Ceramic absorbed with calcium bicarbonate mesoscopic crystals partially inactivate scrapie prions.

Prion diseases are fatal neurodegenerative disorders affecting both humans and animals. The causative agent, prion, is extremely resistant to common disinfection procedures. Thus, effective prion inactivation strategies using relatively safe and less corrosive disinfectants are required. A solution containing CAC-717, mesoscopic crystals of calcium bicarbonate, exerts both antimicrobial and virucidal activities without apparent harmful effects. This study demonstrated that combined treatment with CAC-717 absorbed on ceramic (CAC-717 ceramic) and sodium dodecyl sulfate (SDS) substantially reduced the protein misfolding cyclic amplification (PMCA) seeding activity of Chandler strain scrapie mouse-brain homogenates (ScBH). Additionally, bioassays demonstrated that ScBH-inoculated mice treated with CAC-717 ceramic in combination with sodium dodecyl sulfate (SDS) did not develop disease. Furthermore, this combination effectively inactivated PMCA seeding activity on ScBH-coated stainless-steel wires below the detection limit. Overall, the findings suggest that combined treatment with CAC-717 ceramic and SDS represents a promising and less damaging approach for prion inactivation.

Complete Genome Sequence of a Genotype 3 Atypical Porcine Pestivirus Strain (OKN/2021) from Okinawa Prefecture, Japan.

We report the complete genome sequence of atypical porcine pestivirus (APPV) OKN/2021, which was sampled in the Okinawa Prefecture, Japan. The sequence bears the closest resemblance to another previously detected Japanese genotype 3 APPV sequence. This genome sequencing will help researchers in Japan learn more about the virus epidemiology and evolutionary characteristics.

Calcium bicarbonate as an antimicrobial, antiviral, and prion-inhibiting agent (Review).

Calcium bicarbonate does not act as a disinfectant at neutral pH; however, it exerts strong antimicrobial activity after it is placed in a high-voltage electric field, whereby it assumes an alkaline pH (12.4). Moreover, the microbicidal activity of the resulting solution (named CAC-717) is not influenced by the presence of organic material or resistance of the agent to inactivation. When sprayed on the skin surface, the pH of CAC-717 decreases rapidly to 8.84. CAC-717 comprises fine particles of 50-500 nm. When these mesoscopic crystals are dissolved in water, they destroy the genomes of bacteria or viruses and neutralize the infectious properties of abnormal prion proteins produced in ScN2a cells. The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic has resulted in unprecedented international demand for disinfectants. A small titer of SARS-CoV-2 remains infectious even after 30 sec in growth medium at pH 12.4. CAC-717 has exhibited a strong virucidal effect (3.6 to 4.4 log10 decrease) against all examined SARS-CoV-2 isolates, including mutant forms. Similarly, human noroviruses also remain intact at pH 12.4; however, CAC-717 has been shown to cause a 3.25 log10 reduction in norovirus genomic RNA compared to untreated samples. Existing evidence suggests that an unidentified mechanism controls the virucidal activity of CAC-717.

Spontaneous generation of distinct prion variants with recombinant prion protein from a baculovirus-insect cell expression system.

Prion diseases are transmissible and progressive neurodegenerative disorders characterized by abnormal prion protein (PrPSc) accumulation in the central nervous system. Generation of synthetic PrPSc in a cell-free conversion system and examination of its transmissibility to animals would facilitate testing of the protein-only hypothesis and the understanding of the molecular basis of sporadic prion diseases. In this study, we used recombinant prion protein from a baculovirus-insect cell expression system (Bac-rPrP) and insect cell-derived cofactors to determine whether Bac-rPrPSc is spontaneously produced in intermittent ultrasonic reactions. No spontaneous generation of Bac-rPrPSc was observed at 37 °C, but when the reaction temperature was increased to 45 °C, Bac-rPrPSc was generated in all trials. Some Bac-rPrPSc variants were transmissible to mice, but when the reaction was repeated for 40 rounds, the transmissibility was lost. Notably, a variety of Bac-rPrPSc variants, including non-transmissible ones, differing in resistance to proteinase K and cofactor dependence during amplification, was generated under the same experimental conditions, including the same sonication settings and cofactors. However, their characteristics also disappeared after 40 reaction rounds and the variety converged onto a single variant. These results indicate that various Bac-rPrPSc variants with different transmissibility to mice and structural properties are generated, which compete with each other and gradually converge onto a variant with a slightly faster amplification rate.

Extended application of the rapid post-mortem test kit for bovine spongiform encephalopathy to chronic wasting disease.

Chronic wasting disease (CWD) is a prion disease affecting cervid species primarily in the United States of America and Canada; however, it is now emerging in Scandinavian countries. Although CWD cases have not been reported in Japan, in case of a CWD outbreak occuring, it is critical to prepare for testing a large number of specimens. The present study showed that a rapid post-mortem test kit, which is used for bovine spongiform encephalopathy surveillance in Japan, is valid for the detection of CWD prion.

