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From: TSS (216-119-143-122.ipset23.wt.net)
Subject: Characterization of two distinct prion strains derived from bovine spongiform encephalopathy transmissions to inbred mice [FULL TEXT]
Date: July 24, 2004 at 10:53 am PST
-------- Original Message --------
Subject: Characterization of two distinct prion strains derived from bovine spongiform encephalopathy transmissions to inbred mice [FULL TEXT]
Date: Sat, 24 Jul 2004 12:39:04 -0500
From: "Terry S. Singeltary Sr."
To: Bovine Spongiform Encephalopathy
CC: cjdvoice@yahoogroups.com
Characterization of two distinct prion strains
derived from bovine spongiform encephalopathy
transmissions to inbred mice
Sarah E. Lloyd, Jacqueline M. Linehan, Melanie Desbruslais,
Susan Joiner, Jennifer Buckell, Sebastian Brandner,
Jonathan D. F. Wadsworth and John Collinge Correspondence John Collinge
j.collinge@prion.ucl.ac.uk
MRC Prion Unit and Department of Neurodegenerative Disease, Institute of
Neurology,
University College, London WC1N 3BG, UK Received 9 December 2003
Accepted 27 April 2004 Distinct prion strains can be distinguished by differences in incubation
period, neuropathology
and biochemical properties of disease-associated prion protein (PrPSc)
in inoculated mice.
Reliable comparisons of mouse prion strain properties can only be
achieved after passage in
genetically identical mice, as host prion protein sequence and genetic
background are known
to modulate prion disease phenotypes. While multiple prion strains have
been identified in
sheep scrapie and CreutzfeldtJakob disease, bovine spongiform
encephalopathy (BSE) is
thought to be caused by a single prion strain. Primary passage of BSE
prions to different lines
of inbred mice resulted in the propagation of two distinct PrPSc types,
suggesting that two
prion strains may have been isolated. To investigate this further, these
isolates were
subpassaged in a single line of inbred mice (SJL) and it was confirmed
that two distinct prion
strains had been identified. MRC1 was characterized by a short
incubation time (110±3 days),
a mono-glycosylated-dominant PrPSc type and a generalized diffuse
pattern of PrP-immunoreactive
deposits, while MRC2 displayed a much longer incubation time (155±1 days),
a di-glycosylated-dominant PrPSc type and a distinct pattern of
PrP-immunoreactive deposits
and neuronal loss. These data indicate a crucial involvement of the host
genome in modulating
prion strain selection and propagation in mice. It is possible that
multiple disease phenotypes
may also be possible in BSE prion infection in humans and other animals. INTRODUCTION Prion diseases or transmissible spongiform encephalopathies
are a group of fatal neurodegenerative disorders that
include CreutzfeldtJakob disease (CJD) in humans and
scrapie and bovine spongiform encephalopathy (BSE) in
animals. Prion diseases are characterized by their prolonged
incubation periods and distinctive neuropathology, which
includes an accumulation in affected brains of an abnormal
isomer (PrPSc) of host-encoded cellular prion protein (PrPc).
The conversion of PrPc to PrPSc involves conformation
change resulting in increased b-sheet secondary structure
(Pan et al., 1993) and is associated with detergent insolubility
and the acquisition of partial resistance to protease
digestion (Meyer et al., 1986). According to the protein-only
hypothesis (Griffith, 1967), PrPSc is the principal, if not sole,
component of the infectious agent (Prusiner, 1991).
