dsRNA Elements Research Articles

Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF028.

Molecular characterization of the most common dsRNA element from Trichoderma atroviride indicated that it comprised 8,566 bp and encoded two large open reading frames (ORF1 and 2). The two ORFs were found to overlap by 46 bp with a typical (-1) slippery heptanucelotide sequence. The deduced protein sequences of ORF1 and ORF2 showed significant similarities to those of known mycoviral structural proteins and RNA-dependent RNA polymerases, respectively. Phylogenetic analysis indicated that this dsRNA is a member of a distinct species related to a group of unclassified mycoviruses; therefore, it was named Trichoderma atroviride mycovirus 1 (TaMV1).

Identification and sequence determination of a new chrysovirus infecting the entomopathogenic fungus Isaria javanica.

A new double-stranded RNA (dsRNA) mycovirus has been identified in the isolate NB IFR-19 of the entomopathogenic fungus Isaria javanica. Isaria javanica chrysovirus-1 (IjCV-1) constitutes a new member of the Chrysoviridae family, and its genome is made up of four dsRNA elements designated dsRNA1, 2, 3 and 4 from largest to smallest. dsRNA1 and dsRNA2 encode an RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. dsRNA3 and 4 encode hypothetical proteins of unknown function. IjCV-1 constitutes the first report of a chrysovirus infecting the entomopathogenic fungus Isaria javanica.

Hypovirulence of Sclerotium rolfsii Caused by Associated RNA Mycovirus.

Mycoviruses associated with hypovirulence are potential biological control agents and could be useful to study the pathogenesis of fungal host pathogens. Sclerotium rolfsii, a pathogenic fungus, causes southern blight in a wide variety of crops. In this study, we isolated a series of dsRNAs from a debilitated S. rolfsii strain, BLH-1, which had pronounced phenotypic aberrations including reduced pathogenicity, mycelial growth and deficient sclerotia production. Virus-curing and horizontal transmission experiments that eliminated or transmitted, respectively, all dsRNA elements showed that the dsRNAs were involved in the hypovirulent traits of BLH-1. Ultrastructure examination also showed hyphae fracture and cytoplasm or organelle degeneration in BLH-1 hyphal cells compared to the virus-free strain. Three assembled cDNA contigs generated from the cDNA library cloned from the purified dsRNA indicated that strain BLH-1 was infected by at least three novel mycoviruses. One has similarity to the hypovirulence-associated Sclerotinia sclerotiorum hypovirus 2 (SsHV2) in the family Hypoviridae, and the other two are related to two different unclassified dsRNA mycovirus families. To our knowledge, this is the first report of S. rolfsii hypovirulence that was correlated with its associated dsRNA.

Deep Sequencing Analysis Reveals the Mycoviral Diversity of the Virome of an Avirulent Isolate of Rhizoctonia solani AG-2-2 IV.

Rhizoctonia solani represents an important plant pathogenic Basidiomycota species complex and the host of many different mycoviruses, as indicated by frequent detection of dsRNA elements in natural populations of the fungus. To date, eight different mycoviruses have been characterized in Rhizoctonia and some of them have been reported to modulate its virulence. DsRNA extracts of the avirulent R. solani isolate DC17 (AG2-2-IV) displayed a diverse pattern, indicating multiple infections with mycoviruses. Deep sequencing analysis of the dsRNA extract, converted to cDNA, revealed that this isolate harbors at least 17 different mycovirus species. Based on the alignment of the conserved RNA-dependent RNA-polymerase (RdRp) domain, this viral community included putative members of the families Narnaviridae, Endornaviridae, Partitiviridae and Megabirnaviridae as well as of the order Tymovirales. Furthermore, viruses, which could not be assigned to any existing family or order, but showed similarities to so far unassigned species like Sclerotinia sclerotiorum RNA virus L, Rhizoctonia solani dsRNA virus 1, Aspergillus foetidus slow virus 2 or Rhizoctonia fumigata virus 1, were identified. This is the first report of a fungal isolate infected by 17 different viral species and a valuable study case to explore the diversity of mycoviruses infecting R. solani.

A novel monopartite dsRNA virus isolated from the entomopathogenic and nematophagous fungus Purpureocillium lilacinum.

