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[This corrects the article DOI: 10.1371/journal.pone.0244464.].
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Kimoto-type Japanese rice wine (sake) has a wide variety of flavors, as the predominant microbes, including lactic acid bacteria (LAB) and nitrate-reducing bacteria, that spontaneously proliferate in the fermentation starter vary depending on the brewery. In this study, we traced the microbiota in four lots of starters manufactured in a newly established brewery and evaluated the lot-to-lot variation and characteristics of the microbiota in the brewery. The results of a 16S ribosomal RNA amplicon analysis showed that the starters brewed in the second brewing year had a more diverse microbiota than those in the first brewing year. Among the LAB predominated at the middle production stage, lactococci, including Leuconostoc spp., were detected in all the lots, while lactobacilli predominated for the first time in the second year. These results suggest that repeated brewing increased microbial diversity and altered the microbial transition pattern in the kimoto-style fermentation starters. Phylogenetic analyses for the LAB isolates from each starter identified Leuconostoc suionicum, Leuconostoc citreum, and Leuconostoc mesenteroides as predominant lactococci as well as a unique lactobacillus in place of Latilactobacillus sakei. We also found that a rice koji-derived Staphylococcus gallinarum with nitrate-reducing activity was generally predominant during the early production stage, suggesting that there was a case in which staphylococci played a role in nitrite production in the starters. These findings are expected to contribute to the understanding of the diversity of microbiota in kimoto-type sake brewing and enable control of the microbiota for consistent sake quality.
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Metatranscriptome sequencing expanded the known diversity of the bacterial RNA virome, suggesting that additional riboviruses infecting bacterial hosts remain to be discovered. Here we employed double-stranded RNA sequencing to recover complete genome sequences of two ribovirus groups from acidic hot springs in Japan. One group, denoted hot spring riboviruses (HsRV), consists of viruses with distinct RNA-directed RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. This group forms a distinct phylum, Artimaviricota, or even kingdom within the realm Riboviria. We identified viruses encoding HsRV-like RdRPs in marine water, river sediments and salt marshes, indicating that this group is widespread beyond extreme ecosystems. The second group, denoted hot spring partiti-like viruses (HsPV), forms a distinct branch within the family Partitiviridae. The genome architectures of HsRV and HsPV and their identification in bacteria-dominated habitats suggest that these viruses infect thermoacidophilic bacteria.
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Fontes Termais , Vírus de RNA , Fontes Termais/microbiologia , RNA de Cadeia Dupla , Ecossistema , Filogenia , Japão , Archaea/genética , Bactérias/genética , Vírus de RNA/genéticaRESUMO
BACKGROUND: Inflammatory myofibroblastic tumor (IMT) is a rare stromal tumor, often found in children and young adults, and most commonly occurs in the lungs. Surgical resection is considered the standard treatment for localized IMT, although only limited data exist. Gastric IMT in adults is extremely rare, and there are no established guidelines for its treatment. CASE PRESENTATION: A 69-year-old male presented with persistent fatigue and weakness. Laboratory examination revealed severe anemia and inflammation. Upper gastrointestinal endoscopy at admission revealed a 40-mm type I softish tumor in the lesser curvature of the gastric body, without apparent hemorrhage. Repeated biopsies, including partial resection with snare, failed to give a definitive diagnosis. Computed tomography (CT) revealed a massive lesion at the gastric body, protruding into the gastric lumen, which was consistent with the gastric tumor. After admission, the patient developed anemia refractory to frequent blood transfusions despite the absence of apparent gastrointestinal bleeding. In addition, the patient had recurrent fevers of 38 °C or higher, and persistent high inflammatory levels. Fluorodeoxyglucose-positron emission tomography (FDG-PET) CT 1 month after the first visit exhibited an increased FDG uptake in the gastric tumor. In addition, this CT scan revealed a rapid increase in tumor size to 75 mm. It was suspected that the undiagnosed gastric tumor caused these serious clinical symptoms, and he underwent distal gastrectomy and cholecystectomy. The gross image of the tumor showed an 80-mm cauliflower-like shape with a gelatinous texture. The histopathological diagnosis was IMT. The postoperative course was uneventful, and the patient's symptoms subsided drastically, improving both anemia and systemic inflammation. The patient has shown no recurrence or relapse of the symptoms over one and a half years. CONCLUSIONS: In this case, the tumor resection finally enabled the diagnosis of IMT and resolved the clinical symptoms. Despite its predominantly benign morphological nature, some cases of IMT present clinically adverse courses. Surgical treatment may lead to its final diagnosis and improvement of clinical symptoms.
