Omnipresence of Partitiviruses in Rice Aggregate Sheath Spot Symptom-Associated Fungal Isolates from Paddies in Thailand.

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae, a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

Characterization of Four Novel dsRNA Viruses Isolated from Mucor hiemalis Strains.

We previously screened the total nucleic acid extracts of 123 Mucor strains for the presence of dsRNA molecules without further molecular analyses. Here, we characterized five novel dsRNA genomes isolated from four different Mucor hiemalis strains with next-generation sequencing (NGS), namely Mucor hiemalis virus 1a (MhV1a) from WRL CN(M) 122; Mucor hiemalis virus 1b (MhV1b) from NRRL 3624; Mucor hiemalis virus 2 (MhV2) from NRRL 3616; and Mucor hiemalis virus 3 (MhV3) and Mucor hiemalis virus (MhV4) from NRRL 3617 strains. Genomes contain two open reading frames (ORF), which encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In MhV1a and MhV1b, it is predicted to be translated as a fusion protein via -1 ribosomal frameshift, while in MhV4 via a rare +1 (or-2) ribosomal frameshift. In MhV2 and MhV3, the presence of specific UAAUG pentanucleotide motif points to the fact for coupled translation termination and reinitialization. MhV1a, MhV2, and MhV3 are part of the clade representing the genus Victorivirus, while MhV4 is seated in Totivirus genus clade. The detected VLPs in Mucor strains were from 33 to 36 nm in diameter. Hybridization analysis revealed that the dsRNA molecules of MhV1a-MhV4 hybridized to the corresponding molecules.

A novel mycovirus isolated from the plant-pathogenic fungus Botryosphaeria dothidea.

A novel virus, Botryosphaeria dothidea bipartite mycovirus 1 (BdBMV1), was isolated from the plant-pathogenic fungus Botryosphaeria dothidea strain HNDT1, and the complete nucleotide sequence of its genome was determined. BdBMV1 consists of two genomic segments. The first segment is 1,976 bp in length and contains a single open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp) (68.95 kDa). The second segment is 1,786 bp in length and also contains a single ORF encoding a hypothetical protein of 35.19 kDa of unknown function. Based on the sequence of its RdRp, BdBMV1 is phylogenetically related to several other unclassified dsRNA mycoviruses, including Cryphonectria parasitica bipartite mycovirus 1 (CpBV1), and has a distant relationship to members of the family Partitiviridae.

Molecular characterization of a novel partitivirus isolated from the phytopathogenic fungus Aplosporella javeedii.

Aplosporella javeedii is a pathogenic fungus that causes canker and dieback of jujube in China. In this study, we report a new mycovirus, Aplosporella javeedii partitivirus 1 (AjPV1), isolated from A. javeedii strain NX55-3. The AjPV1 genome contains two double-stranded RNA elements (dsRNA1 and dsRNA2). The size of dsRNA1 is 2,360 bp, and it encodes a putative RNA-dependent RNA polymerase (RdRp), while dsRNA2 is 2,301 bp in length and encodes a putative capsid protein (CP). The sequences of RdRp and CP have significant similarity to those of members of the family Partitiviridae. Sequence alignment and phylogenetic analysis showed that AjPV1 is a new member of the family Partitiviridae that is related to members of the genus Betapartitivirus. To our knowledge, AjPV1 is the first mycovirus reported from A. javeedii.

Molecular characterization of a novel partitivirus hosted by the false morel mushroom Gyromitra esculenta.

Virus populations of uncultivated fungi remain scarcely studied. In the present study, we characterized a new partitivirus isolated from the false morel mushroom Gyromitra esculenta, named "Gyromitra esculenta partitivirus 1" (GePV1). The complete genome of GePV1, whose sequence was determined by combining high-throughput sequencing and RLM-RACE approaches, comprises two dsRNA segments of 1971 bp and 1799 bp, respectively. Each dsRNA genome segment contains a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. The sequences of the RdRp and CP exhibited the highest similarity (69.77% and 47.00% identity, respectively) to those of Rosellinia necatrix partitivirus 2 (RnPV2). Phylogenetic analysis based on the CP and RdRp sequences demonstrated that GePV1 clusters within a clade that includes members of the genus Alphapartitivirus, family Partitiviridae. We propose that GePV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified in the false morel mushroom Gyromitra esculenta.

