Animal experiments were conducted in strict accordance with recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioral Research with Animals (Directive 86/609EC). All efforts were made to minimize detrimental effects on animals. Experiments were approved by the Committee on the Ethics of Animal Experiments of the INIA Institute (permit no. CEEA2009/004).

The open reading frame (ORF) of the bovine Prnp gene was isolated by PCR amplification and cloned in a pGEM-T vector as described (20). The ORF was mutated by using the QuikChange II-XL Kit (Stratagene, La Jolla, CA, USA) with specific oligonucleotides (5′-CGGTCAGTGGAACAAGCTCAGTAAGCCGAAAACC-3′ and 5′-GGTTTTCGGCTTACTGAGCTTGTTCCACTGACCG-3′) according to procedures of the manufacturer. The P113L-PrP ORF was excised from the cloning vector by using restriction enzyme XhoI and inserted into MoPrP.Xho vector (21), which was also digested with XhoI. This vector contains the murine PrP promoter and exon-1, intron-1, exon-2, and 3′-untranslated sequences. Transgenic mice were generated by microinjection of DNA according to a published procedure (22).

Brains were rapidly harvested from the skulls and fixed in 4% paraformaldehyde in phosphate buffer. Coronal slabs were embedded in paraffin and 5-μm sections of cerebrum, cerebellum, brain stem, and spinal cord were obtained by using a sliding microtome. De-waxed sections were stained with hemotoxylin and eosin, Congo red, or thioflavin, or processed for immunohistochemical analysis. Immunohistochemical analysis for detection of glial fibrillary acidic protein (GFAP) and cleaved caspase-3 was conducted by using a modified labeled streptavidin technique (LSAB2-System peroxidase; Dako, Glostrup, Denmark). The rabbit polyclonal antibody to GFAP (Dako) was used at a dilution of 1:600. Cleaved caspase-3 rabbit polyclonal antibody (D175, cell signaling) was used at a dilution of 1:50. Microglial cells were stained with the biotinylated lectin from Lycopericon esculentum (L-0651; Sigma, St. Louis, MO, USA) and used at a dilution of 1:100. PrP was immunolabeled with monoclonal antibody (mAb) 6H4 (Prionics, Schlieren, Switzerland) and used at a dilution of 1:30 in sections pretreated with 35% HCl for 2 min at 100°C and then with 96% formic acid for 10 min at room temperature. These procedures have been detailed elsewhere (23).

For transmission studies, we used the Tg110 mouse line (20) that expresses wild-type bovine-PrP^C in a mouse Prnp null background. Inocula were prepared from brain tissues as 10% (wt/vol) homogenates. Individually identified 6–10-week-old mice were anesthetized and inoculated with 2 mg of 10% brain homogenate in the right parietal lobe by using a 25-gauge disposable hypodermic needle. Mice were observed daily and neurologic status was assessed weekly. When progression of TSE was evident or evident at 650 days postinoculation (dpi), animals were euthanized for ethical reasons. Once mice were euthanized, brains were collected, frozen, and analyzed by Western blotting. Samples fixed in buffered 10% formalin underwent histologic analysis, immunohistochemical analysis, or histoblotting. Spleens were frozen for Western blot analysis.

Frozen mouse brain samples were prepared as 10% (wt/vol) homogenates in 5% glucose in distilled water in grinding tubes (Bio-Rad, Hercules, CA, USA) by using a TeSeE Precess 48 homogenizer (Bio-Rad) following the manufacturer’s instructions. Samples were analyzed by Western blotting as described (24). For immunoblotting experiments, mAbs Sha31 (25), 9A2 (26), 12B2 (26), Saf84 (25), and R145 (Vetyerinary Laboratories Agency, Weybridge, UK) were used at concentrations of 1 μg/mL. Sha31 recognizes ₁₅₆YEDRYYRE₁₆₃ epitope, 9A2 recognizes ₁₁₀WNK₁₁₂ epitope, 12B2 recognizes ₁₀₁WGQGG₁₀₅ epitope, Saf84 recognizes ₁₇₅RPVDQY₁₈₀ epitope, and R145 recognizes ₂₃₁RESQUA₂₃₅ epitope of the bovine PrP sequence.

All procedures involving brains from infected mice were performed as described (27). Samples were fixed in neutral-buffered 10% formalin (4% formaldehyde) before being embedded in paraffin. Once deparaffinated, 2-µm tissue sections were stained with hematoxylin and eosin. Lesion profiles of brains were established according to the standard method described by Fraser and Dickinson (28). For paraffin-embedded tissue blots, the protocol described by Andréoletti et al. (29) was used.

