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.

Mouse lines which are congenic for Sinc, the major gene controlling scrapie incubation period, have been produced by selective breeding from the inbred C57BL(Sincs7) and VM(Sincp7) 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 Sincs7 and Sincp7 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.

Age at death from natural scrapie in a flock of Suffolk sheep.

This study deals with natural scrapie in a flock of Suffolk sheep being bred to maximise the incidence of the disease and shows that with succeeding generations the incubation period of the disease became progressively shorter until the flock died out. The most likely explanation for this phenomenon is considered to be an increase in the load of infection.

Linkage of the gene for the scrapie-associated fibril protein (PrP) to the Sip gene in Cheviot sheep.

The gene Sip with two alleles, sA and pA, is the major gene determining the incubation period of scrapie in its natural host, sheep. Two lines of Cheviot sheep have been bred which differ in their response to experimental infection with SSBP/1 scrapie. The negative line have a decreased incidence of disease caused by SSBP/1 and are SippApA. The positive line have an increased incidence of disease and the majority are either SipsAsA or SipsApA; it is not possible to distinguish between the two genotypes on the basis of scrapie incubation time because the sA allele is fully dominant with SSBP/1 scrapie. There are also rare SippApA segregants in the positive line. The major protein (PrP) of scrapie-associated fibrils is encoded by a cellular gene and a cDNA copy of the hamster PrP mRNA has been used to analyse the restriction fragment length polymorphism of the two lines of Cheviot sheep. Two polymorphisms of the sheep PrP gene were found, by using HindIII and EcoRI, which appear to act as markers for the alleles of Sip. Using these polymorphisms it is now possible to assign a Sip genotype to the sheep in the Cheviot flock. Preliminary results from Anglo-Nubian goats and a cow are also reported.

The unusual properties of CH1641, a sheep-passaged isolate of scrapie.

An isolate of scrapie designated CH1641 was identified from a natural case of scrapie in a Cheviot sheep by passage in sheep and goats. It has not been possible to transmit scrapie to mice from this source. The Sip gene which controls the incubation periods of experimental scrapie in Cheviot sheep has two alleles; sA which shortens and pA which lengthens the incubation periods of most strains of scrapie after the first experimental injection in sheep (the A group of strains). The CH1641 isolate differs from them in that the alleles of Sip act in the opposite way, with incubation being shorter in the pA homozygotes. There is some evidence that one or more genes, in addition to Sip, may be implicated in the control of scrapie incubation in sheep and the possibility of a carrier infection with CH1641 is also discussed.

Genetic aspects of unconventional virus infections: the basis of the virino hypothesis.

The properties of genes involved directly or indirectly in the pathogenesis of scrapie and other unconventional (UCV) virus infections are reviewed. Reasons are presented for assigning paramount importance to the Sinc gene in mice and the Sip gene in sheep (the likely homologue of Sinc). The rationale is given for concluding that the agents of UCV infections have their own genomic molecules coding for strain differences. The virino hypothesis, which proposes that the infective form of the agent is an informational hybrid between the agent's genome and protective host proteins, is presented in detail, with an explanation of the postulated role of Sinc.

Linkage of the scrapie-associated fibril protein (PrP) gene and Sinc using congenic mice and restriction fragment length polymorphism analysis.

Sinc, with two alleles p7 and s7, is the major gene determining the incubation period of all strains of scrapie in mice. The major protein (PrP) of scrapie-associated fibrils is encoded by a cellular gene and we have used a cDNA copy of the hamster PrP mRNA to carry out restriction fragment length polymorphism (RFLP) analysis of different inbred mouse strains including VM(Sincp7) and VM(Sincs7) congenic mice. In VM(Sincp7) mice, a 5.5 kb XbaI fragment hybridized to the PrP cDNA sequence whereas VM(Sincs7) congenic mice had a 3.8 kb XbaI fragment. The VM X VM(Sincs7) congenic F1 mice had both the 5.5 kb and the 3.8 kb fragments. The Sincs7 donor mouse strain, C57BL, had the 3.8 kb fragment suggesting that the Sinc gene and the gene coding for PrP are linked, and could even be the same gene. Other Sincp7 inbred mice (IM and MB) had the 5.5 kb fragment but so too did some Sincs7 strains (RIII and VL), implying that the XbaI site polymorphism is not functionally involved in the difference between the two Sinc alleles. We have mapped the polymorphic XbaI site to the 3' flanking region of the PrP gene. TaqI and HhaI were also found to show polymorphisms in the inbred mouse strains studied. The apparent RFLP with HhaI may be a result of differences in methylation rather than in sequence.

