Pathogenicity of genotype 2.1 classical swine fever virus isolated from Japan in 2019 in pigs.

Classical swine fever (CSF) re-emerged in Japan in 2018 for the first time in 26 years. The disease has been known to be caused by a moderately pathogenic virus, rather than the highly pathogenic virus that had occurred in the past. However, the underlying pathophysiology remains unknown. This study conducted an experimental challenge on specific pathogen-free (SPF) pigs in a naïve state for 2, 4, and 6 weeks and confirmed the disease state during each period by clinical observation, virus detection, and pathological necropsy. We revealed the pathological changes and distribution of pathogens and virus-specific antibodies at each period after virus challenge. These results were comprehensively analyzed and approximately 70% of the pigs recovered, especially at 4- and 6-week post-virus challenge. This study provides useful information for future countermeasures against CSF by clarifying the pathogenicity outcomes in unvaccinated pigs with moderately pathogenic genotype 2.1 virus.

Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

Systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus).

Systemic amyloid light-chain (AL) amyloidosis is an infrequent disease in which amyloid fibrils derived from the immunoglobulin light chain are deposited in systemic organs, resulting in functional impairment. This disease has been notably uncommon in animals, and nonhuman primates have not been reported to develop it. In this study, we identified the systemic AL kappa chain amyloidosis in a captive Bornean orangutan (Pongo pygmaeus) and analyzed its pathogenesis. Amyloid deposits were found severely in the submucosa of the large intestine, lung, mandibular lymph nodes, and mediastinal lymph nodes, with milder lesions in the liver and kidney. Mass spectrometry-based proteomic analysis revealed an abundant constant domain of the immunoglobulin kappa chain in the amyloid deposits. Immunohistochemistry further confirmed that the amyloid deposits were positive for immunoglobulin kappa chains. In this animal, AL amyloidosis resulted in severe involvement of the gastrointestinal submucosa and lymph nodes, which is consistent with the characteristics of AL amyloidosis in humans, suggesting that AL amyloid may have a similar deposition mechanism across species. This report enhances the pathological understanding of systemic AL amyloidosis in animals by providing a detailed characterization of this disease based on proteomic analysis.

Detection and genetic analysis of bovine rhinitis B virus in Japan.

Bovine rhinitis B virus (BRBV) (genus Aphthovirus, family Picornaviridae) is a significant etiological agent of the bovine respiratory disease complex. Despite global reports on BRBV, genomic data for Japanese strains are not available. In this study, we aimed to obtain genomic information on BRBV in Japan and analyze its genetic characteristics. In nasal swabs from 66 cattle, BRBV was detected in 6 out of 10 symptomatic and 4 out of 56 asymptomatic cattle. Using metagenomic sequencing and Sanger sequencing, the nearly complete genome sequences of two Japanese BRBV strains, IBA/2211/2 and LAV/238002, from symptomatic and asymptomatic cattle, respectively, were determined. These viruses shared significant genetic similarity with known BRBV strains and exhibited unique mutations and recombination events, indicating dynamic evolution, influenced by regional environmental and biological factors. Notably, the leader gene was only approximately 80% and 90% identical in its nucleotide and amino acid sequence, respectively, to all of the BRBV strains with sequences in the GenBank database, indicating significant genetic divergence in the Japanese BRBV leader gene. These findings provide insights into the genetic makeup of Japanese BRBV strains, enriching our understanding of their genetic diversity and evolutionary mechanisms.

Genomic diversity of group A rotaviruses from wild boars and domestic pigs in Japan: wide prevalence of NSP5 carrying the H2 genotype.

Group A rotavirus (RVA) sequences were detected in 10.8% (23/212) and 20.7% (87/421) of fecal samples collected in 2017-2022 from wild boars and domestic pigs, using next-generation sequencing. Complete genome sequence analysis of one wild boar and 13 domestic pig RVAs revealed that six of them carried the rare H2 NSP5 genotype. Out of the 39 samples for which the NSP5 genotype could be determined, 23 (59.0%) were of genotype H2. H2 porcine RVAs consist exclusively of Japanese porcine RVAs and exhibit sequence diversity in each segment, suggesting that H2 porcine RVAs may have evolved through reassortment within the Japanese pig population.

