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 analysis of 12 genome segments of mud crab reovirus (MCRV)

Mud crab reovirus (MCRV) is the causative agent of a serious disease with high mortality in cultured mud crab (Scylla serrata). This study sequenced and analyzed 12 genome segments of MCRV. The 12 genome segments had a total length of 24.464 kb, showing a total G + C content of 41.29% and predicted 15 ORFs. Sequence analysis showed that the majority of MCRV genes shared low homology with the counterpart genes of other reoviruses, e.g., the amino acid identity of RNA-dependent RNA polymerase (RdRp) was lower than 13.0% compared to the RdRp sequences of other reoviruses. Nucleotide and amino acid sequences of RdRp and capping enzyme suggested MCRV as a single group. Further genome-based phylogenetical analysis of conserved termini and reovirus polymerase motif indicates that this MCRV belongs to a new genus of the Reoviridae family, tentatively named as Crabreovirus.     

Reptilian reovirus: a new fusogenic orthoreovirus species

The fusogenic subgroup of orthoreoviruses contains most of the few known examples of non-enveloped viruses capable of inducing syncytium formation. The only unclassified orthoreoviruses at the species level represent several fusogenic reptilian isolates. To clarify the relationship of reptilian reoviruses (RRV) to the existing fusogenic and nonfusogenic orthoreovirus species, we undertook a characterization of a python reovirus isolate. Biochemical, biophysical, and biological analyses confirmed the designation of this reptilian reovirus (RRV) isolate as an unclassified fusogenic orthoreovirus. Sequence analysis revealed that the RRV S1 and S3 genome segments contain a novel conserved 5'-terminal sequence not found in other orthoreovirus species. In addition, the gene arrangement and the coding potential of the bicistronic RRV S1 genome segment differ from that of established orthoreovirus species, encoding a predicted homologue of the reovirus cell attachment protein and a unique 125 residue p14 protein. The RRV S3 genome segment encodes a homologue of the reovirus sigma-class major outer capsid protein, although it is highly diverged from that of other orthoreovirus species (amino acid identities of only 16-25%). Based on sequence analysis, biological properties, and phylogenetic analysis, we propose this python reovirus be designated as the prototype strain of a fifth species of orthoreoviruses, the reptilian reoviruses.     

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die-off of grass carp fingerlings and has therefore been called 'American grass carp reovirus' (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (>92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species 'Aquareovirus G' by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an 'evolutionary jump,' involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses.     

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.     

Complete genomic sequence of a reovirus isolated from grass carp in China

A reovirus was isolated from sick grass carp in Guangdong, China in 2009, and tentatively named ‘grass carp reovirus Guangdong 108 strain’ (GCRV-GD108). This reovirus was propagated in grass carp snout fibroblast cell line PSF with no obvious cytopathic effects. Its genome was 24,703 bp in length with a 50% G + C content and 11 dsRNA segments encoding 11 proteins instead of 12 proteins. It has been classified as an Aquareovirus (AQRV). Sequence comparisons showed that it possessed only 7 homologous proteins to grass carp reovirus (GCRV) (with 17.6-45.8% identities), but 9 homologous proteins to mammalian orthoreoviruses (MRV) (with 15-46% identities). GCRV-GD108 lacked homology to VP7, NS4&NS5 and NS3 of GCRV, while it had sigma1 and sigma NS homology to MRV. VP2 of GCRV-GD108 shared high amino acid sequence identity (44-47%) with AQRVs, whereas VP5 did not exhibit much identity (24-25%) to AQRVs. Conserved terminal sequences, 5′-GUAAUUU and UUCAUC-3′, were found in all of the 11 genomic segments of GCRV-GD108 at the 5′ and 3′ non-coding regions (NCRs) of the segments. The 5′ NCRs of GCRV-GD108 was similar to GCRV, but differed from other species of AQRV or Orthoreoviruses (ORV). Phylogenetic analysis of coat proteins belonging to Reoviridae, VP1-VP6, showed that GCRV-GD108 clustered with AQRVs and grouped with ORVs, suggesting that GCRV-GD108 belonged to the genus Aquareovirus but was distinctive from any known species of AQRV. Morphological and pathological analyses, and genetic characterization of GCRV-GD108 suggested that it may be a new species of AQRV and it was more closely related with ORVs than other AQRVs. In addition, RT-PCR analysis of diseased grass carp samples collected from different regions of China indicated that these viruses displayed high similarities to each other (95.3-99.4%). They also shared high sequence similarities to GCRV-GD108 (96.7-99.4%), indicating that GCRV-GD108 is representative of the prevalence strain in southern China.     