Multiple prion strains have been described that are distinguished
by their incubation periods and patterns of neuropathology
when passaged in inbred lines of mice, and these
distinctive phenotypes are preserved on multiple passage
in the same host (for review, see Bruce et al., 1992). The
existence of prion strains challenges the protein-only
hypothesis of prion propagation. However, it is clear that
prion strains are associated with biochemical differences in
PrPSc itself including differences in conformation (Hill et al.,
1997, 2003; Bessen &Marsh, 1992, 1994; Collinge et al., 1996;
Telling et al., 1996; Wadsworth et al., 1999), glycosylation
(Hill et al., 1997, 2003; Collinge et al., 1996) and overall
protease resistance (Kuczius & Groschup, 1999). That these
strain-associated biochemical differences in PrPSc fragment
sizes and glycoform ratios following proteolysis can be
transmitted to PrP in an experimental host argues that they
may be responsible for encoding strain diversity. While the
precise nature of the molecular basis of prion strain diversity
is unclear, that prion strains may be distinguished by the
differing molecular mass of fragments following partial
proteinase K digestion and by differing ratios of di-, monoand
unglycosylated PrPSc is clear. Using this approach, we
described four common PrPSc types in humans (Collinge
et al., 1996; Wadsworth et al., 1999; Hill et al., 2003). PrPSc
types 13 are seen in sporadic and iatrogenic CJD, while type
4 PrPSc is exclusively associated with variant CJD (vCJD)
and is associated with a di-glycosylated-dominant PrPSc
0007-9889 G 2004 SGM Printed in Great Britain 2471
Journal of General Virology (2004), 85, 24712478 DOI 10.1099/vir.0.79889-0
pattern on a Western blot (Collinge et al., 1996; Hill et al.,
2003). This characteristic glycoform ratio is also seen in
BSE-infected cattle brain and these observations, together
with bioassay data from wild-type and transgenic mice and
non-human primates, have proved critical in establishing
a link between BSE and vCJD (Hill et al., 1997; Bruce et al.,
1997; Lasme´zas et al., 1996; Collinge et al., 1996; Asante et al.,
2002). This characteristic molecular signature of BSEderived
prion isolates is seen in all UK cattle BSE cases
examined and, together with biological strain typing studies
in inbred and transgenic mice, suggests that BSE is caused
by a single strain of agent (Hill et al., 1997; Bruce et al., 1994,
2002; Collinge et al., 1996; Kuczius & Groschup, 1999; and
our unpublished data). This molecular pattern, in additional
to biological characteristics, is also maintained on transmission
to other hosts such as domestic cat, sheep, macaque and
other exotic animals, either by natural exposure or by
experimental transmission (Bruce et al., 1994; Fraser et al.,
1994; Collinge et al., 1996; Hill et al., 1998; Lasmezas et al.,
2001). Recent data from French and Italian screening
programmes suggest that more than one strain of BSE
may exist in cattle (Biacabe et al., 2004; Casalone et al.,
2004). We have also shown that on BSE transmission to
a line of transgenic mice expressing only human PrP Met-
129, Tg(HuPrP129M+/+Prnp0/0)-35, two distinct molecular
phenotypes can be produced: one that mirrors the vCJD
phenotype with type 4 PrPSc and an additional molecular
phenotype that is indistinguishable from that of sporadic
CJD with PrPSc type 2 (Asante et al., 2002). These transgenic
mice were generated on a mixed genetic background, and
one possibility was that the different patterns were determined
by background effects in individual mice (Asante
et al., 2002). This interpretation was supported by the
demonstration that vCJD and BSE prions on primary
passage to a series of inbred lines of mice were also able
to produce two distinct molecular phenotypes on Western
blotting, dependent only on the genetic background of the
mice (Asante et al., 2002). These data argued that two distinct
strains had been propagated fromcattle BSE. However,
since the parameters that distinguish prion strains (incubation
time, neuropathology and PrPSc type) are also known
to be modulated by host genetic background, these cannot
be adequately compared after primary passage in different
lines of inbred mice (Bruce, 1993; Moore et al., 1998;
Somerville, 1999). We therefore subpassaged these mouseadapted
BSE prions in the same inbred mouse line to
determine whether distinctive biological characteristics
resulted, indicative of different strains, and whether these
correlated with the different PrPSc types propagated in the
animals. METHODS Transmission studies. SJL/OlaHsd (SJL) and C57BL/6JOlaHsd
(C57BL/6) mice were obtained from Harlan UK Ltd (Bicester, UK).
BSE tissues were collected under strict aseptic conditions, using sterile
instrumentation specifically for transmission studies, by the UK
Central Veterinary Laboratory (now the Veterinary Laboratories
Agency). I783 is derived from a single natural BSE-affected cow
brainstem and I038 is derived from a pool of five natural BSEaffected
brainstems. DNA sequence analysis of the bovine Prnp gene
from I783 showed that this animal was homozygous for the polymorphic
repeat sequence (R6/R6). These inocula have all been previously
used in transmission studies (Hill et al., 1997; Collinge et al.,
1995, 1996; Asante et al., 2002).
