Irene McConnell

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.

Detection of variant Creutzfeldt-Jakob disease infectivity in extraneural tissues

Abnormal accumulations of prion protein (PrP) can be detected in the spleen, lymph nodes, and tonsils of patients with variant Creutzfeldt-Jakob disease (vCJD). Therefore, it has been assumed, but not shown, that these tissues harbour infectivity, which in turn presents the potential for iatrogenic spread through surgery. Here, we show and measure levels of infectivity in spleen and tonsil from two patients with vCJD, by bioassay in intracerebrally inoculated RIII mice. Similar bioassays failed to detect infectivity in buffy coat and plasma.

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann–Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild‐type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days. Disease was transmitted from these mice to both wild‐type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild‐type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease‐associated PrP.

Strain characterization of natural sheep scrapie and comparison with BSE.

Scrapie was transmitted to mice from ten sheep, collected in the UK between 1985 and 1994. As in previous natural scrapie transmissions, the results varied between scrapie sources in terms of the incidence of disease, incubation periods and neuropathology in challenged mice. This contrasted with the uniformity seen in transmissions of BSE to mice. The scrapie and BSE isolates were characterized further by serial passage in mice. Different TSE strains were isolated from each source according to the Sinc or PrP genotype of the mouse used for passage. The same two mouse-passaged strains, 301C and 301V, were isolated from each of three BSE sources. Despite the variation seen in the primary transmissions of scrapie, relatively few mouse-passaged scrapie strains were isolated and these were distinct from the BSE-derived strains. The ME7 scrapie strain, which has often been isolated from independent sheep sources in the past, was identified in isolates from four of the sheep. However, a new distinct strain, 221C, was derived from a further four scrapie sheep. These results suggest that there is agent strain variation in natural scrapie in sheep and that the spectrum of strains present may have changed over the last 20 years. The tested sample is too small to come to any conclusions about whether the BSE strain is present in sheep, but the study provides a framework for further more extensive studies.

Transmission of bovine spongiform encephalopathy and scrapie to mice

Transmission from four cases of bovine spongiform encephalopathy (BSE) to mice resulted in neurological disease in 100% of recipient animals, after incubation periods of between 265 and 700 days post-injection. The results from the four cases were very similar to one another. There were major differences in the incubation period between the four inbred strains of mice tested, and even between strains of the same Sinc genotype, and the incubation periods of Sinc heterozygote mice were much longer than those for any of the inbred strains. Transmission from a case of natural scrapie differed in two important respects: there were no differences in the incubation period between mouse strains of the same Sinc genotype, and that of the heterozygotes was between those of the Sinc homozygotic parental strains. The distribution of vacuolar degeneration in the brains of mice infected with scrapie also differed from those infected with the BSE isolates. Transmission was also achieved from formol-fixed BSE brain. These results show that the same strain of agent caused disease in the BSE cases, and that the relationship of BSE to scrapie in sheep is unclear.

Mice with gene targetted prion protein alterations show that Prnp, Sinc and Prni are congruent.

Classical genetic analysis has identified Sinc/Prni as the major gene controlling mouse scrapie incubation time. Sinc/Prni is linked to Prnp, the gene encoding the prion protein (PrP). Prnp alleles express distinct PrP protein variants, PrP A and PrP B, which arise from codon 108L/F and 189 T/V dimorphisms. Prnp genotype segregates with incubation time length which suggests, but does not prove, that incubation time is controlled by PrP dimorphisms, and that the Sinc/Prni and Prnp loci are congruent. We have used gene targetting to construct mice in which the endogenous Prnp allele has been modified to express PrP B instead of PrP A. Challenge with a mouse-adapted BSE strain results in dramatically shortened incubation times and demonstrates that PrP dimorphisms at codon 108 and/or 189 control incubation time, and that Sinc/Prni and Prnp are congruent.