Full-length genome characterization of a novel alphapartitivirus detected in the ectomycorrhizal fungus Hygrophorus penarioides.

Virus populations of ectomycorrhizal fungi remain poorly studied. In the present study, we characterized a new partitivirus isolated from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides, named "Hygrophorus penarioides partitivirus 1" (HpPV1). The whole genome of HpPV1, determined by merging deep sequencing and RLM-RACE approaches, comprised two dsRNA segments of 2053 bp and 2072 bp, respectively. Both dsRNA genome segments included a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP exhibits the highest similarity (67.49% and 75.61% identity, respectively) to those of partitiviruses identified from an ascomycetous ectomycorrhizal fungus Sarcosphaera coronaria. Phylogenetic analyses performed based on the CP and RdRp sequences demonstrated that HpPV1 clusters within a clade that includes members of the genus Alphapartitivirus, belonging to the family Partitiviridae. Here, we propose that HpPV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides.

Co-infection of a novel fusagravirus and a partitivirus in a Korean isolate of Rosellinia necatrix KACC40168.

We report here the presence of dsRNA mycoviruses in a Korean isolate of Rosellinia necatrix. A multiple band pattern of double-stranded RNA (dsRNA) from R. necatrix suggested mixed mycovirus infection. Next-generation sequencing analysis of purified dsRNAs indicated the presence of two dsRNA mycoviruses related to the members of families "Fusagraviridae" (proposed) and Partitiviridae. The first dsRNA virus revealed that the complete genome sequence was 8868 bp in size and contained two large open reading frames (ORFs 1 and 2), overlapped by 22 bp containing a canonical (- 1) slippery heptanucelotide sequence of UUUAAAC. The deduced amino acid sequence of ORF1 and ORF2 showed highest similarity to the hypothetical protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 3 (RnFGV3). Phylogenetic analysis showed that this dsRNA virus clustered with RnFGV3 and other fusagraviruses. Gene organization, sequence similarity, and phylogenetic analysis indicate that this virus seems to belong to a novel species of "Fusagraviridae", which we have named Rosellinia necatrix fusagravirus 4. The second virus has two dsRNA segments with sizes of 1907 bp and 1918 bp, each of which encoded a single ORF showing highest similarity to the RdRp and capsid protein of known members of Partitiviridae. Evaluation of genome structure, sequence similarity, and phylogeny indicate this to be a new member of the genus Alphapartitivirus in the family Partitiviridae, hereafter designated as Rosellinia necatrix partitivirus 26. This is the first report of the presence of a fusagravirus in an Asian R. necatrix isolate and of its mixed infection with a partitivirus.

A Novel Mycovirus Evokes Transcriptional Rewiring in the Fungus Malassezia and Stimulates Beta Interferon Production in Macrophages.

