* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download The disease characteristics of different strains of scrapie in Sinc
Genetic engineering wikipedia , lookup
Gene expression programming wikipedia , lookup
Gene desert wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Gene expression profiling wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Saethre–Chotzen syndrome wikipedia , lookup
Gene therapy wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Gene nomenclature wikipedia , lookup
Neuronal ceroid lipofuscinosis wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Pathogenomics wikipedia , lookup
Microevolution wikipedia , lookup
History of genetic engineering wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Designer baby wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Public health genomics wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
595 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 596 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) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 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 O I 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 0 "~20 . . . (b) . . 'v . . 'l , . i . . ' . . . . . . . . i Z 0 ........... (e) 10 o L 1 iiiiiii i ~ 20 D- s7s7 ! ..... sTp7 200 250 300 Incubation period (days) . 150 . . . . . . . . 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 598 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 Scrapie strains in Sinc congenic mice i t i D i i (a) 600 500 4 -(a) • i~X • (b) lAX © 400 2 . . . . I. . , .. "..- . . 300 200 I 600 © © © I I I I (b) I [] I o o t i I I I I I . . . . . . . I ' ' ' ' ' "-' " i --(d) " ~2 " '~ -" / 400 ~ 0'1 I 4 (c) o° 3 [] • 1 "~ O 500 .~ 300 I:. / o 100 599 ".. -" ".t:"f f \ "-, ~ ,"/ ,~.., "", ." ~ : o 200 100 I I I I I I i i i t , I i 600 " (c) (•) 500 • 400 (D) 0 4 -(e) ~ I "( f ) 2 ~,z \ ..A / \ 300 o Ii I I 200 100 0 l I ME7 22C I 22L 79A 139A Scrapie strain i I 22A 87V 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 600 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- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 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, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 602 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. References BAZAN, J. F., FLETTERICK, R. J., McKINLEY, M. P. & PRUSINER, S. B. (1987). Predicted secondary structure and membrane topology of the prion protein. Protein Engineering l, 125-135. BOLTON, D. C. & BENDHEIM, P. E. (1988). A modified host protein model of scrapie. In Novel Infectious Agents and the Central Nervous System, Ciba Foundation Symposium 135, pp. 164-181. Edited by G. Buck & J. Marsh. Chichester: John Wiley. BOLTON, D. C., MCKINLEY, M. P. & PRUSINER, S. B. (1982). Identification of a protein that purifies with the scrapie agent. Science 218, 1309-1311. BRUCE, M. E. & DICKINSON, A. G. (1985). Genetic control of amyloid plaque production and incubation period in scrapie-infected mice. Journal of Neuropathology and Experimental Neurology 44, 285-294. BRUCE, M. E. & DICKINSON, A. G. (1987). Biological evidence that scrapie agent has an independent genome. Journal of General Virology 68, 79-89. BRUCE, M. E., DICKINSON, A. G. & FRASER, H. (1976). Cerebral amyloidosis in scrapie in the mouse : effect of agent strain and mouse genotype. Neuropathology and Applied Neurobiology 2, 471-478. BRUCE, M. E., MCBRIDE, P. A. & FARQUHAR, C. F. (1989). Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie. Neuroscience Letters 102, 1-6. CARLSON, G. A., KINGSBURY, D. T., GOODMAN, P. A., COLEMAN, S., MARSHALL, S. T., DEARMOND, S., WESTAWAY,O. & PRUSINER, S. B. (1986). Linkage of prion protein and scrapie incubation time genes. Cell 46, 503-511. CARLSON, G. A., GOODMAN, P. A., LOVETT, M., TAYLOR, B. A., MARSHALL, S. T., PETERSON-TORCHIA, i . , WESTAWAY, n . t~ PRUSlNEI',, S. B. (1988). Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time. Molecular and Cellular Biology 8, 5528-5540. CARP, R. I. & CALLAHAN, S. (1986). Scrapie incubation periods and end-point titers in mouse strains differing at the H-2D locus. Intervirology 26, 85 92. CARP,R. I., CALLAHAN,S. M., SERSEN,E. A. & MORETZ, R. C. (1984). Preclinical changes in weight of scrapie-infected mice as a function of scrapie agent-mouse strain combination. Intervirology 21, 61-69. CARP, R. I., MORETZ, R. C., NATELLI, M. & DICKINSON, A. G. (1987). Genetic control of scrapie : incubation period and plaque formation in I mice. Journal of General Virology 68, 401-407. DEARMoND, S. J., MOBLEY, W. C., DEMOar, D. L., BARRY, R. A., BECKSTEAD, J. H. & PRUSINER, S. B. (1987). Changes in the localization of brain prion proteins during scrapie infection. Neurology 37, 1271-1281. DICKINSON, A. G. (1976). Scrapie in sheep and goats. In Slow Virus Diseases of Animals and Man, pp. 209-241. Edited by R. H. Kimberlin. Amsterdam: North-Holland. DICKINSON, A. G. & FRASER, H. (1977). Scrapie pathogenesis in inbred mice: an assessment of host control and response involving many strains of agent. In Slow Virus Infections of the Central Nervous System, pp. 3-14. Edited by V. ter Meulen & M. Katz. New York: Springer-Verlag. DICKINSON, A. G. & FRASER, H. (1979). An assessment of the genetics of scrapie in sheep and mice. In Slow Transmissible Diseases of the Nervous System, vol. 1, pp. 367-385. Edited by S. B. Prusiner & W. J. Hadlow. New York: Academic Press. DICKINSON, A. G. & MACKAY,J. M. K. (1964). Genetical control of the incubation period in mice of the neurological disease, scvapie. Heredity 19, 279 288. DICKINSON, A. G. & MEIKLE, V. M. H. (1971). Host-genotype and agent effects in scrapie incubation: change in allelic interaction with different strains of agent. Molecular and General Genetics 112, 73-79. DICKINSON, A. G. & OUTRAN, G. W. (1979). The scrapie replicationsite hypothesis and its implications for pathogenesis. In Slow Transmissible Diseases of the Nervous System, vol. 2, pp. 13-31. Edited by S. B. Prusiner & W. J. Hadlow. New York: Academic Press. DICKINSON, A. G. & OUTRAN,G. W. (1983). Operational limitations in the characterisation of the infective units of scrapie. In Virus Nonconventionnels et Affections du Systdme Nerveux Central, pp. 3-16. Edited by L. A. Court & F. Cathala. Paris: Masson. DICKINSON, A. G. • OUTRAN, G. W. (1988). Genetic aspects of unconventional virus infections: the basis of the virino hypothesis. In Novel Infectious Agents and the Central Nervous System, Ciba Foundation Symposium 135, pp. 63-83. Edited by G. Buck & J. Marsh. Chichester: John Wiley. DICKINSON, A. G., MEIKLE, V. M. H. & FRASER, H. (1968). Identification of a gene which controls the incubation period of some strains of scrapie in mice. Journal of Comparative Pathology 78, 293299. DICKINSON, A. G., OUTRAM, G. W., TAYLOR, D. M. & FOSTER, J. D. (1986). Further evidence that scrapie agent has an independent genome. In Unconventional Virus Diseases of the Central Nervous System, pp. 446-460. Edited by L. A. Court, D. Dormont, P. Brown & D. T. Kingsbury. Fontenay-aux-Roses: Commissariat fi l'Energie Atomique. DIRINGER, H., GELDERBLOM, H., HILMERT, H., OZEL, i . , ELDERBLUTH,C. & KIMBERLIN, R. H. (1983). Scrapie