Double-stranded RNA sequencing reveals distinct riboviruses associated with thermoacidophilic bacteria from hot springs in Japan.

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.

DsRNA sequencing revealed a previously missed terminal sequence of a +ssRNA virus that infects dinoflagellate Heterocapsa circularisquama.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus cultured. However, only two strains of HcRNAV have been registered with complete genome sequences (strains 34 and 109 for UA and CY types, respectively). To extend the genomic information of HcRNAV, we performed full-genome sequencing of an unsequenced strain of HcRNAV (strain A8) using the fragmented and primer-ligated double-stranded RNA (dsRNA) sequencing (FLDS) method. The complete genome of HcRNAV A8 with 4457 nucleotides (nt) was successfully determined, and sequence alignment of the major capsid protein gene suggested that A8 was a UA-type strain, consistent with its intraspecific host specificity. The complete sequence was found to be 80 nt longer at the 5' terminus than the registered sequences of HcRNAV strains (34 and 109), suggesting that FLDS is more reliable for determining the terminal sequence than conventional methods (5' Rapid Amplification of cDNA End). Our study contributes to a better understanding of dinoflagellate-infecting viruses with limited sequence data.

Distinct groups of RNA viruses associated with thermoacidophilic bacteria.

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.

Experimental verification of strain-dependent relationship between mycovirus and its fungal host.

Mycoviruses are viruses that infect fungi. Unlike mammalian infectious viruses, their life cycle does not generally have an extracellular stage, and a symbiosis-like relationship is maintained between virus and host fungi. Recently, mycoviruses have been reported to show effects on host fungi, altering biological properties such as growth rate, virulence, drug resistance, and metabolite production. In this study, we systematically elucidated the effects of viruses on host cells by comparing host phenotypes and transcriptomic responses in multiple sets of virus-infected and -eliminated Aspergillus flavus strains. The comparative study showed that mycoviruses affect several cellular activities at the molecular level in a virus- and host strain-dependent manner. The virus-swapping experiment revealed that difference with only three bases in the virus genome led to different host fungal response at the transcriptional level. Our results highlighted highly specific relationship between viruses and their host fungi.

The First Identification of a Narnavirus in Bigyra, a Marine Protist.

Current information on the diversity and evolution of eukaryotic RNA viruses is biased towards host lineages, such as animals, plants, and fungi. Although protists represent the majority of eukaryotic diversity, our understanding of the protist RNA virosphere is still limited. To reveal untapped RNA viral diversity, we screened RNA viruses from 30 marine protist isolates and identified a novel RNA virus named Haloplacidia narnavirus 1 (HpNV1). A phylogenetic ana-lysis revealed that HpNV1 is a new member of the family Narnaviridae. The present study filled a gap in the distribution of narnaviruses and implies their wide distribution in Stramenopiles.

Identification of novel RNA mycoviruses from wild mushroom isolates in Japan.

The characterization of viruses from environmental samples could aid in our understanding of their ecological significance and potential for biotechnological exploitation. While there has been much focus on pathogenic fungi or commercially cultivated mushrooms, attention to viruses from wild Basidiomycota mushrooms is lacking. Therefore, in this study, we conducted viral screening of fungal mycelia isolated from wild basidiocarps using agarose gel electrophoresis (AGE) and fragmented and primer-ligated dsRNA sequencing (FLDS). Among the 51 isolates, seven isolates were detected with virus-like bands during the initial screening with AGE, but only five isolates were detected with viruses after long-term storage. Using the FLDS method, we obtained seven viral genome sequences, including five double-stranded RNA (dsRNA) viruses belonging to Partitiviridae and Curvulaviridae, one positive-sense single-stranded RNA (ssRNA) virus belonging to Endornaviridae and one negative-sense ssRNA virus belonging to Tulasviridae (Bunyavirales). All viruses characterized in this study are novel species. These findings greatly expanded our knowledge of the diversity of RNA viruses from environmental samples.

Discovery, Genomic Sequence Characterization and Phylogenetic Analysis of Novel RNA Viruses in the Turfgrass Pathogenic Colletotrichum spp. in Japan.

