The emergence of classical BSE from atypical/ Nor98 scrapie Alvina Huora,1, Juan Carlos Espinosab,1, Enric Vidalc,1, Hervé Cassarda, Jean-Yves Doueta, Séverine Lugana, Naima Arona, Alba Marín-Morenob, Patricia Lorenzob, Patricia Aguilar-Calvob, Juan Badiolad, Rosa Bolead, Martí Pumarolae, Sylvie L. Benestadf, Leonore Orgeg, Alana M. Thackrayh, Raymond Bujdosoh, Juan-Maria Torresb, and Olivier Andreolettia,2 a UMR Institut National de la Recherche Agronomique (INRA)/École Nationale Vétérinaire de Toulouse (ENVT) 1225, Interactions Hôtes Agents Pathogènes, 31076 Toulouse, France; bCentro de Investigación en Sanidad Animal–Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, 28130 Madrid, Spain; c Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona (UAB)–Institut de Recerca i Tecnologia Agroalimentàries, Barcelona, Spain; dCentro de Encefalopatías y Enfermedades Transmisibles Emergentes, Universidad de Zaragoza, 50013 Zaragoza, Spain; eUnit of Murine and Comparative Pathology, UAB, 08193 Barcelona, Spain; fNorwegian Veterinary Institute, N-0106 Oslo, Norway; gLaboratory of Pathology, National Institute for Agrarian and Veterinary Research, 2780-157 Oeiras, Portugal; and hDepartment of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom Edited by Michael B. A. Oldstone, Scripps Research Institute, La Jolla, CA, and approved November 15, 2019 (received for review September 11, 2019) Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. original prion strain or can display different biological properties Currently there are no efficient measures to control this form of compared to the original inoculum (7, 8). This complex set of prion disease, and, importantly, the zoonotic potential and the risk outcomes for interspecies prion challenge are collectively referred that AS might represent for other farmed animal species remains to as the transmission barrier phenomenon. largely unknown. In this study, we investigated the capacity of AS to After identification of the gene encoding PrP, it was soon dis- propagate in bovine PrP transgenic mice. Unexpectedly, the trans- covered that differences in amino acid sequence between host MEDICAL SCIENCES mission of AS isolates originating from 5 different European coun- PrPC and donor PrPSc constitutes the principal determinant of the tries to bovine PrP mice resulted in the propagation of the classical transmission barrier. For example, the resistance of wild-type mice BSE (c-BSE) agent. Detection of prion seeding activity in vitro by to clinical prion disease induced by hamster scrapie is abrogated protein misfolding cyclic amplification (PMCA) demonstrated that by transgenic expression of hamster PrPC in mice (9, 10). As a low levels of the c-BSE agent were present in the original AS isolates. consequence, mice genetically engineered to express particular C-BSE prion seeding activity was also detected in brain tissue of species forms of PrP sequence, in the absence of endogenous ovine PrP mice inoculated with limiting dilutions (endpoint titration) mouse PrP, have emerged as relevant models to experimentally of ovine AS isolates. These results are consistent with the emergence characterize the outcome of prion strain transmission between and replication of c-BSE prions during the in vivo propagation of AS species (11). It is also now well established that strain properties Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. isolates in the natural host. These data also indicate that c-BSE have a significant impact on the ability of prions to cross the prions, a known zonotic agent in humans, can emerge as a dominant species barrier. For instance, human vCJD can be transmitted prion strain during passage of AS between different species. These readily to conventional mice, but it is extremely difficult for sCJD findings provide an unprecedented insight into the evolution of to propagate in the same mouse lines (12, 13). Furthermore, the mammalian prion strain properties triggered by intra- and interspe- amino acid sequence of PrPSc influences the efficacy of interspecies cies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues Significance and products could lead to new occurrences of c-BSE. prion | atypical scrapie | c-BSE The origin of transmissible BSE in cattle remains unestablished. Sheep scrapie is a potential source of this known zoonotic. Here we investigated the capacity of sheep scrapie to propagate in T ransmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders that affect a large spectrum of mammalian species. These conditions include bovine PrP transgenic mice. Unexpectedly, transmission of atypical but not classical scrapie in bovine PrP mice resulted in propagation of classical BSE prions. Detection of prion seeding scrapie in small ruminants, classical bovine spongiform enceph- activity by in vitro protein misfolding cyclic amplification dem- alopathy (c-BSE) in cattle, and sporadic Creutzfeldt–Jakob dis- onstrated BSE prions in the original atypical scrapie isolates. BSE ease (sCJD) or variant CJD (vCJD) in humans. prion seeding activity was also detected in ovine PrP mice in- The fundamental event in prion propagation is the conversion of oculated with limiting dilutions of atypical scrapie. Our data the normal cellular prion protein (PrPC) into an abnormal disease- demonstrate that classical BSE prions can emerge during intra- associated isoform (PrPSc) in tissues of infected individuals. PrPC is and interspecies passage of atypical scrapie and provide an un- completely degraded by digestion with proteinase K (PK), whereas precedented insight into the evolution of mammalian prions. PrPSc is N-terminally truncated, resulting in a PK-resistant core termed PrPres (1). According to the prion concept, PrPSc is the Author contributions: E.V. and O.A. designed research; A.H., J.C.E., E.V., H.C., J.-Y.D., S.L., principal, if not sole, component of the transmissible prion agent (2), N.A., A.M.-M., P.L., P.A.-C., J.B., R. Bolea, M.P., and O.A. performed research; S.L.B. and and PrPres is a disease marker for prion diseases (1, 3). Particular L.O. contributed new reagents/analytic tools; A.H., J.C.E., E.V., S.L.B., L.O., A.M.T., biochemical properties of PrPSc, such as detergent solubility, PK R. Bujdoso, J.M.T., and O.A. analyzed data; and A.H., S.L.B., L.O., A.M.T., R. Bujdoso, and O.A. wrote the paper. resistance, and electromobility evidenced by Western blot can be The authors declare no competing interest. used to distinguish between different prion agents or strains (4, 5). Intraspecies transmission of prion disease between individuals This article is a PNAS Direct Submission. is typically quite efficient. In contrast, interspecies transmission Published under the PNAS license. 