For second passage to SJL mice, a single brain from a terminally sick
mouse from the primary passage to SJL group (I783) was used to
produce inoculum I1590 [cattle BSE (I783)RSJL mice (I1590)RSJL
mice]. For second passage to C57BL/6 mice, a single brain from a
subclinically
infected mouse from the primary passage to C57BL/6 group
(I038) was used to produce inoculum I656 [cattle BSE (I038)RC57BL/6
mice (I656)RC57BL/6 mice].
For third passage, the SJL-passaged inoculum was generated from a
single mouse brain from the SJL second passage group (I1891) and
was used to inoculate a group of SJL mice [cattle BSE (I783)RSJL
(I1590)RSJL (I1891)RSJL mice]. To generate the C57BL/6-passaged
BSE inoculum, 11 mouse brains from the second passage were pooled
to generate a larger volume of homogenate (I874). The inoculum was
generated in this way as it was originally intended for use in a survey
of incubation times in inbred lines and for use in a large mapping study
to identify genes that influence prion disease incubation time (Lloyd
et al., 2002). This pool (I874) was used to inoculate SJL mice [cattle BSE
(I038)RC57BL/6 (I656)RC57BL/6 (I874)RSJL mice].
All inocula were prepared by homogenizing brain samples (1% w/v
in PBS) using disposable equipment for each inoculum in a microbiological
containment level 3 laboratory and inoculations were performed
within a class I microbiological safety cabinet. All mice were
uniquely identified with a subcutaneous transponder tag. Disposable
cages were used throughout and lids and water bottles were also
uniquely tagged.
Mice were anaesthetized with halothane/O2 and inoculated intracerebrally
into the right parietal lobe with 30 ml inoculum. Incubation time
was defined as the number of days from inoculation to the onset of
clinical signs. This was assessed by daily examination for neurological
signs of disease. Criteria for clinical diagnosis of prion disease were as
described (Carlson et al., 1986). Animals were killed as soon as clinical
scrapie was confirmed or if showing signs of distress.
Western blotting. Brain homogenates (10% w/v in PBS) were prepared,
proteinase K-digested (100 mg proteinase K ml21 for 1 h at
37 uC) and Western-blotted as described previously (Wadsworth
et al., 2001). Blots were probed with a biotinylated anti-PrP monoclonal
antibody ICSM-35 (Asante et al., 2002) in conjunction with an
avidinbiotinalkaline phosphatase conjugate (Dako) and developed
in chemiluminescent substrate (CDP-Star; Tropix Inc.). For quantifi-
cation and analysis of PrP glycoforms, blots were developed in chemi-
fluorescent substrate (AttoPhos; Promega) and visualized on a Storm
840 PhosphorImager (Molecular Dynamics). Quantification of PrPSc
glycoforms was performed using ImageQuaNT software (Molecular
Dynamics). Sodium phosphotungstic acid pre-concentration of PrPSc
was performed as described previously (Wadsworth et al., 2001).
Neuropathology and immunohistochemistry. Mouse brains
were fixed in 10% buffered formol-saline, immersed in 98% formic
acid for 1 h, formalin post-fixed and paraffin wax-embedded. Serial
sections of 4 mm nominal thickness were pre-treated with Tris/citrate/
EDTA buffer (1?3 mMEDTA, 2?
1 mMTris, 1?
1 mMcitrate, pH 7?
8)
for antigen retrieval. PrP deposition was visualized using ICSM-35 as
the primary antibody (diluted 1 : 3000) and gliosis was detected with
anti-glial fibrillary acidic protein rabbit polyclonal antiserum (diluted
1 : 1000; Dako), using an automated immunostaining system (www.
ventanamed.com). Sections of brains were examined by the same
2472 Journal of General Virology 85
S. E. Lloyd and others
person, who was blind to the identity of the animal and genotype.
Sections were scored for spongiosis, neuronal loss, gliosis and PrPSc
deposition. Photographs were taken on an ImageView digital camera
(www.soft-imaging.de) and composed with Adobe Photoshop.