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Recent massive metatranscriptome mining substantially expanded the diversity of the bacterial RNA virome, suggesting that additional groups of riboviruses infecting bacterial hosts remain to be discovered. We employed full length double-stranded (ds) RNA sequencing for identification of riboviruses associated with microbial consortia dominated by bacteria and archaea in acidic hot springs in Japan. Whole sequences of two groups of multisegmented riboviruses genomes were obtained. One group, which we denoted hot spring riboviruses (HsRV), consists of unusual viruses with distinct RNA-dependent RNA polymerases (RdRPs) that seem to be intermediates between typical ribovirus RdRPs and viral reverse transcriptases. We also identified viruses encoding HsRV-like RdRPs in moderate aquatic environments, including marine water, river sediments and salt marsh, indicating that this previously overlooked ribovirus group is not restricted to the extreme ecosystem. The HsRV-like viruses are candidates for a distinct phylum or even kingdom within the viral realm Riboviria. The second group, denoted hot spring partiti-like viruses (HsPV), is a distinct branch within the family Partitiviridae. All genome segments in both these groups of viruses display the organization typical of bacterial riboviruses, where multiple open reading frames encoding individual proteins are preceded by ribosome-binding sites. Together with the identification in bacteria-dominated habitats, this genome architecture indicates that riboviruses of these distinct groups infect thermoacidophilic bacterial hosts.
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Phagocytosis is one of the methods used to acquire symbiotic bacteria to establish intracellular symbiosis. A deep-sea mussel, Bathymodiolus japonicus, acquires its symbiont from the environment by phagocytosis of gill epithelial cells and receives nutrients from them. However, the manner by which mussels retain the symbiont without phagosome digestion remains unknown. Here, we show that controlling the mechanistic target of rapamycin complex 1 (mTORC1) in mussels leads to retaining symbionts in gill cells. The symbiont is essential for the host mussel nutrition; however, depleting the symbiont's energy source triggers the phagosome digestion of symbionts. Meanwhile, the inhibition of mTORC1 by rapamycin prevented the digestion of the resident symbionts and of the engulfed exogenous dead symbionts in gill cells. This indicates that mTORC1 promotes phagosome digestion of symbionts under reduced nutrient supply from the symbiont. The regulation mechanism of phagosome digestion by mTORC1 through nutrient signaling with symbionts is key for maintaining animal-microbe intracellular nutritional symbiosis.
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Bivalves , Simbiose , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina , Fagossomos , Bactérias , DigestãoRESUMO
Post-mega-earthquake geochemical and microbiological properties in subseafloor sediments of the Japan Trench accretionary wedge were investigated using core samples from Hole C0019E, which was drilled down to 851| |m below seafloor (mbsf) at a water depth of 6,890 m. Methane was abundant throughout accretionary prism sediments; however, its concentration decreased close to the plate boundary decollement. Methane isotope systematics indicated a biogenic origin. The content of mole-cular hydrogen (H2) was low throughout core samples, but markedly increased at specific depths that were close to potential faults predicted by logging-while-drilling ana-lyses. Based on isotopic systematics, H2 appeared to have been abundantly produced via a low-temperature interaction between pore water and the fresh surface of crushed rock induced by earthquakes. Subseafloor microbial cell density remained constant at approximately 105| |cells| |mL-1. Amplicon sequences revealed that predominant members at the phylum level were common throughout the units tested, which also included members frequently found in anoxic subseafloor sediments. Metabolic potential assays using radioactive isotopes as tracers revealed homoacetogenic activity in H2-enriched core samples collected near the fault. Furthermore, homoacetogenic bacteria, including Acetobacterium carbinolicum, were isolated from similar samples. Therefore, post-earthquake subseafloor microbial communities in the Japan Trench accretionary prism appear to be episodically dominated by homoacetogenic populations and potentially function due to the earthquake-induced low-temperature generation of H2. These post-earthquake microbial communities may eventually return to the steady-state communities dominated by oligotrophic heterotrophs and hydrogenotrophic and methylotrophic methanogens that are dependent on refractory organic matter in the sediment.
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Terremotos , Expedições , Sedimentos Geológicos/microbiologia , Japão , Metano/metabolismo , ÁguaRESUMO
Anaerobic ammonium-oxidizing (anammox) bacteria are slow-growing and fastidious bacteria, and limited numbers of enrichment cultures have been established. A metagenomic ana-lysis of our 5 established anammox bacterial enrichment cultures was performed in the present study. Fourteen high-quality metagenome-assembled genomes (MAGs) were obtained, including those of 5 anammox Planctomycetota (Candidatus Brocadia, Ca. Kuenenia, Ca. Jettenia, and Ca. Scalindua), 4 Bacteroidota, and 3 Chloroflexota. Based on the gene sets of metabolic pathways involved in the degradation of polymeric substances found in Chloroflexota and Bacteroidota MAGs, they are expected to be scavengers of extracellular polymeric substances and cell debris.