1 of prions can be unpredictable, with apparent failure of disease A.H., J.C.E., and E.V. contributed equally to the work. transmission on many occasions. In other cases, clinical prion 2 To whom correspondence may be addressed. Email: o.andreoletti@envt.fr. disease may not be evident but, rather, there is the presence of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ subclinical infection (6). When interspecies prion transmission doi:10.1073/pnas.1915737116/-/DCSupplemental. does occur, the propagating agent can remain identical to the First published December 16, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1915737116 PNAS | December 26, 2019 | vol. 116 | no. 52 | 26853–26862 prion transmissions, since studies in human PrP transgenic mouse cases (26). Consequently, the origin of atypical scrapie (sponta- models indicate that the human species barrier is more permeable neous disorder versus acquired disease) remains an open question. to sheep-passaged BSE compared to its cattle counterpart (14). In this study, we used mice transgenic for bovine PrP (tgBov From a public health perspective, the transmission barrier phe- mouse line) to characterize the capacity of sheep AS isolates to cross nomenon and its capacity to limit the interspecies propagation of the bovine transmission barrier. Unexpectedly, the TSE agent that prion disease has long been considered as an effective protection of propagated in tgBov mice was indistinguishable from the prion strain humans against animal TSEs (15). However, in 1996, the new hu- that was responsible for the c-BSE epizootic in cattle. In addition, man prion disease vCJD was observed in people in the United our sensitive detection of c-BSE by in vitro PMCA methodology Kingdom. Multiple lines of evidence indicated that vCJD was the indicated that this bovine prion strain was present as a minor prion likely consequence of dietary exposure of humans to the agent strain in the original sheep AS isolates, and that AS prion strain responsible for c-BSE in cattle, an epizootic prion disease that has replication in an ovine PrP host was accompanied by the generation spread in bovine hosts through the recycling of prion-contaminated of c-BSE prions. Collectively, these data provide compelling evidence animal carcasses in the animal food chain (16). Since the emer- for the emergence and the propagation of zoonotic mammalian gence of vCJD, considerable efforts have been deployed to char- prions during intra- and interhost transmission of the AS prion strain. acterize not only the zoonotic potential of animal prions but also Results their capacity to propagate in farmed animal species. Atypical/Nor98 scrapie (AS) probably represents the largest A panel of 8 atypical scrapie (AS) cases collected from sheep and goats in 5 different European countries was obtained (Table 1). geographically spread known animal prion disease. Since its original All of the AS isolates displayed a multiband abnormal PrP discovery in 1998 in Norway, AS has been identified in most EU (PrPres) Western blot profile that was considered to be specific member states, in Asia, and in North and South America (17). AS for small ruminant AS (SI Appendix, Fig. S1). This panel of AS has also been detected in Australia and New Zealand, 2 countries isolates was transmitted to the VRQ ovine PrP transgenic mice that were believed to be free of animal TSEs (18, 19). Retrospective (tg338). The transmission properties such as incubation period studies carried out in archived animal tissues identified an AS case (SI Appendix, Table S1), vacuolar lesion profile (Fig. 1A), and in a sheep that died in 1972 in the United Kingdom, demonstrating PrPres Western blot profile in the brain (SI Appendix, Fig. S1) of that the disease has been present in small ruminant populations for the propagated AS isolates observed after 2 or 3 iterative pas- many decades (20). sages in tg338 were similar and were the same as previously Bioassay in ovine PrP transgenic mice provided evidence that AS reported for AS passage in tg338 mice (21–23). comprised a single prion strain (21–23). The AS prion strain was as- The panel of 8 AS isolates was individually serially transmitted sociated with a multiband PrPres signature that contrasted with those (2 or 3 iterative passages) in bovine PrP transgenic mice (tgBov; normally observed in small ruminant TSE cases (24). Since there is no Table 1). On first passage, signs of clinical prion disease were statistical difference in the apparent prevalence of atypical scrapie observed in a low proportion of inoculated tgBov mice. PrPres was between sheep flocks in general and those flocks where a positive case Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. detected by Western blot in the brains of clinically affected mice had been identified, atypical scrapie is considered by many as a and in some mice that displayed no apparent clinical signs of prion noncontagious prion disease that arises sporadically in sheep and disease when euthanized at the end of their life expectancy. No goats (25). However, atypical scrapie can be experimentally PrPres accumulation was observed in tgBov mice after inoculation transmitted via the oral route in small ruminants, resulting in a with several of the AS isolates (AS 2, AS 3, AS 5, AS 6, and AS 7), similar clinicopathological phenotype to that observed in natural as was the case with classical scrapie PS42 (Table 1). Table 1. Inoculation of atypical scrapie isolates in bovine PrP (tgBov) expressing mice TgBov Isolates First passage Second passage Third passage Positive Incubation, Positive Incubation, Positive Incubation, Identifiant Origin Genotype mice mean ± SD mice mean ± SD mice mean ± SD AS 1 Fr ARQ*/ARQ 1/6 533 3/5 317 ± 63 7/7 235 ± 16 AS 2 Sp ARR/ARQ 0/6 >650 7/9 354 ± 26 5/5 273 ± 5 AS 3 Sp ARQ/ARH 0/6 >650 2/11 504, 525‡ 12/12 269 ± 13 AS 4 Nor ARQ*/ARQ* 3/4 395 ± 44 6/6 230 ± 17 6/6 271 ± 18 AS 5 Sp ARQ/ARQ 0/6 >650 0/4 >650 NA — AS 6 Sp ARQ/ARH 0/6 >650 1/4 >650‡ NA — AS 7† It ARQ/AHQ 0/6 >650 1/7 424 6/6 286 ± 14 AS 8 Po ARQ/ARQ 1/5 439 5/5 297 ± 14 6/6 250 ± 4 PS42 Fr VRQ/VRQ 0/6 >650 0/6 >650 0/6 >650 Ovine c-BSE Fr ARQ/ARQ 6/6 254 ± 19 6/6 234 ± 12 6/6 232 ± 6 Cattle c-BSE Fr — 6/6 295 ± 12 6/6 265 ± 35 6/6 243 ± 7 Transgenic mice that express the bovine PrP (tgBov) were inoculated intracerebrally (6 to 12 mice, 20 μL per mouse) with 8 sheep or goat (†) atypical scrapie (AS) isolates originating