Strain Traits of Intracranially Administered L‐Type Bovine Spongiform Encephalopathy Prions Are not Significantly Modified During Intraspecies Transmission in Cynomolgus Monkeys
Ken'ichi Hagiwara, Hiroaki Shibata, Minoru Tobiume, Yuko Sato, Keiko Ohto, Sachi Okabayashi, Nozomi Nakano, Motohiro Horiuchi, Fumiko Ono
TL;DR
This study shows that L-type BSE prions retain their key traits after being transmitted within cynomolgus monkeys, supporting their zoonotic potential and stable pathogenesis.
Contribution
Demonstrates the stability of L-BSE prion strain traits after intraspecies transmission in nonhuman primates.
Findings
L-BSE prions caused disease in monkeys with similar endpoints and brain pathology as in primary transmission.
L-BSE prions remained non-transmissible to mice after monkey passage, unlike C-BSE prions.
Prion traits such as vacuolation and PrPSc distribution were consistent across transmission rounds.
Abstract
Among the three prion strains of bovine spongiform encephalopathy (BSE), classical BSE (C‐BSE) prions are known causative agents of variant Creutzfeldt–Jakob disease. By contrast, human infections with L‐type (L‐) or H‐type (H‐) BSE prions have not been reported. Nonetheless, the zoonotic potential of L‐BSE prions is supported by their successful primary transmission from cattle to cynomolgus macaque (Macaca fascicularis) monkeys via intracranial challenge. To assess whether the defining strain traits of L‐BSE prions remain stable following secondary intraspecies transmission, we prepared brain homogenates from a diseased macaque that had previously undergone primary transmission of L‐BSE prions, and intracranially administered them into two naïve macaques. Both animals succumbed to the disease within humane endpoints comparable to those observed in the primary transmission.…
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Figure 1
Figure 2
Figure 3| Transmission | Primary | Secondary | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Inoculum | Cattle C‐BSE | Cattle L‐BSE | Macaque C‐BSE | Macaque L‐BSE | |||||
| Macaque ID# | 007 | 010 | 011 | 014 | 015 | 016 | 017 | 022 | 023 |
| Age at inoculation (months) | 24 | 28 | 29 | 25 | 24 | 25 | 24 | 17 | 17 |
| Humane endpoints (mpi) | 37 | 35 | 58 | 25 | 24 | 19 | 19 | 24 | 24 |
| Abnormal behaviors | |||||||||
| Depression | — | +++ | — | — | — | + | — | — | — |
| Self‐harm | + | — | + | — | — | — | — | — | — |
| Anorexia | + | — | + | — | + | — | + | ± | — |
| Hyperekplexia | +++ | +++ | ++ | — | — | ++ | ++ | — | — |
| Neuronal symptoms | |||||||||
| Ataxia | +++ | +++ | +++ | ++ | ++ | ++ | +++ | — | + |
| Tremor | +++ | +++ | +++ | +++ | +++ | + | ++ | ++ | ++ |
| Limb paralysis | +++ | ++ | +++ | +++ | +++ | + | ++ | + | + |
| Myoclonus | ++ | ++ | ++ | ++ | ++ | — | ++ | + | + |
| PrP‐coding sequence | a | — | — | — | b | — | b | — | b |
| Reference | 12 | 15 | 12 | This study | |||||
| Inoculum | PK digestion prior to inoculation | Affected | Duration to humane endpoints (dpi) | Median duration (dpi) |
|---|---|---|---|---|
| Macaque C‐BSE prions (#017) | No | 4/4 | 268, 275, 288, 307 | 281.5 |
| Yes | 8/8 | 261, 261, 262, 264, 264, 264, 291, 303 | 264.0 | |
| Macaque L‐BSE prions (#022) | No | 0/8 | – (706) | — |
| Yes | 0/11 | – (751) | — |
- —Ministry of Health, Labour and Welfare, Japan
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Taxonomy
TopicsPrion Diseases and Protein Misfolding · Neurological diseases and metabolism · Zoonotic diseases and public health
Introduction
1
Transmissible spongiform encephalopathy, often referred to as prion disease, is a fatal neurodegenerative disorder caused by prions, which primarily consist of pathogenic amyloid aggregates of prion protein (PrP). The disease‐associated isoforms (PrP^Sc^) arise from conformational conversion of normal cellular PrP (PrP^C^). A mature form of PrP^C^ is expressed on the outer leaflet of the plasma membrane of cells, being tethered by a glycophosphatidyl inositol (GPI) anchor, and has two potential N‐glycosylation sites. Replicative conversion of PrP^C^ to PrP^Sc^, seeded by PrP^Sc^ molecules, is a key event in prion propagation in which the PrP^Sc^ seeds may originate from endogenous PrP^C^ through unknown mechanisms, mutations in the PrP gene (Prnp), or uptake of exogenous PrP^Sc^. Accordingly, prion diseases have emerged as sporadic, familial, and infectious disorders [1]. Biochemically, PrP^C^ and aggregated PrP^Sc^ differ in their susceptibility to digestion with proteinase K (PK). While PrP^C^ is PK‐digestible, aggregated PrP^Sc^ is partially resistant, leaving two‐thirds of the carboxyl‐terminal (C‐terminal) region of PrP^Sc^ undegraded. PK digestion thereby enables preferential detection of PrP^Sc^ in samples; however, there are exceptions [2].
