A point mutation in GPI-attachment signal peptide accelerates the development of prion disease.

A missense variant from methionine to arginine at codon 232 (M232R) of the prion protein gene accounts for ~ 15% of Japanese patients with genetic prion diseases. However, pathogenic roles of the M232R substitution for the induction of prion disease have remained elusive because family history is usually absent in patients with M232R. In addition, the clinicopathologic phenotypes of patients with M232R are indistinguishable from those of sporadic Creutzfeldt-Jakob disease patients. Furthermore, the M232R substitution is located in the glycosylphosphatidylinositol (GPI)-attachment signal peptide that is cleaved off during the maturation of prion proteins. Therefore, there has been an argument that the M232R substitution might be an uncommon polymorphism rather than a pathogenic mutation. To unveil the role of the M232R substitution in the GPI-attachment signal peptide of prion protein in the pathogenesis of prion disease, here we generated a mouse model expressing human prion proteins with M232R and investigated the susceptibility to prion disease. The M232R substitution accelerates the development of prion disease in a prion strain-dependent manner, without affecting prion strain-specific histopathologic and biochemical features. The M232R substitution did not alter the attachment of GPI nor GPI-attachment site. Instead, the substitution altered endoplasmic reticulum translocation pathway of prion proteins by reducing the hydrophobicity of the GPI-attachment signal peptide, resulting in the reduction of N-linked glycosylation and GPI glycosylation of prion proteins. To the best of our knowledge, this is the first time to show a direct relationship between a point mutation in the GPI-attachment signal peptide and the development of disease.

Potential for transmission of sporadic Creutzfeldt-Jakob disease through peripheral routes.

Five sporadic Creutzfeldt-Jakob disease (CJD) strains have been identified to date, based on differences in clinicopathological features of the patients, the biochemical properties of abnormal prion proteins, and transmission properties. Recent advances in our knowledge about iatrogenic transmission of sporadic CJD have raised the possibility that the infectivity of sporadic CJD strains through peripheral routes is different from that of intracranial infection. To test this possibility, here we assessed systematically the infectivity of sporadic CJD strains through the peripheral route for the first time using a mouse model expressing human prion protein. Although the infectivity of the V2 and M1 sporadic CJD strains is almost the same in intracerebral transmission studies, the V2 strain infected more efficiently than the M1 strain through the peripheral route. The other sporadic CJD strains examined lacked infectivity. Of note, both the V2 and M1 strains showed preference for mice with the valine homozygosity at the PRNP polymorphic codon. These results indicate that the V2 strain is the most infectious sporadic CJD strain for infection through peripheral routes. In addition, these findings raise the possibility that individuals with the valine homozygosity at the PRNP polymorphic codon might have higher risks of infection through peripheral routes compared with the methionine homozygotes. Thus, preventive measures against the transmission of the V2 sporadic CJD strain will be important for the eradication of iatrogenic CJD transmission through peripheral routes.

Eliminating transmissibility of bovine spongiform encephalopathy by dry-heat treatment.

Bovine spongiform encephalopathy (BSE) prion is more resistant to heat inactivation compared to other prions, but the effect of heat inactivation has been reported to differ depending on the BSE-contaminated tissue state or heating type. We aimed to evaluate the secure level of inactivation of original BSE transmissibility by dry-heating. Cattle tissues affected with BSE were subjected to dry-heat treatment for 20 min at various temperatures ranging from 150 to 1000 °C. To assess the inactivation effect, we conducted protein misfolding cyclic amplification (PMCA) and follicular dendritic cell (FDC) assays in transgenic mice expressing bovine prion protein genes. Under dry-heating at 600 °C or higher, BSE cattle tissues lost their transmissibility in transgenic mice. In contrast, transmissibility was detected in the cattle tissues treated at temperatures of 400 °C or lower through the FDC assay combined with PMCA. In this study, we confirmed that transmissibility was eliminated in BSE-affected cattle tissues by dry-heating at 600 °C or higher.

Ganglioside Synthase Knockout Reduces Prion Disease Incubation Time in Mouse Models.

Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.

A Novel Combination of Prion Strain Co-Occurrence in Patients with Sporadic Creutzfeldt-Jakob Disease.

