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Intron retention (IR) is a regulatory mechanism that can retard protein production by acting at the level of mRNA processing. We recently demonstrated that IR occurs at the pre-symptomatic state during the aging process of a mouse model of aging, providing a promising biomarker for that state, and can be restored to the normal state by juzentaihoto (JTT), a Japanese herbal medicine (Kampo) (Okada et al. 2021). Here we characterized the genes that accumulate retained introns, examined the biological significance of increased IR in these genes for the host, and determined whether drugs other than JTT can have this effect. By analyzing RNA-sequencing data generated from the hippocampus of the 19-week-old SAMP8 mouse, a model for studying age-related depression and Alzheimer's disease, we showed that genes with increased IR are generally involved in multiple metabolic pathways and have pivotal roles in sensing homeostasis. We thus propose that IR is a stress response and works to fine-tune the expression of many downstream target genes, leading to lower levels of their translation under stress conditions. Interestingly, Kampo medicines, as well as other organic compounds, restored splicing of a specific set of retained introns in these sensor genes in accordance with the physiological recovery conditions of the host, which corresponds with the recovery of transcripts represented by differentially expressed genes. Thus, analysis of IR genes may have broad applicability in evaluating the pre-symptomatic state based on the extent of IR of selective sensor genes, opening a promising early diagnosis of any diseases and a strategy for evaluating efficacies of several drugs based on the extent of IR restoration of these sensor genes., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. Using klotho mice at the pre-symptomatic state, we discovered that retained-introns accumulated in several organs including the liver and that among these retained introns in the liver a subset was recovered to the normal state by a Japanese traditional herbal medicine. This is the first report of IR recovery by a medicine. IR-recovered genes fell into two categories: those involved in liver-specific metabolism and in splicing. Metabolome analysis of the liver showed that the klotho mice were under starvation stress. In addition, our differentially expressed gene analysis showed that liver metabolism was actually recovered by the herbal medicine at the transcriptional level. By analogy with the widespread accumulation of intron-retained pre-mRNAs induced by heat shock stress, we propose a model in which retained-introns in klotho mice were induced by an aging stress and in which this medicine-related IR recovery is indicative of the actual recovery of liver-specific metabolic function to the healthy state. Accumulation of retained-introns was also observed at the pre-symptomatic state of aging in wild-type mice and may be an excellent marker for this state in general., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The bacterium Campylobacter insulaenigrae was first isolated from marine mammals of Scotland in 2004. Only one case of C. insulaenigrae infection in humans has been previously reported., Case Presentation: An 89-year-old Japanese man without dementia was admitted to our hospital, because he presented with a fever of 38 °C and weakness in right leg since 5 days. He had organized chronic subdural hematoma (CSH), and no history of pre-infection. At the time of admission, he had paralysis of the extraocular muscle, ataxia, and low manual muscle test score of the right side. He was suspected to have Miller Fisher syndrome; however, these symptoms improved without any treatment. On day 22 in the hospital, the patient presented a fever of 38.8 °C, left cranial nerve disorder, and hemiplegia. On day 25, the patient presented with signs of meningeal irritation; cerebrospinal fluid examination indicated an increase in the number of apocytes and a low glucose level. A contrast magnetic resonance imaging (MRI) scan of the patient's head indicated a contrast enhancement effect in his right meninges. The blood culture showed presence of spirillums; 16S rRNA gene sequencing confirmed that the spirillums in the blood culture were Campylobacter insulaenigrae (C. insulaenigrae). We started treatment with meropenem for bacteremia and meningitis. When the symptoms improved, meropenem was replaced with ampicillin, based on the result of the drug sensitivity test. The treatment continued for 4 weeks., Conclusions: We report the first case of meningitis caused by C. insulaenigrae bacteremia in humans, and the second clinical report of C. insulaenigrae infection in humans. The bacterial strains isolated from humans and marine mammals had different genotypes. This suggests that different genotypes could be responsible for differences in the hosts. Further case studies are needed to establish the reasons behind the difference in the manifestations of C. insulaenigrae infections reported so far.

Background: Bacteroides dorei is an anaerobic gram-negative bacterium first described in 2006. Because of the high similarity in mass spectra between B. dorei and Bacteroides vulgatus, discriminating between these species is arduous in clinical practice. In recent decades, 16S rRNA gene sequencing has been a complementary method for distinguishing taxonomically close bacteria, including B. dorei and B. vulgatus, at the genus and species levels. Consequently, B. dorei has been shown to contribute to some diseases, including type 1 autoimmune diabetes mellitus and atherosclerotic diseases. However, there are no reports on invasive infectious diseases caused by B. dorei. This report describes the first case of direct invasion and colonisation of human tissue by B. dorei, thus providing a warning regarding the previously proposed application of B. dorei as a live biotherapeutic for atherosclerotic diseases., Case Presentation: A 78-year-old Japanese man complained of intermittent chest/back pain and was diagnosed with a mycotic thoracic aortic aneurysm by enhanced computed tomography on admission. Despite strict blood pressure control and empirical antibiotic therapy, the patient's condition worsened. To prevent aneurysmal rupture and eliminate infectious foci, the patient underwent surgical treatment. The resected specimen was subjected to tissue culture and 16S rRNA gene sequencing analysis to identify pathogenic bacteria. A few days after the surgery, culture and sequencing results revealed that the pathogen was B. dorei/B. vulgatus and B. dorei, respectively. The patient was successfully treated with appropriate antibacterial therapy and after improvement, was transferred to another hospital for rehabilitation on postoperative day 34. There was no recurrence of infection or aneurysm after the patient transfer., Conclusions: This report describes the first case of invasive infectious disease caused by B. dorei, casting a shadow over its utilisation as a probiotic for atherosclerotic diseases.

NTRK fusion-positive tumors are known to be highly sensitive to TRK inhibitors, such as larotrectinib and entrectinib. Therefore, identification of patients who can potentially benefit from these inhibitors is important; however, the frequency of NTRK fusions in Japanese patients with colorectal cancer (CRC) is unknown. We performed pan-TRK staining using TMA-based immunohistochemistry (IHC) on samples from 971 consecutive Japanese CRC cases from a single institution. Positive cases were further analyzed using NanoString and subsequent targeted RNA sequencing. We found three positive cases using TRK-IHC. Furthermore, the Nanostring assay supported the presence of NTRK fusion in these cases. Subsequent targeted RNA-sequencing and RT-PCR revealed two cases with TPM3-NTRK1 and one with TPR-NTRK1. The TNM stages of these cases were stage I, stage IIA, and stage IIIB, and two showed microsatellite instability-high status. Next-generation sequencing analysis using Cancer hotspot panel revealed TP53 and SMAD4 mutations in separate cases. IHC of β-catenin did not show nuclear accumulation. We found three cases (0.31%) of CRC with NTRK1 fusion among 971 consecutive Japanese CRC cases. No potential driver alterations other than NTRK fusion were identified in these three patients.

