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.

Diet-related factors strongly shaped the gut microbiota of Japanese macaques.

Although knowledge of the functions of the gut microbiome has increased greatly over the past few decades, our understanding of the mechanisms governing its ecology and evolution remains obscure. While host genetic distance is a strong predictor of the gut microbiome in large-scale studies and captive settings, its influence has not always been evident at finer taxonomic scales, especially when considering among the recently diverged animals in natural settings. Comparing the gut microbiome of 19 populations of Japanese macaques Macaca fuscata across the Japanese archipelago, we assessed the relative roles of host genetic distance, geographic distance and dietary factors in influencing the macaque gut microbiome. Our results suggested that the macaques may maintain a core gut microbiome, while each population may have acquired some microbes from its specific habitat/diet. Diet-related factors such as season, forest, and reliance on anthropogenic foods played a stronger role in shaping the macaque gut microbiome. Among closely related mammalian hosts, host genetics may have limited effects on the gut microbiome since the hosts generally have smaller physiological differences. This study contributes to our understanding of the relative roles of host phylogeography and dietary factors in shaping the gut microbiome of closely related mammalian hosts.

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.

Isolation and characterization of mammalian orthoreovirus type 3 from a fecal sample from a wild boar in Japan.

Mammalian orthoreoviruses (MRVs) have been identified in various mammalian species, including humans, bats, and pigs. However, isolation and complete genome sequences of MRVs from wild boars have not yet been reported. In this study, we isolated, sequenced, and analyzed an MRV from a free-living wild boar in Japan using the porcine-sapelovirus-resistant cell line N1380. Complete and empty virus particles were obtained from the N1380 cell culture supernatants, and complete genome sequences were obtained from complete virus particles. Sequence analysis revealed that the isolated MRV, named TY-14, could be classified as MRV3 and had a close genetic relationship to an MRV2 isolate from a lion in a Japanese zoo (L2, L3, and M3 genes) and a human MRV2 isolate from Japan (S2 gene). Phylogenetic analysis showed that TY-14 clustered only with bat MRVs in the M1 phylogenetic tree but formed a cluster with several animal MRVs in the M2 and S3 phylogenetic trees and branched independently in the L1, S1, and S4 phylogenetic trees, suggesting a genetic relationship to viruses of unknown origin. Recombination events were identified in the M2 gene. These results suggest that TY-14 was generated by reassortment and recombination events involving MRVs circulating in Japan, viruses from bats, and other viruses of unknown origin.

Inter-individual variation in the diet within a group of Japanese macaques and its relationship with social structure investigated by stable isotope and DNA analyses.

We investigated individual variation in diet in relation to age-sex class and kin relationship in 28 of 40 members of a small group of wild Japanese macaques (Macaca fuscata). We used stable isotope ratios from hair as an index of individual dietary profiles, genetic relatedness as an index of kin relationship, and mitochondrial DNA (mtDNA) haplotype as a marker of being an immigrant or native member of the group. The range of carbon and nitrogen stable isotope ratios from hair of individual macaques (δ13C: -24.1‰ to -22.6‰, δ15N:3.8-5.5‰), which reflected their diet over a period of ~ 6 months, implied small individual variation in diet. The results of PERMANOVA implied that there were no significant effects of age class, sex, or mtDNA haplotype on hair stable isotope ratios between individuals, or on the variation in individual diet. However, the isotope values of males with mtDNA haplotypes that differed from those of the native females appeared to differ from those of other group members, which implies that immigrant males might have had a different diet profile from that of native group members. Furthermore, there was a weak correlation trend between genetic relatedness and differences in stable isotope ratios between pairs of individuals. Differences in stable isotope values were more marked in pairs with a more distant genetic relationship. This implies that within the group, closely related kin tended to forage together to avoid competing for food. However, this effect might have been weak because the size of the group was small relative to the size of the food patches, thereby reducing competition.

Multiple genotypes of enterovirus G carrying a papain-like cysteine protease (PL-CP) sequence circulating on two pig farms in Japan: first identification of enterovirus G10 carrying a PL-CP sequence.

Two and three genotypes of enterovirus G (EV-G) carrying a papain-like cysteine protease (PL-CP) sequence were detected on two pig farms and classified into genotypes G1 and G10, and G1, G8, and G17, respectively, based on VP1 sequences. A G10 EV-G virus bearing a PL-CP sequence was detected for the first time. Phylogenetic analysis of the P2 and P3 regions grouped the viruses by farm with high sequence similarity. Furthermore, clear recombination break points were detected in the 2A region, suggesting that PL-CP EV-G-containing strains gained sequence diversity through recombination events among the multiple circulating EV-G genotypes on the farms.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

First identification of Sapoviruses in wild boar.

