Mouse-adapted scrapie strains 139A and ME7 overcome species barrier to induce experimental scrapie in hamsters and changed their pathogenic features

Brain homogenates of C57 mice infected with mouse-adapted scrapie strains 139A and ME7 were intracerebrally inoculated into hamsters. Except three hamsters in the group of agent 139A died suddenly in the first week after inoculation, all other animals were alive. Animals infected with strain 139A showed the clinical manifestations 385 to 405 days after inoculation, with the average incubation of 395 ± 8.5 days (Figure 1). The most predominant and observable symptoms of the infected hamsters were itchy and scratchy in the later stage, most animals having obvious skin claws. The clinical courses from onset to death persisted from 25 to 30 days (Table 1). Animals infected with strain ME7 became ill 460 to 530 days after inoculation, with the average incubation of 496.25 ± 27.22 days (Figure 1). The clinical manifestations were severe thin and sluggish. The clinical courses of ME7-infected hamsters were 30 to 40 days (Table 1). It confirms again that the mouse-adapted scrapie strains can overcome the species barrier to infected hamsters.

Previous study revealed that PrP^Sc and SAF appeared almost simultaneously in brains of the hamsters infected with scrapie 263 K earlier than the onset of clinical symptoms [11]. To see the possible appearances in the hamsters' brains infected with scrapie agents 139A and ME7 during incubation period, one animal from each group was randomly selected and the brain was removed surgically 200 days after inoculation, with interval of 20 days till the onset of clinical manifestations. In line with the observation of agent 263 K-infected hamsters, both SAFs and PrP^Sc were detected in the brain tissues earlier than the appearance of clinical symptoms. Interestingly, SAFs were observed in brain tissues much earlier than PrP^Sc, that in the group of agent 139A, SAF was firstly detected in the hamster 224 days, while PrP^Sc in the 286 days after inoculation (Table 2), and in the group of agent ME7, SAF was firstly detected in the animal 250 days, while PrP^Sc in the 330 days after inoculation (Table 3). Both PrP^Sc and SAF were persistently observed in the following tested animals. It indicates that prion-related events, especially SAF, appear much earlier than the onset of clinical manifestations.

Screening more than 5 brain samples of each three strains at the terminal clinical stage identified many long, ramose and roughly 25 nm in diameter fibrils, whose surfaces were shaggy (Figure 2). Compared with the observations of brains infected with agents 263 K (Figure 2A), the amounts of SAFs in 139A-ha (2B) and ME7-ha (2 C) infected hamsters seemed to be smaller, while the SAFs in the ME7-infected brains were relatively shorter. To address the major components of SAFs, brain homogenates were incubated with mAb 3 F4 and subsequently with SPA-coupled gold. EM assays showed large amounts of fibrils covered with black dots (Figure 2D) in the brain homogenates infected with agent 263 K, suggesting that PrP are the main elements of fibrils.

To see the neuropathological characteristics caused by infections with the three scrapie strains in hamsters, histopathological analyses were comparatively performed on the regions of cortex and cerebellum. Spongiform degeneration was more intensive and severe in the cortex infected by strain 263 K, featured with many vacuoles distributed in the field of vision, while moderate spongiform degeneration was observed in the cortex infected by strains 139A and ME7 (Figure 3A, upper panels). The lesion scores of in the cortex regions of strains 263 K, 139A-ha and ME7-ha were evaluated as 4, 2.0 and 2.5, respectively (Figure 3B). Contrast to the changes in the cortex regions, typical spongiform was rarely observed in cerebellum regions of all three strains (Figure 3A, middle panels). Furthermore, gliosis in brain cortex regions were tested by IHC with a commercial mAb against GFAP. Abundant large GFAP-positively stained astrocytes were detected, in which the gliosis in the agent ME7-ha infected brains were comparably severe as that in agent 263 K-infected one, while gliosis in the agent 139A-ha infected brains was significantly mild (Figure 3A, lower panels).

