Genome-scale CRISPR-Cas9 screen identifies host factors as potential therapeutic targets for SARS-CoV-2 infection.

Although many host factors important for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been reported, the mechanisms by which the virus interacts with host cells remain elusive. Here, we identified tripartite motif containing (TRIM) 28, TRIM33, euchromatic histone lysine methyltransferase (EHMT) 1, and EHMT2 as proviral factors involved in SARS-CoV-2 infection by CRISPR-Cas9 screening. Our result suggested that TRIM28 may play a role in viral particle formation and that TRIM33, EHMT1, and EHMT2 may be involved in viral transcription and replication. UNC0642, a compound that specifically inhibits the methyltransferase activity of EHMT1/2, strikingly suppressed SARS-CoV-2 growth in cultured cells and reduced disease severity in a hamster infection model. This study suggests that EHMT1/2 may be a therapeutic target for SARS-CoV-2 infection.

Combination of pathological, biochemical and behavioral evaluations for peripheral neurotoxicity assessment in isoniazid-treated rats.

In drug development, assessment of non-clinical peripheral neurotoxicity is important to ensure human safety. Clarifying the pathological features and mechanisms of toxicity enables the management of safety risks in humans by estimating the degree of risk and proposing monitoring strategies. Published guidelines for peripheral neurotoxicity assessment do not provide detailed information on which endpoints should be monitored preferentially and how the results should be integrated and discussed. To identify an optimal assessment method for the characterization of peripheral neurotoxicity, we conducted pathological, biochemical (biomaterials contributing to mechanistic considerations and biomarkers), and behavioral evaluations of isoniazid-treated rats. We found a discrepancy between the days on which marked pathological changes were noted and those on which biochemical and behavioral changes were noted, suggesting the importance of combining these evaluations. Although pathological evaluation is essential for pathological characterization, the results of biochemical and behavioral assessments at the same time points as the pathological evaluation are also important for discussion. In this study, since the measurement of serum neurofilament light chain could detect changes earlier than pathological examination, it could be useful as a biomarker for peripheral neurotoxicity. Moreover, examination of semi-thin specimens and choline acetyltransferase immunostaining were useful for characterizing morphological neurotoxicity, and image analysis of semi-thin specimens enabled us to objectively show the pathological features.

Detection of chicken chapparvovirus 2 in chickens with hemorrhagic hepatitis in Japan.

Chicken chaphamaparvovirus causes diarrheal symptoms and can be detected in fecal samples. This study reports the detection of chicken chapparvovirus 2 in debilitated chickens with hemorrhagic hepatitis at a broiler farm in Japan. After euthanasia and necropsy, liver hemorrhage was observed. Nuclear inclusion bodies in the hepatocytes were identified using histological analysis. High-throughput sequencing analysis using RNA from livers of three affected chickens revealed infection by chicken chapparvovirus 2 and chicken anemia virus. Polymerase chain reaction analysis showed that all three chickens were positive for chicken chapparvovirus 2, and only one was positive for both chicken chapparvovirus 2 and chicken anemia virus. In conclusion, chicken chapparvovirus 2 causes infection in chickens in Japan and might be involved in hemorrhagic hepatitis.

A canine case of Ehrlichia canis infection without a history of being in an endemic area in Japan.

A mixed-breed, 8-year-old male dog developed neutropenia, thrombocytopenia, and hyperglobulinemia. Bone marrow hyperplasia and splenic plasmacytosis were cytologically observed. The dog had never been outside of Tokyo or Shizuoka Prefecture. Splenectomy was performed to confirm and remove the cause of splenic plasmacytosis. A histopathological diagnosis of splenic plasmacytoma was made; however, serum protein electrophoresis showed polyclonal gammopathy. Further screening was performed, and Ehrlichia canis infection was confirmed. The dog was treated with doxycycline for 5 weeks. After the antibiotic therapy, no relapse of neutropenia, thrombocytopenia, hyperglobulinemia, or positive polymerase chain reaction result of E. canis infection was observed for 3 years. Careful attention should be given to ehrlichiosis when exploring the cause of pancytopenia or hyperglobulinemia, regardless of the travel history.

Raillietina cesticillus infection causes reduced egg production in chickens in a windowless poultry house.

In a windowless poultry house raising layer chickens in Kanagawa prefecture, Japan, a slight increase in the mortality of chickens and a decrease in egg production were observed. Necropsy revealed numerous tapeworms and proglottids in chicken intestines. Histopathologically, gut-associated lymphoid tissues were observed in the lamina propria of the jejunum; however, no significant changes were observed in the other organs. Numerous hide beetles, Dermestes maculatus DeGeer, intermediate hosts of Raillietina cesticillus, were observed in the poultry house. Following a decline in beetle numbers, egg production increased and chicken mortality decreased. The life cycle of a tapeworm was easily established in a closed space, such as a windowless house, which led to severe infections.