Prions are devoid of nucleic acids, and the term ‘strain’ in prion biology does not imply a genetic lineage; rather, it describes phenotypic disease variations and/or biochemical profiles of PrP^Sc^ [3, 4]. Bovine spongiform encephalopathy (BSE) is a prion disease of cattle, involving three known strains: classical‐type (C‐BSE), L‐type (L‐BSE, also known as bovine amyloidotic spongiform encephalopathy [BASE]), and H‐type (H‐BSE) [5, 6, 7, 8]. C‐BSE prions are zoonotic, causing variant Creutzfeldt‐Jakob disease (vCJD). The etiological link between C‐BSE prions and vCJD is supported by experiments using monkeys of the cynomolgus macaque species (Macaca fascicularis), which share 95.7% amino acid sequence identity with human PrP^C^, comprising 200 of the 209 amino acids present in the mature forms of both human and macaque PrP^C^. Following challenge with cattle‐derived C‐BSE prions, macaques develop a neuropathological phenotype similar to that observed in patients with vCJD [9, 10, 11, 12].
By comparison, L‐ and H‐BSE are rarely detected in cattle, predominantly in aged individuals [5, 6, 8]. Surveillance data from the European Food Safety Authority indicate that the number of cattle affected by L‐ or H‐BSE has remained low over the past decade, averaging ~10 cases annually, with four L‐type and six H‐type cases reported in 2023 [13]. The infrequent occurrence of the cases, combined with the implementation of BSE control measures, suggests a low risk of human exposure to L‐ and H‐BSE prions through the consumption of beef products. Indeed, no cases of human infection with L‐ or H‐BSE prions have been reported to date. Nevertheless, L‐BSE prions are efficiently transmitted to cynomolgus macaques via intracranial administration [14, 15], producing neuropathological features distinct from those induced by cattle‐derived C‐BSE prions.
In general, transmission of prions between different animal species is constrained by a substantial barrier, largely owing to differences in the amino acid sequences of host PrP^C^ and prion PrP^Sc^, and the structural compatibility between them. Interspecies transmission varies in efficiency and occasionally leads to the emergence of new prion strains [3]. For example, L‐BSE prions have been shown to acquire phenotypic features resembling those of C‐BSE prions during interspecies transmission from cattle to hamsters [16], or to transgenic mice expressing hamster PrP^C^ or ovine PrP^C^ [17, 18]. Thus, although L‐BSE prions transmitted from cattle to macaques induce a neuropathology phenotype distinct from those of C‐BSE prions in macaques [14, 15, 19], it remains unclear whether the defining strain traits of L‐BSE prions are preserved following secondary intraspecies transmission. A previous study on primary transmission of cattle L‐BSE prions to macaques highlighted the importance of addressing this question [14].
We have conducted an intraspecies secondary transmission study in macaques to examine whether their strain properties and associated neuropathology are retained or altered following primary transmission.
