Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb

Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain (V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5'- and 3'-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5'-UTR) and -UUUACUACU (in the 3'-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).     

The complete genomic sequence of a novel mycovirus from Rhizoctonia solani AG-1 IA strain B275

The complete genome of a novel mycovirus, Rhizoctonia solani dsRNA virus 1 (RsRV1) was sequenced and analyzed. It is composed of two dsRNA genome segments, 2379 bp and 1811 bp in length, which were referred to as RsRV1-1 and RsRV1-2, respectively. RsRV1-1 contains a single open reading frame (ORF1), which has a conserved RNA-dependent RNA polymerase (RdRp) domain, whereas RsRV1-2 contains a single ORF2, which might encode a multifunctional protein. The genome organization of RsRV1 is similar to that of members of the family Partitiviridae. However, phylogenetic analysis indicated that RsRV1 formed a distinct clade together with three other unclassified viruses, suggesting that RsRV1 may belong to a new family of dsRNA mycoviruses. This is the first report of the full-length nucleotide sequence of a novel dsRNA mycovirus, RsRV1, infecting R. solani AG-1 IA strain B275, the causal agent of rice sheath blight.     

Identification of novel double-stranded RNA mycoviruses of Fusarium virguliforme and evidence of their effects on virulence

Virulence and double-stranded RNA (dsRNA) profiles of 44 isolates of Fusarium virguliforme were compared. When grouped according to dsRNA profiles, isolates with large dsRNAs were significantly (P≤0.05) less virulent than isolates without dsRNAs. High-throughput sequence analysis of total RNA prepared from cultures with large dsRNAs identified two novel RNA viruses with genome sequences of approximately 9.3 kbp, which were named Fusarium virguliforme dsRNA mycovirus 1 and Fusarium virguliforme dsRNA mycovirus 2. The new viruses were most closely related to a group of unclassified viruses that included viruses of F. graminearum and Phlebiopsis gigantea and are related to members of the family Totiviridae.     

Complete genome sequence of a novel dsRNA mycovirus isolated from the phytopathogenic fungus Verticillium dahliae Kleb.

A novel double-stranded RNA (dsRNA) mycovirus, designated Verticillium dahliae partitivirus 1 (VdPV1), was isolated from a strain of the fungus Verticillium dahliae. The VdPV1 genome has two dsRNA genome segments. The larger segment (1768 bp) has a single open reading frame (ORF) with a conserved RNA-dependent RNA polymerase (RdRP) domain. The smaller segment (1587 bp) contains a single ORF encoding a putative coat protein. Analysis of its genomic structure indicated that VdPV1 is a new member of the genus Partitivirus. We report the full-length sequence of this partitivirus that infects Verticillium dahliae, the causal agent of verticillium wilt of cotton.     

A novel mycovirus identified from the rice false smut fungus <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis>

The complete sequence of a novel mycovirus infecting Ustilaginoidea virens, the causal agent of false smut of rice, is reported here and designated as Ustilaginoidea virens unassigned RNA virus HNND-1 (UvURV-HNND-1). This virus has an undivided dsRNA genome of 2903 nt in length and contains two non-overlapping open reading frames (ORF1 and 2), with the small ORF1 encoding a protein of unknown function that showed sequence similarity to the comparable protein in virus Alternaria longipes dsRNA virus 1(AlRV1) and a larger ORF2 encoded the protein showing identities to the RNA-dependent RNA polymerases of AlRV1 and some other unassigned dsRNA viruses. Phylogenetic analysis showed that UvURV-HNND-1 is more closely related to unclassified viruses such as AlRV1 and distinct from distantly related members of the family Partitiviridae. Here, we propose in accordance with previous reports that UvURV-HNND-1 might belong to a new mycovirus genus together with AlRV1 and other similar viruses.     

Molecular characterization of two mitoviruses co-infecting a hypovirulent isolate of the plant pathogenic fungus Sclerotinia sclerotiorum [corrected

The complete nucleotide sequences of two double-stranded RNA (dsRNA) segments, isolated from the same hypovirulent strain (KL-1) of Sclerotinia sclerotiorum, were determined. Sequence analysis showed that dsRNAs 1 to be 2513 nts long and is A-U rich (61.7%). Excluding the poly(A) tail, dsRNAs2 is 2421 nts long and its AU content is 53.1%. The 5′ and 3′-terminal sequences of the positive-strand of each dsRNA could be folded into predicted stable stem-loop structures. Mitochondrial codon usage revealed that each dsRNA has a single large open reading frame coding for a protein containing RNA-dependent RNA polymerase conserved motifs. Furthermore, dsRNAs 1 and 2 share sequence similarities with other mitoviruses. These results suggest that dsRNAs 1 and 2 represent two distinct new mitoviruses, designated Sclerotinia sclerotiorum mitovirus 1 (SsMV1/KL-1) and SsMV2/KL-1, respectively. The hypovirulence traits of strain KL-1 and the two mitoviruses could be co-transmitted to a virus-free virulent strain via hyphal anastomosis.     