Potent immunogenicity and neutralization of recombinant adeno-associated virus expressing the glycoprotein of severe fever with thrombocytopenia virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by a tick-borne virus called severe fever with thrombocytopenia syndrome virus (SFTSV). In recent years, human infections through contact with ticks and through contact with the bodily fluids of infected dogs and cats have been reported; however, no vaccine is currently available. SFTSV has two glycoproteins (Gn and Gc) on its envelope, which are vaccine-target antigens involved in immunogenicity. In the present study, we constructed novel SFTS vaccine candidates using an adeno-associated virus (AAV) vector to transport the SFTSV glycoprotein genome. AAV vectors are widely used in gene therapy and their safety has been confirmed in clinical trials. Recently, AAV vectors have been used to develop influenza and SARS-CoV-2 vaccines. Two types of vaccines (AAV9-SFTSV Gn and AAV9-SFTSV Gc) carrying SFTSV Gn and Gc genes were produced. The expression of Gn and Gc proteins in HEK293T cells was confirmed by infection with vaccines. These vaccines were inoculated into mice, and the collected sera produced anti-SFTS antibodies. Furthermore, sera from AAV9-SFTSV Gn infected mice showed a potent neutralizing ability, similar to previously reported SFTS vaccine candidates that protected animals from SFTSV infection. These findings suggest that this vaccine is a promising candidate for a new SFTS vaccine.

Natto extract inhibits infection caused by the Aujeszky's disease virus in mice.

Aujeszky's disease virus (ADV), also known as Suid alphaherpesvirus 1, which mainly infects swine, causes life-threatening neurological disorders. This disease is a serious global risk factor for economic losses in the swine industry. The development of new anti-ADV drugs is highly anticipated and required. Natto, a traditional Japanese fermented food made from soybeans, is a well-known health food. In our previous study, we confirmed that natto has the potential to inhibit viral infections by severe acute respiratory syndrome coronavirus 2 and bovine alphaherpesvirus 1 through their putative serine protease(s). In this study, we found that an agent(s) in natto functionally impaired ADV infection in cell culture assays. In addition, ADV treated with natto extract lost viral infectivity in the mice. We conducted an HPLC gel-filtration analysis of natto extract and molecular weight markers and confirmed that Fraction No. 10 had ADV-inactivating ability. Furthermore, the antiviral activity of Fraction No. 10 was inhibited by the serine protease inhibitor 4-(2-Aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF). These results also suggest that Fraction No. 10, adjacent to the 12.5 kDa peak of the marker in natto extract, may inactivate ADV by proteolysis. Our findings provide new avenues of research for the prevention of Aujeszky's disease.

Antisera Produced Using an E. coli-Expressed SARS-CoV-2 RBD and Complemented with a Minimal Dose of Mammalian-Cell-Expressed S1 Subunit of the Spike Protein Exhibits Improved Neutralization.

E. coli-expressed proteins could provide a rapid, cost-effective, and safe antigen for subunit vaccines, provided we can produce them in a properly folded form inducing neutralizing antibodies. Here, we use an E. coli-expressed SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as a model to examine whether it yields neutralizing antisera with effects comparable to those generated by the S1 subunit of the spike protein (S1 or S1 subunit, thereafter) expressed in mammalian cells. We immunized 5-week-old Jcl-ICR female mice by injecting RBD (30 µg) and S1 subunit (5 µg) according to four schemes: two injections 8 weeks apart with RBD (RBD/RBD), two injections with S1 (S1/S1), one injection with RBD, and the second one with S1 (RBD/S1), and vice versa (S1/RBD). Ten weeks after the first injection (two weeks after the second injection), all combinations induced a strong immune response with IgG titer > 105 (S1/RBD < S1/S1 < RBD/S1 < RBD/RBD). In addition, the neutralization effect of the antisera ranked as S1/RBD~RBD/S1 (80%) > S1/S1 (56%) > RBD/RBD (42%). These results indicate that two injections with E. coli-expressed RBD, or mammalian-cell-produced spike S1 subunit alone, can provide some protection against SARS-CoV-2, but a mixed injection scheme yields significantly higher protection.