from 5 different countries, France (Fr), Spain (Sp), Norway (Nor), Italy (It) or Portugal (Po). The AS affected animals displayed a different PRNP genotype at codons 136, 154, and 171. Some also displayed a F/L dimorphism at codon 141 (*). Cattle classical BSE (c-BSE), ovine c-BSE (first passage of cattle c-BSE in an ARQ/ARQ sheep by the intracerebral route), and classical scrapie (PS42) isolates were inoculated in the same mouse model. After first and second passages, clinically affected or asymptomatic mice that had lived for more than 500 d post inoculation were pooled and used for subsequent passage in the same line. Mice were considered positive when abnormal PrP deposition was detected in the brain. Incubation periods (in days) are shown as mean ± SD except when less than 50% of the mice were found to be positive. In that case, the incubation periods of the positive mice are individually presented. NA, not available. Cattle c-BSE transmission in tgBov data from ref. 51. PS42 transmission data from ref. 15. ‡ Abnormal PrP positive and found dead animals (without symptoms). 26854 | www.pnas.org/cgi/doi/10.1073/pnas.1915737116 Huor et al. MEDICAL SCIENCES Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. Fig. 1. Brain lesion profile and PrPres Western blot profiles in tgBov and tg338 mice inoculated with atypical/Nor98 scrapie (AS) or c-BSE. Groups of mice (n ≥ 6) that express either ovine VRQ PrP (tg338 mice) or bovine PrP (tgBov mice) were intracerebrally challenged with atypical scrapie isolates (AS) or an ovine classical BSE isolate (c-BSE). (A) After 2 or 3 iterative passages in each mouse line, a standard lesion profile was established by scoring the vacuolar changes observed in predefined brain areas. In c-BSE graphs, ●, ovine c-BSE; ○, cattle c-BSE. In AS graphs, ○, AS 1; △, AS 2; ▽, AS 3. (B) The accumulation of PK- resistant PrP (PrPres) in the brain of tgBov mice was established by Western blot using anti-PrP monoclonal antibodies Sha31 (epitope 145-YEDRYYRE-152) and/or 12B2 (epitope 89-WGQGG-93). The same Western blot PrPres control (classical scrapie isolate) was used on all of the gels labeled as WB control. Cattle c-BSE and an ovine c-BSE (original isolate and isolate passaged in tgBov) were included as controls. (C) Vacuolar lesions (thalamus level, conventional histology; hematoxylin-eosin) and abnormal PrP deposition (mesencephalon: tegmentum, immunohistochemistry using 6H4 anti-PrP antibody, epitope 147-DYEDRYYRE-155) in tgBov mice inoculated with AS3 and cattle BSE. (Scale bars: vacuolar lesions, 25 μm; abnormal PrP deposition, 50 μm.) Huor et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26855 Second passage of the AS isolates in tgBov mice was performed laboratories, we exclude the possibility of a cross-contamination using either first-passage PrPres-positive brains or pooled PrPres- of the original AS isolates by the c-BSE agent. negative brains as inoculum. During this process, 7 out of the 8 AS Two hypotheses could explain the emergence of the c-BSE isolates caused the occurrence of clinical prion disease in a pro- agent in tgBov mice after their inoculation with AS isolates. portion of animals in each group inoculated. On third passage First, c-BSE prions could be present at a low level in the original (available for AS 1, 2, 3, 4, and 7), 100% attack rates and mean AS isolates. The high sensitivity of tgBov mice for detection of incubation periods that ranged between 235 and 286 dpi were the c-BSE agent could allow this potentially low level of bovine recorded (Table 1). prions to be identified during passage of the original AS isolates At each passage stage, a 3-band PrPres Western blot profile in the bovine PrP host. Alternatively, the occurrence of c-BSE in characterized by a prominent di-glycosylated PrP band was ob- AS-inoculated tgBov mice could result from a mutation of AS served in the brains of the clinically positive tgBov mice (Fig. 1B). strain properties triggered by passage across the bovine trans- Strikingly, the lesion profile (Fig. 1A), the PrPres Western blot mission barrier for this particular ovine prion strain. profile (Fig. 1B), and the histopathological lesions (Fig. 1C) in the In order to explore the origin of the c-BSE agent observed in brains of AS inoculated tgBov mice were identical to those ob- tgBov mice inoculated with AS, we employed in vitro protein served for transmission of c-BSE (sheep or cattle origin) to tgBov misfolding cyclic amplification (PMCA), a methodology that mice. Importantly, the inoculation of one classical scrapie isolate mimics prion replication in vitro, but in an accelerated form, (PS42) to the same mouse models resulted in the occurrence of a allowing amplification of minute amounts of PrPSc and prion 100% attack rate for prion disease with a short incubation period infectivity (27). In PMCA, a PrPC-containing substrate is com- in tg338 mice (SI Appendix, Table S1), but no clinical disease or bined with a seed that contains PrPSc. Following repeated cycles PrPres accumulation in the brains of tgBov (Table 1). of incubation and sonication, the amount of PrPSc increases. In order to further characterize the nature of the TSE agent PMCA has been previously reported to amplify the c-BSE agent with great efficacy using either tgARQ or tgBov mouse brain that propagated in tgBov mice inoculated with the AS isolates, homogenate as substrate (28). Using this protocol, 2 (tgARQ sub- prions obtained after second passage (isolate AS 2) or third strate) or 3 (tgBov substrate) amplification rounds were suffi- passage (isolate AS 3) in this mouse line were transmitted (2 cient to reach the detection limit for c-BSE prion seeding activity iterative passages) to VRQ (tg338) and ARQ (tgARQ) ovine (SI Appendix, Fig. S2). The level of prion infectivity and prion PrP transgenic mice (Table 2). The incubation periods (Table 2 seeding activity of a reference sheep-passaged c-BSE isolate and SI Appendix, Table S1), the lesion profile (Fig. 2A), and the were endpoint-titrated by both bioassays in tgBov mice and PrPres Western blot profile in the brain (Fig. 2B) of tg338 mice PMCA, respectively (SI Appendix, Table S2). The infectious inoculated with tgBov-adapted AS isolates clearly differed from prion titer of the sheep-passaged c-BSE isolate was ∼107.2 LD50/g those observed in the same mouse line inoculated with the IC in tgBov mice. The prion seeding titer (SA50) was estimated original AS isolates. to be ∼1011.1 SA50/g using tgARQ mouse tissue as substrate and No PrPres deposition could be detected in the spleen of tg338 Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. 