Red-Backed Vole Brain Promotes Highly Efficient In Vitro Amplification of Abnormal Prion Protein from Macaque and Human Brains Infected with Variant Creutzfeldt-Jakob Disease Agent

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.

Blood reference materials from macaques infected with variant Creutzfeldt‐Jakob disease agent

“…The sample is subjected to cycles of incubation and sonication during which PrP TSE converts normal prion protein into new PrP TSE . In previous studies we have shown that normal red‐backed vole brain homogenate expressing prion protein with serine/serine (S/S) at Codon 170 is an efficient substrate for amplification of PrP TSE from vCJD‐infected macaque brain . Figure A shows Western blot results of PMCA‐generated PrP TSE from 10 −9 to 10 −11 duplicate dilutions of vCJD‐infected macaque brain homogenate in normal human plasma (Fig.…”

“…All supernatants were discarded and all liquid was carefully removed. The pellets were thoroughly resuspended with 100 μL of 10% red‐backed vole brain homogenates with 170S/S genotype . Duplicate samples were then subjected to protein misfolding cyclic amplification (PMCA) as previously described.…”

Efficient propagation of variant Creutzfeldt‐Jakob disease prion protein using the cell‐protein misfolding cyclic amplification technique with samples containing plasma and heparin

“…As the PrP res amounts in brains have not been reported in previous studies, and as animal models suggest that prion levels in plasma are much lower than those in whole blood or WBCs, it remains unclear whether our PMCA methods can be used to detect blood‐borne prions in vCJD patients. It is possible that higher detection sensitivity can be achieved by changing the substrate to red‐backed vole brain …”

“…Quaking‐induced conversion and its improved versions, in which agitation induced PrP amyloid formation, were reported to detect prion from cerebrospinal fluids and nasal brushing of sporadic CJD patients . In PMCA, protease resistant isoform of PrP (PrP res ), along with infectivity, are very efficiently propagated by repeating sonication and incubation with, if necessary, an addition of seed‐dependent cofactors or using a specific substrate . Recently, PMCA was shown to detect PrP Sc in white blood cells (WBCs) from a vCJD patient .…”

Ultrasensitive detection of PrPSc in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion

“…We first proved the existence of PrP Sc in the CSF and blood of BSE-infected macaques by PMCA, and showed that cynomolgus macaque BSE PrP Sc , and non-macaque PrP C , effectively converted mouse PrP C to a proteinase K (PK)-resistant form. It is well known that PMCA of several xenogeneic combinations of PrP Sc seed and PrP C substrate can overcome the species barrier (Kurt et al, 2007(Kurt et al, , 2011Green et al, 2008;Castilla et al, 2008;Yoshioka et al, 2011;Murayama et al, 2012;Nemecek et al, 2013), despite the divergent amino acid sequence of prion proteins. Since the BSE prion was transmissive to ICR (WT) mice (Masujin et al, 2008), the cynomolgus macaque PrP Sc generated by the cross-species transmission of BSE prion may retain the original characteristics of BSE PrP Sc , including structural compatibility with mouse PrP C and DSP dependency in PMCA reactions.…”