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Discriminating Scrapie and Bovine Spongiform Encephalopathy Isolates by Infrared Spectroscopy of Pathological Prion Protein

2004; Elsevier BV; Volume: 279; Issue: 32 Linguagem: Inglês

10.1074/jbc.m403730200

1083-351X

Achim Thomzig, Sashko Spassov, Manuela Friedrich, Dieter Naumann, Michael Beekes,

Trace Elements in Health

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 β-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. 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 β-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. Transmissible spongiform encephalopathies (TSEs) 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; BSE, bovine spongiform encephalopathy; FT-IR, Fourier-transform infrared; mAb, monoclonal antibody; TBS, Tris-buffered saline; BE, brain equivalents; TME, transmissible mink encephalopathy; dpi, days postinfection. 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. After the initial reports on the emergence of BSE and variant Creutzfeldt-Jakob disease, in 1986 and 1996, respectively, compelling evidence has gradually accumulated that the latter can most likely be attributed to transmissions, presumably via contaminated food, of BSE agent from cattle to man (1Bruce M.E. Will R.G. Ironside J.W. McConnell I. Drummond D. Suttie A. McCardle L. Chree A. Hope J. Birkett C. Cousens S. Fraser H. Bostock C.J. Nature. 1997; 389: 498-501Crossref PubMed Scopus (1727) Google Scholar, 2Cousens S.N. Linsell L. Smith P.G. Chandrakumar M. Wilesmith J.W. Knight R.S.G. Zeidler M. Stewart G. Will R.G. Lancet. 1999; 353: 18-21Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 3Hill A.F. Desbruslais M. Joiner S. Sidle K.C. Gowland I. Collinge J. Doey L.J. Lantos P. Nature. 1997; 389: 448-526Crossref PubMed Scopus (1208) Google Scholar, 4Scott M.R. Will R. Ironside J. Nguyen H.O.B. Tremblay P. DeARmond S.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15137-15142Crossref PubMed Scopus (462) Google Scholar). Therefore, effective infection control measures for the containment and repression of BSE have become a matter of crucial importance to public health. According to the present state of knowledge, the countermeasures implemented in response to the BSE epidemic are expected to minimize or even eliminate the risk of new primary variant Creutzfeldt-Jakob disease infections of humans directly originating from bovines (5Bradley R. Rabenau H.F. Cinatl J. Doerr H.W. Prions: A Challenge for Science, Medicine, and the Public Health System. S. Karger AG, Basel, Switzerland2004: 146-185Google Scholar). However, further challenges in the area of infection control arise from the hypothetical risk that the BSE agent might have spread via contaminated feed such as meat and bone meal to sheep (5Bradley R. Rabenau H.F. Cinatl J. Doerr H.W. Prions: A Challenge for Science, Medicine, and the Public Health System. S. Karger AG, Basel, Switzerland2004: 146-185Google Scholar) and that BSE, like scrapie, might now be sustained in the ovine population. The clinical symptoms of scrapie, which has been endemic in sheep for centuries without any apparent association with human disease, cannot be reliably distinguished from those exhibited by experimentally challenged BSE-infected ovines. "While it is possible to demonstrate the presence of a TSE by several laboratory techniques using microscopy, electron microscopy, or immunological methods which detect the abnormal form of the prion protein, distinguishing between one strain of scrapie and another, and between BSE and scrapie, is not straightforward" (6, Spongiform Encephalopathy Advisory Committee (1999) http://www.seac.gov.uk/publicats/sub-rep.pdf,Google Scholar). So far, reliable differentiation of BSE and scrapie in sheep has required time-consuming and expensive strain-typing in mice using lesion profiles (7Fraser H. Dickinson A.G. J. Comp. Pathol. 1973; 83: 29-40Crossref PubMed Scopus (279) Google Scholar). Therefore, the development of new methods for an inexpensive, robust, and rapid discrimination between BSE and scrapie constitutes a topical challenge in the surveillance of ovine TSEs addressed in a variety of studies (8Baron T.G. Madec J.Y. Calavas D. J. Clin. Microbiol. 