Significantly low level of small RNA accumulation derived from an encapsidated mycovirus with dsRNA genome

The role of RNA silencing as an antiviral defence has been well elucidated in plants and invertebrates, but not in filamentous fungi. We have previously determined the complete genome sequence of Magnaporthe oryzae virus 2 (MoV2), a dsRNA virus that infects the rice blast fungus Magnaporthe oryzae. In this study, we detected small interfering RNAs (siRNAs) from both positive- and negative-strand MoV2 viral RNA, suggesting that the RNA silencing machinery in M. oryzae functions against the mycovirus. Cloning and characterisation of MoV2 siRNAs indicated that, in MoV2, the ratio of virus-derived siRNAs to total small RNA is significantly lower than that in either plant viruses or Cryphonectria hypovirus 1 (CHV1), another mycovirus. Nevertheless, any MoV2-encoded proteins did not exhibit RNA silencing suppressor activity in both the plant and fungal systems. Our study suggests the existence of a novel viral strategy employed to evade host RNA silencing.     

Complete genome sequence of a novel endornavirus in the wheat sharp eyespot pathogen Rhizoctonia cerealis

We report here the presence of a novel double-stranded RNA (dsRNA) virus in an isolate (R0959) of the fungus Rhizoctonia cerealis, the causal agent of sharp eyespot of wheat in China. Sequence analysis showed that the dsRNA segment is 17,486 bp long and contains a single open reading frame (ORF) with the potential to encode a protein of 5,747 amino acids. The predicted protein contains conserved motifs of putative viral methyltransferase, helicase 1, and RNA-dependent RNA polymerase. Sequence similarity and phylogenetic analysis clearly place it in a distinct species within the genus Endornavirus, family Endornaviridae, and therefore we propose its name to Rhizoctonia cerealis endornavirus 1 (RcEV1). This is the first report of the full-length genomic sequence of a dsRNA mycovirus in R. cerealis.     

Genomic characterization of a novel partitivirus infecting Aspergillus ochraceus

Three double-stranded RNA (dsRNA) segments from Aspergillus ochraceus isolate FA 0611, designated as AoR1, AoR2, and AoR3, were cloned and sequenced. AoR1 was identical with AoV dsRNA 1 previously reported from A. ochraceus ATCC 28706, which putatively encoded an RNA-dependent RNA polymerase of an unidentified mycovirus. AoR2 was found to encode a putative viral capsid protein (CP) with 63% similarity to that of Penicillium stoloniferum virus S, which was detected from P. stoloniferum ATCC 14586. The function of AoR3 was unknown. The three segments were found to contain a conserved sequence at their 5′ termini, while an identical sequence was only found at the 3′ termini of AoR1 and AoR2. It is suggested that AoR1, AoR2, and AoR3 originate from an independent partitivirus infecting A. ochraceus. The novel virus is suggested to be Aspergillus ochraceus virus 1, AoV1. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank Accession numbers of the sequences reported in this article are EU118277, EU118278, and EU118279.     

A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata

Four double-stranded RNAs (dsRNAs), referred to as dsRNA 1 (3617 bp), dsRNA 2 (2794 bp), dsRNA 3 (2576 bp) and dsRNA 4 (1420 bp), were detected in the EGS 35-193 strain of Alternaria alternata at high concentration (3 μg/g dried mycelium). This strain had an impaired growth phenotype. By exposing the strain to cycloheximide during hyphal tip isolation, we isolated strains which had normal mycelial growth and pigmentation, in which decreased levels of the dsRNAs were observed (0.3 μg/g dried mycelium). These results indicate that this dsRNA mycovirus might be involved in modulating traits of its fungal host, A. alternata. The buoyant density of isometric virus particles (about 33 nm in diameter) containing these dsRNAs in CsCl was 1.35–1.40 g/cm³ depending on the size of the packaged dsRNAs. The dsRNA 1 encodes a single open reading frame (3447 nt) containing the conserved motifs of viral RNA-dependent RNA polymerase (RdRp), which is related to the ORF encoded by dsRNA 1 of Aspergillus mycovirus 341. It is noteworthy that all of the coding strands of the four dsRNA genomes have 3′-poly (A) tails ranging from 33 to 50 nt in length. We named this novel dsRNA mycovirus in the EGS 35-193 strain A. alternata virus-1 (AaV-1).     

