Download The disease characteristics of different strains of scrapie in Sinc

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Journal of General Virology (1991), 72, 595~503. Printed in Great Britain
The disease characteristics of different strains of scrapie in Sinc congenic
mouse lines: implications for the nature of the agent and host control of
pathogenesis
M. E. Bruce,* I. McConnell, H. Fraser and A. G. Dickinson
Institute for Animal Health, A F R C & M R C Neuropathogenesis Unit, Ogston Building, West Mains Road,
Edinburgh EH9 3JF, U.K.
Mouse lines which are congenic for Sinc, the major
gene controlling scrapie incubation period, have been
produced by selective breeding from the inbred
C57BL(Sinc sT) and V M ( S i n c p7) strains; the s7 allele of
Sinc has been introduced into a VM background by 18
serial backcrosses, at each generation selecting on the
basis of the incubation period with the ME7 scrapie
strain. The characteristics of the disease produced by
seven scrapie strains have been compared in Sinc ~7 and
Sinc p7 congenic mice and in the F1 cross between them.
As previously found in non-congenic mice, each scrapie
strain has a characteristic, precisely reproducible
incubation period pattern in the three Sinc genotypes.
The Sinc gene controls the incubation period for all
scrapie strains tested but the direction of allelic action
and the apparent dominance pattern differs between
scrapie strains. Comparison with non-congenic mice
shows that other genes also have a minor effect on
incubation period. The distribution of vacuolar degeneration in the brain depends mainly on the scrapie
strain but is also influenced by Sinc and other
unspecified mouse genes. Restriction fragment length
polymorphism analysis has already shown that the
close linkage between Sinc and the gene encoding PrP
has been maintained in the Sinc congenic lines,
strengthening the possibility that PrP is the Sinc gene
product. The present study confirms that scrapie
strains carry information which is independent of the
host but nevertheless suggests that host PrP protein
interacts with this information to regulate the progression of the disease.
Introduction
models, but is remarkably predictable. Its length
depends on both the strain of scrapie agent and genetic
factors in the host. The major gene controlling incubation period in mice is the Sinc gene (scrapie incubation),
two alleles of which have been identified (s7 and p7)
(Dickinson et al., 1968; Dickinson & Meikle, 1971;
Dickinson & Fraser, 1979; Dickinson & Outram, 1988).
Other genes also modify the incubation period to some
extent but their effects are small compared with that of
Sinc (Outram, 1976; Kingsbury et al., 1983; Bruce &
Dickinson, 1985; Carp & Callahan, 1986; Carlson et al.,
1988; Race et a l., 1990); the Sinc gene closely regulates
the incubation period for all scrapie strains so far tested
(Dickinson & Outram, 1979, 1988). However, scrapie
strains differ in which of the two Sinc homozygotes has
the shorter incubation period and also in the apparent
type of dominance shown by the two alleles (Dickinson &
Meikle, 1971 ; Dickinson & Outram, 1979). It is not yet
known how the Sinc gene exerts these effects but it is
clear that its gene product plays a key role in the
pathogenesis of the disease.
The Sinc gene was first recognized about 25 years ago
Scrapie, a transmissible neurological disease of sheep
and goats, is the best understood member of a group of
closely similar diseases which includes CreutzfeldtJakob disease and Gerstmann-Str/iussler syndrome in
humans and bovine spongiform encephalopathy. Scrapie
has been studied extensively in a range of laboratory
models in mice and hamsters. Many distinct strains of
scrapie have been identified in mice, differing in their
incubation period characteristics, pathology, clinical
features and physicochemical properties (Dickinson &
Meikle, 1971; Dickinson & Fraser, 1977; Dickinson &
Outram, 1988; Bruce & Dickinson, 1987; Fraser, 1976;
Bruce et al., 1976; Carp et al., 1984; Kimberlin et al.,
1983). Scrapie strain variation has also been observed in
other experimental species such as goats (Pattison &
Millson, 1961), sheep (Foster & Dickinson, 1988) and
hamsters (Kimberlin & Walker, 1978a; Kimberlin et al.,
1989).