Six subgroups of sporadic Creutzfeldt-Jakob disease have been identified by distinctive clinicopathologic features, genotype at polymorphic codon 129 [methionine (M)/valine (V)] of the PRNP gene, and type of abnormal prion proteins (type 1 or 2). In addition to the pure subgroups, mixed neuropathologic features and the coexistence of two types of abnormal prion proteins in the same patient also have been reported. Here, we found that a portion of the patients previously diagnosed as MM1 had neuropathologic characteristics of the MM2 thalamic form (ie, neuronal loss of the inferior olivary nucleus of the medulla). Furthermore, coexistence of biochemical features of the MM2 thalamic form also was confirmed in the identified cases. In addition, in transmission experiments using prion protein-humanized mice, the brain material from the identified case showed weak infectivity and generated characteristic abnormal prion proteins in the inoculated mice resembling those after inoculation with brain material of MM2 thalamic form. Taken together, these results show that the co-occurrence of MM1 and MM2 thalamic form is a novel entity of sporadic Creutzfeldt-Jakob disease prion strain co-occurrence. The present study raises the possibility that the co-occurrence of the MM2 thalamic form might have been overlooked so far because of the scarcity of abnormal prion protein accumulation and restricted neuropathology.

Protective Effect of Val129-PrP against Bovine Spongiform Encephalopathy but not Variant Creutzfeldt-Jakob Disease.

Bovine spongiform encephalopathy (BSE) is the only known zoonotic prion that causes variant Creutzfeldt-Jakob disease (vCJD) in humans. The major risk determinant for this disease is the polymorphic codon 129 of the human prion protein (Hu-PrP), where either methionine (Met129) or valine (Val129) can be encoded. To date, all clinical and neuropathologically confirmed vCJD cases have been Met129 homozygous, with the exception of 1 recently reported Met/Val heterozygous case. Here, we found that transgenic mice homozygous for Val129 Hu-PrP show severely restricted propagation of the BSE prion strain, but this constraint can be partially overcome by adaptation of the BSE agent to the Met129 Hu-PrP. In addition, the transmission of vCJD to transgenic mice homozygous for Val129 Hu-PrP resulted in a prion with distinct strain features. These observations may indicate increased risk for vCJD secondary transmission in Val129 Hu-PrP-positive humans with the emergence of new strain features.

Multiple affinity purification of a baculovirus-derived recombinant prion protein with in vitro ability to convert to its pathogenic form.

We previously showed that baculovirus-derived recombinant prion protein (Bac-PrP) can be converted to the misfolded infectious form (PrPSc) by protein misfolding cyclic amplification, an in vitro conversion technique. Bac-PrP, with post-translational modifications, would be useful for various applications such as using PrP as an immunogen for generating anti-PrP antibody, developing anti-prion drugs or diagnostic assays using in vitro conversion systems, and establishing an in vitro prion propagation model. For this purpose, highly purified Bac-PrP with in vitro conversion activity is necessary for use as a PrPC source, to minimize contamination. Furthermore, an exogenous affinity tag-free form is desirable to avoid potential steric interference by the affinity tags during the conversion process. In this study, we established purification methods for the untagged Bac-PrP under native conditions by combining exogenous double-affinity tags, namely, a polyhistidine-tag and a profinity eXact tag, with an octarepeat sequence of the N-terminal region of PrP, which has metal ion-binding affinity. The untagged Bac-PrP with near-homogeneity was obtained by three-step affinity purification, and it was shown that the final, purified Bac-PrP could convert to its pathogenic form. The presented purification procedure could be applied not only to PrP but also to other eukaryotic, recombinant proteins that require high purity and intact physiological activity.

Transmission properties of atypical Creutzfeldt-Jakob disease: a clue to disease etiology?