Turfgrass used in various areas of the golf course has been found to present anthracnose disease, which is caused by Colletotrichum spp. To obtain potential biological agents, we identified four novel RNA viruses and obtained full-length viral genomes from turfgrass pathogenic Colletotrichum spp. in Japan. We characterized two novel dsRNA partitiviruses: Colletotrichum associated partitivirus 1 (CaPV1) and Colletotrichum associated partitivirus 2 (CaPV2), as well as two negative single-stranded (ss) RNA viruses: Colletotrichum associated negative-stranded RNA virus 1 (CaNSRV1) and Colletotrichum associated negative-stranded RNA virus 2 (CaNSRV2). Using specific RT-PCR assays, we confirmed the presence of CaPV1, CaPV2 and CaNSRV1 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-264, as well as CaNSRV2 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-288. This is the first time mycoviruses have been discovered in turfgrass pathogenic Colletotrichum spp. in Japan. CaPV1 and CaPV2 are new members of the newly proposed genus "Zetapartitivirus" and genus Alphapartitivirus, respectively, in the family Partitiviridae, according to genomic characterization and phylogenetic analysis. Negative sense ssRNA viruses CaNSRV1 and CaNSRV2, on the other hand, are new members of the family Phenuiviridae and the proposed family "Mycoaspirividae", respectively. These findings reveal previously unknown RNA virus diversity and evolution in turfgrass pathogenic Colletotrichum spp.

Efficient elimination of RNA mycoviruses in aspergillus species using RdRp-inhibitors ribavirin and 2'-C-methylribonucleoside derivatives.

RNA viruses in fungi (mycoviruses) are model systems for understanding the relationships between eukaryotic microorganisms and RNA viruses. To reveal the effects of mycoviruses on host fungi, it is essential to compare the phenotypes between isogenic fungal isolates with or without RNA virus infection. Since active entry machinery for RNA mycoviruses has never been identified, introducing mycoviruses to fungi is a difficult and time-consuming process. Therefore, most studies have tried to generate virus-free isolates from infected strains by eliminating the mycovirus. However, methods of elimination have not been evaluated in a quantitative and comparative manner. In this study, we established a method to remove mycoviruses from host cells using the antiviral drugs ribavirin, 2'-C-methylcytidine (2CMC), 2'-C-methyladenosine (2CMA), and 7d2CMA, and compared the efficiency of removal in virus-infected strains of Aspergillus fumigatus. The results indicated that treatment with the drugs removed RNA viruses of diverse proportions in the families Chrysoviridae, Mitoviridae, Partitiviridae, Polymycoviridae, and an unclassified RNA virus group. Viruses belonging to Narnaviridae were hardly eliminated by these antiviral treatments when they were the sole infectious agents. We found that 2CMC showed activity against a wider range of RNA mycoviruses compared to ribavirin, 2CMA, and 7d2CMA, although 7d2CMA also efficiently removed dsRNA viruses from the families Chrysoviridae, Partitiviridae, and Polymycoviridae. These results indicated that removal of mycoviruses depends on the specific viral species and antiviral drug. This is the first report demonstrating a preferential antiviral effect against mycoviruses, which will enhance research on microbial RNA viruses and support their elimination from economically important fungi such as edible mushrooms.

Eukaryotic Microbial RNA Viruses-Acute or Persistent? Insights into Their Function in the Aquatic Ecosystem.

Isolated RNA viruses mainly parasitize eukaryotes. RNA viruses either expand horizontally by infecting hosts (acute type) or coexist with the host and are vertically inherited (persistent type). The significance of persistent-type RNA viruses in environmental viromes (the main hosts are expected to be microbes) was only recently reported because they had previously been overlooked in virology. In this review, we summarize the host-virus relationships of eukaryotic microbial RNA viruses. Picornavirales and Reoviridae are recognized as representative acute-type virus families, and most of the microbial viruses in Narnaviridae, Totiviridae, and Partitiviridae are categorized as representative persistent-type viruses. Acute-type viruses have only been found in aquatic environments, while persistent-type viruses are present in various environments, including aquatic environments. Moreover, persistent-type viruses are potentially widely spread in the RNA viral sequence space. This emerging evidence provides novel insights into RNA viral diversity, host-virus relationships, and their history of co-evolution.