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Compostos de Amônio , Metagenoma , Compostos de Amônio/metabolismo , Oxidação Anaeróbia da Amônia , Anaerobiose , Bactérias/genética , Bactérias/metabolismo , Metagenômica , Oxirredução , RNA Ribossômico 16S/genéticaRESUMO
RNA virus populations are not clonal; rather, they comprise a mutant swarm in which sequences are slightly different from the master sequence. Genetic diversity within a population (intrapopulation genetic diversity) is critical for RNA viruses to survive under environmental stresses. Disinfection has become an important practice in the control of pathogenic viruses; however, the impact of intrapopulation genetic diversity on the sensitivity of disinfection, defined as -log10 (postdisinfected infectious titer/predisinfected titer), has not been elucidated. In this study, we serially passaged populations of rhesus rotavirus. We demonstrated that populations with reduced chlorine sensitivity emerged at random and independently of chlorine exposure. Sequencing analysis revealed that compared with sensitive populations, less-sensitive ones had higher non-synonymous genetic diversity of the outer capsid protein gene, suggesting that changes in the amino acid sequences of the outer capsid protein were the main factors influencing chlorine sensitivity. No common mutations were found among less-sensitive populations, indicating that rather than specific mutations, the diversity of the outer capsid protein itself was associated with the disinfection sensitivity and that the disinfection sensitivity changed stochastically. Simulation results suggest that the disinfection sensitivity of a genetically diverse population is destabilized if cooperative viral clusters including multiple sequences are formed. These results advocate that any prevention measures leading to low intrapopulation genetic diversity are important to prevent the spread and evolution of pathogenic RNA viruses in society.
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Zooplankton and viruses play a key role in marine ecosystems; however, their interactions have not been examined in detail. In the present study, the diversity of viruses associated with zooplankton collected using a plankton net (mesh size: 100| |µm) in the subtropical western North Pacific was investigated by fragmented and primer ligated dsRNA sequencing. We obtained 21 and 168 operational taxonomic units (OTUs) of ssRNA and dsRNA viruses, respectively, containing RNA-dependent RNA polymerase (RdRp). These OTUs presented average amino acid similarities of 43.5 and 44.0% to the RdRp genes of known viruses in ssRNA viruses and dsRNA viruses, respectively. Dominant OTUs mainly belonged to narna-like and picorna-like ssRNA viruses and chryso-like, partiti-like, picobirna-like, reo-like, and toti-like dsRNA viruses. Phylogenetic ana-lyses of the RdRp gene revealed that OTUs were phylogenetically diverse and clustered into distinct clades from known viral groups. The community structure of the same zooplankton sample was investigated using small subunit (SSU) rRNA sequences assembled from the metatranscriptome of single-stranded RNA. More than 90% of the sequence reads were derived from metazoan zooplankton; copepods comprised approximately 70% of the sequence reads. Although this ana-lysis provided no direct evidence of the host species of RNA viruses, these dominant zooplankton are expected to be associated with the RNA viruses detected in the present study. The present results indicate that zooplankton function as a reservoir of diverse RNA viruses and suggest that investigations of zooplankton viruses will provide a more detailed understanding of the role of viruses in marine ecosystems.
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Vírus de RNA , Água do Mar/virologia , Zooplâncton , Animais , Ecossistema , Oceano Pacífico , Filogenia , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genéticaRESUMO
DNA chemical modifications, including methylation, are widespread and play important roles in prokaryotes and viruses. However, current knowledge of these modification systems is severely biased towards a limited number of culturable prokaryotes, despite the fact that a vast majority of microorganisms have not yet been cultured. Here, using single-molecule real-time sequencing, we conducted culture-independent 'metaepigenomic' analyses (an integrated analysis of metagenomics and epigenomics) of marine microbial communities. A total of 233 and 163 metagenomic-assembled genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTase genes were not genetically linked with the endonuclease genes predicted to be involved in defense mechanisms against extracellular DNA. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, 5 of which showed novel specificities and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative specificities in MTases that are highly conserved in Alphaproteobacteria, which may enhance our understanding of the co-evolutionary history of the methylation systems and the genomes. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses in nature.
