Sensitive detection of pathological seeds of α-synuclein, tau and prion protein on solid surfaces.

Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.

Quantitative analysis of viremia and viral shedding in pigs infected experimentally with classical swine fever virus isolates obtained from recent outbreaks in Japan.

Although classical swine fever occurred in September 2018 for the first time in 26 years, its virulence is thought to be moderate based on field observations by veterinary authorities and our previous experimental infections. We quantified viremia and viral shedding in pigs infected with recent Japanese classical swine fever virus isolates, as well as a highly virulent strain. The results show that pigs infected with the Japanese strains exhibited lower viremia and viral shedding than those infected with the highly virulent strain. However, horizontal transmission occurred in pigs infected with the Japanese strains, similar to those infected with the highly virulent strain. Additionally, viremia and neuralization antibodies coexisted in pigs infected with the Japanese strains, presenting challenges for control measures.

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.

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.

Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathy.

H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in cattle. During passaging of H-BSE in transgenic bovinized (TgBoPrP) mice, a novel phenotype of BSE, termed BSE-SW emerged and was characterized by a short incubation time and host weight loss. To investigate the biological and biochemical properties of the BSE-SW prion, a transmission study was conducted in cattle, which were inoculated intracerebrally with brain homogenate from BSE-SW-infected TgBoPrP mice. The disease incubation period was approximately 15 months. The animals showed characteristic neurological signs of dullness, and severe spongiform changes and a widespread, uniform distribution of disease-associated prion protein (PrPSc) were observed throughout the brain of infected cattle. Immunohistochemical PrPSc staining of the brain revealed the presence of intraglial accumulations and plaque-like deposits. No remarkable differences were identified in vacuolar lesion scores, topographical distribution patterns, and staining types of PrPSc in the brains of BSE-SW- vs H-BSE-infected cattle. PrPSc deposition was detected in the ganglia, vagus nerve, spinal nerve, cauda equina, adrenal medulla, and ocular muscle. Western blot analysis revealed that the specific biochemical properties of the BSE-SW prion, with an additional 10- to 12-kDa fragment, were well maintained after transmission. These findings indicated that the BSE-SW prion has biochemical properties distinct from those of H-BSE in cattle, although clinical and pathologic features of BSW-SW in cattle are indistinguishable from those of H-BSE. The results suggest that the 2 infectious agents, BSE-SW and H-BSE, are closely related strains.

Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining.

We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

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

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.

Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice.

L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE that is transmissible to cattle and several lines of prion protein (PrP) transgenic mice, but not to wild-type mice. In this study, we examined the transmissibility of sheep-passaged L-BSE prions to wild-type mice. Disease-associated prion protein (PrP(Sc)) was detected in the brain and/or lymphoid tissues during the lifespan of mice that were asymptomatic subclinical carriers, indicating that wild-type mice were susceptible to sheep-passaged L-BSE. The morphological characteristics of the PrP(Sc) of sheep-passaged L-BSE included florid plaques that were distributed mainly in the cerebral cortex and hippocampus of subsequent passaged mice. The PrP(Sc) glycoform profiles of wild-type mice infected with sheep-passaged L-BSE were similar to those of the original isolate. The data indicate that sheep-passaged L-BSE has an altered host range and acquired transmissibility to wild-type mice.

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.

Different antigenicities of the N-terminal region of cellular and scrapie prion proteins.

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP(Sc) ) and cellular prion protein (PrP(C) ) under native conditions. To clarify conformational differences between these two isoforms, PrP-deficient mice were immunized with brain homogenates of normal and scrapie-infected animals. All mice generated anti-PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP(Sc) consisting of QGSPGGN (PrP41-47) at the N-terminus. This study demonstrated a conformational dissimilarity at the N-terminus between PrP(Sc) and PrP(C) , a finding that may provide novel information about conformational features of PrP(Sc) .

Development of a Novel Indirect ELISA for the Serological Diagnosis of African Swine Fever Using p11.5 Protein as a Target Antigen.

