Classical Bovine Spongiform Encephalopathy by Transmission of H-Type Prion in Homologous Prion Protein Context

TOC Summary: An epidemic agent could have originated from such a cattle prion.

T ransmissible spongiform encephalopathies, or prion diseases, are a group of neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, and bovine spongiform encephalopaty (BSE) in cattle. Prion diseases are characterized by specifi c histopathologic lesions and deposits of an abnormal conformational isoform (PrP Sc ) of the host-encoded physiologic prion protein (PrP C ) in the central nervous system. PrP Sc but not PrP C is partially resistant to digestion by proteinase K, resulting in an N terminally truncated prion protein termed PrP res that can be detected by Western blot and showing a characteristic banding pattern that refl ects the 3 PrP res glycoforms. The apparent molecular masses and relative quantities of these glycoforms are used in biochemical PrP res typing as the criteria to differentiate between prion diseases.
BSE is a prion epidemic that has caused the deaths of ≈200,000 cattle in Europe, mainly in the United Kingdom, since it emerged in 1985. Although multiple agent strains have been identifi ed in sheep scrapie (1,2) and human CJD (3,4), early evidence showed that BSE was caused by a single major strain (5,6) with the ability to effi ciently cross the species barriers and showing stable features even when transmitted to other species. Transmission of BSE to humans through contaminated food is believed to be responsible for variant CJD (vCJD) (7,8). Several authors reported that BSE and vCJD prions share similar strain-specifi c features, including a unique PrP res molecular signature (6,9,10), after transmission to mice or macaques,. However, other studies described the production of different PrP res molecular signature after BSE and vCJD prions transmission in wild-type (11) and human PrP transgenic mice (12,13).
Epidemiologic investigations identifi ed contaminated meat and bone meal as the vehicle that recycled the BSE agent in the cattle population (14). However, the origin of BSE remains under debate, and the disease has been hypothesized to have derived either from sheep scrapie or from a spontaneous bovine prion disease analogous to sporadic forms of CJD in human (15) or even from human transmissible spongiform encephalopathy (16).
More recently, 2 atypical forms of BSE have been identifi ed in several European countries (17), Japan (18,19), the United States (20), and Canada (21). Several studies suggest that these atypical disorders are associated with 2 distinct prion strains that are mainly characterized by distinct PrP res profi les, named high-type (H-type) and lowtype (L-type) according to the electrophoretic migration of the unglycosylated PrP res , which is higher (BSE-H) or lower (BSE-L) than classical BSE (BSE-C) (22). An additional distinctive signature of H-type and L-type PrP res is the smaller proportion of the diglycosylated PrP res compared with the classical-type (C-type) PrP res , more obvious in L-type BSE (23)(24)(25).
All epidemiologic and biologic evidence strongly suggests that BSE-H and BSE-L represent sporadic forms of BSE (23,24) associated with 2 distinct prion strains. Transmission experiments in different mouse models, including transgenic mice expressing bovine PrP, showed that BSE-H and BSE-L exhibited strain-specifi c features clearly distinct between each other that also differed from BSE-C (13,(25)(26)(27)(28). However, BSE-L isolates unexpectedly showed transmission of a disease with some phenotypic features that resembled those of the BSE-C agent when inoculated in either transgenic mice expressing ovine PrP (28) or inbred wild-type mouse lines (25), suggesting that atypical bovine strains can modify their properties, at least after species barrier passages, converging with those of BSE-C.
We show that the transmission of atypical BSE-H isolates in transgenic mice expressing homologous bovine prion protein (PrP) led to emergence of a clearly distinct prion with strain features similar to those of the BSE-C agent and that such similarities were maintained on subsequent passages. These observations provide new insights into the nature of the events that could have led to the BSE epizootic.

Transgenic Mice
We used Tg110 transgenic mice in all inoculation experiments. This mouse line expresses bovine PrP C (≈8× that of the level of PrP C in cattle brain) under the control of the mouse prnp gene promoter in a mouse PrP 0/0 background (29).

