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Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains.

Orrú CD, Groveman BR, Raymond LD, Hughson AG, Nonno R, Zou W, Ghetti B, Gambetti P, Caughey B - PLoS Pathog. (2015)

Bottom Line: Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification.Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans.Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Montana, United States of America.

ABSTRACT
Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

No MeSH data available.


Related in: MedlinePlus

Western blot of BV rPrPRes from RT-QuIC reactions seeded with rodent, bovine, cervine and sheep prion types.PK-treated RT-QuIC products from mouse (A), hamster (B), cattle (C), deer (C), elk (C) and sheep (D) prion seeds were probed with R20 (hamster PrP epitope residues 218–231). In (D), the classical scrapie-seeded reactions include those seeded with samples from PRNP VRQ/VRQ and ARQ/ARQ sheep (not designated). The Nor98-seeded reactions were seeded with samples from ARR/ARR, ARQ/AHQ and ARQ/ARQ sheep. RT-QuIC reactions and immunoblotting analysis for each of these types of prions were performed at least twice with similar results. The banding profiles shown are representative of multiple (n) independently tested biological replicates.
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ppat.1004983.g006: Western blot of BV rPrPRes from RT-QuIC reactions seeded with rodent, bovine, cervine and sheep prion types.PK-treated RT-QuIC products from mouse (A), hamster (B), cattle (C), deer (C), elk (C) and sheep (D) prion seeds were probed with R20 (hamster PrP epitope residues 218–231). In (D), the classical scrapie-seeded reactions include those seeded with samples from PRNP VRQ/VRQ and ARQ/ARQ sheep (not designated). The Nor98-seeded reactions were seeded with samples from ARR/ARR, ARQ/AHQ and ARQ/ARQ sheep. RT-QuIC reactions and immunoblotting analysis for each of these types of prions were performed at least twice with similar results. The banding profiles shown are representative of multiple (n) independently tested biological replicates.

Mentions: We further compared the BV rPrPRes products of reactions seeded with different rodent, bovine, cervine and ovine prion strains (Table 2). As with the human prion seeds, we observed distinct strain-dependent BV rPrPRes banding profiles from reactions seeded with different prion types. Mouse 22L scrapie-seeded BV rPrPRes products consistently showed a ~10 and ~12 kDa PK-resistant band, whereas BV rPrPRes products from reactions seeded with Chandler, ME7, 87V and anchorless 22L (22L GPI-) scrapie displayed a predominant ~10 kDa band (Fig 6A). The lack of the GPI anchor in the 22L GPI- scrapie seed resulted in an RT-QuIC product that was distinct from the wild-type GPI-anchored 22L scrapie. Additionally, closely related hamster prion strains (Hyper and 263K; Fig 6B) showed similar BV rPrPRes banding profiles (~10 and ~12 kDa PK-resistant bands) which were distinct from the Drowsy-seeded BV rPrPRes products (primarily a ~10 kDa band; Fig 6B). Deer and elk CWD-seeded reactions each gave ~8, 9, 10, and 12 kDa bands, but differed in the relative intensities of the top two bands between the two (Fig 6C). Furthermore, distinct strain-dependent BV rPrPRes banding profiles were observed between classical (C-BSE) and atypical (L-BSE) (~10 kDa vs. ~9, 10, and 12 kDa bands, respectively; Fig 6C), as well as between classical and atypical Nor98 sheep scrapie (~10 kDa vs. ~9, 10, and 12 kDa bands, respectively; Fig 6D). Collectively, these immunoblotting results suggested that certain human and animal prion diseases can be discriminated in part based on analysis of the rPrPRes products of BV rPrPSen-based RT-QuIC reactions.


Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains.

Orrú CD, Groveman BR, Raymond LD, Hughson AG, Nonno R, Zou W, Ghetti B, Gambetti P, Caughey B - PLoS Pathog. (2015)

Western blot of BV rPrPRes from RT-QuIC reactions seeded with rodent, bovine, cervine and sheep prion types.PK-treated RT-QuIC products from mouse (A), hamster (B), cattle (C), deer (C), elk (C) and sheep (D) prion seeds were probed with R20 (hamster PrP epitope residues 218–231). In (D), the classical scrapie-seeded reactions include those seeded with samples from PRNP VRQ/VRQ and ARQ/ARQ sheep (not designated). The Nor98-seeded reactions were seeded with samples from ARR/ARR, ARQ/AHQ and ARQ/ARQ sheep. RT-QuIC reactions and immunoblotting analysis for each of these types of prions were performed at least twice with similar results. The banding profiles shown are representative of multiple (n) independently tested biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4472236&req=5

ppat.1004983.g006: Western blot of BV rPrPRes from RT-QuIC reactions seeded with rodent, bovine, cervine and sheep prion types.PK-treated RT-QuIC products from mouse (A), hamster (B), cattle (C), deer (C), elk (C) and sheep (D) prion seeds were probed with R20 (hamster PrP epitope residues 218–231). In (D), the classical scrapie-seeded reactions include those seeded with samples from PRNP VRQ/VRQ and ARQ/ARQ sheep (not designated). The Nor98-seeded reactions were seeded with samples from ARR/ARR, ARQ/AHQ and ARQ/ARQ sheep. RT-QuIC reactions and immunoblotting analysis for each of these types of prions were performed at least twice with similar results. The banding profiles shown are representative of multiple (n) independently tested biological replicates.
Mentions: We further compared the BV rPrPRes products of reactions seeded with different rodent, bovine, cervine and ovine prion strains (Table 2). As with the human prion seeds, we observed distinct strain-dependent BV rPrPRes banding profiles from reactions seeded with different prion types. Mouse 22L scrapie-seeded BV rPrPRes products consistently showed a ~10 and ~12 kDa PK-resistant band, whereas BV rPrPRes products from reactions seeded with Chandler, ME7, 87V and anchorless 22L (22L GPI-) scrapie displayed a predominant ~10 kDa band (Fig 6A). The lack of the GPI anchor in the 22L GPI- scrapie seed resulted in an RT-QuIC product that was distinct from the wild-type GPI-anchored 22L scrapie. Additionally, closely related hamster prion strains (Hyper and 263K; Fig 6B) showed similar BV rPrPRes banding profiles (~10 and ~12 kDa PK-resistant bands) which were distinct from the Drowsy-seeded BV rPrPRes products (primarily a ~10 kDa band; Fig 6B). Deer and elk CWD-seeded reactions each gave ~8, 9, 10, and 12 kDa bands, but differed in the relative intensities of the top two bands between the two (Fig 6C). Furthermore, distinct strain-dependent BV rPrPRes banding profiles were observed between classical (C-BSE) and atypical (L-BSE) (~10 kDa vs. ~9, 10, and 12 kDa bands, respectively; Fig 6C), as well as between classical and atypical Nor98 sheep scrapie (~10 kDa vs. ~9, 10, and 12 kDa bands, respectively; Fig 6D). Collectively, these immunoblotting results suggested that certain human and animal prion diseases can be discriminated in part based on analysis of the rPrPRes products of BV rPrPSen-based RT-QuIC reactions.

Bottom Line: Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification.Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans.Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Montana, United States of America.

ABSTRACT
Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

No MeSH data available.


Related in: MedlinePlus