Genome Organization and Expression of the <Emphasis Type="Italic">Penicillium stoloniferum</Emphasis> Virus F

The complete sequences of three double-stranded (ds) RNAs (referred to F1, F2 and F3) of Penicillium stoloniferum virus F (PsV-F) were established. The F1 dsRNA was 1677 bp in length, and it contained one open reading frame (ORF) of 538 amino acids (molecular weight of 63 kDa, referred to P63), The F2 dsRNA was 1500 by in length, and also it contained one ORF of 420 amino acids (molecular weight of 46 kDa, referred to P46). The F3 dsRNA was 677 bp in length, but contained a small ORF with unknown function. A sequence motif of (5′-CGTAAAA-3′) was found only at the 5′ termini of the F1 and F2 dsRNAs, and a sequence motif of (5′-TAAAAAAAAA-3′) was found at the 3′ termini of all three dsRNA segments. The predicted amino acid sequence of F1 showed 38–48% sequence homology with the putative dsRNA-dependent RNA polymerases (RdRp) of dsRNA viruses, but the predicted amino acid of F2 showed no homology. Phylogenetic analysis using the RdRp sequences of the various Partitiviruses and Alphacryptoviruses revealed that PsV-F clustered well with Partitiviruses, but showed remote relationship with PsV-S. Near full-length and positive-sense single-stranded (ss) RNAs derived from the Fl, F2 and F3 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P63 and P46 showed a positive reaction against PsV-F antiserum, indicating P63 and P46 as RdRp and capsid protein, respectively. These results suggest that PsV-F can be a member of Partitivirus, but it is quite distinct from PsV-S electrophoretically, serologically and genetically, though both viruses coexist in the same cell.     

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus

Summary. A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with ³²P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg²⁺ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3′ ends, but different 5′ ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.     

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).     

A double-stranded RNA as the genome of a potential virus infecting <Emphasis Type="Italic">Vicia faba</Emphasis>

Preparations of double-stranded (ds) RNAs extracted from naturally infected Vicia faba Linn. growing in Hangzhou, Zhejiang Province, Eastern China displayed 3 dominant bands (FaR1, FaR2, and FaR3). FaR2 and FaR3 were found to be identical to the genomic dsRNAs of a recently reported Vicia cryptic virus (VCV). The positive strand of FaR1 contained two large open reading frames (ORFs), ORF1 and ORF2. The putative proteins encoded by these ORFs were found to have certain similarities to the putative capsid protein [ABO36237] and RNA-dependent RNA polymerase [ABC96788], respectively, of Tomato yellow stunt virus. Thus, FaR1 may represent the genome of a new dsRNA virus, which we have named Vicia cryptic virus M. The GenBank Accession numbers of the sequences reported in this paper are EU605883, EU605884, and EU371896.     

Molecular characterisation of two novel double-stranded RNA elements from <Emphasis Type="Italic">Phlebiopsis gigantea</Emphasis>

The incomplete sequences of two large, 10–12 kbp, double-stranded RNAs (dsRNAs) found in the TW-2 isolate of the saprophytic fungus, Phlebiopsis gigantea (Pg) are reported. Both PgV-TW2 dsRNA1 and dsRNA2 potentially encode fusion proteins which are apparently expressed by a translational frameshifting mechanism. The C-terminal region of both predicted proteins was 21% identical and contained the eight motifs conserved in RNA-dependent RNA polymerases of dsRNA mycoviruses and had highest similarity with members of the family Totiviridae, but possibly do not form virions. The remainder of the N-terminal protein sequences predicted from the PgV-TW2 dsRNA1 and dsRNA2 sequences and the 3′-terminal nucleotide sequences of both dsRNAs had no homology with one another or any sequence in the database suggesting that individually both may be members of novel families of mycoviruses. The nucleotide sequence data reported in this article has been assigned the accession numbers AM111O96 and AM111097 for PgV-TW2 dsRNA1 and PgV-TW2 dsRNA2 respectively.     

Molecular characterization and detection of Vicia cryptic virus in different Vicia faba cultivars

Summary After extraction of double-stranded (ds) RNAs from Vicia faba, dsRNA1 and dsRNA2 of Vicia cryptic virus (VCV), a member of the genus Alphacryptovirus (family Partitiviridae), were detected in six out of seven different cultivars by agarose gel electrophoresis. In attempts to sequence the complete VCV genome, the dsRNA1 and dsRNA2 sequences from a total of five different V. faba cultivars were determined. Analysis of these sequences indicated that V. faba cultivars contain almost indistinguishable VCV sequences. The larger dsRNA1 was 2012 bp in length and contained a major open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp). The smaller dsRNA2 was 1779 bp in length and comprised a single ORF on its plus-strand encoding the coat protein (CP). The sequences of the dsRNA1 and dsRNA2 ORFs shared highest amino acid sequence identities (84 and 56%, respectively) with the corresponding gene products of the alphacryptovirus white clover cryptic virus 1 (WCCV-1). The 5′-terminal untranslated regions of dsRNA1 and dsRNA2 of VCV were highly conserved and were strikingly similar to the corresponding regions of WCCV-1. RdRp amino acid sequence alignments revealed conserved motifs, which correlate with the phylogenetic clustering of the family Partitiviridae.     