Purpureocillium lilacinum is a ubiquitous saprophytic fungus commonly isolated from soils and widely known as a biological control agent against phytopathogenic nematodes and pest insects. Mycoviruses infect a wide number of fungal species, but the study of viruses infecting entomopathogenic fungi is still quite recent. In this study, a total of 86 P. lilacinum isolates collected from soil in natural and cultivated habitats throughout the Czech Republic were analyzed; 22% of the isolates harbored double-stranded RNA (dsRNA) elements with viral characteristics. These results suggest that mycoviruses are common in P. lilacinum. One of the most common dsRNA elements detected in the survey was completely sequenced and corresponded to the 2,864-bp genome of a previously undescribed mycovirus, designated Purpureocillium lilacinum nonsegmented virus 1 (PlNV-1). Phylogenetic analysis of the RNA-dependent RNA polymerase of PlNV-1 indicated that this virus might belong to a new taxon related to the family Partitiviridae.

Genome sequence of a novel mitovirus identified in the phytopathogenic fungus Alternaria arborescens.

The phytopathogenic fungus Alternaria spp. contains a variety of double-stranded RNA (dsRNA) elements of different sizes. Detailed analysis of next-generation sequencing data obtained using dsRNA purified from Alternaria arborescens, from which we had previously found Alternaria arborescens victorivirus 1, revealed the presence of another mycoviral-like dsRNA of approximately 2.5kbp in length. When using the fungal mitochondrial genetic code, this dsRNA has a single open reading frame that potentially encodes an RNA-dependent RNA polymerase (RdRp) with significant to sequence similarity to those of viruses of the genus Mitovirus. Moreover, both the 5'- and 3'-untranslated regions have the potential to fold into stable stem-loop structures, which is characteristic of mitoviruses. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of RdRp indicated that the virus we identified in A. arborescens is a distinct member of the genus Mitovirus in the family Narnaviridae, designated as "Alternaria arborescens mitovirus 1" (AaMV1).

Genome sequence of a novel victorivirus identified in the phytopathogenic fungus Alternaria arborescens.

Strains of the phytopathogenic fungus Alternaria spp. have been found to contain a variety of double-stranded RNA (dsRNA) elements indicative of mycovirus infection. Here, we report the molecular characterization of a novel dsRNA mycovirus, Alternaria arborescens victorivirus 1 (AaVV1), from A. arborescens, the tomato pathotype of A. alternata. Using next-generation sequencing of dsRNA purified from an A. arborescens strain from the United States of America, we found that the AaVV1 genome is 5203bp in length and contains two open reading frames (ORF1 and 2) that overlap at the tetranucleotide AUGA. Proteins encoded by ORF1 and ORF2 showed significant similarities to the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively, of dsRNA mycoviruses of the genus Victorivirus. Pairwise comparisons and phylogenetic analysis of the deduced amino acid sequences of both CP and RdRp indicated that AaVV1 is a member of a distinct species of the genus Victorivirus in the family Totiviridae.

Seed-borne viral dsRNA elements in three cultivated Raphanus and Brassica plants suggest three cryptoviruses.

Since the 1970s, several dsRNA viruses, including Radish yellow edge virus, Raphanus sativus virus 1, Raphanus sativus virus 2, and Raphanus sativus virus 3, have been identified and reported as infecting radish. In the present study, in conjunction with a survey of seed-borne viruses in cultivated Brassica and Raphanus using the dsRNA diagnostic method, we discovered 3 novel cryptoviruses that infect Brassica and Raphanus: Raphanus sativus partitivirus 1, which infects radish (Raphanus sativus); Sinapis alba cryptic virus 1, which infects Sinapis alba; and Brassica rapa cryptic virus 1 (BrCV1), which infects Brassica rapa. The genomic organization of these cryptoviruses was analyzed and characterized. BrCV1 might represent the first plant partitivirus found in Gammapartitivirus. Additionally, the evolutionary relationships among all of the partitiviruses reported in Raphanus and Brassica were analyzed.