Presences of PrP^Sc in brain homogenates of the hamsters infected with strains 139A and ME7 at the terminal stage of diseases were tested by Western blot assays with PrP specific monoclonal antibody 3 F4 after PK treatment. Protease resistant bands (PrP^res) were seen in all diseased animals, which mobilized roughly at 19-25 kDa. Interestingly, the PrP^res from the brains infected with strains 139A and ME7 showed identical electrophoretic and glycosylation profiles, in which the diglycosyl form of PrP^res was the most predominant, followed by monoglycosyl and nonglycosyl forms, showing similar profiles as that in the preparation of the hamsters infected with strain 263 K (Figure 4A). Calculating the relative gray values of each glycosyl PrP^Sc bands in the reactions with mAb 3 F4 showed that the percentages of diglycosyl, monoglycosyl and nonglycosyl forms in the brains of agent 263 K were 56.8%, 27.9%, 15.3%, the forms of 139A-ha were 52.8%, 32.8%, 14.4% and the forms of ME7-ha were 52.1%, 33.1%, 14.8%. Compared with PrP^res in the mice brains infected by the individual parent mouse-adapted strains in Western blot with PrP-specific mAb 1E4, in which the monoglycosyl PrP^res were predominant (Figure 4B), the diglycosyl PrP^res in hamsters brains of agents 139A and ME7 were predominant (Figure 4A), suggesting that mouse-adapted scrapie strains may change their PrP^Sc molecular profiles after replicating in hamsters brains.

To get more information of PrP^res profiles, same amounts of hamsters' brain homogenates infected with three different scrapie agents at the terminal stage of diseases were comparatively assayed by Western blots with other commercial PrP-specific mAbs, including 1E4, 6D11, 6H4 and 8H4. Like that in the reactions with mAb 3 F4, similar electrophoretic and glycosylated PrP^res patterns were observed in the preparations reacted with the tested antibodies, all showing predominately diglycosal PrP^res (Figure 4D). In line with the observations in Figure 4A reacted with mAb 3 F4, clear small molecule-weight bands at the position of Mr 21 were detected in all three homogenates in the reaction with mAb 6D11, indicating presences of the truncated C2 fragments (Figure 4D). Additionally, at our experimental condition, relatively weaker signals of PrP^res were observed in 139A infected hamsters' brains in the Western blots with various PrP mAbs.

To test the PK-resistances of PrP^Sc from the three scrapie strains, brain homogenates from 3 infected animals at the terminal stage of diseases were pooled as the representative samples and exposed to the digestion with different amounts of PK from 20 to 1000 μg/ml. The input PrP amounts in various homogenates were equilibrated by Western blots prior to PK-treatment. Figure 5A revealed PrP^res signals in all preparations at the experimental condition. Compared with the input PrP, the intensities of PrP^res signals of the three scrapie strains in the preparations of 20 μg/ml PK decreased slightly. The PrP^res signals of scrapie agents 263 K and ME7 remained fairly stable in the reactions containing 20 to 1000 μg/ml PK, but that of agent 139A started to drop down from the preparation of 200 μg/ml PK (Figure 5B). These results indicate that the PrP^Sc in hamsters' brains by infections of hamster-adapted agent 263 K or mouse-adapted agents 139A and ME7 have similar PK-resistant features.

