Circulation of Nor98 Atypical Scrapie in Portuguese Sheep Confirmed by Transmission of Isolates into Transgenic Ovine ARQ-PrP Mice

Portugal was among the first European countries to report cases of Atypical Scrapie (ASc), the dominant form of Transmissible Spongiform Encephalopathy (TSE) in Portuguese small ruminants. Although the diagnostic phenotypes observed in Portuguese ASc cases seem identical to those described for Nor98, unequivocal identification requires TSE strain-typing using murine bioassays. In this regard, we initiated characterization of ASc isolates from sheep either homozygous for the ARQ genotype or the classical scrapie-resistant ARR genotype. Isolates from such genotypes were transmitted to TgshpXI mice expressing ovine PrPARQ. Mean incubation periods were 414 ± 58 and 483 ± 107 days in mice inoculated with AL141RQ/AF141RQ and AL141RR/AL141RR sheep isolates, respectively. Both isolates produced lesion profiles similar to French ASc Nor98 ‘discordant cases’, where vacuolation was observed in the hippocampus (G6), cerebral cortex at the thalamus (G8) level, cerebellar white matter (W1) and cerebral peduncles (W3). Immunohistochemical PrPSc deposition was observed in the hippocampus, cerebellar cortex, cerebellar white matter and cerebral peduncles in the form of aggregates and fine granules. These findings were consistent with previously reported cases of ASc Nor98 transmitted to transgenic TgshpXI mice, confirming that the ASc strain present in Portuguese sheep corresponds to ASc Nor98.


Introduction
Scrapie belongs to a group of diseases called transmissible spongiform encephalopathies (TSEs) or prion diseases. TSEs are caused by conversion of a natural prion protein (PrP C ) into an abnormal prion protein (PrP Sc ), which accumulates in affected tissues of the infected host, leading to neural degeneration [1]. While PrP C is completely hydrolyzed by proteinase K, PrP Sc has a proteinase K-resistant core (PrP res ), constituting the diagnostic hallmark of transmissible prion diseases. Prion differentiation is performed through evaluation of detergent solubility, as well as biochemical properties and electromobility of PrP res [2,3].
TSEs include Creutzfeldt-Jakob disease (CJD) in humans, Bovine Spongiform Encephalopathy (BSE) in cattle, Scrapie in small ruminants, Feline Spongiform Encephalopathy (FSE) in felids, and Chronic Wasting Disease (CWD) in cervids [4,5]. Currently, there are two known forms of scrapie infecting small ruminants, classical scrapie (CSc) and atypical scrapie (ASc). CSc is a transmissible form of scrapie that was first diagnosed nearly 300 years ago [6]. Sheep susceptibility to CSc is determined by polymorphisms in the prion protein gene (prnp), particularly at codons 136, 154 and 171. V136R154Q171 and A136R154Q171 sheep are the most susceptible genotypes, whereas A136R154R171 and A136H154Q171 are associated with relative resistance to the disease [7]. ASc has been reported in older sheep and in sheep with the prnp CSc-resistant AHQ and ARR alleles, as well as the AF141RQ allele [8][9][10]. ASc was first diagnosed in 1998 in Norway, but there is evidence it has existed since as early as 1972 [11]. Unlike CSc, outbreaks of ASc appear to be spontaneous. In addition, ASc tends to disseminate poorly within a flock [1,12].
The European Union active scrapie surveillance plan was implemented in 2002 following the European BSE crisis [13]. This program consists of testing a representative number of healthy slaughtered and fallen stock animals older than 18 months (active surveillance). Animals with suspected clinical signs of disease must also be tested (passive surveillance). Testing involves collection of brainstem samples, at the level of the obex, to be tested by an EU wide approved TSE rapid test. All positive results must be submitted for confirmatory tests, including Western immunoblotting, histopathology or immunohistochemistry (IHC). Additionally, genotyping must be performed [14].
ASc was first diagnosed in Portuguese sheep in 2003. The first seven cases were identified following the EU active surveillance plan, leading to testing of approximately 30,000 small ruminants for TSEs [15]. Unlike other European countries, ASc is the dominant form of scrapie in Portuguese sheep, and CSc was only identified, for the first time, in 2008. Until the end of 2020, Portugal reported 779 ASc cases (713 Portuguese sheep, 45 imported sheep; 15 Portuguese goats and 6 imported goats) and 45 CSc cases (39 Portuguese sheep and 6 imported sheep) in small ruminants.
Following confirmation of the first Portuguese ASc cases, this study aimed to straintype the disease using murine bioassays. Two brainstem isolates from sheep belonging to AL 141 RQ/AF 141 RQ and AL 141 RR/AL 141 RR genotypes were selected for this study. Each isolate was transmitted to a cohort of transgenic ovine mice expressing ovine PrPARQ (TgshpXI).