Novel mitoviruses in Rhizoctonia solani AG-3PT infecting potato

Double-stranded RNA (dsRNA) elements are ubiquitous in Rhizoctonia solani. Total dsRNA was randomly amplified from a R. solani isolate (RS002) belonging to anastomosis group-3PT (AG-3PT), associated with black scurf in potato. Assembly of resulting cDNA sequences identified a nearly complete genome of a novel virus related to the genus Mitovirus (family Narnaviridae), herein named Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002). The 2797 nucleotide partial genome of RMV-1-RS002 is A-U rich (59.06 %), and can be folded into stable stem-loop structures at 5′ and 3′ ends. Universal and mold mitochondrial codon usages revealed a large open reading frame in the genome, putatively encoding an 826 amino acid polypeptide, which has conserved motifs for mitoviral RNA-dependent RNA polymerase. The full length putative polypeptide shared 25.6 % sequence identity with the corresponding region of Tuber excavatum mitovirus (TeMV). The partial genome of a second mitovirus (proposed name Rhizoctonia mitovirus 2 RS002 (RMV-2-RS002)) was also amplified from RS002. A nearly identical copy of RMV-1-RS002 was detected in two additional AG-3PT isolates. These data indicate that multiple mitoviruses can exist in a single isolate of R. solani AG-3PT, and that mitoviruses such as RMV-1-RS002 are probably widespread in this pathogen. The roles of mitoviruses in the biology of R. solani AG-3PT remain unknown.

Characterization of a novel Sclerotinia sclerotiorum RNA virus as the prototype of a new proposed family within the order Tymovirales

Recent studies have shown that Sclerotinia sclerotiorum, an important plant pathogen fungus, harbors diverse mycoviruses. A new mycovirus, tentatively named as Sclerotinia sclerotiorum deltaflexivirus 1 (SsDFV1), was isolated from a S. sclerotiorum strain AX19 containing multiple dsRNA elements. The complete genome of SsDFV1 was shown to be 8178 nucleotides long excluding the poly (A) tail. SsDFV1 has a large putative open reading frame (ORF1) and three smaller ORFs (2–4). ORF1 encodes a putative methyltransferase-helicase-RdRp polyprotein of 2075 amino acids. ORFs (2–4) encode three putative small hypothetical proteins (<40kDa) with unknown biological functions. No evidence for a coat protein encoded by SsDFV1 was obtained. Multiple alignment suggested that three conserved domains, RdRp, methyltransferase, and helicase, from SsDFV1 have lower identity (approximately 25%) with all the reported viruses of four approved families, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae and Tymoviridae in the order Tymovirales. Moreover, a phylogenetic tree also suggested that the SsDFV1 could not be phylogenetically placed in any of the approved families, and forms a separated cluster distinct from other known viruses. Therefore, these combined results suggest that SsDFV1 could represent a new positive-sense single-stranded RNA virus with some unique molecular features, and we propose to create a tentative family Deltaflexiviridae that accommodates SsDFV1.

Sequence determination of a satellite RNA isolated from Aspergillus foetidus.

Aspergillus foetidus virus (AfV) has at least two distinct particle types, designated as AfV-fast (F) and AfV-slow (S). AfV-S includes AfV-S1, a victorivirus; AfV-S2, an unclassified satellite RNA; and AfV-S3, a previously uncharacterized dsRNA element. Here, we describe the complete sequence of AfV-S3, which is a short non-coding RNA with no known homologs. AfV-S3 is predicted to form an extended secondary structure, shares a 5' terminus with AfV-S2, and is a satellite RNA possibly dependent on both AfV-S1 and AfV-S2. This work concludes the sequencing of the A. foetidus virome.

Identification and sequence determination of a novel double-stranded RNA mycovirus from the entomopathogenic fungus Beauveria bassiana.

An isolate of the entomopathogenic fungus Beauveria bassiana was found to contain five double-stranded (ds) RNA elements ranging from 1.5 to more than 3 kbp. The complete sequence of the largest dsRNA element is described here. Analysis of the RdRp nucleotide sequence reveals its similarity to unclassified dsRNA elements, such as Alternaria longipes dsRNA virus 1, and its distant relationship to the RNA-dependent RNA polymerases of members of the family Partitiviridae.

