Molecular and biological characterization of a novel partitivirus from Talaromyces pinophilus.

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

Co-infection of Aspergillus ochraceopetaliformis strain RCEF7483 by a novel chrysovirus and a known partitivirus.

An airborne microflora isolate, Aspergillus ochraceopetaliformis RCEF7483, was found to harbor seven dsRNA elements, indicating co-infection with a novel chrysovirus and a known partitivirus. Sequence analysis and RT-PCR confirmed dsRNA5-7 as components of Aspergillus ochraceous virus (AOV), a member of the Partitiviridae family. In light of its distinct host, we have designated it Aspergillus ochraceopetaliformis partitivirus 1 (AoPV1). The dsRNA segments, named dsRNA1-4, with lengths of 3706 bp, 3410 bp, 3190 bp, and 3158 bp, respectively, constitute the genome of a novel chrysovirus designated Aspergillus ochraceopetaliformis chrysovirus 1 (AoCV1). The dsRNA1-4 segments contain five open-reading frames (ORF1-5). Specifically, ORF1 encodes a putative RNA-dependent RNA polymerase (RdRp) with a length of 1112 amino acids, and ORF2 encodes a putative coat protein (CP) spanning 976 amino acids. Additionally, ORF3-5 encode hypothetical proteins (HP1, HP2, and HP3) with lengths of 108, 843, and 914 amino acids, respectively. Comparative analysis revealed the highest similarity of dsRNA1-4 with corresponding proteins in Aspergillus terreus chrysovirus 1 (AtCV1) (RdRp, 66.58%; CP, 51.02%; HP2, 61.80%; and HP3, 41.30%). Due to falling below the threshold for a new species in the Chrysoviridae, we propose that dsRNA1-4 in A. ochraceopetaliformis strain RCEF7483 constitute the novel chrysovirus AoCV1. Moreover, phylogenetic analysis using RdRp amino acid sequences placed AoCV1 within the Alphachrysovirus genus of the Chrysoviridae family, clustering with AtCV1 and other alphachrysoviruses. Our study contributes to the understanding of mycoviruses in A. ochraceopetaliformis and expands our knowledge of the diversity and evolution of chrysoviruses in fungal hosts.

Mixed infection of ITPase-encoding potyvirid and secovirid in Mercurialis perennis: evidences for a convergent euphorbia-specific viral counterstrike.

BACKGROUND: In cellular organisms, inosine triphosphate pyrophosphatases (ITPases) prevent the incorporation of mutagenic deaminated purines into nucleic acids. These enzymes have also been detected in the genomes of several plant RNA viruses infecting two euphorbia species. In particular, two ipomoviruses produce replicase-associated ITPases to cope with high concentration of non-canonical nucleotides found in cassava tissues. METHOD: Using high-throughput RNA sequencing on the wild euphorbia species Mercurialis perennis, two new members of the families Potyviridae and Secoviridae were identified. Both viruses encode for a putative ITPase, and were found in mixed infection with a new partitivirid. Following biological and genomic characterization of these viruses, the origin and function of the phytoviral ITPases were investigated. RESULTS: While the potyvirid was shown to be pathogenic, the secovirid and partitivirid could not be transmitted. The secovirid was found belonging to a proposed new Comovirinae genus tentatively named "Mercomovirus", which also accommodates other viruses identified through transcriptome mining, and for which an asymptomatic pollen-associated lifestyle is suspected. Homology and phylogenetic analyses inferred that the ITPases encoded by the potyvirid and secovirid were likely acquired through independent horizontal gene transfer events, forming lineages distinct from the enzymes found in cassava ipomoviruses. Possible origins from cellular organisms are discussed for these proteins. In parallel, the endogenous ITPase of M. perennis was predicted to encode for a C-terminal nuclear localization signal, which appears to be conserved among the ITPases of euphorbias but absent in other plant families. This subcellular localization is in line with the idea that nucleic acids remain protected in the nucleus, while deaminated nucleotides accumulate in the cytoplasm where they act as antiviral molecules. CONCLUSION: Three new RNA viruses infecting M. perennis are described, two of which encoding for ITPases. These enzymes have distinct origins, and are likely required by viruses to circumvent high level of cytoplasmic non-canonical nucleotides. This putative plant defense mechanism has emerged early in the evolution of euphorbias, and seems to specifically target certain groups of RNA viruses infecting perennial hosts.