1011.05 SA50/g using tgBov mouse tissue as substrate. Considering mice inoculated with the original AS isolates (Fig. 3A). Conversely, the fact that mice were inoculated using 20 μL of sample and the transmission in tg338 of tgBov-adapted AS isolates was associated PMCA reactions were seeded using 5 μL of the same sample, the with a PrPres accumulation in the spleen, as was transmission of PMCA can be considered to be about 1,500 fold more sensitive ovine and cattle c-BSE (Fig. 3A). PrPres WB profile in the spleen of than the bioassay in tgBov. This also means that 1 c-BSE LD50 in tg338 mice that were inoculated with c-BSE and AS isolates pas- tgBov mice corresponds to ∼1,500 SA50 assessed by PMCA. saged in tgBov were identical (Fig. 3B). In addition to its high sensitivity, in vitro PMCA can reproduce, Transmission of tgBov-adapted AS isolates in both tg338 and at least partly, the transmission barrier phenomenon observed tgARQ mice resulted in prion incubation periods (Table 2), brain during the in vivo prion bioassay (29). Therefore, amplification of vacuolar lesion profiles (Fig. 2A), PrPres Western blot profile prion seeding activity in AS isolates by PMCA using tgBov mouse patterns (Fig. 2B), and PrPres distribution patterns in the brain tissue as substrate offered an opportunity to characterize the po- (Fig. 3A) that were similar to c-BSE passaged in tg338 and tgARQ tential impact of the bovine transmission barrier on AS strain mice. Collectively, these results demonstrate beyond reasonable properties. doubt that our transmission of AS in a bovine PrP host resulted in The AS isolates that were originally transmitted to tgBov mice the propagation of the c-BSE agent. Since the bioassays reported (except AS8) and 18 additional AS isolates (originating from here were performed in 3 independent institutes (located in Norway, France, and Portugal) were subjected to PMCA (Table 3). France and Spain) that used inoculum prepared by 5 distinct Each AS isolate was used to seed reactions containing either Table 2. Inoculation of atypical scrapie adapted in tgBov and c-BSE isolates in ovine PrP expressing mouse models (tg338 and tgARQ) Tg338 TgARQ Isolates First passage Second passage First passage Second passage Positive Incubation, Positive Incubation, Positive Incubation, Positive Incubation, Identifiant Origin mice mean ± SD mice mean ± SD mice mean ± SD mice mean± SD AS 2 Second pass in TgBov 6/6* >650 6/6 617 ± 75 6/6 350 ± 9 6/6 260 ± 3 AS 3 Third pass in TgBov 6/6* >650 6/6 672 ± 83 6/6 354 ± 21 6/6 257 ± 2 Ovine c-BSE Second pass in TgBov 6/6* >750 6/6 653 ± 32 6/6 270 ± 12 6/6 259 ± 4 Cattle c-BSE Cattle 6/6* >700 6/6 682 ± 52 6/6 321 ± 16 6/6 263 ± 7 Transgenic mice that express the VRQ (tg338) or ARQ (tgARQ) variants of ovine PrP were inoculated intracerebrally (6 mice, 20 μL per mouse) with atypical scrapie isolates or ovine c-BSE isolate that had previously been adapted in tgBov (2 iterative passages). Cattle BSE was also included as control. After first passage, clinically affected or asymptomatic mice that had lived for more than 500 d post inoculation were pooled and used for second passage in the same line. Mice were considered positive when abnormal PrP deposition was detected in the brain. Incubation periods (in days) are shown as mean ± SD. *Abnormal PrP positive and found dead animals (without symptoms). 26856 | www.pnas.org/cgi/doi/10.1073/pnas.1915737116 Huor et al. MEDICAL SCIENCES Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. Fig. 2. Brain lesion profile and PrPres Western blot in the brain of tgARQ and tg338 mice inoculated with AS scrapie adapted in tgBov. Groups of mice (n ≥ 6) that express ovine VRQ PrP (tg338 mice) or ovine ARQ PrP (tgARQ mice) were intracerebrally challenged with atypical scrapie isolates (AS) or an ovine c-BSE isolate that had previously been adapted (2 iterative passages) in tgBov mice. (A) After 2 iterative passages in each mouse line, a standard lesion profile was established by scoring the vacuolar changes observed in predefined brain areas. In AS graphs, △, AS 2; ▽, AS 3. (B) The Western blot profile of PK-resistant PrP (PrPres) in the original AS isolates and in the brain of inoculated mice was established by Western blot using anti PrP monoclonal antibody Sha31 (epitope 145-YEDRYYRE-152). The same Western blot PrPres control (classical scrapie isolate) was used on all of the gels labeled as WB control. bovine PrP or ovine ARQ PrP substrate (10 to 18 replicates per number of PrPres-positive reactions were observed when bovine substrate). After amplification, PrPres was detected by Western PrP was used as substrate (in the case of AS 10) or when ovine blot in a low proportion of the reactions seeded, with 19 out of ARQ PrP was used as substrate (in the cases of AS 9 and AS 25). the 25 AS isolates for tgBov and tgARQ combined (Table 3). In Whatever combination of AS isolate and substrate PrP was used, most instances, a similar proportion of PrPres-positive PMCA the PrPres Western blot profile in PMCA-positive reaction prod- reactions were observed when either bovine PrP or ovine ARQ ucts and its reactivity with 12B2 antibody were indistinguishable PrP was used as substrate. However, in some cases (n = 3), a low from those observed for PMCA reaction products seeded with Huor et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26857 brains of these endpoint-titration tg338 mice were subsequently subjected to PMCA. Irrespective of the substrate used for PMCA, either bovine or ovine ARQ PrP, PrPres was observed in a similar proportion of the PMCA reactions seeded with either the original AS isolates or AS isolates passaged in tg338 mice (Table 4). The PrPres Western blot profile observed in all of the PMCA-positive reactions was identical to that seen in reactions seeded with authentic c-BSE prions (Fig. 5). PMCA reactions seeded with brain homogenate prepared from age-matched noninoculated tg338 mice remained PrPres-negative (Table 4 and Fig. 5). Considering the level of c-BSE seeding activity originally pre- sent in isolates AS 25 and AS 26 (less than 100 SA50/mL; Table 4), there is an extremely low level of probability that one of the 6 tg338 mice inoculated with 20 μL of a 10−6 diluted AS isolate (<2 10−6 SA50 per dose of inoculum) could be exposed to 1 infectious dose of c-BSE agent (1 c-BSE LD50 is ∼1,500 SA50). Conse- quently, the presence of c-BSE prion seeding activity in the brains of tg338 mice inoculated with a 10−6 log10 dilution of original AS isolate implies that a low titer of c-BSE