RESULTS
Primary passage of BSE to SJL and C57BL/6
mice
As part of a study to map prion disease incubation time
genes (Lloyd et al., 2001), we inoculated a range of inbred
mouse lines with BSE prions (isolate I783). Coding polymorphisms
of the mouse prion protein gene (Prnp) are
known to influence incubation time (Westaway et al., 1987;
Moore et al., 1998) and therefore all mice tested were Prnpa
(Leu-108, Thr-189). While BSE transmits readily to wildtype
mice, there is nevertheless a substantial transmission
barrier (Wells et al., 1998; Wadsworth et al., 2001), which
results in long and variable incubation times and an
incomplete attack rate on primary passage (Hill et al., 1997;
Asante et al., 2002) (Table 1) compared with the passage
of mouse adapted-prions in mice. Primary passage of BSE
prions in mice usually results in a di-glycosylated-dominant
PrPSc pattern on a Western blot that closely resembles the
PrPSc types seen in cattle BSE and human vCJD (type 4)
(Hill et al., 1997; Somerville et al., 1997; Collinge et al.,
1996). Most of the inbred lines tested (C57BL/6JOlaHsd,
FVB/NHsd, NZW/OlaHsd, SM/J and SWR/OlaHsd) corresponded
to this pattern (Fig. 1 and unpublished data;
Asante et al., 2002). However, two strains (SJL/OlaHsd
and RIIIS/J) produced an alternative PrPSc type where the
fragment sizes appeared the same but the glycoform ratios
were different such that the mono-glycosylated glycoform
was dominant (Fig. 1; Asante et al., 2002). Passage of cattle
BSE (I783) in C57BL/6 mice gave, as reported previously, a
prolonged incubation time (710±15 days); however,
passage of cattle BSE (I783) in SJL mice gave a very much
shorter incubation time (196±13 days) (Table 1; Asante
et al., 2002). Neuropathological findings were unremarkable
with only diffuse PrP staining in both inbred mouse lines
(Asante et al., 2002). We also inoculated C57BL/6 mice with
another cattle BSE inoculum (I038) (Collinge et al., 1996).
On Western blots, both I783 and I038 show the same diglycosylated-
dominant PrPSc pattern (Fig. 1a), which has,
Table 1. BSE transmissions to SJL and C57BL/6 mice
Inoculation Incubation time (days±SEM) Clinical signs (no./total)
Primary passage
Cattle BSE (I783)RSJL mice* 196±13 25/40
Cattle BSE (I783)RC57BL/6 mice* 710±15 6/25
Cattle BSE (I038)RC57BL/6 mice >839 0/12
Second passage
BSE (I783)RSJL (I1590)RSJL 125±3 8/8
BSE (I038)RC57BL/6 (I656)RC57BL/6 189+2 11/11D
Third passage
BSERSJLRSJL (I1891) RSJL 110±3 8/8
BSERC57BL/6RC57BL/6 (I874) RSJL 155±1 36/36d
*Asante et al. (2002).
DAn additional two animals were found dead at 174 and 168 days. No
samples were available for Western
blotting or histology.
dAn additional two animals were found dead at 150 and 173 days. No
samples were available for Western
blotting or histology.
Fig. 1. Western blots of proteinase K-treated brain homogenates
from cattle BSE and BSE transmission and subpassage
in inbred mice. Western blots were analysed by high-sensitivity
ECL using biotinylated anti-PrP monoclonal antibody ICSM-35.
All lanes show PrPSc present in 5 or 10 ml 10% brain homogenate
with the exception of lane 2 of (a), and lane 1 of (b),
which show PrPSc derived from 100 ml 10% brain homogenate
following pre-concentration with sodium phosphotungstic acid.
(a) Cattle BSE isolates. Lane 1, isolate I038; lane 2, isolate I783.
(b) Primary transmission of BSE prions to inbred mice. Lane 1,
transmission of BSE (isolate I038) to C57BL/6 mice; lanes 2 and
3, transmission of BSE (isolate I783) to C57BL/6 mice (lane 2)
or SJL mice (lane 3). (c) Secondary passage of BSE prions in
inbred mice. Lane 1, secondary passage of BSE (isolate I038) in
C57BL/6 mice; lane 2, secondary passage of BSE (isolate I783)
in SJL mice. (d) Third passage of BSE in inbred mice. Lane 1,
BSE (isolate I038) was passaged twice in C57BL/6 mice and
then passaged in SJL mice; lane 2, BSE (isolate I783) was
passaged three times in SJL mice.
http://vir.sgmjournals.org 2473
Two BSE-derived prion strains
to the best of our knowledge, been observed in all UK
BSE cattle brain isolates reported to date. C57BL/6 mice
inoculated with I038 did not show clinical signs of disease
(0/12) at >839 days (Table 1; Collinge et al., 1996). However,
I038 has been used in many transmissions in our
laboratory and has transmitted efficiently with a consistent
di-glycosylated-dominant PrPSc pattern on Western blots
following transmission to various lines of inbred mice
(Fig. 1b; Hill et al., 1997; Collinge et al., 1995, 1996).