African swine fever is a hemorrhagic viral disease with a mortality rate of nearly 100% in pigs. Hence, it is classified as a notifiable disease by the World Organization for Animal Health. Because no field-available vaccine exists, African swine fever virus (ASFV) control and eradication solely depend on good farm biosecurity management and rapid and accurate diagnosis. In this study, we developed a new indirect serological enzyme-linked immunosorbent assay (ELISA) using recombinant p11.5 protein from ASFV as a solid-phase target antigen. The cutoffs were determined by receiver operating curve analysis performed with serum samples obtained from naïve and infected pigs. Based on the results of a commercially available serological ELISA, the relative sensitivity and specificity of our assay were 93.4% and 94.4% (N = 166; area under the curve = 0.991; 95% confidence interval = 0.982-0.999), respectively. Furthermore, to compare the performance of the serological ELISAs, we conducted the assays on a panel of sera collected from pigs and boars experimentally infected with different ASFV isolates. The results indicated the greater sensitivity of the newly developed assay and its ability to detect anti-ASFV antibodies earlier after virus inoculation.

A Spontaneously Occurring African Swine Fever Virus with 11 Gene Deletions Partially Protects Pigs Challenged with the Parental Strain.

African swine fever (ASF) is an infectious Suidae disease caused by the ASF virus (ASFV). Adaptation to less susceptible, non-target host cells is one of the most common techniques used to attenuate virulent viruses. However, this may induce many mutations and large-scale rearrangements in the viral genome, resulting in immunostimulatory potential loss of the virus in vivo. This study continuously maintained the virulent ASFV strain, Armenia2007 (Arm07), to establish an attenuated ASFV strain with minimum genetic alteration in a susceptible host cell line, immortalized porcine kidney macrophage (IPKM). A mutant strain was successfully isolated via repeated plaque purification in combination with next-generation sequencing analysis. The isolated strain, Arm07ΔMGF, which was obtained from a viral fluid at a passage level of 20, lacked 11 genes in total in the MGF300 and MGF360 regions and showed marked reduction in virulence against pigs. Moreover, all the pigs survived the challenge with the parental strain when pigs were immunized twice with 105 TCID50 of Arm07ΔMGF, although viremia and fever were not completely prevented after the challenge infection. These findings suggest that this naturally attenuated, spontaneously occurring ASFV strain may provide a novel platform for ASF vaccine development.

Comparative genomic and transcriptomic analyses of African swine fever virus strains.

African swine fever (ASF) is the most devastating disease caused by the African swine fever virus (ASFV), impacting the pig industry worldwide and threatening food security and biodiversity. Although two vaccines have been approved in Vietnam to combat ASFV, the complexity of the virus, with its numerous open reading frames (ORFs), necessitates a more diverse vaccine strategy. Therefore, we focused on identifying and investigating the potential vaccine targets for developing a broad-spectrum defense against the virus. This study collected the genomic and/or transcriptomic data of different ASFV strains, specifically from in vitro studies, focusing on comparisons between genotypes I, II, and X, from the National Center for Biotechnology Information (NCBI) database. The comprehensive analysis of the genomic and transcriptomic differences between high- and low-virulence strains revealed six early genes, 13 late genes, and six short genes as potentially essential ORFs associated with high-virulence. In addition, many other ORFs (e.g., 14 multigene family members) are worth investigating. The results of this study provided candidate ORFs for developing ASF vaccines and therapies.

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.

Usability of Immortalized Porcine Kidney Macrophage Cultures for the Isolation of ASFV without Affecting Virulence.

Immortalized porcine kidney macrophage (IPKM) cells are highly susceptible to major African swine fever virus (ASFV) isolates. To clarify the compatibility of this cell line for ASFV isolation from biomaterials, animal experiments and in vitro isolation were performed. Pork products seized at international airports were subjected to virus inoculation in pigs (in vivo) and IPKM cell cultures (in vitro) to examine the viability and virulence of the contaminating viruses. Moreover, the viruses isolated using IPKM cells were inoculated into pigs to assess the virulence shift from the original materials. All pigs that were inoculated with either homogenate samples of seized pork product or IPKM-isolated ASFVs developed typical symptoms of ASF and died (or were euthanized) within the term of the animal experiments. The success rate of virus isolation in IPKM cells was comparable to that observed in porcine primary alveolar macrophage (PAM) cells. The IPKM cell line would be an ideal tool for the isolation and propagation of live ASFVs with high efficiency and enhanced usability, such as immortal, proliferative, and adhesive properties. The isolated viruses retained biologically similar characteristics to those of the original ones during isolation in vitro.

