Fig 1.
In vitro propagation assay of different BSE sources on TgNN6h substrate (A), normal cattle brain substrate (B) and normal human brain substrate (C). The color scale represents the proportion of positive tubes (showing proteinase K-resistant PrP by Western blot) out of the total number of tubes subjected to PMCA (n = 4).
Fig 2.
Biochemical analysis of PMCA-propagated BSE prions on TgNN6h and normal cattle and human brain substrates.
Original inocula: brain homogenates from BSE-infected cattle, BSE-infected sheep (sBSE), BSE-infected pig (pBSE), or a vCJD patient; PMCA in TgNN6h substrate: isolates generated in vitro after 19 rounds of PMCA in TgNN6h substrate; PMCA in cattle substrate: isolates generated in vitro after 13 rounds of PMCA in wild-type bovine substrate; PMCA in human substrate: isolates generated in vitro after 16 rounds of PMCA in wild-type human substrate. Original inocula and samples propagated in cattle and human substrate were digested with 85 μg/mL of proteinase K (PK), while TgNN6h-propagated samples were digested with 170 μg/mL PK, and analyzed by Western blot using monoclonal antibody 6H4 (1:10,000); bands corresponding to incomplete digestion are marked with asterisks. Undigested TgNN6h, cattle, and human substrates were loaded as controls.
Table 1.
Inoculation of TgNN6h and Tg340 mice with BSE (and BSE-derived) prions.
Fig 3.
PrPSc detection from first and second-passage BSE-PMCA, sBSE-PMCA, pBSE-PMCA, and vCJD-PMCA inoculated TgNN6h mice, as well as direct vCJD-inoculated Tg340 mice.
10% brain homogenates from challenged mice were digested with 170 μg/mL of proteinase K and analyzed by Western blot using 3F4 antibody (1:10000). Undigested 10% brain homogenate from TgNN6h (non-glycosylated human PrPC) and TgWV (normally glycosylated human PrPC) were included as controls.
Fig 4.
Neuropathological features of TgNN6h mice inoculated with PMCA-propagated BSE prions.
A Spongiosis and PrPSc deposition profiles. Spongiform lesions and PrPSc deposition were evaluated semiquantitatively on a scale of 0 (absence of lesions/deposits) to 5 (high intensity lesion/deposition) in the following brain areas: frontal cortex (Fc), parietal cortex (Pc), septal area (Sa), corpus callosum (Cc), hippocampus (Hc), thalamus (T), hypothalamus (Ht), mesencephalon (Mes), pons (Po), cerebellum (Cbl), and medulla oblongata (Mo). All BSE inocula showed almost identical neuropathological profiles when transmitted to TgNN6h mice. B Hematoxylin and eosin staining and immunohistochemical analysis of the brains of BSE-PMCA, sBSE-PMCA, pBSE-PMCA or vCJD-PMCA affected mice showed the presence of conspicuous plaque-like deposits, surrounded by areas of severe spongiform change (florid plaques). Scale: x20, insert pictures: x60. Immunohistochemistry was performed using the 3F4 antibody (1:1,000). Brain areas: Parietal cortex (BSE-PMCA), mesencephalon (sBSE-PMCA), and thalamus (pBSE-PMCA and vCJD-PMCA).
Table 2.
Inoculation of BoTg110 mice with non-glycosylated BSE isolates and cattle BSE.
Fig 5.
Recovery of BSE banding pattern on passage from TgNN6h to BoTg110 of BSE, sBSE and pBSE isolates.
Brain homogenates at 1% from challenged mice were digested with 85 μg/mL (for original isolates) or 170 μg/mL (for TgNN6h and BoTg110 brains) of proteinase K and analyzed by Western blot using 6H4 antibody (1:10,000). A transition from a classical three-banded BSE pattern in the original isolates to a single 19-kDa band-containing pattern in TgNN6h mice was observed, followed by a complete recovery of the three-banded pattern in BoTg110 mice. Note that the banding pattern of the pBSE original isolate differs from the classical BSE pattern in being predominantly monoglycosylated (a hallmark imposed by porcine PrPC), while after passage to BoTg110 the pattern is identical to those of other BSE sources. Red arrows indicate passage history.
Fig 6.
Neuropathological features of BoTg110 mice inoculated with non-glycosylated BSE isolates and normally glycosylated cattle BSE.
A Spongiosis and PrPSc deposition profiles. Spongiform lesions and PrPSc deposition were evaluated semiquantitatively on a scale of 0 (absence of lesions/deposits) to 5 (high intensity lesion/deposition) in the following brain areas: frontal cortex (Fc), parietal cortex (Pc), septal area (Sa), corpus callosum (Cc), hippocampus (Hc), thalamus (T), hypothalamus (Ht), mesencephalon (Mes), pons (Po), cerebellum (Cbl), and medulla oblongata (Mo). Non-glycosylated isolates generated very similar neuropathological features to those of cattle BSE when transmitted to BoTg110 mice. B Immunohistochemical analysis of the brains of BoTg110 mice inoculated with the different isolates (pons). All inocula produced intense vacuolation and PrPSc plaques especially intense at the pons level. Immunohistochemistry was performed using the 6H4 antibody (1:1,000).