Stability properties of PrPScfrom cattle with experimental transmissible spongiform encephalopathies: use of a rapid whole homogenate, protease-free assay

Vrentas, Catherine E.; Greenlee, Justin J.; Baron, Thierry; Caramelli, Maria; Czub, Stefanie; Nicholson, E. M.

doi:10.1186/1746-6148-9-167

Cited by 13 publications

(19 citation statements)

References 40 publications

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“…Determination of PrP Sc stability by the use of the IDEXX HerdChek BSE ELISA (in the absence of proteinase K digestion) was performed as described previously ( 29 ) with modifications to improve buffering with high concentrations of brain homogenate ( 30 ). Briefly, whole homogenates (lacking PK treatment) were prepared by beadbeating in Dulbecco’s PBS with 10X buffering strength, and homogenate samples were incubated with increasing concentrations of guanidine hydrochloride (GdnHCl; 0.25–4 or 0.25–5 M) for 1 h in 10× PBS.…”

Section: Methodsmentioning

confidence: 99%

A Comparison of Classical and H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism in Wild-Type and EK211 Cattle Following Intracranial Inoculation

et al. 2016

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In 2006, a case of H-type bovine spongiform encephalopathy (BSE-H) was diagnosed in a cow that was associated with a heritable polymorphism in the bovine prion protein gene (PRNP) resulting in a lysine for glutamate amino acid substitution at codon 211 (called E211K) of the prion protein. Although the prevalence of this polymorphism is low, cattle carrying the K211 allele may be predisposed to rapid onset of BSE-H when exposed or to the potential development of a genetic BSE. This study was conducted to better understand the relationship between the K211 polymorphism and its effect on BSE phenotype. BSE-H from the US 2006 case was inoculated intracranially (IC) in one PRNP wild-type (EE211) calf and one EK211 calf. In addition, one wild-type calf and one EK211 calf were inoculated IC with brain homogenate from a US 2003 classical BSE case. All cattle developed clinical disease. The survival time of the E211K BSE-H inoculated EK211 calf (10 months) was shorter than the wild-type calf (18 months). This genotype effect was not observed in classical BSE inoculated cattle (both 26 months). Significant changes in retinal function were observed in H-type BSE challenged cattle only. Cattle challenged with the same inoculum showed similar severity and neuroanatomical distribution of vacuolation and disease-associated prion protein deposition in the brain, though differences in neuropathology were observed between E211K BSE-H and classical BSE inoculated animals. Western blot results for brain tissue from challenged animals were consistent with the inoculum strains. This study demonstrates that the phenotype of E211K BSE-H remains stable when transmitted to cattle without the K211 polymorphism, and exhibits a number of features that differ from classical BSE in both wild-type and heterozygous EK211 animals.

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Section: Methodsmentioning

confidence: 99%

A Comparison of Classical and H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism in Wild-Type and EK211 Cattle Following Intracranial Inoculation

et al. 2016

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“…The relationship between PrP Sc fibril stability in guanidinium hydrochloride (GdnHCl) and incubation time varies between host species and prions strains [23][24][25][26][27][28][29][30] . In CWD-affected elk, longer incubation periods were associated with higher fibril stability and fibril stabilities of PrP Sc from MM132 and LM132 elk were similar 5 .…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, dilutions of mouse brain samples were incubated at concentrations of guanidine hydrochloride (GdnHCl) over a range from 0.25 M to 4.0 M. Brain homogenate was diluted up to 8X into PBS in order to bring the final EIA optical density (OD 450 ) in the ELISA well to 1.0 or below. A final concentration of brain homogenate of ≤ 2% (w/v) was used in all samples to avoid exceeding the buffering capacity, as was observed to occur at higher concentrations of brain homogenate 23 . The relative amount of PrP Sc remaining, as assessed by the OD 450 after dilution to 0.25 M GdnHCl, was plotted against GdnHCl concentration.…”

Section: Western Blottingmentioning

confidence: 99%

“…The midpoint of the curve, or [GdnHCl] 1/2 , is defined as the concentration of GdnHCl at which the PrP Sc signal was reduced by half of the signal at 0.25 M GdnHCl; PrP Sc with a smaller [GdnHCl] 1/2 is less stable. As described previously 23 , due to variations in the upper baseline shape, the Smooth Line function in Microsoft Excel was used to connect data points in each curve (for each individual animal) and visualize the midpoint. In order to assess statistical differences, Welch's ANOVA (accounting for unequal variances) was used, with the Games-Howell test for (non-parametric) post-hoc analysis.…”

Section: Western Blottingmentioning

confidence: 99%

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Novel Strain of the Chronic Wasting Disease Agent Isolated From Experimentally Inoculated Elk With LL132 Prion Protein