To determine whether the two different PrPSc types corresponded
to distinguishable prion strains, we completed
additional passages so that they could be compared on the
same host genetic background.
Second passage of BSE to SJL and C57BL/6
mice
On second passage to either SJL or C57BL/6 mice, incubation
times for both groups were substantially reduced,
with a 100% attack rate (Table 1), showing the expected
adaptation on second passage to the mouse. It was not
appropriate to compare the incubation times between
inocula in this passage because of host genetic background
effects. On Western blotting, the PrPSc type bred true,
maintaining the pattern seen on primary passage, with the
SJL-derived strain giving a mono-glycosylated-dominant
pattern and the C57BL/6-derived strain showing a diglycosylated-
dominant pattern (Fig. 1c).
Third passage of BSE to SJL and C57BL/6 mice
All third passages were carried out in SJL mice and were
therefore appropriate for comparison of all criteria of prion
strains: incubation time, PrPSc type and neuropathology.
The incubation time on third passage was reduced for both
the SJL- and C57BL/6-derived BSE, suggesting further
adaptation to mouse (Table 1). The incubation times of
110±3 days for SJL-derived BSE and 155±1 days for
C57BL/6-derived BSE were highly significantly different
(P<0?0001, MannWhitney test). As inoculation was intracerebral
with brain homogenate from terminally affected
animals, there should be no titre effects, also evidenced by
the remarkably consistent incubation periods, and there are
no genetic background effects since all transmissions were to
inbred SJL mice. These data are therefore consistent with
propagation of two distinct prion strains in these SJL mice.
Brains from each of these groups were examined by
Western blotting. The PrPSc type observed on primary
and secondary passage was faithfully maintained on
third passage. The SJL-passaged BSE showed a monoglycosylated-
dominant pattern and the C57BL/6-passaged
BSE showed a di-glycosylated-dominant pattern (Fig. 1 and
Fig. 2) on further subpassage in SJL.
Further evidence that we have isolated two distinct mouse
prion strains from cattle BSE came from neuropathological
studies of SJL brains from both groups. For the C57BL/6-
derived BSE (group 1) and the SJL-derived BSE (group 2),
five and seven individual brains were examined, respectively.
Findings were consistent within each group. The level and
distribution of spongiosis (vacuolation) was similar for both
groups (Fig. 3a and b, and Fig. 4a and b). Spongiosis was
most prominent in the cortex, hippocampus and thalamus
with some involvement of the brain stem and basal ganglia,
while the cerebellum was spared. However, consistent
differences were observed with regard to neuronal cell loss
in the hippocampus where the granule cell layer of the CA1
region of the dentate gyrus showed significant loss of cells
with the C57BL/6-derived inoculum but remained intact
with the SJL-derived BSE (Fig. 3a and b). Patterns of PrPSc
deposition were strikingly different between the two groups
(Fig. 3cf, Fig. 4c and d). For the C57BL/6-derived BSE
(group 1), hippocampus, thalamus and brain stem were all
heavily involved but less affected in SJL-derived BSE (group
2). In group 1, the cortex had a strongly pronounced
ribbon of PrPSc, which followed the cortical lamination,
but the rest of the cortex was mainly negative. However, in
group 2, the cortex had a uniform distribution of diffuse
staining for PrPSc (Fig. 3c and d; Fig. 4c and d). Many
plaques were found in the corpus callosum and on the
surface of the brain in group 1, but very few were seen in
group 2 (Fig. 3c and d; Fig. 4c and d). Differences were
also observed in the cerebellum, which was only slightly
involved in group 1, with occasional plaques found in
the granular layer. However, in group 2, the cerebellum
exhibited a uniform diffuse staining in both the molecular
and granular layer with no plaques (Fig. 3e and f). DISCUSSION Several strains of sheep scrapie and human CJD have been
described, yet BSE in the UK is thought to be caused by a
single prion strain (Bruce et al., 1994, 1997; Collinge et al.,
Fig. 2. Bar graph showing relative proportions of di- (white), mono-
(black) and unglycosylated (grey) PrP following partial digestion
with proteinase K. Data are plotted as mean±SEM (n=5).