BSE Isolates
The 5 BSE-H isolates used in this study comprised brainstem samples from naturally affected cows, diagnosed as atypical H-type BSE on the basis of the molecular analyses of PrP res (23). All cows were healthy and killed at 8-15 years of age. Four (isolates 07-644, 03-440, 03-2095, and 02-2695) were provided by the Agence Française de Sécurité Sanitaire des Aliments (Lyon, France). Isolate 45 was obtained from the Polish National Veterinary Research Institute (Pulawy, Poland). For comparative studies, material obtained from brainstem of 1 cow naturally infected with BSE-C (RQ 225:PG1199/00), supplied by the Veterinary Laboratories Agency (New Haw, Addlestone, Surrey, UK), was used as BSE-C control. For mouse inoculations, all isolates were prepared from brain tissues as 10% (wt/vol) homogenates. For subpassages, 10% brain homogenates from Tg110 mice collected from primary passage were used as inocula.
All inocula were prepared in sterile 5% glucose as 10% homogenates. Each inoculum was prepared separately in a biosafety cabinet according to a strict protocol to avoid cross-contamination. To diminish the risk for bacterial infection, the inocula were preheated for 10 min at 70°C before inoculation.

Mouse Transmission Studies
Groups of 6-12 mice (6-7 weeks of age, weighing ≈20 g) were inoculated with 20 μL of the appropriate sample in the right parietal lobe by using 25-gauge disposable hypodermic syringes. UNO MICRO ID-8 ISO transponders (Roestvaststaal BV, Zevenaar, the Netherlands) were used for individual identifi cation of mice. After inoculation, mice were observed daily and their neurologic status were assessed 2×/wk. When progression of the disease was evident, animals were euthanized. All animals were housed in accordance with guidelines of the Code for Methods and Welfare Considerations in Behavioral Research with Animals of the European Union directive 86/609EC). Necropsy was performed, and brain and spleen were taken. Part of the sample was frozen at −20°C for biochemical analysis, and the remaining part was fi xed for histopathologic studies.
Survival times were calculated for each inoculum as the time between inoculation and euthanasia in days and expressed as the mean of the survival days postinoculation (dpi) of all the inoculated mice with its correspondent standard error of the mean. Data were processed by using SigmaPlot 2001 software (Systat Software, San Jose, CA, USA).

PrP res Western Blotting
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. All samples were analyzed by Western blot by using the kit TeSeE Western Blot 355 1169 (Bio-Rad) but with some adjustments for the different amounts of samples used. To achieve the volume proposed in the manufacturer's recommendations, 100 μL of the brain homogenates to be tested were supplemented with 100 μL of a 10% brain homogenate from PrP null mice (30). Processed samples were loaded on Criterion 12% polyacrylamide gels from Bio-Rad (165.6001) and electrotransferred to immobilon membranes (IPVH 000 10 [Millipore, Billerica, MA, USA]). For the immunoblotting experiments, Sha31 (31), Saf84 (Cayman Chemical, Ann Arbor, MI, USA) and 12B2 (32) monoclonal antibodies (mAbs) were used at a concentration of 1 μg/ mL. Sha31 recognizes 156 YEDRYYRE 163 epitope, and Saf84 recognizes 171 QVYYRPVDQYS 181 epitope and 12B2 recognizes 101 WGQGG 105 epitope of the bovine PrP sequence. Immunocomplexes were detected by horseradish peroxidase-conjugated antimouse immunoglobulin G (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Immunoreactivity was visualized by chemiluminescence (Amersham Pharmacia Biotech) and obtained after exposition with medical radiographic fi lm (Agfa, Mortsel, Belgium).

Histopathologic Analysis
All procedures involving mouse brains and spleens were performed as described (33). Briefl y, samples were fi xed in neutral-buffered 10% formalin (4% formaldehyde) before embedding in paraffi n. Once deparaffi nized, 2-μm-thick tissue sections were stained with hematoxylin and eosin. Lesion profi les of the brains were established according to the standard method described by Fraser and Dickinson (34). For paraffi n-embedded tissue blots, the protocol described by Andréoletti et al. (35) was used.

Results
The transmission dynamic of BSE-H agent into Tg110 mice is similar to that of BSE-C. The 4 BSE-H isolates from France and 1 from Poland that were intracerebrally inoculated into transgenic mice expressing bovine PrP (Tg110 mice) induced a typical neurologic disease on primary transmission, with a 100% attack rate ( Figure 1). Remarkably, the survival times (mean ± SD 274 ± 3 to 346 ± 6 dpi) were similar than those produced by several BSE-C isolates ≈300 days) on the same Tg110 mouse line (29,36,37). The longest mean survival times observed for mice infected with isolates 03-440 (mean ± SD 346 ± 6 dpi) and 02-2695 (330 ± 14 dpi) could refl ect a lower infectivity of these isolates, consistent with its comparatively lower PrP res content (data not shown). Moreover, the survival time of mice infected with these 2 isolates was reduced on subpassage, approaching that for BSE-C or BSE-H isolates of presumably higher titer (i.e., producing no substantial reduction of survival time on subpassage: isolates 07-644, 03-2095, and 45).