Diversity of mutations in the dystrophin gene and details of muscular lesions in porcine dystrophinopathies.

During meat inspections in pigs, dystrophinopathies are among the muscle lesions targeted for disposal. In this study, the authors examined the lesions and the distribution of dystrophin expression in 25 pigs with dystrophinopathy. In addition, complementary deoxyribonucleic acid (cDNA) sequencing and western blotting were performed in 6 of the 25 cases, all of which were characterized by degeneration, necrosis, and fat replacement of muscle fibers. Comparing the results of immunohistochemistry with anti-dystrophin antibodies that recognized at different sites in the protein, the authors noted that the loss of dystrophin expression was most pronounced in the C-terminus-recognizing antibody (19/25 cases). The authors detected 5 missense mutations and 3 types of shortened transcripts generated by the skipping of exons in the cDNA, which were associated with the pathogenesis. One missense mutation had been reported previously, whereas the remaining mutations identified had not been previously documented in pigs. In the cases with shortened transcripts, normal-sized transcripts were detected together with the defective transcripts, suggesting that these mutations were caused by splicing abnormalities. In addition, they were in-frame mutations, all of which have similar pathogeneses of Becker muscular dystrophy in humans. These cases were 6 months of age and exhibited macroscopic discoloration, fatty replacement, and muscle degeneration, suggesting that the effect of these mutations on skeletal muscle was significant.

Antemortem diagnosis of external iliac arterial thromboembolism in a calf using contrast-enhanced computed tomography: a case report.

A 28-day-old Holstein calf with astasia (i.e., right hind limb flaccid paralysis) was referred to Large Animal Veterinary Educational Center. Thoracic radiographs showed an alveolar pattern and air bronchograms indicating pneumonia, whereas skeletal radiography showed muscle atrophy of the right hind limb but no abnormalities in the coxofemoral and stifle joints. Contrast-enhanced computed tomography revealed the pulmonary abscess in the right cranial pulmonary lobe and a thrombus (5 × 5 × 20 mm) in the right external iliac artery distal to the bifurcation of the abdominal aorta. These findings were confirmed via pathological examination. Streptococcus bovis/equinus complex colonies were isolated from the thrombus and pulmonary abscess. Thus, the pulmonary abscess was considered the infectious source of the thromboembolism in the right external iliac artery. Arterial thromboembolism should be included in the differential diagnoses in calves with astasia but without radiographic skeletal abnormalities.

Cor triatriatum sinister with dextrocardia in association with ostium secundum atrial septal defect, subpulmonary ventricular septal defect and bicuspid pulmonary valve in a pig.

Necropsy of a 52-day-old Camborough pig revealed numerous cardiac malformations. The positional relationship of the atria, ventricles and great vessels was a mirror image type (I, L and L): inverted arrangement of the atria, with a left-sided right atrium and right-sided left atrium (situs inversus); inverted arrangement of the ventricles, with a left-sided morphological right ventricle and right-sided morphological left ventricle (L-loop); and aortic valve to the front left relative to the pulmonary valve (L-malposed). The major malformations included an ostium secundum atrial septal defect, cor triatriatum sinister (CTS), a subpulmonary ventricular septal defect and a bicuspid pulmonary valve. Histological examination revealed myocyte hypertrophy, focal myocardial necrosis and calcification in the left morphological right ventricle of the heart. To the best of our knowledge, this is the first report of CTS in pigs. Although the individual malformations found in the present case are not unique, an unusual combination of these cardiac malformations has not been described in animals.

Prebiotic effect of poly-D-3-hydroxybutyrate prevents dyslipidemia in obese mice.

Obesity is a global health problem caused by genetic, environmental, and psychological factors and is associated with various health disorders. As such, there is a growing focus on the prevention of obesity and related diseases. The gut microbiota plays a crucial role in these diseases and has become a therapeutic target. Prebiotics, such as poly-d-3-hydroxybutyric acid (PHB), have gained attention for their potential to alter the gut microbiota, promote beneficial bacterial growth, and alleviate obesity. In this study, we examined the prebiotic effects of PHB in obese mice. We found that, in C57BL/6N mice, PHB reduced blood lipid levels. Analysis of the intestinal microflora also revealed an increase in short-chain fatty acid-producing bacteria. When PHB was administered to obese mice, subcutaneous fat and dyslipidemia were reduced, and the number of beneficial bacteria in the intestinal microflora increased. Furthermore, fatty degradation and oxidative stress were suppressed in the liver. PHB regulates gut bacterial changes related to obesity and effectively inhibits dyslipidemia, suggesting that it could be a prebiotic agent for curing various obesity-related diseases. In summary, PHB increases the beneficial gut microbiota, leading to an alleviation of obesity-associated dyslipidemia.

