Two unrelated double-stranded RNA molecule patterns in <Emphasis Type="Italic">Gremmeniella abietina</Emphasis> type A code for putative viruses of the families <Emphasis Type="Italic">Totiviridae</Emphasis> and <Emphasis Type="Italic">Partitiviridae</Emphasis>

Summary. Two double stranded (ds) RNA molecule patterns, probably of viral origin, were sequenced from Gremmeniella abietina var. abietina type A. The genome of Gremmeniella abietina RNA virus L1 (GaRV-L1) from isolate HR2 was 5133bp and contained two open reading frames (ORFs). The 5-proximal ORF coded for a putative coat protein (CP) and the 3-proximal ORF encoded putative RNA-dependent RNA polymerase (RdRp). GaRV-L1 had sequence similarities with a previously described totivirus (Helminthosporium victoriae 190S virus) and two unclassified members of family Totiviridae (Sphaeropsis sapinea RNA virus 1 and Sphaeropsis sapinea RNA virus 2). The genome of Gremmeniella abietina RNA virus MS1 (GaRV-MS1) from isolate C5 was composed of three dsRNA molecules coding for a putative RdRp (dsRNA1), a putative CP (dsRNA2) and protein of unknown function (dsRNA3). The lengths of these dsRNA molecules were 1782, 1586 and 1181bp, respectively. Sequence comparisons indicated that the GaRV-MS1 dsRNA pattern comprises a putative virus that is highly similar to Discula destructiva virus 1, Discula destructiva virus 2 and Fusarium solani virus 1 of the family Partitiviridae.     

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.     

<Emphasis Type="Italic">Gremmeniella abietina</Emphasis> mitochondrial RNA virus S1 is phylogenetically related to the members of the genus <Emphasis Type="Italic">Mitovirus</Emphasis>

Summary. A double stranded (ds) RNA genome of Gremmeniella abietina mitochondrial RNA virus S1 (GaMRV-S1) was sequenced. The length of the genome was 2572 base pairs, it had a very low GC content (30.6%), and sequence and length variations occurred in both ends of it. The genome coded for a putative 741 amino acid long RNA-dependent RNA polymerase (RdRp) using a mitochondrial translation code. Comparison of the putative amino acid sequences suggested that GaMRV-S1 is a putative member of the genus Mitovirus.     

Genomic organization of a novel partitivirus from the phytopathogenic fungus Ustilaginoidea virens

From the plant pathogen Ustilaginoidea virens, four double-stranded RNA (dsRNA) segments designated Uv-dsRNA1, -2, -3, and -4 were isolated, cloned, and sequenced. Uv-dsRNA1 (1775 bp) and -2 (1588 bp) potentially encode an RNA-dependent RNA polymerase (RdRp) and a viral coat protein (CP), respectively. Since the RdRp and CP sequences encoded by Uv-dsRNA1 and -2, respectively, are most closely related to, but clearly distinct from, those of viruses of the genus Partitivirus, they appear to be the two genome segments of a new partitivirus, for which the name Ustilaginoidea virens partitivirus 1 is proposed. In contrast, Uv-dsRNA3 (1352 bp) did not share significant sequence similarity with GenBank sequences, and the ORF of Uv-dsRNA4 (1119 bp) was only 32 % identical to a functionally unknown protein (GaRVMS2s3gp1) encoded by Gremmeniella abietina RNA virus MS2.     

Intergeneric relationship between the Aspergillus ochraceous virus F and the Penicillium stoloniferum virus S

It was reported that the ‘slow’ component (PsV-S) of Penicillium stoloniferum virus complex also occurred in a second genus, Aspergillus ochraceous. The responsible virus for this intergeneric occurrence was considered to be the ‘fast’ component (AoV-F) of A. ochraceous virus complex. In this investigation, AoV dsRNA 1, that was previously shown to cross-hybridize with PsV-S dsRNA, has been cloned. It was 1754 bp in length and contained one open reading frame of 539 amino acids (p63), had the same genome organization as PsV-S dsRNA S1 and also had the conserved sequence motif of the PsV-S dsRNAs (5′-GCGCAAAA-3′) at the 5′ terminus. A BLAST search indicated that p63 was a putative dsRNA-dependent RNA polymerase (RdRp), had 81% of sequence homology to members of the genus Partitivirus, and grouped together with PsV-S in phylogenetic analysis. But immunoblot analysis showed that the capsid protein (P3) of AoV-F virus component did not reacted against PsV-S antiserum. These evidences suggest that the cross serological relationship between AoV-F and PsV-S previously observed may have been due to the RdRps of the respective viruses rather than between their respective capsid proteins as was assumed in 1985.     

