Isolation and genetic characterization of novel bovine parechoviruses from Japanese black cattle.

Novel bovine parechoviruses (Bo ParVs) were isolated from the feces of Japanese black cattle. Phylogenetic analysis revealed that the novel Bo ParVs formed an independent cluster, exhibiting 72.2-75.6% nucleotide sequence identity to previous Bo ParVs, suggesting that they represent a new genotype. Bo ParVs, including the novel Bo ParVs, shared sequence similarity with each other in the 3' untranslated region (3'UTR) and exhibited low sequence similarity (<38.9% identity) to other parechoviruses. However, a secondary structure prediction of the 3'UTR revealed that the Bo ParVs shared conserved motifs in domain 2 with parechovirus B and E, suggesting some evolutionary constrains in this region.

A zebrafish gene with sequence similarities to human uromodulin and GP2 displays extensive evolutionary diversification among teleost and confers resistance to bacterial infection.

In the process of investigating synaptic changes happening to mutants lacking postsynaptic receptors in the neuromuscular junction, we focused on a hitherto uncharacterized zebrafish gene zgc153932 whose expression was increased in the RNAseq and droplet digital PCR (ddPCR) analysis of a paralyzed mutant sofa potato. The zgc153932 gene which we named omcin5 (omc5) showed amino acid sequence similarity to human uromodulin and GP2, which are expressed in epithelial cells of the kidney and the gut respectively and bind to bacteria pili. omc5 had 14 paralogues in a ∼400 KB region on the chromosome 12 of the zebrafish genome. These genes, named omcin1 through 15, constitute a gene cluster which presumably arose from recent gene duplications in the zebrafish lineage. An antibody raised against the epitope common to 6-9 genes in the omcin family revealed expression in the cloaca of 1 day post fertilization (dpf) embryos which broadened to the urinary and digestive tracts by 5 dpf. Expression of omc5 was increased by exposure of embryos to Escherichia coli (E. coli). Survival of omc5 mutant embryos was shortened in the presence of E. coli, or when they were not maintained in germ-free conditions. Adults omc5 mutants also exhibited susceptibility to infection. Other teleost species which had omcin-like genes in their genomes showed a range of gene duplication, resulting in clusters of 1 to >15 omcin-like genes. We hereby identified a new gene family specific to teleost that include a microbial induced gene which confers resistance to bacterial infection.

Identification of multiple inter- and intra-genotype reassortment mammalian orthoreoviruses from Japanese black cattle in a beef cattle farm.

Mammalian orthoreoviruses (MRVs), belonging to the genus Orthoreovirus in the family Spinareoviridae, possess a double-stranded RNA segmented genome. Due to the segmented nature of their genome, MRVs are prone to gene reassortment, which allows for evolutionary diversification. Recently, a genotyping system for each MRV gene segment was proposed based on nucleotide differences. In the present study, MRVs were isolated from the fecal samples of Japanese Black cattle kept on a farm in Japan. Complete genome sequencing and analysis of 41 MRV isolates revealed that these MRVs shared almost identical sequences in the L1, L2, L3, S3, and S4 gene segments, while two different sequences were found in the S1, M1, M2, M3, and S2 gene segments. By plaque cloning, at least six genetic constellation patterns were identified, indicating the occurrence of multiple inter- (S1 and M2) and intra- (M1, M3, and S2) reassortment events. This paper represents the first report describing multiple reassortant MRVs on a single cattle farm. These MRV gene segments exhibited sequence similarity to those of MRVs isolated from cattle in the U.S. and China, rather than to MRVs previously isolated in Japan. Genotypes consisting solely of bovine MRVs were observed in the L1, M1, and M2 segments, suggesting that they might have evolved within the cattle population.

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.

Morphological analysis for two types of viral particles in vacuoles of SARS-CoV-2-infected cells.