Materials and Methods
2
Inoculation, Care, and Prnp Analysis of Macaques
2.1
Studies using cynomolgus macaques were conducted at the Tsukuba Primate Research Center (TPRC) of the National Institutes of Biomedical Innovation, Health and Nutrition (NIBN), Japan. Homogenates of the cerebral frontal cortex of macaque #015, which was euthanised at humane endpoints following infection with L‐BSE prions from a cow [15], were prepared in phosphate‐buffered saline (PBS, pH 7.2) at 10% (w/v) tissue concentration. Aliquots of 0.2 mL (equivalent to 20 mg of tissue) were intracranially injected into the left temporal region of recipient male macaques (#022 and #023) at an approximate depth of 4 mm from the skull surface, under ketamine/xylazine anaesthesia. The animals were housed individually in ventilated cages of appropriate size and observed daily, in accordance with TPRC guidelines [19]. They were euthanised at humane endpoints to minimise distress, and tissue samples were collected for analysis. DNA sequence analysis of Prnp was performed as previously described [19].
Histopathology and Immunohistochemistry
2.2
Brain tissues were fixed in 10% formalin in PBS, immersed in 98% formic acid to inactivate prion infectivity, and embedded in paraffin. Coronal tissue sections (3‐µm thickness) were prepared and mounted on glass slides. To visualise PrP^Sc^, the sections were treated with 0.1 N NaOH containing 2% NaCl and 0.1% N‐lauroylsarcosine for antigen retrieval [20], and were subjected to immunohistochemical staining using the rabbit polyclonal anti‐PrP antibody T4. Antibody T4 was raised against a synthetic peptide corresponding to amino acid residues 221–239 of bovine PrP [21, 22], and also binds to the homologous epitope in macaque PrP. Immunopositive signals were detected with the EnVision+ system (Dako Agilent, California, USA), using 3,3'‐diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) as the substrate [19, 21]. Alternatively, sections were stained with hematoxylin and eosin (H&E). Stained sections were observed under a BX51 microscope (Olympus Co. Tokyo, Japan).
Western Blot Analysis
2.3
Samples were prepared as previously described [23]. Briefly, an aliquot of 20% (w/v) tissue homogenate in PBS (typically 25 µL) was mixed with an equal volume of buffer containing 4% (w/v) zwittergent 3–14 (Merck KGaA, Darmstadt, Germany), 1% (w/v) lauroylsarcosine sodium salt (Merck KgaA), 0.5 mg/mL collagenase (Fujifilm Wako Pure Chemical Co., Osaka, Japan), and 60 units/mL benzonase (Merck KgaA) in 100 mM NaCl and 50 mM HEPES‐NaOH (pH 7.4). After a 30‐min incubation at 37°C, the samples were treated with PK (Roche Diagnostics GmbH, Vienna, Austria) at 50 μg/mL for 45 min at 37°C, unless otherwise stated. To each sample, half the sample volume of a mixture of 2‐butanol and methanol (5:1, v/v) containing 10 mM phenylmethanesulfonyl fluoride was added, followed by centrifugation at 17,500 × g for 15 min at 18°C. The resulting precipitates were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), followed by western blot analysis using anti‐PrP antibody clones 3F4 (Merck KgaA) at 200 ng/mL [24] and SAF84 (Bertin Technologies, Montigny‐le‐Bretonneux, France) at 70 ng/mL [25]. Detection was performed using horseradish peroxidase (HRP)‐conjugated AffiniPure F(ab′)2 anti‐mouse IgG (Jackson ImmunoResearch Laboratories Inc., Pennsylvania, USA) at 80 ng/mL. Chemiluminescent signals were visualised using Immobilon Forte Western HRP substrate (Merck KgaA) and captured on Super RX films (Fujifilm, Tokyo, Japan) or with a CCD camera‐equipped imaging system (WSE‐6200 LuminoGraph II; ATTO Co. Tokyo, Japan). Signal intensities were fitted to one‐phase exponential decay curves using Prism 6 software (GraphPad Software Inc., Massachusetts, USA).
Mouse Bioassays
2.4
Murine bioassay experiments were conducted at the National Institute of Infectious Diseases, Japan (NIID). Cerebral frontal cortex specimens from macaques undergoing secondary transmission with C‐BSE prions (#017) [12] and L‐BSE prions (#023) were homogenised in PBS at a 20% tissue concentration (w/v). To normalize PrP^Sc^ levels based on semi‐quantitative western blot analysis, the homogenates were further diluted with PBS to 0.5% (w/v) for #017% and 1% (w/v) for #023. Aliquots (25 µL) of the diluted homogenates were intracranially injected into C57BL/6 J mice.