Intracellular production of reactive oxygen species and a DAF-FM-related compound in Aspergillus fumigatus in response to antifungal agent exposure.

Fungi are ubiquitously present in our living environment and are responsible for crop and infectious diseases. Developing new antifungal agents is constantly needed for their effective control. Here, we investigated fungal cellular responses to an array of antifungal compounds, including plant- and bacteria-derived antifungal compounds. The pathogenic fungus Aspergillus fumigatus generated reactive oxygen species in its hyphae after exposure to the antifungal compounds thymol, farnesol, citral, nerol, salicylic acid, phenazine-1-carbonic acid, and pyocyanin, as well as under oxidative and high-temperature stress conditions. The production of nitric oxide (NO) was determined using diaminofluorescein-FM diacetate (DAF-FM DA) and occurred in response to antifungal compounds and stress conditions. The application of reactive oxygen species or NO scavengers partly suppressed the inhibitory effects of farnesol on germination. However, NO production was not detected in the hyphae using the Greiss method. An LC/MS analysis also failed to detect DAF-FM-T, a theoretical product derived from DAF-FM DA and NO, in the hyphae after antifungal treatments. Thus, the cellular state after exposure to antifungal agents may be more complex than previously believed, and the role of NO in fungal cells needs to be investigated further.

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.

Detection and Characterization of RNA Viruses in Red Macroalgae (Bangiaceae) and Their Food Product (Nori Sheets).

Persistent RNA viruses, which have been suggested to form symbiotic relationships with their hosts, have been reported to occur in eukaryotes, such as plants, fungi, and algae. Based on empirical findings, these viruses may also be present in commercially cultivated macroalgae. Accordingly, the present study aimed to screen red macroalgae (family Bangiaceae conchocelis and Neopyropia yezoensis thallus) and processed nori sheets (N. yezoensis) for persistent RNA viruses using fragmented and primer-ligated dsRNA sequencing (FLDS) and targeted reverse transcription PCR (RT-PCR). A Totiviridae-related virus was detected in the conchocelis of Neoporphyra haitanensis, which is widely cultivated in China, while two Mitoviridae-related viruses were found in several conchocelis samples and all N. yezoensis-derived samples (thallus and nori sheets). Mitoviridae-related viruses in N. yezoensis are widespread among cultivated species and not expected to inhibit host growth. Mitoviridae-related viruses were also detected in several phylogenetically distant species in the family Bangiaceae, which suggests that these viruses persisted and coexist in the family Bangiaceae over a long period of time. The present study is the first to report persistent RNA viruses in nori sheets and their raw materials.

The Intrapopulation Genetic Diversity of RNA Virus May Influence the Sensitivity of Chlorine Disinfection.

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.

Antibacterial diphenyl ether production induced by co-culture of Aspergillus nidulans and Aspergillus fumigatus.

Fungi are a rich source of secondary metabolites with potent biological activities. Co-culturing a fungus with another microorganism has drawn much attention as a practical method for stimulating fungal secondary metabolism. However, in most cases, the molecular mechanisms underlying the activation of secondary metabolite production in co-culture are poorly understood. To elucidate such a mechanism, in this study, we established a model fungal-fungal co-culture system, composed of Aspergillus nidulans and Aspergillus fumigatus. In the co-culture of A. nidulans and A. fumigatus, production of antibacterial diphenyl ethers was enhanced. Transcriptome analysis by RNA-sequencing showed that the co-culture activated expression of siderophore biosynthesis genes in A. fumigatus and two polyketide biosynthetic gene clusters (the ors and cic clusters) in A. nidulans. Gene disruption experiments revealed that the ors cluster is responsible for diphenyl ether production in the co-culture. Interestingly, the ors cluster was previously reported to be upregulated by co-culture of A. nidulans with the bacterium Streptomyces rapamycinicus; orsellinic acid was the main product of the cluster in that co-culture. In other words, the main product of the ors cluster was different in fungal-fungal and bacterial-fungal co-culture. The genes responsible for biosynthesis of the bacterial- and fungal-induced polyketides were deduced using a heterologous expression system in Aspergillus oryzae. The molecular genetic mechanisms that trigger biosynthesis of two different types of compounds in A. nidulans in response to the fungus and the bacterium were demonstrated, which provides an insight into complex secondary metabolic response of fungi to microorganisms. KEY POINTS: • Co-culture of two fungal species triggered antibiotic diphenyl ether production. • The co-culture affected expression levels of several genes for secondary metabolism. • Gene cluster essential for induction of the antibiotics production was determined.