The incubation period of the disease in mice is long,
ranging from 4 months to over 2 years for the various
0000-9895 © 1991 SGM
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596
M. E. Bruce and others
in a r a n d o m - b r e d mouse colony at the M o r e d u n
Institute, E d i n b u r g h , U . K . ( D i c k i n s o n & M a c k a y , 1964;
D i c k i n s o n et al., 1968). Selective i n b r e e d i n g from this
colony o n the basis of scrapie i n c u b a t i o n period resulted
in the p r o d u c t i o n of the V M mouse strain, w h i c h is
homozygous for the p7 allele of Sinc. A l m o s t all other
laboratory mouse strains tested have b e e n s h o w n to carry
the s7 allele of Sinc ( D i c k i n s o n & Mackay, 1964;
O u t r a m , 1976; K i n g s b u r y et al., 1983; C a r p & C a l l a h a n ,
1986; Carp et al., 1987) [recently some authors have
referred to the Sinc gene by the unofficial d e s i g n a t i o n
Prn-i; see Mouse Newsletter, July 1987, no. 78, p. 3]. T h e
present paper describes the p r o d u c t i o n of congenic
mouse lines which differ only in the vicinity of the Sinc
locus. This has been achieved by i n t r o d u c i n g the s7 allele
into a V M b a c k g r o u n d , selecting on the basis of the
i n c u b a t i o n period of the disease p r o d u c e d by a particular
scrapie strain, ME7.
Strains of scrapie differ in the p a t t e r n of their
i n c u b a t i o n periods in the three Sinc genotypes of mouse.
They also differ m a r k e d l y in the severity a n d d i s t r i b u t i o n
o f vacuolar d e g e n e r a t i o n they produce in the brain. This
has led to an i n d e p e n d e n t m e t h o d of strain d i s c r i m i n a tion in which vacuolar d e g e n e r a t i o n is scored in a
n u m b e r of specified b r a i n areas to construct a 'lesion
profile' (Fraser & D i c k i n s o n , 1968; Fraser, 1976). T h e
lesion profile is characteristic for the strain of scrapie b u t
also d e p e n d s to some extent on the strain of the mouse
(Fraser & D i c k i n s o n , 1973; Fraser, 1976). The availability of Sinc congenic lines has m a d e it possible to
m i n i m i z e the m i n o r effects of other genes o n i n c u b a t i o n
periods a n d lesion profiles. I n this report we describe the
biological properties of seven scrapie strains in the Sinc
congenic lines.
Methods
Mouse strains. Two fullyinbred strains were used in the production of
the VM-Sincs7 strain. These were VM/Dk (referred to as VM in this
paper), which is homozygous for the p7 allele of Sine, and
C57BL/FaBtDk (referred to as C57BL), which is homozygousfor the s7
allele. Both of these mouse strains are of the H-2b major histocompatibility haplotype. The VM strain was at the 21st inbred generation at the
start of the selectivebreeding procedure, described in detail in Results.
The characteristics of the VM-Sincs7 congenic strain were later
compared with those of VM, C57BL and the F 1 crosses, VM x VMSine~7 and VM x C57BL. Male and female mice were used in
approximately equal numbers.
Scrapie strains. Seven scrapie strains were used, ME7, 22A, 22C, 22L,
79A, 87V and 139A (Table 1). These strains have been isolated and
maintained by serial intracerebral passage of brain from terminally
affected mice. They have previouslybeen wellcharacterized in terms of
their incubation periods and pathology in C57BL, VM and the F1 cross
between these two strains and are stable on repeated mouse-to-mouse
passage in the genotype specified in Table 1.
Injections. To prepare inocula, brain tissue from terminally affected
mice was homogenizedin physiologicalsaline at 19/ooconcentration and
Table 1. Details o f the seven scrapie strains used in this
study
Scrapie strain
Origin*
ME7$
Suffolk sheep,
natural scrapie
Cheviot sheep, SSBP/1
experimental scrapie
Cheviot sheep, SSBP/1
experimental scrapie
Goat, "drowsy'
experimental scrapie
'Chandler' mouse isolate
originally from 'drowsy'
goat scrapie
Cheviot sheep, SSBP/1
experimental scrapie
Cheviot × Border Leicester
sheep, natural scrapie
22Ct
22L
79A~
139A
22At
87Vt
Mouse strain
used for
serial passage
C57BL
C57BL
C57BL
C57BL
C57BL
VM
VM
* Animal that the scrapie strain was isolated from and the type of
scrapie that it had (Dickinson, 1976).
t Strains previously cloned by between two and four sequential
passages at limiting dilution for infectivity (Dickinson & Outram,
1983).
centrifuged at 500g for 10 min. Aliquots were stored at -20 °C and
thawed and reground before injection. Mice were injected either
intracerebrally (i.c.) or intraperitoneally (i.p.) with 0.02 ml of inoculum
when they were 3 to 8 weeks old.
