Transmissibility of H-Type Bovine Spongiform Encephalopathy to Hamster PrP Transgenic Mice

<div><p>Two distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE) can be distinguished from classical (C-) BSE found in cattle based on biochemical signatures of disease-associated prion protein (PrP^Sc). H-BSE is transmissible to wild-type mice—with infected mice showing a long survival period that is close to their normal lifespan—but not to hamsters. Therefore, rodent-adapted H-BSE with a short survival period would be useful for analyzing H-BSE characteristics. In this study, we investigated the transmissibility of H-BSE to hamster prion protein transgenic (TgHaNSE) mice with long survival periods. Although none of the TgHaNSE mice manifested the disease during their lifespan, PrP^Sc accumulation was observed in some areas of the brain after the first passage. With subsequent passages, TgHaNSE mice developed the disease with a mean survival period of 220 days. The molecular characteristics of proteinase K-resistant PrP^Sc (PrP^res) in the brain were identical to those observed in first-passage mice. The distribution of immunolabeled PrP^Sc in the brains of TgHaNSE mice differed between those infected with H-BSE as compared to C-BSE or L-BSE, and the molecular properties of PrP^res in TgHaNSE mice infected with H-BSE differed from those of the original isolate. The strain-specific electromobility, glycoform profiles, and proteolytic cleavage sites of H-BSE in TgHaNSE mice were indistinguishable from those of C-BSE, in which the diglycosylated form was predominant. These findings indicate that strain-specific pathogenic characteristics and molecular features of PrP^res in the brain are altered during cross-species transmission. Typical H-BSE features were restored after back passage from TgHaNSE to bovinized transgenic mice, indicating that the H-BSE strain was propagated in TgHaNSE mice. This could result from the overexpression of the hamster prion protein.</p></div

Vacuolar lesion scores and molecular features of TgBoPrP mice inoculated with H-BSE prions passaged in TgHaNSE mice.

<p>(A) Lesion profiles of first-passage TgBoPrP mice inoculated with H-BSE from cattle (H-BSE/cattle; blue), and H-BSE passaged once in TgHaNSE mice (H-BSE/TgHa/TgBo; red). Vacuolation was scored on a scale of 0–5 in the following brain areas: 1, dorsal medulla; 2, cerebellum; 3, midbrain; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septal nuclei of the paraterminal body; 8 caudal cerebral cortex; and 9, rostral cerebral cortex. Data represent mean ± standard deviation (<i>n</i> = 6). (B) TgBoPrP mice inoculated with H-BSE passaged in TgHaNSE mice (TgHaNSE/TgBoPrP) yielded an additional fragment representing the traceback when probed with the SAF84 mAb. TgBoPrP mice were subdivided into two groups (<i>n</i> = 5 and 7) and inoculated on different days with the same brain homogenate prepared from first-passage TgHaNSE mice infected with H-BSE. Cattle, cattle infected with H-BSE; TgHaNSE, third-passage TgHaNSE mice infected with H-BSE. Molecular markers are shown to each side (kDa).</p

Molecular typing of PrP^res in brains of third-passage, H-BSE-infected TgHaNSE mice.

<p>(A) PrP^res expression was analyzed with the mAbs 6H4 and SAF84 before or after PNGase F deglycosylation. The unglycosylated fragment from TgHaNSE mice inoculated with H-BSE was similar in size to that of C-BSE and higher than that of L-BSE. Molecular markers are shown to the left (kDa). (B) PrP^res glycoform percentages in C-BSE-, L-BSE-, and H-BSE-infected mice were analyzed with the mAb T2. Glycoform ratios were similar between TgHaNSE mice inoculated with H-BSE and those inoculated with C- or L-BSE. Results are shown as mean ± standard deviation of triplicate experiments. Bar graph shows diglycosylated (white columns), monoglycosylated (gray columns), and unglycosylated (black columns) forms of the protein.</p

PrP^res profiles of H-BSE prions analyzed with mAbs 3F4, 6H4, T2, and SAF84.

<p>(A) Triple bands observed for TgHaNSE mice inoculated with H-BSE were smaller than those of cattle, C57BL/6 mice, and TgBoPrP mice analyzed with mAbs 3F4 and 6H4. TgHaNSE mice infected with H-BSE did not exhibit the 10–12-kDa band detected in cattle, C57BL/6 mice, and TgBoPrP mice infected with H-BSE, which were analyzed with the mAb SAF84. P1, P2, and P3, first, second, and third passage, respectively. TgHaNSE mouse-passaged H-BSE in TgBoPrP mice (H-BSE/TgHa/TgBoPrP) showed the molecular signatures of the H-BSE prion. (B) H-BSE (H), C-BSE (C), and L-BSE (L) strains showed similar molecular features in TgHaNSE mice. Molecular markers are shown to the left (kDa).</p

Neuroanatomical PrP^Sc distribution patterns in the brains of TgHaNSE mice infected with H-BSE, mouse-passaged C-BSE, or L-BSE.

<p>Representative images of coronal brain sections are shown. From left to right: septal level, hippocampus and thalamic level, midbrain, and medulla with cerebellum. PrP^Sc labeled with the mAb 3F4 was mainly distributed in the brainstem, thalamus, and hippocampus of TgHaNSE mice infected with H-BSE, but was deposited throughout the brain of mice infected with C-BSE. In L-BSE infected mice, PrP^Sc immunoreactivity was dense; the protein formed plaques in the periventricular and subcallosal regions. P1 and P2, first and second passage, respectively.</p

Western blot analysis of brains from first-passage, H-BSE-infected TgHaNSE mice.

<p>(A) Three of five mice showed positive immunoreactivity using the mAb T2. (B) PrP^res expression in the brains of TgHaNSE or C57BL/6 mice infected with H-BSE, as detected using mAbs 4E10, 3F4, and SAF84. Sc, mouse-adapted Obihiro scrapie control strain. Molecular markers are shown to the left (kDa).</p

Characteristics of monoclonal antibodies used in this study.

<p>Characteristics of monoclonal antibodies used in this study.</p

Features and types of PrP^Sc in TgHaNSE mice infected with H-BSE or mouse-passaged C-BSE.

<p>(A, B) Stellate-type PrP^Sc deposits (arrows) were observed in the cerebral cortex of H-BSE-infected mice (A), whereas particulate, punctuate, and stellate-type PrP^Sc were distributed in the cortex of C-BSE-infected mice (B). (C and D) Particulate (C) and fine punctuate (D) PrP^Sc immunoreactivity was observed in the red nuclei of the midbrain. (E–H) In the cerebellum, PrP^Sc immunoreactivity was particulate in the granular layer (E) and streaked in the molecular layer of H-BSE infected mice (G). In contrast, PrP^Sc was less prevalent in the molecular layer of C-BSE-infected mice; however, coarse particulate and stellate-type PrP^Sc accumulation was detected in the granular layer of cerebellar cortex and in the cerebellar medulla, respectively (F and H). P1 and P3, first and third passage, respectively.</p

Infection of mice with L- and S-type prions with or without passaging in GT1-7 cells.

<p>Infection of mice with L- and S-type prions with or without passaging in GT1-7 cells.</p

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells

<div><p>In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrP^Sc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 10⁵ times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.</p></div