Alternatively, 20% (w/v) brain homogenates were digested with PK (50 μg/mL), followed by the addition of one‐third volume of ethanol. The mixtures were centrifuged at 17,500 × g for 15 min at 4°C, and the pellets were resuspended in PBS to reconstitute a 4% (w/v) tissue concentration. Aliquots (25 µL) of the resuspended material were then intracranially injected into mice. All experiments were terminated at humane endpoints. Survival data were analyzed using the Kaplan‐Meier method with Prism 6 software.
Results
3
Primary and Secondary Transmission of L‐BSE Prions in Macaques
3.1
Table 1 summarizes key information for the macaques used in the present study (macaques #022 and #023) and those from our previous studies (#007, #010, #011, #014, #015, #016, and #017) [12, 15]. Regarding previously examined cattle‐derived C‐BSE prions, the time to humane endpoints following primary intracranial administration was 35, 37, and 58 months post‐inoculation (mpi) in macaques #010, #007, and #011, respectively. The following secondary transmission using macaque‐derived C‐BSE prions resulted in significantly shortened durations (19 months for both #016 and #17), reflecting prion adaptation during serial passage in the macaque host [10, 12]. In contrast, macaques challenged by cattle‐derived L‐BSE prions in our primary transmission study developed disease more rapidly (euthanized at humane endpoints at 24 mpi (#015) and 25 mpi (#014)) than those inoculated with C‐BSE prions in primary transmission (#007, #010, and #011), indicating a lower transmission barrier for L‐BSE prions from cattle to macaques. In the present study, secondary transmission of L‐BSE prions in macaques #022 and #023 resulted in comparable time to human endpoints as that of L‐BSE primary transmission macaques #014 and #015 (#014, 25 mpi; #015, 24 mpi; #022, 24 mpi; #023, 24 mpi) (Table 1). During secondary transmission, hyperekplexia and ataxia were moderate in macaques infected with L‐BSE prions compared to those infected with C‐BSE prions (Table 1).
Histopathological and Immunohistochemical Brain Examination
3.2
H&E staining of brain tissue sections revealed that secondary L‐BSE prion transmission induced intense spongiosis in the cerebral cortex (Figure 1a–c, upper panels), and mild spongiosis in the thalamus and hippocampus (Figure 1d–f, upper panels). Confluent spongiosis in the cerebral cortex extended from cortical layers II to VI (Figure 1a), with vacuole diameters typically ranging from 5 to 25 µm. Immunohistochemical staining showed a widespread, fine, and diffuse synaptic distribution of PrP^Sc^ in the cerebrum (Figure 1b–e, lower panels), consistent with previous reports of primary transmission of L‐BSE and BASE prions to macaques [14, 15]. The diffuse synaptic distribution of PrP^Sc^ was clearly different from the plaque‐type distribution of PrP^Sc^ observed in the cerebrum infected with C‐BSE prions (Figure 1l,m). In the hippocampus, diffuse synaptic distribution of PrP^Sc^ was observed in the molecular layer, in proximity to the granular cell layer (Figure 1f, lower panel). In contrast, florid plaques surrounded by proximate vacuoles were observed in the cerebrum of a macaque infected with C‐BSE prions (Figure 1n) [9, 10, 11, 12], resembling those found in patients with vCJD [1, 26]. Such plaques were absent in macaques infected with L‐BSE or BASE prions in previous primary transmission experiments [14, 15], and likewise absent in macaques with secondary transmission of L‐BSE prions in this study (Figure 1b–f). Pathological features were also conserved in the cerebellum, where secondary L‐BSE prion transmission produced mild spongiosis in the granular layer (Figure 1g,h, upper panel) and sparse PrP^Sc^ deposition as small plaques (typically < 10 µm) in the molecular layer (Figure 1g,h, lower panel) [15]. Such plaques in the cerebellum were characteristically observed in macaques infected with L‐BSE prions, but not in macaques infected with C‐BSE prions (Figure 1o, upper and lower panels) [12]. In accordance with western blot analysis, which showed lower accumulation of PrP^Sc^ in medulla oblongata (see section 3.3), immunohistochemical analysis revealed sparse and weak signals of PrP^Sc^ in medulla oblongata (Figure 1i).