prions was generated during the propagation of ovine AS prions in a host that expressed ovine PrP, namely tg338 mice. Discussion The mechanism(s) that lead to an alteration in the phenotype of prion strains as these transmissible entities undergo transmission between different host species remain uncertain. This is despite the identification that differences in amino acid sequence between host PrPC and donor PrPSc, together with prion strain identity, are principal determinants of the transmission barrier (9, 10). Based on the concept that conformation of PrPSc molecules/aggregates encode prion strain information (2, 4, 5, 30, 31), at least 2 non- exclusive hypotheses, “deformed templating” (32, 33) and the Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. “conformational selection model” (32–35), have been proposed to explain the mutation of prion strains. Fig. 3. Brain and spleen PrPres accumulation in tg338 mice inoculated with AS The deformed templating hypothesis postulates that a prion scrapie adapted in tgBov. Groups of mice (n ≥ 6) that express ovine VRQ PrP strain replicates as a clone of PrPSc molecules/aggregates. When (tg338 mice) were intracerebrally challenged with atypical scrapie isolates (AS) confronted by a transmission barrier that does not allow clonal and AS that had previously been adapted (2 iterative passages) in tgBov mice. prion replication, the propagation process is modified so that “al- In parallel, cattle c-BSE isolate and ovine BSE isolate (adapted in tgBov) were transmitted (2 iterative passages) in tg338 mice. (A) In tg338 mice (second tered” PrPSc structural variants are generated in an attempt to passage), the PrPres distribution pattern in the brain (thalamic coronal section) convert the new host PrPC. While the majority of these presumably and in the spleen was established by paraffin-embedded tissue blot using anti- fail to replicate efficiently in the new host, variants eventually PrP monoclonal antibody Sha31 (epitope 145-YEDRYYRE-152). (Scale bars: emerge that are successful and adapt to the new PrP environment brain, 160 μm; spleen, 100 μm.) (B) The Western blot profile of PK-resistant PrP through multiple trial-and-error replication events. In this de- (PrPres) in the spleen of tg338 mice (second passage) inoculated with AS iso- formed templating model, confrontation of the transmission bar- lates and AS isolates passaged in tgBov and c-BSE (cattle and ovine origin) was rier serves as the triggering event that initiates the generation of established using anti-PrP monoclonal antibody Sha31. new prion variant(s) and as a filter for their selection (35). The conformational selection model proposes that a prion strain naturally propagates in its host as an ensemble of PrPSc conformers authentic ovine c-BSE prions (Fig. 4). No PrPres was observed in dominated by a stable energetically favorable conformation re- PMCA reactions that were unseeded (n = 120) or in those reac- sponsible for the observed prion strain phenotype. Furthermore, tions seeded (n = 60) with prion-free sheep brain homogenate this model predicts that the number of stable PrPSc conformers is (representative samples shown in Fig. 4). It should be noted that limited for each PrP amino acid sequence, which would explain the the PrP amino sequence was 100% homologous between certain existence of a finite number of stable prion strains that can prop- AS isolates (AS 5, AS 26) and the ovine PrP substrate (tgARQ) agate in a given species. It is further proposed that, during trans- used in PMCA reactions. Therefore, in vitro amplification of mission of a prion strain to a new host, one of the less dominant c-BSE prions in PMCA reactions seeded with these AS isolates using PrPSc conformers of those present in the ensemble is selected with ovine ARQ PrP as substrate cannot be a consequence of mutation a resultant change, or mutation, in the properties of the newly of prion strain properties triggered by a transmission barrier. propagating prion strain. In the conformational selection model, Taken together, the tgBov mouse bioassay and PMCA results the transmission barrier acts simply as a selective filter for new strongly support the view that a low level of c-BSE prions was prion variants, and ease of permeation of the barrier results from initially present in at least 21 out of the 26 AS isolates tested. the extent of overlap of PrPSc conformers that exist between the To further clarify the origin of the c-BSE agent detected in AS interacting species (32, 33). isolates, 2 of these isolates (AS 25 and AS 26) were endpoint- Our data reported here showed that c-BSE prions are present titrated in tg338 mice (1/10 dilution series, 6 or 7 tg338 mice in- as a minor variant in natural isolates of ovine AS. In addition, oculated per dilution). For both isolates, the last positive trans- transmission of ovine AS to bovine PrP mice demonstrated that missions were observed in mice that received a 10−6 log10 dilution of c-BSE can emerge during these transmissions as the dominant the original 10% w/vol brain material (SI Appendix, Table S3). The prion strain. These results provide a cogent argument in favor of 26858 | www.pnas.org/cgi/doi/10.1073/pnas.1915737116 Huor et al. Table 3. Protein Misfolding Cyclic Amplification seeding activity different ovine prion strains is associated with a unique and in atypical scrapie isolates stable PrPSc conformer and a distinct set of minor variants. The Isolates PMCA positive reactions tgBov mouse line has previously been reported to support the propagation of a variety of natural ovine prions, of which several Identifiant Origin Genotype TgBov substrate TgARQ substrate displayed significantly shorter incubation periods than c-BSE AS 1 Fr ARQ*/ARQ 3/12 5/12 (15). Strikingly, in our experiments, the diversity of prion vari- AS 2 Sp ARR/ARQ 2/12 3/12 ants in the AS isolates (7 different cases) revealed by the serial AS 3 Sp ARQ/ARH 3/12 4/18 passage in tgBov was restricted to the c-BSE agent (Table 1). AS 4 No ARQ*/AFRQ 12/12 9/12 This consistent emergence of a single prion strain argues against AS 5 Sp ARQ/ARQ 4/12 3/12 the view that AS prion replication in sheep can randomly gen- AS 6 Sp ARQ/ARH 5/12 1/12 erate all of the existing stable PrPSc variants associated with a AS 7 It ARQ/AHQ 1/12 2/12 particular ovine PrPC amino acid sequence. Instead, our data AS 8 Po ARQ/ARQ ND ND support the view that individual prion strains are associated with AS 9 Nor ARR/ARQ 0/12 2/10 a restricted repertoire of stable PrPSc variants in a given host. AS 10 — ARQ*/AHQ 1/12 0/10 Whether AS is