Inactivation of Scrapie Prions by the Electrically Charged Disinfectant CAC-717.

Previous studies have revealed that the electrically charged disinfectant CAC-717 has strong virucidal and bactericidal effects but is safe for humans and animals. In this study, CAC-717 was further evaluated for its potential effects as a disinfectant against scrapie prions. Western blotting showed that CAC-717 reduced the amount of the abnormal isoform of prion protein (PrPSc) in prion-infected cell (ScN2a) lysates. Furthermore, the reduction of prion transmissibility was confirmed by a mouse bioassay, in which mice injected with scrapie prions pre-treated with CAC-717 survived longer than those injected with untreated scrapie prions. Lastly, to evaluate the seeding activity of ScN2a cell lysates treated with CAC-717, quantitative protein misfolding cyclic amplification (PMCA) was performed directly on ScN2a cell lysates treated with CAC-717, which showed that the median dose of PMCA (PMCA50) dropped from log9.95 to log5.20 after CAC-717 treatment, indicating more than a 4 log reduction. This suggests that the seeding activity of PrPSc is decreased by CAC-717. Collectively, these results suggest that CAC-717 has anti-prion activity, reducing both PrPSc conversion activity and prion transmissibility; thus, CAC-717 will be useful as a novel disinfectant in prion diseases.

Influence of Interspecies Transmission of Atypical Bovine Spongiform Encephalopathy Prions to Hamsters on Prion Characteristics.

Bovine spongiform encephalopathy (BSE) is a prion disease in cattle and is classified into the classical type (C-BSE) and two atypical BSEs, designated as high type (H-BSE) and low type (L-BSE). These classifications are based on the electrophoretic migration of the proteinase K-resistant core (PrPres) of the disease-associated form of the prion protein (PrPd). In a previous study, we succeeded in transmitting the H-BSE prion from cattle to TgHaNSE mice overexpressing normal hamster cellular PrP (PrPC). Further, Western blot analysis demonstrated that PrPres banding patterns of the H-BSE prion were indistinguishable from those of the C-BSE prion in TgHaNSE mice. In addition, similar PrPres glycoprofiles were detected among H-, C-, and L-BSE prions in TgHaNSE mice. Therefore, to better understand atypical BSE prions after interspecies transmission, H-BSE prion transmission from TgHaNSE mice to hamsters was investigated, and the characteristics of classical and atypical BSE prions among hamsters, wild-type mice, and mice overexpressing bovine PrPC (TgBoPrP) were compared in this study using biochemical and neuropathological methods. Identical PrPres banding patterns were confirmed between TgHaNSE mice and hamsters in the case of all three BSE prion strains. However, these PrPres banding patterns differed from those of TgBoPrP and wild-type mice infected with the H-BSE prion. In addition, glycoprofiles of TgHaNSE mice and hamsters infected with the L-BSE prion differed from those of TgBoPrP mice infected with the L-BSE prion. These data indicate that the PrPC amino acid sequences of new host species rather than other host environmental factors may affect some molecular aspects of atypical BSE prions. Although three BSE prion strains were distinguishable based on the neuropathological features in hamsters, interspecies transmission modified some molecular properties of atypical BSE prions, and these properties were indistinguishable from those of C-BSE prions in hamsters. Taken together, PrPres banding patterns and glycoprofiles are considered to be key factors for BSE strain typing. However, this study also revealed that interspecies transmission could sometimes influence these characteristics.

PrPSc with Seeding Activity Extensively Overlaps with Proteinase-Resistant PrPSc Rather than Infectious PrPSc.

The disease-associated prion protein (PrPSc) has the ability to seed the conformational conversion of normal prion proteins into the amyloid fibril form. This prion seeding activity can be measured using an in vitro amplification assay termed real-time quaking-induced conversion (RT-QuIC). There is a strong correlation between RT-QuIC positivity and prion infection; however, the relationship between seeding activity and infectivity remains elusive. In this study, we used endpoint dilution RT-QuIC on the brain homogenates from wild-type mice with mouse-adopted bovine spongiform encephalopathy (mBSE) at defined intervals during the incubation period and evaluated the temporal relationship among prion seeding dose, levels of proteinase-resistant PrPSc (PrPres), and infectious titer. We found that the infectious titer reached a plateau by 100 days postinfection, whereas seeding dose and PrPres levels were continuously elevated. Our calculation showed that the doubling time (dt) for seeding dose from 40 to 100 days postinoculation was closer to the dt for PrPres levels than to the dt for prion titer. Although an uncoupling of seeding doses and PrPres levels was observed at end-stage disease in this model, our findings suggest that there is substantial but not complete overlap between PrPSc with seeding activity and PrPres rather than infectious PrPSc.

