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.     

Molecular characterization of a novel victorivirus from the entomopathogenic fungus Beauveria bassiana

New Zealand isolates of the entomopathogenic fungus Beauveria were examined for the presence of dsRNAs and virus-like particles. Seven out of nine isolates contained one or more high-molecular-weight dsRNAs and all seven contained isometric virus particles ranging in size from 30 to 50 nm. B. bassiana isolate ICMP#6887 contained a single dsRNA band of ~6 kb and isometric virus-like particles of ~50 nm in diameter. Sequencing revealed that the virus from ICMP#6887 had a genome of 5,327 nt with two overlapping ORFs coding for a putative coat protein (CP) and an RNA-dependent RNA-polymerase (RdRp). The sequence showed a highest CP identity of 58.3 % to Tolypocladium cylindrosporum virus 1 (TcV1) and a highest RdRp identity of 48.8 % to Sphaeropsis sapinea RNA virus 1 (SsRV1). Since both TcV1 and SsRV1 belong to the genus Victorivirus, the new virus from B. bassiana ICMP#6887 was tentatively assigned the name Beauveria bassiana victorivirus 1 (BbVV1-6887).     

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.     

A novel double-stranded RNA mycovirus from Fusarium graminearum: nucleic acid sequence and genomic structure

Ten Fusarium graminearum isolates from China were screened for dsRNA mycoviruses. Five dsRNAs (2.4 to 3.5 kbp) were purified from isolate China 9, cloned, and sequenced. BLAST analysis showed that the proteins encoded by dsRNA1 possess motifs that are conserved in RNA-dependent RNA polymerases, dsRNA2 resembles the hypothetical protein encoded by dsRNA3 of Magnaporthe oryzae chrysovirus 1, dsRNA4 shares no significant similarity to any published protein, and dsRNA5 has a C2H2 zinc finger domain. Tandem mass spectrophotometry, surface protein labeling of virus-like particles, SDS-PAGE, and protein BLAST results supports the notion that three of the virus segments code for structural proteins, of which dsRNA3 possibly codes for the capsid protein. Relative quantitative RT-PCR studies of the 5 dsRNAs suggested that the segments are encapsidated separately in unequal amounts. Genomic structure and phylogenic studies support the possibility that this virus may be a candidate for the type species of a novel genus in the family Chrysoviridae.     

cDNA probes for detection of specific dsRNAs from the fungal pathogen, Monosporascus cannonballus

Monosporascus cannonballus is an ascomycete fungus that is the causative agent of Monosporascus root rot/vine decline, a serious disease of muskmelon and watermelon. Double-stranded RNA (dsRNA) was identified in approximately 60% of M. cannonballus isolates recovered from infected muskmelon plants in 1993. After repeated laboratory transfer on culture media, the majority of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to muskmelon. Initially, dsRNA purification and cDNA synthesis were attempted in three M. cannonballus isolates harboring dsRNAs. However, numerous difficulties were encountered due to the stable, double-stranded nature of the dsRNAs and contamination of the preparations by fungal rRNA. Several purification and cDNA protocols were evaluated and eventually modified into methods that were ultimately highly effective for cloning dsRNAs from M. cannonballus. The cDNAs derived from purified dsRNA preparations were cloned into a pUC119 plasmid vector and amplified in Escherichia coli. Nine cDNA clones were identified that are specific for medium-sized (ca. 3 kbp) dsRNAs associated with M. cannonballus isolate Ca91-17(96+). The methods used to make the cDNA clones of the dsRNAs in M. cannonballus may be useful for those working on fungal dsRNAs. In addition, these cDNAs may be useful for identifying dsRNAs associated with the hypovirulence phenotype.     

A totivirus infecting the mutualistic fungal endophyte Epichloë festucae

Epichlo? festucae (Ascomycota) infects the grass Festuca rubra. Infected plants may be more resistant to herbivores and obtain other benefits. The 5109bp dsRNA genome of a virus which infects E. festucae was sequenced, and its incidence in natural populations and transmission were studied. The viral genome has characteristics of the family Totiviridae. Its two ORFs are overlapped by four nucleotides; ORF1 codes a 765 amino acid putative coat protein (CP); ORF2 is in a -1 frameshift with respect to ORF1, and codes a 826 amino acid RNA dependent RNA polymerase (RdRp). This virus, denominated Epichlo? festucae virus 1 (EfV1), is closely related to members of the genus Totivirus which infect filamentous fungi, as deduced from phylogenetic analyses of CPs and RdRps. In two natural populations of Epichlo? festucae, 36.4% of the isolates were infected by EfV1. The virus was efficiently transmitted to asexual fungal spores. However, when ascospore progeny of matings between virus-free and infected strains was analyzed, it was found that the virus was not transmitted to progeny of sexual spores.     