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.     

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.     

Detection and sequence analysis of two novel co-infecting double-strand RNA mycoviruses in Ustilaginoidea virens

Four novel double-stranded RNA molecules, named dsRNA 1 (5124 bp), dsRNA 2(1711 bp), dsRNA 3 (1423 bp) and dsRNA 4 (855 bp), were detected in strain HNHS-1 of Ustilaginoidea virens, the causal agent of rice false smut disease. Sequence analysis showed that the dsRNA1 contains two overlapping open reading frames (ORF) potentially encoding proteins with modest levels of sequence similarity to the coat protein (CP) and putative RNA-dependent RNA polymerase (RdRp), respectively, of viruses of the family Totiviridae. The deduced gene product of the ORF encoded by dsRNA2 is homologous to putative RdRp of viruses in the family Partitiviridae; the ORF encoded by dsRNA3 shares some similarity to a hypothetical protein with unknown function. It is noteworthy that the dsRNA4 lacked integrated ORFs. Isomeric viral particles of about 40 nm in diameter were observed by transmission electron microscopy in a mycelium tissue preparation of strain HNHS-1-R1, a single-spore subculture of strain HNHS-1 containing only the dsRNA1 segment. Phylogenetic analysis and examination of the organization of the two putative RdRp sequences both indicated that there are at least two novel virus species present in strain HNHS-1. We named the two novel viruses Ustilaginoidea virens RNA virus 2 and Ustilaginoidea virens partitivirus 4, respectively.     

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.     

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.     

Description of a new putative virus infecting the conifer pathogenic fungus Heterobasidion parviporum with resemblance to Heterobasidion annosum P-type partitivirus

The complete sequences of two double-stranded RNA segments from the fungus Heterobasidion parviporum were characterized. The larger segment (2,290 bp) contained an open reading frame encoding a putative RNA-dependent RNA polymerase (RdRp, 722 aa), while the smaller one (2,238 bp) encoded a putative coat protein of 659 aa. Based on phylogenetic analysis, the dsRNA segments constitute the genome of a new virus assigned to the family Partitiviridae and named Heterobasidion RNA virus 2 (HetRV2). The RdRp segment was clearly related to H. annosum P-type partitivirus (aa similarity of 59%) but was only distantly related to previously described viruses of H. parviporum (aa similarity 26–35%). The dsRNA could be experimentally transmitted to all five species of the Heterobasidion annosum sensu lato complex and two species of the H. insulare complex, indicating that horizontal transfer between these intersterile fungal species is possible.     

Co-infection by two distinct totivirus-like double-stranded RNA elements in Chalara elegans (Thielaviopsis basicola)

A full-length cDNA clone was developed from a 5.3 kb double-stranded (ds) RNA element present in strain CKP of the plant pathogenic fungus Chalara elegans. The complete nucleotide sequence was 5310 bp in length and sequence analysis revealed that it contained three large putative open reading frames (ORFs). ORF1 was initiated at nucleotide position 329 and encoded a putative coat protein, which shared some homology (35-45% amino acid identity) to other dsRNAs in the family Totiviridae. Both ORF2 and ORF3 were initiated at nucleotide positions 2619 and 4071, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp). Sequence comparison using deduced amino acid sequences of both ORF2 and ORF3 revealed that all RdRp conserved motifs shared highest homology (41% identity) to that of SsRNA1 of Totiviridae. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 1 (CeRV1). During the development of the full-length cDNA clone of CeRV1, several partial cDNA clones from an additional dsRNA fragment in strain CKP were obtained, which when aligned with each other, produced one linear fragment which was 2336 bp long. Northern blot and sequence analysis of this second clone showed it differed in sequence composition from CeRV1. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 2 (CeRV2). Sequence analysis of CeRV2 showed it contained all conserved motifs and shared some homology (45% amino acid identity) to RdRp regions of Totiviridae. The nucleotide and amino acid sequences of the conserved motifs of the RdRp regions between CeRV1 and CeRV2 showed an identity of 56% and 50%, respectively. These findings suggest that co-infection of two distinct totivirus-like dsRNAs (CeRV1 and CeRV2) in C. elegans, a first report in this fungus. Transmission electron microscopy of strain CKP of C. elegans revealed the presence of putative virus-like particles in the cytoplasm, which were similar both in shape and size to viruses in the Totiviridae.     

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.     

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.     