Spongiform degeneration and immunohistochemical detection of PrPSc in macaque brains following secondary transmission of L‐BSE and C‐BSE prions. Representative histological and immunohistochemical images from macaque #023 with L‐BSE prions (this study; a–k) and macaque #017 with C‐BSE prions (prior study; l–o). (a) Haematoxylin and eosin (H&E) staining of the cerebral cortex (coronal parietal lobe), comprising three contiguous images showing cortical layers I‐VI. (b‐o) H&E staining (upper panels) and immunohistochemical staining of PrPSc with anti‐prion antibody T4 (lower panels; counterstaining with hematoxylin). (n) A representative florid plaque. Scale bars: 200 µm in (a) and 50 µm in all other panels. Objectives: ×4 for (a); ×10 for (b), (g), (j) and (l); ×20 for (c), (d), (h), (m), and (o); and ×40 for (e), (f), (i), (k), and (n). BSE: bovine spongiform encephalopathy; PrP prion protein.
Western Blot Analysis of Neural Tissues, the Spleen, and Tonsils
3.3
Western blot analysis of macaques undergoing primary and secondary transmission of C‐BSE or L‐BSE prions revealed positive PrP^Sc^ signals in the frontal, parietal, and occipital cortices (Figure 2a), as well as the hippocampus (Figure 2b). The three characteristic bands observed in the western blots represent the non‐, mono‐, and di‐glycosylated forms of PrP^Sc^, corresponding to the absence, presence at one site, or presence at both of the two potential N‐glycosylation sites (Asn^181^ and Asn^197^) where glycans may be attached. Signal intensities of PrP^Sc^ were markedly weaker in the cerebellum, pons, and medulla oblongata than in the frontal lobe during both primary and secondary L‐BSE transmission, and 250‐µg tissue equivalents from the cerebellum, pons, and medulla oblongata versus 5 µg from the frontal lobe showed comparable signal intensities of PrP^Sc^ in western blot analysis (Figure 2a,b; see right panel of 2b). In the spinal cord, PrP^Sc^ accumulation following C‐BSE infection was higher in the cervical and lumbar portions during both primary (#007) and secondary (#017) transmission, and lower in the thoracic region, consistent with regional differences in spinal cord diameter and grey matter content (Figure 2c). In contrast, L‐BSE transmission (primary: #015; secondary: #023) resulted in overall lower PrP^Sc^ accumulation, with signal intensities relatively uniform across the cervical, thoracic, and lumbar regions (Figure 2c). The reduced PrP^Sc^ signals in the cerebellum and spinal cord following L‐BSE transmission indicated slower or less efficient infiltration and/or propagation of L‐BSE prions in these regions, supporting distinct neurotropism between L‐BSE and C‐BSE prions in macaques. In primary transmission studies using C‐BSE and L‐BSE prions, PrP^Sc^ was not detected in the spleen or tonsils by immunohistochemistry or western blotting [12, 15]. Consistent with previous findings, western blotting showed no PrP^Sc^ signal in these tissues during secondary transmission in the present study (Figure 2d).
Western blot analysis of PrPSc accumulation in macaque tissues. (a, b) Western blots of proteinase K (PK)‐digested brain tissue samples from the indicated macaques undergoing primary or secondary transmission with C‐BSE or L‐BSE prions. Samples correspond to 5 µg or 250 µg pre‐digestion tissue equivalents (Tissue eq.), probed with anti‐PrP antibodies 3F4 and/or SAF84. Three glycoforms of PrPSc (di‐, mono‐, and non‐glycosylated) are indicated (a). Relative molecular mass (Mr) is shown in the right panel (b). (c) PK‐digested spinal cord samples from the same macaques. (d) Western blots of spleen and tonsil tissues from the indicated macaques, showing no detectable PrPSc signals even with 5 mg and 2.5 mg Tissue eq. Frt, frontal lobe; Par, parietal lobe; Occ, occipital lobe; Cblm, cerebellum; Med obl, medulla oblongata; Hipp, hippocampus; Cerv sp, cervical spinal cord; Thor sp, thoracic spinal cord; Lumb sp, lumbar spinal cord; grey, grey matter; white, white matter.