Incidence of Phlebiopsis gigantea large virus-1 in a collection of Phlebiopsis gigantea isolates

Eighty six Phlebiopsis gigantea isolates from at least 9 different tree species from various locations in 12 different European countries and North America were screened for the presence of large molecular weight dsRNA >10 kbp in size. In 7 isolates, which contained large dsRNAs, the presence of Phlebiopsis gigantea large virus-1 (PgLV-1) was suggested following the sequencing of the RT-PCR amplicons generated with PgLV-1 specific oligonucleotide primers which also revealed little genetic diversity between the virus isolates.     

Nucleotide sequence relationships of double-stranded RNAs in flax rust,Melampsora lini

Flax rust, Melampsora lini strain SP6, contains 11 double-stranded (ds) RNA molecules with a total length of about 25 kbp. The dsRNAs are inherited in three genetic units: the L unit comprising a single 5.2 kbp dsRNA and contained within a 40-nm virus-like particle, and the A and B units each consisting of five dsRNAs (A1–A5, and B1–B5) ranging in size from 1.2 to 2.7 kbp. This paper reports the isolation of a cDNA library representing 10 of the 11 dsRNAs. By nucleic-acid hybridization techniques it has been shown that all ten sequences are unique showing no detectable cross-hybridization with any other dsRNA present in the rust. A near fulllength sequence of 1932 bp of the B3 dsRNA is reported and contains several open reading frames, the largest of which comprises most of the molecule.     

Identification of a satellite double-stranded RNA in the parasitic protozoan Trichomonas vaginalis infected with T. vaginalis virus T1

Co-infection by a 0.5-kb small double-stranded (ds) RNA together with Trichomonas vaginalis virus (TVV) genomic 4.6-kb dsRNA is commonly observed in a number of T. vaginalis isolates. By molecular cloning and primer extension experiments, the 497-bp cDNA sequence of a 0.5-kb dsRNA co-infecting with TVV-T1 in T vagina/is T1 isolate was elucidated. Consistent with the replication cycle of a typical dsRNA virus, a plus-strand viral RNA beginning at +1 of the 0.5-kb dsRNA was identified in infected T. vaginalis T1 cells by primer extension and Northern hybridization studies. The 0.5-kb dsRNA was separately encased in TVV capsids from the viral genomic dsRNA, as shown by protein analysis and electron microscopic examination of viral particles purified by multiple rounds of CsCl gradient centrifugation. The riboprobes transcribed from a cloned cDNA of the 0.5-kb dsRNA exhibited strong hybridization to a small dsRNA in a T vaginalis T9 isolate, which harbors a TVV-T9 distantly related to TVV-T1, but the same probes showed very little hybridization to the viral genomic dsRNA of both TVV-T1 and TVV-T9. Very little sequence homology between the 0.5-kb dsRNA and the 4.6-kb dsRNA in TVV-T1 was found by computer-assisted analysis, suggesting that the small dsRNA in T. vaginalis T1 is not derived from the genome of TVV-T1 or other distantly related T. vaginalis viruses. These results suggest that the small dsRNAs in T vaginalis are satellite RNAs of T. vaginalis 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.     

Searching for a New Putative Cryptic Virus in Pinus sylvestris L

Double-stranded RNAs (dsRNAs) were detected in different pine populations in Germany and Hungary. Two dsRNA species of 1.5 and 1.58 kbp, respectively, persisted in the same trees for at least 2 years and their presence was not associated with any symptoms. The dsRNAs were found to sediment in the VLP (virus-like particles) fraction and to be protected by protein(s) against RNase A digestion at low salt. cDNA cloning and sequencing of the smaller segment (dsRNA2) led to the identification of a putative RNA-dependent RNA-polymerase (RdRp) containing the GDD, as well as three other, conserved motifs. Sequence comparison with different RNA viruses and phylogenetic analysis indicates that the putative RdRp from pine shows highest similarity to the homologous proteins of Beet cryptic virus 3 and of a cryptic virus of Pyrus pyrifolia. On the basis of these results we suggest that the 1.5 and 1.58 kbp dsRNAs in P. sylvestris may represent the genomic segments of a new plant cryptic virus, Cryptoviruses have not yet been reported to occur in Gymnosperms.     

Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples

Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTV1SA), was used as template to optimise a polymerase chain reaction (PCR) for the detection of bluetongue virus RNA. Pairs of oligonucleotides complementary to the 3' termini of eight of the ten genome segments were tested. Those representing the 5' termini of genome segment 7 gave the best amplification results producing a single DNA band with the same mobility during agarose gel electrophoresis as genome segment 7. It was confirmed by cloning and sequence analysis, that this PCR-amplified DNA contained both terminal regions of genome segment 7 and therefore represented full length cDNA. Using these segment 7 oligonucleotides it was not only possible to detect routinely as few as 6 molecules of segment 7 dsRNA per sample, but also to detect purified dsRNAs from isolates of other BTV serotypes (1 Australia (AUS), 2, 3, 4, 10, 16 and 20). However, with the exception of Tilligery virus, isolates from other Orbivirus serogroups tested all gave negative results (African horse sickness, epizootic haemorrhagic disease, Palyam, Warrego and Eubenangee). The PCR was also used to analyse red blood cells (RBC) and buffy coat samples from cattle infected with BTV4. Positive results were obtained from samples taken 7 days post-infection (p.i.) (containing 1.6 x 10(3) TCID50 of virus/ml of whole blood) and from the RBC sample only, taken 14 days p.i. (16 TCID50/ml). However, at 28 days p.i. (less than 1.6 TCID50/ml) BTV RNA was not detected using the PCR in either sample.     

