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Presence of Subclinical Infection in Gene-Targeted Human Prion
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Protein Transgenic Mice Exposed to Atypical BSE
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Running title: Subclinical Infection in HuTg Mice Exposed to BASE
Rona Wilson1, Karen Dobie1, Nora Hunter1, Cristina Casalone2, Thierry Baron3 and
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Rona M Barron1*
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Midlothian, UK
Neurobiology Division, The Roslin Institute and R(D)SVS, University of Edinburgh, Roslin,
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Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy
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Agence Nationale de Sécurité Sanitaire, Lyon, France
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*
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Neurobiology Division
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The Roslin Institute and R(D)SVS, University of Edinburgh
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Roslin
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Midlothian, EH25 9PS
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UK
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Tel 0131 527 4200
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Fax 0131 440 0434
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rona.barron@roslin.ed.ac.uk
Corresponding Author
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Contents Category: TSE Agents
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Word count summary: 220
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Word count main text: 4806
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Number of tables and figures: 6
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Presence of Subclinical Infection in Gene-Targeted Human Prion Protein
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Transgenic Mice Exposed to Atypical BSE
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Summary
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The transmission of bovine spongiform encephalopathy (BSE) to humans, leading to variant
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Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform
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encephalopathies (TSEs) can pose a risk to human health. Until recently, TSE disease in
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cattle was thought to be caused by a single agent strain, BSE, also known as classical BSE,
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or BSE-C. However, due to the initiation of a large scale surveillance programme throughout
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Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE,
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also named BSE-L) and BSE-H have since been discovered. To model the risk to human
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health, we previously inoculated these two forms of atypical BSE (BASE and BSE-H) into
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gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP) (HuTg) but
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were unable to detect any signs of TSE pathology in these mice. However, despite the
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absence of TSE pathology, upon subpassage of some BASE challenged HuTg mice, a TSE
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was observed in recipient gene-targeted bovine PrP Tg (Bov6) mice, but not in HuTg mice.
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Disease transmission from apparently healthy individuals indicates the presence of
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subclinical BASE infection in mice expressing human PrP that cannot be identified by current
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diagnostic methods. However, due to the lack of transmission to HuTg mice on subpassage,
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the efficiency of mouse to mouse transmission of BASE appears to be low when mice
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express human rather than bovine PrP.
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Introduction
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Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle,
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and belongs to a group of diseases known as transmissible spongiform encephalopathies
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(TSEs) or prion diseases. The main characteristic of BSE is the accumulation in the brain of
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PrPTSE, which is a protease resistant conformational variant of the normal host encoded
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cellular prion protein (PrPC). BSE was first reported in the UK in 1987 (Bruce et al., 1997;
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Wells et al., 1987) and its transmission to humans through the consumption of contaminated
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food is thought to be the cause of the variant form of Creutzfeldt-Jakob disease (vCJD)
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(Bruce et al., 1997; Hill et al., 1997). While a number of other animal TSEs exist, including
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scrapie in sheep and goats and chronic wasting disease (CWD) in cervids, BSE is the only
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TSE known to be naturally transmissible from animals to humans. Previously, TSE disease
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in cattle was believed to be caused by a single prion strain, known as classical BSE (BSE-
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C).
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Europe, two atypical BSE agents were reported (Biacabe et al., 2004; Casalone et al., 2004;
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Jacobs et al., 2007; Stack et al., 2009), and identified as BSE-H and bovine amyloidotic
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spongiform encephalopathy (BASE, also named BSE-L). BSE-H and BASE were originally
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described in 2004 in France (Biacabe et al., 2004) and Italy (Casalone et al., 2004)
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respectively, however these atypical BSE strains have since been identified in other
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European countries (Jacobs et al., 2007), Japan (Hagiwara et al., 2007) and North America
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(Dudas et al., 2010; Richt et al., 2007). Several studies have shown that following
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transmission into transgenic mice that overexpress the bovine prion protein, these atypical
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BSE agents show neuropathological and molecular phenotypes which are distinct from BSE-
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C, indicating they are different BSE strains (Beringue et al., 2007; Béringue et al., 2006;
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Buschmann et al., 2006; Capobianco et al., 2007; Okada et al., 2010). Indeed, BASE and
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BSE-H can be distinguished by the electrophoretic migration of their protease-resistant
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PrPTSE isoforms and their different patterns of glycosylation (Biacabe et al., 2004;
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Buschmann et al., 2006; Casalone et al., 2004; Jacobs et al., 2007). Interestingly however,
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studies have shown the conversion of both BASE and BSE-H to classical BSE when
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passaged through wildtype mice (Baron et al., 2011; Capobianco et al., 2007).