Complete nucleotide sequences of three dsRNA segments from Raphanus sativus-root cv. Yipinghong with leaf yellow edge symptoms

Summary. The two minor dsRNA bands, previously detected in symptomatic leaves of Raphanus sativus-root cv. Yipinghong [5], were subjected to further analysis. cDNA cloning and sequencing revealed that the smaller of the two dsRNA bands is actually a doublet consisting of two co-migrating dsRNA segments and the resulting three segments were designated as RasR 3, RasR 4, and RasR 5. RasR 3 was 1717 bp in length and potentially encoded a protein of about 55.3 kDa, containing all of the six conserved motifs shared by the RNA dependent RNA polymerases of members of the family Partitiviridae. RasR 4 and RasR 5, which co-migrated in the 5% polyacrylamide gel, were 1521 and 1485 bp in length and each encoded a putative protein of unknown function. Their molecular masses, as calculated from the deduced amino acid, were 38.2 and 38.8 kDa, respectively. The 5′ UTRs of all three segments shared regions of high sequence similarities, but were distinct from those of the RasR 1 and RasR 2. Taken together, these results along with those described in the previous report [5], suggest that more than one partitivirus was co-infecting radish leaves.     

Complete nucleotide sequence and genome organization of a dsRNA partitivirus infecting Pleurotus ostreatus

The nucleotide sequences of the genomic dsRNA mycovirus infecting Pleurotus ostreatus (P. ostreatus virus 1; PoV1) were determined and compared to the sequences of the other mycoviruses belonging to partitiviruses and totivirues. PoV1 dsRNA-1 and dsRNA-2 had genomes of 2296 and 2223 nucleotides, respectively. The purified virus preparations contained isometric particles of 28-30 nm in diameter, and also the same two dsRNAs were isolated from purified virus preparations. The sequences of PoV1 dsRNA-1 and dsRNA-2 had GC contents of 48.4 and 51.5%, respectively. dsRNA-1 had 78 and 97 nucleotides of 5'- and 3'-untranslated region (UTR) while dsRNA-2 had 114 and 198 nucleotides of 5'- and 3'-UTR, respectively. Computer analysis of putative open reading frame (ORF) shows that dsRNA-1 and dsRNA-2 contain a single ORF encoding proteins of 82.2 and 71.1 kDa that show high sequence identity with RNA-dependent RNA polymerase and capsid protein of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis they were found to form a distinct virus clade with partitiviruses, and were more distantly related to totiviruses.     

Detection of a Double-Stranded RNA Virus from a Strain of the Violet Root Rot Fungus Helicobasidium mompa Tanaka

Three double-stranded (ds) RNA species (ca. 1.30, 1.27 and 1.23×10⁶) were isolated by CF-11 cellulose chromatography from a strain of the violet root rot fungus Helicobasidium mompa recovered from apple roots. Purified virion preparations contained isometric particles about 25nm in diameter, and also the same three species of dsRNA isolated from total extracts by CF-11 cellulose chromatography. The molecular mass of the coat protein was about 67K when estimated by SDS-PAGE. The largest dsRNA (referred to as dsRNA1) contains a single, long open reading frame of 1794 nucleotides that encodes a putative polypeptide containing 598 amino acid residues with a molecular mass of 69.9K. This polypeptide contains amino acid sequence motifs conserved in putative RNA-dependent RNA polymerases of RNA viruses. Phylogenetic analysis revealed similarities to RNA-dependent RNA polymerases from Atkinsonella hypoxylon 2H virus, a member of the family Partitiviridae.     

The H1 double-stranded RNA genome of Ustilago maydis virus-H1 encodes a polyprotein that contains structural motifs for capsid polypeptide, papain-like protease, and RNA-dependent RNA polymerase.

The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light). The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences. As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype. The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins. The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa. The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses. The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomyces cerevisiae viruses ScV-L1 and -La.     

Analysis of the terminal sequences of the genome segments of four orbiviruses

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.     

Electrophoresis of the complementary strands of the double-stranded Kemerovo virus RNAs in agarose-urea gel

Single-stranded (ss)RNAs derived from 10 double-stranded (ds)RNA segments of Kemerovo virus (KV) were separated into 13 RNA bands by agarose-urea gel electrophoresis. The complementary strands of the dsRNA segments 1, 9 and 10 displayed different electrophoretic mobility. An attempt was made to determine the origin of the ssRNA bands. The ssRNA bands originating from the dsRNA segments 1, 2, 3, 9 and 10 were identified unequivocally, while those originating from the dsRNA segments 4, 5, 6, 7 and 8 were characterized partially. The minus RNA strands of the dsRNA segments 9 and 10 exhibited higher electrophoretic mobilities as their complementary plus RNA strands.     

Mycoviruses are common among different species of endophytic fungi of grasses

A survey of mycoviruses was made in a collection of 103 isolates belonging to 53 different species of endophytic fungi of grasses. Double-stranded RNA (dsRNA) elements were detected in isolates of 12 of the species analyzed. The banding characteristics and sizes of some of the dsRNA elements suggest that they might belong to previously described mycovirus families. The observed incidence (22.6%) indicates that the presence of mycoviruses could be common among species of this group of ubiquitous fungi.