A Method for In Situ Localization of Single-Strand and Double-Strand RNA Elements Contained in the Hepatitis C Virus Genome 3'-Untranslated Region

The ability to resolve secondary and tertiary structures, as present in their native state within the genome of RNA viruses, is an essential first step in elucidating the replication process of these viruses. The hepatitis C virus (HCV) genome is composed of a single (+) strand RNA whose replication is controlled primarily by the 3'-untranslated region (3'UTR). The 3'UTR comprises a genotype-specific variable region (VR), a poly (U/UC) repeat and a conserved 98-base sequence called the X-tail. Although structural models of the 3'UTR from in vitro biochemical analysis have been proposed, its native structure as part of the full-length viral genome existing within the intracellular environment is uncertain. As a means to map the native double-strand (ds) and single-strand (ss) RNA structures contained in the 3'UTR of the full-length genome, we performed in situ chemical tagging in combination with site-directed RT-qPCR. Results indicate that ssRNA is predominant in the VR-poly (U/UC) repeat sequences, whereas dsRNA elements are restricted to the X-tail. In situ chemical tagging also identified a unique dsRNA element located in the VR-poly (U/ UC) repeat region comprised in part of sequences located outside the 3'UTR. The results described here constitute a first report for in situ probing and detection of secondary structures present within the 3'UTR sequence of the HCV genome. The use of intracellular RNA modifying agents to demarcate dsRNA and ssRNA, in combination with PCR to amplify trace amounts of the targeted RNA, has potential application in mapping native secondary structures within complex intracellular RNAs.

Rapid detection of Magnaporthe oryzae chrysovirus 1-A from fungal colonies on agar plates and lesions of rice blast

Mycoviruses associated with hypovirulent phenotypes have been reported for many plant pathogenic fungi. Common techniques to detect mycoviruses depend on the presence of dsRNA elements. These techniques require cultivation of the host fungus, extraction of nucleic acids and purification of dsRNA. These procedures are time-consuming steps and use organic solvents. Here we developed a simple and rapid method detect Magnaporthe oryzae chrysovirus 1-A by direct one-step RT-PCR in samples picked using a sterilized toothpick from fungal colonies growing on plate media. This method could be applied for direct detection of mycoviruses from lesions caused by virus-infected plant pathogenic fungi.

Prevalence and diversity of mycoviruses infecting the plant pathogen Ustilaginoidea virens

Rice false smut caused by Ustilaginoidea virens is a destructive disease in many rice-growing areas. Mycoviruses have been described in many fungal species, but there is little information regarding mycoviruses in U. virens. In this study, double-stranded (ds) RNA banding patterns were assessed in 198 wild-type isolates of U. virens obtained from different geographical regions in China. The presence of viral infections was unusually common in U. virens: 188 of the 198 isolates contained dsRNA elements with viral characteristics, and the presence of mixed infections with two or more related or unrelated mycoviruses was commonly detected. The GX-1 isolate contained four dsRNA mycoviruses: Ustilaginoidea virens RNA virus 1 (UvRV1) belonging to Totiviridae, Ustilaginoidea virens RNA virus 4 (UvRV4) belonging to an unclassified family which includes the Curvularia thermal tolerance virus, and the last two probably belonging to Partitiviridae. Biological comparisons of virus-free and infected fungal isolates revealed that UvRV1 strain GX-1 and UvRV4 were likely cryptic, since the infected strains did not show apparent symptoms or debilitation. Northern blotting experiments revealed that UvRV1 strain GX-1 and UvRV4 were frequently found in U. virens, irrespective of the place of origin, and similarly sized dsRNA bands were not always of similar sequence. Thus, our findings suggest that mycoviruses infecting U. virens in China are widespread and highly diverse.

Characterization of a novel dsRNA endornavirus in the plant pathogenic fungus Thielaviopsis basicola

Double-stranded (ds) RNA elements are commonly present in strains of the plant pathogenic fungus Thielaviopsis basicola which infects a wide range of plant species. To characterize a novel 12 kb dsRNA in strain NC1527 of this fungus, reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain an 11,602 bp cDNA sequence from overlapping cDNA clones. Northern blot analysis confirmed that 15 individual cDNA clones that covered the entire length of the cDNA sequence all hybridized to the 12 kb dsRNA. An open reading frame (ORF) search revealed that the 5′non-coding region of this sequence spans 27 nucleotides, followed by one large ORF of 11,575 bp nucleotides, which potentially encodes a large putative polyprotein of 3858 amino acid residues. Specialized Basic Local Alignment Search Tool (BLAST) searches of conserved domains indicated that the putative polyprotein contained a viral RNA helicase1 (Hel), glycosyl transferase (GT) and RNA-dependent RNA polymerase (RdRp) domain regions. BLASTp searches in the protein database using translated nucleotides showed that the cloned dsRNA had homology to endornaviruses, which are present in a few fungi as well as some plant species. The amino acid homologies ranged from 28% to 34% in the RdRp domain region, 23–32% in the helicase domain region and 29–30% in GT region. We designate this dsRNA element as a new endornavirus, Chalara elegans endornavirus 1 (CeEV1). Phylogenetic comparison of the sequence of RdRP with other endornavirus indicated that CeEV1 was relatively distant and may be an ancestral form.