Cannabis Virome Reconstruction and Antiviral RNAi Characterization through Small RNA Sequencing.

Viral infections pose an emerging threat to hemp (Cannabis sativa) cultivation. We used Illumina small (s)RNA sequencing for virome reconstruction and characterization of antiviral RNA interference (RNAi) in monoecious and dioecious hemp varieties, which exhibited different virus-like symptoms. Through de novo and reference-based sRNA assembly, we identified and reconstructed Cannabis cryptic virus (family Partitiviridae), Cannabis sativa mitovirus 1 (Mitoviridae) and Grapevine line pattern virus (Bromoviridae) as well as a novel virus tentatively classified into Partitiviridae. Members of both Partitiviridae and Bromoviridae were targeted by antiviral RNAi, generating 21 nt and, less abundant, 22 nt sRNAs from both strands of the entire virus genome, suggesting the involvement of Dicer-like (DCL) 4 and DCL2 in viral sRNA biogenesis, respectively. Mitovirus sRNAs represented predominantly the positive-sense strand and had a wider size range, with the 21 nt class being most abundant on both strands. For all viruses, 21 and 22 nt sRNAs had predominantly 5'-terminal uridine or cytosine, suggesting their binding to antiviral Argonaute (AGO) 1 and AGO5, respectively. As no clear association of any virus with symptoms was observed, further studies should clarify if these viruses individually or in combination can cause hemp diseases.

Molecular and biological characterization of a partitivirus from Paecilomyces variotii.

Paeciliomyces variotii is a thermo-tolerant, ubiquitous fungus commonly found in food products, indoor environments, soil and clinical samples. It is a well-known biocontrol agent used against phytopathogenic fungi and its metabolites have many industrial applications. Rare reports of P. variotii-related human infections have been found in the medical literature. In this study, we report for the first time the infection of P. variotii isolated from a soil sample collected in a rice field with a double-stranded RNA virus, Paeciliomyces variotii partitivirus 1 (PvPV-1) in the family Partitiviridae. P. variotii harboured icosahedral virus particles 30 nm in diameter with two dsRNA segments 1758 and 1356 bp long. Both dsRNA1 and dsRNA2 have a single open reading frame encoding proteins of 63 and 40 kDa, respectively. These proteins have significant similarity to the RNA-dependent RNA polymerase and capsid protein encoded by the genomic segments of several viruses from the family Partitiviridae. Phylogenetic analysis revealed that PvPV-1 belongs to the family Partitiviridae but in an unclassified group/genus, tentatively nominated Zetapartitivirus. PvPV-1 was found to increase the growth rate of the host fungus, as indicated by time course experiments performed on a range of different media for virus-infected and virus-free isogenic lines. Further, dual-culture assays performed for both isogenic lines confirmed the antagonistic potential of P. variotii against other phytopathogenic fungi. The findings of this study assist us in understanding P. variotii as a potential biocontrol agent, together with plant-fungus-virus interactions.

Genomic characterization and seed transmission of a novel unclassified partitivirus infecting Polygonatum kingianum Coll. et Hemsl.

This study identified a novel virus in the family Partitiviridae infecting Polygonatum kingianum Coll. et Hemsl, which is tentatively named polygonatum kingianum cryptic virus 1 (PKCV1). PKCV1 genome has two RNA segments: dsRNA1 (1926 bp) has an open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 581 amino acids (aa), and dsRNA2 (1721 bp) has an ORF encoding a capsid protein (CP) of 495 aa. The RdRp of PKCV1 shares 20.70-82.50% aa identity with known partitiviruses, and the CP of PKCV1 shares 10.70-70.80% aa identity with known partitiviruses. Moreover, PKCV1 phylogenetically clustered with unclassified members of the Partitiviridae family. Additionally, PKCV1 is common in P. kingianum planting regions and has a high infection rate in P. kingianum seeds.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima.

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

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.

Rose Virome Analysis and Identification of a Novel Ilarvirus in Taiwan.