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747-Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA-sequencing, RAD-sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA-sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO-lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage-specific selection on mitochondria-related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population., (© 2020 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.)

Somatic embryogenesis (SE), which is in vitro regeneration of plant bodies from somatic cells, represents a useful means of clonal propagation and genetic engineering of forest trees. While protocols to obtain calluses and induce regeneration in somatic embryos have been reported for many tree species, the knowledge of molecular mechanisms of SE development is still insufficient to achieve an efficient supply of somatic embryos required for the industrial application. Cryptomeria japonica, a conifer species widely used for plantation forestry in Japan, is one of the tree species waiting for a secure SE protocol; the probability of normal embryo development appears to depend on genotype. To discriminate the embryogenic potential of embryonal masses (EMs) and efficiently obtain normal somatic embryos of C. japonica, we investigated the effects of genotype and transcriptome on the variation in embryogenic potential. Using an induction experiment with 12 EMs each from six genotypes, we showed that embryogenic potential differs between/within genotypes. Comparisons of gene expression profiles among EMs with different embryogenic potentials revealed that 742 differently expressed genes were mainly associated with pattern forming and metabolism. Thus, we suggest that not only genotype but also gene expression profiles can determine success in SE development. Consistent with previous findings for other conifer species, genes encoding leafy cotyledon, wuschel, germin-like proteins, and glutathione-S-transferases are likely to be involved in SE development in C. japonica and indeed highly expressed in EMs with high-embryogenic potential; therefore, these proteins represent candidate markers for distinguishing embryogenic potential., Competing Interests: The authors have declared that no competing interests exist.

Many macrofungal cryptic species remain unidentified. A possible solution is to increase the number of loci analyzed and use rigorous statistics for macrofungal species delimitation. To validate this assumption, cryptic species of the Hypholoma fasciculare complex, a group of common wood-decomposing fungi, were attempted to be delineated. Massively parallel sequencing of mitochondrial ribosomal RNA (mt_rRNA), nuclear ribosomal internal transcribed spacer (ITS) region, and 24 single-copy genes were performed for 96 specimens collected in Japan. Then, the species boundaries were inferred using comparative gene genealogies (mt_rRNA vs. ITS), Bayesian Poisson tree process (bPTP) model for the phylogeny of concatenated nuclear sequences, and analysis of molecular variance (AMOVA) for single nucleotide polymorphisms. In both the mt_rRNA and ITS phylogenies, the H. fasciculare complex was not divided into well-supported clades. Nevertheless, based on the bPTP, two mitochondrial haplotypes were inferred to represent distinct species (H. fasciculare and H. subviride). The results of AMOVA also indicated that the differentiation of nuclear loci can be explained mostly by differences between haplotype. These results suggest that it is necessary to increase the number of target loci to 20 or more and use both phylogeny-based and population genetics-based statistics for the accurate delimitation of macrofungal species.

Expression quantitative trait locus (eQTL) analyses have enabled us to predict the function of disease susceptibility SNPs. However, eQTL for the effector memory T cells (TEM) located in the lamina propria mononuclear cells (LPMCs), which play an important role in Crohn's disease (CD), are not yet available. Thus, we conducted RNA sequencing and eQTL analyses of TEM cells located in the LPMCs from IBD patients (n = 20). Genome-wide association study (GWAS) was performed using genotyping data of 713 Japanese CD patients and 2,063 controls. We compared the results of GWAS and eQTL of TEM, and also performed a transcriptome-wide association study using eQTL from Genotype Tissue Expression project. By eQTL analyses of TEM, correlations of possible candidates were confirmed in 22,632 pairs and 2,463 genes. Among these candidates, 19 SNPs which showed significant correlation with tenascin-XA (TNXA) expression were significantly associated with CD in GWAS. By TWAS, TNFSF15 (FDR = 1.35e-13) in whole blood, ERV3-1 (FDR = 2.18e-2) in lymphocytes, and ZNF713 (FDR = 3.04e-2) in the sigmoid colon was significantly associated with CD. By conducting integration analyses using GWAS and eQTL data, we confirmed multiple gene transcripts are involved in the development of CD.

Use of anaerobic sludge digester is a common practice around the world for solids digestion and methane generation from municipal sewage sludge. Understanding microbial community structure is vital to get better insight into the anaerobic digestion process and to gain better process control. However, selective analysis of viable microorganisms is limited by DNA-based assays. In this study, propidium monoazide (PMA)-PCR with 16S rRNA gene sequencing analysis was used to distinguish live and dead microorganisms based on cell membrane integrity. Microbial community structures of PMA-treated and PMA-untreated anaerobic digester sludge samples were compared. Quantitative PCR revealed that 5-30% of the rRNA genes were derived from inactive or dead cells in anaerobic sludge digesters. This caused a significant decrease in the numbers of operational taxonomic units and Chao1 and Shannon indices compared with that of the PMA-untreated sludge. Microbial community analysis showed that majority of the viable microbiome consisted of Euryarchaeota, Bacteroidetes, Deltaproteobacteria, Chloroflexi, Firmicutes, WWE1, Spirochaetes, Synergistetes, and Caldiserica. On the other hand, after the PMA treatment, numbers of Alphaproteobacteria and Betaproteobacteria declined. These were considered residual microbial members. The network analysis also revealed a relationship among the OTUs belonging to WWE1 and Bacteroidales. PMA-PCR-based 16S rRNA gene sequencing analysis is an effective tool for uncovering viable microbiome in complex environmental samples.

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site for migratory ducks and endangered cranes. We have surveyed avian influenza viruses (AIVs) in this area since 2012 and isolated low-pathogenic AIVs (LPAIVs) of various subtypes every winter season. H3N8 LPAIVs were isolated during the 2012/13 and 2016/17 seasons, and H4N6 LPAIVs were isolated during the 2012/13 and 2013/14 seasons. In the 2017/18 season, one H3N8 and two H4N6 LPAIV strains were isolated from environmental water samples. Genetic and phylogenetic analysis for each gene segment from these H3N8 and H4N6 LPAIVs suggested that our isolates were genetic reassortants generated by intermixing between AIVs circulating not only in Eurasia but also in Africa and/or North America. Comparison of the genetic constellations of our three isolates with their counterparts isolated during previous seasons from the Izumi plain revealed a drastic transition in the genetic constellations of both subtypes. These findings emphasize the importance of continuous surveillance of AIVs on the Izumi plain.