Isolation and immortalization of macrophages derived from fetal porcine small intestine and their susceptibility to porcine viral pathogen infections.

Macrophages are a heterogeneous population of cells that are present in all vertebrate tissues. They play a key role in the innate immune system, and thus, in vitro cultures of macrophages provide a valuable model for exploring their tissue-specific functions and interactions with pathogens. Porcine macrophage cultures are often used for the identification and characterization of porcine viral pathogens. Recently, we have developed a simple and efficient method for isolating primary macrophages from the kidneys and livers of swine. Here, we applied this protocol to fetal porcine intestinal tissues and demonstrated that porcine intestinal macrophages (PIM) can be isolated from mixed primary cultures of porcine small intestine-derived cells. Since the proliferative capacity of primary PIM is limited, we attempted to immortalize them by transferring the SV40 large T antigen and porcine telomerase reverse transcriptase genes using lentiviral vectors. Consequently, immortalized PIM (IPIM) were successfully generated and confirmed to retain various features of primary PIM. We further revealed that IPIM are susceptible to infection by the African swine fever virus and the porcine reproductive and respiratory syndrome virus and support their replication. These findings suggest that the IPIM cell line is a useful tool for developing in vitro models that mimic the intestinal mucosal microenvironments of swine, and for studying the interactions between porcine pathogens and host immune cells.

Establishment and characterization of the immortalized porcine lung-derived mononuclear phagocyte cell line.

Mononuclear phagocytes (MNP), including monocytes, dendritic cells (DC), and macrophages, play critical roles in innate immunity. MNP are abundant in the lungs and contribute to host defense against airborne agents and pulmonary immune homeostasis. In this study, we isolated porcine lung-derived MNP (PLuM) from primary cultures of parenchymal lung cells and then immortalized them by transferring the SV40 large T antigen gene and porcine telomerase reverse transcriptase gene using lentiviral vectors. The established cell line, immortalized PLuM (IPLuM), expressed DC/macrophage markers; i.e., CD163, CD172a, and major histocompatibility complex class II, whereas they did not express a porcine monocyte-specific marker, CD52. The expression patterns of these cell surface markers indicate that IPLuM originate from the DC/macrophage lineage rather than the monocyte lineage. The bacterial cell wall components muramyl dipeptide and lipopolysaccharide induced the production of the interleukin-1 family of pro-inflammatory cytokines in IPLuM. Phagocytotic activity was also detected by time-lapse fluorescence imaging of live cells when IPLuM were cultured in the presence of pHrodo dye-conjugated E. coli BioParticles. It is worth noting that IPLuM are susceptible to African swine fever virus infection and support the virus' efficient replication in vitro. Taken together, the IPLuM cell line may be a useful model for investigating host-agent interactions in the respiratory microenvironments of the porcine lung.

Administration of the antiviral agent T-1105 fully protects pigs from foot-and-mouth disease infection.

Foot-and-mouth disease (FMD) is a contagious disease affecting cloven-hoofed animals. Its transmissibility and antigenic variety make this disease difficult to control. Antiviral agents are expected to have an immediate effect that is independent of viral antigenicity; thus, they can serve as effective tools for inhibiting the spread of the causative agent, the FMD virus (FMDV), from infected animals. In this study, we investigated the antiviral activity of a pyrazinecarboxamide derivative, T-1105, against FMDV. Cytopathic effect inhibition assays revealed that T-1105 strongly inhibited the replication of 28 reference strains of all seven FMDV serotypes at non-cytotoxic concentrations. The antiviral effect of T-1105 against FMDV was also evaluated by experimental infection of domestic pigs. T-1105 was administered orally to pigs starting 1 h before or 6 h after the inoculation of a porcinophilic FMDV serotype O, topotype CATHAY. None of the pigs administered with T-1105 showed clinical signs of FMD. Moreover, no infectious FMDVs or FMDV-specific genes were detected in their sera, oral and nasal discharges, or tissues collected 48 h after virus inoculation. These findings strongly suggest that administration of T-1105 is effective in controlling the spread of FMDV in pigs.

Tracing conformational transition of abnormal prion proteins during interspecies transmission by using novel antibodies.

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