PrP res Molecular Profi les of BSE-H-Inoculated Tg110 Mice
The brains of inoculated mice were examined for PrP res by Western blot analysis with Sha31 mAb. Consistent with the effi cient transmission observed, PrP res was readily detected from the fi rst passage in all Tg110 mice inoculated with the different BSE-H isolates. In 3 BSE-H isolates (07-644, 03-440, and 03-2095), the totality (100%) of the inoculated Tg110 mice produced a PrP res profi le similar to that in cattle (H-type PrP res ) but clearly distinct from that produced by BSE-C agent (C-type PrP res ) in cattle ( Figure  2, panel A; data not shown). Compared with C-type PrP res , H-type PrP res was characterized by a signifi cantly higher apparent molecular mass of the unglycosylated band. The results obtained with these 3 isolates were comparable with those obtained by other authors with 2 BSE-H isolates inoculated in a different bovine PrP mouse line (27), where they concluded that the H-type agent essentially retained its biochemical phenotype upon serial transmission to bovine PrP transgenic mice.
We observed a different situation for the other 2 BSE-H isolates (02-2695 from France and 45 from Poland), where 3 and 2, respectively, of infected mice ( Figure 1) showed a PrP res profi le clearly distinct from that of BSE-H in cattle (Figure 2, panel B). These 5 mice produced a PrP res Figure 1. Overview of transmission of BSE-H isolates in tg110 mice. Five different isolates were intracerebrally inoculated into groups of 6-12 mice per isolate. Survival times at different serial passages are indicated as mean ± SD days postinoculation. Molecular profi les exhibited in the brains of inoculated mice are indicated as H-type, C-type, or C-like PrP res , and proportion of mice showing each profi le. Previously reported data on BSE-C transmission in these mice (36) are included here only for comparison. BSE, bovine spongiform encephalitis; BSE-H, unglycosylated PrP res that is higher than BSE-C; H-type, high-type Western blot profi le of PrP res ; C-type, classicaltype Western blot profi le of PrP res ; C-like, classical BSElike; PrP res , protease-resistant prion protein; BSE-C, classical BSE. with lower ≈1.5 kDa) apparent molecular mass of the 3 PrP res glycoforms, which was indistinguishable from that produced by BSE-C agent in these mice (Figure 2, panel B, and data not shown). Further characterization of this PrP res with other antibodies showed that the PrP res produced by these mice was not recognized by 12B2 mAb (Figure 3). However, 12B2 immunoreactivity against the H-type PrP res produced by other mice (inoculated at the same time with the same isolates) remains essentially similar to that in cattle BSE-H ( Figure 3). Furthermore, PrP res immunolabeling with Saf84 mAb showed that these mice, contrary to mice with H-type PrP res , did not retain the characteristic PrP res band profi le (4 bands) of cattle BSE-H but showed a PrP res profi le (3 bands) similar to that of BSE-C in Tg110 mice ( Figure 4).
In addition, the proportion of the diglycosylated PrP res increased in comparison with the mice with H-type features, as shown by using Sha31 mAb (Figures 2, 3).
Another difference was that the PrP res level in brain was much higher than that in mouse brains with H-type PrP res but similar to that in mouse brains inoculated with BSE-C, as shown by comparative Western blot analysis by using Sha31 mAb ( Figure 5) and by the different equivalent brain tissue masses loaded to obtain equivalent PrP res signals (Figures 2-4). A 10-fold equivalent brain tissue mass was loaded for brains from mice showing H-type PrP res molecular profi le than from those with a classicallike (C-like) PrP res molecular profi le to obtain equivalent PrP res signals (Figures 2-4). These mice thus showed PrP res molecular features indistinguishable from those in Tg110 mice infected with C-like features.
For these 2 isolates, a second passage was performed in Tg110 mice by using a brain homogenate derived from a mouse with either H-type or C-like PrP res ( Figure  1). Survival times did not differ substantially when the different inocula were compared (H-type vs. C-like PrP res brain homogenate) or when compared with second passages  of BSE-C in these mice. All the mice inoculated with the H-type brain homogenates showed H-type PrP res features, whereas all the mice inoculated with the C-like brain homogenates exhibited C-type PrP res molecular features indistinguishable from that of BSE-C (data not shown).