However, due to the initiation of a large-scale surveillance programme throughout
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Previously, we modelled the possible susceptibility of humans to BASE and BSE-H using
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gene-targeted human PrP transgenic (HuTg) mice (Wilson et al., 2012b). In humans,
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susceptibility to TSE infection is linked to a polymorphism in the human PrP gene at codon
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129 (Zeidler et al., 1997). The UK population is approximately 50% MV, 40% MM and 10%
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VV, however all clinical cases of vCJD to date have occurred in codon 129 MM individuals.
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Using gene targeting we have produced two unique lines of transgenic mice (HuTg) in which
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the endogenous murine PrP gene has been replaced with the human PrP gene encoding
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either methionine (HuMM) or valine (HuVV) at codon 129 (Bishop et al., 2006). By crossing
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the homozygous 129MM and 129VV lines we can also produce a true 129MV heterozygote
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(HuMV). Following inoculation of these atypical BSE agents in HuTg mice, we did not detect
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any signs of TSE disease pathology (Wilson et al., 2012b), suggesting that the transmission
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barrier was significant in the presence of human PrP, and that the risk of disease
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transmission from atypical BSE was low. However we could not rule out the possibility that
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disease transmission in the presence of human PrP may be inefficient, and that the HuTg
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mice which survive to lifespan may be able to maintain low level agent replication in the
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CNS. Indeed, primary transmission of prion diseases between different species is often
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challenging, and although this subclinical infection does not result in TSE disease during the
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lifespan of the animal, the replication of low levels of infectivity in an animal may pose a risk
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of accidental transmission through routes such as surgery and blood transfusion. Therefore,
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the presence of subclinical forms of TSE infection resulting from atypical BSE could present
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a major public health risk, and warrants further investigation.
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To address the potential for subclinical infection in animals expressing human PrP following
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exposure to atypical BSE, we performed subpassage experiments from several HuTg brains
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challenged with BASE or BSE-H into HuTg mice and Bov6 Tg mice (expressing bovine PrP).
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In the present study we found evidence of subclinical infection in one HuMM and one HuVV
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Tg mouse challenged with BASE. These findings suggest that that low level replication of
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BASE can occur in hosts expressing human PrP, however the efficiency of any potential
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mouse to mouse transmission in hosts expressing human PrP appears to be low.
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Results
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Subpassage of brain tissue from BASE challenged HuPrP Tg mice into Bov6 Tg mice
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Brain tissue harvested from previous transmission studies of BASE and BSE-H into HuTg
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mice (Wilson et al., 2012b) were examined for evidence of TSE agent replication by
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bioassay in HuTg or Bov6 mice. Several different tissues were analysed (see Table 1),
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which included six BASE-challenged HuTg brains, three BSE-H-challenged HuTg brains and
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two Bov6 brains challenged with either BASE or BSE-H (included as controls). All HuTg
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tissues had previously been shown to lack any signs of TSE pathology by
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immunohistochemistry and vacuolation profiling. Each brain homogenate was inoculated into
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HuTg mice of the same genotype as the inoculum (or HuMM mice in the case of the bovine
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control tissue) and Bov6 mice. Details of genotype, clinical status and age of the
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subpassage tissues used are shown in Table 1. BASE-challenged HuMM Tg brain (C18985-
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HuMM) transmitted a TSE to 4/10 Bov6 mice and was defined by the presence of either PrP
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deposition (using immunohistochemistry) in the brain or vacuolar pathology (Table 2). No
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clinical signs of TSE disease were detected in these mice. Unexpectedly, BASE-challenged
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HuVV Tg brain (C19409-HuVV) also transmitted a TSE to 1/11 Bov6 mice (Table 2), and
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again PrP deposition and vacuolar pathology were present without clinical signs of disease.
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No other BASE- or BSE-H-challenged HuTg mouse brains transmitted disease in either
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Bov6 or HuTg mice. As expected a TSE was identified, by the presence of PrPTSE deposition
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and vacuolar pathology, in 10/11 and 11/11 of Bov6 Tg mice inoculated with BASE-
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challenged Bov6 brain (C18275-Bov6) and BSE-H-challenged Bov6 brain (C19414-Bov6)
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respectively (both controls).
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The lesion profiles, which define areas of vacuolation and their degree of severity in the
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brain, were determined for Bov6 mice inoculated with controls C18275-Bov6 and C19414-
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Bov6 (Figure 1).
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standard practice, all mice challenged with C18275-Bov6 and C19414-Bov6 which scored
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positively for vacuolation pathology were included to give an indication of vacuolation profile.