Amyloid scrapie plaques in mice, and Alzheimer senile plaques, share common antigens with tau, a microtubule-associated protein.

Immunolabelling was performed on brain sections of scrapie-infected mice with antibodies against tau, a microtubule-associated protein, and against the paired helical filaments of Alzheimer's disease. Both kinds of antibodies have been shown previously to label paired helical filaments in neurons and in abnormal neurites associated with the senile plaques of Alzheimer's disease. Amyloid plaques in murine scrapie were labelled by both antisera. The structural substrate for the immunolabelling was probably the dystrophic neurites in the periphery of the plaques, leaving the amyloid core unstained. These results emphasize that similar mechanisms are likely to be involved in the pathogenesis of plaques in both diseases.

Genetic control of scrapie: incubation period and plaque formation in I mice.

The host component of control of scrapie incubation period in the mouse is manifested largely through the action of the Sinc gene. Only one mouse strain (VM) has been found that is p7p7 (prolonged incubation for ME7 agent) and two other strains have been derived from VM. All other strains, designated s7s7, have a short incubation for ME7. In the present study, the I strain was shown to fulfil the criteria that are characteristic of mouse strains with the p7 allele of Sinc: a comparatively long incubation period for ME7 and a short incubation period for 22A, the incubation period for F1 hybrid mice (s7s7 X p7p7) either fell between the incubation periods for the parental strains (with ME7) or were longer than either parent (with 139A and 22A), amyloid plaques occurred following injection of ME7 and 87V but not after 22A or 139A, lesion profiles for four scrapie strains were similar in I mice and p7p7 mouse strains, and injection of 87V led to disease in less than 300 days. Finally, allelism tests using F1 hybrid mice (I X a p7p7 mouse strain) and progeny of backcrosses between these F1 mice and I mice failed to reveal the segregation of additional major genes affecting scrapie incubation period.

Biological evidence that scrapie agent has an independent genome.

There are many distinct strains of scrapie agent, identified by their relative incubation periods and quantitative and qualitative neuropathological properties in inbred mice of particular genotypes. When serially passaged under specified conditions of mouse strain, route of infection and dose of infectivity these properties are stable. However, they may change in a predictable manner if the passage strategy is altered. The scrapie strain 87A shows what has previously been defined as Class III stability; it is stable when passaged at low dose in C57BL mice, but often suddenly changes its properties in the course of a single passage if high doses are used, always resulting in the same new strain. The latter, designated 7D, has shorter incubation periods and more extensive pathology than 87A, properties which are subsequently stable on serial passage even at high dose. This phenomenon has been seen repeatedly using scrapie isolates from six different natural cases in five different breeds of sheep. These isolates are closely similar in all their properties, showing them to be independent isolations of the 87A strain; there have been no isolations of 87A in which the phenomenon did not occur. On the other hand, none of the many other scrapie strains used in the same laboratory have shown this change. 87A brain samples consistently behave as if they contain 87A together with a smaller amount of 7D. This is so even after 87A has previously been passaged at high dilution, well beyond the limiting dilution for 7D, a procedure which would eliminate any minor agent strain originally present in the isolate. Therefore it is highly likely that the 7D in tissues of mice infected with 87A is generated de novo at each passage by mutational change from 87A during the incubation period. The established fact that many different strains exist and the considerable evidence that mutation can occur lead to the conclusion that scrapie agent has its own independently replicating genome.

Evidence that transmissible mink encephalopathy agent is biologically inactive in mice.

Transmissible mink encephalopathy (TME) is probably a form of the sheep disease, scrapie, introduced by accidentally feeding mink with scrapie-infected sheep tissues. Although no successful transmissions of TME to mice have been achieved previous work has involved various limitations. To maximize the possibility of transmission, 176 mice, representing 14 different genotypes mostly not previously tested with TME, were injected with TME-infected mink brain from three sources with different histories. No scrapie-like disease was detected clinically or histologically in these mice or in a further 111 which were subsequently injected with brain or spleen material from 10 of the TME-injected mice killed when senile. Furthermore, a series of experiments involving seven strains of scrapie, demonstrated that prior injection of mice with TME failed to affect the normal progress of scrapie infection indicating that TME agent had not occupied scrapie replication sites or otherwise influenced the pathogenesis of scrapie. The overall conclusion from these experiments is that TME is biologically inactive in mice. Although many strains of natural scrapie can be transmitted to laboratory mice, this has not been possible with all strains and it is concluded that one or more of such strains is likely to be the cause of TME in mink.

Prolongation of scrapie incubation period by an injection of dextran sulphate 500 within the month before or after infection.