Lesion Profi les and PrP Sc Deposition Patterns in BoPrP-Tg110 Mice
We next examined vacuolation and PrP Sc distribution in the brain, which are known to vary by strain (10,34,38). In general, mice brains exhibiting H-type PrP res correlated with overall more intensive vacuolation that is pronounced in areas such as the hypothalamus, medial thalamus, and mesencephalic tegmentum ( Figure 6). However, a different situation was observed when we studied the brains of BSE-H-infected mice exhibiting C-like PrP res features. All these mice showed a lesion pattern comparable with that in BSE-C-infected mice, in which slight differences are found only in the mesencephalic tegmentum ( Figure 6). These differences consisted of moderate lesions, whereas in BSE-C-infected mice, no lesions were found in this area. These features were conserved on secondary transmissions where no remarkable differences were found when compared with primary transmissions (data not shown).
Moreover, PrP Sc deposits were distinctly distributed after both primary and secondary transmissions when BSE-H-infected mice exhibiting C-type PrP res features were compared with those with H-type PrP res , as assessed by paraffi n-embedded tissue blot on brain coronal sections (Figure 7). However, the PrP Sc deposition patterns were clearly similar when these mice were compared with those infected with BSE-C, after both primary and secondary transmission.
Transmission experiments (27) showed that, contrary to BSE-C, BSE-H is poorly lymphotropic in mouse models. The comparative study of PrP Sc accumulation in spleen from our Tg110 mice infected with both agents showed that BSE-H infected mice exhibiting H-type PrP res in their brain were consistently scored as negative for PrP Sc detection by paraffi n-embedded tissue blot. In contrast, clear PrP Sc deposits were always detected in BSE-H-infected mice exhibiting C-like features, as in mice infected with BSE-C (Figure 7). Similar results were obtained after secondary transmissions.