Moore

Tatum

Hwang

et al. 2020

Sci Rep

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Chronic wasting disease (CWD) is a fatal, progressive disease that affects cervid species, includingRocky mountain elk (Cervus elaphus nelsoni). There are 2 allelic variants in the elk prion protein gene: L132 (leucine) and M132 (methionine). Following experimental oral challenge with the CWD agent incubation periods are longest in LL132 elk, intermediate in ML132 elk, and shortest in MM132 elk. In order to ascertain whether such cWD-infected elk carry distinct prion strains, groups of Tg12 mice that express M132 elk prion protein were inoculated intracranially with brain homogenate from individual CWD-infected elk of various genotypes (LL132, LM132, or MM132). Brain samples were examined for microscopic changes and assessment of the biochemical properties of disease-associated prion protein (prp Sc ). On first passage, mice challenged with LL132 elk inoculum had prolonged incubation periods and greater prp Sc fibril stability compared to mice challenged with MM132 or LM132 inoculum. On second passage, relative incubation periods, western blot profiles, and neuropathology were maintained. These results suggest that the CWD prion isolated from LL132 elk is a novel CWD strain and that M132 PrP c is able to propagate some biophysical properties of the L132 PrP Sc conformation.Chronic wasting disease (CWD) is a fatal, progressive, neurodegenerative disease that has been reported in a number of cervid species including Rocky Mountain elk (Cervus elaphus nelsoni) and white-tailed deer (Odocoileus virginianus). Chronic wasting disease belongs to a group of diseases known as the transmissible spongiform encephalopathies (TSEs), or prion diseases, that are a group of neurodegenerative diseases in which a key feature is the accumulation of disease-associated prion protein (PrP Sc ) in the the brain.The prion protein gene (PRNP) of Rocky Mountain elk encodes for either methionine (M) or leucine (L) at codon 132 1,2 . The amino acid expressed at codon 132 greatly affects incubation periods in elk after experimental oral inoculation with the chronic wasting disease agent: elk expressing prion protein homozygous for leucine at codon 132 (hereafter referred to as LL132 elk) incubate approximately 1.5 times longer than LM132 elk and 3 times longer than MM132 elk 3,4 . We recently demonstrated that these variations in incubation period may be influenced by genotype-associated differences in the relative amount of PrP Sc in the brain and biochemical properties of PrP Sc , including fibril stability and amyloid formation rate 5 . TSE strains may be differentiated by host range, incubation period, clinical presentation, patterns of immunoreactivity or microscopic lesions in the brain, or the physiochemical properties of the PrP Sc itself (reviewed in 6 ). Prions lack genomic DNA to propagate strain information, and the strain properties are believed to be enciphered by the structure of the PrP Sc conformer 7-11 . Two different strains of CWD have been identified when CWD from white-tailed deer was passaged into Syrian hamsters 12 , ...

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“…The IDEXX HerdChek EIA test kit was used to selectively detect the presence of disease associated misfolded prion protein. The IDEXX HerdCheck Assay can be used to test various mammalian tissues [18]. Any disease-associated conformer, PrP Sc , can bind to the ligand on the surface which is immobilized on the surface and captured with an antigen.…”

Section: Enzyme Immunoassay (Eia) Of Brain Homogenates From Cervidsmentioning

confidence: 99%

Role of donor genotype in RT-QuIC seeding activity of chronic wasting disease prions using human and bank vole substrates

2020

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Chronic wasting disease is a transmissible spongiform encephalopathy of cervids. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein (PrP C) to pathogenic conformers (PrP Sc), and the pathogenic forms accumulate in the brain and other tissues. Real-time Quaking Induced Conversion (RT-QuIC) can be used for the detection of prions and for prion strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how either PrP Sc from cervids of different genotypes or PrP Sc from different sources of CWD influence the fibril formation of recombinant bank vole (BV) or human prion proteins using RT-QuIC. We found that reaction mixtures seeded with PrP Sc from different genotypes of white-tailed deer or reindeer brains have similar conversion efficiency with both substrates. Also, we observed similar results when assays were seeded with different sources of CWD. Thus, we conclude that the genotypes of all sources of CWD used in this study do not influence the level of conversion of PrP C to PrP Sc .

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Stability properties of PrPScfrom cattle with experimental transmissible spongiform encephalopathies: use of a rapid whole homogenate, protease-free assay

Cited by 13 publications

References 40 publications

A Comparison of Classical and H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism in Wild-Type and EK211 Cattle Following Intracranial Inoculation

A Comparison of Classical and H-Type Bovine Spongiform Encephalopathy Associated with E211K Prion Protein Polymorphism in Wild-Type and EK211 Cattle Following Intracranial Inoculation

Novel Strain of the Chronic Wasting Disease Agent Isolated From Experimentally Inoculated Elk With LL132 Prion Protein

Role of donor genotype in RT-QuIC seeding activity of chronic wasting disease prions using human and bank vole substrates

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