unique in its ability to generate c-BSE prion AS 11 — AHQ/ARQ 3/12 7/12 particles during its replication process remains to be established. AS 12 — ARR/ARQ 0/12 0/12 Classical BSE was first recognized in 1984 and 1985 as a novel AS 13 — ARR/AHQ 0/12 0/12 prion disease affecting cattle in the United Kingdom (40). Epi- AS 14 — ARQ/AHQ 1/12 1/12 demiological data clearly established that the number of cases of AS 15 — ARR/ARR 3/12 1/12 c-BSE was amplified by the recycling of infected animal carcasses AS 16 — ARR/AHQ 1/12 3/10 into cattle feed in the form of meat and bone meal (MBM) (41). AS 17 — ARQ*/AHQ 1/12 1/10 Since bovine prion disease had not been recognized in cattle AS 18 — ARQ*/AHQ 0/12 0/10 prior to the c-BSE epizootic and the disease is apparently non- MEDICAL SCIENCES AS 19 Po ARR/ARR 0/12 0/12 contagious between cattle, several hypotheses were proposed to AS 20 — ARR/AHQ 3/12 1/12 explain its emergence. These range from the spontaneous occurrence AS 21 — ARR/ARR 0/12 0/10 AS 22 — ARQ*/AHQ 0/12 0/12 AS 23 — ARQ*/ARQ 1/12 1/12 AS 24 — ARQ*/ARQ 2/12 4/12 AS 25 Fr AHQ/AHQ 0/12 1/12 AS 26 — ARQ/ARQ 1/12 1/12 TSE free sheep — ARQ/ARQ 0/12 0/12 Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. Unseeded — — 0/120 0/120 Twenty-six AS scrapie cases (1/50 diluted 10% brain homogenates) originating from 5 different countries (France [Fr], Spain [Sp], Italy [It], Portugal [Po] and Norway [Nor]) were used to seed PMCA reactions (5 μL of seed per reaction). The AS affected animals displayed different Prnp geno- types at codons 136, 154, and 171. Two different PMCA substrates were used. The first one was prepared using brains from transgenic mice overexpressing the ARQ variant of the sheep prion protein (tgARQ). The second was prepared using brains from transgenic mice overexpressing the bovine prion protein (tgBov). For each isolate and substrate, 10 to 18 indi- vidual replicates were tested. Reactions were subjected to 3 amplification rounds. After each round, reaction products (1 volume) were mixed with fresh substrate (9 volumes) to seed the following round. PMCA reaction products (third amplification round) were analyzed by Western blot for the presence of PrPres. The number of PrPres Western blot positive reac- tions/total number of reactions are reported. Unseeded reactions and reac- tions seeded with brain homogenate prepared from a TSE-free sheep were included as specificity controls. ND, not done. *F/L dimorphism displayed at codon 141. the conformational selection model as the mechanism for prion strain mutation during interspecies prion transmission. This would be expected to occur by selection of a preexisting PrPSc Fig. 4. PrPres detection in PMCA reactions seeded with atypical/Nor98 scrapie variant in AS isolates, one best suited to the new replicative isolates. Protein misfolding cyclic amplification (PMCA) reactions were seeded environment. Within this conceptual framework, the occurrence with atypical/Nor98 scrapie (AS) isolates (1/50 diluted 10% brain homogenate) of prion strain mutation is dependent upon the particular rep- that had been identified in 5 European countries (Table 3). PMCA reactions seeded with brain homogenate from a TSE-free sheep (originating from New ertoire of PrPSc variants associated with distinct prion strains. Zealand) and unseeded PMCA reactions were included as specificity controls. This notion is supported by our observation that c-BSE prions PMCA substrate consisted of brain homogenate from either bovine PrP (tgBov) emerged during serial transmission of ovine AS in tgBov mice or ovine PrP (tgARQ) mice. PMCA reactions were subjected to 3 (tgARQ sub- but not from serial passage of classical scrapie in the same mouse strate) or 4 (tgBov substrate) amplification rounds, each comprising 96 cycles line [Table 1 and Cassard et al. (15)]. (10 s sonication, 14 min and 50 s incubation at 39.5 °C) in a Qsonica700 device. The diversity of prion strains that exist in small ruminants re- The PMCA reactions were analyzed by Western blot for the presence of ab- normal PK-resistant PrP (PrPres) using anti-PrP monoclonal antibodies Sha31 mains undefined, although it is established that at least 5 different (epitope 145-YEDRYYRE-152) and/or 12B2 (epitope 89-WGQGG-93). Each natural ovine prion strains exist, including AS (6, 36–39). Western blot included a classical scrapie isolate (labeled as WB control) and an According to the conformational selection model, each of these ovine c-BSE isolate as controls. Huor et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26859 Table 4. PMCA seeding activity in atypical scrapie passaged in tg338 PMCA seeds PrPres positive PMCA reactions Seeding activity, SA50/mL Case Origin TgBov substrate TgARQ substrate TgBov substrate TgARQ substrate 1.86 AS 25 Sheep 0/12 1/12 0 (0–10 )* 101.40 First passage in tg338 (neat) Mouse 1 2/12 1/12 101.72 101.40 Second passage in tg338 (neat) Mouse 1 2/12 1/12 101.72 101.40 End-point titration in tg338 (10−6 dilution) Mouse 1 2/12 3/12 101.72 101.92 Mouse 2 1/12 3/12 101.40 101.92 Mouse 3 2/12 2/12 101.72 101.72 AS 26 Sheep 1/12 1/12 101.40 101.40 First passage in tg338 (neat) Mouse 1 2/12 0/12 101.72 0 (0–101.86) * Second passage in tg338 (neat) Mouse 1 2/12 0/12 101.72 0 (0–101.86) * End-point titration in tg338 (10−6 dilution) Mouse 1 2/12 1/12 101.72 101.40 Mouse 2 1/12 1/12 101.40 101.40 Noninoculated tg338 Mouse 1 0/12 0/12 — — Mouse 2 0/12 0/12 — — Mouse 3 0/12 0/12 — — Two sheep atypical scrapie (AS) isolates were selected. The 10% w/vol brain homogenates were inoculated into tg338 mice (2 iterative passages). Groups of 6 tg338 mice were inoculated intracerebrally with 20 μL of serial 10-fold dilutions of the same homogenates. Transmission was observed in 3 (AS 25) and 2 (AS 26) mice inoculated with 10−6 brain homogenate. No transmission was observed at lower dilutions. PMCA reactions (12 replicates) were seeded with 1/50 diluted brain homogenate (10% w/vol) from 1) the original sheep, 2) the second passage tg338 mice (pool of brains), and 3) individual brain from positive tg338 in the endpoint titration experiment. Brain homogenates (10% w/vol) from age-matched, noninoculated tg338 mice were also used as seeds (1/50 diluted). Two different PMCA substrates were used. The first one was prepared using brains from transgenic mice overexpressing the ARQ variant of the sheep prion protein. The second was prepared using brains from transgenic mice overexpressing the bovine prion protein (tgBov). Reactions were subjected to up to 4 amplification rounds. After each round, reaction products (1 volume) were