Results
Four out of the initial 30 animals died less than 100 days post-inoculation and were excluded from analysis. Three of these mice had been inoculated with the ARR isolate and one mouse inoculated with the ARQ isolate. This resulted in a total of 26 brains submitted for Western immunoblot analysis, 14 mice of AL 141 RQ/AF 141 RQ genotype and 12 mice of AL 141 RR/AL 141 RR genotype. Of the 14 mice analyzed after challenge with the ARQ isolate, 10 were confirmed PrP Sc positive, while all 13 mice inoculated with the ARR isolate were PrP Sc positive. Mean incubation periods of these positive mice were 418 ± 55 days for AL 141 RQ/AF 141 RQ cohort and 483 ± 102 days in AL 141 RR/AL 141 RR cohort.
Lesion profiles of TgshpXI mice inoculated with both genotypes are provided in Figure 1. The blue line refers to mice inoculated with AL 141 RQ/AF 141 RQ genotype and the red line refers to mice inoculated with AL 141 RR/AL 141 RR genotype. Both groups of mice exhibited vacuolation in the hippocampus (G6), cerebral cortex at the level of the thalamus (G8), cerebellar white matter (W1) and cerebral peduncles (W3) (Figure 2a-c). However, in comparison with the AL 141 RR/AL 141 RR genotype, mice belonging to the AL 141 RQ/AF 141 RQ cohort demonstrated overall increased severity in their lesions, despite shorter incubation periods.      The immunohistochemical features of both isolates revealed the same PrP Sc deposition patterns. PrP Sc deposition was observed in the hippocampal fissure and corpus callosum in the form of moderate aggregates and fine granular PrP Sc structures (Figure 2d), in the cerebellum (molecular layer) as mild fine granules (Figure 2e), as well as in the cerebellar white matter in the form of punctuate (Figure 2f).
Western immunoblot analysis of brains of TgshpXI showed the typical multi-band pattern of immunoreactive bands of PrP Sc between 12-60 kDa with the prominent low molecular mass band of approximately 12 kDa (Figure 3).
The immunohistochemical features of both isolates revealed the same PrP Sc deposition patterns. PrP Sc deposition was observed in the hippocampal fissure and corpus callosum in the form of moderate aggregates and fine granular PrP Sc structures (Figure 2d), in the cerebellum (molecular layer) as mild fine granules (Figure 2e), as well as in the cerebellar white matter in the form of punctuate (Figure 2f).
Western immunoblot analysis of brains of TgshpXI showed the typical multi-band pattern of immunoreactive bands of PrP Sc between 12-60 kDa with the prominent low molecular mass band of approximately 12 kDa (Figure 3).

Discussion
The results of this study indicate the ASc strain present in Portuguese sheep is indistinguishable from ASc Nor98. Both Portuguese isolates (from sheep of AL141RR/AL141RR and AL141RQ/AF141RQ genotypes) caused vacuolation peaks in regions G6 (hippocampus), G8 (cerebral cortex at the level of the thalamus) and W3 (cerebral peduncles). These findings coincided with the characteristic lesion profiles observed in other transgenic PrP mouse models inoculated with atypical scrapie [2,11,16,17]. Additionally, there was severe vacuolation in the cerebellar white matter (W1), as previously described for Tg338 mice inoculated with ASc French 'discordant' cases [2,17]. Previous studies using mouse bioassays showed similar Western immunoblot mobility patterns, but also having slight variations in lesion profiles, incubation periods, and PrP Sc deposition, depending on the mouse line used for determination of lesion profiles. Collectively, these findings suggest ASc is caused by a single prion strain [18].
The isolate from the AL141RQ/AF141RQ genotype promoted an increase in lesion severity with shorter incubation periods. Similarly, a previous study found inoculation of isolates from donors with prnp alleles linked to higher susceptibility to ASc lead to shorter incubation periods for lesion development in transgenic TgOvPrP4 mice, in comparison with those belonging to sheep with prnp alleles associated with ASc resistance [19]. Notably, in this context, is having the ARQ/ARQ genetic background in transgenic Tgshp XI mice renders them more susceptible to develop disease with the AL141RQ/AF141RQ isolate.