The novel sustained 3-hydroxybutyrate donor poly-D-3-hydroxybutyric acid prevents inflammatory bowel disease through upregulation of regulatory T-cells.

Inflammatory bowel disease (IBD) is a chronic persistent intestinal disorder, with ulcerative colitis and Crohn's disease being the most common. However, the physio-pathological development of IBD is still unknown. Therefore, research on the etiology and treatment of IBD has been conducted using a variety of approaches. Short-chain fatty acids such as 3-hydroxybutyrate (3-HB) are known to have various physiological activities. In particular, the production of 3-HB by the intestinal microflora is associated with the suppression of various inflammatory diseases. In this study, we investigated whether poly-D-3-hydroxybutyric acid (PHB), a polyester of 3-HB, is degraded by intestinal microbiota and works as a slow-release agent of 3-HB. Further, we examined whether PHB suppresses the pathogenesis of IBD models. As long as a PHB diet increased 3-HB concentrations in the feces and blood, PHB suppressed weight loss and histological inflammation in a dextran sulfate sodium-induced IBD model. Furthermore, PHB increased the accumulation of regulatory T cells in the rectum without affecting T cells in the spleen. These results indicate that PHB has potential applications in treating diseases related to the intestinal microbiota as a sustained 3-HB donor. We show for the first time that biodegradable polyester exhibits intestinal bacteria-mediated bioactivity toward IBD. The use of bioplastics, which are essential materials for sustainable social development, represents a novel approach to diseases related to dysbiosis, including IBD.

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.

Macroglossia in a pig diagnosed as Becker muscular dystrophy due to dystrophin pseudoexon insertion derived from intron 26.

We report a case of Becker muscular dystrophy in a 6-month-old, mixed-breed, castrated male pig detected with macroglossia at a meat inspection center. The pig presented a severely enlarged tongue extending outside its mouth. The tongue was firm and pale with discolored muscles. Histologically, there was severe fibrosis, fatty replacement, and myofiber necrosis, degeneration, and regeneration. Immunofluorescence showed focal and severely weak labeling for dystrophin at the sarcolemma of myocytes in the tongue. Analysis of dystrophin mRNA showed a 62 base pair insertion between exons 26 and 27. The insertion was derived from intron 26. Based on these findings, we diagnosed the case as Becker muscular dystrophy-the first known muscular dystrophy case induced by pseudoexon insertion in animals.

Glucosylceramide in T cells regulates the pathology of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease in the colon characterized by excessive activation of T cells. Glycosphingolipids (GSLs) are composed of lipid rafts in cellular membranes, and their content is linked to immune cell function. In the present study, we investigated the involvement of GSLs in IBD. Microarray data showed that in IBD patients, the expression of only UDP-glucose ceramide glucosyltransferase (UGCG) decreased among the GSLs synthases. Ad libitum access to dextran sulfate sodium (DSS) resulted in decreased UGCG and glucosylceramide (GlcCer) content in mesenteric lymph nodes and T cells from the spleen. Furthermore, the knockdown of Ugcg in T cells exacerbated the pathogenesis of colitis, which was accompanied by a decrease in Treg levels. Treatment with GlcCer nanoparticles prevented DSS-induced colitis. These results suggested that GlcCer in T cells is involved in the pathogenesis of IBD. Furthermore, GlcCer nanoparticles are a potential efficacious therapeutic target for IBD patients.

Isolation and whole-genome sequencing of a novel aviadenovirus from owls in Japan.

Adenoviruses have been reported to infect a variety of birds. Here, we isolated a novel adenovirus from the liver of a dead owl chick (Bengal eagle owl; Bubo bengalensis) at a raptor-breeding facility in Japan and determined the complete genome sequence of the virus. We performed necropsies on the dead owl chicks and found that they had enlarged livers, pericardial edema, and focal necrosis of the liver tissue. Transmission electron microscopy of the liver tissue revealed a virus-like structure, appearing as paracrystalline arrays in the nucleus, and immunohistochemical staining with anti-adenovirus antibodies showed positive reactions in hepatocytes and other cells. Attempts to isolate the virus from homogenized liver tissue of a dead owl chick showed a cytopathic effect on chicken-derived cultured cells after multiple blind passages. Further, we determined the complete genome sequence of this virus and performed phylogenetic analysis, revealing that this adenovirus belongs to the genus Aviadenovirus, forming a cluster with fowl and turkey aviadenoviruses. The amino acid sequence divergence between the DNA polymerase of this virus and its closest known adenovirus relative is approximately 29%, implying that this virus can be assigned to a new species in the genus Aviadenovirus. Based on our data, this novel owl adenovirus is a likely cause of fatal infections in owls, which may threaten wild and captive owl populations. Further, this virus is unique among raptor adenoviruses in that it infects chicken-derived cultured cells, raising the importance of further investigations to evaluate interspecies transmission of this virus.