infectivity, fibrils and low molecular weight protein. Nature, London 306, 476-478. DOH-URA, K., TATEISHI, J., KITAMOTO, T., SASAKI, H. & SAKAKI, Y. (1990). Creutzfeldt-Jakob disease patients with congophilic kuru plaques have the missense variant prion protein common to Gerstmann-StrS.ussler syndrome. Annals of Neurology 27, 121-126. FOSTER, J. D. & DICKINSON, A. G. (1988). The unusual properties of CH 1641, a sheep-passaged isolate of scrapie. Veterinary Record 123, 5-8. FRASER, H. (1976). The pathology of natural and experimental scrapie. In Slow Virus Diseases of Animals and Man, pp. 267 305. Edited by R. H. Kimberlin. Amsterdam: North-Holland. FRASER, H. & DICKINSON, A. G. (1968). The sequential development of the brain lesions of scrapie in three strains of mice. Journal of Comparative Pathology 78, 301 311. FRASER, H. & DICKINSON, A. G. (1973). Scrapie in mice: agent-strain differences in the distribution and intensity of grey matter vacuolation. Journal of Comparative Pathology 83, 29-40. FRASER, H. & DICKINSON, A. G. (1985). Targeting of scrapie lesions and spread of agent via the retino-tectal projection. Brain Research 346, 32-41. GOLDGABER,D., GOLDFARB,L. G., BROWN, P., ASHER, D. M., BROWN, W. T., LIN, S., TEENER, J. W., FEINSTONE, S. M., RUBENSTEIN, R., KASCSAK, R. J., BOELLAARD, J. W. & GA/DUSEK, D. C. (1989). Mutations in familial Creutzfeldt-Jakob disease and GevstmannStvfiussler-Sheinker's syndrome. Experimental Neurology 106, 204206. GREGOIRE, N., NEZRI, C., GORDE-DURAND, J. M., BOURAS,C., BERT, J. ~; SALAMON,G. (1984). Cerebral metabolic changes induced by an unconventional agent: experimental model for some human degenerative diseases of the central nervous system. Monographs in Neural Science 11, 193-203. HOPE, J., MORTON, L. J. D., FARQUHAR, C. F., MULTHAUP, G., BEYREUTHER,K. & KIMBERLIN, R. H. (1986). The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein. EMBO Journal 5, 2591-2597. HSIAO, K., BAKER, H. F., CROW, T. J., POULTER, M., OWEN, F., TERWILLIGER, J. D., WESTAWAY, D., Oar, J. & PRUSlNER, S. B. (1989). Linkage of a pylon protein missense variant to GerstmannStrgussler syndrome. Nature, London 338, 342-345. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23 Scrapie strains in S i n c congenic mice HUNTER, N., HOPE, J., McCONNELL, I. & DICKINSON, A. G. (1987). Linkage of the scrapie-associated fibril protein (PrP) gene and Sine using congenic mice and restriction fragment length polymorphism analysis. Journal of General Virology 68, 2711-2716. HUNTER, N., FOSTER, J. D., DICKINSON, A. G. & HOPE, J. (1989). Linkage of the gene for the scrapie associated fibril protein (PrP) to the Sip gene in Cheviot sheep. Veterinary Record 124, 364~366. KIMBERLIN, R. H. & WALKER, C. A. (1978a). Evidence that the transmission of one source of scrapie agent to hamsters involves separation of agent strains from a mixture. Journal of General Virology 39, 487-496. KIMBERLIN, R. H. & WALKER, C. A. (1978b). Pathogenesis of mouse scrapie: effect of route of inoculation on infectivity titres and doseresponse curves. Journal of Comparative Pathology 88, 39-47. KIMBERLIN, R. H. & WALKER,C. A. (1979). Pathogenesis of mouse scrapie: dynamics of agent replication in spleen, spinal cord and brain after infection by different routes. Journal of Comparative Pathology 89, 551 562. KIMBERLIN, R. H. &. WALKER, C. m. (1986). Transport, targeting and replication of scrapie in the CNS. In UnconventionalVirusDiseasesof the Central Nervous System, pp. 547-562. Edited by L. A. Court, D. Dormont, P. Brown & D. T. Kingsbury. Fontenay-aux-Roses: Commissariat A l'Energie Atomique. KIMBERLIN, R. H. & WALKER, C. A. (1988). Pathogenesis of experimental scrapie. In Novel Infectious Agents and the Central Nervous System, Ciba Foundation Symposium 135, pp. 37 62. Edited by G. Bock & J. Marsh. Chichester: John Wiley. KIMBERLIN, R. H., WALKER, C. A., MILLSON, G. C., TAYLOR, D. M., ROEERTSON, P. A., TOMLINSON, A. H. & DICKINSON, A. G. (1983). Disinfection studies with two strains of mouse-passaged scrapie agent. Journal of Neurological Sciences 59, 355-369. KIMSERLIN, R. H., COLE, S. & WALKER, C. A. (1987). Pathogenesis of scrapie is faster when infection is intraspinal instead of intracerebral. Microbial Pathogenesis 2, 405-414. KIMBERLIN, R. H., WALKER, C. A. & FRASER, H. (1989). The genomic identity of different strains of mouse scrapie is expressed in hamsters and preserved on reisolation in mice. Journal of General Virology 70, 2017-2025. KINGSBURY, D. T., KASPER, K. C., STITES, D. P., WATSON, J. D., HOGAN, R. N. & PRUSlNER, S. B. (1983). Genetic control of scrapie and Creutzfeldt-Jakob disease in mice. Journal of Immunology 131, 491-496. MCBRIDE, P. A., BRUCE, i . E. & FRASER, H. (1988). Immunostaining of scrapie cerebral amyloid plaques with antisera raised to scrapieassociated fibrils (SAF). Neuropatholology and Applied Neurobiology 14, 325-336. 603 MCKINLEY, M. P., BOLTON, D. C. & PRUSINER, S. B. (1983). A protease-resistant protein is a structural component of the scrapie prion. Cell 35, 57-62. MERZ, P. A., SOMERVILLE, R. A., WISNIEWSKI, H. M. & ]QBAL,K. (1981). Abnormal fibrils from scrapie-infected brain. Acta neuropathologica 54, 63~4. OESCH, B., GROTH, D. F., PRUSINER, S. B. 8~. WEISSMANN, C. (1988). Search for a scrapie-specific nucleic acid : a progress report. In Novel Infectious Agents and the Central Nervous System, Ciba Foundation Symposium 135, pp. 209-223. Edited by G. Bock & J. Marsh. Chichester: John Wiley. OUTRAM,G. W. (1976). The pathogenesis of scrapie in mice. In Slow Virus Diseases of Animals and Man, pp. 325-357. Edited by R. H. Kimberlin. Amsterdam: North-Holland. OWEN, F., POULTER, M., LOFTHOUSE, R., COLLINGE, J., CROW, T. J., RISBY, D., BAKER, H. F., RIDLEY, R. M., HSIAO, K. • PRUSINER, S. B. (1989). Insertion in prion protein gene in familial CreutzfeldtJakob disease. Lancet i, 51-52. PATTISON, I. H. & MILLSON, G. C. (196I). Scrapie produced experimentally in goats with special reference to the clinical syndrome. Journal of Comparative Pathology 71, 101-108~ PRUSINER, S. B. (1982). Novel proteinaceous infectious particles cause scrapie. Science 216, 136-144. RACE, R. E., GRAHAM, K., ERNST, D., CAUGHEY, B. & CHESEBRO, B. (1990). Analysis of linkage between scrapie incubation period and the prion protein gene in mice. Journalof General Virology 71, 493497. ScoTr, M., FOSTER, D., MIRENDA, C., SERBAN, D., COUFAL, F., W~,LCHLI, M., TORCHIA, M., GROTH, D., CARLSON, G., DEARMOND, S. J., WESTAWAY, D. & PRUSINER, S. B. (1989). Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59, 847-857. SOMERVILLE, R. A., MERZ, P. A. & CARP, R. I. (i986). Partial copurification of scrapie-associated fibrils and scrapie infectivity. Intervirology 25, 48-55. WESTAWAY,D., GOODMAN,P. A., MIRENDA, C. A., MCKINLEY, M. P., CARLSON, G. A. & PRUSINER, S. B. (1987). Distinct prion proteins in short and long scrapie incubation period mice. Cell 51, 651662. WILLS, P. R. (1989). Induced frameshifting mechanism of replication for an information-carrying scrapie prion. MicrobialPathogenesis 6, 235-249. (Received 31 August 1990; Accepted 29 November 1990) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 02:10:23