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Metilases de Modificação do DNA , Epigenômica , DNA/genética , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Metiltransferases/genética , Células Procarióticas/metabolismoRESUMO
In this study, we extracted essential oils from four species of plants with lemony scents (Melissa officinalis L., Aloysia citriodora Palau (= Lippia citriodora (Palau) Kunth), Thymus × citriodorus, Perilla citriodora (Makino) Nakai). We then examined the components of extracts using gas chromatography (GC) and GC-mass spectrometry (GC-MS). A comparison of components indicated that the largest proportions of essential oils were caryophyllene (25%) in M. officinalis, geraniol (50%) in T. citriodorus, and citral (61 and 82%) in A. citriodora and P. citriodora. Moreover, we used a sensory evaluation method using dilute aqueous solutions of extract components, citral, linalool, d-limonene, and geraniol, to select the mixture with a flavor that mostly resembled lemon. The participants in the study felt that an aqueous citral solution flavored more like lemon than aqueous d-limonene. Furthermore, an open field study of sedative effects of citral and d-limonene, when inhaled, on mice demonstrated that citral exhibited a sedative effect at a lower concentration than that of d-limonene.
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Lippia , Óleos Voláteis , Monoterpenos Acíclicos , Animais , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , OdorantesRESUMO
Abyssal plains cover more than half of Earth's surface, and the main food source in these ecosystems is phytodetritus, mainly originating from primary producers in the euphotic zone of the ocean. Global climate change is influencing phytoplankton abundance, productivity, and distribution. Increasing importance of picoplankton over diatom as primary producers in surface oceans (especially projected for higher latitudes) is projected and hence altering the quantity of organic carbon supplied to the abyssal seafloor as phytodetritus, consequences of which remain largely unknown. Here, we investigated the in situ responses of abyssal biota from viruses to megafauna to different types of phytoplankton input (diatoms or cyanobacteria which were labeled with stable isotopes) at equatorial (oligotrophic) and temperate (eutrophic) benthic sites in the Pacific Ocean (1°N at 4277 m water depth and 39°N at 5260 m water depth, respectively). Our results show that meiofauna and macrofauna generally preferred diatoms as a food source and played a relatively larger role in the consumption of phytodetritus at higher latitudes (39°N). Contrarily, prokaryotes and viruses showed similar or even stronger responses to cyanobacterial than to diatom supply. Moreover, the response of prokaryotes and viruses was very rapid (within 1-2 days) at both 1°N and 39°N, with quickest responses reported in the case of cyanobacterial supply at higher latitudes. Overall, our results suggest that benthic deep-sea eukaryotes will be negatively affected by the predicted decrease in diatoms in surface oceans, especially at higher latitudes, where benthic prokaryotes and viruses will otherwise likely increase their quantitative role and organic carbon cycling rates. In turn, such changes can contribute to decrease carbon transfer from phytodetritus to higher trophic levels, with strong potential to affect oceanic food webs, their biodiversity and consequently carbon sequestration capacity at the global scale.
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Mudança Climática , Cianobactérias , Biota , Ecossistema , Oceanos e MaresRESUMO
Fragmented and primer ligated dsRNA sequencing (FLDS) is a sequencing method applicable to long double-stranded RNA (dsRNA) that enables the complete genome sequencing of both double- and single-stranded RNA viruses. However, the application of this method on a low amount of dsRNA has been hindered by adaptor dimer formation during cDNA amplification and sequence library preparation. We herein developed FLDS ver. 3 by optimizing the terminal modification of an oligonucleotide adaptor and the conditions of adaptor ligation. We also examined the concentration of Mg2+ in the PCR reaction for cDNA amplification and the purification method of amplified cDNA. Fine sequence reads were successfully obtained from metagenomic shotgun sequencing libraries constructed from 10 and 100 pg dsRNA, and these libraries exhibited weaker detection sensitivity for low-abundance dsRNAs (viral genomes and genome segments) than that constructed from 1| |ng of dsRNA. We also report the utility of capillary electrophoresis for dsRNA quantification. The FLDS ver. 3 package expands the frontiers of our knowledge in RNA virus diversity and evolution.
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Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Análise de Sequência de RNA/métodos , Primers do DNA/genética , Genoma Viral , Metagenômica , Reação em Cadeia da Polimerase , Vírus de RNA/isolamento & purificaçãoRESUMO
Marine sediments represent a vast habitat for complex microbiomes. Among these, ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are one of the most common, yet little explored, inhabitants, which seem extraordinarily well adapted to the harsh conditions of the subsurface biosphere. We present 11 metagenome-assembled genomes of the most abundant AOA clades from sediment cores obtained from the Atlantic Mid-Ocean ridge flanks and Pacific abyssal plains. Their phylogenomic placement reveals three independently evolved clades within the order Nitrosopumilales, of which no cultured representative is known yet. In addition to the gene sets for ammonia oxidation and carbon fixation known from other AOA, all genomes encode an extended capacity for the conversion of fermentation products that can be channeled into the central carbon metabolism, as well as uptake of amino acids probably for protein maintenance or as an ammonia source. Two lineages encode an additional (V-type) ATPase and a large repertoire of DNA repair systems that may allow to overcome the challenges of high hydrostatic pressure. We suggest that the adaptive radiation of AOA into marine sediments occurred more than once in evolution and resulted in three distinct lineages with particular adaptations to this extremely energy-limiting and high-pressure environment.