Rose (Rosa spp.), especially R. hybrida, is one of the most popular ornamental plants in the world and the third largest cut flower crop in Taiwan. Rose mosaic disease (RMD), showing mosaic, line patterns and ringspots on leaves, is a common rose disease caused by the complex infection of various viruses. Due to pests and diseases, the rose planting area in Taiwan has been decreasing since 2008; however, no rose virus disease has been reported in the past five decades. In the spring of 2020, rose samples showing RMD-like symptoms were observed at an organic farm in Chiayi, central Taiwan. The virome in the farm was analyzed by RNA-seq. Rose genomic sequences were filtered from the obtained reads. The remaining reads were de novo assembled to generate 294 contigs, 50 of which were annotated as viral sequences corresponding to 10 viruses. Through reverse transcription-polymerase chain reaction validation, a total of seven viruses were detected, including six known rose viruses, namely apple mosaic virus, prunus necrotic ringspot virus, rose partitivirus, apple stem grooving virus, rose spring dwarf-associated virus and rose cryptic virus 1, and a novel ilarvirus. After completing the whole genome sequencing and sequence analysis, the unknown ilarvirus was demonstrated as a putative new species, tentatively named rose ilarvirus 2. This is the first report of the rose virus disease in Taiwan.

Molecular characterization of a novel partitivirus isolated from Rhizoctonia solani.

Rhizoctonia solani is a widely distributed plant pathogen that can damage many crops. Here, we identified a novel mycovirus tentatively named Rhizoctonia solani partitivirus 433 (RsPV433) from an R. solani (AG-3) strain which caused tobacco target spot disease on flue-cured tobacco. RsPV433 was consisted of two dsRNA segments with lengths of 2450 and 2273 bp, which encoded an RNA-dependent RNA polymerase and a coat protein, respectively. BLASTP results of RsPV433 showed that the closest relative of RsPV433 was Sarcosphaera coronaria partitivirus (QLC36830.1), with an identity of 60.85% on the RdRp amino sequence. Phylogenetic analysis indicated that RsPV433 belonged to the Betapartitivirus genus in the Partitiviridae family. The virus transmission experiment revealed that RsPV433 can be transmitted horizontally. We further tested the biological effect of RsPV433 on R. solani strains and found that the RsPV433-infected R. solani strain grew slower than the RsPV433-free strain on the PDA medium and RsPV433 seemed to have no obvious impact on the lesion inducing ability of R. solani.

Molecular characterization of a novel double-stranded RNA virus infecting the entomopathogenic fungus Metarhizium brunneum.

There are four dsRNAs segments present in the entomopathogenic fungus Metarhizium brunneum strain RCEF0766. The genomic segments dsRNA1 and dsRNA3 are of a novel virus, "Metarhizium brunneum bipartite mycovirus 1" (MbBV1), while dsRNA2 and dsRNA4 are the components of the Metarhizium brunneum partitivirus 2 (MbPV2), a member in genus Gammapartitivirus of the family Partitiviridae based on molecular analysis and RT-PCR. This suggests that the strain RCEF0766 was co-infected by two different mycoviruses. The complete genome sequence of MbBV1 was elucidated by high-throughput sequencing and RLM-RACE. MbBV1 consists of two dsRNAs (1987 and 1642 bp) encode open-reading frames (ORFs). The ORF1 in dsRNA 1 encode is a putative RNA-dependent RNA polymerase (RdRp) with the molecular weight of 68.08 kDa, while ORF2 in dsRNA 2 encodes a hypothetical protein with the molecular weight of 33.07 kDa. The deduced proteins of ORF1 and ORF2 have the highest identity to those of Erysiphe necator-associated bipartite virus 1 (76.88% and 65.30%). Based on the amino acid sequence of RdRp, MbBV1 is phylogenetically clustered together with the unassigned mycoviruses and represents a distinct lineage. Our study proposes that MbBV1 is a novel mycovirus with bisegmented dsRNA genomes and should be considered a new member of the unassigned group.

RNA-seq Analysis of Rhizoctonia solani AG-4HGI Strain BJ-1H Infected by a New Viral Strain of Rhizoctonia solani Partitivirus 2 Reveals a Potential Mechanism for Hypovirulence.