Sapoviruses (SaVs) are enteric viruses that have been detected in human and animals previously; however, SaVs have not been identified in wild boar yet. Using a metagenomics approach, we identified SaVs in fecal samples of free-living wild boars in Japan for the first time. Six of the 48 specimens identified belonged to one genogroup (G)III, one GV and four GVI SaV sequence reads. We successfully determined complete genome of GV and GVI SaV strains using the long reverse transcription PCR strategy and the 5' rapid amplification of cDNA end method. Phylogenetic tree analysis and pairwise distance calculation revealed that GV SaV detected from wild boar was related to recently assigned GV.5 strains from pig, while GVI SaV was assigned to a new genotype within GVI. Moreover, wild boar may act as a reservoir for transmission of SaVs to the pig population (and vice versa) because GIII, GV, and GVI SaVs were all detected in pigs previously.

Tumor Resection May Improve Survival in Patients With Soft Tissue Sarcoma Aged 75 Years and Older.

BACKGROUND/AIM: This study aimed to analyze the decision-making process of treatment and the clinical outcomes of patients with STS aged 75 years and older. PATIENTS AND METHODS: Overall, 72 patients were reviewed. The mean and median ages were 82 and 81 years, respectively. RESULTS: Of the 72 patients, 17 did not undergo surgical resection. Age was related to the decision to pursue surgical treatment. Resection was performed in 55 patients. The 5-year overall survival was 51.8%. Patients with an American Society of Anesthesiologists physical status (ASA-PS) score of 3 had a worse survival. Twenty-five patients developed metastasis. The median survival time after metastasis in 12 patients who received metastasectomy and/or lung radiofrequency ablation was superior (18.2 months) to that of the remaining 13 patients without any treatment (3.2 months). CONCLUSION: Surgical treatment should be considered in elderly patients if their physical condition is not poor. ASA-PS score may be a tool to indicate physical condition.

STAT3 inhibitor, cucurbitacin I, is a novel therapeutic agent for osteosarcoma.

The development of clinical agents remains a costly and time-consuming process. Although identification of new uses of existing drugs has been recognized as a more efficient approach for drug discovery than development of novel drugs, little screening of drugs that might be used for a rare malignant tumor such as osteosarcoma (OS) has been performed. In this study, we attempted to identify new molecular targeted agents for OS by employing Screening Committee of Anticancer Drugs (SCADS) kits. To screen compounds for OS treatment, their effect on cell viability of the OS cell lines 143B, MG63, HOS, SAOS-2, and HUO9 were evaluated. Candidate drugs were narrowed down based on a global anti-proliferative effect against these five OS cell lines. After excluding cytotoxic compounds and compounds unsuitable for in vivo administration, cucurbitacin I was extracted. Cucurbitacin I has been found to have cytotoxic and anti-proliferative properties against several tumors through inhibition of signal transducer and activator of transcription 3 (STAT3) activation. Cucurbitacin I dose- and time-dependently inhibited the proliferation of all five OS cell lines. Following cucurbitacin I treatment, STAT3 was inactivated and analysis of Mcl-1, cleaved PARP and caspase-3 indicated apoptosis induction. Expression of cell cycle regulator proteins, such as phospho-cyclin D1, c-Myc and survivin, were suppressed. Finally, cucurbitacin I potently inhibited the tumor growth of human OS 143B cells in nude mice. Our in vitro and in vivo results suggest that STAT3 inhibition by cucurbitacin I will be an effective and new approach for the treatment of OS.

Impact of plasma fibrinogen levels in benign and malignant soft tissue tumors.

BACKGROUND: Fibrinogen, a 340 kDa glycoprotein synthesized in the liver, is known to be involved in tumor angiogenesis, enlargement, and metastasis. Elevated plasma fibrinogen levels are associated with tumor progression in many cancer patients. However, there are no reports about differences in fibrinogen levels between benign and malignant soft tissue tumors. OBJECTIVES: The purpose of this study was to clarify whether preoperative plasma fibrinogen levels can be used for differential diagnosis of benign or malignant soft tissue tumors. METHODS: The plasma fibrinogen levels from 102 primary soft tissue tumor patients were measured before biopsy or treatment. Fibrinogen levels were analyzed and compared to various clinical parameters. RESULTS: According to receiver operating characteristic (ROC) curve analysis, a threshold of serum fibrinogen of 315 mg/dL identified malignant patients with 60.9% sensitivity and 87.5% specificity. The diagnostic accuracy was evaluated by area under the curve (AUC: 0.805). Over 315 mg/dL of fibrinogen was associated with a significantly increased risk of malignancy by multiple logistic regression analysis (OR: 6.452, p= 0.0004). CONCLUSIONS: We demonstrated that plasma fibrinogen levels have a relationship with tumor malignancy of soft tissue tumors. High fibrinogen levels can be a helpful subsidiary tool for the prediction of malignant soft tissue tumors with other diagnostic tools.

Characteristics of stable isotope signature of diet in tissues of captive Japanese macaques as revealed by controlled feeding.