Seven lines (founders) of 113LBoPrP^C heterozygous transgenic mice carrying the endogenous murine Prnp gene (Prnp mu^+/− 113Lbo^+/−) were obtained. Lines 113LBoPrP-Tg037 and 113LBoPrP-Tg009 were selected on the basis of their expression levels, and bred to homozygosity in a murine Prnp null background. To achieve this expression, selected lines were crossed with Prnp null mice (Prnp mu^−/−) to achieve transgene-hemizygous lines (Prnp mu^−/− 113Lbo^+/−). Absence of the murine Prnp gene was determined by using PCR with specific primers. Transgene expression levels were then determined in brain homogenates by serial dilution and compared with PrP^C levels found in bovine brain homogenates. Transgene expression levels for the two 1-month-old mice with hemizygous Tg lines 113LBoPrP-Tg037 and 113LBoPrP-Tg009 were found to be ≈3× and 0.5×, respectively. Mutant 113LBoPrP expressed in 009 and 037 transgenic lines showed an electrophoretic profile similar to that of wild-type bovine PrP^C from BoPrP-Tg110 mice or cow brain, although only small differences in glycoform ratios were observed (Figure 1). Next, by crossing hemizygous animals, we obtained transgene-homozygous animals (Prnp mu^−/− 113Lbo^+/+) (30).

Spontaneous neurologic disease developed in 113LBoPrP-Tg037 mice expressing mutant 113LBoPrP. These mice had reduced lifespans compared with either non-Tg (Prnp^−/−) mice or transgenic mice expressing similar or higher levels of wild-type BoPrP (Table 1). However, disease did not develop in 113LBoPrP-Tg009 mice expressing low levels of mutant protein, and these mice had survival times similar to non-Tg (Prnp^−/−) mice or BoPrP-Tg110 mice. Onset of clinical signs and survival times were dependent on the expression level of 113LBoPrP (Table 1) (i.e., transgene-homozygous 113LBoPrP-Tg037 mice showed an earlier onset of clinical signs and reduced survival times than hemizygous mice of the same line). Neurologic alterations generally involved motor impairment with ataxia affecting mainly the hind limbs. Mice showed a rough coat and prominent hunch at the early stages of clinical signs. Most mice had a wobbling gait and slight paralysis in the back limbs. Some mice had conjunctivitis and showed compulsive scratching in the head area. At the end stage of the disease, mice had highly restricted movement and lethargy. No signs of hyperactivity were detected in these mice.

All 113LBoPrP-Tg037 mice at the terminal disease stage showed spongiosis in the cerebral cortex, thalamus, and hilus of the dentate gyrus, but not in the CA1 region of the hippocampus and granule cell layer of the dentate gyrus, compared with age-matched control BoPrP-Tg110 mice (Figure 2). Marked granule cell loss, spongiosis in the molecular layer, granule cell layer, subcortical white matter, and Bergmann glia hypertrophy and hyperplasia were also observed in these mice at the terminal disease stage. However, at the early stages of clinical signs, no spongiform changes were found, although neuronal loss was observed when 113LBoPrP-Tg037 mice were compared with age-matched control BoPrP-Tg110 mice. These findings were particularly evident in the hippocampus proper (including hilus) and granular cell layer of the cerebellum.

Changes were more pronounced in animals with severe clinical manifestations. Neurons with a shrunken cytoplasm and nucleus were observed in all 113LBoPrP-Tg037 mice, and these appeared in the molecular layer of the cerebellum, hippocampus (mainly plexiform layers and hilus), thalamus, and pons. Morphologic PrP aggregates, congophilic materials, or thioflavin-positive deposits were not detected in 113LBoPrP-Tg037 mice. Astrocyte gliosis was observed throughout the brain in 113LBoPrP-Tg037 mice, even at the early stage of the disease. The number and size of reactive astrocytes increased, as shown by immunolabeling with antibody against GFAP, in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum, and brain stem. Microglial proliferation, as visualized with Lycopericum esculentum lectin, was evident in brains of 113LBoPrP-Tg037 mice but not in age-matched control mice.

To explore changes in biochemical properties of mutant 113LBoPrP, we solubilized brain homogenates from control BoPrP-Tg110, 113LBoPrP-Tg037, and 113LBoPrP-Tg009 mouse lines in extraction buffer and ultracentrifuged them at 100,000 × g for 1 h. Western blotting of soluble and insoluble fractions indicated differential biochemical properties of mutant 113LBoPrP compared with wild-type BoPrP (Figure 3); the 113L mutation resulted in a more insoluble protein. This insolubility was detected in 113LBoPrP-Tg037 and 113LBoPrP-Tg009 mouse lines. However, when the 113LBoPrP-Tg009 mouse line was compared with the other 2 mouse lines, the expression level was lower and insolubility was detectable only when an 8-fold equivalent brain tissue mass was used (Figure 3, panel B) to obtain equivalent PrP signal.