mixed with fresh substrate (9 volumes) to seed the following round. PMCA reaction products were analyzed by Western blot for the presence of PrPres. The number of PrPres Western blot-positive reactions/total number of reactions are reported. Seeding activity titers were estimated using the Spearman–Karber’s limiting dilution titration method (most likely value) or, when no positive reaction was observed, by the Poisson’s probabilistic model. Titers are given as the number of PMCA SA50 per milliliter of 10% brain homogenate. *Most likely value and IC 95% as described by Brown et al. (66). of c-BSE in cattle to the passage and adaptation of a prion originating With the decline of the c-BSE epizootic in cattle and the com- Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. from another species (42, 43). Our studies here that show the pres- bined increase in pressure from industry, EU authorities have begun ence of c-BSE prions in AS isolates, combined with the demon- to consider discontinuing certain TSE control measures. The ab- strated presence of AS in the United Kingdom long before the rogation of the SRM measures for small ruminants and the partial appearance of the c-BSE epizootic in cattle, suggests that the reauthorization of the use of processed animal protein, formerly recycling of AS cases in MBM might be a source of bovine prion known as MBM, in animal feed are part of the EU authorities’ disease (20). In addition to its potential role in the initial emer- agenda. Our observation of the presence of the c-BSE agent in gence of c-BSE in cattle, the presence of c-BSE prions in natural AS-infected small ruminants suggests that modification of the cases of AS has current and direct implications for both the TSE control measures could result in an increased risk of ex- continued risk of this ovine prion disease to other farmed animals posure to c-BSE prions for both animals and humans. Whether and for human exposure risks. The distribution of AS cases are or not this exposure will result in further c-BSE transmission in widespread across the world (17–19). A recent retrospective analysis cattle and/or humans remains an open and important question. of surveillance data collected over a period exceeding 10 y in the European Union (EU) concluded that the prevalence of detected Methods AS cases has remained relatively stable in the different member Ethics Statement. All animal experiments were performed in compliance with states, with between 2 and 6 positive cases per 10,000 tested animals institutional and French national guidelines and in accordance with the per year. This implies that a substantial number of AS-infected ani- European Directives 86/609/EEC and 2010/63/EU. In France, the animal ex- periments that are part of this study (national registration 01734.01) were mals could enter either the animal or human food chain each year approved by the local ENVT ethics committee. Experiments developed in (44, 45), and each case represents a potential source of exposure to CISA-INIA (Madrid, Spain) were approved by the Committee on the Ethics of the c-BSE agent for farmed animals (MBM derived from rendered Animal Experiments of the Instituto Nacional de Investigación y Tecnología small ruminants) and human consumers (consumption of healthy Agraria y Alimentaria and the General Directorate of the Madrid Commu- slaughtered animals), respectively. The epidemiological features of nity Government (permit numbers: CEEA 2009/004 and PROEX 228–16). AS within the EU is likely to reflect the situation of the disease in Mouse inoculations were performed under anesthesia (isoflurane). other countries that breed and maintain small ruminants. Experiments developed in IRTA-CReSA (Barcelona, Catalonia) involving In Europe, the c-BSE crisis and the emergence of vCJD resulted animals were approved by the animal experimentation ethics committee of in the implementation of a strong and coherent policy (EU regu- the Autonomous University of Barcelona (reference number 585–3487) in lation 999/2001) aimed at control and eradication of this animal agreement with Article 28, sections (a), (b), (c), and (d) of the “Real Decreto prion disease. The total feed ban on the use of MBM in animal feed 214/1997 de 30 de Julio,” European Directive 86/609/CEE, and the European Council Guidelines included in the European Convention for the Protection and the systematic retrieval from the food chain of ruminant tissues of Vertebrate Animals used for Experimental and Other Scientific Purposes. that have the potential to contain high levels of prion infectivity, so- Mice that displayed clinical signs were anesthetized with isoflurane before called Specified Risk Material (SRM) measures, were instrumental sacrifice using CO2 inhalation. for control of c-BSE in cattle and prevention of dietary human exposure to these bovine prions (46, 47). As a side effect, these Atypical/Nor98 Scrapie Cases and Control Sheep. Natural atypical scrapie (AS) measures also strongly limited the exposure of farmed animals cases identified through active or passive surveillance programs were selected and human consumers to the other TSE agents circulating in according to their geographical origin (France, Spain, Italy, Norway, and Por- farmed animal species, including AS. tugal) and PRNP genotypes (SI Appendix, Table S1 and Table 1). These cases 26860 | www.pnas.org/cgi/doi/10.1073/pnas.1915737116 Huor et al. Groups of 6- to 10-wk-old female mice (n ≥ 6) were anesthetized and in- oculated with 20 μL of a 10% tissue homogenate in the right parietal lobe using a 25-gauge disposable hypodermic needle. Mice were observed daily, and their neurological status was assessed weekly. When clinically progressive TSE disease was evident, the animals were euthanized and their brains harvested. Half of the brain was fixed by immersion in 10% formol saline and the other half was frozen at −20 °C. Tissues from animals found dead were frozen (no formalin fixation). In animals where no clinical signs were observed, mice were killed at the end of their natural lifespan (650 to 750 d). In those cases, in- cubation periods reported in Tables 1 and 2 as >650 dpi corresponded to the survival time observed in at least 3 out of the 6 mice. PMCA Amplification. Brains from tgBov, tgARQ, and tg338 mice were used to prepare the PMCA substrates. PMCA was performed as previously described (28, 56). Briefly, PMCA reactions (50 μL final volume) were seeded with 5 μL of sample to be tested. PMCA reactions were then subjected to 3 amplifi- cation rounds, each comprising 96 cycles (10 s