[Elucidation of the Molecular Basis of Abnormal Prion Protein (PrP) Formation in a Cell-Free System Using Baculovirus and Insect Cell-derived Recombinant PrP].

The molecular basis underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) has not been fully elucidated. The protein-misfolding cyclic amplification (PMCA) technique, which can amplify PrPSc in vitro with the use of intermittent sonication, mimics the process of in vivo PrPSc replication. Accumulating evidence suggests that co-factors other than PrP may play a crucial role in the faithful replication of PrPSc. In conventional PMCA, brain homogenates (BHs) from normal animals are used as the PrPC substrate. Since BHs contain many impurities, it is difficult to identify the co-factors using conventional PMCA. Thus, we developed a modified PMCA system using baculovirus and insect cell-derived recombinant PrP as a substrate (insect cell PMCA; iPMCA). We demonstrated that nucleic acids and glycosaminoglycans (GAGs) such as heparan sulfate (HS) or its analogue heparin (HP) are critical for PrPSc amplification in iPMCA. Of note, the addition of HS or HP restored the conversion efficiency in iPMCA under nucleic acid-depleted conditions. Moreover, the iPMCA products were infectious and preserved the strain properties of the input seed PrPSc. These data suggest that not only nucleic acids but also some GAGs play an important role in facilitating faithful replication of prions, at least in vitro.

First case of atypical scrapie in a goat in Japan.

Atypical scrapie in goats has only been reported in European countries. The present study reports the identification of the first case of atypical scrapie in goats in Japan. The genotype of the animal was ALRQ/ALHQ at codons 136, 141, 154, and 171 in prion protein (PrP). Western blot analysis showed a multiplex proteinase K-resistant prion protein (PrP-res) band pattern with a band <15 kDa that was clearly distinguishable from the triplet PrP-res band pattern observed in classical scrapie cases. Histopathological and immunohistological examination showed mild vacuolation and fine granular to globular immunolabelling of disease-associated PrP in the dorsal horn of cervical spinal cord. Collectively, our results confirmed that this goat was affected by atypical scrapie.

Estimation of prion infectivity in tissues of cattle infected with atypical BSE by real time-quaking induced conversion assay.

Atypical bovine spongiform encephalopathy (BSE), first identified in 2004, poses a threat due to the potential to spread the disease to cattle and other animals, including humans. Here, we estimated prion titers in various tissues of cattle infected with atypical BSE using a real-time quaking-induced conversion assay that detects amyloid seeding activity of a disease-specific prion protein, PrPSc, a major component of prions. PrPSc was detected both in and outside of nerve tissues, and some of the peripheral nerve tissues contained relatively high prion titers. Low titers of prions were also observed in masseter, jejunum, and adrenal glands. Quantitative data on prion infectivity in tissues of atypical BSE-affected cattle is useful to assess the risk of atypical BSE.

Epidermal growth factor-nanoparticle conjugates change the activity from anti-apoptotic to pro-apoptotic at membrane rafts.

The proliferation epidermal growth factor (EGF) is known to acquire contradictory apoptotic activities upon conjugation with gold nanoparticles (GNPs) through hitherto unknown mechanisms. Here, we identified an essential role of membrane rafts in the drastic activity switching of EGF-GNPs through the following intracellular signaling. (1) In contrast to the rapid diffusion of activated EGF receptor after the soluble EGF stimulation, the receptor is confined within membrane rafts upon binding to the EGF-GNPs. (2) This initial receptor confinements switch its endocytosis process from normal clathrin-mediated endocytosis to caveolin-mediated one, changing the phosphorylation dynamics of essential downstream kinases, i.e., extracellular signal-regulated kinase and AKT. Importantly, the destruction of membrane rafts by ß-cyclodextrin reversed this trafficking and signaling, restoring EGF-GNPs to lost anti-apoptotic property. These results reveal the importance of GNP-mediated signal condensation at membrane rafts in conferring the unique apoptotic activity on EGF-nanoparticle conjugates. STATEMENT OF SIGNIFICANCE: Epidermal growth factor (EGF) is a small secretory protein that induces cell proliferation upon binding to its receptor existed on cellular plasma membranes. One interesting feature of the protein in the nanobiology field is, its acquisition of apoptosis-inducing (cellular suicide) activity rather than proliferative one upon conjugation to gold nanoparticles through hitherto unknown mechanisms. Here, we identified the involvement of membrane rafts, plasma membrane nanodomains enriched with cholesterol, in the apoptosis processes by changing the receptor trafficking and downstream signal transduction pathways. Moreover, the destruction of lipid rafts restored the EGF-nanoparticle conjugates with lost anti-apoptotic activity. These finding highlight potential applications of EGF-nanoparticle conjugates to cancer therapy, as the EGF receptor are highly expressed in cancer cells.