Discussion
The results of this study indicate the ASc strain present in Portuguese sheep is indistinguishable from ASc Nor98. Both Portuguese isolates (from sheep of AL 141 RR/AL 141 RR and AL 141 RQ/AF 141 RQ genotypes) caused vacuolation peaks in regions G6 (hippocampus), G8 (cerebral cortex at the level of the thalamus) and W3 (cerebral peduncles). These findings coincided with the characteristic lesion profiles observed in other transgenic PrP mouse models inoculated with atypical scrapie [2,11,16,17]. Additionally, there was severe vacuolation in the cerebellar white matter (W1), as previously described for Tg338 mice inoculated with ASc French 'discordant' cases [2,17]. Previous studies using mouse bioassays showed similar Western immunoblot mobility patterns, but also having slight variations in lesion profiles, incubation periods, and PrP Sc deposition, depending on the mouse line used for determination of lesion profiles. Collectively, these findings suggest ASc is caused by a single prion strain [18].
The isolate from the AL 141 RQ/AF 141 RQ genotype promoted an increase in lesion severity with shorter incubation periods. Similarly, a previous study found inoculation of isolates from donors with prnp alleles linked to higher susceptibility to ASc lead to shorter incubation periods for lesion development in transgenic TgOvPrP4 mice, in comparison with those belonging to sheep with prnp alleles associated with ASc resistance [19]. Notably, in this context, is having the ARQ/ARQ genetic background in transgenic Tgshp XI mice renders them more susceptible to develop disease with the AL 141 RQ/AF 141 RQ isolate.
Additionally, the PrP Sc profile showing marked deposition in the hippocampal fissure, corpus callosum, cerebellum, and cerebellar white matter, is similar to those reported cases of ASc transmitted to transgenic TgshpXI mice [2].
Both ASc affected sheep were diagnosed after observation of discrete vacuolation of the spinal tract nucleus of the trigeminal nerve, in addition to globular PrP Sc deposits in the white matter at the level of the obex. Unfortunately, in archived sheep tissues examined for this study, collection of the cerebellum was not a standard procedure at that time; thus, it was not possible to verify the PrP Sc deposition in this region. The variability in PrP Sc neuroanatomical distribution in the natural host is well-known [9,20]. However, the impact of such variations on the lesion and PrP Sc profile in transgenic mice remains uncertain. Hence, it is important to study the influence of such variations in murine bioassays, by selecting cases with different neuroanatomical distribution, in order to ascertain presence of differing atypical scrapie strains, as well as other factors, which may determine such variability.
Recent work revealed the possibility of the development of a classical-BSE (BSE-C) prion after inoculation of bovine PrP transgenic mice with ASc isolates [3]. That study found that BSE-C may be present in natural ASc isolates as a minor variant, and transmission of such isolates to transgenic bovine mice resulted in emergence of BSE-C as a dominant variant. The same phenomenon was not observed after inoculation of CSc isolates. Hence, there is concern regarding the possibility of ASc having a role in the emergence of BSE-C in cattle, and a possible role in the origin of the 1980s BSE crisis, resulting from inclusion of rendered small ruminants in cattle feed [3]. Furthermore, archival ASc isolates reveal ASc was present in the United Kingdom years before BSE [11]. Another study found oral transmission of ASc into sheep has resulted in a phenotype shift to CH1641, a classical scrapie strain showing an immunoblot profile similar to bovine BSE. Although CH1641 has not been diagnosed in Portuguese sheep as of yet, it is prudent to maintain vigilant systematic analysis of lesion profiles, PrP Sc immunolabelling types and patterns, as well as PrP Sc electrophoretic profiles in natural hosts for evidence of any phenotypic shift and strain conversion. Such surveillance is particularly relevant in a country such as Portugal, where, in contrast to other EU countries, ASc was first diagnosed in the absence of previous CSc cases.