Novel ollusvirus detected in a solitary wild bee species (Osmia taurus) in Japan.

Pathogens of wild bees in Japan remain largely unknown. We examined viruses harbored by solitary wild Osmia bees, including Osmia cornifrons and Osmia taurus. Interestingly, the full-length genome of a novel virus (designated as "Osmia-associated bee chuvirus", OABV) was identified in three Osmia taurus bees collected in Fukushima prefecture. The sequences and genomic features are similar to those of Scaldis River bee virus. Phylogenetic analysis based on RNA-dependent RNA polymerase, glycoprotein, and nucleoprotein sequences showed that OABV formed a subcluster within ollusviruses and was closely related to strains identified in European countries. This study extends our knowledge of wild bee parasites in Japan.

Prevalence and genetic diversity in bovine parechovirus infecting Japanese cattle.

The first bovine parechovirus (Bo_ParV) was reported in 2021, and currently, only two nearly complete genome sequences of Bo_ParV are available. In this study, we detected Bo_ParVs in 10 out of 158 bovine fecal samples tested using real-time RT-PCR, and Bo_ParVs were isolated from three of these samples using MA104 cells. Analysis of the P1 region revealed that Bo_ParVs shared high pairwise amino acid sequence similarity (≥ 95.7% identity), suggesting antigenic similarity among Bo_ParVs, whereas nucleotide sequence identity values (≥ 84.8%) indicated more variability. A recombination breakpoint was identified in the 2B region, which may influence the evolution of this virus.

Development of a one-run real-time PCR detection system for pathogens associated with poultry infectious diseases.

Infectious diseases are an important issue in the poultry industry, requiring early diagnosis and countermeasures. To address this, we present a system based on TaqMan real-time PCR to detect pathogen genome in specimens collected from chickens. We designed 12 primer-probe sets for pathogens causing respiratory or systemic symptoms. In field samples, we detected three viruses, including DNA and RNA viruses, and three bacteria. The chicken anemia virus and Avibacterium paragallinarum were detected only in young and laying hens, respectively. Bacteria were detected only in throat swabs, and gallid alphaherpesvirus 2 was detected in different specimens at each developmental stage. Our novel TaqMan real-time PCR system effectively detects pathogen's gene in chickens, while taking age into account.

Novel recombinant porcine enterovirus G viruses lacking structural proteins are maintained in pig farms in Japan.

Type 1 recombinant enterovirus G (EV-G), which carries the papain-like cysteine protease (PLCP) gene of torovirus between its 2C/3A regions, and type 2 recombinant EV-G, which carries the torovirus PLCP gene with its flanking regions having non-EV-G sequences in place of the viral structural genes, have been detected in pig farms in several countries. In a previous study, we collected 222 fecal samples from 77 pig farms from 2104 to 2016 and detected one type 2 recombinant EV-G genome by metagenomics sequencing. In this study, we reanalyzed the metagenomic data and detected 11 type 2 recombinant EV-G genomes. In addition, we discovered new type 2 recombinant EV-G genomes of the two strains from two pig farms samples in 2018 and 2019. Thus, we identified the genomes of 13 novel type 2 recombinant EV-Gs isolated from several pig farms in Japan. Type 2 recombinant EV-G has previously been detected only in neonatal piglets. The present findings suggest that type 2 recombinant EV-G replicates in weaning piglets and sows. The detection of type 1 recombinant EV-Gs and type 2 recombinant EV-Gs at 3-year and 2-year intervals, respectively, from the same pig farm suggests that the viruses were persistently infecting or circulating in these farms.

Development of a new quantification method of Sarcocystis cruzi through detection of the acetyl-CoA synthetase gene.