Ovarian cancer has the highest mortality rate among gynecological cancers. Gene mutations are involved in the carcinogenesis, metastasis, and therapeutic response in ovarian cancer. However, the variety and proportion of gene mutation is not fully analyzed in Japanese ovarian cancer patients, especially, in those with recurrent tumors. In the present study, RNA-sequencing was performed for 32 clinical ovarian specimens obtained from 24 Japanese patients (24 primary cancer specimens and 8 recurrent specimens paired with corresponding primary cancer specimens). Mutations in 24 primary specimens were analyzed by comparing the sequence data mapped on RefSeq genes with those in the public online databases BRCA Exchange, COSMIC, ClinVar, and cBioportal. Mutations were observed in TP53 in 16 specimens (67%), BRCA1 in 9 (38%), BRCA2 in 13 (54%), ARID1A in 3 (13%), PIK3CA in 2 (8%), KRAS in 1 (4%), PTEN in 1 (4%), and CTNNB1 in 1 (4%), excluding synonymous mutations. Among those identified muations, 13 of 14 mutations in TP53, 10 of 11 mutations of BRCA1, 10 of 23 mutation positions of BRCA2, none of 7 mutations of ARID1A, 1 mutation of PIK3CA, and 1 mutation of CTNNB1 were consistent with those reported in the public online databases; however, the other mutations identified were novel. Comparison between matched-paired specimens of primary and recurrent tumors revealed the changes of mutational status in expressed RNAs. RNA-sequencing-based mutation analysis will be useful to reveal ethnic differences of gene mutations in ovarian cancer and to understand the contribution of gene mutations to recurrence.

Leptospira was isolated from environmental water in central Japan using selective medium comprising five antibiotics, namely sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5-fluorouracil. Of 100 water samples 57 (57%) were culture-positive and 50 pure cultures were isolated. Of the 50 cultures isolated from water 48 were classified into a saprophytic clade on the basis of 16S ribosomal RNA gene sequences. However, it was previously reported that isolates from soil in Japan belonged to pathogenic, intermediate, and saprophytic clades, the current findings suggest less diversity of Leptospira species in environmental water than that in soil in Japan., (© 2019 The Societies and John Wiley & Sons Australia, Ltd.)

Background: Kawasaki disease (KD) is a systemic vasculitis which may be associated with coronary artery aneurysms. A notable risk factor for the development of coronary artery aneurysms is resistance to intravenous immunoglobulin (IVIG) therapy, which comprises standard treatment for the acute phase of KD. The cause of IVIG resistance in KD is largely unknown; however, the contribution of genetic factors, especially variants in immune-related genes, has been suspected., Methods: To explore genetic variants related to IVIG-unresponsiveness, we designated KD patients who did not respond to both first and second courses of IVIG therapy as IVIG-unresponsive patients. Using genomic DNA from 30 IVIG-unresponsive KD patients, we performed pooled genome sequencing targeting 39 immune-related cytokine receptor genes., Results: The single nucleotide variant (SNV), rs563535954 (located in the IL4R locus), was concentrated in IVIG-unresponsive KD patients. Individual genotyping showed that the minor allele of rs563535954 was present in 4/33 patients with IVIG-unresponsive KD, compared with 20/1063 individuals in the Japanese genome variation database (odds ratio = 7.19, 95% confidence interval 2.43-21.47). Furthermore, the minor allele of rs563535954 was absent in 42 KD patients who responded to IVIG treatment (P = 0.0337), indicating that a low-frequency variant, rs563535954, is associated with IVIG-unresponsiveness in KD patients. Although rs563535954 is located in the 3'-untranslated region of IL4R, there was no alternation in IL4R expression associated with the mior allele of rs563535954. However, IVIG-unresponsive patients that exhibited the minor allele of rs563535954 tended to be classified into the low-risk group (based on previously reported risk scores) for prediction of IVIG-resistance. Therefore, IVIG-unresponsiveness associated with the minor allele of rs563535954 might differ from IVIG-unresponsiveness associated with previous risk factors used to evaluate IVIG-unresponsiveness in KD., Conclusion: These findings suggest that the SNV rs563535954 could serve as a predictive indicator of IVIG-unresponsiveness, thereby improving the sensitivity of risk scoring systems, and may aid in prevention of coronary artery lesions in KD patients.

Purpose: The diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20-40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants., Methods: Aberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES., Results: We identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases., Conclusion: These results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.

Between January and May in 2018, 17 male cases of hepatitis A were reported in Yokohama, Japan. Of these, 14 identified as men who have sex with men. The viral sequence in this outbreak was same as that of the recent European and Taiwanese outbreaks strain.

Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Ixodid ticks transmit several important viral pathogens. We isolated a new virus (Tofla virus: TFLV) from Heamaphysalis flava and Heamaphysalis formsensis in Japan. The full-genome sequences revealed that TFLV belonged to the genus Nairovirus, family Bunyaviridae. Phylogenetic analyses and neutralization tests suggested that TFLV is closely related to the Hazara virus and that it is classified into the Crimean-Congo hemorrhagic fever group. TFLV caused lethal infection in IFNAR KO mice. The TFLV-infected mice exhibited a gastrointestinal disorder, and positron emission tomography-computed tomography images showed a significant uptake of (18)F-fluorodeoxyglucose in the intestinal tract. TFLV was able to infect and propagate in cultured cells of African green monkey-derived Vero E6 cells and human-derived SK-N-SH, T98-G and HEK-293 cells. Although TFLV infections in humans and animals are currently unknown, our findings may provide clues to understand the potential infectivity and to develop of pre-emptive countermeasures against this new tick-borne Nairovirus.