1999; 37: 3701-3704Crossref PubMed Google Scholar, 9Hope J. Wood S.C. Birkett C.R. Chong A. Bruce M.E. Cairns D. Goldmann W. Hunter N. Bostock C.J. J. Gen. Virol. 1999; 80: 1-4Crossref PubMed Scopus (145) Google Scholar). During the past few years, considerable progress has been achieved in this field of TSE research, predominantly by using immunobiochemical techniques (10Kuczius T. Groschup M.H. Mol. Med. 1999; 5: 406-418Crossref PubMed Google Scholar, 11Stack M.J. Chaplin M.J. Clark J. Acta Neuropathol. 2002; 104: 279-286Crossref PubMed Scopus (182) Google Scholar, 12Lezmi S. Martin S. Simon S. Comoy E. Bencsik A. Deslys J.P. Grassi J. Jeffrey M. Baron T. J. 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The causative agent of TSEs is widely considered to represent a new biological principle of infection. The prion hypothesis (15Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar) holds that TSE agents ("prions") consist essentially if not entirely of misfolded prion protein PrPSc. The normal cellular isoform of this protein (PrPC) is expressed in neurons, lymphoid cells, and other tissues of mammals. According to the "protein only" model of the prion hypothesis, TSE agents replicate through a molecular mechanism in which abnormally folded PrPSc acts as a catalyst or template nucleus, which recruits cellular prion protein and transforms it into its own "infectious" spatial structure (for a review see Ref. 16Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar). This etiological model implies that the characteristic phenotypic features of different scrapie and other TSE agents, of which more than 20 have been isolated in different breeds of mice alone (17Bruce M.E. Br. Med. Bull. 2003; 66: 99-108Crossref PubMed Scopus (219) Google Scholar), must be coded in the secondary, tertiary, or quaternary structure of PrPSc or in its specific glycosylation. While PrPC contains about 42% α-helical and only 3% β-sheet structure, PrPSc is substantially made up of β-sheets and exhibits a markedly reduced α-helical proportion (18Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar, 19Gasset M. Baldwin M.A. Lloyd D.H. Gabriel J.M. Holtzman D.M. Cohen F. Fletterick R. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10940-10944Crossref PubMed Scopus (316) Google Scholar, 20Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2076) Google Scholar). Therefore, within the framework of the "protein only" model of the prion hypothesis, the secondary structure and, more specifically, the β-sheets of PrPSc molecules appear as key candidates for coding the phenotypic characteristics of TSE strains. During the past few years it has been conclusively shown that several different TSE strains can be distinguished by immunobiochemical typing of the electrophoretic mobilities and glycosylation characteristics of PrPSc (or its protease-resistant core, PrP27–30) in the Western blot (21Collinge J. Sidle K.C. Meads J. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Crossref PubMed Scopus (1591) Google Scholar, 22Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (722) Google Scholar, 23Parchi P. Capellari S. Chen S.G. Petersen R.B. Gambetti P. Kopp N. Brown P. Kitamoto T. Tateishi J. Giese A. Kretzschmar H. Nature. 1997; 386: 232-234Crossref PubMed Scopus (231) Google Scholar, 24Somerville R.A. Chong A. Mulqueen O.U. Birkett C.R. Wood S.C. Hope J. Nature. 1997; 386: 564Crossref PubMed Scopus (117) Google Scholar). When comparing two strains of hamster-adapted transmissible mink encephalopathy (TME), termed hyper (HY) and drowsy (DY), the unglycosylated fraction of PrP27–30 derived from HY- and DY-PrPSc by digestion with proteinase K exhibited different apparent molecular masses of 21 and 19 kDa, respectively (25Bessen R.A. Marsh R.F. J. Virol. 