Sarcocystis cruzi is a member of the genus Sarcocystis, infecting bovine animals such as cattle and bison as intermediate hosts, and canids such as dogs and raccoon dogs as definitive hosts. Acute sarcocystosis of S. cruzi causes occasional symptoms in cattle, including weight loss, reduced milk production, abortions, and death, and similar to other Sarcocystis species can potentially cause food poisoning in humans when raw or undercooked infected cattle meat is consumed. Despite these issues, genetic information on S. cruzi is scarce, and there is no specific quantitative method for the detection and quantification of the parasite in infected cattle. In this study, we aimed to develop a method based on high-throughput sequencing of S. cruzi genome and transcriptome that specifically and quantitatively detects the S. cruzi acetyl-CoA synthetase gene (ScACS). Cardiac muscles were collected from slaughterhouses in Saitama Prefecture to obtain sarcocysts from which DNA and RNA were extracted for the high-throughput sequencing. Using the sequences, we developed a specific quantitative PCR assay which could distinguish S. cruzi ACS from that of Toxoplasma gondii by taking advantage of the differences in their exon/intron organizations and validated the assay with the microscopic counting of the S. cruzi bradyzoites. Thus, this assay will be useful for future studies of S. cruzi pathogenesis in cattle and for the surveillance of infected animals, thereby easing public health concerns.

An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model.

A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD's eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell's receptor, with a binding constant of 7.9 × 10-9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.

First Isolation and Identification of Homologous Recombination Events of Porcine Adenovirus from Wild Boar.

Porcine adenoviruses (PAdVs) are distributed in pig populations and classified into five immunologically distinct serotypes (PAdV-1 to 5). In this study, a PAdV was isolated from a fecal sample of wild boar for the first time. Whole-genome analysis revealed that this strain (Ino5) has sequence homology (approximately > 93%) throughout the genome with the PAdV-5 strain HNF-70 that was isolated from a pig in Japan in 1987, except for the hexon, E3 612R, and fiber coding regions. Two possible recombination breakpoints were detected in the hexon and E3 612R regions, which were found to have reduced GC content. Structural prediction analysis showed that a part of the hexon protein corresponding to the tower region of Ino5 had structural differences when compared with HNF-70, suggesting antigenic heterogeneity between these strains. PAdVs were detected in 1.77% (2/113) and 12% (12/100) of the fecal samples from wild boars and pigs collected in Japan by PCR, respectively. Phylogenetic analyses of the hexon and fiber genes revealed that some samples showed different grouping in the hexon and fiber genes, suggesting that these viruses have recombination events. These findings suggest that the PAdV-5 has evolved with homologous recombination events in the same manner as human adenoviruses among not only pig populations, but also wild boars in Japan.

Genetic diversity, reassortment, and recombination of mammalian orthoreoviruses from Japanese porcine fecal samples.

Mammalian orthoreoviruses (MRVs) are non-enveloped double-stranded RNA viruses with a broad host range. MRVs are prevalent worldwide, and in Japan, they have been isolated from various hosts, including humans, dogs, cats, wild boars, and pigs, and they have also been found in sewage. However, Japanese porcine MRVs have not been genetically characterized. While investigating porcine enteric viruses including MRV, five MRVs were isolated from the feces of Japanese pigs using MA104 cell culture. Genetic analysis of the S1 gene revealed that the Japanese porcine MRV isolates could be classified as MRV-2 and MRV-3. Whole genome analysis showed that Japanese porcine MRVs exhibited genetic diversity, although they shared sequence similarity with porcine MRV sequences in the DDBJ/EMBL/GenBank database. Several potential intragenetic reassortment events were detected among MRV strains from pigs, sewage, and humans in Japan, suggesting zoonotic transmission. Furthermore, homologous recombination events were identified in the M1 and S1 genes of Japanese porcine MRV. These findings imply that different strains of Japanese porcine MRV share a porcine MRV genomic backbone and have evolved through intragenetic reassortment and homologous recombination events.

First isolation and genomic characterization of bovine parechovirus from faecal samples of cattle in Japan.