Mycoviruses ofBotrytis cinereaisolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide-resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty-five percent of the B. cinerea isolates analysed contained double-stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.

Two novel mitoviruses from a Canadian isolate of the Dutch elm pathogen Ophiostoma novo-ulmi (93–1224)

BackgroundOphiostoma novo-ulmi is the causative agent of Dutch elm disease (DED). It is an ascomycetous filamentous fungus that ranks as the third most devastating fungal pathogen in Canada. The disease front has spread eastward and westward from the epicentre in Ontario and Quebec and is threatening elm populations across the country. Numerous mitigation strategies have been tried to eradicate this pathogen, but success has thus far been limited. An alternative approach might utilize double-stranded RNA (dsRNA) mycoviruses which have been reported to induce hypovirulence in other fungi.MethodsUsing a modified single primer amplification technique (SPAT) in combination with chromosomal walking, we have determined the genome sequence of two RdRp encoding dsRNA viruses from an O. novo-ulmi isolate (93–1224) collected from the disease front in Winnipeg.ResultsWe propose that these viruses, which we have named OnuMV1c and OnuMV7 based on sequence similarity to other Ophiostoma mitoviruses, are two new members of the genus Mitovirus in the family Narnaviridae.ConclusionsThe discovery of such dsRNA elements raises the potential for engineering these viruses to include other genetic elements, such as anti-sense or interfering RNAs, to create novel and highly specific biological controls. Naïve fungal hosts could be infected with both the engineered molecule and a helper mitovirus encoding an RdRp which would provide replication capacity for both molecules.

A novel dsRNA element isolated from the Aspergillus foetidus mycovirus complex

Aspergillus foetidus virus (AfV) contains at least two icosahedral particle types named AfV-fast (-F) and AfV-slow (-S), based on relative electrophoretic mobility. AfV-F is a quadripartite double-stranded RNA (dsRNA) virus, and AfV-S contains AfV-S1, which is a member of the genus Victorivirus in the family Totiviridae, and AfV-S2, which may be a satellite RNA or satellite virus and is described here. Analysis of the complete AfV-S2 nucleotide sequence reveals it to be significantly similar to an unclassified RNA from the fungus Rosellinia necatrix and distantly related to the RNA-dependent RNA polymerases of several single-stranded RNA genomes.

A novel virus in the family Hypoviridae from the plant pathogenic fungus Fusarium graminearum

A double-stranded (ds) RNA element, sized at approximately 13kb pairs, was purified from a field isolate, HN10, of Fusarium graminearum. The coding strand of the dsRNA was 13,023 nucleotides (nt) long (excluding the 3′ poly(A) tail) and was predicted to contain two discontiguous open reading frames (ORF A and ORF B). The 5′ proximal ORF A of 531nt encoded a protein of 176 amino acids (aa), and a BLAST search showed it to be similar to the putative papain-like protease domains encoded by Valsa ceratosperma hypovirus 1 (35% identity) and Cryphonectria hypovirus 4 (CHV4) (31% identity). The 3′ proximal ORF B of 11,118nt encoded a large polyprotein with three conserved domains, including papain-like protease, RNA-dependent RNA polymerase and RNA helicase domains. The polyprotein shared significant aa identities with CHV1 (32%) and CHV2 (32%). Both the genome organization and phylogenetic analysis suggested that the characterized RNA represented a novel hypovirus, designated “Fusarium graminearum hypovirus 1 (FgHV1)”, which was closely related to CHV1 and CHV2 in the Hypoviridae family. Elimination of the virus resulted in no dramatic phenotypic alteration of the fungus.