Discussion
We studied the behavior and stability of the atypical BSE-H during propagation into a bovine PrP background, thus in the absence of a species barrier. We used Tg110 mice (29,36) because they express a PrP C homologous to that of the donors, thus providing a relevant context for comparing atypical BSE-H and epizootic BSE-C isolates.
Our results showed that all BSE-H isolates induced a typical neurologic disease on primary transmission, with a 100% attack rate and survival times similar to those produced by several BSE-C isolates in this mouse line (29,36) (Figure 1). The longer survival times for some mice infected with BSE-H isolates could refl ect a lower 1640 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 9, September 2011  infectivity of this isolate consistent with the reduction of survival time observed on subpassages, approaching that for BSE-C or BSE-H isolates of presumably higher titer (i.e., producing no substantial reduction of survival time on subpassage). These results are also consistent with another comparative study of BSE-H and BSE-C transmissions in a different bovine PrP mouse line (27). These data suggest that atypical BSE-H and BSE-C agents have similar transmission features into Tg110 mice. Although all BSE-H-inoculated mice showed homogeneous survival times, a phenotypic divergence was observed in a few animals infected with 2 of the BSE-H isolates. Surprisingly, these few mice showed phenotypic features clearly distinct from those in most of the BSE-Hinfected mice but similar to those of BSE-C propagated onto the same mice, according to various criteria. First, a PrP res profi le indistinguishable from that produced by BSE-C agent in these mice but clearly distinct from that of BSE-H in cattle, in terms of 1) apparent molecular mass of PrP res , 2) PrP res glycosylation pattern, 3) immunoreactivity with 12B2 mAb, and 4) pattern of labeling with Saf84 antibody. Second, the vacuolation profi le essentially overlapped that in mice infected with BSE-C, with slight differences only in the mesencephalic tegmentum area. Third, the spatial distribution of PrP res in the brain was clearly similar to that of mice infected with BSE-C. Fourth, PrP Sc was consistently detected in the spleen, similar to mice infected with BSE-C. These similarities with BSE-C were fully retained after a second passage by using brain homogenate from mice with C-like features, whereas a BSE-H strain phenotype was maintained in mice inoculated with mouse brains homogenates containing H-type PrP res .
However, C-like features emerged in only 2 of the 5 isolates tested. Because only a low proportion of the mice inoculated with these 2 isolates exhibited these novel features (3/12 and 2/10, respectively), the lack of such observation in the other 3 isolates, and in 2 other independent studies of 3 BSE-H isolates in different bovine transgenic mouse lines (27), could be due to the low number of inoculated mice (6 per isolate), which could be statistically insuffi cient for such an event. No variability was ever observed in the PrP res profi les of >100 Tg110 mice inoculated with 4 different BSE-C isolates (29,36) ( Figure  1). However, a divergent evolution of the BSE agent has been reported after trans-species transmission in both wildtype (11) and human PrP transgenic mice (12,39,40).
Although further studies are required to clarify the mechanisms associated with the emergence of distinct phenotypes among individual mice, several factors would be expected to infl uence the probability of detecting such a variant through mouse bioassay. These factors are 1) amount or regions of cattle brain tissue taken for inoculum preparation, 2) physicochemical treatment during inoculum preparation (e.g., temperature, homogenization buffer), 3) the precise site of mouse inoculation, 4) the infectious titer of the inoculum, and 5) others unknown mouse factor affecting prion propagation and disease evolution. Because samples used in this study were prepared from the same region (brainstem) following the same precise protocol and under identical conditions, differences in inoculum preparation and conditions are unlikely. However, the possibility that the observations might be infl uenced by the precise neuroanatomic origin of the inoculated bovine brainstem homogenate or by other mouse bioassay-related factors cannot be excluded.
The possible cross-contamination of the BSE-H isolates material (02-2695 and 45 from 2 laboratories in different countries) by a BSE-C infectious source was judged highly improbable for several reasons. These reasons are 1) the strict biosafety procedures followed for sample collection, preparation of the inocula, inoculation scheme, and care of mice; 2) the absence of C-type PrP Sc in the BSE-H inocula used for transmissions as deduced by Western blot analysis; and 3) 2 independent transmission experiments, involving separate batches of both incriminated isolates, all produced consistent results.
Together, these observations support 2 possible hypotheses. First, a minor strain component might be showing either H-type PrP res phenotype (black triangles, n = 6 animals) or C-like PrP res phenotype (blue squares, n = 3 animals). Lesion profi le in brains from Tg110 inoculated with BSE-C (fi rst passage) is also included for comparison (red circles, n = 6 animals). Lesion scoring was undertaken for 9 areas of gray matter (G) and white matter (W) in mouse brains: dorsal medulla (G1), cerebellar cortex (G2), superior colliculus (G3), hypothalamus (G4) medial thalamus (G5), hippocampus (G6), septum (G7), medial cerebral cortex at the level of the thalamus (G8) and at the level of the septum (G9), cerebellum (W1), mesencephalic tegmentum (W2), and pyramidal tract (W3). Error bars indicate SE. BSE, bovine spongiform encephalopathy; BSE-H, unglycosylated PrP res that is higher than BSE-C; PrP res , protease-resistant prion protein; H-type, high-type Western blot profi le of PrP res ; C-type, classical-type Western blot profi le of PrP res ; C-like, classical BSE-like; BSE-C, classical BSE. present in BSE-H isolates that could emerge on subsequent transmission in Tg110 mice. Second, a new strain component has been generated during propagation of BSE-H agent in Tg110. In both instances, emergence of the new strain, either in the original cattle or during propagation in Tg110 mice, could be promoted by specifi c propagation conditions or by physicochemical treatment of the inoculum. In this regard, acquisition of novel properties by a sporadic cattle transmissible spongiform encephalopathy agent by a physicochemical treatment, such as that applied to carcass-derived products, has been invoked as a possible origin for the BSE epidemic (7).
Contrary to BSE-H, the atypical BSE-L agent retained unique and distinct phenotypic features, compared with BSE-C agent, on transmission to both bovine and human PrP transgenic mice (26)(27)(28). This agent, however, acquired phenotypic traits intriguingly similar to those of the BSE agent during trans-species transmission in either transgenic mice expressing ovine PrP (28)   been speculated to have originated from atypical BSE-L after conversion in an intermediate host such as a sheep. However, the capacity of these BSE-L-derived agents to retain BSE phenotypic traits after reinoculation to bovine PrP transgenic mice is a key question, remaining to be demonstrated, to show whether the observed convergence truly refl ects a permanent strain shift of the BSE-L agent rather than a phenotypic convergence in an experimental model. In contrast, our results suggest that prion strain divergence might occur on propagation of atypical BSE-H in a homologous bovine PrP context and that this strain divergence could result from a permanent strain shift of the BSE-H agent toward a C-like agent that is stable in subsequent passages. These fi ndings emphasize the potential capacity of prion diversifi cation during propagation, even in the absence of any species barrier, and represent an experimental demonstration of the capability of an atypical, presumably sporadic, bovine prion to acquire C-like properties during propagation in a homologous bovine PrP context.
Results in transgenic mouse models cannot be directly extrapolated to the natural host. However, our observations are consistent with the view that the BSE agent could have originated from a cattle prion, such as BSE-H, and provide new insights into the nature of the events that could have led to the appearance of this agent.