Cucumber mosaic virus (CMV) is a damaging pathogen of over 200 mono- and dicotyledonous crop species worldwide. It has the broadest known host range of any virus, but the timescale of its evolution is unknown. To investigate the evolutionary history of this virus, we obtained the genomic sequences of 40 CMV isolates from brassicas sampled in Iran, Turkey and Japan, and combined them with published sequences. Our synonymous ('silent') site analyses revealed that the present CMV population is the progeny of a single ancestor existing 1550-2600 years ago, but that the population mostly radiated 295-545 years ago. We found that the major CMV lineages are not phylogeographically confined, but that recombination and reassortment is restricted to local populations and that no reassortant lineage is more than 251 years old. Our results highlight the different evolutionary patterns seen among viral pathogens of brassica crops across the world., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Human G8 Rotavirus A (RVA) strains are commonly detected in Africa but are rarely detected in Japan and elsewhere in the world. In this study, the whole genome sequence of the first human G8 RVA strain designated AU109 isolated in a child with acute gastroenteritis in 1994 was determined in order to understand how the strain was generated including the host species origin of its genes. The genotype constellation of AU109 was G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analyses of the 11 genome segments revealed that its VP7 and VP1 genes were closely related to those of a Hungarian human G8P[14] RVA strain and these genes shared the most recent common ancestors in 1988 and 1982, respectively. AU109 possessed an NSP2 gene closely related to those of Chinese sheep and goat RVA strains. The remaining eight genome segments were closely related to Japanese human G2P[4] strains which circulated around 1985-1990. Bayesian evolutionary analyses revealed that the NSP2 gene of AU109 and those of the Chinese sheep and goat RVA strains diverged from a common ancestor around 1937. In conclusion, AU109 was generated through genetic reassortment event where Japanese DS-1-like G2P[4] strains circulating around 1985-1990 obtained the VP7, VP1 and NSP2 genes from unknown ruminant G8 RVA strains. These observations highlight the need for comprehensive examination of the whole genomes of RVA strains of less explored host species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The pathogenesis of multiple sclerosis (MS), an autoimmune disease affecting the brain and spinal cord, remains poorly understood. Patients with MS typically present with recurrent episodes of neurological dysfunctions such as blindness, paresis, and sensory disturbances. Studies on experimental autoimmune encephalomyelitis (EAE) animal models have led to a number of testable hypotheses including a hypothetical role of altered gut microbiota in the development of MS. To investigate whether gut microbiota in patients with MS is altered, we compared the gut microbiota of 20 Japanese patients with relapsing-remitting (RR) MS (MS20) with that of 40 healthy Japanese subjects (HC40) and an additional 18 healthy subjects (HC18). All the HC18 subjects repeatedly provided fecal samples over the course of months (158 samples in total). Analysis of the bacterial 16S ribosomal RNA (rRNA) gene by using a high-throughput culture-independent pyrosequencing method provided evidence of a moderate dysbiosis in the structure of gut microbiota in patients with MS. Furthermore, we found 21 species that showed significant differences in relative abundance between the MS20 and HC40 samples. On comparing MS samples to the 158 longitudinal HC18 samples, the differences were found to be reproducibly significant for most of the species. These taxa comprised primarily of clostridial species belonging to Clostridia clusters XIVa and IV and Bacteroidetes. The phylogenetic tree analysis revealed that none of the clostridial species that were significantly reduced in the gut microbiota of patients with MS overlapped with other spore-forming clostridial species capable of inducing colonic regulatory T cells (Treg), which prevent autoimmunity and allergies; this suggests that many of the clostridial species associated with MS might be distinct from those broadly associated with autoimmune conditions. Correcting the dysbiosis and altered gut microbiota might deserve consideration as a potential strategy for the prevention and treatment of MS.

Background: In Japanese Red Cross (JRC) blood centers, blood collected from donors with serum alanine aminotransferase (ALT) levels of more than 60 U/L are disqualified even if serologically negative for transfusion-transmitted infections (TTIs). To assess potential risks of TTIs in plasma with elevated serum ALT levels in the current donor screening program of the JRC, we conducted a metagenomic analysis (MGA) of virome profiles in the plasma of blood donors with or without elevated serum ALT levels., Study Design and Methods: Based on serum ALT levels, donors were classified into three groups: "high," more than 79 U/L; "middle," 61 to 79 U/L; and "low," less than 61 U/L. We individually analyzed 100 plasma samples from each group by MGA, employing shotgun sequencing. Viral sequences detected using MGA were partly confirmed using real-time polymerase chain reaction (PCR)., Results: Donors with high and middle ALT levels were significantly younger than those with low ALT levels, and more than 90% were males. Herpesviridae, Anelloviridae, Picornaviridae, and Flaviviridae sequences were identified in plasma samples, and their distribution and frequency were not significantly different among the three groups., Conclusion: The serum ALT test may be unsuitable for monitoring for additional risks of TTIs in blood donors who were negative for typical TTIs using serologic and nucleic acid tests. Although MGA is less sensitive than PCR, it remains the best technology to detect known viruses in these donors., (© 2015 AABB.)

Black queen cell virus (BQCV) has been found in honey bees worldwide. By using the reverse transcription polymerase chain reaction (RT-PCR) technique, BQCV was detected in a non-native species, Apis mellifera L., collected in both Thailand and Japan, and three other honey bee species (Apis cerana indica F., Apis dorsata F., and Apis florae F.) native to Thailand and Apis cerana japonica F. native to Japan. Based on the capsid coding region, the phylogenetic analysis showed that the BQCV strains found in A. cerana indica and A. cerana japonica were similar within the group and closer to BQCV in Asia. It is interesting to note that the genetic variation of the BQCV isolates was more associated with geographic origin than the host bee species from which the isolates were obtained., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Physiological studies of a unique endocrine gland in fish, named corpuscles of Stannius (CS), described a Ca2(+)-regulatory function for this gland mediated by stanniocalcin-1, a hypocalcemic polypeptide hormone. However, to date, the endocrine functions of the glands have not been completely elucidated.We hypothesized that other unidentified active principles in the glands are involved in the regulation of plasma ion (Na(+), Ca2(+)) and/or blood pressure. In this study, transcriptome sequencing of CS glands was performed using Japanese eels (Anguilla japonica) adapted to freshwater (FW) or seawater (SW) to reveal the presence and differential expression of genes encoding proteins related to the ion-osmoregulatory and pressor functions. We acquired a total of 14.1 Mb and 12.1 Mb quality-trimmed reads from the CS glands collected from FW and SW adapted eels, respectively. The de novo assembly resulted in 9254 annotated genes. Among them, 475 genes were differentially expressed with 357 up- and 118 down-regulated in the SW group. Gene ontology analysis further demonstrated the presence of natriuresis and pressor related genes. In summary, ours is the first study using high-throughput sequencing to identify gene targets that could explain the physiological importance of the CS glands.

Japanese flounder (Paralichthys olivaceus) is one of the economic important fish in China. Sexual dimorphism, especially the different growth rates and body sizes between two sexes, makes this fish a good model to investigate mechanisms responsible for such dimorphism for both fundamental questions in evolution and applied topics in aquaculture. However, the lack of "omics" data has hindered the process. The recent advent of RNA-sequencing technology provides a robust tool to further study characteristics of genomes of nonmodel species. Here, we performed de novo transcriptome sequencing for a double haploid Japanese flounder individual using Illumina sequencing. A single lane of paired-end sequencing produced more than 27 million reads. These reads were assembled into 107,318 nonredundant transcripts, half of which (51,563; 48.1%) were annotated by blastx to public protein database. A total of 1051 genes that had potential alternative splicings were detected by Chrysalis implemented in Trinity software. Four of 10 randomly picked genes were verified truly containing alternative splicing by cloning and Sanger sequencing. Notably, using a doubled haploid Japanese flounder individual allow us to analyze gene duplicates. In total, 3940 "single-nucleotide polymorphisms" were detected form 1859 genes, which may have happened gene duplicates. This study lays the foundation for structural and functional genomics studies in Japanese flounder., (Copyright © 2014 Wang et al.)