Incubation period measurement. Injected mice were coded and scored
weekly for neurological signs of scrapie. They were killed at a defined
clinical endpoint, when they were in extremis or when they had
exhibited severe clinical signs for 3 consecutiveweeks (Dickinson et al.,
1968). After decoding, incubation periods were calculated as the
interval between injection and this defined endpoint. This method of
incubation period measurement has been shown to give highly
repeatable results for a wide range of combinations of scrapie strain
and mouse strain.
Histopathologieal procedures. Mice were sacrificed by cervical
fracture and their brains were immersion-fixedin 10~ formol saline.
Sections (6 lam) at four standard coronal levels were stained with
haematoxylin and eosin. Vacuolardegeneration was scored from coded
sections on a scale of 0 to 5 in nine defined grey matter areas of brain,
and on a scale of 0 to 3 in three white matter areas. For presentation
purposes, white matter scores were plotted over the same range as the 0
to 5 scale to make them visually comparable with grey matter scores.
After decoding, 'lesion profiles' were constructed from the average
score in each area, as previouslydescribed (Fraser & Dickinson, 1968;
Fraser, 1976).
Results
Production o f S i n c congenic lines
To produce Sinc congenic lines, the s7 allele from the
C57BL d o n o r strain was i n t r o d u c e d into V M by serially
backcrossing Sinc Svp7 heterozygotes to the V M ( S i n c pT)
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S c r a p i e s t r a i n s in Sinc congenic m i c e
Generation
C57BL(s7s7) x VM(p7pT)
I
s7p7 x VM(p7p7)
/11
p7p7/s7~7
597
(a)
30
1
x VM(p7p7)
2010
2 (lst backcross)*
p7p7"~ s71p7 x VM(p7p7)
p7p7
s7p7
3 (2nd backcross)*
!o
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4 to 17 (3rd to 16th backcross)*
I
I
Z
p7p7"/~s71p7 x VM(p7p7)
p7p7s - ' ~ T p ~ 7
p7p7
s7p7
18 (17th backcross)*
19 (18th backcross)*
s7s7 x s7s7
[
20
i
10
0
20t
VM-Sinc s7
Fig. 1. Breeding strategy used for the production of the VM-Sinc~7
congenic line, showing the Sinc genotype of mice at each generation.
Genotypes of individual mice in generations marked with an asterisk
were determined by crossing them with VM mice (Sinc pT) and testing
their progeny with ME7 scrapie. Genotypes of individual mice in the
generation marked t were determined by crossing them with Sinc~
mice and testing their progeny with ME7 scrapie.
parental strain (Fig. 1). Mice carrying the s7 allele were
selected on the basis of the incubation periods seen after
i.c. infection with the M E 7 strain o f scrapie. This was
possible because there are large differences between the
incubation periods in the three S i n c genotypes with this
scrapie strain; for example, in a previous experiment in
which mice were injected i.c. with 1 ~o M E 7 inoculum,
the m e a n incubation periods +__S.E.M., based on 12 to 16
animals, were 171 + 2 days for C 5 7 B L ( S i n c ~ 7 ) , 328 + 4
days for V M ( S i n c pT) and 251 + 2 days for the F1 cross,
V M x C57BL(SincSTp7).
A t each backcross generation, approximately 30 mice
of as yet u n k n o w n genotype (either S i n c p7 or S i n c svpT)
were m a t e d with V M ( S i n c pT) mice. T h e first ten
offspring o f each m a t i n g were injected i.c. with M E 7
scrapie after weaning. S i n e sTp7 parents were clearly
identified f r o m the bimodal distribution o f incubation
periods in this first set o f offspring (e.g. Fig. 2). The
subsequent progeny of S i n e sTp7 x V M matings identified
in this way were included in the next backcrossing to V M
and the whole procedure was repeated up to the 18th
backcross generation.
