Molecular and transmission characteristics of primary-passaged ovine scrapie isolates in conventional and ovine PrP transgenic mice

Abstract

A more complete assessment of ovine prion strain diversity will be achieved by complementing biological strain typing in conventional and ovine PrP transgenic mice with a biochemical analysis of the resultant PrPSc. This will provide a correlation between ovine prion strain phenotype and the molecular nature of different PrP conformers associated with particular prion strains. Here, we have compared the molecular and transmission characteristics of ovine ARQ/ARQ and VRQ/VRQ scrapie isolates following primary passage in tg338 (VRQ) and tg59 (ARQ) ovine PrP transgenic mice and the conventional mouse lines C57BL/6 (Prnp(a)), RIII (Prnp(a)), and VM (Prnp(b)). Our data show that these different genotypes of scrapie isolates display similar incubation periods of >350 days in conventional and tg59 mice. Facilitated transmission of sheep scrapie isolates occurred in tg338 mice, with incubation times reduced to 64 days for VRQ/VRQ inocula and to </=210 days for ARQ/ARQ samples. Distinct genotype-specific lesion profiles were seen in the brains of conventional and tg59 mice with prion disease, which was accompanied by the accumulation of more conformationally stable PrPSc, following inoculation with ARQ/ARQ compared to VRQ/VRQ scrapie isolates. In contrast, the lesion profiles, quantities, and stability of PrPSc induced by the same inocula in tg338 mice were more similar than in the other mouse lines. Our data show that primary transmission of different genotypes of ovine prions is associated with the formation of different conformers of PrPSc with distinct molecular properties and provide the basis of a molecular approach to identify the true diversity of ovine prion strains.

FIG. 1.

Western blot analysis of ARQ/ARQ and VRQ/VRQ ovine scrapie isolates. Homogenates of ARQ/ARQ and VRQ/VRQ ovine scrapie isolates were prepared as described in Materials and Methods and analyzed by SDS-PAGE and Western blotting using anti-PrP monoclonal antibody P4 at 1 μg/ml. (Top) Homogenates were treated with various concentrations of PK or not treated with PK. (Bottom) Tracks 1 to 4, 10% homogenate; track 5, 5% homogenate; track 6, 2.5% homogenate; tracks 1 and 3, no PK; tracks 2 and 4 to 6, PK at 32 μg/ml. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots.

FIG. 2.

Lesion profiles induced by primary transmissions of ovine scrapie isolates. Prion-infected brains were harvested from mice that had developed terminal prion disease and were subjected to neuropathological examination for the presence and severity of spongiform neurodegeneration. The lesion profiles for C57BL/6 (a and f), RIII (b and g), VM (c and h), tg59 (d and i), and tg338 (e and j) mice inoculated with ARQ/ARQ (a to e) or VRQ/VRQ (f to j) sheep scrapie isolates are shown. The lesion profiles were induced by SE1848/0007 (ARQ/ARQ) (thick line in panels a to e), SE1848/0008 (ARQ/ARQ) (dashed line in panels a to e), and SE1848/0005 (VRQ/VRQ) (thick line in panels f to j), and SE1848/0006 (VRQ/VRQ) (dashed line in panels f to j). The data shown are mean lesion profile scores (three or more brains examined) for the following areas of the brain: for grey (G) matter, G1, dorsal medulla nuclei; G2, cerebellar cortex of the folia, including the granular layer, adjacent to the fourth ventricle; G3, cortex of the superior colliculus; G4, hypothalamus; G5, thalamus; G6, hippocampus; G7, septal nuclei of the paraterminal body; G8, cerebral cortex (at the level of G4 and G5); G9, cerebral cortex (at the level of G7); for white (W) matter, W1, cerebellar peduncles; W2, white matter in lateral tegmentum; W3, cerebellar peduncle/internal capsule. No data were obtained for SE1848/0007 in VM mice (c).

FIG. 3.