UNLABELLED: The genotype at polymorphic codon 129 of the PRNP gene has a profound influence on both phenotypic expression and prion strain susceptibility in humans. For example, while the most common sporadic Creutzfeldt-Jakob disease (CJD) subtype, sporadic CJD-MM1 (M1 strain), induces a single phenotype after experimental transmission regardless of the codon 129 genotype of the recipient animal, the phenotype elicited by sporadic CJD-VV2 (V2 strain), the second most common subtype, varies according to the host codon 129 genotype. In particular, the propagation of the V2 strain in codon 129 methionine homozygotes has been linked only to acquired forms of CJD such as plaque-type dura mater graft-associated CJD (dCJD), a subgroup of iatrogenic CJD with distinctive phenotypic features, but has never been observed in sporadic CJD cases. In the present report, we describe atypical CJD cases carrying codon 129 methionine homozygosity, in a neurosurgeon and in a patient with a medical history of neurosurgery without dural grafting, showing the distinctive phenotypic features and transmission properties of plaque-type dCJD. These findings raise the possibility that the two cases, previously thought to represent sporadic CJD, might actually represent acquired CJD caused by infection with the V2 strain. Thus, careful analyses of phenotypic features and transmission properties in atypical cases may be useful to distinguish acquired from sporadic cases of CJD. IMPORTANCE: Susceptibility to and phenotypic expression of Creutzfeldt-Jakob disease (CJD) depend on both the prion strain and genotype at polymorphic codon 129 of the PRNP gene. For example, propagation of the second most common sporadic CJD strain (V2 strain) into codon 129 methionine homozygotes has been linked to plaque-type dura mater graft-associated CJD (dCJD), a subgroup of iatrogenic CJD with distinctive phenotypic features, but has never been observed in sporadic CJD. In the present report, we describe atypical CJD cases in a neurosurgeon and in a patient with a medical history of neurosurgery without dural grafting, showing the distinctive phenotypic features and transmission properties of plaque-type dCJD. These findings raise the possibility that the two cases, previously considered to represent sporadic CJD, might actually represent acquired CJD caused by infection with the V2 strain.

Neuropathological and biochemical criteria to identify acquired Creutzfeldt-Jakob disease among presumed sporadic cases.

As an experimental model of acquired Creutzfeldt-Jakob disease (CJD), we performed transmission studies of sporadic CJD using knock-in mice expressing human prion protein (PrP). In this model, the inoculation of the sporadic CJD strain V2 into animals homozygous for methionine at polymorphic codon 129 (129 M/M) of the PRNP gene produced quite distinctive neuropathological and biochemical features, that is, widespread kuru plaques and intermediate type abnormal PrP (PrP(Sc) ). Interestingly, this distinctive combination of molecular and pathological features has been, to date, observed in acquired CJD but not in sporadic CJD. Assuming that these distinctive phenotypic traits are specific for acquired CJD, we revisited the literature and found two cases showing widespread kuru plaques despite the 129 M/M genotype, in a neurosurgeon and in a patient with a medical history of neurosurgery without dura mater grafting. By Western blot analysis of brain homogenates, we revealed the intermediate type of PrP(Sc) in both cases. Furthermore, transmission properties of brain extracts from these two cases were indistinguishable from those of a subgroup of dura mater graft-associated iatrogenic CJD caused by infection with the sporadic CJD strain V2. These data strongly suggest that the two atypical CJD cases, previously thought to represent sporadic CJD, very likely acquired the disease through exposure to prion-contaminated brain tissues. Thus, we propose that the distinctive combination of 129 M/M genotype, kuru plaques, and intermediate type PrP(Sc) , represents a reliable criterion for the identification of acquired CJD cases among presumed sporadic cases.

The influence of PRNP polymorphisms on human prion disease susceptibility: an update.

Two normally occurring polymorphisms of the human PRNP gene, methionine (M)/valine (V) at codon 129 and glutamic acid (E)/lysine (K) at codon 219, can affect the susceptibility to prion diseases. It has long been recognized that 129M/M homozygotes are overrepresented in sporadic Creutzfeldt-Jakob disease (CJD) patients and variant CJD patients, whereas 219E/K heterozygotes are absent in sporadic CJD patients. In addition to these pioneering findings, recent progress in experimental transmission studies and worldwide surveillance of prion diseases have identified novel relationships between the PRNP polymorphisms and the prion disease susceptibility. For example, although 219E/K heterozygosity confers resistance against the development of sporadic CJD, this genotype is not entirely protective against acquired forms (iatrogenic CJD and variant CJD) or genetic forms (genetic CJD and Gerstmann-Sträussler-Scheinker syndrome) of prion diseases. In addition, 129M/V heterozygotes predispose to genetic CJD caused by a pathogenic PRNP mutation at codon 180. These findings show that the effects of the PRNP polymorphisms may be more complicated than previously thought. This review aims to summarize recent advances in our knowledge about the influence of the PRNP polymorphisms on the prion disease susceptibility.