At the 19th generation (i.e. the 18th backcross
generation), genotypes o f individual mice were determined as above. N i n e S i n e sTp7 mice were identified, six
females and three males, and these were m a t e d amongst
themselves to produce progeny representing all three
S i n c genotypes. The individual genotypes o f these 20th
generation mice were in turn determined by m a t i n g t h e m
150
200
250
300
Incubation period (days)
350
Fig. 2. Distribution of ME7 i.c. incubation periods in the first set of
progeny ofmatings between VM and 26 mice from the 14th generation.
(a) The offspring of 14 SincsTp7 parents were either SincsTp7 or Sincp7
and showed a bimodal distribution of incubation periods. Subsequent
offspring of these matings were selected for the next backcrossing to
VM. (b) The offspring of 12 Sincp7 parents were all SincP7 and showed a
unimodal distribution of incubation periods.
20
(a)
lo
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"~20
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.
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(b)
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200
250
300
Incubation period (days)
.
150
.
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350
Fig. 3. Distribution of ME7 i.c. incubation periods in the progeny of
matings between inbred Sinc~7 mice and 15 mice from the 20th
generation. (a) The offspring of three Sincs7 parents were all Sine~7 and
showed a unimodal distribution of incubation periods. Of the three
Sinc~7 parents identified, two were mated with each other to form the
basis of the VM-Sincs7 line. (b) The offspring of 11 S/ncs7p7parents were
either Sincs7 or Sinc~7p7 and showed a bimodal distribution of
incubation periods. (c) The offspring of one Sincp7 parent were all
SincsTp7 and showed a unimodal distribution of incubation periods.
with inbred S i n c s7 mice and testing their offspring with
M E 7 scrapie (Fig. 3). In this w a y we identified three
S i n c ~7 mice in the 20th generation, two o f w h i c h were
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598
M . E. Bruce and others
m a t e d with each other to form the basis of the V M - S i n c s7
line; the line was subsequently maintained by full-sibling
mating. A t no stage during the whole selective breeding
procedure were mice which were incubating scrapie used
for breeding purposes.
Disease characteristics o f scrapie strains in Sinc congenic
lines
(i) Incubation periods
Previous experiments have shown that each of the seven
scrapie strains used, injected i.c. as 1 ~ brain inocula
under standard experimental conditions, has a characteristic pattern o f incubation periods in C57BL, V M and
V M × C57BL mice (Table 2). I n a later set o f experiments, V M - S i n c sT, C57BL, VM, V M x C57BL and
V M x V M - S i n c s7 mice were injected i.c. or i.p. with
these scrapie strains under the same standard conditions
but using 1 ~o inocula prepared from different ' d o n o r '
mouse brains. W i t h this dose all mice develop clinical
scrapie except Sinc ~7 and Sinc s7p7 mice injected with 87V
scrapie (see below). The incubation periods observed in
these experiments are listed in Table 2, together with
previous results in VM, C 5 7 B L and V M x C57BL mice.
F o r ease o f comparison between groups, the data in
Table 2 are also presented graphically in Fig. 4. The i.c.
incubation periods in C57BL, V M and V M x C57BL
mice were closely similar to those in the previous
experiments, illustrating the high degree of repeatability
o f incubation period measurement.. F o r each scrapie
strain the incubation period in V M - S i n c s7 was closest
to that in C57BL mice. However, there were clear
differences between these two mouse strains and
between the two FI crosses, V M x V M - S i n c s7 and
V M x C57BL, particularly with the 79A, 139A and 22A
scrapie strains. These differences were within the range
previously reported for unrelated mouse strains of the
same Sinc genotype (Outram, 1976; K i n g s b u r y et al.,
1983; Bruce & Dickinson, 1985; C a r p & Callahan, 1986).
This adds to existing evidence that genes other than Sinc
can make some contribution to the differences seen
between non-congenic mouse strains. T h e availability o f
V M - S i n c s7 congenic mice makes it possible to study the
effect o f the Sinc gene, minimizing the complication o f
effects of these other genes.