The biological and genetic diversity of Wheat yellow mosaic virus (WYMV) isolates in Japan was characterized. On the basis of wheat cultivar reactions, 14 WYMV isolates from various places were classified into pathotypes I, II, or III. These were distributed in central, northern, and southern areas of Japan, respectively. WYMV isolates comprised three genotypes (A, A' and B) based on amino acid differences in RNA1 and two genotypes (a and b) based on amino acid differences in RNA2. A correlation was found between the WYMV RNA1-based genotype and pathotype, suggesting that factors associated with pathogenicity map to RNA1. Genotype Aa and A'a were distributed mainly in the central to southern areas of Japan, and genotype Bb was found in northern areas of Japan, as shown by reverse-transcription polymerase chain reaction restriction fragment length polymorphism analysis. Chinese isolates YA and YZ were closely related to genotypes Bb and Aa, respectively. Wheat was introduced from China to Japan in the 4th and 5th centuries, and the two genotypes of WYMV might also have been introduced with the crop from China and later adapted to local wheat cultivars in Japan.

Boron is an essential micronutrient for plants, but can above certain concentrations be toxic to living organisms. A major environmental concern is the removal of boron from contaminated water and fly ash. For this purpose, the samples were collected from a fly ash dumping site, Nagasaki prefecture, Japan. The chemical characteristics and heavy metal concentration of the samples were performed by X-ray fluorescent analysis and leaching test. For bacterial analysis, samples were collected in sterile plastic sheets and isolation was carried out by serial dilution method. The boron tolerant isolates that showed values of maximum inhibitory concentration toward boron ranging from 100 to 260 mM level were screened. Based on 16S rRNA sequencing and phylogenetic analysis, the isolates were most closely related to the genera Bacillus, Lysinibacillus, Microbacterium and Ralstonia. The boron tolerance of these strains was also associated with resistant to several heavy metals, such as As (III), Cr (VI), Cd, Cu, Pb, Ni, Se (III) and Zn. Indeed, these strains were arsenic oxidizing bacteria confirmed by silver nitrate test. These strains exhibited their salt resistances ranging from 4 to 15 % were determined in Trypticase soy agar medium. The boron tolerant strains were capable of removing 0.1-2.0 and 2.7-3.7 mg l(-1) boron from the medium and fly ash at 168 h. Thus, we have successfully identified the boron tolerant and removal bacteria from a fly ash dumping site for boron remediation.

Culex flavivirus (CxFV) is an insect-specific flavivirus that was first reported in 2007 in Japan. CxFV strains were isolated from Culex tritaeniorhynchus Giles and Culex pipiens L. group mosquitoes and genetically characterized in Toyama Prefecture, Japan, from 2004 to 2009, to reveal host specificity, mode of transmission, and seasonal and geographical distribution. The minimum infection rate (MIR) of CxFV within Cx. tritaeniorhynchus populations was 0.3 and much lower than that within Cx. pipiens group (17.9). The complete genome sequences of 11 CxFV isolates (four from Cx. tritaeniorhynchus and seven from Cx. pipiens group) consisted of 10,835-10,837 nucleotides. When these 11 isolates and five reference strains (NIID-21-2 and Tokyo strains from Japan, Iowa07 and HOU24518 strains from the United States, H0901 strain from China) were compared, there were 95.2-99.2% nucleotide and 98.1-99.8% amino acid identities. Phylogenetic analysis showed that the 11 isolates were divided into four clusters. One cluster consisted of five isolates from Cx. pipiens group and Cx. tritaeniorhynchus from one site and their nucleotide sequences almost completely matched. One cluster consisted of an isolate with a unique sequence from a Cx. pipiens group mosquito captured in an aircraft from Taiwan, suggesting that it was introduced from abroad. CxFV strains were divided into several groups according to countries when nucleotide sequences of CxFV available in GenBank and 11 Toyama isolates were compared. These results suggest that CxFV is maintained in nature among Culex mosquitoes in a mosquito habitat-specific but not a species-specific manner.

Porcine rotavirus B (RVB) has frequently been detected in the diarrhea of suckling and weaned pigs. Because it is difficult to propagate RVBs serially in cell culture, little genetic information about RNA segments other than VP7, NSP1 and NSP2 is available for porcine RVBs. We conducted a phylogenetic analysis focusing on nonstructural protein 5 (NSP5) using 22 porcine RVB strains, which were identified in fecal samples collected around Japan. Sequence analysis showed that NSP5 genes of RVBs contain one ORF, in contrast to the corresponding gene of RVAs that have two ORFs. Comparison of NSP5 amino acid sequences from porcine RVBs with other RVBs revealed that eight serine and serine/threonine residues at the N- and C-terminal regions are highly conserved among RVBs. Phylogenetic analysis also indicated the presence of six clusters (H1-H6) including rat, human, porcine and bovine plus ovine clusters with cut-off values of 78% at the nucleotide level. Moreover, the NSP5 genes of porcine RVBs were divided to three clusters. The data presented here demonstrates that several porcine RVBs with distinctive genotypes are circulating among farms throughout Japan. Our findings provide important new insights into the evolutionary dynamics of RVBs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Actinomycete strains K10-0485(T) and K10-0528(T) were isolated from the roots of Ophiopogon japonicus collected in Yokohama, Kanagawa Prefecture, Japan. The 16S ribosomal RNA (rRNA) gene sequences, morphological characteristics and chemotaxonomic data indicated that these strains belonged to the genus Actinoallomurus. Strain K10-0485(T) showed high similarity of the 16S rRNA gene sequence with A. luridus TT02-15(T) (99.1%), but the DNA-DNA hybridization relatedness values between strain K10-0485(T) and A. luridus TT02-15(T) were below 70%. Three species showed similarities of 16S rRNA gene sequences with K10-0528(T), namely A. spadix JCM 3146(T) (98.0%), A. purpureus TTN02-30(T) (98.0%) and A. luridus TT02-15(T) (97.9%), but all similarity values of the 16S rRNA gene sequences were lower than the boundary value (98.7%) for distinguishing as different species. Based on phylogenetic analyses, DNA-DNA hybridization relatedness and physiological characteristics, the two isolated strains should be classified as two new species in the genus Actinoallomurus, and we propose the names Actinoallomurus liliacearum sp. nov. and Actinoallomurus vinaceus sp. nov. The type strain of Actinoallomurus liliacearum is K10-0485(T) (=JCM 17938(T), BCC 49424(T), NBRC 108672(T)) and that of Actinoallomurus vinaceus is K10-0528(T) (=JCM 17939(T), BCC 49425(T), NBRC 108763(T)).

