PNGase digestion of vCJD-infected Tg340 and vCJD-PMCA-TgNN6h mice brains.

Samples were digested with 170 μg/mL PK and 3 ng/μL PNGase F, loaded on SDS-PAGE and subjected to Western Blot using monoclonal antibody 3F4 (1:10,000). Note that PNGase treatment reduced vCJD-infected Tg340 three-band pattern to a single band of approximately 19 kDa, but did not affect the banding pattern of vCJD-PMCA-infected TgNN6h, which in addition retained the ~15-kDa band that we have previously observed in this unglycosylated model, assumed to be an endogenous proteolytic fragment. PNGase digestion did not cause the emergence of the 15-kDa band in Tg340 samples, indicating that it is not the unglycosylated form of a fragment present in Tg340 brains. (DOCX)</p

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.</p

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).</p

PrP^Sc 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.</p

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).</p

Hematoxylin-eosin and Congo Red staining of a florid plaque observed in the thalamus of a sBSE-PMCA inoculated TgNN6h mouse (x40).

The core and the radiating spicules of the florid plaques observed in TgNN6h mice were Congo Red positive, indicating an amyloid fibrils organization. (DOCX)</p

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.</p

Inoculation of BoTg110 mice with non-glycosylated BSE isolates and cattle BSE.

Inoculation of BoTg110 mice with non-glycosylated BSE isolates and cattle BSE.</p

Inoculation of TgNN6h and Tg340 mice with BSE (and BSE-derived) prions.

Inoculation of TgNN6h and Tg340 mice with BSE (and BSE-derived) prions.</p

<i>In vitro</i> titration of BSE infectivity present in the original BSE inoculum and the PMCA-adapted, then TgNN6h-passaged BSE, sBSE and pBSE inocula.

Two serial PMCA rounds of these inocula were performed on BoTg110 substrate, and the PMCA products were digested with 85 μg/mL PK, loaded on SDS-PAGE and subjected to Western Blot using monoclonal antibody 6H4 (1:10,000); only the second PMCA round is shown. Note that, while the original inoculum (from cattle) was able to propagate down to a 10−5 dilution in two rounds of serial PMCA, the TgNN6h-passaged inocula were able to amplify only at a 10−1 dilution, indicating that their infectious titers were significant lower. (DOCX)</p