sonication, 14 min and 50 s incubation at 39.5 °C) in a Qsonica700 device. After each round, reaction products (1 volume) were mixed with fresh substrate (9 volumes) to seed the following round. The PMCA reaction products were analyzed by Western blot for the presence of PK-resistant PrP. Each WB line was loaded with the equivalent of 20 μL of PK-digested PMCA product. Each PMCA run included a reference ovine BSE sample (10% brain homogenate) as a control for the amplification efficiency. Unseeded controls (2 unseeded controls for 8 seeded reactions) were also included in each run. MEDICAL SCIENCES Western Blot Detection of Abnormal PrP. PK-resistant abnormal PrP (PrPres) extraction and Western blot were performed as previously described (57). Immunodetection was performed using 2 different PrP-specific monoclonal antibodies, Sha31 (1 μg/mL) (58) and 12B2 (4 μg/mL) (59), which recognize the amino acid sequences YEDRYYRE (145–152) and WGQGG (89–93), respectively (60). Paraffin-Embedded Tissue Blot. Paraffin-embedded brain tissue from inocu- lated mice was analyzed as previously described (61–63). Fig. 5. PMCA seeding activity detection in 2 bioassay endpoint-titrated Downloaded from https://www.pnas.org by 158.109.106.178 on July 12, 2022 from IP address 158.109.106.178. atypical/Nor98 isolates. Two atypical/Nor98 isolates, AS 25 and AS 26 (Ta- Lesion Profiling and Abnormal PrP Immunohistochemistry. Vacuolar brain le- ble 3), were endpoint-titrated in tg338 mice (1/10 dilution series, 6 tg338 sion profiles were established following the method described by Fraser et al. mice per dilution). For both isolates, the last positive transmissions were (64). In situ PrPSc immune labeling was performed as previously described observed in mice that received a 10−6 dilution of the original 10% w/vol (63) using 6H4 anti-PrP antibody (epitope: 147DYEDRYYRE155 of the bovine brain material (SI Appendix, Table S3). The original AS isolates and the brains PrP concentration 3 μg/mL). of clinically affected mice inoculated with neat and 10−6 diluted isolates were used to seed PMCA reactions that either used tgARQ or tgBov as Infectious and Seeding Activity Titer Estimates. A series of 1/10 dilutions of a substrate. PMCA reactions seeded with age-matched inoculated tg338 mice reference 10% w/vol brainstem from an ovine-BSE (ARQ/ARQ) isolate and 2 AS and unseeded reactions were included as specificity controls. Reactions were isolates (AS 25 and AS 26) were prepared. Successive 1/10 dilutions of brain subjected to 3 (tgARQ substrate) or 4 (tgBov substrate) amplification rounds, homogenate were inoculated intracerebrally (20 μL) into tgBov or tg338 mice each comprising 96 cycles (10 s sonication, 14 min and 50 s incubation at (n = 6 per inoculum). Dilutions of the same c-BSE isolate were used to seed 39.5 °C) in a Qsonica700 device. The PMCA reactions were analyzed by PMCA reactions that used brain tissue from either bovine PrP (tgBov mice) or Western blot for the presence of abnormal PK-resistant PrP (PrPres) using the ovine ARQ PrP (tgARQ mice) as substrate. Twelve individual replicates of each Sha31 (epitope 145-YEDRYYRE-152) and/or the 12B2 (89-WGQGG-93) anti- sample dilution were tested. Reactions were then subjected to 3 amplification PrP antibodies. Each Western blot included a classical scrapie isolate (labeled rounds. PMCA reaction products (third amplification round) were analyzed by as WB control) and an ovine c-BSE isolate as controls. Western blot for the presence of PrPres. The titer of prion seeding activity was estimated by the Spearman–Kärber’s method (65). For AS and AS passaged in tg338 isolates (10% brain homogenate), 1/50 have been originally classified as AS by TSE national reference laboratories in diluted material was used to seed 12 individual reactions (tgBov or tgARQ each country. All of the cases corresponded to sheep except the AS 7 case (goat). substrates). After 3 amplification rounds, the number of PrPres Western In all cases, PrP genotype was checked by sequencing exon 3 of the PRNP gene as blot-positive reactions per total number of reactions was established. previously described (48). The polymorphisms at codons 136 (A/V), 154 (H/R), and These ratios were used to estimate seeding activity titers (SA/μL of 10% 171 (R/Q/H), which have been demonstrated to strongly influence the susceptibility brain homogenate) by the limiting dilution titration method (application to TSE in sheep, are indicated (49). Additionally, the presence of a phenylalanine at of Poisson’s probabilistic model) described by Brown et al. (66) or by codon 141 (F/L), which has been shown to impact on the susceptibility to atypical/ Spearman–Kärber’s method. According to Fisher et al. (67) and as pre- Nor98 scrapie, are also indicated (Tables 1 and 4) (23, 48). Brain material collected viously used for prion infectivity comparisons (68), 1 SA50 was considered in TSE-free Poll–Dorset sheep (APHA) was used as control (50). to be equivalent to 0.693 SA. c-BSE Isolates. Cattle and ovine classical BSE (c-BSE) isolates were used as Data Availability. All data are available in the manuscript and SI Appendix. control. The cattle c-BSE isolate was a natural case originating from France. This isolate was used in previous studies aimed at the characterization of ACKNOWLEDGMENTS. This work was funded by Fonds Europeens de Devel- c-BSE strain properties through transmission to mice overexpressing the PrP oppement Regional Programme Operationnel de Cooperation Territoriale sequence of various host species (51). The ovine c-BSE isolate was obtained by Espagne France Andorre TRANSPRION (EFA282/13) and REDPRION (EFA148/ the intracerebral inoculation of the same cattle c-BSE isolate in ARQ/ARQ 16); the UK Food Standards Agency Exploring permeability of the species barrier TSE-free sheep (first passage) as described in Andréoletti et al., 2004 (50). (M03043 and FS231051); the European Union through FP7 222887 “Priority;” the Spanish Ministerio de Economía y Competitividad (AGL2016-78054-R [AEI/ FEDER, UE]); and Fundació La Marató de TV3 (201821-31). A.M.-M. was sup- Mouse Bioassays. Bioassays were carried out using mice expressing bovine PrP ported by a fellowship from the INIA (FPI-SGIT-2015-02), and P.A.-C. was (tgBov/tg110) (52, 53) and/or mice expressing ovine ARQ (tgARQ) (54) or VRQ supported by a fellowship from the Spanish Ministerio de Economía y (tg338) PrP (55). Competitividad (BES-2010-040922). Huor et al. PNAS | December 26, 2019 | vol. 116 | no. 52 | 26861 1. M. P. McKinley, D. C. 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