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      Highly Infectious Prions Generated by a Single Round of Microplate-Based Protein Misfolding Cyclic Amplification

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          ABSTRACT

          Measurements of the presence of prions in biological tissues or fluids rely more and more on cell-free assays. Although protein misfolding cyclic amplification (PMCA) has emerged as a valuable, sensitive tool, it is currently hampered by its lack of robustness and rapidity for high-throughput purposes. Here, we made a number of improvements making it possible to amplify the maximum levels of scrapie prions in a single 48-h round and in a microplate format. The amplification rates and the infectious titer of the PMCA-formed prions appeared similar to those derived from the in vivo laboratory bioassays. This enhanced technique also amplified efficiently prions from different species, including those responsible for human variant Creutzfeldt-Jakob disease. This new format should help in developing ultrasensitive, high-throughput prion assays for cognitive, diagnostic, and therapeutic applications.

          IMPORTANCE

          The method developed here allows large-scale, fast, and reliable cell-free amplification of subinfectious levels of prions from different species. The sensitivity and rapidity achieved approach or equal those of other recently developed prion-seeded conversion assays. Our simplified assay may be amenable to high-throughput, automated purposes and serve in a complementary manner with other recently developed assays for urgently needed antemortem diagnostic tests, by using bodily fluids containing small amounts of prion infectivity. Such a combination of assays is of paramount importance to reduce the transfusion risk in the human population and to identify asymptomatic carriers of variant Creutzfeldt-Jakob disease.

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          Most cited references54

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          An analytical solution to the kinetics of breakable filament assembly.

          We present an analytical treatment of a set of coupled kinetic equations that governs the self-assembly of filamentous molecular structures. Application to the case of protein aggregation demonstrates that the kinetics of amyloid growth can often be dominated by secondary rather than by primary nucleation events. Our results further reveal a range of general features of the growth kinetics of fragmenting filamentous structures, including the existence of generic scaling laws that provide mechanistic information in contexts ranging from in vitro amyloid growth to the in vivo development of mammalian prion diseases.
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            Generating a prion with bacterially expressed recombinant prion protein.

            The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the attributes of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in approximately 130 days and reached the terminal stage of disease in approximately 150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease; these findings confirmed that the mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.
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              Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.

              The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).
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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                31 December 2013
                Jan-Feb 2014
                : 5
                : 1
                : e00829-13
                Affiliations
                [ a ]INRA (Institut National de la Recherche Agronomique), UR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France
                [ b ]Franklab, Montigny-le-Bretonneux, France
                [ c ]UMR INRA ENVT 1225, Interactions Hôtes Agents Pathogènes, École Nationale Vétérinaire de Toulouse, Toulouse, France
                Author notes
                Address correspondence to Vincent Béringue, vincent.beringue@ 123456jouy.inra.fr , or Mohammed Moudjou, mohammed.moudjou@ 123456jouy.inra.fr .

                M.M. and P.S. contributed equally to this work.

                Invited Editor Byron Caughey, NIH/NIAID Rocky Mountain Laboratories Editor Reed Wickner, National Institutes of Health

                Article
                mBio00829-13
                10.1128/mBio.00829-13
                3884057
                24381300
                68eca6c6-afcf-4b8d-8df5-e31aa3390efa
                Copyright © 2013 Moudjou et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 November 2013
                : 25 November 2013
                Page count
                Pages: 10
                Categories
                Research Article
                Custom metadata
                January/February 2014

                Life sciences
                Life sciences

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