Abstract
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 (PrPTSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrPTSE in tissues and blood. Macaque vCJD PrPTSE 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 PrPTSE. 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 PrPTSE. 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 PrPTSE 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 PrPTSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrPTSE was more permissive than human PrPTSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrPTSE from brains of humans and macaques with vCJD. PrPTSE signals were reproducibly detected by Western blot in dilutions through 10-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrPTSE 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 PrPTSE in vCJD-infected human and macaque blood.
Highlights
Transmissible spongiform encephalopathies (TSEs), known as prion diseases, are incurable and inevitably fatal neurodegenerative infections with long asymptomatic incubation periods
We investigated the ability of PrP in normal macaque brain homogenate to amplify PrPTSE present in 10% Variant Creutzfeldt-Jakob disease (vCJD)-infected macaque brain suspension diluted 10-2 and 10-3 (Figure 1)
We showed that red-backed vole (RBV) brain with 170S/S PrP genotype was an excellent substrate for in vitro amplification of PrPTSE from human and macaque brains infected with the vCJD agent
Summary
Transmissible spongiform encephalopathies (TSEs), known as prion diseases, are incurable and inevitably fatal neurodegenerative infections with long asymptomatic incubation periods. A recent survey of appendix tissue in U.K. estimated that as many as 1 in 2,000 people had accumulation of abnormal prion protein PrPTSE in lymphoid follicles [8]. These individuals are currently asymptomatic but might be incubating vCJD, if the presence of PrPTSE implies vCJD infection; they may either remain in a silent carrier state and never develop clinical vCJD, or eventually progress to have overt vCJD. Despite the decline in the number of food-borne vCJD cases, TTvCJD remains a concern for public health
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