In this study, we analyzed the morphological structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells. We identified the two types of viral particles present within the vacuoles of infected cells. Using transmission electron microscopy, we observed that SARS-CoV-2 particles exhibited both low- and high-electron-density structures, which was further confirmed through three-dimensional reconstruction using electron tomography. The budding of these particles was exclusively observed within these vacuoles. Intriguingly, viral particles with low-electron-density structures were confined to vacuoles, whereas those with high-electron-density structures were found in vacuoles and on the cell membrane surface of infected cells. Notably, high-electron-density particles found within vacuoles exhibited the same morphology as those outside the infected cells. This observation suggests that the two types of viral particles identified in this study had different maturation status. Our findings provide valuable insights into the molecular details of SARS-CoV-2 particles, contributing to our understanding of the virus.

The International Virus Bioinformatics Meeting 2023.

The 2023 International Virus Bioinformatics Meeting was held in Valencia, Spain, from 24-26 May 2023, attracting approximately 180 participants worldwide. The primary objective of the conference was to establish a dynamic scientific environment conducive to discussion, collaboration, and the generation of novel research ideas. As the first in-person event following the SARS-CoV-2 pandemic, the meeting facilitated highly interactive exchanges among attendees. It served as a pivotal gathering for gaining insights into the current status of virus bioinformatics research and engaging with leading researchers and emerging scientists. The event comprised eight invited talks, 19 contributed talks, and 74 poster presentations across eleven sessions spanning three days. Topics covered included machine learning, bacteriophages, virus discovery, virus classification, virus visualization, viral infection, viromics, molecular epidemiology, phylodynamic analysis, RNA viruses, viral sequence analysis, viral surveillance, and metagenomics. This report provides rewritten abstracts of the presentations, a summary of the key research findings, and highlights shared during the meeting.

Changes in salivary microbiota due to gastric cancer resection and its relation to gastric fluid microbiota.

Gastric cancer is one of the leading causes of death worldwide, and resections are performed to cure the disease. We have previously reported the changes in the gastric microbiota after gastric cancer resection, which may be associated with the oral microbiota; however, the changes in the oral microbiota remain uncharacterized. This study aimed to characterize the changes in the salivary microbiota caused by gastric cancer resection and to evaluate their association with the gastric fluid microbiota. Saliva and gastric fluid samples were collected from 63 patients who underwent gastrectomy before and after surgery, and a 16S rRNA metagenomic analysis was performed to compare the microbiota composition. The number of bacterial species in the salivary microbiota decreased, and the bacterial composition changed after the resection of gastric cancer. In addition, we identified several bacterial genera that varied significantly in the salivary microbiota, some of which also showed similar changes in the gastric fluid microbiota. These findings indicate that changes in the gastric environment affect the oral microbiota, emphasizing the close association between the oral and gastric fluid microbiota. Our study signifies the importance of focusing on the oral microbiota in the perioperative period of gastrectomy in patients with gastric cancer.

Current trends in RNA virus detection through metatranscriptome sequencing data.

With advances in sequencing technology, metatranscriptome sequencing from a variety of environmental and biological sources has revealed the existence of various previously unknown RNA viruses. This review presents recent major RNA virome studies sampled from invertebrate and vertebrate species as well as aquatic environments. In particular, we focus on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and related RNA virus identification through metatranscriptome sequencing analyses. Recently developed bioinformatics software and databases for RNA virus identification are introduced. A relationship between newly identified RNA viruses and endogenous viral elements in host genomes is also discussed.

Characterization of Oral Microbiota Following Chemotherapy in Patients With Hematopoietic Malignancies.