The results shown in Table 2 and Fig. 4 confirm that
the Sinc gene has an overwhelming influence on the
timing of events in scrapie pathogenesis. The largest
effect was seen with the 87V strain o f scrapie, which had
an incubation period o f 290 + 4 days in i.c. infected
Sinc p7 mice but did not produce clinical disease within
the lifespan of mice of other Sinc genotypes. However,
two o f seven V M - S i n c s7 mice and one o f seven C 5 7 B L
mice surviving longer than 700 days after injection with
87V had characteristic scrapie brain lesions when killed
Table 2. Mean incubation period for different combinations o f scrapie strain and mouse strain or
cross
Mean incubation period (days) + S.E.M.
Route of
infection
Scrapie
strain
I.c.
(previous
data)
ME7
22C
22L
79A
139A
22A
87V
ME7
22C
22L
79A
139A
22A
87V
ME7
22C
22L
79A
I.c.
I.p.
VM
328+__4(14)*
458 + 3(11)
208 + 1(16)
301 _ 6(10)
201 + 3(20)
203 + 3(16)
290 + 3(15)
346 + 3(84)
447 + 9(7)
210 + 3(11)
309 + 4(16)
208 + 2(12)
199 _ 3(39)
290 + 4(5)
524 + 0(2)
603 + 5(9)
431+9(11)
464 + 7(14)
C57BL
VM x C57BL
171 +2(16)
182 ___1(18)
148 + 1(17)
158 + 2(14)
155 + 1(17)
466 + 4(23)
> 700(20)t
168 + 2(19)
170 + 1(6)
157 + 1(11)
161 + 2(10)
166 + 2(20)
474 + 7(14)
> 700(7)t"
275 + 3(19)
235 -+ 3(15)
234+7(11)
224 + 5(12)
251 +2(12)
269 + 4(16)
189 + 1(17)
280 + 4(24)
249 + 3(19)
587 _ 7(18)
> 700(27)t
252 + 3(19)
269 + 3(6)
192 + 2(8)
276 + 5(11)
270 + 4(9)
567 + 7(9)
ND
408 + 6(17)
362 + 17(4)
312+7(16)
348 + 4(12)
VM-Sinc s7
178 + 1(84)
163 + 1(20)
147 _+1(16)
153 + 2(29)
137 + 2(12)
441 + 2(84)
> 700(7)'I"
310 + 3(32)
248 ___2(27)
260 _ 4(16)
211 _+3(14)
VM x VM-Sinc~7
259 + 4(19)
ND:~
ND
230 __ 1(6)
224 _+4(12)
504 + 5(18)
ND
425 __7(6)
ND
r~
320_+5(3)
* Figures in parentheses show number of mice in each group tested.
t No mice in these groups developed clinical disease within their lifespan; the numbers of mice surviving longer
than 700 days post-injection are shown.
:~ND, Not done.
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Fig. 4. Graphical representation of the incubation period data in Table
2. (a) Mean incubation periods in previous experiments in which VM,
C57BL and VM x C57BL mice were injected i.c. with 1 ~ inocula. (b)
Mean incubation periods in later experiments in which VM-Sin¢s7 and
VM x VM-Sincs7 mice were also included; mice were again injected
i.c. with 1 ~ inocula. (c) Mean incubation periods in the same mouse
genotypes injected i.p. with 1 ~ inocula. For each scrapie strain the
same inoculum was used in (b) and (c), but that used in (a) was from a
different mouse source. Incubation periods are shown in V M (ilt), VMSinc s7 ( 0 ) , VM x V M - S i n c s7 ( i ) , C57BL (C)) and VM x C57BL ([3)
mice. Note that mice of the same Sinc genotype are represented by the
same shape of symbol and that the VM and VM-Sincs7 congenic lines
and the cross between them are shown as solid symbols. Where symbols
are missing, the particular combination of scrapie strain and mouse
genotype was not tested, except in the case of 87V with which the
projected incubation periods in Sinc ~7 and Sinc sTp7 mice were longer
than lifespan. For standard errors and numbers of mice/group, see
Table 2; groups of less than six mice are shown in parentheses.
in senility. Other work (M. E. Bruce, unpublished
results) suggests that all of these Sinc $7 mice were
incubating scrapie but that the projected incubation
period was longer than their lifespan.