Mycoviruses infect fungi, and while most persist asymptomatically, there are examples of mycoviruses having both beneficial and detrimental effects on their host. Virus-infected Saccharomyces and Ustilago strains exhibit a killer phenotype conferring a growth advantage over uninfected strains and other competing yeast species, whereas hypovirus-infected Cryphonectria parasitica displays defects in growth, sporulation, and virulence. In this study, we identify a double-stranded RNA (dsRNA) mycovirus in five Malassezia species. Sequence analysis reveals it to be a totivirus with two dsRNA segments: a larger 4.5-kb segment with genes encoding components for viral replication and maintenance, and a smaller 1.4-kb segment encoding a novel protein. Furthermore, transcriptome sequencing (RNA-seq) of virus-infected versus virus-cured Malassezia sympodialis revealed an upregulation of dozens of ribosomal components in the cell, suggesting the virus modifies the transcriptional and translational landscapes of the cell. Given that Malassezia is the most abundant fungus on human skin, we assessed the impact of the mycovirus in a murine epicutaneous infection model. Although infection with virus-infected strains was not associated with an increased inflammatory response, we did observe enhanced skin colonization in one of two virus-infected M. sympodialis strains. Noteworthy, beta interferon expression was significantly upregulated in bone marrow-derived macrophages when challenged with virus-infected, compared to virus-cured, M. sympodialis, suggesting that the presence of the virus can induce an immunological response. Although many recent studies have illuminated how widespread mycoviruses are, there are relatively few in-depth studies about their impact on disease caused by the host fungus. We describe here a novel mycovirus in Malassezia and its possible implications in pathogenicity.IMPORTANCEMalassezia species represent the most common fungal inhabitant of the mammalian skin microbiome and are natural skin commensal flora. However, these fungi are also associated with a variety of clinical skin disorders. Recent studies have reported associations of Malassezia with Crohn's disease and pancreatic cancer, further implicating this fungal genus in inflammatory and neoplastic disease states. Because M. sympodialis has lost genes involved in RNA interference (RNAi), we hypothesized Malassezia could harbor dsRNA mycoviruses. Indeed, we identified a novel mycovirus of the totivirus family in several Malassezia species and characterized the MsMV1 mycovirus of M. sympodialis We found conditions that lead to curing of the virus and analyzed isogenic virus-infected/virus-cured strains to determine MsMV1 genetic and pathogenic impacts. MsMV1 induces a strong overexpression of transcription factors and ribosomal genes, while downregulating cellular metabolism. Moreover, MsMV1 induced a significantly higher level of beta interferon expression in cultured macrophages. This study sheds light on the mechanisms of pathogenicity of Malassezia, focusing on a previously unidentified novel mycovirus.

A Novel Taxon of Monosegmented Double-Stranded RNA Viruses Endemic to Triclad Flatworms.

Freshwater planarians, flatworms from order Tricladida, are experimental models of stem cell biology and tissue regeneration. An aspect of their biology that remains less well studied is their relationship with viruses that may infect them. In this study, we identified a taxon of monosegmented double-stranded RNA (dsRNA) viruses in five planarian species, including the well-characterized model Schmidtea mediterranea Sequences for the S. mediterranea virus (abbreviated SmedTV for S. mediterranea tricladivirus) were found in public transcriptome data from multiple institutions, indicating that SmedTV is prevalent in S. mediterranea lab colonies, though without causing evident disease. The presence of SmedTV in discrete cells was shown through in situ hybridization methods for detecting the viral RNA. SmedTV-staining cells were found to be concentrated in neural structures (eyes and brain) but were also scattered in other worm tissues as well. In contrast, few SmedTV-staining cells were seen in stem cell compartments (also consistent with RNA sequencing data) or early blastema tissue. RNA interference (RNAi) targeted to the SmedTV sequence led to apparent cure of infection, though effects on worm health or behavior were not observed. Efforts to transmit SmedTV horizontally through microinjection were unsuccessful. Based on these findings, we conclude that SmedTV infects S. mediterranea in a persistent manner and undergoes vertical transmission to progeny worms during serial passage in lab colonies. The utility of S. mediterranea as a regeneration model, coupled with the apparent capacity of SmedTV to evade normal host immune/RNAi defenses under standard conditions, argues that further studies are warranted to explore this newly recognized virus-host system.IMPORTANCE Planarians are freshwater flatworms, related more distantly to tapeworms and flukes, and have been developed as models to study the molecular mechanisms of stem cell biology and tissue regeneration. These worms live in aquatic environments, where they are likely to encounter a variety of viruses, bacteria, and eukaryotic organisms with pathogenic potential. How the planarian immune system has evolved to cope with these potential pathogens is not well understood, and only two types of planarian viruses have been described to date. Here, we report discovery and inaugural studies of a novel taxon of dsRNA viruses in five different planarian species. The virus in the best-characterized model species, Schmidtea mediterranea, appears to persist long term in that host while avoiding endogenous antiviral or RNAi mechanisms. The S. mediterranea virus-host system thus seems to offer opportunity for gaining new insights into host defenses and their evolution in an important lab model.

Viral RNA Genomes Identified from Marine Macroalgae and a Diatom.

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.