Oral microbiota may be associated with serious local or systemic medical conditions resulting from chemotherapy. This study was conducted to evaluate the changes in the oral microbiota following the initiation of chemotherapy in patients with hematopoietic malignancies and to identify the characteristics of the oral microbiota associated with oral mucositis. Oral samples were collected from 57 patients with hematopoietic malignancies at 2 time points: before the start of chemotherapy and 8 to 20 days after the start of chemotherapy, when chemotherapy-induced oral mucositis often occurs, and 16S rRNA metagenomic analyses were performed. Comparative and linear discriminant analysis effect size (LEfSe) analyses were used to determine the characteristic bacterial groups before and after the initiation of chemotherapy and in those who developed oral mucositis. The alpha and beta diversities of oral microbiota before and after the initiation of chemotherapy differed significantly (operational taxonomic unit index, P < .001; Shannon's index, P < .001; unweighted UniFrac distances, P = .001; and weighted UniFrac distances, P = .001). The LEfSe analysis revealed a group of bacteria whose abundance differed significantly before and after the initiation of chemotherapy. In the group of patients who developed oral mucositis, a characteristic group of bacteria was identified before the start of chemotherapy. In conclusion, we characterized the oral microbiota associated with the initiation of chemotherapy in patients with hematopoietic malignancies. In addition, our findings suggest that oral microbiota composition before the start of chemotherapy may be associated with oral mucositis. The results of this study emphasize the importance of oral management focusing on the oral microbiota during chemotherapy in patients with hematologic malignancies.

Prevalence and genetic diversity in bovine parechovirus infecting Japanese cattle.

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

Salivary Microbiota Associated with Peripheral Microvascular Endothelial Dysfunction.

AIMS: Oral health is associated with atherosclerotic cardiovascular disease (ACVD). We previously identified the salivary microbiota characteristics of patients with ACVD. However, whether salivary microbiota is characteristic under impaired vascular endothelial function before ACVD onset remains unclear. Therefore, we aimed to evaluate the characteristics of salivary microbiota associated with peripheral microvascular endothelial dysfunction. METHODS: We collected saliva samples from 172 community-dwelling elderly individuals without a history of ACVD and performed 16S rRNA metagenomic analysis. We assessed the peripheral microvascular endothelial function using reactive hyperemia index (RHI) and compared the salivary microbiota in the groups with normal (RHI ≥ 2.10), borderline, and abnormal (RHI ＜1.67) peripheral endothelial function. Furthermore, we applied machine learning techniques to evaluate whether salivary microbiota could discriminate between individuals with normal and abnormal endothelial function. RESULTS: The number of operational taxonomic units (OTUs) was higher in the abnormal group than in the normal group (p=0.037), and differences were found in the overall salivary microbiota structure (unweighted UniFrac distances, p=0.038). The linear discriminant analysis (LDA) effect size (LEfSe) algorithm revealed several significantly differentially abundant bacterial genera between the two groups. An Extra Trees classifier model was built to discriminate between groups with normal and abnormal vascular endothelial function based on the microbial composition at the genus level (AUC=0.810). CONCLUSIONS: The salivary microbiota in individuals with endothelial dysfunction was distinct from that in individuals with normal endothelial function, indicating that the salivary microbiota may be related to endothelial function.

Identification of the corticotropin-releasing factor receptor 1 antagonists as inhibitors of Chikungunya virus replication using a Gaussia luciferase-expressing subgenomic replicon.

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular replication of the replicon genome. Through a chemical compound library screen using the Gluc reporter CHIKV replicon, we identified several compounds that suppressed CHIKV infection in Vero cells. Among the hits identified, CP-154,526, a non-peptide antagonist of the corticotropin-releasing factor receptor type-1 (CRF-R1), showed the strongest anti-CHIKV activity and inhibited CHIKV infection in Huh-7 cells. Interestingly, other CRF-R1 antagonists, R121919 and NGD 98-2, also exhibited inhibitory effects on CHIKV infection. Time-of-drug addition and virus entry assays indicated that CP-154,526 suppressed a post-entry step of infection, suggesting that CRF-R1 antagonists acted on a target in the intracellular replication process of CHIKV. Therefore, the Gluc reporter replicon system established in this study would greatly facilitate the development of antiviral drugs against CHIKV infection.

NeoRdRp: A Comprehensive Dataset for Identifying RNA-dependent RNA Polymerases of Various RNA Viruses from Metatranscriptomic Data.