Exposure of PrP^Sc to increasing concentrations of GdnHCl leads to a transition from the native to denatured state, measured as loss of resistance to protease digestion. To test the consistency of three kinds of PrP^Sc in the PK-resistance after exposing to GdnHCl, the representative samples of these three infected hamsters were incubated with a series of amounts of GdnHCl from 1 to 6 M separately. The input PrP amounts in various homogenates were equilibrated by Western blots prior to GdnHCl- treatment. Western blots with mAb 3 F4 revealed that the total PrP signals from preparations of the agent 263 K remained comparatively constant among the reactions with different concentrations of GdnHCl while PrP signals of the agents 139A-ha and ME7-ha weakened clearly only in the reactions of 6 M GdnHCl (Figure 6A and 6B). The GdnHCl-treated products were subsequently exposed to the proteolysis with 50 μg/ml PK. PrP^res signals were detectable in preparations of the agents 139-ha and ME7-ha incubated with GdnHCl below 4 M and in that of the agent 263 K incubated with GdnHCl below 5 M (Figure 6C). Calculations of the relative gray values of PrP^res signals showed that PrP^Sc in the preparations of agents 139A-ha and ME7-ha started to drop down remarkably after exposed with 3 M GdnHCl and disappeared since 4 M GdnHCl, whereas PrP^Sc of agent 263 K became notable weak after exposed with 4 M GdnHCl and undetectable since 5 M GdnHCl (Figure 6D). These data indicate that the mouse-adapted scrapie strains 139A- and ME7-induced PrP^Sc in the brains of hamsters have the similar conformational stabilities as that of the agent 263 K-induced PrP^Sc.

To see the potential influences of GdnHCl on the morphology of SAFs, brain homogenates of 263 K, 139A-ha and ME7-ha-infected hamsters were individually incubated with 3 M GdnHCl. EM assays identified the clusters of small round particles and short clubs stained by phosphomolybdic acid in the three scrapie strains (Figure 7). No SAF-like structures with long, ramose or straight fibrils were observed after exposing to GdnHCl.

Brain materials from hamster scrapie strain 263 K, mouse scrapie strains 139A and ME7 were homogenized (1:10) prior to challenging as inoculum. 1 μl of individual brain homogenate was intracerebrally injected into 15-day old Golden hamsters under halothane anaesthesia respectively. Each group consisted of 16 hamsters. For equilibrating the injected amounts of the different strains, we analyzed the absolute PrP^Sc grey value of different strains by Western blot method with the same loading volume after totally PK digestion. The clinical symptoms and signs were scored as described previously [11] and the incubation was calculated from the inoculation to the terminal stage of the disease individually. Then the animals were euthanized at the end of clinical phase and brains were taken surgically for further studies during the incubation period and after the onset of illness.

The individual brain homogenate of hamster scrapie strain 263 K, hamster scrapie strains 139A and ME7 were absorbed onto copper nets covered with carbon membrane and stained with 2% phosphomolybdic acid for 2 min at room temperature. Scrapie-associated fibril (SAF) was observed with transmitted electron microscopy at the condition of 80 KV (Philips, JEOL1200EX). For colloidal gold immunoelectron microscopy assays, samples were absorbed onto copper nets and incubated with 1:100 diluted mAb 3 F4 for 8 hr. After washed with PBS for three times, the copper nets were exposed to 1:50 diluted 5 nm SPA-immunogold for 1 hr and stained with 2% phosphomolybdic acid as described above.

Brain tissues of different hamster-adapted strains were fixed in 10% buffered formalin solution. Before histological processes, all the fixed tissues were immersed in 98% formic acid for at least 1 h for inactivation. Paraffin sections (5 μm in thickness) were subjected to conventional staining with hematoxylin and eosin (HE). The spongiform degeneration for the three strains was monitored by light-microscopy and the severity and distribution of vacuolation were measured according to the protocol described elsewhere [26], briefly, 0, no lesions; 0.5, minimum vacuolation (2-3 vacuoles in half a × 40 objective field); 1.0, little vacuolation (3-5 vacuoles in half a field); 2.0, moderate vacuolation (several vacuoles evenly scattered); 3.0, extensive vacuolation (many vacuoles distributed in half a field); 4.0, severe vacuolation (numerous vacuoles often coalescing). For glial fibrillary acidic protein (GFAP), the sections were incubated with 1:500 diluted anti-GFAP mAb at 4°C overnight. Subsequently, the goat anti-mouse IgG biotinylated antibody diluted 1/200 in 10% normal goat serum was incubated for 30 min at room temperature, and an avidin-biotin-peroxidase complex was applied using diaminobenzidine (DAB) as a substrate. Finally, sections were counterstained with hematoxylin for 1 min, dehy-drated, and routinely mounted [11].