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Amônia , Archaea , Archaea/genética , Sedimentos Geológicos , Metagenoma , Oxirredução , FilogeniaRESUMO
The 4704-nt genome sequence of Sikte waterborne virus (SWV), determined by fragmented and primer ligated dsRNA sequencing and by direct Sanger sequencing, is linear, nonsegmented and has the five ORFs of other tombusviruses. The 5' and 3' untranslated regions (UTRs) are 150 and 335 nt long, respectively. Phylogenetic analysis of the coat protein revealed that SWV is related to CymRSV and PNSV, but that of the SWV replicase protein, the p92 readthrough protein, indicated a close relationship to CNV. These phylogenetic analyses suggest the occurrence of recombination events in SWV, as reported previously for other tombusviruses.
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Chenopodium quinoa/virologia , Genoma Viral/genética , RNA Viral/genética , Tombusvirus/classificação , Tombusvirus/genética , Sequência de Bases , Proteínas do Capsídeo/genética , Alemanha , Fases de Leitura Aberta/genética , Rios/virologia , Análise de Sequência de RNA , Sequenciamento Completo do GenomaRESUMO
Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases' behaviors and functions in daily genetic engineering experiments.
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Enzimas de Restrição do DNA/metabolismo , DNA/análise , Engenharia Genética/métodos , Imagem Individual de Molécula/métodos , Sistemas CRISPR-Cas/genética , Sistema Livre de Células/enzimologia , DNA/metabolismo , Microscopia de Fluorescência/métodosRESUMO
The lichen is a microbial consortium that mainly consists of fungi and either algae (Viridiplantae) or cyanobacteria. This structure also contains other bacteria, fungi, and viruses. However, RNA virus diversity associated with lichens is still unknown. Here, we analyzed RNA virus diversity in a lichen dominated by fungi and algae using dsRNA-seq technology and revealed that partitiviruses were dominant and active in the microbial consortium. The Partitiviridae sequences found in this study were classified into two genera, which have both plant- and fungi-infecting partitiviruses. This observation suggests that the lichen provides an opportunity for horizontal transfer of these partitiviruses among microbes that form the lichen consortium.
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Protists provide insights into the diversity and function of RNA viruses in marine systems. Among them, marine macroalgae are good targets for RNA virome analyses because they have a sufficient biomass in nature. However, RNA viruses in macroalgae have not yet been examined in detail, and only partial genome sequences have been reported for the majority of RNA viruses. Therefore, to obtain further insights into the distribution and diversity of RNA viruses associated with marine protists, we herein examined RNA viruses in macroalgae and a diatom. We report the putative complete genome sequences of six novel RNA viruses from two marine macroalgae and one diatom holobiont. Four viruses were not classified into established viral genera or families. Furthermore, a virus classified into Totiviridae showed a genome structure that has not yet been reported in this family. These results suggest that a number of distinct RNA viruses are widespread in a broad range of protists.
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Diatomáceas/virologia , Vírus de RNA de Cadeia Dupla/genética , Genoma Viral/genética , Água do Mar/virologia , Alga Marinha/virologia , Biodiversidade , Vírus de RNA de Cadeia Dupla/classificação , Vírus de RNA de Cadeia Dupla/isolamento & purificação , Filogenia , Proteínas Virais/genética , ViromaRESUMO
RNA viruses form a dynamic distribution of mutant swarms (termed "quasispecies") due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission exemplifies a bottleneck effect, in that only part of a viral population can reach the next susceptible hosts. In the present study, two lineages of the rhesus rotavirus (RRV) strain of rotavirus A were serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001, and three phenotypes (infectious titer, cell binding ability, and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and nonsynonymous substitutions on rotavirus genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate a stronger bottleneck effect can create more sequence spaces for minor sequences.IMPORTANCE In this study, we investigated a bottleneck effect on an RRV population that may drastically affect the viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck created a new space in a population for minor mutants originally existing in a hidden layer, which includes minor mutations that cannot be distinguished from a sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected according to molecular epidemiology using next-generation sequencing in which the viral genetic diversity within a viral population is investigated.