Characterization of variant Creutzfeldt-Jakob disease prions in prion protein-humanized mice carrying distinct codon 129 genotypes.

To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.

Prion replication elicits cytopathic changes in differentiated neurosphere cultures.

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.

Quantitative analysis of wet-heat inactivation in bovine spongiform encephalopathy.

The bovine spongiform encephalopathy (BSE) agent is resistant to conventional microbial inactivation procedures and thus threatens the safety of cattle products and by-products. To obtain information necessary to assess BSE inactivation, we performed quantitative analysis of wet-heat inactivation of infectivity in BSE-infected cattle spinal cords. Using a highly sensitive bioassay, we found that infectivity in BSE cattle macerates fell with increase in temperatures from 133°C to 150°C and was not detected in the samples subjected to temperatures above 155°C. In dry cattle tissues, infectivity was detected even at 170°C. Thus, BSE infectivity reduces with increase in wet-heat temperatures but is less affected when tissues are dehydrated prior to the wet-heat treatment. The results of the quantitative protein misfolding cyclic amplification assay also demonstrated that the level of the protease-resistant prion protein fell below the bioassay detection limit by wet-heat at 155°C and higher and could help assess BSE inactivation. Our results show that BSE infectivity is strongly resistant to wet-heat inactivation and that it is necessary to pay attention to BSE decontamination in recycled cattle by-products.

Rapid assessment of bovine spongiform encephalopathy prion inactivation by heat treatment in yellow grease produced in the industrial manufacturing process of meat and bone meals.

BACKGROUND: Prions, infectious agents associated with transmissible spongiform encephalopathy, are primarily composed of the misfolded and pathogenic form (PrPSc) of the host-encoded prion protein. Because PrPSc retains infectivity after undergoing routine sterilizing processes, the cause of bovine spongiform encephalopathy (BSE) outbreaks are suspected to be feeding cattle meat and bone meals (MBMs) contaminated with the prion. To assess the validity of prion inactivation by heat treatment in yellow grease, which is produced in the industrial manufacturing process of MBMs, we pooled, homogenized, and heat treated the spinal cords of BSE-infected cows under various experimental conditions. RESULTS: Prion inactivation was analyzed quantitatively in terms of the infectivity and PrPSc of the treated samples. Following treatment at 140°C for 1 h, infectivity was reduced to 1/35 of that of the untreated samples. Treatment at 180°C for 3 h was required to reduce infectivity. However, PrPSc was detected in all heat-treated samples by using the protein misfolding cyclic amplification (PMCA) technique, which amplifies PrPScin vitro. Quantitative analysis of the inactivation efficiency of BSE PrPSc was possible with the introduction of the PMCA50, which is the dilution ratio of 10% homogenate needed to yield 50% positivity for PrPSc in amplified samples. CONCLUSIONS: Log PMCA50 exhibited a strong linear correlation with the transmission rate in the bioassay; infectivity was no longer detected when the log PMCA50 of the inoculated sample was reduced to 1.75. The quantitative PMCA assay may be useful for safety evaluation for recycling and effective utilization of MBMs as an organic resource.

Glycosylphosphatidylinositol anchor-dependent stimulation pathway required for generation of baculovirus-derived recombinant scrapie prion protein.

The pathogenic isoform (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP(Sc) seed catalyzes the structural conversion of endogenous PrP(C) into nascent PrP(Sc) in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP(res)) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP(Sc) by PMCA. However, the biological properties of PrP(Sc) obtained by in vivo transmission of recPrP(res) are unique or different from those of PrP(Sc) used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP(Sc). In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP(C) substrate for amplification of the mouse scrapie prion strain Chandler, and PrP(Sc) that accumulated in mice inoculated with Bac-PrP(res) had biochemical and pathological properties very similar to those of the PrP(Sc) seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP(res), the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP(Sc). Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.