Characterization of two different apricot latent virus variants associated with peach asteroid spot and peach sooty ringspot diseases

Summary.  Peach asteroid spot (PAS) and peach sooty ringspot (PSRS) are two diseases of stone fruit trees of unknown aetiology. The use of a cRNA probe of the newly described Apricot latent virus (ApLV), a tentative member of the Foveavirus genus, indicated the presence of cross-hybridizing agents in PAS isolate LA2 and in PSRS isolates Caserta 12 and Clava J4. Analysis of dsRNA patterns revealed in each case the presence of a major dsRNA band of about 9.6 kbp. The purified dsRNAs were used to obtain cDNA clones for isolates LA2 and Caserta 12. Sequence analysis of a 1.1 kbp cDNA clone from isolate LA2 showed very high homology with the known ApLV sequence, indicating that this isolate represents a closely related variant of ApLV. Sequence analysis of a 3.06 kbp Caserta 12 cDNA clone representing the 3′ region of the genome revealed a genomic organization similar to that reported for other members of the Foveavirus genus, including the triple gene block and a large, 43.6 kDa coat protein. Sequence comparison with the CP gene of ApLV, the only sequenced region so far for this virus, showed an overall homology of 78%. These results indicate that the foveavirus represented by the Caserta 12 isolate of PSRS disease may be regarded as a distant variant of ApLV. The present results indicate that the viral agents associated with peach asteroid spot and peach sooty ringspot diseases might be variants of the recently described ApLV. Received November 23, 2000/Accepted April 25, 2001     

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).     

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.     

Evidence for equimolar synthesis of double-strand RNA and minus-strand RNA in rotavirus-infected cells

The genome of the rotaviruses consists of eleven segments of double-strand RNA (dsRNA). Each segment is replicated asymmetrically with viral plus-strand RNA, i.e. messenger (m)RNA, serving as the template for the synthesis of minus-strand RNA to produce dsRNA. To examine the relative frequency of replication of each of the eleven genome segments, MA104 cells were infected with low (3rd) and high (12th) passage stocks of simian rotavirus SA11. The total cytoplasmic RNA of the infected cell was radiolabeled either by maintaining the infected cells in the presence [3H]uridine prior to harvest or by 3'-endlabeling the purified RNA with [32P]pCp and T4 RNA ligase. The RNA was then analyzed for the presence of 3H- and 32P-labeled dsRNA by electrophoresis on 10% polyacrylamide gels. Total cytoplasmic RNA from infected cells was also 3'-end-labeled with [32P]pCp and T4 RNA ligase and examined for the presence of minus-strand RNA by electrophoresis on low pH agarose-urea gels. Bands representing dsRNAs and minus-strand RNAs on autoradiographs of the gels were analyzed for intensity by densitometry. The results showed that the eleven segments of viral dsRNA were present in equimolar concentrations in cells either infected with low or high passage stocks of virus. Like intracellular dsRNAs, full-length minus-strand RNAs were also present in equimolar concentration in cells either infected with low or high passage rotavirus. These data indicate that, despite the non-equimolar levels of viral RNAs in the cell, the eleven genome segments of rotavirus are replicated with equal frequencies in vivo.     

Blueberry latent virus: an amalgam of the Partitiviridae and Totiviridae

A new, symptomless virus was identified in blueberry. The dsRNA genome of the virus, provisionally named Blueberry latent virus (BBLV), codes for two putative proteins, one without any similarities to virus proteins and an RNA-dependent RNA polymerase. More than 35 isolates of the virus from different cultivars and geographic regions were partially or completely sequenced. BBLV, found in more than 50% of the material tested, has high degree of homogeneity as isolates show more than 99% nucleotide identity between them. Phylogenetic analysis clearly shows a close relationship between BBLV and members of the Partitiviridae, although its genome organization is related more closely to members of the Totiviridae. Transmission studies from three separate crosses showed that the virus is transmitted very efficiently by seed. These properties suggest that BBLV belongs to a new family of plant viruses with unique genome organization for a plant virus but signature properties of cryptic viruses including symptomless infection and very efficient vertical transmission.     

A novel monopartite dsRNA virus from rhododendron

A dsRNA molecule of 3.4 kbp was extracted from two great rhododendron samples from Great Smoky Mountains National Park. Sequencing of this molecule suggests that it represents the genome of an undescribed virus, for which the provisional name rhododendron virus A (RhVA) is proposed. In phylogenetic analyses, this virus clustered together with southern tomato virus and related viruses, forming a coherent and distinct clade among dsRNA viruses. RhVA likely belongs to a yet-to-be-established taxon of viruses with a non-segmented dsRNA genome.