Molecular characterization of the largest and smallest genome segments,S1 and S12, of <Emphasis Type="Italic">Rice gall dwarf virus</Emphasis>

The nucleotide sequences of segments S1 and S12 of a Chinese isolate of Rice gall dwarf virus (RGDV) were determined. This provides the first complete sequences of these segments. The complete sequence of S1, the largest genome segment of RGDV, was 4,505 nucleotides in length and was predicted to encode a large protein of 1,458 amino acids with a calculated molecular mass of nearly 166.2 kDa. The protein was related to that encoded by S1 of Rice dwarf virus (RDV; 50% identity and 67% similarity) and (to a lesser extent) to some large proteins of other reoviruses. It appears to be an RNA-dependent RNA polymerase (RdRp) and is probably present in particles as a minor core protein. S12, the smallest genome segment of RGDV, was 853 nucleotides in length, encoding a single major protein of 206 amino acids with a calculated molecular mass of nearly 23.6 kDa. This protein, though a little larger than those of RDV S11 and Wound tumor virus (WTV) S12 in size, showed some similarity to them, especially in the conserved N-terminal region and may have RNA-binding properties. Despite having a common host plant, RDV and RGDV were not more closely related to one another than either of them was to WTV. Phylogenetic analysis of the RdRp showed that members of the genus Phytoreovirus were more closely related to those of the genus Rotavirus than to any other genus within the family Reoviridae. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ333946 and DQ494209.     

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

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

Comparative sequence analyses of a new mammalian reovirus genome and the mammalian reovirus S1 genes from six new serotype 2 human isolates

We previously described isolation of a potentially new mammalian reovirus, designated BYD1, which can cause clinical symptoms similar to that of severe acute respiratory syndrome (SARS) in guinea pigs and macaques, from throat swabs of one SARS patient of Beijing, in 2003. For this study, we determined the genome sequences of BYD1 and the S1 gene sequences of other five mammalian reovirus isolates (BLD, JP, and BYL were isolated from different SARS patients during the outbreak, 302I and 302II were isolated from fecal specimens of two children of Beijing in 1982) to allow molecular comparison with other previously reported mammalian reoviruses (MRVs). Comparative analyses of the BYD1 genome with those of three prototype mammalian reovirus strains demonstrated that BYD1 is a novel reassortant virus, with its S1 gene segment coming from a previously unidentified serotype 2 isolate and other nine segments coming from ancestors of homologous T1L and T3D segments, which supports the hypothesis that mammalian reovirus gene segments reassort in nature. Further analyses of the S1 segments of the six isolates showed that all the isolates are novel serotype 2 MRVs based on their S1 gene sequences, which are markedly different from those of all previously reported, and the S1 genes of the four new isolates share more than 99% identity with each other, proving that they diverged from a common ancestor most recently, and the S1 genes of the four new isolates share about 65% identity with those of the two strains isolated in 1982.     

Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 °C at pH 6.0 in the presence of 3 mM MgCl_2. Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.     

Sequence and phylogenetic analysis of the S1 genome segment of turkey-origin reoviruses

Based on previous reports characterizing the turkey-origin avian reovirus (TRV) σB (σ2) major outer capsid protein gene, the TRVs may represent a new group within the fusogenic orthoreoviruses. However, no sequence data from other TRV genes or genome segments has been reported. The σC protein encoded by the avian reovirus S1 genome segment is the cell attachment protein and a major antigenic determinant for avian reovirus. The chicken reovirus S1 genome segment is well characterized and is well conserved in viruses from that species. This report details the amplification, cloning and sequencing of the entire S1 genome segment from two and the entire coding sequences of the σC, p10 and p17 genes from an additional five TRVs. Sequence analysis reveals that of the three proteins encoded by the TRV S1 genome segment, σC shares at most 57% amino acid identity with σC from the chicken reovirus reference strain S1133, while the most similar p10 and p17 proteins share 72% and 61% identity, respectively, with the corresponding S1133 proteins. The most closely related mammalian reovirus, the fusogenic Nelson Bay reovirus, encodes a σC protein that shares from 25% to 28% amino acid identity with the TRV σC proteins. This report supports the earlier suggestion that the TRVs are a separate virus species within the Orthoreovirus genus, and may provide some insight into TRV host specificity and pathogenesis. NC/SEP–R44/03 S1 genome segment DQ525419 NC/98 S1 genome segment DQ995806 TRV sigmaC sequences DQ996601–DQ996605 TRV p10 sequences DQ996606–DQ996610 TRV p17 sequences DQ996611–DQ996615     

Mycoreovirus 1 S4-coded protein is dispensable for viral replication but necessary for efficient vertical transmission and normal symptom induction

Rearrangements of two segments, S6 and S10, of Mycoreovirus 1 (MyRV1), a member of the family Reoviridae, were previously shown to be induced at a high rate by the multifunctional protein p29 encoded by a distinct ssRNA virus, the prototype hypovirus CHV1-EP713 (Sun and Suzuki, RNA 14, 2557-2571, 2008). Here we report the occurrence of rearrangements of MyRV1 S4, albeit at a very low frequency, in the absence of CHV1 p29, resulting in internal 80-90% deletions of the open reading frame (ORF) in S4. Comparative analyses of fungal strains infected by wild-type MyRV1 and its variants carrying rearrangements of S4, S4 plus S10 and S10 indicated that S4-encoded VP4, like VP10, is non-essential for virus replication but required for efficient vertical transmission and symptom expression caused by MyRV1. This is the first example of a reovirus variant that carries deletions of over 75% of the ORFs in two genome segments and is still replication-competent.     