The complete nucleotide sequence and genome organization of Fig cryptic virus,a novel bipartite dsRNA virus infecting fig,widely distributed in the Mediterranean basin

Two double-stranded RNA (dsRNA) segments of a virus with a bipartite genome identified in fig (Ficus carica L.) and denoted Fig cryptic virus (FCV) were cloned and sequenced. Viral dsRNAs are 1696 bp (RNA-1) and 1415 bp (RNA-2) in size. RNA-1 contains a single ORF (1419 nt) potentially encoding a 54 kDa protein and comprising the conserved amino acid motifs of the RNA-dependent RNA polymerase (RdRp) domain of species of the genus Alphacryptovirus. Its full-length amino acid sequence has the highest identity with Raphanus sativus cryptic virus 2 (RsCV-2) (36%), Beet cryptic virus 3 (BCV-3) (36%) and Fragaria chiloensis cryptic virus (FCCV) (34%). RNA-2 has also a single ORF (1014 nt) coding for a polypeptide with a predicted molecular mass of 38 kDa, identified as the viral coat protein (CP). In a phylogenetic tree constructed with the amino acid sequences of the RdRp domain, FCV clusters in a clade comprising BCV-3 and a number of tentative species of the genus Alphacryptovirus. FCV is not mechanically transmissible. It was detected in fig orchards of six Mediterranean countries (Albania, Algeria, Italy, Lebanon, Syria and Tunisia) where it does not seem to induce a visible disease.     

Mycoviruses infecting the endophytic and entomopathogenic fungus Tolypocladium cylindrosporum

A mixed virus infection in a strain of the endophytic and entomopathogenic fungus Tolypocladium cylindrosporum was deduced from a study of the transmission to conidia of several double-stranded RNA (dsRNA) elements. The transmission rates of each dsRNA were different, and monosporic isolates harbouring different combinations of the original set of six dsRNAs were obtained. A 5196 bp dsRNA element was sequenced and represents the genome of T. cylindrosporum virus 1 (TcV1), a new member of the genus Victorivirus in the Totiviridae family. This virus was transmitted to 81.4% of the conidia; in contrast, four dsRNAs of 3.1-3.7 kbp were transmitted only to 4.7% of the monosporic isolates obtained from the infected parental strain. These four dsRNAs did not show segregation during transmission, and one of them was shown by sequence analysis to encode an RdRp, suggesting that the four molecules might represent the whole genome of a quadripartite chrysovirus. A third possible virus with a genome of approximately 4.2 kbp was transmitted to 79.1% of the monosporic isolates produced by the infected strain. Ribavirin was used to cure T. cylindrosporum from viruses, and TcV1 was sensitive to this drug. All monosporic cultures derived from the infected strain treated with 80 and 100 μM concentrations of the drug were free of TcV1.     

Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses

Novel double-stranded RNAs (∼ 8 kbp) were isolated from threecornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA polymerase (RdRp). Expression of the 3’-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.     

Sequence of RNA-dependent RNA Polymerase Genes Provides Evidence for Three More Distinct Mitoviruses in Ophiostoma Novo-ulmi Isolate Ld

Three of the twelve double-stranded (ds) RNAs, dsRNAs 1a, 1b and 3b, which are located in the mitochondria of a diseased isolate, Ld, of the Dutch elm disease fungus, Ophiostoma novo-ulmi have been cDNA cloned and sequenced. Examination of the sequences of the RdRp genes predicted from the nucleotide sequences of the three dsRNAs suggest that they constitute the genome of three new mitoviruses.     

A new putative alphapartitivirus recovered from the powdery mildew fungus <Emphasis Type="Italic">Erysiphe palczewskii</Emphasis>

Two double-stranded RNAs (dsRNA) likely representing the genome of a novel alphapartitivirus which we provisionally named Erysiphe palczewskii alphapartitivirus 1 (EpV1) were recovered from the powdery mildew fungus E. palczewskii infecting Sophora japonica in Jingzhou, Hubei province of China. The two dsRNAs, 1955 (dsRNA1) and 1917 (dsRNA2) bp in size, respectively, each contains a single open reading frame (ORF) encoding a 585- and 528-aa protein, respectively. The 585-aa protein contains a conserved RNA-dependent RNA polymerase (RdRp) domain and shows significant homology to RdRps of approved or putative partitiviruses, particularly those belonging to the genus Alphapartitivirus. However, it shares an aa sequence identity lower than 80% with its closest relative, the RdRp of the putative alphapartitivirus Grapevine partitivirus, and lower than 60% with the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRp aa sequences of selected partitiviruses, the putative virus EpV1 clustered with Grapevine partitivirus and formed a well-supported monophyletic clade with known or putative alphapartitiviruses.     