Cytochalasin D enhances the accumulation of a protease-resistant form of prion protein in ScN2a cells: involvement of PI3 kinase/Akt signalling pathway.

The conversion of a host-encoded PrPsen (protease-sensitive cellular prion protein) into a PrPres (protease-resistant pathogenic form) is a key process in the pathogenesis of prion diseases, but the intracellular mechanisms underlying PrPres amplification in prion-infected cells remain elusive. To assess the role of cytoskeletal proteins in the regulation of PrPres amplification, the effects of cytoskeletal disruptors on PrPres accumulation in ScN2a cells that were persistently infected with the scrapie Chandler strain have been examined. Actin microfilament disruption with cytochalasin D enhanced PrPres accumulation in ScN2a cells. In contrast, the microtubule-disrupting agents, colchicine, nocodazole and paclitaxel, had no effect on PrPres accumulation. In addition, a PI3K (phosphoinositide 3-kinase) inhibitor, wortmannin and an Akt kinase inhibitor prevented the cytochalasin D-induced enhancement of PrPres accumulation. Cytochalasin D-induced extension of neurite-like processes might correlate with enhanced accumulation of PrPres. The results suggest that the actin cytoskeleton and PI3K/Akt pathway are involved in the regulation of PrPres accumulation in prion-infected cells.

Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification.

Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.

Experimental verification of a traceback phenomenon in prion infection.

The clinicopathological phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD) correlate with the allelotypes (M or V) of the polymorphic codon 129 of the human prion protein (PrP) gene and the electrophoretic mobility patterns of abnormal prion protein (PrP(Sc)). Transmission of sCJD prions to mice expressing human PrP with a heterologous genotype (referred to as cross-sequence transmission) results in prolonged incubation periods. We previously reported that cross-sequence transmission can generate a new prion strain with unique transmissibility, designated a traceback phenomenon. To verify experimentally the traceback of sCJD-VV2 prions, we inoculated sCJD-VV2 prions into mice expressing human PrP with the 129M/M genotype. These 129M/M mice showed altered neuropathology and a novel PrP(Sc) type after a long incubation period. We then passaged the brain homogenate from the 129M/M mouse inoculated with sCJD-VV2 prions into other 129M/M or 129V/V mice. Despite cross-sequence transmission, 129V/V mice were highly susceptible to these prions compared to the 129M/M mice. The neuropathology and PrP(Sc) type of the 129V/V mice inoculated with the 129M/M mouse-passaged sCJD-VV2 prions were identical to those of the 129V/V mice inoculated with sCJD-VV2 prions. Moreover, we generated for the first time a type 2 PrP(Sc)-specific antibody in addition to type 1 PrP(Sc)-specific antibody and discovered that drastic changes in the PrP(Sc) subpopulation underlie the traceback phenomenon. Here, we report the first direct evidence of the traceback in prion infection.

Experimental H-type bovine spongiform encephalopathy characterized by plaques and glial- and stellate-type prion protein deposits.

Atypical bovine spongiform encephalopathy (BSE) has recently been identified in Europe, North America, and Japan. It is classified as H-type and L-type BSE according to the molecular mass of the disease-associated prion protein (Pr(PSc)). To investigate the topographical distribution and deposition patterns of immunolabeled Pr(PSc), H-type BSE isolate was inoculated intracerebrally into cattle. H-type BSE was successfully transmitted to 3 calves, with incubation periods between 500 and 600 days. Moderate to severe spongiform changes were detected in the cerebral and cerebellar cortices, basal ganglia, thalamus, and brainstem. H-type BSE was characterized by the presence of PrP-immunopositive amyloid plaques in the white matter of the cerebrum, basal ganglia, and thalamus. Moreover, intraglial-type immunolabeled Pr(PSc) was prominent throughout the brain. Stellate-type immunolabeled Pr(PSc) was conspicuous in the gray matter of the cerebral cortex, basal ganglia, and thalamus, but not in the brainstem. In addition, Pr(PSc) accumulation was detected in the peripheral nervous tissues, such as trigeminal ganglia, dorsal root ganglia, optic nerve, retina, and neurohypophysis. Cattle are susceptible to H-type BSE with a shorter incubation period, showing distinct and distinguishable phenotypes of Pr(PSc) accumulation.