Transmissions of native and Tg(BoPrP)-passaged BSE, vCJD, and scrapie prions to Tg(BoPrP)4125 (A, C, E) and Tg(BoPrP^+/+)4092 (B, D, F) mice. After inoculation with 1% homogenates of brain tissue, Tg mice were placed under observation for appearance of clinical signs of neurologic disease. The data represent a plot of the number of animals surviving without presenting signs of clinical disease versus time in days. The derivation of BSE, vCJD, and scrapie inocula used were described previously (57, 58). (A) Survival curves after inoculation with BSE prions. BSE brain (filled diamonds) indicates that brain tissue was derived from five independent cases of BSE: GJ248/85, SE1809/11, 97/1612, 97/1997, and PG31/90. Tg(BoPrP) BSE (open squares) indicates that brain tissue was derived from four separate Tg(BoPrP) mice infected with BSE prions. (B) Survival curves after inoculation with BSE case PG31/90 (filled diamonds). Tg(BoPrP) BSE (open squares) indicates that brain tissue was derived from two separate Tg(BoPrP) mice infected with BSE prions. (C and D) Survival curves after inoculation with vCJD prions. vCJD brain (filled diamonds) indicates that brain tissue was derived from five independent cases of vCJD: RU96/02, RU96/07, RU96/70, RU96/80, and RU96/110. Tg(BoPrP) vCJD (open squares) indicates that brain tissue was derived from three separate Tg(BoPrP) mice infected with vCJD prions. (E) Survival curve after inoculation with scrapie prions. Suffolk scrapie (filled diamonds) indicates that brain tissue was derived from five independent cases of scrapie: Oreo, T97-1104, no. 15, no. 139, and no. 340. (F) Survival curves after inoculation with scrapie prions. Suffolk scrapie (filled diamonds) indicates that brain tissue was derived from two independent cases of scrapie: no. 139 and no. 340. Tg(BoPrP) Suffolk scrapie (open squares) indicates that brain tissue was derived from four separate Tg(BoPrP) mice infected with sheep scrapie prions. For each panel, the mean incubation periods ± standard errors of the means are indicated; the values in parentheses signify the number of ill mice over the number of inoculated mice.

Survival curves from serial transmissions of BSE prions (A) and scrapie prions (B) to Tg(Bo3M,YIQII)23953 mice. Tg mice were inoculated with 1% brain homogenates. (A) BSE brain (filled diamonds) indicates inocula derived from BSE case PG31/90. Tg(Bo3M,YIQII) BSE (open squares) indicates inocula derived from two Tg(Bo3M,YIQII) mice infected with BSE prions. (B) Sc brain (filled diamonds) indicates inocula derived from two cases of scrapie: no. 139 and no. 27. Case no. 27 was described previously (64). Tg(Bo3M,YIQII) Sc (open squares) indicates inocula derived from two Tg(Bo3M,YIQII) mice infected with scrapie prions. Mean incubation periods ± standard errors of the means are indicated for each inoculum; the values in parentheses signify the number of ill mice over the number of inoculated mice.

Neuropathology and vacuolation in Tg(Bo3M,YIQII)23953 mice after passage of BSE, vCJD, and scrapie prions. (A to F) Neuropathology as seen by immunohistochemistry for PrP^Sc in Tg(Bo3M,YIQII)23953 mice after inoculation with BSE (A, B), scrapie (C, D), and vCJD prions (E, F) on first (left column) and second (right column) passage. (A, B, E) Hippocampal sections show massive aggregates of amyloid plaques in the subcallosal region after inoculation with BSE (A, B) and vCJD (E) prions. The BoPrP-specific 9095 antibody with a hematoxylin counterstain was used (58). The bar in panel B represents 100 μm and also applies to panels A and E. Hp, hippocampus; NC, neocortex. (C, D) Sections of the zona incerta stained with hematoxylin and eosin shows mild-to-moderate vacuolation in multiple brain regions after inoculation with scrapie prions. No amyloid plaques were found. The bar in panel D represents 70 μm and also applies to panel C. (F) Higher magnification of the large amyloid plaque at the far right edge of panel E. This plaque is ∼100 μm in diameter, has a radially arranged fibrous substructure like a kuru plaque, and is partially surrounded by vacuoles. Bar, 50 μm. CC, corpus callosum. (G to J) Histograms comparing the intensity and neuroanatomic distribution of vacuolation in the neuropil after inoculation with BSE (G, H) and scrapie (I, J) prions on first passage (G, I) and second passage (H, J). (G, H) Primary and secondary transmissions of BSE prions reveal virtually identical distributions of vacuolation in the brain. Little or no vacuolation was seen in the zona incerta and hypothalamus. (I, J) With scrapie prions, vacuolation was more widely spread. Upon serial transmission, vacuolation was absent in the thalamus proper and the habenula. Abbreviations are defined in the legend to Fig. 5.