For each scrapie strain tested the ranking of incubation periods in the three Sinc genotypes was the same
whether the comparison was made in congenic or noncongenic mice. Scrapie strains differed in their absolute
incubation periods in any single mouse genotype, in the
ranking of incubation periods in the two Sinc homozygotes and in the apparent dominance shown between the
1 234
56
7 891"13"12
2*
34567
891"13*
2*
Scoring areas in brain
Fig. 5. Lesion profiles for six strains of scrapie injected i.c. into VM), VM ( - - - ) and C57BL (.--) mice (n = eight to 25
mice/group). Mice were injected with (a) ME7, (b) 22C, (c) 22L, (d)
79A, (e) 139A and (f) 22A. Vacuolar degeneration was scored in nine
grey matter and three white matter areas of brain (Fraser & Dickinson,
1968; Fraser, 1976). The grey matter areas are: 1, dorsal medulla; 2,
cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, medial
thalamus; 6, hippocampus; 7, septum; 8, medial cerebral cortex at the
level of the thalamus; 9, medial cerebral cortex at the level of the
septum. The white matter areas are 1% cerebellar white matter; 2",
white matter of the mesencephalic tegmentum; 3", pyramidal tract.
Sinc s7 (
two alleles in the F1 heterozygote. Thus, the incubation
period was shorter in Sinc s7 mice than in Sinc p7 mice for
strains ME7, 22C, 22L, 79A and 139A but the reverse
was true for 22A and 87V. With strains ME7, 22C, 22L
and 79A the incubation period in the heterozygote lay
between those of the two parental strains, whereas with
22A and 139A it was beyond the parental range. As
expected from previous studies (Kimberlin & Walker,
1978b), incubation periods following i.p. infection were
approximately 50~ longer than those following i.c.
infection with the same inocula. This is mainly because
the agent replicates in the periphery before entering the
central nervous system (CNS) after an i.p. injection,
whereas infection is established directly in the brain by
an i.c. injection (Kimberlin & Walker, 1979). Despite
this difference in pathogenesis, the ranking of incubation periods in the three Sinc genotypes was the same
regardless of route of infection.
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600
M . E. Bruce and others
(ii) Lesion profiles
The lesion profiles in i.c. infected C57BL and VM mice
were as expected from previous studies (Fig. 5).
Although they tended to share the same general features,
the profiles in these two mouse strains differed from each
other in some respects, particularly with the 22A strain of
scrapie. The profiles in V M - S i n c s7 mice were broadly
similar to those in C57BL and VM mice but did not
consistently correspond to either; for example, the VMSinc ~v grey matter profile was similar to the VM profile
for 79A and 139A but more closely resembled the C57BL
profile for 22C and ME7. As previously demonstrated
(Fraser, 1976; Kimberlin et al., 1987), profiles were
generally lower for the i.p than for the i.c. route but
showed the same general shape (data not shown).
Therefore, the distribution of vacuolar degeneration in
the brain, as represented by the lesion profile, depended
primarily on the strain of scrapie but was also influenced
to a lesser extent by the Sinc gene and other unspecified
mouse genes.
Discussion
The present study demonstrates that, without any doubt,
there are distinct strains of scrapie which have their own
characteristic and reproducible properties. This confirms the conclusions of a large number of previous
studies in non-congenic mice, in which about 20 scrapie
strains have been identified using the same strain-typing
methods (Dickinson & Fraser, 1977; Dickinson &
Outram, 1988). This scrapie strain diversity is not simply
imposed by the mouse strain used for passage as
numerous different strains with stable properties have
been isolated in the same mouse genotype; for example,
five of the strains used in the present study were
maintained by passage in C57BL mice. We have also
confirmed in congenic mice that the Sinc gene controls
the incubation period for all scrapie strains tested. These
results highlight two central questions concerning scrapie and related diseases. Firstly, what is the nature of the
causative agent? And, secondly, what is the mechanism
of the host control of pathogenesis ?
