Kentaro Masujin

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrPSc, a protease-resistant misfolded isoform of the cellular prion protein PrPC. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrPSc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrPSc in BSE-affected cattle therefore remains unknown. Methodology/Principal Findings We report here that PrPSc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrPSc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrPSc in blood was not substantiated in the BSE-affected cattle examined. Conclusions/Significance The distribution of PrPSc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrPSc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.

Efficient in vitro amplification of a mouse-adapted scrapie prion protein

Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie prion protein (PrP(Sc)), a major protein component of the infectious agents associated with prion diseases. Although exponential in vitro amplification of hamster scrapie PrP(Sc) has been established, the PMCA used was unsuccessful in achieving good amplification of PrP(Sc) from other animals. Here, we have investigated the cause of the insufficient PrP(Sc) amplification in mice and have developed an improved method suitable for amplification of the PrP(Sc) of the mouse-adapted scrapie prion strain Chandler. Mouse PrP(C), the cellular form of the prion protein, tends to become resistant to proteases during incubation independent of sonication. By adding digitonin to the reaction buffer as a lipid detergent, accumulation of the protease-resistant PrP(C) was inhibited; hence, mouse PrP(Sc) could be amplified to infinite levels. The present study is the first report describing effective amplification of PrP(Sc) of the mouse-adapted scrapie prion and this improved PMCA technique will contribute to prion research that uses mice as experimental animals.

Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle

A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 ± 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 ± 13.5 days). Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion inoculated mice showed no clinical signs up to 649 days. Severe vacuolation and a widespread and uniform distribution of PrPSc were pathologically observed in the brain of BSE/JP24 prion affected TgBoPrP mice. The molecular weight and glycoform ratio of PrPSc in BSE/JP24 were different from those in C-BSE, and PrPSc in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE. These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties reported for that of C-BSE. Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 ± 3.1 days). This result implies that BSE/JP24 prion has newly emerged and showed the possibility that L-type BSE prion might be classified into multiple strains.

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 (PrPSc). To investigate the topographical distribution and deposition patterns of immunolabeled PrPSc, 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 PrPSc was prominent throughout the brain. Stellate-type immunolabeled PrPSc was conspicuous in the gray matter of the cerebral cortex, basal ganglia, and thalamus, but not in the brainstem. In addition, PrPSc 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 PrPSc accumulation.

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

ABSTRACT Prion diseases of cattle include the classical bovine spongiform encephalopathy (C-BSE) and the atypical H-type BSE (H-BSE) and L-type BSE (L-BSE) strains. Although the C- and L-BSE strains can be detected and discriminated by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays, no such test has yet been described for the detection of H-BSE or the discrimination of each of the major bovine prion strains. Here, we demonstrate an RT-QuIC assay for H-BSE that can detect as little as 10−9 dilutions of brain tissue and neat cerebrospinal fluid samples from clinically affected cattle. Moreover, comparisons of the reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K-treated RT-QuIC reaction products indicated that H-, L-, and C-BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis.

Intraspecies Prion Transmission Results in Selection of Sheep Scrapie Strains

Background Sheep scrapie is caused by multiple prion strains, which have been classified on the basis of their biological characteristics in inbred mice. The heterogeneity of natural scrapie prions in individual sheep and in sheep flocks has not been clearly defined. Methodology/Principal Findings In this study, we intravenously injected 2 sheep (Suffolk and Corriedale) with material from a natural case of sheep scrapie (Suffolk breed). These 3 sheep had identical prion protein (PrP) genotypes. The protease-resistant core of PrP (PrPres) in the experimental Suffolk sheep was similar to that in the original Suffolk sheep. In contrast, PrPres in the Corriedale sheep differed from the original PrPres but resembled the unusual scrapie isolate, CH1641. This unusual PrPres was not detected in the original sheep. The PrPres distributions in the brain and peripheral tissues differed between the 2 breeds of challenged sheep. A transmission study in wild-type and TgBoPrP mice, which overexpressing bovine PrP, led to the selection of different prion strains. The pathological features of prion diseases are thought to depend on the dominantly propagated strain. Conclusions/Significance Our results indicate that prion strain selection occurs after both inter- and intraspecies transmission. The unusual scrapie prion was a hidden or an unexpressed component in typical sheep scrapie.

Tracing Conformational Transition of Abnormal Prion Proteins during Interspecies Transmission by Using Novel Antibodies

Conformational differences in abnormal prion proteins (PrPSc) have been postulated to produce different prion phenotypes. During the interspecies transmission of prions, the conformation of PrPSc may change with passage; however, little is known about the mechanism of PrPSc transition. In this study, novel PrPSc-specific monoclonal antibodies (mAbs) were developed that could detect the PrPSc of mouse but not that of sheep. By using these mAbs, we attempted to examine PrPSc accumulated in mice inoculated with sheep scrapie serially up to five passages. The presence of PrPSc in the mice was confirmed at all passages; however, mAb-bound PrPSc conformer was detected only from the third passage onward. The generated mAb enabled tracing of a particular conformer during adaptation in sheep-to-mice transmission of prion, suggesting that the conformational transition of PrPSc was caused by propagation of this conformer. Such mAbs capable of discriminating conformational differences may allow us to address questions concerning PrPSc conformation and strain diversity.

Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues.

Experimental Transmission of H-Type Bovine Spongiform Encephalopathy to Bovinized Transgenic Mice

To characterize the biological and biochemical properties of H-type bovine spongiform encephalopathy (BSE), a transmission study with a Canadian H-type isolate was performed with bovinized transgenic mice (TgBoPrP), which were inoculated intracerebrally with brain homogenate from cattle with H-type BSE. All mice exhibited characteristic neurologic signs, and the subsequent passage showed a shortened incubation period. The distribution of disease-associated prion protein (PrPSc) was determined by immunohistochemistry, Western blot, and paraffin-embedded tissue (PET) blot. Biochemical properties and higher molecular weight of the glycoform pattern were well conserved within mice. Immunolabeled granular PrPSc, aggregates, and/or plaque-like deposits were mainly detected in the following brain locations: septal nuclei, subcallosal regions, hypothalamus, paraventricular nucleus of the thalamus, interstitial nucleus of the stria terminalis, and the reticular formation of the midbrain. Weak reactivity was detected by immunohistochemistry and PET blot in the cerebral cortex, most thalamic nuclei, the hippocampus, medulla oblongata, and cerebellum. These findings indicate that the H-type BSE prion has biological and biochemical properties distinct from those of C-type and L-type BSE in TgBoPrP mice, which suggests that TgBoPrP mice constitute a useful animal model to distinguish isolates from BSE-infected cattle.

Ultrasensitive detection of PrP(Sc) in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrP(Sc)) in the central nervous system. The pathological features and biochemical properties of PrP(Sc) in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt-Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrP(Sc). This method involves amplifying PrP(Sc) by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrP(C) substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrP(Sc) in the CSF by serial PMCA, and the CSF levels of PrP(Sc) tended to increase with disease progression. In addition, PrP(Sc) was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrP(Sc) in non-human primate models of CJD.