Insolubility was detected early in the lifespan (30 days after birth) of mice (Figure 3, panel C), which suggested that quantification of 113LBoPrP would reflect a cumulative effect. PK resistance was not found in mutant 113LBoPrP or in wild-type BoPrP, which were digested at the PK concentration used (Figure 3).

To test potential infectivity of brains of mutant 113LBoPrP-Tg037 mice, we intracerebrally inoculated brain homogenates from sick animals into transgenic mice expressing wild-type bovine PrP (BoPrP-Tg110). In the first experiment (Table 2), we used a brain homogenate from a unique terminally sick animal. In this instance, neurologic signs developed in only 1 of 5 (attack rate 20%) inoculated Tg110 mice. This mouse died at 333 dpi and contained a considerable amount of PrP^res in its brain, as shown by Western blot (Figure 4).

When brain homogenate from this mouse was reinoculated into 6 Tg110 mice (second passage), neurologic signs developed in all recipients, and these mice showed a shorter mean ± SEM incubation period (272 ± 38 dpi), which was suggestive of an increased infectious titer that was maintained in subsequent passages (Table 2). These results were confirmed in a second independent experiment with a brain homogenate derived from a pool of 5 terminally sick 113LBoPrP-Tg037 mice. In this instance, 2 of 6 (attack rate 33%) inoculated mice were infected (Tg110 mice) (33% attack rate) and had incubation periods of 322 and 406 dpi, respectively. As in the first experiment, second passage (using a pool containing both mouse brains) produced an attack rate of 100% and a shorter incubation period (291 ± 23 dpi), which were maintained in subsequent passages (Table 2).

Brain homogenate from Tg110 mice expressing comparable amounts of wild-type BoPrP, as well as brain homogenate from healthy mice, was also used to inoculate uninfected mice, which served as negative controls. Neurologic signs did not develop in any of the inoculated mice (Table 2). These mice were euthanized at 650 dpi and did not show any PrP^res in their brains by Western blot.

For comparative studies, material from the brainstem of cows that contained classical bovine spongiform encephalopathy (BSE), atypical BSE-H, and atypical BSE-L prions was also inoculated into mice by the same procedure. These 3 inocula induced a typical neurologic disease after primary transmission and showed an attack rate of 100% (Table 2). Survivals times of mice inoculated with brainstem of 113LBoPrP-Tg037 mice on second and third passages were similar to those produced by the classical BSE-C isolate (Table 2), as well as by other isolates reported for the same Tg110 mouse line (20,22,32).

Western blot analysis with mAb 9A2 against brain-PrP^res produced by BoPrP-Tg110 mice infected with the new 113L-BSE prion (Figure 4, panel A) showed a typical BSE PrP banding pattern characterized by small fragments (19-kDa fragment for the aglycosyl band) and prominent diglycosylated species in all challenged PrP^res-positive mice. This result was indistinguishable from that produced by classical BSE-C prion in these mice but differed from that observed after inoculation with atypical BSE-H or BSE-L prions (Figure 4, panel A).

Further characterization of PrP^res with other mAbs showed that the new 113L-BSE prion was not recognized by mAb 12B2 (Figure 4, panel B), whose epitope (₁₀₁WGQGG₁₀₅ according to the bovine PrP sequence) is known to be poorly protected from PK digestion (26,32) in the classical BSE-derived prion but well preserved in the atypical BSE-H prion (Figure 4, panel B) (31). Furthermore, PrP^res immunolabeling with mAbs Saf84 and R145 showed that mice infected with the new 113L-BSE prion, in contrast to mice infected with the H-type prion, did not show the characteristic PrP^res band profile (4 bands) of cattle BSE-H, but showed a PrP^res-profile (3 bands) similar to that of the BSE-C prion (Figure 4, panels C, D).

Comparative study of PrP^Sc accumulation in spleen from Tg110-infected mice showed that mice infected with 113L-BSE or BSE-C prions consistently showed positive results for presence of PrP^res by Western blot. In contrast, no PrP^Sc deposits were detected in mice infected with either BSE-L or BSE-H prions. Similar results were obtained in subsequent passages.

We next examined vacuolation and PrP^Sc distribution in the brain, which are known to vary by strains/TSE prions (28,33). In general, we observed that PrP^Sc deposition patterns in brains of 113L-BSE–infected mice were different from mice infected with BSE-H or BSE-L prions, but these overlapped mostly with those infected with BSE-C prion (Figure 5). However, 113L-BSE–infected mice at the terminal stage of disease showed only spongiform changes that remained limited to the thalamus even after 3 passages (Figure 6). This finding was in contrast with lesion profiles observed in the mice infected with BSE-C, BSE-H or BSE-L prions (Figure 6) in which substantial vacuolar changes were observed in various brain areas. These results indicate that 113L-BSE is an authentic infectious prion that phenotypically differs from BSE-H and BSE-L prions but has biochemical characteristic and histopathologic features similar to those of the classical BSE-C prion.