The brain samples of the infected hamsters and mice were homogenized in 10% lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH 7.5) according to the protocol described elsewhere [27]. PrP^Sc-enriched fractions of brain homogenate prepared from the different strains were centrifuged at 1000 rpm for 10 min and the cell debris was removed. The supernatants were collected and centrifuged at 20 000 g for 90 min, and the pellets were treated again as above. Then the pellets were mixed and stored at -80°C as the purified insoluble PrP^Sc.

The brain homogenates from three biological samples of each strain collected at the terminal stage were pooled as the representative samples of individual strains. For detection of PK-resistant PrP (PrP^res) in brain tissues, the samples were incubated with 50 μg/ml of PK (Merck) at 37°C for 60 min. For evaluation of PK-resistances of PrP^Sc from various hamster-adapted strains, the brain specimens were treated with different amounts of PK at the final concentrations of 20, 50, 100, 200, 500 and 1000 μg/ml at 37°C for 60 min. The digestions were stopped with 3 mM PMSF (Sigma) for Western blot. Different strain samples were undergone with 3 separate PK treatments.

Samples were separated in 12% SDS-PAGE and electronically transferred to a nitrocellulose membrane according to the protocol described elsewhere [28]. For the mouse-derived specimen, PrP-specific monoclonal antibody (mAb) 1E4 (dilution 1:1000) was used as the primary antibody. For the hamster-derived samples, PrP-specific mAb 1E4, 6D11, 3 F4, 6H4 and 8H4 (dilution 1:1000) were used respectively. The reactions were conducted in TBS-T (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.05% Tween 20) containing 5% (wt/vol) nonfat milk at 4°C overnight and subsequently washed three times with TBS-T. Then the membranes were incubated with horseradish peroxidase-conjugated (HRP)-conjugated goat anti-mouse immunoglobulin G (Santa Cruz) at 37°C for 1 h and PrP-specific signals was detected with an ECL detection kit (Amersham-Pharmacia Biotech).

After mixed with equal volume of glycoprotein denaturing buffer (New England Biolabs), various PrP^Sc preparations were heated at 100°C for 10 min. Subsequently, 50 mM sodium phosphate, pH 7.5, containing 1% NP-40 and 2 μl of N-glycosidase F (1,800,000 U/mg, New England Biolabs) were added into the samples and the mixtures were incubated at 37°C for 2 h. PrP signals in each preparation were detected by Western blot as described above.

100 μl of the representative sample each strain mentioned above were mixed with five volumes of cold methanol at -20°C for 2 h. After centrifuged at 20 000 g for 30 min, the pellets were resuspended with 100 μl of different concentration of GdnHCl, including 1, 2, 3, 4, 5 and 6 M and incubated at 37°C for 12 h as described elsewhere [29]. Subsequently, five volumes of cold methanol was added into each preparation and maintained at -20°C for 2 h. After centrifuged at 20 000 g for 30 min, the pellet was resuspended with 100 μl TN buffer (10 mM Tris, 130 mM NaCl, pH 7.0). Half part was employed directly into SDS-PAGE and the rest was subjected into PK-digestion (50 μg/ml) at 37°C for 1 h Different strain samples were undergone with 3 separate GdnHCl and PK treatments.

Quantitative analysis of immunoblot images was carried out using computer-assisted software Image Total Tech (Pharmacia). Briefly, the image of immunoblot was scanned with Typhoon (Pharmacia) and digitalized, saved as TIF format. The values of each target blot were evaluated. All data are presented as the mean ± SD.