A novel picornavirus was isolated from the faeces of a diarrhoeic cow using MA-104 cells at the third blind passage. This virus, named Den1/2021/JPN, was completely sequenced using total RNA from the cell culture supernatant by deep sequencing. The genome of Den1/2021/JPN had a standard picornavirus genome organisation with conserved picornaviral motifs. The 5' untranslated region harboured a type-II internal ribosomal entry site. Den1/2021/JPN was most closely related to a bovine parechovirus (Bo_ParV) named cow/2018/4, which has been recently identified in publicly available databases. Phylogenetic analyses and pairwise sequence comparison revealed that Den1/2021/JPN and Bo_ParV cow/2018/4 clustered with parechoviruses and were most closely related to Parechovirus E identified in birds of prey, exhibiting nucleotide sequence similarity of 64.2-64.5 %, 58.6-59.7 % and 66.3-66.4 % in the polyprotein, P1 and 2C+3 CD coding regions, respectively. This study presents the first report on the isolation of Bo_ParV. Den1/2021/JPN and Bo_ParV cow/2018/4, which are candidates for a novel species in the genus Parechovirus.

Genomic diversity and intragenic recombination of species C rotaviruses.

Rotavirus C (RVC) is a major cause of diarrhoea in swine, cattle, and humans worldwide. RVC exhibits sequence diversity in all 11 genes, especially in VP4 and VP7, and all segment-based genotyping has been performed similar to rotavirus A. To date, recombination events have been reported in rotavirus A and B. However, there are no reports describing gene recombination of RVC, except for recombination in NSP3 between RVC and rotavirus H. In this study, nine porcine RVC strains identified in Japanese pigs were completely sequenced and analysed together with RVC sequences from the GenBank database. The analyses showed that sequences of the VP4, VP2, and NSP1 of several porcine RVC strains did not branch with any of those of the RVC strains in the GenBank database, suggesting new genotypes. Several homologous recombination events, between or within genotypes, were identified in the VP4, VP7, VP2, NSP1, and NSP3 genes. Of these, nine, one, and one intergenotypic recombination events in the VP4, VP2, and NSP3 genes, respectively, were supported with sufficient statistical values. Although these findings suggest occurrences of the intragenic recombination events in the RVC genome, potential sequence errors and poor sequence assemblies in the databases should be watched with care. The results in this study present data about the important recombination events of the RVCs, which influence evolution of the virus by aiding them to gain genetic diversity and plasticity, although further sequence data will be necessary to obtain more comprehensive understanding of such mechanisms.

Validation of the usefulness of 26S rDNA D1/D2, internal transcribed spacer, and intergenic spacer 1 for molecular epidemiological analysis of Macrorhabdus ornithogaster.

Macrorhabdus ornithogaster (MO) is an infectious fungus that causes gastric damage in birds. In this study, we established nested and seminested polymerase chain reaction (PCR) methods that specifically amplify the domain D1/D2 region (D1/D2) of 26S ribosomal DNA (rDNA), internal transcribed spacer (ITS) of rDNA, and intergenic spacer (IGS) 1 region from avian feces. Phylogenetic analysis of MO collected from Japanese pet birds showed little genetic variation; analysis based on these regions did not distinguish between host species order, differences in MO shape, or host gastrointestinal symptoms. These regions were found to be unsuitable for molecular epidemiological studies of MO and further investigation into other genetic regions is required.

Natto extract, a Japanese fermented soybean food, directly inhibits viral infections including SARS-CoV-2 in vitro.

Natto, a traditional Japanese fermented soybean food, is well known to be nutritious and beneficial for health. In this study, we examined whether natto impairs infection by viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as bovine herpesvirus 1 (BHV-1). Interestingly, our results show that both SARS-CoV-2 and BHV-1 treated with a natto extract were fully inhibited infection to the cells. We also found that the glycoprotein D of BHV-1 was shown to be degraded by Western blot analysis and that a recombinant SARS-CoV-2 receptor-binding domain (RBD) was proteolytically degraded when incubated with the natto extract. In addition, RBD protein carrying a point mutation (UK variant N501Y) was also degraded by the natto extract. When the natto extract was heated at 100 °C for 10 min, the ability of both SARS-CoV-2 and BHV-1 to infect to the cells was restored. Consistent with the results of the heat inactivation, a serine protease inhibitor inhibited anti-BHV-1 activity caused by the natto extract. Thus, our findings provide the first evidence that the natto extract contains a protease(s) that inhibits viral infection through the proteolysis of the viral proteins.