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Lesion profile patterns of primary and secondary passages of BASE or BSE-H in Bov6 mice
Although the production of lesion profiles in pre-clinical mice is not
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were similar between the same BSE agent (Figure 1), although higher levels of vacuolation
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were observed in areas 8 and 9 of the grey matter and in the while matter in BASE
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subpassage mice. This difference may be due to these mice being non-clinical, and culled at
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different stages of pre-clinical disease, or adaptation of the agent to the murine host on
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subpassage. Due to low number of animals which scored positively for vacuolation
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pathology and lack of statistical significance, lesion profiles from Bov6 mice challenged with
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C18985-HuMM or C19409-HuVV were not included.
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Neuropathology
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PrP deposition in the thalamus, medulla (Figure 2a, 2b and Figure S1), midbrain and
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caudate was detected using immunohistochemistry in Bov6 mice challenged with C18985-
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HuMM. Similarly, one Bov6 mouse challenged with C19409-HuVV showed PrP deposition in
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the same brain regions, however staining was not as heavy (Figure 2c, 2d). Control Bov6
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mice challenged with C18275-Bov6 showed widespread heavy PrP deposition (Figure 2e, 2f
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and Figure S1), unlike control Bov6 mice challenged with C19414-Bov6 which only showed
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sparse PrP deposition (Figure 2g, 2h). Bov6 mice challenged with C18985-HuMM, C19409-
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HuVV and C18275-Bov6 showed plaque-like PrP deposits, which were stained dark brown.
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While Bov6 mice challenged with C19414-Bov6 also showed plaque-like PrP deposition,
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these deposits were not as heavily stained. To control for age-related effects, gliosis was
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first assessed in brain tissue obtained from a previous aging study of Bov6 mice (Wilson et
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al., 2012a). Mild gliosis was present throughout the brains of these mice (Figure 3a 3b). In
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contrast to gliosis due to aging, an obvious increase in the appearance of astrogliosis was
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clearly evident throughout the brains of Bov6 mice inoculated with C18985-HuMM, C18275-
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Bov6 and C19414-Bov6 (Figure 3d, 3g, 3j). While Bov6 mice challenged with C18275-Bov6
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showed a marked increase in microgliosis (Figure 3h) as compared to the uninfected aged
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Bov6 control, the increase in microgliosis in Bov6 challenged with C18985-HuMM was not as
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pronounced (Figure 3e) and interestingly the microgliosis observed in Bov6 mice challenged
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with C19414-Bov6 (Figure 3k) was not dissimilar to that seen in the uninfected aged Bov6
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control mice. Interestingly, the presence of PrP deposition correlated with astrogliosis and
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microgliosis (Figure 3i) in Bov6 mice challenged with C18275-Bov6. However, despite
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obvious astrogliosis, none or very little PrP deposition was observed in the hippocampus of
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Bov6 mice challenged with C18985-HuMM (Figure 3f) or C19414-Bov6 (Figure 3l)
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respectively.
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Molecular Profile of PrPTSE in Bov6 mice inoculated with C18985, C18275 and C19414
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Brains from Bov6 mice challenged with C18985-HuMM, C18275-Bov6 and C19414-Bov6 (2
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brains per isolate) were examined for the presence of PrPTSE by western blot. Brains from
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primary transmissions of Bov6 mice challenged with BASE, BSE-H or BSE-C were also
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included for comparison. All brains selected for analysis were from mice which survived
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challenge to ≥418 dpi and immunohistochemical analysis showed PrP deposition in all
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selected animals.
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present in the brains of Bov6 mice challenged with C18275-Bov6, C19414-Bov6 (Fig 4a, b)
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and C18985-HuMM (Fig 4b). Furthermore, these agents produced distinct PrPTSE profiles.
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Similarly to primary transmissions of BASE into Bov6 mice, we found that the C18275-Bov6
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PrPTSE unglycosylated isoform had a lower molecular weight than BSE-C and a distinct
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PrPTSE glycoform pattern (Fig 4a). Likewise, we found that the C19414-Bov6 PrPTSE
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unglycosylated isoform had a higher molecular weight than BSE-C or BASE and was similar
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to the PrPTSE molecular profile from primary transmissions of BSE-H into Bov6 mice (Fig 4a).
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These results are consistent with studies using transgenic overexpressing bovine PrP mice
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(Beringue et al., 2007; Béringue et al., 2006; Buschmann et al., 2006; Capobianco et al.,
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2007; Okada et al., 2010). Due to low levels of C18985-HuMM PrPTSE, it was difficult to fully
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resolve the glycoform pattern, although we did observe a heavier diglycosylated band which
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may be suggestive of a BSE-like PrPTSE profile (Fig 4b). However, further subpassage
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experiments in Bov6 mice would be required to establish the presence of BSE-C or BASE.