Much of the research on scrapie in recent years has
centred on a host-coded protein, PrP (BoRon et al., 1982;
McKinley et al., 1983). Relatively protease-resistant
forms of this protein accumulate in the brain during
scrapie pathogenesis and aggregate into scrapie-associated fibrils (SAF) and amyloid (Merz et al., 1981 ; Diringer
et al., 1983; Hope et al., 1986; DeArmond et al., 1987;
McBride et al., 1988; Bruce et al., 1989). Infectivity in
tissue extracts has been shown to copurify with SAF, at
least to some extent (McKinley et al., 1983; Diringer et
al., 1983; Somerville et al., 1986), but no scrapie-specific
nucleic acids have yet been identified, even in the
relevant protein fractions (Oesch et al., 1988). This
failure to detect scrapie-specific nucleic acids and the
resistance of scrapie to treatments which would usually
inactivate nucleic acids have prompted the 'prion'
hypothesis, that the replicating infectious agent is
composed only of a protein, PrP, and is devoid of nucleic
acid (Prusiner, 1982).
The major obstacle to believers in the protein-only
model is the existence of many strains of scrapie, which
carry information independent of the host. We have
shown elsewhere that scrapie strains can be copassaged
as mixtures and still retain their separate identities
(Dickinson et al., 1986) and that certain strains predictably give rise to mutants, even when passaged in a single
host genotype (Bruce & Dickinson, 1987). Although it is
possible to elaborate theoretical models in which
proteins encode some form of strain specificity (Bolton &
Bendheim, 1988; Wills, 1989), it is difficult to account
for these aspects of strain variation on a protein-only
basis; there is also no direct experimental evidence for
replicating proteins. The simplest explanation remains
that the scrapie informational molecule is an as yet
undetected nucleic acid which is protected by its close
association with host tissue components, as detailed in
the 'virino' hypothesis (Dickinson & Outram, 1983,
1988). PrP (or abnormal PrP) could well be a host
component in such a model.
On the other hand, there is growing evidence that PrP
is involved in the host's control of pathogenesis, as well
as being pathologically modified in the course of the
disease. Using restriction fragment length polymorphism analysis and nucleotide sequencing a close linkage
has been demonstrated previously between the gene
encoding PrP and the Sinc gene (Carlson et al., 1986;
Westaway et al., 1987; Hunter et al., 1987). Our Sinc
congenic lines have already been compared using the
XbaI and TaqI endonucleases, which detect polymorphisms in the non-coding or flanking regions of the PrP
gene; V M - S i n c s7 mice were found to resemble C57BL
mice but differ from VM mice in their restriction pattern
(Hunter et al., 1987). This confirms the close linkage of
the PrP gene and the Sinc gene, a linkage which has been
maintained through the 18 successive backcrosses
carried out to produce the congenic lines; either the PrP
gene lies within the stretch of DNA transferred with the
Sinc gene or PrP is in fact the Sinc gene product.
In linkage studies in other laboratories, a few possible
individual recombinants have been identified in crosses
between non-congenic mouse strains in which incubation period and PrP did not segregate together (Carlson
et al., 1988; Race et al., 1990). However, there is doubt as
to whether these really were recombinants, firstly
because of possible effects of genes other than Sinc on
incubation period in these non-congenic strains. Second-
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Scrapie strains in Sinc congenic mice
ly, in both studies an uncloned scrapie isolate was used,
possibly containing a mixture of strains which would
produce confusing incubation period results. Recently, a
more definitive study has strengthened the possibility
that PrP is indeed the Sinc gene product; the hamster
PrP gene, inserted in multiple copies into the Sinc s7
mouse genome, conferred hamster-like incubation period properties on the transgenic mouse line when infected
with a hamster-passaged scrapie isolate (Scott et al.,
1989).
Two polymorphic sites have been identified within the
coding region of the mouse PrP gene, both of which are
predicted to result in amino acid substitutions in the
protein (Westaway et al., 1987). All of the Sinc ~7 mouse
strains tested so far are predicted from their gene
sequence to have leucine at codon 108 and threonine at
codon 189, whereas all Sinc p7 strains are predicted to
have phenylalanine at codon 108 and valine at codon
189. If PrP proves to be the Sinc gene product, these
differences in the primary structure of the protein could
be responsible for the Sinc gene effects on scrapie
incubation period and pathology.