Immunohistochemical loss of keratin (K)13 is one of the most valuable diagnostic criteria for discriminating carcinoma in situ (CIS) from non-malignancies in the oral mucosa while K13 is stably immunolocalized in the prickle cells of normal oral epithelium. To elucidate the molecular mechanism for the loss of K13, we compared the immunohistochemical profiles for K13 and K16 which is not expressed in normal epithelia, but instead enhanced in CIS, with their mRNA levels by in-situ hybridization in formalin-fixed paraffin sections prepared from 23 CIS cases of the tongue, which were surgically removed. Reverse transcriptase-PCR was also performed using RNA samples extracted from laser-microdissected epithelial fragments of the serial paraffin sections in seven of the cases. Although more enhanced expression levels for K16 were confirmed at both the protein and gene levels in CIS in these seven cases, the loss of K13 was associated with repressed mRNA levels in four cases, but not in the other three cases. The results suggest that the loss of K13 is partly due to its gene repression, but may also be due to some unknown post-translational events.

The motile actinomycete strain RI50-RCA114(T) was isolated using rehydration and centrifugation method from a soil sample obtained from Rishiri Island in Japan. The taxonomic status of this organism was established using a polyphasic approach. Cells of strain RI50-RCA114(T) were Gram positive, aerobic, motile and formed irregular sporangia. The strain grew in the presence of 0-2% (w/v) NaCl, between pH 5 and 8, and over a temperature range of 20-37°C, with optimal growth at 30°C. Whole-cell hydrolysates of the strain contained meso-diaminopimelic acid, galactose, glucose and mannose, in addition to one unidentified O-methyl-hexose. The predominant menaquinone was MK-9(H(4)), and the major polar lipids comprised phosphatidylethanolamine, diphosphatidylglycerol and phosphatidyl-N-methylethylethanolamine. The major cellular fatty acids were iso-C(16:0), iso-C(15:0) and anteiso-C(17:0). Comparative 16S ribosomal RNA gene sequence analysis revealed that strain RI50-RCA114(T) had the closest sequence similarity with Actinoplanes globisporus JCM 3186(T) (97.6%). However, DNA-DNA hybridization assays as well as physiological and biochemical analyses differentiated strain RI50-RCA114(T) from its closest phylogenetic relative. On the basis of these data, we propose that strain RI50-RCA114(T) (=NBRC 108556(T)=BCC 49184(T)) be classified as the type strain of a novel Actinoplanes species and named Actinoplanes rishiriensis sp. nov.

To better understand the biogeography and relationship between temperature and community structure within microbial mats, the bacterial diversity of mats at a slightly alkaline, sulfide-containing hot spring was explored. Microbial mats that developed at temperatures between 75-52°C were collected from an area of approximately 1 m(2) in Nakabusa, Nagano, Japan. Bacterial 16S rRNA genes from these samples were examined by terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis. T-RFLP profiles revealed 66 unique fragments (T-RFs). Based on total T-RFs observed in environmental profiles and clone libraries, a temperature effect on diversity was determined, with complexity in the community increasing as temperature decreased. The T-RF pattern indicated four distinct community assemblages related to temperature. Members of the Aquificales and particularly the sulfur-oxidizing bacterium Sulfurihydrogenibium were present at all temperatures and were the dominant component of mats taken at 75-67°C. Sulfide oxidation, which persisted throughout the temperature gradient, was the presumed dominant pathway of primary production above 67°C. As temperature decreased, successive additions of anoxygenic and oxygenic phototrophs increased primary productivity, allowing for diversification of the community.

Interferon (IFN) induces the double-stranded RNA-dependent protein kinase (PKR) to inhibit viral replication. Two motifs of the PKR-binding domain exist in the E2 and the NS5A regions of the hepatitis C virus (HCV). These regions are called the PKR-eukaryotic transcription factor (elF2-alpha) phosphorylation homology domain (PePHD), and the IFN sensitivity-determining region (ISDR). Both regions are inhibited by PKR. Thus, several studies have reported the relationship between these regions and IFN responsiveness and the HCV viral load. However, the data obtained from these studies remain controversial. The aim of this study was to investigate the genomic heterogeneity of the PePHD and the ISDR in patients with genotype 3a and how this impacts HCV replication and the response to IFN therapy. Twenty-one male patients infected with HCV genotype 3a were studied. The PePHD was well conserved, and mutations were found in only one amino acid position in two patients. Patients with three or more mutations in the ISDR had lower viral loads than those with fewer than two mutations (192.2 ± 176.7 vs. 1279.4 ± 997.6 KIU/ml, P = 0.0277). Ten (71.4%) of 14 patients achieved a sustained virological response to IFN therapy. No specific amino acid substitutions in the PePHD and the ISDR were associated with IFN responsiveness; however, the number of mutations in the ISDR was significantly associated with the HCV viral load. The findings from this study suggest that the ISDR plays an important role in regulating viral replication in patients infected with HCV genotype 3a., (Copyright © 2011 Wiley-Liss, Inc.)

We previously showed the possibility that Gag A146P, which is an escape mutant from HLA-B∗57-restricted CTLs, was selected by HLA-B∗48:01-restricted Gag138-147(LI10)-specific CTLs in a Japanese cohort in which HLA-B∗57 individuals were not detected. We herein demonstrated Gag140-147(GI8) to be the optimal epitope rather than LI10 and that GI8-specific T cells failed to recognize the A146P mutant virus-infected cells. The sequence analysis of Gag146 in 261 chronically HIV-1-infected Japanese showed the accumulation of the A146P mutation in HLA-B∗48:01(+) individuals. These findings together indicate that the A146P mutant is accumulating in Japanese by selection by GI8-specific CTLs., (Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

An actinomycete strain K07-0460(T) producing new antitrypanosomal antibiotics, spoxazomicins, was isolated from the roots of a variety of orchid collected in the subtropical Okinawa prefecture. The 16S ribosomal RNA gene sequence analysis indicated that the strain belonged to the genus Streptosporangium and showed high similarities with S. amethystogenes subsp. amethystogenes DSM 43179(T) (99.4%), S. amethystogenes subsp. fukuiense IFO 15365(T) (99.2%) and S. longisporum DSM 43180(T) (98.7%). The DNA-DNA hybridization relatedness values between strain K07-0460(T) and the three strains were below 70%. On the basis of phylogenetic analysis, DNA-DNA hybridization relatedness and physiological characteristics, the strain should be classified as a new species Streptosporangium oxazolinicum sp. nov. in the genus Streptosporangium. The type strain of S. oxazolinicum is K07-0460(T) (=JCM 17388(T)).