PK Resistance of PrPSc
3.4
We previously demonstrated that cattle‐derived C‐BSE prions exhibited greater resistance to PK digestion than L‐BSE prions (Figure 3a), and both prion types showed comparable PK resistance following primary transmission to macaques (Figure 3b) [19]. In the present study, we confirmed that L‐BSE prions again had PK resistance comparable to that of C‐BSE after secondary transmission to macaques (compare Figure 3b,c). In addition, we compared PK resistance between L‐BSE prions after primary and secondary transmissions to macaques (Figure 3d). Fitting the data to one‐phase exponential decay curves demonstrated a higher resistance after secondary transmission compared to after primary transmission, although the difference was slight.
Proteinase K (PK) sensitivity profile of disease‐associated forms of prion protein (PrPSc). (a, b) Relative signal intensities of PrPSc following PK digestion of brain homogenates, plotted against PK concentration, from cattle‐derived C‐ and L‐bovine spongiform encephalopathy (BSE) prions before (a) and after (b) primary transmission in macaques. Data in these panels were adapted from western blot data shown in our previous study [19] for comparison. Plots represent the results of three independent analyses after normalization of signal intensities to those obtained at 50 µg/mL PK (set to 100.0%). (c) Western blot (right panel) of PrPSc in frontal lobe homogenates prepared from macaques #017 and #023 following secondary transmission. The homogenates were digested with increasing concentrations of PK (50–1000 μg/mL), and subjected to western blot analysis using anti‐PrP antibody 3F4 to visualize PrPSc bands. The sum of signal intensities of di‐, mono‐, and non‐glycosylated forms of PrPSc in each lane was plotted after normalization to that obtained at 50 µg/mL PK (set to 100.0%) as in (a) and (b) (left panel). Mr, Relative molecular mass. The data represent the results of three independent analyses. (d) PK sensitivity of PrPSc in frontal lobe homogenates prepared from macaques #014 and 015 (primary transmission), and #022 and #023 (secondary transmission). Taking account of approximately 1.5‐fold abundance of PrPSc in the frontal lobe after secondary transmission than after primary transmission (Figure 2a), aliquots of the homogenates corresponding to 12 µg frontal lobe of #014 and #015, and 8 µg frontal lobe of #022 and #023, were incubated with PK at the concentrations indicated. A representative western blot image of macaques #015 and #023 is shown on the right.
Mouse Bioassays
3.5
As shown in Figure 3b, C‐and L‐BSE prions show comparable PK resistance following primary transmission to macaques. However, our previous study showed that their transmissibility to C57BL/6 J mice differed: C‐BSE prions were transmissible, whereas L‐BSE prions were not, consistent with the findings observed in earlier studies for cattle origin [19]. In the present study, L‐BSE prions after secondary transmission were challenged in C57BL/6 J mice. As a result, they again failed to transmit disease to the mice, mirroring the transmission barrier observed with cattle‐derived L‐BSE. In contrast, C‐BSE prions from a macaque (#017) remained transmissible to the mice (Table 2), despite L‐ and C‐BSE prions showing similar PK resistance (Figure 3c) and potent transmissibility between macaques (Table 1).
Given that prion infectivity resides in the PK‐resistant C‐terminal region of PrP^Sc^ [27], the unsuccessful transmission led us to hypothesize that the N‐terminal region of PrP^Sc^ in L‐BSE prions interferes with transmission to mice. To test the hypothesis, we subjected L‐BSE prions to PK digestion to truncate the N‐terminal region of PrP^Sc^; however, we observed no change in transmissibility, indicating that N‐terminal trimming is insufficient to overcome the cross‐species barrier between macaques and mice (Table 2).
Discussion
4
This study aimed to extend our understanding of L‐BSE prion behavior in nonhuman primates by evaluating secondary intraspecies transmission in cynomolgus macaques. After intracranial inoculation as the most straightforward approach, we observed disease progression, neuropathology, and neurotropism consistent with primary intracranial transmission. Notably, L‐BSE prions propagated in macaque brains remained non‐transmissible to C57BL/6 J mice, mirroring the transmission barrier observed with cattle‐derived L‐BSE. These findings indicate that L‐BSE prions of cattle origin are intrinsically permissive to cynomolgus macaques via intracranial administration, and retain their strain characteristics during secondary transmission to macaques without significant modification. The most probable scenario for human‐to‐human transmission of L‐BSE prions is via iatrogenic exposure, including blood transfusion, organ transplantation, or the use of contaminated surgical equipment. The present study, which demonstrates the stability of L‐BSE strain traits in nonhuman primates following intracranial administration, has implications for potential iatrogenic infection through neurosurgical procedures.