Rhizoctonia solani partitivirus 2 (RsPV2), in the genus Alphapartitivirus, confers hypovirulence on R. solani AG-1-IA, the causal agent of rice sheath blight. In this study, a new strain of RsPV2 obtained from R. solani AG-4HGI strain BJ-1H, the causal agent of black scurf on potato, wasidentified and designated as Rhizoctonia solani partitivirus 2 strain BJ-1H (RsPV2-BJ). An RNA sequencing analysis of strain BJ-1H and the virus RsPV2-BJ-free strain BJ-1H-VF derived from strain BJ-1H was conducted to investigate the potential molecular mechanism of hypovirulence induced by RsPV2-BJ. In total, 14,319 unigenes were obtained, and 1,341 unigenes were identified as differentially expressed genes (DEGs), with 570 DEGs being down-regulated and 771 being up-regulated. Notably, several up-regulated DEGs were annotated to cell wall degrading enzymes, including ß-1,3-glucanases. Strain BJ-1H exhibited increased expression of ß-1,3-glucanase after RsPV2-BJ infection, suggesting that cell wall autolysis activity in R. solani AG-4HGI strain BJ-1H might be promoted by RsPV2-BJ, inducing hypovirulence in its host fungus R. solani AG-4HGI. To the best of our knowledge, this is the first report on the potential mechanism of hypovirulence induced by a mycovirus in R. solani.

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.

Genomic and biological characterization of a novel partitivirus infecting Fusarium equiseti.

This study describes a new mycovirus infecting a strain from the Fusarium incarnatum-equiseti species complex. Based on phylogenetic and genomic analyses, this virus belongs to the recently proposed genus "Zetapartitivirus" in the family Partitiviridae. The name "Fusarium equiseti partitivirus 1″ (FePV1) is therefore suggested for this novel viral species. Similar to other partitiviruses, FePV1 genome is composed by two dsRNA segments that exhibit each one large ORF encoding for an RdRp and a CP, respectively. A smaller dsRNA was also detected in infected mycelium and could be a satellite RNA of FePV1. In addition to characterized zetapartitiviruses, other FePV1-related sequences were retrieved from online databases and their significance is discussed. Following conidial isolation, an FePV1-free isogenic line of the fungal host was obtained. In comparison with the original infected strain, this line showed higher growth, biomass production and pathogenicity on tomato, advocating that FePV1 induces hypovirulence on its host.

Novel and divergent bipartite mycoviruses associated with the ectomycorrhizal fungus Sarcosphaera coronaria.

Members of the family Partitiviridae are reported from a variety of fungal and plant taxa. After dsRNA-preparation, deep sequencing, and bioinformatics, we here reveal the existence of various divergent partitiviruses co-infecting the ectomycorrhizal fungus Sarcosphaera coronaria, symbiotically associated with the pine species Pinus brutia in Turkey. A total of 75 complete or nearly complete sequences related to the members of Alphapartitivirus and Betapartitivirus, were detected from the ascocarp sample of the fungal isolate. Two of the identified partitivirus genome segments encoding for partitiviral capsid protein represent evolutionarily distinct members of Alphapartitivirus, indicating that they may have diverged in the presence of long spatial isolation. In an attempt to match the two genome segments of the identified partitiviruses and distinguish individual species co-inhabiting a single host, nine possible genome segment pairs were identified.

Mixed infection by a partitivirus and a negative-sense RNA virus related to mymonaviruses in the polypore fungus Bondarzewia berkeleyi.

Virus communities of forest fungi remain poorly characterized. In this study, we detected two new viruses co-infecting an isolate of the polypore fungus Bondarzewia berkeleyi using high-throughput sequencing. One of them was a putative new partitivirus designated as Bondarzewia berkeleyi partitivirus 1 (BbPV1), with two linear dsRNA genome segments of 1928 and 1863 bp encoding a putative RNA-dependent RNA polymerase (RdRP) of 591 aa and a putative capsid protein of 538 aa. The other virus, designated as Bondarzewia berkeleyi negative-strand RNA virus 1 (BbNSRV1), had a non-segmented negative-sense RNA genome of 10,983 nt and was related to members of family Mymonaviridae. The BbNSRV1 genome includes six predicted open reading frames (ORFs) of 279, 425, 230, 174, 200 and 1970 aa. The longest ORF contained conserved regions corresponding to Mononegavirales RdRP and mRNA-capping enzyme region V constituting the mononegavirus Large protein. In addition, a low level of sequence identity was detected between the putative nucleocapsid protein-coding ORF2 of Lentinula edodes negative-strand RNA virus 1 and BbNSRV1. The viruses characterized in this study are the first ones described in Bondarzewia spp., and BbNSRV1 is the second mymona-like virus described in a basidiomycete host.