RNA viruses are distributed throughout various environments, and most have recently been identified by metatranscriptome sequencing. However, due to the high nucleotide diversity of RNA viruses, it is still challenging to identify novel RNA viruses from metatranscriptome data. To overcome this issue, we created a dataset of RNA-dependent RNA polymerase (RdRp) domains that are essential for all RNA viruses belonging to Orthornavirae. Genes with RdRp domains from various RNA viruses were clustered based on amino acid sequence similarities. A multiple sequence alignment was generated for each cluster, and a hidden Markov model (HMM) profile was created when the number of sequences was greater than three. We further refined 426 HMM profiles by detecting RefSeq RNA virus sequences and subsequently combined the hit sequences with the RdRp domains. As a result, 1,182 HMM profiles were generated from 12,502 RdRp domain sequences, and the dataset was named NeoRdRp. The majority of NeoRdRp HMM profiles successfully detected RdRp domains, specifically in the UniProt dataset. Furthermore, we compared the NeoRdRp dataset with two previously reported methods for RNA virus detection using metatranscriptome sequencing data. Our methods successfully identified the majority of RNA viruses in the datasets; however, some RNA viruses were not detected, similar to the other two methods. NeoRdRp may be repeatedly improved by the addition of new RdRp sequences and is applicable as a system for detecting various RNA viruses from diverse metatranscriptome data.

Comparison of the gastric microbiome in Billroth I and Roux-en-Y reconstructions after distal gastrectomy.

The changes in gastric microbiota following reconstruction after gastrectomy have not been reported. This study aimed to compare the gastric microbiota following Billroth I and Roux-en-Y reconstructions after distal gastrectomy. We enrolled 71 gastrectomized patients with gastric cancer; 31 and 40 underwent Billroth I and Roux-en-Y reconstructions, respectively. During upper gastrointestinal endoscopy, gastric fluid was collected immediately before and 6 months after distal gastrectomy. Deoxyribonucleic acid isolated from each sample was evaluated using 16S ribosomal ribonucleic acid metagenomic analysis. Analysis revealed that the gastric microbiota's species richness (expressed as the alpha diversity) was significantly lower after than before distal gastrectomy (operational taxonomic units, p = 0.001; Shannon index, p = 0.03). The interindividual diversity (beta diversity) was significantly different before and after distal gastrectomy (unweighted UniFrac distances, p = 0.04; weighted UniFrac distances, p = 0.001; Bray-Curtis, p = 0.001). Alpha and beta diversity were not significantly different between Billroth I and Roux-en-Y reconstructions (observed operational taxonomic units, p = 0.58; Shannon index, p = 0.95; unweighted UniFrac distances, p = 0.65; weighted UniFrac distances, p = 0.67; Bray-Curtis, p = 0.63). Our study demonstrated significant differences in gastric microbiota diversity, composition, and community before and after distal gastrectomy but no difference between Billroth I and Roux-en-Y reconstruction after distal gastrectomy.

Genomic diversity and intragenic recombination of species C rotaviruses.

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

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

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

Three-dimensional reconstruction by electron tomography for the application to ultrastructural analysis of SARS-CoV-2 particles.

SARS-CoV-2 is the cause of COVID-19. The three-dimensional morphology of viral particles existing and multiplying in infected cells has not been established by electron tomography, which is different from cryo-electron tomography using frozen samples. In this study, we establish the morphological structure of SARS-CoV-2 particles by three-dimensional reconstruction of images obtained by electron tomography and transmission electron microscopy of biological samples embedded in epoxy resin. The characteristic roots of spike structures were found to be arranged at the surface of a virion covered with an envelope. A high-electron-density structure that appears to be a nucleocapsid was observed inside the envelope of the virion on three-dimensional images reconstructed by electron tomography. The SARS-CoV-2 particles that budded in the vacuoles in the cytoplasm were morphologically identical to those found outside the cells, suggesting that mature and infectious SARS-CoV-2 particles were already produced in the vacuoles. Here, we show the three-dimensional morphological structure of SARS-CoV-2 particles reconstructed by electron tomography. To control infection, inhibition of viral release from vacuoles would be a new target in the development of prophylactic agents against SARS-CoV-2.

Characterization of Salivary Microbiota in Patients with Atherosclerotic Cardiovascular Disease: A Case-Control Study.