1994; 68: 7859-7868Crossref PubMed Google Scholar). This experimental evidence for the presence of differentially accessible cleavage sites in HY- and DY-PrPSc could most plausibly be accounted for by differences in the conformation of the misfolded prion protein molecules that were derived from PrPC with an identical amino acid sequence. Consistent with these findings, a conformation-dependent immunoassay has provided further indirect evidence that PrPSc molecules from HY- and DY-TME as well as from six other hamster-adapted TSE strains have differences in the three-dimensional structure of their polypeptide chains (26Safar J. Wille H. Itri V. Groth D. Serban H. Torchia M. Cohen F.E. Prusiner S.B. Nat. Med. 1998; 4: 1157-1165Crossref PubMed Scopus (1078) Google Scholar). When Fourier transform-infrared (FT-IR) spectroscopy was used to directly investigate the structure of prion proteins associated with different TSE strains, the conclusions described above were confirmed and substantially expanded by showing that PrPSc extracts from HY- and DY-TME in hamsters exhibit different β-sheet structures (27Caughey B. Raymond G.J. Bessen R.A. J. Biol. Chem. 1998; 273: 32230-32235Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Thus, conceptual as well as experimental clues strongly point to strain-specific phenotypic information of TSE agents either being encoded or at least mirrored in the secondary structure of PrPSc. This suggests FT-IR spectroscopic structural profiling of PrPSc or PrP27–30, a technique that should efficiently resolve differences in the secondary structure of the protein, as a promising tool for the molecular typing and differentiation of TSE agents. With this rationale, the experiments described in this report pursued two aims. First, we intended to identify in laboratory animals TSE agents that cause similarly presenting TSEs and thereby mirror the diagnostic challenge of distinguishing infectious isolates that can neither be differentiated by the clinical symptoms or neuropathological changes they produce nor by the immunobiochemical properties of their associated PrPSc. For this purpose, Syrian hamsters were chosen as model animals because, as outlined above, they have provided key insights into the relationship between phenotypically different TSE strains and PrPSc structure. Second, upon successful identification and passage of such isolates in the hamster model, we investigated whether it is possible to reliably distinguish them and other TSE agents by FT-IR spectroscopic structural characterization of PrP27–30 extracted from the diseased individuals. In the following, we report on an experimental proof-of-concept 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. Because the model animals used in our study have frequently provided base-line information about natural TSEs, this proof-of-concept may well be indicative of a diagnostic approach that allows the rapid and reliable discrimination of strains in nonexperimental TSEs of animals and humans. TSE Agents and Animal Experiments—Serial passaging of hamster-adapted 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-μl aliquots of 1% (w/v) hamster-brain homogenates in TBS (10 mm Tris-HCl, 133 mm NaCl, pH 7.4) from terminally ill donors. Hamster scrapie strain 263K (28Kimberlin R.H. Walker C.A. J. Gen. Virol. 1977; 34: 295-304Crossref PubMed Scopus (228) Google Scholar) was originally provided by R. H. Kimberlin and has been serially passaged for more than 20 years in our laboratory. Strains ME7-H and 22A-H generated by R.H. Kimberlin after 4 and 7 passages in hamsters, respectively (29Kimberlin R.H. Walker C.A. Fraser H. J. Gen. Virol. 1989; 70: 2017-2025Crossref PubMed Scopus (178) Google Scholar), were kindly provided by the Institute for Animal Health, Edinburgh, UK. Isolates "RIV/8" of ME7-H and "RIV/12" of 22A-H were used for the first passage of these TSE agents at the Robert Koch-Institute. BSE-H was newly derived in our laboratory after one passage of BSE agent from cattle in mice and subsequent transmission to hamsters. 2A. Thomzig, H. Diringer, and M. Beekes, manuscript in preparation. Briefly, 20-μl aliquots of 1% (w/v) brain tissue homogenate in TBS from a German BSE case in a cow imported from the UK were injected intracerebrally into C57Bl/10 mice; between 529 and 697 days postinfection (dpi), 4/9 recipients succumbed to fatal neurological disease with clinical symptoms strongly reminiscent of murine transmissible spongiform encephalopathy. 