Following western blot analysis, proteinase-K resistant PrPTSE was
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Investigating presence of PrPTSE in the brains of BASE-challenged HuMM mice
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Previously we performed primary inoculations of BASE into HuMM mice, but did not detect
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any signs of TSE neuropathology (Wilson et al., 2012b). Due to the observed transmission of
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TSE from one of the HuMM mice selected for subpassage, and the possibility of subclinical
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infection, brain tissue from 15 remaining HuMM mice that received the primary BASE
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inoculum were assayed for accumulation of PrP using a rapid TSE diagnostic assay. The
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IDEXX HerdChek* Bovine BSE Antigen Test Kit, which is an antigen capture enzyme
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immunoassay (EIA), utilises a unique Seprion ligand capture technology (Microsens
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Biotechnologies) to identify the presence of aggregated PrP in the brain, and has been used
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successfully on HuTg tissue in previous experiments (Plinston et al., 2011). All assay
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readouts for the 15 brain tissues examined were negative, indicating the lack of PrPTSE
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within these brains.
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Discussion
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In this study, knock-in transgenic mice expressing human PrP were utilised to model the
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potential risks posed to humans from exposure to atypical BSE agents. While the use of
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transgenic mice does not accurately mimic infection of humans, they provide a model
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system in which TSE infection in the presence of human PrP can be examined in
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comparison with genetically identical control lines expressing either bovine PrP or wild-type
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murine PrP. Data cannot be fully extrapolated to humans, but can provide some indication of
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potential risk. Three gene targeted human PrP Tg (HuTg) mouse lines were utilised,
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representing the genetic diversity in the human population due to the PrP codon 129-
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methionine/valine polymorphism (HuMM, HuMV and HuVV). As these mice are produced by
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gene replacement, they do not suffer from any adverse phenotypes observed in standard
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transgenic lines and may more accurately model what happens in nature. Our previous
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studies showed that following primary transmissions of BASE and BSE-H into HuTg mice,
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we could not detect any pathological signs of TSE disease (PrP deposition and vacuolar
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pathology) in any of the mice (Wilson et al., 2012b). In the current study we re-examined all
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remaining tissues from the BASE inoculated HuMM Tg mice using a rapid TSE diagnostic
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assay (IDEXX) to investigate the presence of PrPTSE in the brains of HuTg mice challenged
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with BASE. This assay was employed to ensure no PrP deposition was missed due to the
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level of sectioning of the tissue block, and to assay for any forms of abnormal PrP which
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may not have been identified by IHC. However, all IDEXX results on the 15 HuMM tissues
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examined were negative. Although the identification of PrPTSE by IHC or IDEXX assay is an
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indication of TSE disease, the only method by which TSE infectivity can be identified is
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bioassay. To address the potential for subclinical infection and low level agent replication in
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animals expressing human PrP, we performed subpassage experiments from HuTg brains
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following primary challenge with BASE or BSE-H into HuTg mice and Bov6 Tg mice. Upon
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subpassage we found evidence of subclinical infection in both a HuMM and a HuVV Tg
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mouse challenged with BASE, demonstrated by the pathological signs of TSE disease (PrP
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deposition and/or vacuolar pathology) observed in 5 Bov6 Tg mice (4 in MM challenge, 1 in
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VV challenge). However no TSE disease transmission was observed in the HuTg mice
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following subpassage.
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The existence of subclinical TSE infection in humans and animals has been documented
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previously (Hill & Collinge, 2003a, b; Race et al., 2001; Race et al., 2002). One such study
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showed that the cross-species passage of hamster 263K scrapie into wild-type mice,
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appeared not to transmit, with the mice appearing clinically normal with no PrPTSE
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detectable. However upon subpassage of brain tissue from a PrPTSE negative, clinically
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normal mouse to wild-type mice, PrPTSE was detectable in the brain tissue (Race et al.,
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2002). Indeed, while less than 200 people have developed clinical vCJD, it is likely that
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millions were exposed by consumption of BSE-contaminated beef and it is possible a
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number of these individuals may act as asymptomatic carriers of TSE infectivity. In the
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present study we found evidence of subclinical TSE infection in two HuTg mice challenged
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with BASE (C18985-HuMM, C19409-HuVV; both negative for neuropathology and clinical
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signs of TSE disease). In these studies, our experiments may suggest that during primary
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passage low level replication of BASE may occur in the HuTg mice, however once within a
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susceptible host (Bov6) it is able to replicate efficiently. Indeed, the inefficient replication of
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the infectious TSE agent may explain why we were unable to detect any signs of TSE
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pathology in our HuTg mice challenged with BASE. Furthermore, our current experimental
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methods of detection may not be sensitive enough to detect low levels of PrPTSE. However, it
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is also possible that PrPTSE is not the infectious agent and that we are simply not searching
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for the correct markers for TSE infection in these animals. Indeed, the dissociation between
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PrPTSE and TSE infectivity has been shown in both natural and experimental cases
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(Andreoletti et al., 2011; Barron et al., 2007; Race et al., 2002). Nevertheless, as it is known
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that PrP expression in the host is necessary for the development of neurodegeneration,
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PrPTSE remains an important diagnostic marker of TSE infection.
