Nabanita Nag
Christina M. Carlson
Jay R. Schneider
Dennis M. Heisey
Christopher J. Johnson
David M. Asher
Luisa Gregori
Julie Nemecek
2013
<p><span>Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrP</span><span>TSE</span><span>) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP</span><span>TSE</span><span> in tissues and blood. Macaque vCJD PrP</span><span>TSE</span><span> did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrP</span><span>TSE</span><span>. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP</span><span>TSE</span><span>. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP</span><span>TSE</span><span> was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP</span><span>TSE</span><span> demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP</span><span>TSE</span><span> was more permissive than human PrP</span><span>TSE</span><span> in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP</span><span>TSE</span><span> from brains of humans and macaques with vCJD. PrP</span><span>TSE</span><span> signals were reproducibly detected by Western blot in dilutions through 10</span><span>-12</span><span> of vCJD-infected 10% brain homogenates. This is the first report showing PrP</span><span>TSE</span><span> from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrP</span><span>TSE</span><span> in vCJD-infected human and macaque blood.</span></p>
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10.1371/journal.pone.0078710
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PLOS
Red-backed vole brain promotes highly efficient <i>in vitro</i> amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.
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