Abstract
For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species.
Highlights
Transmissible spongiform encephalopathies (TSEs)1 such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans are invariably fatal neurodegenerative disorders of the central nervous system
For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27–30
We report on an experimental proof-ofconcept that FT-IR profiling of PrP27–30 potentially provides a biophysical method for the swift differentiation of TSE agents, including those that are difficult or even impossible to discriminate by a variety of approaches for fast strain typing
Summary
TSE Agents and Animal Experiments—Serial passaging of hamsteradapted scrapie strains 263K, ME7-H, and 22A-H and of a new hamster-adapted BSE isolate, BSE-H, was performed by intracerebral infection of outbred Syrian hamsters with 50-l aliquots of 1% (w/v) hamster-brain homogenates in TBS (10 mM Tris-HCl, 133 mM NaCl, pH 7.4) from terminally ill donors. 50-l aliquots of 1% (w/v) brain homogenate in TBS from a diseased C57Bl/10 mouse sacrificed at 606 dpi were intracerebrally inoculated into hamsters This produced terminal TSElike symptoms between 360 and 467 dpi in 10/10 animals. After stopping the digestion by adding 2ϫ sample buffer and boiling for 5 min, sample aliquots were subjected to SDS-PAGE, Western blotting, and PrP immunostaining with mAb 3F4 as described above. Second derivatives were calculated using a 13-point smoothing function
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