An actinomycete strain, IR73-Li102(T), was isolated from a lichen sample obtained from Iriomote Island, Japan, and subsequently characterized using a polyphasic approach. Comparative 16S rRNA gene sequence analysis revealed that strain IR73-Li102(T) had the highest sequence similarities with Actinomycetospora chiangmaiensis YIM 0006(T) (98.3%), A. corticola 014-5(T) (98.1%) and A. rishiriensis RI109-Li102(T) (98.0%). However, DNA-DNA hybridization assays, as well as physiological and biochemical analyzes, showed that strain IR73-Li102(T) could be clearly differentiated from its closest phylogenetic relatives. The strain contained meso-diaminopimelic acid, and arabinose and galactose were present in whole-cell hydrolysates. The predominant menaquinone was MK-8(H(4)), and the diagnostic phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acid was iso-C(16:0) (58%). The chemotaxonomic properties of strain IR73-Li102(T) were consistent with those shared by members of the genus Actinomycetospora. On the basis of the phenotypic features and DNA-DNA hybridization data, strain IR73-Li102(T) (= NBRC 106365(T) = KCTC 19783(T)) represents a novel species of the genus Actinomycetospora, for which the name Actinomycetospora iriomotensis sp. nov. is proposed.

Objective: To describe outbreaks of nosocomial influenza infection with molecular methods and to elucidate the viral linkages among outbreak case patients including both inpatients and healthcare workers (HCWs)., Setting: A 180-bed acute and long-term care hospital in Japan., Methods: Retrospective observational study of nosocomial outbreaks of infection with influenza A/H3N2. Together with information about onset dates and vaccination history, we obtained nasopharyngeal swab samples from individuals with cases of influenza or influenza-like illness (ILI). The hemagglutinin genes of the recovered viruses were sequenced and compared, along with those of community-circulating strains, for similarity by phylogenetic tree analysis., Results: The outbreaks occurred from February 26 through April 3, 2007, during the 2006-2007 epidemic season, and they involved 11 patients and 13 HCWs. The 2 outbreaks involved 2 different genotypes of influenza A/H3N2 viruses. These virus variants were closely related to the influenza strains that were circulating in the community during the same epidemic season., Conclusion: This study showed the dissemination of highly homologous influenza virus variants among inpatients and HCWs within a short period, as a result of nosocomial transmission. These strains were also similar to influenza strains that were circulating in the community.

The molecular epidemiology of rotavirus infections in non-hospitalized children in five different regions (Sapporo, Saga, Tokyo, Osaka, and Maizuru) of Japan during 2007-2009 was investigated. Overall, rotavirus was detected in 156 out of 1008 (15.5%) specimens. The rotavirus infection in 2007-2008 (19.3%) was higher than those in 2008-2009 (12.1%). G1P[8] was the most prevalent (62.8%), followed by G3P[8] (21.8%), G9P[8] (14.7%), and G2P[4] (0.7%). Interestingly, the number of G3P[8] strains increased threefold from the former season (2006-2007) from 7.3% to 21.8%, whereas G2P[4] and G9P[8] decreased from 11.4% to 0.7% and 20.3% to 14.7%, respectively. In the phylogenetic analysis, G3 rotaviruses were closely related to "the new variant G3" 5091 strain, which previously emerged in Japan and China. G9 viruses isolated in 2007-2008 were genetically close to the Thai strain, while those isolated in 2008-2009 had a close relationship with Chinese strains. G1 viruses appeared to be more similar to the recently reported G1 strain in China. Nucleotide sequence analysis of 33 P[8]-nontypeable strains revealed 5 nucleotide mismatches at the primer binding site. Based on previously reported (2003-2007) and current (2007-2009) data of rotavirus surveillance in the five areas of Japan, it was revealed that in Sapporo, Osaka, and Maizuru, G1P[8] and G3P[8] were detected at high frequencies, ranging from 47.2 to 57.7% and 31.7 to 47.4%, respectively. In Tokyo, G1P[8] (47.4%) was the predominant strain, followed by G9P[8] (20.6%), whereas in Saga, G3P[8] (38.9%) and G9P[8] (36.1%) were identified as the most dominant types. None of G9P[8] was detected in Sapporo. This study highlights the genetic diversity and the significance of rotavirus diarrhea in Japan., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

Norovirus (NoV) and sapovirus (SaV) are important pathogens of human gastroenteritis. Compared to NoV, the transmission route of SaV is unclear. An outbreak of gastroenteritis occurred at a restaurant in June 2008, and SaV and NoV were detected in fecal specimens from 17 people who ate at the restaurant and one asymptomatic food handler and also in stripped shellfish and liquids remaining in the shellfish packages by reverse transcription-polymerase chain reaction (RT-PCR) and/or real-time RT-PCR. Nucleotide sequencing analysis of the RT-PCR products corresponding to the partial capsid region revealed 99.3-100% identities for SaV and 98.6-99.3% identities for NoV among the digestive diverticulum of the frozen stripped shellfish (Ruditapes philippinarum), "Asari," the package liquid, and feces from symptomatic or asymptomatic guests. These results suggested a link between the consumption of contaminated shellfish and clinical features in the patients. While the transmission of NoV by shellfish has been reported, this report shows that SaV can also be transmitted by shellfish., ((c) 2010 Wiley-Liss, Inc.)

The phenotypic and phylogenetic characteristics of an aerobic, Gram-negative, motile, non-pigmented Alteromonas-like bacterium (designated strain KMM 3894(T)), isolated from a sandy sediment sample collected offshore of the Sea of Japan, were investigated. Comparative 16S rRNA gene sequence analysis revealed that strain KMM 3894(T) belonged to the genus Aestuariibacter and was most closely related to Aestuariibacter halophilus JC2043(T) (95.5 % sequence similarity). Fatty acid analysis showed C(16 : 1)omega7c, C(18 : 1)omega7c, and C(16 : 0) as the dominant components. Strain KMM 3894(T) could be differentiated from recognized species of the genus Aestuariibacter by its ability to grow at 4 degrees C and at 30 degrees C, the optimum temperature for growth, and its inability to utilize most carbohydrates. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain KMM 3894(T) is considered to represent a novel species of the genus Aestuariibacter, for which the name Aestuariibacter litoralis sp. nov. is proposed. The type strain is KMM 3894(T) (=NRIC 0754(T)=JCM 15896(T)).