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It is theoretically possible that residual inocula might be the cause of the TSE pathology
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observed in our Bov6 mice challenged with C18985-HuMM or C19409-HuVV, however it
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would seem unlikely in these studies. Indeed, if this were the case, the expectation would be
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to see more cases of TSE pathology in all groups of inoculated mice. Furthermore, previous
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studies have shown the rapid clearance of prions from the brain following intracerebral
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inoculation (Safar et al., 2005a; Safar et al., 2005b), which would not agree with the retention
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of significant levels of BASE 600 days post inoculation. In previous studies we observed that
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following challenge of vCJD into Bov6 mice or HuTg mice, more clinical cases were
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observed in Bov6 mice (personal communication, data unpublished). These findings would
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support our data showing the lack of TSE pathology in the HuTg mice subpassaged with
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BASE- or BSE-H-challenged HuTg mice.
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Transmission of BASE to overexpressing human PrP Tg mice (homozygous for methionine
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at codon 129) has been demonstrated following primary passage (Beringue et al., 2008;
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Kong et al., 2008), however in the present study we were particularly interested in the
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discovery of subclinical infection in a HuVV mouse challenged with BASE, which has not
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previously been documented. Indeed, very few studies have been published investigating
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the transmission of TSEs into HuVV mice. Although all clinical cases of vCJD have been in
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individuals who are methionine homozygous at codon 129, studies in HuTg mice have
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shown that all three genotypes (MM, MV and VV) may be susceptible with differing
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incubation times (Bishop et al., 2006). Indeed transmission experiments have shown that
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only 1/16 HuVV challenged with vCJD displayed TSE pathology as compared to 11/17
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HuMM Tg mice. However, although classical BSE and BASE are both cattle TSE strains, we
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cannot assume that the susceptibility of different human PrP genotypes would be the same
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between HuTg challenged with classical BSE or BASE. Indeed, other studies have
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suggested that the phenotypic features and PrPTSE characteristics of BASE bear
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resemblance to a subtype of sCJD (sCJDMV2) (Casalone et al., 2004). sCJDMV2 has been
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found to affect individual who are methionine/valine heterozygous at codon 129 of the PrP
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gene (Parchi et al., 1999), and this finding has raised the possibility that sCJDMV2 may
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actually not be a truly sporadic disease but may be acquired from the consumption of BASE-
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contaminated meat (Brown et al., 2006; Casalone et al., 2004). Although our previous
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studies showed no TSE pathology on primary passage of BASE (Wilson et al., 2012b) or
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classical cattle BSE (Bishop et al., 2006) to the HuTg mice, others have shown efficient
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transmission of BASE in mice overexpressing 129-Met human PrP, with higher levels of
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transmissibility than observed with classical cattle BSE. We have yet to perform subpassage
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from HuTg mice that received BSE-C to determine whether low level agent replication also
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occurs in these mice. However these data combined suggest that BASE may indeed
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transmit more efficiently to HuTg mice than BSE-C (Beringue et al., 2008).
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The potential existence of subclinical TSE infection in humans has several significant
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implications for public health, especially regarding the possibility of iatrogenic transmission of
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TSE disease from individuals who seem apparently healthy. Therefore, continued efforts
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must be made to ensure public health. However, while our findings suggest that low levels
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replication of BASE can occur in hosts expressing human PrP, the efficiency of any potential
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human to human transmission appears to be low.
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Materials and Methods
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Preparation of Inocula
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Brain tissue from the frontal and parietal cortices of a 15 year old Piemontese cow (fallen
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stock) infected with BASE was supplied by Istituto Zooprofilattico Sperimentale del
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Piemonte, Liguria e Valle D'Aosta, Torino, Italy. The brainstem of a 15 year old BSE-H
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infected Prim Holstein cow (identified from a rendering plant) was supplied by the French
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TSE Reference Laboratory (Agence Nationale de Sécurité Sanitaire (Anses-Lyon), France).