Follicular Dendritic Cell Dedifferentiation by Treatment with an Inhibitor of the Lymphotoxin Pathway Dramatically Reduces Scrapie Susceptibility

ABSTRACT Transmissible spongiform encephalopathies (TSEs) may be acquired peripherally, in which case infectivity usually accumulates in lymphoid tissues before dissemination to the nervous system. Studies of mouse scrapie models have shown that mature follicular dendritic cells (FDCs), expressing the host prion protein (PrPc), are critical for replication of infection in lymphoid tissues and subsequent neuroinvasion. Since FDCs require lymphotoxin signals from B lymphocytes to maintain their differentiated state, blockade of this stimulation with a lymphotoxin β receptor-immunoglobulin fusion protein (LTβR-Ig) leads to their temporary dedifferentiation. Here, a single treatment with LTβR-Ig before intraperitoneal scrapie inoculation blocked the early accumulation of infectivity and disease-specific PrP (PrPSc) within the spleen and substantially reduced disease susceptibility. These effects coincided with an absence of FDCs in the spleen for ca. 28 days after treatment. Although the period of FDC dedifferentiation was extended to at least 49 days by consecutive LTβR-Ig treatments, this had little added protective benefit after injection with a moderate dose of scrapie. We also demonstrate that mature FDCs are critical for the transmission of scrapie from the gastrointestinal tract. Treatment with LTβR-Ig before oral scrapie inoculation blocked PrPSc accumulation in Peyers patches and mesenteric lymph nodes and prevented neuroinvasion. However, treatment 14 days after oral inoculation did not affect survival time or susceptibility, suggesting that infectivity may have already spread to the peripheral nervous system. Although manipulation of FDCs may offer a potential approach for early intervention in peripherally acquired TSEs, these data suggest that the duration of the treatment window may vary widely depending on the route of exposure.

Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice

The incubation period (IP) and the neuropathology of transmissible spongiform encephalopathies (TSEs) have been extensively used to distinguish prion isolates (or strains) inoculated into panels of inbred mouse strains. Such studies have shown that the bovine spongiform encephalopathy (BSE) agent is indistinguishable from the agent causing variant Creutzfeldt–Jakob disease (vCJD), but differs from isolates of sporadic CJD, reinforcing the idea that the vCJD epidemic in Britain results from consumption of contaminated beef products. We present a mouse model for genetic and environmental factors that modify the incubation period of BSE cross-species transmission. We have used two mouse strains that carry the same prion protein (PrP) allele, but display a 100-day difference in their mean IP following intracerebral inoculation with primary BSE isolate. We report genetic effects on IP that map to four chromosomal regions, and in addition we find significant factors of host environment, namely the age of the hosts mother, the age of the host at infection, and an X-cytoplasm interaction in the host.

Scrapie infection can be established readily through skin scarification in immunocompetent but not immunodeficient mice

Scarification of the skin is a possible route of entry for scrapie infectivity in sheep, and for Creutzfeldt-Jakob disease agent in humans within the context of occupational exposure to infected brain in the autopsy room or laboratory. The effectiveness of skin scarification routes as portals of entry for infectivity had not previously been tested experimentally but this study has shown that these are efficient routes for establishing infection in mice using the 139A and ME7 strains of scrapie agent. Scarification had much the same efficiency as inoculation by the intraperitoneal, intravenous or perivenous routes but was not effective in immunocompromised (SCID) mice. It was concluded that replication of infectivity within the lymphoreticular system, which is precluded in SCID mice, is a necessary prerequisite for the development of infection in the central nervous system following inoculation via scarification.

Identification of a putative cellular receptor for the lentivirus visna virus

One mechanism by which viral tropism may be controlled is by the expression of a specific virus receptor on the cell surface. This paper reports the identification of a putative cellular receptor for visna virus, the prototype virus of the family Lentiviridae. Using a virus overlay protein blot assay we identified a group of polypeptides of apparent Mr 30K to 33K which interacts with visna virus and is present on permissive but not non-permissive cells. A rat polyclonal anti-ovine major histocompatibility complex (MHC) class II antigen (Ag) serum raised to immunopurified MHC class II Ag, but not preimmune serum, blocked the interaction of visna virus with these polypeptides. In an ELISA, immunopurified MHC class II Ag bound to visna virus but not to bovine parainfluenza 3 virus. Preincubation of visna virus with immunopurified soluble MHC class II Ag resulted in a marked decrease in virus-induced syncytium formation, i.e. preincubation with class II Ag inhibited infection with visna virus, but we have been unable to inhibit infection using class II Ag-specific antisera. These results suggest that ovine MHC class II Ag acts as a component of a cellular receptor for visna virus. This is of particular interest owing to the close similarities between visna virus and human immunodeficiency virus (HIV), and the relationship between MHC class II and CD4, the cellular receptor for HIV. It is also of relevance to recent reports that a growing number of viruses utilize polypeptides of the Ig supergene family as receptors.