AIMS: Oral bacteria have been reported to be associated with the pathogenesis of atherosclerosis; however, the relationship between the oral microbiota and atherosclerosis remains unclear. The present study aimed to investigate whether or not salivary microbiota of patients with atherosclerotic cardiovascular disease (ACVD) differs from that of subjects without ACVD, and to characterize the salivary microbiota of patients with ACVD. METHODS: This study included 43 patients with ACVD and 86 age- and sex-matched non-ACVD individuals. 16S rRNA metagenomic analysis were performed using DNA isolated from the saliva samples of the participants. To select unique operational taxonomic unit (OTU) sets of ACVD, we conducted the random forest algorithm in machine learning, followed by confirmation via 10-fold cross-validation Results: There was no difference in richness or evenness between the ACVD and non-ACVD groups (alpha diversity; observed OTU index, p=0.503; Shannon's index, p=0.478). However, significant differences were found in the overall salivary microbiota structure (beta diversity; unweighted UniFrac distances, p=0.001; weighted UniFrac distances, p=0.001). The Actinobacteria phylum was highly abundant in patients with ACVD, while the Bacteroidetes phylum was less abundant. The random forest classifier identified 43 OTUs as an optimal marker set of ACVD. In a 10-fold cross validation using the validation data, an area under the curve (AUC) of 0.933 (95% CI, 0.855-1.000) was obtained. CONCLUSIONS: The salivary microbiota in patients with ACVD was distinct from that of non-ACVD individuals, indicating that the salivary microbiota may be related to ACVD.

Characterization of salivary microbiota in elderly patients with type 2 diabetes mellitus: a matched case-control study.

OBJECTIVE: The importance of oral health in type 2 diabetes mellitus (T2DM) is widely recognized; however, oral microbiota characteristics associated with T2DM in the elderly population are not well-understood. This study was conducted to evaluate the characteristics of the salivary microbiota in elderly Japanese patients with T2DM. METHODS: Saliva samples were collected from 42 elderly Japanese patients with T2DM and 42 age- and sex-matched subjects without T2DM (control). 16S ribosomal RNA metagenomic analysis and comparative analysis of both groups were performed. Random forest classification by machine learning was performed to discriminate between the salivary microbiota in the two groups. RESULTS: There were significant differences in the overall salivary microbiota structure between the T2DM and control groups (beta diversity; unweighted UniFrac distances, p = 0.001; weighted UniFrac distances, p = 0.001). The phylum Firmicutes was abundant in patients with T2DM, whereas the phylum Bacteroidetes was abundant in controls. The T2DM prediction model by random forest based on salivary microbiota data was verified with a high predictive potential in five cross-validation tests (area under the curve (AUC) = 0.938 (95% CI, 0.824-1.000)). CONCLUSION: Characterization revealed that the salivary microbiota profile of the elderly patients with T2DM is significantly distinct from that of the controls. CLINICAL RELEVANCE: These data indicate the necessity of oral health management based on the characteristics of the salivary microbiota in elderly patients with T2DM. Our findings will contribute to future research on the development of new diagnostic and therapeutic methods for this purpose.

Identification of Feline Foamy Virus-derived MicroRNAs.

MicroRNAs (miRNAs) classified as non-coding RNAs regulate various metabolic systems and viral life cycles. To date, numerous DNA viruses, many of which are members of the herpesvirus family, and a relatively small number of RNA viruses, including retroviruses, have been reported to encode and express miRNAs in infected cells. A few retroviruses have been shown to express miRNAs, and foamy viruses (FVs) were initially predicted by computational analyses to possess miRNA-coding regions. Subsequent studies on simian and bovine FVs confirmed the presence of functional and biologically active miRNA expression cassettes. We herein identified feline FV-derived miRNAs using a small RNA deep sequencing ana-lysis. We confirmed their repressive functions on gene expression by dual-luciferase reporter assays. We found that the seed sequences of the miRNAs identified in the present study were conserved among all previously reported FFV isolates. These results suggest that FFV-derived miRNAs play a pivotal role in FFV infection.