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Primary transmissions are described in Wilson et al., 2012a. Seven BASE challenged mouse
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brains and four BSE-H-challenged mouse brains were selected for subpassage (Table 1). All
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inocula were prepared from brain tissue in sterile saline at a concentration of 5% (wt/vol). In
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order to prevent any possibility of cross-contamination of samples, all tissues were
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homogenised in clean previously unused dounce glass homogenisers that were discarded
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after single use. Samples were handled individually and the safety cabinet decontaminated
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between each inoculum prep. Positive control bov6 tissues were prepared on a different day
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from the HuMM and HuVV tissues (See Table 1). Full pathological characterisation of source
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tissues (BASE, C.Casalone; BSE-H, T.Baron) was previously performed to confirm disease
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status.
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Subpassage Inoculation of Transgenic Mice
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Gene-targeted Tg mice expressing bovine PrP (Bov6) or human PrP (HuMM, HuMV and
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HuVV) have been described previously (Bishop et al., 2006).
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subpassage covered a range of genotypes, ages and clinical status and included 9 HuTg
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mice inoculated with either BASE (6) or BSE-H (3) and 2 control Bov6 mice inoculated with
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BASE or BSE-H (Table 1). Each of the 11 brains selected for subpassage was used to
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prepare a 5% homogenate for use as inocula. Mice were injected by intracerebral inoculation
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(i.c.) into the right cerebral hemisphere under halothane anaesthesia. Each mouse received
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0·02 mL of 5% brain homogenate and each homogenate was inoculated into a group of 12
Tissues selected for
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HuTg mice of the same genotype as the inoculum, and 12 Bov6 mice as controls.
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Homogenates from the 2 Bov6 control brains were each inoculated into groups of 12 HuMM
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and 12 Bov6 mice. In order to prevent any possible cross-contamination, inoculations were
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performed from one or two inocula only per day, with the safety cabinet cleaned and
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decontaminated between each set of inoculations. Of note, the HuMM and HuVV samples in
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Table 1 shown to transmit disease were inoculated on different days from the two Bov6
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control tissues (C18275 and C19414; Table 1). From 100 days mice were scored each week
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for signs of disease and were killed by cervical dislocation at a pre-defined clinical endpoint,
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or due to welfare reasons (Dickinson et al., 1968). Due to the low number of culls for clinical
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TSE disease, survival times only were calculated for mice showing both PrP deposition and
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vacuolar pathology. Brains were recovered at post mortem and one half of the brain was
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snap-frozen in liquid nitrogen for biochemical analysis and the remaining half brain was fixed
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for histological processing. All mouse experiments were reviewed and approved by the Local
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Ethical Review Committee and performed under licence from the United Kingdom Home
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Office in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986.
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Vacuolation Scoring
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(H&E).
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cerebellum, superior colliculus, hypothalamus, thalamus, hippocampus, septum, retrospinal
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cortex, cingulated and motor cortex) and three regions of white matter (cerebellar white
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matter, midbrain white matter, and cerebral peduncle). Sections were scored on a scale of 0
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(no vacuolation) to 5 (severe vacuolation) for the presence and severity of vacuolation and
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mean vacuolation scores for each mouse group in each experiment were calculated and
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plotted with standard errors of means (SEM) against scoring areas to produce a lesion
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profile, as previously described (Bruce et al., 1997; Fraser & Dickinson, 1967). While the
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production of lesion profiles in pre-clinical mice is not standard practice, all mice which
Sections were cut (6µm) from each mouse brain and stained using hematoxylin and eosin
TSE-related vacuolation was assessed at nine grey-matter regions (medulla,
363
scored positively for vacuolation pathology were included whether or not clinical signs were
364
present, due to the lack of clinical signs observed in any of the mice.
365
366
367
368
Immunohistochemical (IHC) analysis of PrP deposition and glial activation in the brain
PrPTSE localisation in the brain was assessed using immunohistochemistry.
Following
369
fixation in 10% formal saline, brains were treated for 1·5 h in 98% formic acid, dissected to
370
expose several brain regions, and embedded in paraffin.