2474 Journal of General Virology 85
S. E. Lloyd and others
1996; Hill et al., 2003). However, recent data from French
and Italian cattle suggest that more than one strain of BSE
may exist (Biacabe et al., 2004; Casalone et al., 2004). Strain
typing may be carried out in many ways (Safar et al., 1998;
Peretz et al., 2002) but strains were originally defined and
classified by their characteristics on passage to particular
inbred lines of mice based on incubation time and patterns
of neuropathological targeting, and more recently have been
distinguished by PrPSc type on Western blots. A mouse
prion strain difference should only be assessed in the same
line of inbred mouse, as genetic background is known to
modulate all the defining features of a prion strain (Bruce,
1993; Moore et al., 1998). Based on these criteria, we have
isolated two distinct strains from cattle BSE, which we have
designated MRC1 and MRC2, respectively. MRC1 showed a
mono-glycosylated-dominant PrPSc type on Western blots.
It was derived from a primary BSE passage in SJL/OlaHsd
mice and had a relatively short incubation time and a
Fig. 3. Neuropathological analysis of brain from SJL mice inoculated
with BSE following two passages in C57BL/6 (a, c, e)
or SJL (b, d, f) mice. (a, b) Haematoxylin and eosin (H&E)-stained
sections of the hippocampus showing spongiform
neurodegeneration. Arrowheads in (a) indicate neuronal loss. (c, d) PrP
immunohistochemistry showing the distinct laminar
distribution pattern of abnormal PrP in the cortex of
C57BL/6RC57BL/6RSJL-passaged BSE (c), while the PrP immunoreactivity
in SJLRSJLRSJL-passaged BSE is uniformly distributed (d). The cerebellum
of the former group (e) mainly shows
plaques and very little diffuse staining, while the latter group (f)
predominantly shows diffuse staining and no plaques. Bars,
450 mm (ad), 110 mm (e, f).
http://vir.sgmjournals.org 2475
Two BSE-derived prion strains
generalized diffuse pattern of PrP immunostaining in the
brain. MRC2 shows a di-glycosylated-dominant PrPSc type
on Western blots, was derived from a primary BSE passage
in C57BL/6JOlaHsd mice and had a relatively long
incubation time and showed a distinctive pattern of PrP
immunoreactivity and neuronal loss.
SJL mice were able to support both the MRC1 and MRC2
strain patterns. However, it will be important to establish
whether these strains are stable on further passage in SJL and
other strains of mice. Both C57BL/6 and SJL mice share the
same PrP amino acid sequence and therefore the strain
selection must be a feature of other genetic loci. Within
the limits of resolution available from Western blotting,
the fragment sizes following proteinase K digestion were the
same for the PrPSc associated with both strains, raising the
possibility that the differences related to glycosylation and
not to gross conformational differences, at least as differentiated
by proteinase K digestion. Additional studies will
be required to characterize precisely the conformation and
physico-chemical properties of PrPSc associated with these
prion strains.
Although recent reports suggest that alternative strains of
BSE can be found in cattle, previous studies have suggested
that BSE is caused by a single strain of agent (Bruce et al.,
1994; Biacabe et al., 2004; Casalone et al., 2004). 301V
and 301C represent previously reported independent BSE
strains; however, these were propagated in different strains
of mice, which not only had very different genetic backgrounds
(VM/Dk and C57) but also had two amino acid
coding differences in PrP and therefore their strain characteristics
cannot be directly compared. It is possible that
MRC2 represents the same strain as 301C; however, formal
comparisons will be required to investigate this.