Chimeric PrP transgenes. Constructs were created as described in Materials and Methods. The name of each construct, as referenced in the text, is shown at the left of each diagram illustrating the corresponding PrP ORFs. Regions corresponding to BoPrP are shown in gray, and regions derived from MoPrP are shown in white. The five residues that vary in the chimeric ORFs are diagrammed in their respective single-letter amino acid codes.

Conformational stabilities of native and Tg(BoPrP)-passaged BSE and scrapie prions. Sample processing, electrophoresis, and blotting were performed as described in Materials and Methods. Samples were denatured with different concentrations of GdnHCl and then either treated with proteinase K (+) or left untreated (−), as indicated. (A) Tg(BSE) indicates brain tissue derived from two Tg(BoPrP) mice infected with BSE prions, diluted five times with brain tissue from uninfected Tg(BoPrP) mice. Tg(Sc) indicates brain tissue derived from two Tg(BoPrP) mice infected with scrapie prions. The Tg(BSE) brain tissue was diluted with uninfected Tg(BoPrP) brain homogenate because scrapie-infected Tg(BoPrP) mice contain ∼5-times-lower amounts of PrP^Sc than BSE-infected Tg(BoPrP) mice (M. Scott and H.-O. Nguyen, unpublished data). (B) Densitometric analysis of the Western blots from panel A showing the percentage of protease-resistant protein plotted as a function of GdnHCl concentration. (C) Brain homogenate of a cow (case PG31/90) diagnosed with BSE. (D) Brain homogenate of a sheep (case no. 27) diagnosed with scrapie. (E) Brain homogenate derived from two Tg(BoPrP) mice infected with BSE prions and diluted five times with brain tissue from uninfected Tg(BoPrP) mice. (F) Brain homogenate derived from two Tg(BoPrP) mice infected with scrapie prions.

Neuropathology and vacuolation in Tg(BoPrP)4092 mice after inoculation with BSE, vCJD, and scrapie prions. (A to L) Neuropathology as seen by immunohistochemistry for PrP^Sc in Tg(BoPrP)4092 mice inoculated with BSE (A to D), vCJD (E to H), and scrapie (I to L) prions on first passage (left column) and on second passage (right column). (A, B, E, F, I, and J) Sections of the subcallosal region of the hippocampus show small aggregates of PrP amyloid plaques from BSE prions (A, B), large, discontinuous aggregates of PrP amyloid plaques from vCJD prions (E, F), and no amyloid plaques from scrapie prions (I, J). The bar in panel J represents 100 μm and applies to panels A, B, E, F, and I. (C, D, G, H, K, and L) Coronal sections of the raphe of the rostral pons show distribution of PrP^Sc deposits and vacuolation. Note that all sections of the raphe are shown lying horizontally, with the dorsal raphe to the left and the more ventral raphe to the right. Numerous PrP^Sc deposits surround vacuolation of the neuropil from BSE (C, D) and vCJD (G, H) prions. Only minute, punctate PrP^Sc deposits are scattered among the small number of vacuoles from scrapie prions (K, L). The bar in panel L represents 50 μm and applies to panels C, D, G, H, and K. (M to R) Histograms comparing the intensity and neuroanatomic distribution of vacuolation in the neuropil after inoculation with BSE (M, N), vCJD (O, P), and scrapie (Q, R) prions on first passage (M, O, Q) and second passage (N, P, R). Vacuolation is confined largely to the habenula and the raphe of the rostral pons in both the first and second passage of BSE (M, N) and vCJD (O, P) prions. Note the degree of vacuolation in the raphe is twice as intense during the second passage. Inoculation with scrapie prions (Q, R) resulted in widespread, mild-to-moderate vacuolation of multiple brain regions without amyloid plaque formation. NC, neocortex; PrC, pyriform cortex; Hp, hippocampus; Cd, head of caudate nucleus; Sp, septal nuclei; Th, thalamus proper; Hb, habenula; Hy, hypothalamus; Tg, overall tegmentum of the brainstem including the raphe; Rp, raphe of the brainstem; Cb, cerebellum; W, white matter.