50-μl aliquots of 1% (w/v) brain homogenate in TBS from a diseased C57Bl/10 mouse sacrificed at 606 dpi were then intracerebrally inoculated into hamsters. This produced terminal TSE-like symptoms between 360 and 467 dpi in 10/10 animals. For the present study, all examinations were performed on the 2nd serial passages of BSE-H, ME7-H, and 22A-H in our laboratory, which showed incubation times of 287 ± 28, 331 ± 16, and 206 ± 8 days (expressed as mean ± S.D.) until the occurrence of terminal disease, respectively, and on a passage of 263K with an incubation time of 83 ± 5 days. These incubation times remained constant in further serial transmissions (data not shown) indicating stable propagation of BSE-H, ME7-H, and 22A-H after the first passage in our hamsters. Animals were regularly observed for clinical symptoms and humanely sacrificed by CO2 euthanasia at the terminal stage of disease. After sacrificing, the brains were immediately removed and further processed as outlined below. Lesion Profiles—Brains were fixed in 4% formalin for 48 h, treated with formic acid for 60 min at room temperature to reduce infectivity, postfixed in formalin for 24 h, and trimmed coronally into 2–3-mm-thick slices. After processing in an enclosed tissue processor, samples were embedded in paraffin wax. 6-μm microtome slices from the relevant regions were mounted onto slides and stained in ethyl eosin for 5–10 min. The scoring of vacuolar lesions was performed as described elsewhere (7Fraser H. Dickinson A.G. J. Comp. Pathol. 1973; 83: 29-40Crossref PubMed Scopus (279) Google Scholar). PET Blot Mapping of Cerebral PrPSc Distribution—PrPSc-PET blotting (31Schulz-Schaeffer W.J. Tschoke S. Kranefuss N. Drose W. Hause-Reitner D. Giese A. Groschup M.H. Kretzschmar H.A. Am. J. Pathol. 2000; 156: 51-56Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) of hamster brain slices was performed as described elsewhere (30McBride P.A. Schulz-Schaeffer W.J. Donaldson M. Bruce M. Diringer H. Kretzschmar H.A. Beekes M. J. Virol. 2001; 75: 9320-9327Crossref PubMed Scopus (210) Google Scholar). In brief, hamster brains were fixed in 4% formalin for 48 h, treated with formic acid for 60 min at room temperature to reduce infectivity, postfixed in 4% formalin for 24 h, and trimmed coronally into 2–3-mm-thick slices using a brain slicing mold. After processing in an enclosed tissue processor, samples were embedded in paraffin wax. 6-μm microtome slices were mounted on nitrocellulose membranes (0.45 μm pore size; Bio-Rad) and dried flat at 50 °C for 16 h. For PET blotting according to Schulz-Schaeffer et al. (31Schulz-Schaeffer W.J. Tschoke S. Kranefuss N. Drose W. Hause-Reitner D. Giese A. Groschup M.H. Kretzschmar H.A. Am. J. Pathol. 2000; 156: 51-56Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), specimens were washed in TBS, pH 7.8, with 0.05% Tween 20 (TBST), and digested with 25 μg/ml proteinase K (Roche Applied Science) in a buffer containing 10 mm Tris-HCl, pH 7.8, 100 mm NaCl, and 0.1% (w/v) Brij 35 for 2 h at 55 °C. Sections were denatured in 3 m guanidine isothiocyanate, blocked in 0.2% (w/v) casein in TBST, and incubated with mouse α-hamster PrP antibody 3F4 (mAb 3F4; 1:2500 (32Kascsak R.J. Rubenstein R. Merz P.A. Tonna-DeMasi M. Fersko R. Carp R.I. Wisniewski H.M. Diringer H. J. Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar)) overnight at 4 °C. After incubation with the secondary antibody (alkaline phosphatase-labeled rabbit anti-mouse IgG (1:2000; Dako, Denmark)) for 60 min at room temperature, sections were stained with nitro blue tetrazolium (Sigma) and 5-bromo-4-chloro-3-indolylphosphate (Sigma) to visualize the reaction product. The immunostained PET blots were assessed for cerebral PrPSc distribution using a stereo microscope. For negative controls, brain slices were incubated in normal mouse serum (1:25,000) instead of mAb 3F4 prior to incubation with secondary antibody. Western Blot Typing of PrP27–30—50 μl of 10% (w/v) brain homogenates in TBS, pH 7.4, were mixed with 5 μl of 13% (w/v) Sarkosyl and 10 μl of proteinase K stock solution (1 mg/ml; Roche Applied Science) and subsequently digested for 60 min at 37 °C (33Beekes M. Baldauf E. Cassens S. Diringer H. Keyes P. Scott A.C. Wells G.A. Brown P. Gibbs C.J.J. Gajdusek D.C. J. Gen. Virol. 