This study investigated the prevalence of group A, B, and C rotavirus (RAV, RBV, RCV) and adenovirus (AdV) infections in infants and children with acute gastroenteritis in Japan from July 2006 to June 2007. A total of 628 fecal specimens collected from infants and children with acute gastroenteritis in five different places (Maizuru, Tokyo, Sapporo, Saga and Osaka) in Japan during the period of July 2006-June 2007 were examined for RAV, RBV, RCV and AdV by RT-multiplex PCR. RAV was further characterized to G-typing and P-typing by RT-multiplex PCR and sequencing method. It was found that 123 (19.6%) fecal specimens were positive for RAV followed by AdV of 4.5%. RBV and RCV could not be detected in this study. Serotype G1 (58.5%) was identified at high levels followed by G9 (20.3%), G2 (11.4%), and G3 (7.3%). P genotyping revealed P[8] as predominant (84.6%) followed by P[4] (13.8%) and P[6] (1.6%). During the 2006/2007 rotavirus season, G1P[8] strains were most common with G9P[8], G2P[4], G3P[8], G1P[4] and G9P[6] also detected. It is clear from this study that RAV is still the cause of diseases in Japan. To our knowledge, this is the first report of RV P[6] strain in humans in Japan.

Previously, Chinese gajutsu available in Japan was identified, from the chloroplast trnK gene sequence, to be the rhizomes of Curcuma phaeocaulis and two genotypes of C. kwangsiensis. Although we defined the two genotypes, the pl and gl types, on the basis of the nucleotide difference, their external features did not correspond to the two phenotypes described in the literature. In this paper, to investigate the relationship between genotype and phenotype of C. kwangsiensis, a field investigation was carried out in its main cultivation areas of Guangxi Zhuangzu Autonomous Region and Guangdong Province, China, and sequence analysis of the trnK gene and single-nucleotide polymorphism (SNP) analysis of the nuclear 18S rRNA gene were performed on the collected specimens. Four genotypes of C. kwangsiensis were recognized from the combined 18S rRNA gene-trnK gene sequences: homozygote-K(gl)Wtk type, homozygote-K(pl)Ztk type, heterozygote-K(gl)Wtk type, and heterozygote-Ltk type. Among the four genotypes, C. kwangsiensis in a field used for cultivation of gajutsu was of heterozygote-K(gl)Wtk type. Formation of a heterozygote in the 18S rRNA gene might be a result of crossbreeding of C. kwangsiensis with several Curcuma species which had cytosine at nucleotide position 234. GC analysis of the rhizomes revealed that C. kwangsiensis was characterized by camphor and beta-elemene, and by detecting additional components such as curdione and curcumenol Curcuma species involved in the formation of the heterozygote might be speculated upon.

Background: In our recent study of the full-length hepatitis A virus (HAV) genome from some patients with fulminant hepatitis and acute hepatitis, possible associations were suggested between the severity of hepatitis A and the amino acid substitutions in the nonstructural protein 2B. We therefore analyzed HAV 2B from many patients with various clinical disease severities., Methods: Serum samples from 30 Japanese patients with sporadic hepatitis A from five widely separated regions of Japan, comprising nine patients with fulminant hepatitis (FH), six with severe acute hepatitis (AHs), and 15 with acute hepatitis (AH), were examined for HAV RNA. The entire sequences of HAV 2B were analyzed., Results: Compared with the sequence of the wild-type HAV strain GBM, nucleotide sequences of 2B had homology of 94.5 +/- 1.0% in FH, 95.2 +/- 1.2% in AHs, and 95.1 +/- 1.8% in AH. Deduced amino acid sequences had homology of 97.5 +/- 2.1% in FH, 97.9 +/- 2.4% in AHs, and 98.5 +/- 1.3% in AH. Differences were not statistically significant among the three groups. The average number of amino acid mutations between amino acids 100 and 200 was 5.0 +/- 5.2 per case in FH, 4.0 +/- 6.0 in AHs, and 1.9 +/- 2.9 in AH. The differences between FH and AH, AHs and AH, and between severe cases (FH and AHs) and nonsevere cases (AH) were not statistically significant (P = 0.13, P = 0.45, and P = 0.10, respectively)., Conclusions: There were no obvious differences in the sequences among FH, AHs, and AH throughout the 2B region, but there seemed to be more mutations in the strains obtained from FH and AHs patients than in those obtained from AH patients in the central part of HAV 2B.

A total of 752 fecal specimens collected from July 2004 to June 2005 from children with acute gastroenteritis in four localities in Japan (Maizuru, Tokyo, Sapporo, and Osaka) were screened for group A rotavirus by RT-PCR. It was found that 82 (10.9%) specimens were positive for group A rotavirus. The G-(VP7 genotypes) and P-(VP4 genotypes) types were further investigated. The P-types of 18 rotavirus strains, which could not be typed by RT-PCR, were determined by sequencing analysis. Of these, 94% (17/18) were P[8] with multiple point mutations at the VP4 primer-binding site. Another sample turned out to be a rare genotype P[9], which was closely related to feline rotavirus. The predominant genotype was G1P[8] (46.4%), followed by G3P[8] (32.9%) and G2P[4] (12.2%). A number of unusual combinations including, G1P[4] (1.2%), G2P[8] (1.2%), G3P[9] (1.2%), G1G3P[8] (1.2%), and G2G3P[8] (3.7%), were also detected. A new nomenclature of P[8] was proposed, in which worldwide rotavirus P[8] strains were classified into four sub-lineages, namely IA, IB, IIA, and IIB. A wide range of amino acid substitutions (up to 22) specific for P[8] lineages and sub-lineages were also identified. Interestingly, only short amino acid motifs located at positions 32-35, 121-135, and 195-236 of VP4 correctly defined the phylogenetic P[8] lineages and sub-lineages. Of note, at least two distinct clusters of rotavirus P[8] were co-circulating in the Japanese pediatric population studied.

A total of 752 fecal specimens collected during the period of July 2004 to June 2005 from infants and children with acute gastroenteritis from four different regions (Maizuru, Tokyo, Sapporo, and Osaka) of Japan were tested for the presence of norovirus by RT-PCR. It was found that 139 (18.5%) fecal specimens were positive for norovirus. Norovirus infection was detected almost all year round with the highest prevalence in January. Norovirus GII was the most predominant genogroup (98.6%; 137 of 139). The genotypes detected in this study were GI/1, GII/1, GII/3, GII/4, and GII/6. Of these, NoV GII/4 (known as the Lordsdale virus cluster) was re-emerging and became the leading genotype (77.7%). Meanwhile, the incidence of NoV GII/3 (known as the Arg320 virus cluster) has dropped rapidly, accounting for only 15.8%. Another interesting feature of the study was the identification of Picton03/AU-like recombinant NoV for the first time in Japan. Based on the genetic analysis, it was interesting to note that NoV GII/4 in 2004-2005 made a distinct cluster in comparison to other NoV GII/4 circulating in 2002-2003 and 2003-2004. Of note, "new recombinant variant designated GIIb" within NoV GII/3, which was first detected in Saga City, Japan in 2003-2004 in only one case, had increased, spreading widely in Japan and representing 45.5% (10 of 22). Further epidemiological studies should be conducted to determine whether this new recombinant variant strain will be dominant in Japan in the coming year.