Congenital malformations of the external and middle ear accompanied by temporal bone anomaly in a calf.

A 7-month-old female Holstein calf presented with bilateral microtia and absent external acoustic meatus. The real-time polymerase chain reaction test was negative for bovine viral diarrhea virus and bovine leukemia virus. The calf's dam had a normal reproductive history. Computed tomography confirmed bilateral atresia of external auditory canals, aplasia of tympanic cavities and the ossicular chain, and temporomandibular joint abnormality. Necropsy revealed a severe malformation of the temporal bone. In the tympanic region, the external acoustic pore, tympanic bulla, and muscular process were absent bilaterally. The bilateral inner ear structure was normal. Based on these findings, we diagnosed the present case as congenital malformations of the external and middle ear accompanied by temporal bone anomaly.

Detection and genetic analysis of a novel atypical porcine pestivirus from piglets with congenital tremor in Japan.

Atypical porcine pestivirus (APPV), which has been confirmed to be associated with congenital tremor (CT) in pigs, is a newly discovered porcine virus that has been found in the Americas, Europe and Asia; however, no report of APPV in Japan has been published. We identified an APPV in the central nervous system of Japanese piglets with CT and firstly determined and analysed the complete genome sequence. Phylogenetic analysis using the complete genome nucleotide sequence of the Japanese APPV, named Anna/2020, and those of APPVs from the NCBI database showed that APPVs were divided into three genotypes (genotypes 1 to 3), and that Anna/2020 clustered with the genotype 3 APPV strains, but distantly branched from these strains. Pairwise complete coding region nucleotide sequence comparisons revealed that there was 94.0%- 99.7% sequence identity among the genotype 3 strains, while Anna/2020 showed 87.0%-89.3% identity to those genotype 3 strains, suggesting that Anna/2020 represents a novel APPV lineage within genotype 3. Retrospective examinations using RT-PCR revealed one genotype 1 and two novel genotype 3 APPVs from pigs without CT, and that novel genotype 3 APPVs have been prevalent in Japan since at least 2007.

Endothelial ganglioside GM3 regulates angiogenesis in solid tumors.

Cancer cells require oxygen and nutrients for growth, making angiogenesis one of the essential components of tumor growth. Gangliosides, constituting membrane lipid rafts, regulate intracellular signal transduction and are involved in the malignancy of cancer cells. While endothelial cells, as well as cancer cells, express vast amounts of gangliosides, the precise function of endothelial gangliosides in angiogenesis remains unclear. In this study, we focused on gangliosides of vascular endothelial cells and analyzed their functions on tumor angiogenesis. In human breast cancer, GM3 synthase was highly expressed in vascular endothelial cells as well as immune cells. Angiogenesis increased in GM3S-KO mice. In BAEC, RNA interference of GM3S showed increased cellular invasion and oxidative stress tolerance through activation of ERK. In the breast cancer model, GM3-KO mice showed an increase in tumor growth and angiogenesis. These results suggest that the endothelial ganglioside GM3 regulates tumor angiogenesis by suppressing cellular invasion and oxidative stress tolerance in endothelial cells.

Lipid-soluble polyphenols from sweet potato exert antitumor activity and enhance chemosensitivity in breast cancer.

Polyphenols are abundant in vegetables and fruit. They have been shown to have various antitumor, antioxidant, and anti-inflammatory effects. Here, we extracted the lipid-soluble fraction of polyphenols from fermented sweet potato (Ipomoea batatas). These lipid-soluble polyphenols mainly contained caffeic acid derivatives with strong antioxidant ability, which we hypothesized to affect diseases for which oxidative stress is a factor, such as cancer. We therefore investigated the antitumor and chemo-sensitizing effects of lipid-soluble polyphenols on E0771 murine breast cancer cells. The lipid-soluble polyphenols accumulated in the cells' cytoplasm due to its high lipophilicity, and reduced reactive oxygen species through its strong antioxidant activity. The lipid-soluble polyphenols also arrested the cell cycle at G0/G1 by suppressing Akt activity, and enhanced the cytotoxicity of anticancer agents. In this model, lipid-soluble polyphenols inhibited tumor growth and enhanced the efficacy of chemotherapy drugs. These results suggest the potential of lipid-soluble polyphenols as a functional food to support cancer therapy.

Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals.

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with "exceptional" gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these "exceptional" lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.