371
autoclaved for 15 min at 121C and immersed in 95% formic acid for 10 minutes prior to
372
incubation with 0.44 g/ml anti-PrP monoclonal antibody (MAb) 6H4 (Prionics) at room
373
temperature overnight. Secondary anti-mouse biotinylated antibody (Jackson Immuno
374
Research Laboratories, UK) was added at 2.5 g ml-1 and incubated for 1 h at room
375
temperature. Immunolabelling was performed using the ABC Elite kit (Vector Laboratories)
376
and the signal was visualised by a reaction with hydrogen peroxidise-activated
377
diaminobenzidine (DAB). The presence of astrogliosis, a hallmark of prion disease, was
378
assessed by incubating brain sections (6µm) with 1.45 g ml-1 anti-glial fibrillary acidic
379
protein (GFAP; DAKO UK Ltd) antibody at room temperature for 1 hour. To detect microglial
380
activation, brain sections were pretreated using hydrated microwaving for 10 minutes prior to
381
incubation with 0.05 g ml-1 anti-Iba1 antibody (Wako Chemicals GmbH) at room
382
temperature for 1 hour. For both GFAP and anti-Iba-1 antibodies, 2.6 g ml-1 biotinylated
383
secondary anti-rabbit antibody (Jackson Immno Research Laboratories, UK) was added for
384
1 hour at room temperature. Both astrocytes and microglia were visualized by a reaction
385
with hydrogen peroxidise-activated DAB.
386
387
388
389
Sections (6µm) were then
Identification of PrPTSE by immunoblotting
Frozen brain samples from Bov6 mice challenged with C18985-HuMM, C18275-Bov6 and
390
C19414-Bov6 (and also brains from primary inoculations of Bov6 mice challenged with
391
BASE, BSE-H and BSE-C) were homogenised at 10% in an NP40 buffer (0·5% v/v NP40,
392
0·5% w/v sodium deoxycholate, 0·9% w/v sodium chloride, 50mM Tris-HCl pH 7·5) and
393
clarified at 11,000g for 15 minutes. Brain homogenate supernatant from the transgenic mice
394
and controls was incubated with or without 20 g proteinase K ml-1 for 1 hour at 37C. The
395
products were denatured and separated on a 12% Novex Tris/Glycine gel (Invitrogen, UK)
396
before transfer to polyvinylidine difluoride (PVDF) membrane by western blotting.
397
amount of brain tissue loaded onto the gels varied between 0.6 and 3mg).
398
identified with monoclonal antibody 6H4 (0.1 g ml-1) and bands visualized using
399
horseradish peroxidise (HRP)-labelled anti-mouse secondary antibody (Jackson Immuno
400
Research Laboratories, UK) and a chemiluminescence substrate (Roche). Images were
401
captured on radiographic film and with a Kodak 440CF digital imager.
The
PrP was
402
403
Immunoassay for detection of PrPTSE in the brain
404
The IDEXX HerdChek* Bovine Spongiform Encephalopathy (BSE) Antigen Test Kit is an
405
antigen capture enzyme immunoassay (EIA) for detection PrPTSE in post-mortem tissues.
406
Previously we performed primary inoculations of BASE into HuMM mice (Wilson et al.,
407
2012b). Brains derived from these mice were homogenised in sterile saline in a Rybolyser
408
(Hybaid, Middlesex, UK) to achieve a 30% homogenate. The protocol was performed
409
following manufacturer’s instructions.
410
411
PCR genotyping of mouse tail DNA
412
All mice were analysed by PCR post mortem to confirm PrP genotype. Mouse tail DNA was
413
extracted and genotyped as previously described (Bishop et al., 2006; Wemheuer et al.,
414
2011).
415
416
417
418
419
Acknowledgements
420
The authors would like to acknowledge J. Manson for the HuTg and Bov6 mice; S.
421
Cumming, S. Carpenter, R. Greenan and K. Hogan for experimental setup, care and scoring
422
of the animals; A. Boyle, S. Mack, D. Drummond and G. McGregor for histology processing
423
and scoring. These studies were funded by contract M03054 from the Food Standards
424
Agency (FSA) UK. The Roslin Institute receives Institute Strategic Programme Grant (ISP)
425
funding from the Biotechnology and Biological Sciences Research Council (BBSRC), UK.
426
427
428
429
430
431
432
Table 1. Tissues used for subpassage into HuTg and Bov6 Tg mice.
TSE
Agent
Reference
No.
Genotype
of mice
Clinical
Score*
Pathology
Survival
(days)
1
BASE
C17840
HuVV
+
-
395
2
BASE
C18220
HuMM
+
-
539
3
BASE
C18222
HuMM
+
-
527
4
BASE
C18275
Bov6
+
+
541
5
BASE
C18985
HuMM
-
-
652
6
BASE
C19170
HuMM
-
-
687
7
BASE
C19409
HuVV
-
-
749
8
H-type
C18252
HuMV
+
-
421
9
H-type
C19414
Bov6
-
+
623
10
H-type
C19697
HuMM
-
-
708
11
H-type
C19700
HuVV
-
-
708
433
434
435
436
*Animals in all primary passage experiments were scored blind. Animals with clinical TSE
score but no confirmed TSE neuropathology were included to examine whether the
phenotype was transmissible.