Degenerate PCR Targeting the Major Capsid Protein Gene of HcRNAV and Related Viruses.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus that has been isolated to date. We herein investigated the diversity of the major capsid protein gene of HcRNAV and related viruses using degenerate PCR and in silico ana-lyses. Diverse sequences related to HcRNAV were successfully amplified from marine sediments. Amplicons contained conserved and variable regions; the latter were predicted to be located on the outer surface of the capsid. Our approach provides insights into the diversity of viruses that are difficult to isolate in the environment and will enhance rapidly growing metagenome sequence repositories.

Wide distribution of resistance to the fungicides fludioxonil and iprodione in Penicillium species.

Fludioxonil and iprodione are effective fungicides widely used for crop protection and are essential for controlling plant pathogenic fungi. The emergence of fungicide-resistant strains of targeted pathogens is regularly monitored, and several cases have been reported. Non-targeted fungi may also be exposed to the fungicide residues in agricultural fields. However, there are no comprehensive reports on fungicide-resistant strains of non-targeted fungi. Here, we surveyed 99 strains, representing 12 Penicillium species, that were isolated from a variety of environments, including foods, dead bodies, and clinical samples. Among the Penicillium strains, including non-pathogenic P. chrysogenum and P. camembertii, as well as postharvest pathogens P. expansum and P. digitatum, 14 and 20 showed resistance to fludioxonil and iprodione, respectively, and 6 showed multi-drug resistance to the fungicides. Sequence analyses revealed that some strains of P. chrysogenum and Penicillium oxalicum had mutations in NikA, a group III histidine kinase of the high-osmolarity glycerol pathway, which is the mode of action for fludioxonil and iprodione. The single nucleotide polymorphisms of G693D and T1318P in P. chrysogenum and T960S in P. oxalicum were only present in the fludioxonil- or iprodione-resistant strains. These strains also exhibited resistance to pyrrolnitrin, which is the lead compound in fludioxonil and is naturally produced by some Pseudomonas species. This study demonstrated that non-targeted Penicillium strains distributed throughout the environment possess fungicide resistance.

RNA Virosphere in a Marine Zooplankton Community in the Subtropical Western North Pacific.

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.

DsRNA Sequencing for RNA Virus Surveillance Using Human Clinical Samples.

Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.

Intimate genetic relationships and fungicide resistance in multiple strains of Aspergillus fumigatus isolated from a plant bulb.

Fungal infections are increasingly dangerous because of environmentally dispersed resistance to antifungal drugs. Azoles are commonly used antifungal drugs, but they are also used as fungicides in agriculture, which may enable enrichment of azole-resistant strains of the human pathogen Aspergillus fumigatus in the environment. Understanding of environmental dissemination and enrichment of genetic variation associated with azole resistance in A. fumigatus is required to suppress resistant strains. Here, we focused on eight strains of azole-resistant A. fumigatus isolated from a single tulip bulb for sale in Japan. This set includes strains with TR34 /L98H/T289A/I364V/G448S and TR46 /Y121F/T289A/S363P/I364V/G448S mutations in the cyp51A gene, which showed higher tolerance to several azoles than strains harbouring TR46 /Y121F/T289A mutation. The strains were typed by microsatellite typing, single nucleotide polymorphism profiles, and mitochondrial and nuclear genome analyses. The strains grouped differently using each typing method, suggesting historical genetic recombination among the strains. Our data also revealed that some strains isolated from the tulip bulb showed tolerance to other classes of fungicides, such as QoI and carbendazim, followed by related amino acid alterations in the target proteins. Considering spatial-temporal factors, plant bulbs are an excellent environmental niche for fungal strains to encounter partners, and to obtain and spread resistance-associated mutations.

Erratum: Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses.

[This corrects the article DOI: 10.1093/ve/veaa101.].