Molecular characterization and detection of a new closterovirus identified from blackcurrant by high-throughput sequencing

Two large contigs with high sequence similarities to several closteroviruses were identified by high-throughput sequencing from a blackcurrant plant. The complete genome of this new virus was determined to be 17,320 nucleotides. Its genome contains ten open reading frames (ORF) that include, in the 5′–3′ direction, a large ORF encoding a putative viral polyprotein (ORF 1a) and nine ORFs that encode RNA-dependent RNA polymerase (RdRp, ORF 1b), p6 (ORF 2), heat shock protein 70-like protein (Hsp70h, ORF 3), Hsp-90-like protein (p61, ORF 4), CP minor (ORF 5), CP (ORF 6), p17 (ORF 7), p11 (ORF 8), and p26 (ORF 9), respectively. BCCV-1 shares nucleotide sequence identities of 43–45% with other 9 closteroviruses at genome sequences. The amino acid sequence identities between BCCV-1 and the closteroviruses were 49–55% (RdRp), 37–41% (Hsp70h), 19–33% (p61), 26–38% (CPm), and 19–28% (CP), respectively. Phylogenetic analysis of Hsp70h sequences placed the new virus with members of genus Closterovirus in the same group. The results indicate that this new virus, which is provisionally named as Blackcurrant closterovirus 1, should represent a new species of the genus Closterovirus. A RT-PCR was developed and used to detect BCCV-1 in more germplasm accessions of Ribes spp.     

A new endornavirus species infecting Malabar spinach (Basella alba L.)

A putative new endornavirus was isolated from Malabar spinach (Basella alba). The viral dsRNA consisted of 14,027 nt with a single ORF that coded for a polyprotein of 4,508 aa. The genome organization was similar to that of four other endornaviruses. Conserved domains for helicase-1, capsular synthase, UDP-glucose-glycosyltransferase (UGT), and RdRp were detected. Infected plants were phenotypically undistinguishable from healthy ones. The name Basella alba endornavirus is proposed for the virus isolated from Malabar spinach.     

Complete sequence of the Pepino mosaic virus RNA genome

We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical.     

The partial purified RNA-dependent RNA polymerases from bamboo mosaic potexvirus and potato virus X infected plants containing the template-dependent activities

RNA-dependent RNA polymerases (RdRp) isolated from bamboo mosaic potexvirus (BaMV) and potato virus X infected Nicotiana benthamiana plants and solubilized with the detergent NP-40, generated a full-length genomic and two subgenomic double-stranded RNAs of respective viruses in an in vitro RdRp assay containing endogenous RNA templates. Template-dependent and species-specific RdRp activity could be detected after the removal of endogenous RNA templates. The 3′ untranslated regions (UTR) containing a stretch of 40 adenylate residues were shown to be an efficient exogenous RNA template for in vitro RdRp reactions. Solution hybridization and nuclease digestion studies revealed that the products transcribed in vitro were minus-sense. Besides using the 3′ UTR for minus-sense RNA synthesis, the BaMV RdRp can also recognize 3′ terminal 77 nucleotides of the minus-strand for plus-sense RNA synthesis. Promoter studies with BaMV RdRp showed that domain D containing the potexviral hexamer motif of the 3′ UTR would be the major contributor of minus-sense RNA synthesis in vitro. On the other hand, the pseudoknot domain containing the poly(A) sequences would be sufficient for minus-sense RNA synthesis.     

The complete genome sequence of Perina nuda picorna-like virus,an insect-infecting RNA virus with a genome organization similar to that of the mammalian picornaviruses

Perina nuda picorna-like virus (PnPV) is an insect-infecting RNA virus with morphological and physicochemical characters similar to the Picornaviridae. In this article, we determine the complete genome sequence and analyze the gene organization of PnPV. The genome of PnPV consists of 9476 nucleotides (nts) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 8958 nts (2986 codons) flanked by 473 and 45 nt noncoding regions on the 5' and 3' ends, respectively. Northern blotting did not detect the presence of any subgenomic RNA. The PnPV genome codes for four structural proteins (CP1-4), and determination of their N-terminal sequences by Edman degradation, showed that all four are located in the 5' region of the genome. The 3' part of the PnPV genome contains the consensus sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) in that order from the 5' to the 3' end. In all of these characters, the genome organization of PnPV resembles the mammalian picornaviruses and two other insect picorna-like viruses, infectious flacherie virus (IFV) of the silkworm and Sacbrood virus (SBV) of the honeybee. In a phylogenetic tree based on the eight conserved domains in the RdRp sequence, PnPV formed a separate cluster with IFV and SBV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses.