Emergence of a novel bovine spongiform encephalopathy (BSE) prion from an atypical H-type BSE

The H-type of atypical bovine spongiform encephalopathy (H-BSE) was serially passaged in bovinized transgenic (TgBoPrP) mice. At the fourth passage, most challenged mice showed a typical H-BSE phenotype with incubation periods of 223 ± 7.8 days. However, a different phenotype of BSE prion with shorter incubation periods of 109 ± 4 days emerged in a minor subset of the inoculated mice. The latter showed distinct clinical signs, brain pathology, and abnormal prion protein profiles as compared to H-BSE and other known BSE strains in mice. This novel prion was transmitted intracerebrally to cattle, with incubation periods of 14.8 ± 1.5 months, with phenotypes that differed from those of other bovine prion strains. These data suggest that intraspecies transmission of H-BSE in cattle allows the emergence of a novel BSE strain. Therefore, the continuation of feed ban programs may be necessary to exclude the recycling of H-BSE prions, which appear to arise spontaneously, in livestock. Such measures should help to reduce the risks from both novel and known strains of BSE.

Neuropathological examination. The degree of brain vacuolation and neuroanatomical distribution patterns of PrP Sc in TgBoPrP mice with BSE-SW were different from H-BSE and C-BSE (Fig. 2). The lesion scores of BSE-SW were lower than H-BSE scores in most brain areas, with the exception of dorsal medulla, cerebellar cortex, and thalamus (Fig. 2a). Regarding the PrP Sc deposition and distribution patterns in BSE-SW, punctuate and granular PrP Sc deposition was observed in the septal nucleus, corpus callosum, habenular nucleus, hypothalamus, superior colliculus, mesencephalic tegmentum, vestibular nucleus, cochlear nucleus, and reticular formation of the brainstem. Relatively heavy deposition of PrP Sc , consisting mainly of intraglial and intraneuronal staining, was conspicuous in the white matter of the cerebellum of BSE-SW in comparison to H-BSE (Fig. 2e,f,k,l). The PrP Sc staining was minimal in the cerebral and cerebellar cortex (Fig. 2c). PrP-plaques in the subcallosal region were less frequent in BSE-SW than in H-BSE (Fig. 2h,i). In contrast, the most conspicuous feature of PrP Sc staining type in TgBoPrP mice infected with H-BSE comprised heavily deposited plaques and/or large aggregates, which mainly located in the subcallosal and adjacent periventricular area of the brain (Fig. 2h). Such plaque deposits were occasionally detected in the cerebral and cerebellar cortex, but were not present in the thalamus or the brainstem. The plaques displayed birefringence with Congo red under polarized light (data not shown). The minimal PrP Sc staining in the cerebral and cerebellar cortex was similar to BSE-SW (Fig. 2b) (Fig. 3f). These values were not significantly different. Signal intensity of the diglycosylated PrPcore #1 of BSE-SW was weaker than that of H-BSE during 3.0-3.5 M treatment (Fig. 3d). This result was consistent with the mAb 6H4 experiment (Fig. 3c). However, the lower three  bands of PrPcore #2 showed similar signal intensities during 3.5-4.0 M treatment (Fig. 3b,d). This revealed that the conformational stability of PrPcore #1, but not PrPcore #2, is different in PrP Sc from H-BSE and BSE-SW.
Transmission of BSE-SW to cattle. To examine whether the BSE-SW prion could become a threat as a novel prion disease in cattle, three Holstein calves were challenged intracerebrally. All the inoculated animals developed progressive neurological disease. The animals exhibited initial clinical signs of the disease between 11.5 and 12.5 months post-inoculation (mpi), which included disturbance, mild fear or anxiety, mild gait changes, and, occasionally, low head carriage. After one to two months of the initial clinical signs, the animals were leaning towards the floor and rested their heads against the wall, which was followed by ataxia of the hind limbs that progressed to difficulty in getting up without assistance at the clinically terminal stage of the disease. The bodily condition gradually worsened because of a loss of weight during the two to three month clinical duration. 1, dorsal medulla; 2, cerebellar cortex; 3, superior cortex; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septal nuclei; 8, cerebral cortex at the level of the hypothalamus and thalamus; and 9, cerebral cortex at the level of the caudate nuclei. The data are presented as mean ± standard deviation (n = 7). Black circles, H-BSE; Diamonds, BSE-SW; Black triangles, C-BSE. The degree of vacuolation in the brain of BSE-SW was different from H-BSE and C-BSE (a). PET blots with mAb SAF84 corresponding to the brain areas at the level of thalamus (b-d), and the level of medulla and cerebellum (e-g) are shown. IHC was performed with mAb F99/97. 6 None of the animals exhibited anorexia, nervousness, or aggression, and responded to visual, acoustic, and tactile stimuli throughout the course of the disease. The cattle were eventually culled at 13.3 mpi, 15 mpi, and 16.2 mpi before astasia, in accordance with the welfare guidelines for animal experiments. The incubation periods of cattle infected with BSE-SW (14.8 ± 1.5 mpi) were shorter than those for H-BSE, C-BSE, and L-BSE (Table 2). Obex samples from these cattle were subjected to routine BSE confirmatory tests.

Histopathology and PrP Sc immunohistochemistry of the obex of BSE-SW-affected cattle.
Mild vacuolation of the extracellular neuropil was observed in the dorsal motor nucleus of the vagus nerve (DMNV), the solitary nucleus, the nucleus of trigeminal nerve spinal tract, and the olivary nucleus in all animals.
No intraneuronal vacuolation was seen. Spongy changes were not prominent in the gray matter of medulla oblongata at the obex (Fig. 4a). Immunolabeling of PrP Sc with mAb F99/97.6.1 resulted in intraneuronal and intraglial patterns throughout the obex (Fig. 4b). Intraneuronal labeling was less common in DMNV and the hypoglossal nucleus compared to other nuclei. Fine and coarse granular PrP Sc was sparsely distributed throughout the neuropil of reticular formation. No other extracellular types of PrP Sc , such as plaque-like and stellate deposits, were identified in the obex region.  5). The molecular mass of PrPcore #1 of BSE-SW, as determined by mAb 6H4, was lower than H-BSE and similar to C-BSE (Fig. 5b). MAb P4 did not detect PrPcore of BSE-SW (Fig. 5a). PrPcore #2 was also observed in BSE-SW (Fig. 5c,d). These results revealed that the biochemical properties of BSE-SW have indeed been transmitted to cattle.

Discussion
Our previous reports have revealed the usefulness of TgBoPrP mice for characterizing BSE prions 16,21,22 . In this study, a novel BSE, BSE-SW, was detected using this mouse model. The incubation periods of H-BSE, L-BSE, and C-BSE prions in the TgBoPrP mice were approximately 215 days, 150 days, and 190 days, respectively 16,21,22 . The BSE-SW prion showed the shortest incubation period (approximately 90 days) among the known BSE prions. We have previously performed several transmission experiments of sheep scrapie to TgBoPrP mice, but their incubation periods were over 170 days 23 , and we have not observed any prions with ~90-day incubation periods in these mice. In addition, the biochemical and biological properties of PrP Sc from BSE-SW were clearly different from C-BSE, L-BSE, H-BSE, and sheep scrapie (data not shown). PrP Sc of BSE-SW has some similarity to H-BSE on the account of the presence of truncated 12-kDa fragments (PrPcore #2). Fig. 6 shows the putative PK digestion site of PrP Sc from BSE-SW, as assessed by immunoreactivity with mAbs P4, 6H4, and SAF84. These results argue against the possibility that the BSE-SW prion resulted from a contamination of other laboratory prion strains. It is known that sheep scrapie comprises different prion strains 24,25 , and some affected sheep harbor these mixed scrapie prion strains 3 . Numerous scrapie strains had emerged in the course of several passage histories 26 . We have also previously isolated distinct scrapie strains from the brain of a scrapie-affected sheep after primary passage in wild type mice 27 . The different scrapie prion strains appeared after primary passage in sheep scrapie cases, which was considered to be due to prion strain selection. For H-BSE prions, French and Polish cases were reported, which transformed into a C-BSE-like phenotype during mouse passages [18][19][20] , revealing their potential heterogeneity. However, the BSE-SW prions described herein appear to have emerged by a different manner than our previous scrapie case 27 . The average incubation period of H-BSE in TgBoPrP mice was 320 days at first passage, and then shortened to 227 days and 216 days at secondary and third passages, respectively. Based on this observation, H-BSE prions were adapted to TgBoPrP mice at second passage. By the third passage, no multiple prion strains in the H-BSE material were evident. BSE-SW prion with a shorter incubation period emerged after H-BSE has adapted to TgBoPrP mice. The results of this study suggest that BSE-SW emerged through a conformational rearrangement. Alternatively, the BSE-SW prion was only present in a very small titer in H-BSE and required several passages in sensitive animals to emerge after selection from a mixture of preexisting prion strains. The Canadian H-BSE sample that was used in this study was also used to inoculate wild type mice, but   we did not observe the emergence of a C-BSE-like phenotype in those circumstances (unpublished data). The underlying mechanisms of the emergence of new prion strains are important to elucidate prion heterogeneity. This novel BSE strain has never been observed in field BSE cases, but our experiments reveal the potential risk associated with H-BSE. It has been suggested that different conformations of PrP Sc are involved in the prion strain diversity 28 , and that rearrangement of PrP Sc from a uniform conformation causes the emergence of new host-adapted PrP Sc 3 . PrP Sc of BSE-SW exhibited different conformational stability from H-BSE. It is also known that strain "mutation or transformation" may occur upon intraspecies transmission, where the PrP amino acid sequences of the host and the donor are identical 3 . These finding are consistent with the conclusion that PrP Sc of BSE-SW has a different conformation than H-BSE.

BSE-SW C-BSE L-BSE H-BSE
Furthermore, our new strain was successfully transmitted to cattle. Standard diagnostic testing for BSE confirmed the presence of spongiform changes associated with PrP Sc accumulation in the obex, and the challenged cattle fulfilled BSE criteria (Figs 4 and 5). The disease phenotype and features of PrP Sc , different from the known types of BSE, indicated that this prion could cause a novel type of atypical BSE. The shorter incubation periods in cattle were consistent with the relative incubation periods in TgBoPrP mice, and indicate high virulence of this  novel prion. Further analysis of diseased cattle is necessary to clarify its characteristics. Such studies could help to elucidate the mechanisms of conformational change in PrP Sc , which lead to the propagation of new prion strains.
The ban on meat-and-bone meal in livestock feed has contributed to the decline in C-BSE occurrences 6 . Recently, easing of the BSE-related regulations and control measures has been discussed. The origin of atypical BSE remains unknown, but it has been proposed to be spontaneous or sporadic 29 . H-BSE has been reported to transform into a C-BSE-like phenotype during animal passages [18][19][20] . Furthermore, we have shown here that the sequential transmission of H-BSE in TgBoPrP mice, i.e., to a homologous bovine PrP context, generated a novel type of BSE. Considering these observations, a continuous feed ban program may be necessary even after C-BSE is eradicated. Prohibiting the recycling of spontaneously occurring H-BSE prions in cattle should help to prevent both re-emerging and emerging types of BSEs.

