The genome of <Emphasis Type="Italic">Beet cryptic virus</Emphasis> 1 shows high homology to certain cryptoviruses present in phylogenetically distant hosts

This study determined the complete nucleotide sequence of Beet cryptic virus 1 (BCV1). As expected by analogy to previously sequenced alphacryptoviruses, dsRNA1 (2008 bp) encodes a 72.5-kDa protein containing sequence motifs characteristic for RNA-dependent RNA polymerases (RdRp). In addition to the full-length dsRNA1, a truncated form was also detected in dsRNA extracts. dsRNA2 (1783 bp) codes for the viral coat protein (CP) as proven by the identity of the predicted CP sequence to peptide sequences of the purified virion protein. The amino acid sequence of BCV1 RdRp as well as the 5′- and 3′-UTRs show 81–85% identity to the corresponding regions of Vicia cryptic virus (VCV), White clover cryptic virus 1 (WCCV1) and Carrot cryptic virus (CaCV). The amino acid sequence identity of the CP is about 55–62%, moreover, a strong conservation of predicted α-helical regions was observed. The high degree of similarity of these seed- and pollen-transmitted viruses persisting in phylogenetically distant hosts, together with their high similarity to fungal partitiviruses strongly supports the hypothesis that horizontal transfer by a fungus played a role in the emergence of the present cryptovirus species. The change in the distribution of cryptic viruses may also be due to human influence: While earlier BCV1 occurred frequently in sugar beet cultivars, it is very rare in cultivars currently used in agricultural practice and was detected in only one of the 28 cultivars investigated in our experiments.     

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.     

Complete nucleotide sequence of double-stranded RNA viruses from <Emphasis Type="Italic">Fusarium graminearum</Emphasis> strain DK3

The complete genomes two different dsRNA mycoviruses, Fusarium graminearum virus 3 (FgV3) and Fusarium graminearum virus 4 (FgV4), was sequenced and analyzed. The viral genome of FgV3 is 9,098 base pairs (bp) long and contains two open reading frames (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) and a protein of unknown function. The FgV4 genome is composed of two dsRNA genome segments of 2,383 bp and 1,739 bp. FgV4 dsRNA-1 contains a single ORF, which has a conserved RdRp motif, while FgV4 dsRNA-2 contains two putative ORFs coding for products of unknown function. Both the genome organization and phylogenetic analysis indicated that FgV3 was closely related to members of the families Totiviriridae and Chrysoviridae, but it was placed outside of their main clusters, whereas FgV4 formed a distinct clade with the family Partitiviridae. This is the first report of the full-length nucleotide sequences of FgV3 and FgV4 infecting Fusarium graminearum.     

Complete nucleotide sequences and genome characterization of double-stranded RNA 1 and RNA 2 in the Raphanus sativus-root cv. Yipinghong

Summary. Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yipinghong with yellowing at the leaf edge in China. Purified viral particles of 28–30 nm in diameter contained dsRNA segments with the same number and mobility as these extracted directly from radish leaves. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained a single open reading frame (ORF) on their plus-stranded RNA, putatively encoding a RNA dependent RNA polymerase and a capsid protein similar to that encoded by members of the family Partitiviridae. In addition, both RasR 1 and RasR 2 were highly conserved at the 5′ untranslated regions (UTR) and had an adenosine-uracil rich stretch at the 3′ UTR, with an identical terminal motif (5′-AAAAUAAAACC-3′). Taken together, these results suggest that the two major dsRNA segments constitute the genome of a partitivirus infecting radish.     

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.     

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.     

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.     

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.     

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.     

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 Penicillium stoloniferum Virus S

The complete sequences of two double-stranded RNAs (dsRNAs) (referred to S1 and S2) of Penicillium stoloniferum virus S (PsV-S) were established. The S1 dsRNA was 1,690bp in length, and it contained a unique open reading frame (ORF) of 539 amino acids (molecular weight of 62kDa, referred to P62). The S2 dsRNA was 1,523bp in length, and also it contained one ORF of 434 amino acids (molecular weight of 47kDa, referred to P47). Both S1 and S2 ORFs were identified only on the positive strand of each dsRNA segment. A sequence motif of (5-CUG-3) was found at the 3-termini of the positive strands of PsV-S1 and S2 dsRNAs. The predicted amino acid sequences of S1 dsRNA showed high sequence homology with the putative RNA-dependent RNA polymerases of RNA viruses. Near full-length and positive-sense single-stranded RNAs derived from the S1 and S2 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P62 and P47 showed a positive reaction against PsV-S antiserum in Western blot analysis. Phylogenetic analysis using the RDRP sequences and the capsid proteins of the various partitiviruses revealed that PsV-S is a definite member of the partitivirus, the family Partitiviridae, and especially clusters well along with D. destructiva virus 1 and 2.     

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.     

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.     

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.     

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.     

Comparison of the RNA polymerase genes of marine birnavirus strains and other birnaviruses

Summary.  The cDNA nucleotide sequence of the genome segment B encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp), was determined for 5 marine birnavirus (MABV) strains from different host or geographic origins and 1 infectious pancreatic necrosis virus (IPNV) strain AM-98. Segment B of the IPNV AM-98 strain and 4 MABV strains, Y-6, YT-01A, H1 and NC1, contained a 2535 bp ORF, which encoded a protein of 845 amino acid residues with a predicted MW of 94.4 kDa. Only the MABV AY-98 RdRp had 1 amino acid shorter RdRp. Pairwise comparisons were made among our data and 4 other known IPNV sequences. The nucleotide sequences of the 5 MABV strains were very similar each other, with identities of 98.3–99.7%. The highest divergence of the nucleotide level was between MABV strains and IPNV SP strain (serotype A2), with 20.4–20.8% divergences in the coding region, which gave 10.1–11.3% divergence in the amino acid level. The aquabirnavirus RdRp was noticeably conserved in amino acid sequences. Though the identities of the nucleotide sequences of encoding region were 85.1–85.9% between MABV strains and IPNV serotype A1 strains, they shared as high as 95.1–95.9% identities in amino acid level. A phylogenetic tree was constructed based on the amino acid sequences of the RdRp gene from different birnaviruses including avibirnavirus and entomobirnavirus. Ten aquabirnavirus strains were clustered into 3 Genogroups. The Genogroup I consisted of four IPNV A1 serotype strains. All MABV strains were clustered into Genogroup II. Only IPNV SP strain was clustered into an independent Genogroup III. Received August 19, 2002; accepted October 30, 2002     

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants

Summary.  A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated of 34 866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other clostero-viruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot. Accepted January 10, 1997; Received November 12, 1996     

The complete genomic sequence of a novel botybirnavirus isolated from a phytopathogenic <Emphasis Type="Italic">Bipolaris maydis</Emphasis>

Bipolaris maydis is the causal agent of corn southern leaf blight. Here, we report a novel double-stranded RNA (dsRNA) mycovirus designated Bipolaris maydis botybirnavirus 1 (BmBRV1) from B. maydis strain JZ11 in Jingzhou, Hubei province of China. BmBRV1 has a genome consisting of two dsRNAs (dsRNA1 and dsRNA2) with a size of 6435 and 5987 bp, respectively, each of which contains a single open reading frame (ORF). The two polyproteins encoded by dsRNA1 and dsRNA2 share the highest amino acid identities of 81.8 and 75.3%, respectively, with the RdRp and coat protein of Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1), a tentative species of the genus Botybirnavirus. Phylogenetic analysis based on the amino acid sequences of RdRp indicated that BmBRV1 belongs to a distinct species of the newly proposed family Botybirnaviridae.