437
438
Reference numbers highlighted in bold represent the tissue from which disease transmission
was observed (see Table 2)
439
440
441
442
443
444
445
446
Table 2. Subpassage transmissions into HuTg and Bov6 Tg mice
Inoculation
Source
Mouse
Line
Survival*
(Pathol
neg)
C17840
Bov6
HuVV
562±12
464±33
Survival†
(Pathol
Pos)
-
C18220
Bov6
HuMM
538±20
542±14
C18222
Bov6
HuMM
C18275
Clinical
Signs
Vacuolar
Pathology
PrP
deposition
0/12
0/12
0/12
0/12
0/12
0/12
-
0/12
0/12
0/12
0/12
0/12
0/12
512±36
472±42
-
0/11
0/12
0/11
0/12
0/11
0/12
Bov6
HuMM
329
554±21
588±8
-
0/11
0/10
10/11
0/10
10/11
0/10
C18985
Bov6
HuMM
526±35
475±43
483±42
-
0/10
0/10
3/10
0/10
4/10
0/10
C19170
Bov6
HuMM
526±36
498±31
-
0/11
0/12
0/11
0/12
0/11
0/12
C19409
Bov6
HuVV
525±37
507±28
589
-
0/11
0/12
1/11
0/12
1/11
0/12
C18252
Bov6
HuMV
502±27
520±35
-
0/10
0/12
0/10
0/12
0/10
0/12
C19414
Bov6
HuMM
530±43
524±27
-
0/11
0/12
10/11
0/12
11/11
0/12
C19697
Bov6
HuMM
549±26
451±40
-
0/11
0/12
0/11
0/12
0/11
0/12
C19700
Bov6
HuVV
538±29
501±25
-
1/11
0/12
0/11
0/12
0/11
0/12
* Measured as days ± SEM and calculated from mice with no signs of TSE neuropathology
† Measured as days ± SEM and calculated from mice showing vacuolar pathology and/or PrP deposition.
447
Figure 1. Pattern of vacuolation observed in brains of Bov6 mice derived from 1st and 2nd
448
passage of BASE or H-type BSE. A profile was produced from nine grey matter areas (1,
449
medulla; 2, cerebellum; 3, superior colliculus; 4, hypothalamus; 5, thalamus; 6,
450
hippocampus; 7, septum; 8, retrospinal cortex; 9, cingulate and motor cortex) and three
451
white matter areas (11, cerebellar white matter; 12, midbrain white matter; 13, cerebral
452
peduncle. Average scores were taken from a minimum of seven mice per group and plotted
453
against brain area ± SEM.
454
455
456
Figure 2. Comparative analysis of PrPTSE deposition in the thalamus (a,c,e,g) and midbrain
457
(b,d,f,h) regions of brains from Bov6 mice subpassaged with C18985-HuMM (a,b), C19409-
458
HuVV (c,d), C18275-Bov6 (e,f) and C19414-Bov6 (g,h). Images obtained after staining with
459
anti-PrP antibody 6H4 and counterstained with hematoxylin. Magnification is as shown.
460
461
462
Figure 3. Comparative analysis of the hippocampus of Bov6 mice challenged with C18985-
463
HuMM, C18275-Bov6 and C19414-Bov6. Micro- and astrogliosis is present in all mice,
464
detected by anti-GFAP (d,g,i) and anti-Iba1 respectively (e,h,k). PrP deposition is visible by
465
anti-6H4 antibody (i,l). Uninfected aged Bov6 mice showing mild gliosis (a,b) and no PrP
466
deposition (c) were used as controls. Magnification 10x.
467
468
469
Figure 4. Comparative western blot analysis of the proteinase K-resistant fragment (PrPTSE)
470
of the prion protein in Bov6 mice challenged with C18275-Bov6 (lanes 3 and 4) and C19414-
471
Bov6 (lanes 7 and 8). Primary transmissions of BSE-C (lanes 1 and 2), BASE (lanes 5 and
472
6) and BSE-H (lanes 9 and 10) into Bov6 mice have also been included for comparison (a).
473
Western blot analysis of the proteinase K-resistant fragment (PrPTSE) of the prion protein in
474
Bov6 mice challenged with BSE-C (lane 1), C18275-Bov6 (lane 2), C18985-HuMM (lane 3)
475
and C19414-Bov6 (lane 4) (b).All lanes show PK-treated brain homogenate. Anti-PrP mAb
476
6H4 was used to detect bands.
477
478
479
480
481
482
483
484
485
486
487
488
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