Interspecies transmission to bovinized transgenic mice uncovers new features of a CH1641-like scrapie isolate.

In animal prion diseases, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in cervids, and scrapie in sheep and goats, a disease-associated isoform of prion protein (PrPd) accumulates in the brains of affected animals. Although the CH1641 scrapie isolate was experimentally established in the UK, a few natural CH1641-like scrapie cases have been reported in France and the UK. The molecular mass of the unglycosylated protease-resistant core of PrPd (PrPres) is known to be similar between CH1641-like scrapie and experimental BSE in sheep. We previously established an experimental CH1641-like scrapie isolate (Sh294) from a natural classical scrapie case. Here, we demonstrated that the Sh294 isolate was independent of both classical and atypical BSEs by cross-species transmission to bovine PrP overexpressing (TgBoPrP) mice and wild-type mice. Interestingly, we found that the Sh294 isolate altered its host range by the transmission to TgBoPrP mice, and we succeeded in the first stable reproduction of CH1641-like scrapie specific PrPres banding patterns with the ~12-kDa small C-terminal fragment in wild-type mice. This study provides new insight into the relationship between CH1641-like scrapie isolates and BSEs. In addition, interspecies transmission models such as we have demonstrated here could be a great help to investigate the origin and host range of animal prions.

Chronic wasting disease prion infection of differentiated neurospheres.

A possible strategy to develop more diverse cell culture systems permissive to infection with naturally occurring prions is to exploit culture of neurospheres from transgenic mice expressing the normal prion protein (PrP) of the native host species. Accordingly, we developed differentiated neurosphere cultures from the cervid PrP-expressing mice to investigate whether this in vitro system would support replication of non-adapted cervid-origin chronic wasting disease (CWD) prions. Here we report the successful amplification of disease-associated PrP in differentiated neurosphere cultures within 3 weeks after exposure to CWD prions from both white-tailed deer or elk. This neurosphere culture system provides a new in vitro tool that can be used to assess non-adapted CWD prion propagation and transmission.

Evaluation of rapid post-mortem test kits for bovine spongiform encephalopathy (BSE) screening in Japan: Their analytical sensitivity to atypical BSE prions.

A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.

Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle.

To determine oral transmissibility of the L-type bovine spongiform encephalopathy (BSE) prion, we orally inoculated 16 calves with brain homogenates of the agent. Only 1 animal, given a high dose, showed signs and died at 88 months. These results suggest low risk for oral transmission of the L-BSE agent among cattle.

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.

Identification of the first case of atypical scrapie in Japan.

A Corriedale ewe was confirmed as the first atypical scrapie case during an active surveillance program for transmissible spongiform encephalopathies in small ruminants in Japan. The animal was homozygous for the AF141RQ haplotype of PRNP. The animal showed clinical neurological signs possibly due to listeriosis before culling. Western blot analysis showed an unusual multiple banded pattern with a low-molecular fragment at ~7 kDa. Histopathology revealed suppurative meningoencephalitis caused by listeriosis in the brainstem. Fine granular to globular immunostaining of disease-associated prion proteins was mainly detected in the neuropil of the spinal tract of the trigeminal nerve and in the white matter of the spinocerebellar tract. Based on these results, this case was conclusively diagnosed as atypical scrapie with encephalitic listeriosis.

Heparan Sulfate and Heparin Promote Faithful Prion Replication in Vitro by Binding to Normal and Abnormal Prion Proteins in Protein Misfolding Cyclic Amplification.

The precise mechanism underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrPSc, results in PrPSc replication that preserves the strain-specific characteristics of the input PrPSc; thus, PMCA mimics the process of in vivo PrPSc replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrPSc amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrPSc replication in vitro Here, we investigated whether GAGs play a role in the faithful replication of PrPSc by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrPSc These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrPSc through the sulfated groups present on HP and that the N-terminal domain of Bac-PrPSc might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrPC and/or PrPSc through their sulfate groups is critical for the faithful replication of prions.

Transmission of atypical scrapie to homozygous ARQ sheep.

Two Cheviot ewes homozygous for the A136L141R154Q171 (AL141RQ) prion protein (PrP) genotype were exposed intracerebrally to brain pools prepared using four field cases of atypical scrapie from the United Kingdom. Animals were clinically normal until the end of the experiment, when they were culled 7 years post-inoculation. Limited accumulation of disease-associated PrP (PrPSc) was observed in the cerebellar molecular layer by immunohistochemistry, but not by western blot or enzyme-linked immunosorbent assay. In addition, PrPSc was partially localized in astrocytes and microglia, suggesting that these cells have a role in PrPSc processing, degradation or both. Our results indicate that atypical scrapie is transmissible to AL141RQ sheep, but these animals act as clinically silent carriers with long incubation times.