A single intraperitoneal injection of 250 micrograms dextran sulphate 500 (DS500) reduced the susceptibility of mice to scrapie given by the same route. A lower dose (25 micrograms) was less effective but still produced significant incubation period lengthening, while a high dose (2.5 mg) further increased the degree of prolongation. This reduced susceptibility occurred with DS500 administered up to at least 4 weeks prior to intraperitoneal scrapie inoculation and up to at least 2 weeks after scrapie inoculation. A reduced average effect, but more variable between mice, was obtained with DS500 given 1 month or 2 months after scrapie. The effective scrapie titre was reduced by 90% when DS500 was injected either 72 h before or 7 h after ME7 scrapie. Using a relatively lower but normally still fatal dose of the 22A strain of scrapie approximately 50% of the treated mice survived. The effective 90% loss of titre was consistent with either of these strains of scrapie in 11 different inbred strains of mice (BALB/c, BSC, BRVR, C3H, C57BL, IM, LM, MM, RIII, VL and VM). No significant increase in the prolongation effect was obtained using multiple DS500 doses in two different time combinations. DS500 causes long-term interference in both the early processing and the replication of scrapie agent, unlike those immunomodulators which increase susceptibility.

Targeting of scrapie lesions and spread of agent via the retino-tectal projection.

Scrapie infectivity and degenerative vacuolation was initially localized within the contralateral superior colliculus following intraocular injection. The time course of these events was prolonged. With the ME7 strain of scrapie in Sincs7 genotype mice, infectivity began to rise in the superior colliculus from about 70 days, followed by the earliest asymmetrical lesions there from 120 days, with death occurring at about 250 days, at which time vacuolar degeneration was widespread in the brain. With other mouse Sinc genotype mouse/agent strain combinations the process was even further prolonged. With 87V scrapie strain in Sincp7 genotype mice the first lesions to appear were in the contralateral tectum at 300 days. It is concluded that scrapie agent can spread within ganglion cell axons.

Genetic control of amyloid plaque production and incubation period in scrapie-infected mice.

The extent of amyloid plaque production was investigated in three inbred mouse strains carrying the p7 allele of the scrapie incubation (Sinc) gene (VM, IM and MB). With either ME7 or 87V scrapie, many more plaques were seen in the MB strain than in VM or IM mice. A backcrossing experiment using 87V suggested the involvement of more than one gene. Within this backcrossing experiment there was a positive correlation between mean plaque count and mean incubation period for the various strains and crosses. Also male mice tended to have higher plaque counts and longer incubation periods than female mice of the same genotype. These results suggest that some of the genes controlling minor variation in the incubation period also influence plaque production. This is consistent with previous evidence that the number of amyloid plaques depends, to some extent, on the duration of agent replication within the brain. This study has also identified a high plaque model (MB mice infected with 87V) for future investigation of the nature of the amyloid protein.

Disinfection studies with two strains of mouse-passaged scrapie agent. Guidelines for Creutzfeldt-Jakob and related agents.

A variety of disinfection procedures were tested on two strains of scrapie agent, treated either as brain macerates (autoclaving) or as 10% homogenates (chemical treatments). It is suggested that a given treatment should produce a titre loss, of both strains of scrapie, of at least 10(4) units before it be regarded as useful for the disinfection of the agents of scrapie and Creutzfeldt-Jakob disease (CJD). By this criterion, treatment at room temperature with about 4% Hycolin (0.6% chlorinated phenols), 0.2% permanganate, 5% Tego (dodecyl-di(aminoethyl)-glycine) or 5% sodium dodecyl sulphate (SDS) are unsuitable. However, data indicate that SDS might be used to reduce the heat stability of scrapie agent. Hypochlorite (Sterilex) was the only satisfactory chemical reagent tested. At least 10(4)-10(5) units of infectivity were lost by treatment with hypochlorite containing 1,000 ppm available chlorine after a 4-16 h exposure, or containing 10,000 ppm available chlorine after a half-hour exposure. The latter result points to the use of concentrated hypochlorite (about 2% available chlorine; approximately 20% Sterilex) to decontaminate surfaces. We suggest that the cleaning action of SDS, or other strong detergents, might also help to decontaminate surfaces, but studies on this are needed. Autoclaving at 126 degrees C for 1-2 h reduced titres by 10(3)-10(7) units, depending on the strain of agent. However, total disinfection of brain containing high titres of infectivity was approached only at 136 degrees C when titre losses of about 10(6) units were obtained by autoclaving for 4-32 min. Further studies are needed before we can make simple, general recommendations for the disinfection of CJD agents in hospital practice.