Complexity of dsRNA Mycovirus Isolated from Fusarium graminearum

Fusarium graminearum is the causal agent of a serious scab disease of small grains in Korea. We screened 827 isolates of F. graminearum from diseased barley and maize and tested for the presence of double-stranded RNA (dsRNA) mycovirus. Of them, 19 isolates contained various sizes of dsRNAs. A dsRNA associated with pronounced morphological changes including reduction in mycelial growth, increase in dark orange to red pigmentation, reduced sporulation and virulence was previously observed in nine dsRNA-containing Fusarium isolates (Chu et al., Appl Env Microbiol 68, 2529-2534, 2002). Ten additional isolates were found infected with dsRNA mycoviruses. These mycoviruses contain 2-4 different segments of dsRNAs with the size-range of approximately 1.7-10 kbp in length. The presence of dsRNAs did not affect colony morphology and were transmissible through conidia and ascospore with incidence of 30-100%. Interestingly, dsRNA mycovirus found in F. graminearum isolates, JB33 and JNKY19, that show the pattern of mixed infection of two different viruses were transmitted to all progeny conidia and ascospores. These results indicate that there is genomic diversity of dsRNA mycoviruses that infect F. graminearum isolates and that impact of virus infection on host's morphology and virulence is determined by the interaction between dsRNAs and the fungal host, not by the mere presence of the dsRNAs.     

Use of double-stranded RNA templates by the tombusvirus replicase in vitro: Implications for the mechanism of plus-strand initiation

Plus-stranded RNA viruses replicate efficiently in infected hosts producing numerous copies of the viral RNA. One of the long-standing mysteries in RNA virus replication is the occurrence and possible role of the double-stranded (ds)RNA formed between minus- and plus-strands. Using the partially purified Cucumber necrosis virus (CNV) replicase from plants and the recombinant RNA-dependent RNA polymerase (RdRp) of Turnip crinkle virus (TCV), in this paper, we demonstrate that both CNV replicase and the related TCV RdRp can utilize dsRNA templates to produce viral plus-stranded RNA in vitro. Sequence and structure of the dsRNA around the plus-strand initiation site had a significant effect on initiation, suggesting that initiation on dsRNA templates is a rate-limiting step. In contrast, the CNV replicase could efficiently synthesize plus-strand RNA on partial dsRNAs that had the plus-strand initiation promoter "exposed", suggesting that the polymerase activity of CNV replicase is strong enough to unwind extended dsRNA regions in the template during RNA synthesis. Based on the in vitro data, we propose that dsRNA forms might have functional roles during tombus- and carmovirus replication and the AU-rich nature of the terminus could be important for opening the dsRNA structure around the plus-strand initiation promoter for tombus- and carmoviruses and possibly many other positive-strand RNA viruses.     

A novel quadripartite dsRNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix

Here we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45 nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7 kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family.     

Presence of double-stranded RNA and virus-like particles in Phaffia rhodozyma

Four double-stranded RNA (dsRNA) molecules were isolated from Phaffia rhodozyma UCD 67-385. Their molecular sizes were approximately 4.3, 3.1, 0.9 and 0.75 kilobase pairs (kbp) as determined by agarose-gel electrophoresis and they were designated as L, M, S₁ and S₂, respectively. By differential centrifugation in sucrose gradients, these dsRNAs copurified with isometric virus-like particles 36 nm in diameter. A cured strain, UV-S2, lacking the S₂-dsRNA was obtained from P. rhodozyma UCD 67-385 by ultraviolet (UV) light treatment. UV-S2 strain contains identical virus-like particles to those from the wild-type strain, as determined by electron microscopy, suggesting that the S₂-dsRNA was not essential for the expression of mycovirus structural polypeptides. On the other hand, both the UCD 67-385 and UV-S2 strains were able to kill P. rhodozyma UCD 67-383, a strain without dsRNAs. These results suggest that the dsRNA molecules also encode a killer system. Finally, the UV-S2 strain maintains killer ability, which suggests that S₂-dsRNA is not involved in the killer phenotype expression.     