On the other hand, we acknowledge a limitation in using inoculum prepared from the brain tissue of a macaque previously infected intracranially with cattle‐derived L‐BSE prions, because direct intracranial transmission from cattle to humans represents an unrealistic exposure scenario. In this context, a recent study detected prions in asymptomatic macaques that were autopsied at 75 mpi of oral administration of cattle‐derived L‐BSE prions [28]. Using protein misfolding cyclic amplification (PMCA) analysis, the study identified prion propagation in lymphoid tissues, as well as the cervical and thoracic spinal cord. Notably, the PMCA products derived from the ileum, spleen, inguinal lymph nodes, thoracic cord, submaxillary gland, and mesenteric lymph nodes of perorally administered macaques exhibited biochemical signatures that resembled the PMCA products of cattle‐derived C‐BSE prions rather than L‐BSE prions, based on SDS‐PAGE migration patterns and PK sensitivity. On the other hand, the brain tissues were negative for prions at autopsy, possibly reflecting a preclinical incubation stage. Whether L‐BSE prions administered perorally can propagate in the brain with traits similar to those induced by intracranial administration remains an intriguing and unresolved question.
Lastly, we demonstrated in mouse bioassay that L‐BSE prions, following secondary transmission to macaques, remained non‐transmissible to C57BL/6 J mice. The results were consistent with cattle‐derived L‐BSE prions and those subjected to primary transmission in macaques, both of which failed to transmit to the mice. Considering that prion infectivity is known to associate with the PK‐resistant region of a C‐terminal half of PrP^Sc^ [27], we wondered if the N‐terminal region of PrP^Sc^ of L‐BSE prions hampered their transmissibility to mice. We tested whether L‐BSE prions became transmissible to the mice after cleaving off the N‐terminal region by PK digestion, and found PK‐mediated truncation of the N‐terminal region failed to restore infectivity. As it seems unlikely that PK digestion itself impairs transmission, the results strongly support the notion that the PK‐resistant region of PrP^Sc^, but not the N‐terminal region, dictates the differential susceptibility of L‐BSE prions between macaques and mice.
In the present study, we demonstrated that L‐BSE prions exhibit a low barrier to transmission in macaques by intracranial administration, and retain their unique neuropathological traits. The comparable biochemical traits of L‐ and C‐BSE prions following transmission to macaques, including band patterns in western blot analysis and PK resistance, suggest the overall structural differences between the macaque‐adapted two BSE prions are restricted and subtle. Nevertheless, they should have pivotal distinct structures in the PK‐resistant region of PrP^Sc^ that dictate their unique transmission barriers, propagation efficacies, and neuropathological features. It is an intriguing study to consider in the future how macaque PrP^C^ accommodates the two BSE prion strains, allowing for their propagation with different degrees of transmission barriers, propagation efficiency, and neurotropism.
Author Contributions
Conceptualisation and project administration: Fumiko Ono, Hiroaki Shibata, Ken'ichi Hagiwara, Minoru Tobiume, and Motohiro Horiuchi Data curation: Ken'ichi Hagiwara, Minoru Tobiume, Yuko Sato, and Fumiko Ono Formal analysis: Ken'ichi Hagiwara, Hiroaki Shibata, and Fumiko Ono Funding acquisition: Motohiro Horiuchi Investigation: Ken'ichi Hagiwara, Hiroaki Shibata, Minoru Tobiume, Yuko Sato, Keiko Ohto, Sachi Okabayashi, Nozomi Nakano, and Fumiko Ono Writing − original draft: Ken'ichi Hagiwara. All authors reviewed and approved the final manuscript.
Ethics Statement
Cynomolgus macaques were obtained from the breeding colony of the TPRC, and protocols for the macaque experiments were approved by the Animal Welfare, Care and Use Committee, and the Animal Ethics Biosafety Committee of the NIBN (approval numbers DK17‐003, DS18‐069, DS18‐069R1, and DS23‐41R2). Protocols for the mouse experiments were approved by the Animal Welfare, Care and Use Committee of the NIID (approval number 116002). All experiments were conducted in compliance with the biosafety regulations of the TPRC and the NIID.
Conflicts of Interest
The authors declare no conflicts of interest.
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