Late Male-Killing Viruses in Homona magnanima Identified as Osugoroshi Viruses, Novel Members of Partitiviridae.

Late male-killing, a male-specific death after hatching, is a unique phenotype found in Homona magnanima, oriental tea tortrix. The male-killing agent was suspected to be an RNA virus, but details were unknown. We herein successfully isolated and identified the putative male-killing virus as Osugoroshi viruses (OGVs). The three RNA-dependent RNA polymerase genes detected were phylogenetically related to Partitiviridae, a group of segmented double-stranded RNA viruses. Purified dsRNA from a late male-killing strain of H. magnanima revealed 24 segments, in addition to the RdRps, with consensus terminal sequences. These segments included the previously found male-killing agents MK1068 (herein OGV-related RNA16) and MK1241 (OGV-related RNA7) RNAs. Ultramicroscopic observation of purified virions, which induced late male-killing in the progeny of injected moths, showed sizes typical of Partitiviridae. Mathematical modeling showed the importance of late male-killing in facilitating horizontal transmission of OGVs in an H. magnanima population. This study is the first report on the isolation of partiti-like virus from insects, and one thought to be associated with late male-killing, although the viral genomic contents and combinations in each virus are still unknown.

ICTV Virus Taxonomy Profile: Partitiviridae.

The Partitiviridae is a family of small, isometric, non-enveloped viruses with bisegmented double-stranded (ds) RNA genomes of 3-4.8 kbp. The two genome segments are individually encapsidated. The family has five genera, with characteristic hosts for members of each genus: either plants or fungi for genera Alphapartitivirus and Betapartitivirus, fungi for genus Gammapartitivirus, plants for genus Deltapartitivirus and protozoa for genus Cryspovirus. Partitiviruses are transmitted intracellularly via seeds (plants), oocysts (protozoa) or hyphal anastomosis, cell division and sporogenesis (fungi); there are no known natural vectors. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Partitiviridae, which is available at www.ictv.global/report/partitiviridae.

Identification of novel RNA viruses in alfalfa (Medicago sativa): an Alphapartitivirus, a Deltapartitivirus, and a Marafivirus.

Genomic RNA molecules of plant RNA viruses are often co-isolated with the host RNAs, and their sequences can be detected in plant transcriptome datasets. Here, an alfalfa (Medicago sativa) transcriptome dataset was analyzed and three new RNA viruses were identified, which were named Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), and Medicago sativa marafivirus 1 (MsMV1). The RNA-dependent RNA polymerases of MsAPV1, MsDPV1, and MsMV1 showed about 68%, 58%, and 46% amino acid sequence identity, respectively, with their closest virus species. Sequence similarity and phylogenetic analyses indicated that MsAPV1, MsDPV1, and MsMV1 were novel RNA virus species that belong to the genus Alphapartitivirus of the family Partitiviridae, the genus Deltapartitivirus of the family Partitiviridae, and the genus Marafivirus of the family Tymoviridae, respectively. The bioinformatics procedure applied in this study may facilitate the identification of novel RNA viruses from plant transcriptome data.

Two alphapartitiviruses co-infecting a single isolate of the plant pathogenic fungus Rhizoctonia solani.

Seven dsRNA segments were detected from a single Rhizoctonia solani strain HG81. From the full-length cDNA sequences of four smaller dsRNA segments, the genomes of two related partitiviruses, designated as Rhizoctonia solani partitivirus 3 (RsPV3) and RsPV4, were determined. The genomes of RsPV3 and RsPV4 are both composed of two separate dsRNA segments, with each segment possessing a single open reading frame (ORF). ORF1 from RsPV3 and RsPV4 encodes a putative RNA-dependent RNA polymerase, while ORF2 of RsPV3 and RsPV4 encodes a putative capsid protein. RsPV3 and RsPV4 share high sequence identity with viruses classified within the genus Alphapartitivirus, family Partitiviridae.