Ethics statement. Procedures involving animals have been approved by the Animal Care and Use
Committee at the National Institute of Animal Health (approval ID: 11-008, 13-005). Animal experiments were performed in accordance with the Guidelines for Animal Transmissible Spongiform Encephalopathy Experiments of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
Transgenic mice. TgBoPrP mice overexpressing the bovine PrP gene (encoding BoPrP) in a mouse PrP deficient background were used. These mice harbored the cattle PrP gene containing six copies of the octarepeat sequence (EMBL, X55882), and produced approximately eight times more BoPrP per gram of protein than found in the cattle brain 30 . BSE material. Brain samples of Canadian H-BSE cattle, courteously provided by Dr. S. Czub, were used in this study 31 . Mouse-passaged L-BSE and C-BSE prions were also used. These prions were routinely maintained by serial passaging into TgBoPrP mice, as described previously 21,22 . C-BSE and L-BSE prions were adapted to TgBoPrP mice by serial passaging, and their incubation periods were approximately 190 days and 150 days, respectively. Brain samples from C-BSE, L-BSE, and H-BSE-affected cattle were also used 10,32,33 . Mouse transmission study. Brain tissues from BSE-affected animals were homogenized in nine volumes of phosphate buffered saline (PBS) using a multi-bead shocker (Yasui Kikai) and centrifuged at 1,000 × g for 5 min at room temperature (RT). Three-week-old female TgBoPrP mice were inoculated intracerebrally with 20 μl supernatant. Following inoculation, clinical status of the mice was monitored daily to assess the onset of neurological signs. The brains of diseased mice were removed and stored at -80 °C for biochemical analysis or fixed for histopathology. For the fourth passage, eight diseased mice brains from the third passage were used to independently challenge to TgBoPrP mice.
Cattle transmission study. Three female 3-4-month-old Holstein calves were challenged intracerebrally with 1 ml of 10% brain homogenate of BSE-SW-affected TgBoPrP mice, as described previously 10 . Animals were euthanized before ataxia deterioration. The brains of diseased cattle were removed and stored at − 80 °C for western blotting analysis or fixed for pathological examination (i.e., BSE confirmatory tests).
Western blot analysis. Samples were separated by SDS-PAGE and blotted electrically onto a PVDF membrane (Millipore). The blotted membrane was incubated with mAbs P4, 6H4, and SAF84 at RT for 1 h. MAb binding was detected by horseradish peroxidase-conjugated anti-mouse IgG. Signals were developed with a chemiluminescent substrate (SuperSignal; Pierce Biotechnology).

Neuropathology, immunohistochemistry, and PET-blot analysis. Histopathological analysis of
TgBoPrP mice and cattle was performed according to a previously described method 10,16,21 . Briefly, the brains were fixed in 10% buffered formalin solution (pH 7.4) containing 10% methanol. The formalin-fixed brains were immersed in 98% formic acid, and embedded in paraffin wax. Sections (4 μm thick) were cut and stained with hematoxylin and eosin (HE). The lesion profile was determined by scoring the vacuolar changes in nine standard grey matter areas, as previous described 37 . For PrP Sc immunohistochemistry (IHC), sections were Scientific RepoRts | 6:22753 | DOI: 10.1038/srep22753 incubated with mAbs SAF84 or F99/97.61, followed by incubation with anti-mouse universal immunoperoxidase polymer (Histofine Simple Stain MAX-PO (M), Nichirei) as the secondary antibody, and visualized using 3,3′ -diaminobenzidine tetrachloride as the chromogen. Finally, the sections were counterstained with hematoxylin. Paraffin embedded tissue (PET) blot was performed as described previously 21 . Dewaxed membranes were treated with 80 μg/ml of PK for 30 min at 37 °C, and then denatured using 3 M guanidine thiocyanate for 10 min at RT. The blotted membranes were incubated with mAb SAF84 for 90 min at RT. Signals were detected using Histofine MAX-AP (M) Kit (Nichirei) with 5-bromo 4-chloro-3-indolyl phosphate and nitro blue tetrazolium (BCIP/NBT; Roche Diagnostics) as substrates.
Conformational stability assay. Conformation stability assay was performed according to a previously described method with minor modification 38 . Briefly, 50 μl of 10% brain homogenate were added to an equal volume of guanidine hydrochloride (GdnHCl), concentration range 0-8 M. Mixed samples were incubated at 37 °C for 1 h. The samples were diluted by the addition of 850 μl Tris buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton-X, and 0.5% deoxycholate. Following this, 50 μL GdnHCl were added to each sample to obtain 0.4 M final concentration. Next, the samples were digested with 20 μg/ml PK at 37 °C for 1 h. PrP Sc concentration and western blot analysis were carried out as described above. Conformational stability was examined using mAbs 6H4 and SAF84. Denaturation curves were obtained by densitometric analysis using Fluorochem software (Alpha Innotech Co.). GdnHCl concentration at half maximal denaturation ([GdnHCl] 1/2 ) was used as a measure of the relative conformational stability of PrP Sc .