible species by various routes. While the infectious properties of amyloidogenic proteins, other than misfolded prion protein (PrPTSE), are currently under scrutiny, their potential to transmit from cell to cell, one of the intrinsic properties of the prion, has been recently shown in vitro and in vivo. Over the decades, various cell culture and laboratory animal models have been developed to study TSEs. These assays have been widely used in a variety of applications but showed to be time consuming and entailed elevated costs. Novel economic and fast alternatives became available with the development of in vitro assays that are based on the property of conformationally abnormal PrPTSE to recruit normal cellular PrPC to misfold. These include the cell-free conversion assay, protein misfolding cyclic amplification (PMCA) and quaking induced conversion assay (QuIC), of which the PMCA has been the only technology shown to generate infectious prions. Moreover, it allows indefinite amplification of PrPTSE with strain-specific biochemical and biological properties of the original molecules and under certain conditions may give rise to new spontaneously generated prions. The method also allows addressing the species barrier phenomena and assessing possible risks of animal-to-animal and animal-to-human transmission. Additionally, its unprecedented sensitivity has made possible the detection of as little as one infectious dose of PrPTSE and the biochemical identification of this protein in different tissues and biological fluids, including blood, cerebral spinal fluid (CSF), semen, milk, urine and saliva during the pre-clinical and clinical phases of the disease. The mechanistic similarities between TSEs and other conformational disorders have resulted in the adaptation of the PMCA to the amplification and detection of various amyloidogenic proteins. Here we provide a compelling discussion of the different applications of this technology to the study of TSEs and other neurodegenerative diseases.
    Full-text · Article · Nov 2014 · Virus Research
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