1995; 76: 2567-2576Crossref PubMed Scopus (54) Google Scholar). The digestion was stopped by adding 435 μl of 2× sample buffer, i.e. 4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol in 120 mm Tris-HCl, pH 6.8, containing 20% (w/v) glycerol and 0.05% (w/v) bromphenol blue, and boiling for 5 min. 1–5 μl of the solution (corresponding to 1–5 × 10-5 g of homogenized brain tissue) were separated in a 15% SDS-PAGE (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar) or in Tris glycine gels (NOVEX, Invitrogen) and subsequently blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore) using the semidry method (Fast-Blot; Biometra). The PVDF membranes were blocked with 5% (w/v) low-fat milk powder in TBS for 30 min and incubated overnight at 4 °C with mAb 3F4 (1:2000) in 3% bovine serum albumin in TBS. After washing in TBS and incubation for 60 min at room temperature with the secondary antibody (biotinylated goat anti-mouse IgG (1:2000) in 3% bovine serum albumin in TBS), a biotin-streptavidin kit (Dako, Denmark) for signal enhancement was used (30 min; at room temperature). After washing the membranes in TBS, antibody binding to PrP was visualized using a mixture of nitro blue tetrazolium (Sigma) and 5-bromo-4-chloro-3-indolylphosphate (Sigma) as substrate. The relative staining intensities of di-, mono- and nonglycosylated PrP in each sample were determined by densitometry for a graphical representation of the glycosylation profiles; densitometric measurements were performed in triplicate for each blotting membrane with samples from at least four donor animals infected with 263K-, ME7-H-, 22A-H, and BSE-H agent. Assaying the Sensitivity of PrPSc to Proteinase K at Different pH Values—25 μl of 10% (w/v) brain homogenates in TBS from hamsters infected with the four different TSE strains (containing approximately the same amounts of PrPSc) were diluted 1:20 in 50 mm Tris adjusted to pH values of 4.0, 5.0, 6.0, 6.5, 7.0, 7.4, 8.0, 8.5, 9.0, and 10.0. Samples were digested with proteinase K as described above at a protease concentration of 40 μg/ml for 60 min at 37 °C. After stopping the digestion by adding 2× 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. FT-IR Spectroscopic Characterization of PrP27–30—263K-, ME7-H-, 22A-H-, and BSE-H-associated proteinase K-resistant prion protein PrP27–30 was purified from the brains of intracerebrally infected Syrian hamsters at the terminal stage of disease, using a protocol published previously by Diringer et al. (35Diringer H. Beekes M. Ozel M. Simon D. Queck I. Cardone F. Pocchiari M. Ironside J.W. Intervirology. 1997; 40: 238-246Crossref PubMed Scopus (50) Google Scholar) with some modifications: (i) five hamster brains were used instead of 20 brains as starting material. (ii) At each step of the purification procedure that required centrifugation of samples, new polycarbonate vials were used (26.3-ml polycarbonate tubes; Beckman). (iii) Resuspending of pellets by ultrasonification was carried out in glass instead of polycarbonate tubes in order to avoid contamination of the protein with finely dispersed polycarbonate disintegrated from the tube walls. (iv) The final pellet was resuspended in 1 ml of distilled water adjusted to pH 8.5 with 1 m Tris solution and divided into volumes representing 2, 1, and 0.5 "brain equivalents" (BE), i.e. 2, 1, and 0.5 g of starting brain material, and centrifuged in a Beckman TL-100 ultracentrifuge using a TLA-45 rotor at 45,000 rpm for 1 h at 4 °C. The supernatant was discarded, and the protein pellets were stored at -20 °C until use. For determination of the total protein content, 0.5 BE were mixed with 50 μl of sample buffer and boiled for 5 min. The total protein content was determined by staining with Amido Black as described elsewhere (33Beekes M. Baldauf E. Cassens S. Diringer H. Keyes P. Scott A.C. Wells G.A. Brown P. Gibbs C.J.J. Gajdusek D.C. J. Gen. Virol. 1995; 76: 2567-2576Crossref PubMed Scopus (54) Google Scholar). The purity of the extracted protein was monitored by SDS-PAGE and subsequent silver staining as described previously (33Beekes M. Baldauf E. Cassens S. Diringer H. Keyes P. Scott A.C. Wells G.A. Brown P. Gibbs C.J.J. Gajdusek D.C. J. Gen. Virol. 