The pathogenesis of scrapie is likely to involve a
number of stages, including the uptake of infectivity by
cells, possibly a processing step before replication
commences, actual replication within cells and spread to
other cells in which the cycle is repeated (Kimberlin &
Walker, 1986, 1988). At least part of the cell-to-cell
progression within the CNS has been shown to follow
specific neuronal projection pathways (Fraser & Dickinson, 1985; Gregoire et al., 1984). Severe neurological
dysfunction, leading to clinical disease and death, has
been suggested to occur only when infectivity and
damage have reached critical levels in certain 'clinical
target areas' which are responsible for vital functions in
the animal (Kimberlin & Walker, 1986, 1988).
It is not known which step in the above sequence is
regulated by the Sinc gene. However, the effect clearly
does not depend on differences in the efficiency of the
initial infection as there are only minor differences
between estimates of infectivity levels in the same tissue
sample, measured by titration in mice of different Sinc
genotypes; each combination of scrapie strain and
mouse genotype in fact has its own distinct doseresponse curve(Dickinson & Outram, 1988). The studies
described by Dickinson & Outram (1988) have also
shown that the efficiency of infection is not influenced by
whether there is a match between the Sinc genotypes of
the 'donor' and 'recipient' mice; there are also no great
differences between Sinc genotypes in the titre of
infectivity in brain in the terminal phase of the disease,
showing that mice of different genotypes are not simply
becoming ill at different stages in the course of infection.
It follows that the Sinc gene exerts its effect either on the
601
rate of cell-to-cell spread of infection or on the rate of
replication. As discussed above, it is probable that the
Sinc gene encodes PrP, which has the characteristics of a
cell surface glycoprotein and could therefore be some
type of receptor (Bazan et al., 1987). This might suggest
that the Sinc gene effect involves cell-to-cell spread
rather than actual replication.
The interaction of the Sinc gene with the replicable
information carried by the agent shows a number of
peculiar features. Firstly, considering the length of the
incubation periods, the control exerted by Sinc is
remarkably precise; secondly, the direction of allelic
action differs according to the strain of scrapie.
However, perhaps the most intriguing feature of the Sinc
gene is that the dominance characteristics of the two
alleles also appear to differ according to the scrapie
strain. With certain scrapie strains one allele or the other
exhibits apparent overdominance, giving incubation
periods in the heterozygote which are beyond the range
of those in the parents. The fact that this is seen even in
Sinc congenic mice shows that it is not simply a nonspecific result of hybrid vigour. Rather, as discussed by
Dickinson & Outram (1979), the phenomenon may
suggest that the two alleles do not act independently in
the heterozygote. This led them to the hypothesis that the
molecular structure controlling pathogenesis is a complex of subunits which are contributed by both Sinc
alleles. However, the incubation period data can be
viewed from a different perspective. In their discussion
on the action of the Sinc gene, Dickinson & Outram
(1979) also pointed out that when the results for many
scrapie strains are taken into account, the incubation
period in the heterozygote is almost always longer than
that in Sinc ~v mice and there is a high correlation
(r=0.98) between incubation periods in these two
genotypes. On the other hand, there is no significant
correlation between incubation periods in SincOV mice
and either of the other two genotypes. The heterozygote
therefore behaves like a 'slower' version of a Sine s7
mouse, irrespective of the scrapie strain; this raises the
question of why the p7 allele has so little positive effect in
the heterozygote, even though it can have such a large
effect in the p7 homozygote.
Finally, it is clear that host genetic factors are also
important in field scrapie and related infections in other
species. The occurrence of both natural and experimental sheep scrapie is controlled by the Sip gene (a
probable analogue of Sinc), which in sheep may also be
linked to the PrP gene (Dickinson & Fraser, 1979;
Dickinson & Outram, 1988; Hunter et al., 1989). There is
evidence that the allelic effects of Sip, like Sinc, differ
according to the strain of scrapie encountered (Foster &
Dickinson, 1988). Similar host genetic controls are also
likely to operate in the scrapie-like diseases in humans,
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602
M. E. Bruce and others
Creutzfeldt-Jakob disease and Gerstmann-Str/iussler
syndrome. Recently it has been shown that the occurrence of these conditions in some families is linked to the
inheritance of variants of PrP (Owen et al., 1989; Hsiao
et al., 1989; Goldgaber et al., 1989; Doh-ura et al., 1990).
Fundamental studies on the genetics of agent-host
interactions in Sinc congenic mice are therefore relevant
to the whole family of scrapie-like diseases.
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(Received 31 August 1990; Accepted 29 November 1990)
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