We determined the magnitude of isotopic fractionation of carbon and nitrogen stable isotope ratios (as enrichment factors, Δδ(13)C and Δδ(15)N, respectively) between the tissues and diets of captive Japanese macaques (Macaca fuscata) using a controlled feeding experiment, to provide basic data for reconstructing their feeding habits. The Δδ(13)C and Δδ(15)N values, respectively, were 0.9 ± 0.2 ‰ (mean ± standard deviation, SD) and 3.0 ± 0.3 ‰ for whole blood, 1.3 ± 0.2 ‰ and 4.3 ± 0.3 ‰ for plasma, and 0.8 ± 0.2 ‰ and 3.0 ± 0.2 ‰ for red blood cells. However, the Δδ(13)C and Δδ(15)N values for hair were 2.8 ± 0.3 ‰ and 3.4 ± 0.2 ‰, respectively. No difference was detected in the δ(13)C and δ(15)N values of hair sampled from different parts of the body. We investigated the effects of diet on δ(13)C in growing hair by alternating the diet of the macaques each month between two diets that differed markedly in δ(13)C. Hair regrown after shaving repeatedly recorded the δ(13)C of the diet consumed during the time of hair growth. On the other hand, hair naturally grown during the diet-change experiment did not show a clear pattern. One possible reason is that the hair had grown abnormally under unnatural indoor conditions and showed complicated isotope signatures. To reconstruct the long-term feeding history of Japanese macaques, we need to further clarify the relationships between the stable isotope signature of diet and various body tissues.

Estimating the success rate of ovulation and early litter loss rate in the Japanese black bear (Ursus thibetanus japonicus) by examining the ovaries and uteri.

In order to develop a method for estimating the success/failure rates of reproductive processes, especially those of ovulation and neonate nurturing, in the Japanese black bear (Ursus thibetanus japonicus), we examined offspring status, corpora lutea (CLs), placental scars (PSs) and corpora albicantia (CAs) in 159 females (0-23 years old) killed as nuisances on Honshu Island of Japan during 2001-2009. PSs were found to remain in the uterus at least until November of the year of parturition. CA detectability began to decline after September of the year of parturition. Monthly and age-specific proportions of CL-present females revealed that the post-mating season starts in August, and that the age of first ovulation is 4 years. These results indicate that the success rate of ovulation (SRO: the probability that solitary/non-lactating mature females actually succeed in ovulation) can be estimated by calculating the proportion of CL-present females among > or = 4-year-old females without PSs captured from August to November; the early litter loss rate (ELLR: the probability that parenting females lose all of their cubs [0-year-old offspring] before mating season) can be estimated by calculating the proportion of CL-present females among those with PSs and CAs captured in August or later. The estimated values of SRO and ELLR were 0.93 (62/67) and 0.27 (6/22), respectively.

Evaluation of stored body fat in nuisance-killed Japanese black bears (Ursus thibetanus japonicus).

We evaluated the stored body fat of Japanese black bears (Ursus thibetanus japonicus) killed as nuisances in Gifu and Fukushima prefectures, Japan, during 2005-2007. We employed femur marrow fat (FMF), modified kidney fat index (mKFI), and abdominal subcutaneous fat (ASF) as indices for quantitative evaluation. We examined the basic characteristics of these indices, such as seasonality, age and sex dependency, and the quantitative relationship among them. mKFI and ASF increased towards the beginning of the denning period (December), while FMF was relatively stable throughout the sampling period (July-December). In cubs, all indices showed significantly lower values than in the older age classes. There seemed to be a catabolizing order between FMF and mKFI, but not between mKFI and ASF. We also evaluated the yearly change in the indices, and discussed its relevance to the incidence of bear intrusion into human residential areas. Bears nuisance-killed in summer (July-September) 2006 had a significantly larger amount of stored body fat than those killed in summer 2007, although the number of nuisance kills was larger in 2006 than in 2007. This suggests that poor nutritional condition is not a direct cause of bear intrusion.

Postglacial population expansion of Japanese macaques (Macaca fuscata) inferred from mitochondrial DNA phylogeography.

We investigated the diversity and phylogeography of mitochondrial DNA (mtDNA) in Japanese macaques (Macaca fuscata), an endemic species in Japan that has the northernmost distribution of any non-human primate species. DNA samples from 135 localities representing the entire range of this species were compared. A total of 53 unique haplotypes were observed for the 412-bp partial mtDNA control region sequence, with length variation distinguishing the two subspecies. Clustering analyses suggested two putative major haplogroups, of which one was geographically distributed in eastern Japan and the other in western Japan. The populations in the east showed lower mtDNA diversity than those in the west. Phylogeographical relationships of haplotypes depicted with minimum spanning network suggested differences in population structure. Population expansion was significant for the eastern but not the western population, suggesting establishment of the ancestral population was relatively long ago in the west and recent in the east. Based on fossil evidence and past climate and vegetation changes, we inferred that the postulated population expansion may have taken place after the last glacial period (after 15,000 years ago). Mitochondrial DNA showed contrasting results in both variability and phylogenetic status of local populations to those of previous studies using protein variations, particularly for populations in the periphery of the range, with special inference on habitat change during the glacial period in response to cold adaptation.