1995; 76: 2567-2576Crossref PubMed Scopus (54) Google Scholar), and PrP27–30 was also visualized by Western blotting using mAb 3F4 as described above (not shown). The amount of total protein in the final fraction was usually in the range of 50–150 μg/5 BEs. FT-IR Spectroscopic Measurements—FT-IR spectra of PrP27–30 were collected with a Bruker IFS28B FT-IR spectrometer. Spectral resolution applied was 4 cm-1; for apodization a Blackman-Harris 3-Term function was applied, and a zero filling factor of 4 was used yielding an encoding interval of ∼1 data point per wave number. For each spectrum 128 scans were co-added and averaged. Transmission/absorption FT-IR spectra were collected and electronically stored between 4000 and 400 wave numbers, while the instrument was continuously purged with dry air to reduce water vapor absorption. The FT-IR spectra were collected in the front measurement channel of an IFS28B FT-IR spectrometer (Bruker Optics GmbH, Germany) equipped with a DTGS detector. For FT-IR measurements of samples in D2O, the protein from 2 BEs (20–60 μg) was resuspended and centrifuged twice using a TLA-45 rotor at 45,000 rpm for 10 min at 4 °C in 1 ml of 0.1% Zwittergent 3-14 in D2O (Z/D2O) to obtain a finely dispersed protein suspension. Hydrogen-deuterium (H/D) exchange to equilibrium was obtained after 60 min of incubation at room temperature. Aliquots from the supernatant of the second centrifugation were used to collect reference spectra for digital subtraction from the protein Z/D2O suspension. The spectra of PrP27–30 were obtained from samples resuspended in Z/D2O with a final concentration of ∼10 μg/μl. 1.8 μl were transferred to an IR cell constructed from two CaF2 windows, in one of which a cylindrical cavity with a 6-μm path length was engraved. PrP27–30 samples from three independent purification runs of 263K, ME7-H, 22A-H, and BSE-H were each examined. For comparison and graphical representation, the spectra were vector-normalized between 1600 and 1750 wave numbers to get similar intensities of the bands. Second derivatives were calculated using a 13-point smoothing function. With the study background outlined above, we focused on approaches for the characterization and discrimination of TSE agents that, in principle, would also be applicable for a swift strain differentiation under field conditions. This excluded parameters such as the incubation time or techniques such as strain typing by lesion profiling in mice (7Fraser H. Dickinson A.G. J. Comp. Pathol. 1973; 83: 29-40Crossref PubMed Scopus (279) Google Scholar, 17Bruce M.E. Br. Med. Bull. 2003; 66: 99-108Crossref PubMed Scopus (219) Google Scholar) as diagnostic options and produced the following results. Clinical Examination for Neurological and Behavioral Symptoms—Hamsters diseased with 263K scrapie showed head bobbing, generalized tremor, and ataxia of gait. These animals were frequently and persistently in motion, easily irritated by touch and noise, upon which they often twitch, and had difficulties maintaining balance and rising from a supine position. In contrast, clinically ill animals challenged with ME7-H or 22A-H scrapie agent exhibited phlegmatic sluggishness with bradykinesia and kyphosis. Unlike 263K scrapie hamsters, ME7-H- and 22A-H-infected animals were frequently and persistently resting, not obviously irritated by touch or noise, and well able to slowly rise from a supine position until very advanced stages of disease. Head bobbing, generalized tremor, or ataxic gait as in 263K scrapie were not observed, but the animals showed signs of hind limb paralysis. Hamsters diseased with BSE-H were also found to rest frequently and continuously in a kyphotic position and then appeared lethargic. However, in contrast to the phlegmatic sluggishness and apparent unresponsiveness to irritation by touch and noise as observed in ME7-H and 22A-H scrapie, BSE-H hamsters exhibited spontaneous convulsions from a resting position and had an extreme sensibility to touch (especially to the hindlimbs) which easily triggered tetanic responses. When moving, BSE-H hamsters showed ataxia of gait and hind limb paralysis without bradykinesia. Unlike the 263K scrapie hamsters, they were fairly well able to rise from a supine position and did not show head bobbing and generalized tremor. Neurological and behavioral symptoms allowed us to distinguish 263K scrapie and BSE-H from each other as well as both from ME7-H and 22A-H. However, a symptomatic discrimination between the latter two was not possible. Neuropathological Lesion Profiling—Following clinical examination, we analyzed the cerebral lesion profiles caused by 263K, ME7-H, 22A-H, and BSE-H agent. Lesion profiles provide a well