The strain characteristics associated with MRC1 resemble
those seen with passage of Chandler/RML scrapie in these
strains of mice with respect to incubation time (122±1 days),
histology and PrPSc type on Western blotting (unpublished
data). We are confident that our findings are not the result
of contamination with mouse scrapie as these transmissions
were performed in accordance with rigorous biosafety
protocols for preparation of inocula, inoculations and care
of mice. Disposable equipment was used for each inoculum
and each mouse was identified with a unique transponder.
In addition, the MRC1 strain was also seen on primary
passage to RIIIS mice and also on primary passage of vCJD
to SJL mice (Asante et al., 2002). vCJD is caused by a BSElike
prion strain (Hill et al., 1997; Bruce et al., 1997; Collinge
et al., 1996); therefore, it is not surprising that the same
phenomenon is seen with both vCJD and BSE prions
in the same strains of mice. Further transmission studies
are under way to characterize these vCJD-derived mouse
strains. A similar bifurcation of the BSE strain characteristics
was also observed with independent BSE inocula in
transgenic mice expressing human PrP with methionine
at codon 129, Tg(HuPrP129M+/+Prnp0/0)-35 (Asante
et al., 2002).
MRC2 and MRC1 were derived from different sources of
BSE prions, I038 and I783, respectively. I038 originated
from a pool of five infected cow brainstems. However, our
extensive transmission studies with these isolates excluded
the pooling of material from five cows as the source of
the strain variation that we observe in these transmissions.
I038 gave the classical BSE signature and the novel phenotype
was derived from I783, which originated from a single
cow brain. I783 has been used for transmissions in several
inbred lines of mice in our laboratory and consistently
produces the MRC2 strain (as determined by Western
blotting) in C57BL/6JOlaHsd, FVB/NHsd, NZW/OlaHsd,
SM/J and SWR/OlaHsd mice. However, it also consistently
produces the MRC1 strain (as determined by Western
blotting) in SJL/OlaHsd and RIIIS/J mice (Fig. 1b, lane 3
and Asante et al., 2002). DNA sequence analysis of the
bovine Prnp gene in I783 confirmed that the animal was
homozygous for PrP amino acids, thereby excluding PrP
heterozygosity as the source of the strain bifurcation. I038
and other independent BSE inocula have also been shown
to produce two strains in Tg(HuPrP129M+/+Prnp0/0)-35
transgenic mice, which are on a mixed genetic background.
We therefore believe that the prion strain selection or
Fig. 4. Schematic representation of spongiosis
(blue, a, b) and PrPSc deposition (red,
c, d) in the brain for SJL mice inoculated
with BSE following two passages in C57BL/
6 (a, c) or SJL (b, d) mice. For PrPSc
deposition, red represents areas of intense
staining and pink represents areas of lighter
staining.
2476 Journal of General Virology 85
S. E. Lloyd and others
mutation is a feature of the genetic background of the mice
and studies are under way to identify the genes involved.
Although recent data suggest that more than one strain of
BSE may exist in cattle (Biacabe et al., 2004; Casalone et al.,
2004), in this study, both I038 and I783 appeared to be the
same single strain. However, Western blotting was unable to
determine whether a mixed population of strains already
existed in either of these brains or whether the alternative
strains were generated de novo in the mice. The inbred lines
studied to date represent only a small proportion of the
allelic variation that exists in the mouse genome; therefore,
the study of other strains of mice, particularly those derived
from unrelated strains such as trapped wild mice, may reveal
additional BSE-derived strains.
The human population exposed to BSE has a much more
diverse genetic background than can be observed using
laboratory strains of inbred mice. Therefore, it is possible
that BSE infection may be revealed not only as the type 4
PrPSc and associated vCJD clinicopathological phenotype
seen to date (Collinge et al., 1996; Hill et al., 2003), but in
future, may also present with alternative PrPSc types, incubation
times and neuropathology that may not be distinguishable
from sporadic CJD (Hill et al., 1997; Asante et al.,
2002) or represent additional novel phenotypes (Collinge,
1999). BSE may also have infected sheep flocks in the UK
and, given the genetic diversity of sheep breeds, it is also
possible that in some breeds BSE propagates as a strain type
that is indistinguishable from natural sheep scrapie. ACKNOWLEDGEMENTS We thank R. Bradley, D. Matthews, S. A. C. Hawkins and colleagues at
the UK Veterinary Laboratories Agency for providing BSE-infected
material. We also thank Catherine OMalley, Paul Hudson and Judy
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