Selection of Portuguese ASc Isolates
Among the first ASc cases, brainstem samples from two sheep to be AL 141 RQ/AF 141 RQ and scrapie resistant AL 141 RR/AL 141 RR genotypes, confirmed in 2004, were selected for this study. Cerebellum samples were not available as they were not routinely collected from sheep at that time. ASc was diagnosed after subjecting brainstem samples to rapid testing using approved lab tests and protocols as outlined (TeSeE™ kit, Bio-Rad, Munich, Germany) and to confirmatory tests histopathology, immunohistochemistry (anti-PrP 2G11 mouse antibody ovine PrP peptide sequence 146-R 154 -R 171 -182, Institute Pourquier, 1:200) and Western immunoblotting (TeSeE ® western blot kit, Bio-Rad) (see Table 1 for a summarized description of the isolates).

Transgenic Ovine ARQ PrP Mice (TgshpXI) Bioassay
All infection experiments in mice (LALLF 7221.3-2-1-027/02) described in this study were approved by the competent authority of the Federal State of Mecklenburg Western Pomerania, Germany, based on national and European legislation, namely the directive 2010/63/EU on the protection of animals used for scientific purposes.
Transmissions studies were conducted at the Friedrich-Loeffler-Institut (Isle of Riems, Germany). Each brainstem sample was prepared as a 10% homogenate in 0.9% sterile sodium chloride solution for intracerebral inoculation (30 µL). Samples from the two sheep genotypes were inoculated into transgenic TgshpXI mice overexpressing ovine ARQ genotype. Each genotype sample was replicated 15 times, resulting in a total of 30 animals.
All mice were examined for clinical symptoms at least twice weekly. Incubation periods were determined as time between inoculation and death of animals. Mice were culled after manifesting clinical signs of disease, followed by removal of their brains for further examination. Animals dying less than 100 days after inoculation were excluded from the study.

Lesion Profile
Formalin-fixed (4% neutral buffered formalin), paraffin-embedded brains were sectioned coronally at the level of the medulla nuclei, midbrain, thalamus and basal ganglia. All sections (4 µm thick) were stained in hematoxylin and eosin (H&E), according to standard protocols (https://science.vla.gov.uk/tse-lab-net/, accessed on 20 January 2019). Lesion profiles were produced according to vacuolation severity in nine grey matter (GM) and three white matter (WM) regions. The vacuolation score ranged from 0 to 5 in GM regions and from 0 to 3 in WM regions, as previously described [21,22].

Western Immunoblot Analysis
Western immunoblot analysis was performed as described previously [23,24]. Briefly, 10% (w/v) brain homogenates were prepared in 0.42 mM sucrose solution containing 0.5% deoxycholic acid sodium salt (DOC) and 0.5% Nonidet P40 (NP 40). Gross cellular debris was removed by centrifugation (6000 rpm for 5 min) at room temperature followed by addition of Proteinase K (Boehringer Mannheim) to 200 µL of supernatant, to a final concentration of 50 µg/mL PK, then incubated at 55 • C for 1 h. Digestion was terminated by addition of 4 µL Pefabloc (Roche, Mannheim, Germany) and heating for 5 min at 95 • C. Digested homogenates were mixed with phosphotungstic acid (PTA) to a final concentra-tion of 0.3% (w/v) PTA. Samples were incubated at 37 • C for 60 min with constant agitation before centrifugation at 13,300 rpm for 30 min at room temperature. The supernatant was carefully removed, pellets resuspended in sample buffer, heated for 5 min at 95 • C and loaded onto 16% Tris-polyacrylamide gels. Gels were transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) and blocked for 1 h in 5% (w/v) non-fat milk powder in PBS containing 0.1% (v/v) Tween-20 (PBST). Membranes were incubated with L42 monoclonal antibody (recombinant ovine PrP residues 145-163, R-Biopharm, Darmstadt, Germany) at a concentration of 0.4 µg/mL for 1 h at room temperature before washing three times with PBST followed by incubation in a 0.15 µg/mL concentration of alkaline-phosphatase-conjugated anti-mouse immunoglobulin (Dianova) in PBST for 1 h at room temperature. Membranes were finally washed three times with PBST, and bound antibodies were detected using the chemiluminescent substrate CDP Star (Tropix) and visualized directly in an image analysis system (Versa Doc, Quantity One, Bio-Rad, Munich, Germany).