Immunohistochemistry of prion-infected mouse brains. (A and B) PrPSc patterns in the cortex and hippocampus of C57BL/6 mice inoculated with ovine ARQ/ARQ and VRQ/VRQ “good” transmitter isolates, respectively. Granular deposits were evident in both cases, but aggregates and plaques were observed only in the mice inoculated with ARQ/ARQ isolates. (C) PrPSc patterns in the thalamus of tg59 mice inoculated with ARQ/ARQ scrapie isolates showing granular PrPSc deposits and larger aggregates distributed throughout the thalamic region. (D) Granular PrPSc patterns in the hypothalamus of tg59 mice inoculated with ARQ/ARQ scrapie isolates. Large aggregates and plaques were also evident in the ventral thalamus. Plaques were the predominant PrPSc deposits in the ventral thalamus, while only rarely were small granular PrPSc deposits observed in the rest of the thalamic region. (E) PrPSc patterns in the thalamus of tg59 mice inoculated with VRQ/VRQ isolates showing granular PrPSc. Aggregates and plaques were observed only in the mice inoculated with ARQ/ARQ isolates. (F and G) PrPSc patterns in the cortex, hippocampus, thalamus, and dorsal hypothalamus of tg338 mice inoculated with ARQ/ARQ and VRQ/VRQ scrapie isolates, respectively. Fine granular deposits, not obvious at this magnification, were evident in both cases, but aggregates of PrPSc along the corpus callosum were observed only in the mice inoculated with ARQ/ARQ isolates. (H) High magnification of the hypothalamus from the same tg338 animal as in panel G to demonstrate PrPSc deposits located in the neuron, glial cells, and neurophil. Bars, 500 μm (A to G) and 50 μm (H).

FIG. 4.

Relative levels of total PrPSc in prion-infected C57BL/6, VM, tg59, and tg338 mouse brains measured by CDI. Prion-infected brains were harvested from mice that had developed terminal prion disease, and homogenates were prepared in Sarkosyl as described in Materials and Methods. Native PrPSc (not treated with GdnHCl) and denatured PrPSc (treated with 6 M GdnHCl) was captured by anti-PrP monoclonal antibody V24 and detected by biotinylated anti-PrP monoclonal antibody 6H4 followed by europium-labeled streptavidin. The data shown are the mean time-resolved fluorescence (TRF) counts per second (cps) plus standard deviation (error bar) for each treatment group. The brain homogenates were treated with 6 M GdnHCl (black bars) or not treated with GdnHCl (white bars). Statistical analysis of the data, with P values shown in the text, was performed using the two-tailed Student t test (unpaired samples).

FIG. 5.

Western blot detection of PrP. Prion-infected brains were harvested from mice that had developed terminal prion disease, and homogenates were prepared as described in Materials and Methods. Brain homogenates were treated with PK (+) or not treated with PK (−) and analyzed by SDS-PAGE and Western blotting with anti-PrP monoclonal antibody 683 for C57BL/6 and RIII mouse brains and monoclonal antibody P4 for tg59 and tg338 mouse brains. Each track was loaded with 25 μg of total protein. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots.

FIG. 6.

Conformational stability of ARQ- and VRQ-induced PrPSc. Prion-infected mouse brain homogenates were prepared as described in Materials and Methods. Brain homogenates were treated with GdnHCl at the final concentrations shown, incubated in the presence (+) or absence (−) of PK, and subsequently analyzed by SDS-PAGE and Western blotting with anti-PrP monoclonal antibody 683 (a and b) or monoclonal antibody P4 (c to f). The Western blots show PrP from C57BL/6 mice (a and b), tg59 mice (c and d), or tg338 mice (e and f). The mice were inoculated with ARQ/ARQ (a, c, and e) or VRQ/VRQ (b, d, and f) sheep scrapie isolates. The positions of molecular mass markers (in kilodaltons) are shown to the left of the blots.