A novel mycovirus closely related to hypoviruses that infects the plant pathogenic fungus Sclerotinia sclerotiorum

Three dsRNA segments, two similarly sized at 9.5 kbp and a third one of approximately 3.6 kbp, were extracted from a hypovirulent strain SZ-150 of Sclerotinia sclerotiorum. The complete cDNA sequence of one of the two large dsRNA segment (10398 bp, excluding the poly (A) tail) reveals a single ORF that encodes a polyprotein with conserved domains of putative papain-like protease, UDP glucose/sterol glycosyltransferase, RNA-dependent RNA polymerase and viral RNA Helicase. This virus is closely related to Cryphonectria hypovirus (CHV) 3/GH2 and CHV4/SR2 in the family Hypoviridae and designated as Sclerotinia sclerotiorum hypovirus 1 (SsHV1/SZ-150). The satellite-like 3.6 kbp dsRNA segment (S-dsRNA) shares high sequence identity with the 5′-UTR of SsHV1/SZ-150. SsHV1/SZ-150 alone is not the primary causal agent for hypovirulence of strain SZ-150 since strains without the S-dsRNA show normal phenotype. This is the first report of a naturally occurring hypovirus that infects a fungus other than Cryphonectria parasitica.     

Identification and Sequence Analysis of Potato yellow vein virusCapsid Protein Minor Gene

Potato yellow vein virus (PYVV) is a whitefly-transmitted (Trialeurodes vaporariorum) closterovirus (WTC) with an as yet unidentified genome composition. PYVV dsRNA preparations consist of three high molecular weight dsRNA species (dsRNAs 1, 2 and 3) 8.0, 5.5 and 4.0kbp in size respectively, as well as two low molecular weight dsRNA species of 2.0 and 1.8kbp (denoted x and y). The PYVV capsid protein minor (CPm) gene was identified on the dsRNA 3 species, and was subsequently cloned and sequenced. The PYVV CPm gene is 2022 nucleotides long and putatively encodes a protein with estimated size 77.5kDa. The PYVV CPm gene product is considerably larger than the equivalent proteins encoded by the bipartite criniviruses, Lettuce infectious yellows virus (LIYV) and Cucurbit yellow stunting disorder virus (CYSDV) (52 and 53kDa, respectively). The PYVV CPm possesses a centralized domain which is absent from both the LIYV and CYSDV CPm counterparts. Pairwise comparisons as well as phylogenetic analysis based on the available amino acid sequences of the CPm of various WTCs, showed that PYVV is closely related to LIYV, CYSDV and also Beet pseudo-yellows virus.     

Complete sequence of the genome of two dsRNA viruses from Discula destructiva

Complete nucleotide sequences were determined for the four dsRNA segments present in isolate 247 of Discula destructiva from South Carolina. The largest dsRNA (dsRNA 1) was 1787 bp in length with a single open reading frame (ORF) that coded for a putative RNA-dependent RNA polymerase (RdRp). The dsRNA 2 was 1585 bp in length with a single ORF that coded for a putative viral coat protein. Both the dsRNA 3 (1178 bp in length) and dsRNA 4 (308 bp) contained single ORFs. However, neither the nucleotide sequence nor the sequence of the putative translation products, showed any similarity with sequences currently available from GenBank. Although distinct, all 4 dsRNAs showed conserved nucleotides at both the 5′ and 3′ termini. Sequences of the two dsRNAs in an isolate of D. destructiva (331 originating from Idaho) were similar in length to, and shared similarity with, the dsRNA 1 and dsRNA 2 of isolate 247. However, although the putative RdRps of isolates 247 and 331 are closely related, the putative viral coat proteins coded for by the respective dsRNA 2s are distinct. Thus, the dsRNAs in the two fungal isolates appeared to originate from distinct, but related viruses, which we have named D. destructiva virus 1 and D. destructiva virus 2, respectively. Phylogenetic analysis indicated that the two viruses were most closely related to Fusarium solani virus 1 and should be considered members of the genus Partitivirus. Another isolate of D. destructiva (272.1) contains a 12 kb dsRNA in addition to the 4 dsRNAs found in isolate 247. Partial sequence of this 12 kb molecule showed a relationship to other large dsRNA molecules isolated from plants.     

A new cryptic virus belonging to the family Partitiviridae was found in watermelon co-infected with Melon necrotic spot virus

A novel virus was detected in watermelon plants (Citrullus lanatus Thunb.) infected with Melon necrotic spot virus (MNSV) using SOLiD next-generation sequence analysis. In addition to the expected MSNV genome, two double-stranded RNA (dsRNA) segments of 1,312 and 1,118 bp were also identified and sequenced from the purified virus preparations. These two dsRNA segments encode two putative partitivirus-related proteins, an RNA-dependent RNA polymerase (RdRP) and a capsid protein, which were sequenced. Genomic-sequence analysis and analysis of phylogenetic relationships indicate that these two dsRNAs together make up the genome of a novel Partitivirus. This virus was found to be closely related to the Pepper cryptic virus 1 and Raphanus sativus cryptic virus. It is suggested that this novel virus putatively named Citrullus lanatus cryptic virus be considered as a new member of the family Partitiviridae.