Genome characterization of a debilitation-associated mitovirus infecting the phytopathogenic fungus Botrytis cinerea

The full-length sequences of Botrytiscinereamitovirus 1 (BcMV1) and an associated RNA (BcMV1-S) in strain CanBc-1c-78 of Botrytis cinerea were determined. Sequence analysis showed that BcMV1 is 2804 nt long and AU-rich (66.8%). BcMV1 shares 95% nucleotide sequence identity with Ophiostomanovo-ulmimitovirus 3b (OnuMV3b). However, it is 472 nt longer than OnuMV3b. Mitochondrial codon usage revealed that BcMV1 contains one open reading frame encoding RdRp, which is 96% identical to the RdRp of OnuMV3b. These findings confirm that BcMV1 belongs to the genus Mitovirus and is a strain of OnuMV3b. BcMV1-S is 2171 nt long and derived from BcMV1 through a single internal in-frame deletion of 633 nt, suggesting that it is a defective RNA of BcMV1. BcMV1-S was found to suppress the replication of BcMV1 and to be co-transmissible with BcMV1 through hyphal anastomosis. Its presence, however, did not alleviate the BcMV1-associated debilitation phenotypes of B. cinerea.     

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.     

Peruvian horse sickness virus and Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia

During 1997, two new viruses were isolated from outbreaks of disease that occurred in horses, donkeys, cattle and sheep in Peru. Genome characterization showed that the virus isolated from horses (with neurological disorders, 78% fatality) belongs to a new species the Peruvian horse sickness virus (PHSV), within the genus Orbivirus, family Reoviridae. This represents the first isolation of PHSV, which was subsequently also isolated during 1999, from diseased horses in the Northern Territory of Australia (Elsey virus, ELSV). Serological and molecular studies showed that PHSV and ELSV are very similar in the serotype-determining protein (99%, same serotype). The second virus (Rioja virus, RIOV) was associated with neurological signs in donkeys, cattle, sheep and dogs and was shown to be a member of the species Yunnan orbivirus (YUOV). RIOV and YUOV are also almost identical (97% amino acid identity) in the serotype-determining protein. YUOV was originally isolated from mosquitoes in China.     

Assessment of sequence relatedness of double-stranded RNA genes by RNA-RNA blot hybridization

Three well-characterized reovirus serotypes were used to investigate the usefulness of RNA-RNA blot hybridization as a means to assess the genetic relatedness of double-stranded RNA (dsRNA) viruses. [5'-32P]pCp-labeled genomic dsRNAs from reovirus 1, 2 and 3 were used as probes in hybridization experiments in which segments of the three serotypes were separated in 10% polyacrylamide gels and transferred electrophoretically to membranes. Nine of the 10 reovirus genes cross-hybridized between the serotypes. The S1 gene was serotype specific. The L2 gene of reovirus 2 showed a lower level of cross-hybridization with types 1 and 3 when compared to the hybridization signal observed for L2 when types 1 and 3 were hybridized to each other. The data were consistent with previous studies on the relatedness of the three virus serotypes. Since RNA-RNA blot hybridization allows the number and identity of conserved genes to be determined, this approach may prove useful for assessing the genetic relatedness among other viruses in the family Reoviridae.     

Sequence Analysis of the S3 Gene from a Turkey Reovirus

The deduced 2 protein sequence from the S3 gene segment of a novel turkey reovirus, designated NC98, isolated from the bursa of birds exhibiting poult enteritis and mortality syndrome was determined. The isolate, serologically distinct from other avian reoviruses, was isolated in turkey embryo kidney cells and RNA was purified for cDNA synthesis. Oligonucleotide primers were designed based on conserved avian S3 nucleotide sequence data. The NC98 S3 open reading frame comprised 1101 base pairs and encoded 366 amino acids with a predicated molecular mass of 40.5 kDa. Although the S3 nucleotide sequence from several chicken isolates share at least 86% identity, they share only 64% with the NC98 turkey isolate. Interestingly, the S3 nucleotide sequence from a muscovy duck reovirus shares 55% identity with NC98 and 53% identity with chicken isolates. As observed in other avian reovirus 2 protein sequences, a zinc-binding motif and double-stranded RNA binding domain were found within the predicted amino acid sequence of NC98. Phylogenetic analysis of the deduced 2 sequence demonstrated that NC98 separated as a distinct virus relative to other avian strains. The results of this study indicate that NC98 is a novel turkey reovirus that shares limited genomic sequence identity to isolates of chicken and duck origin and should be considered a separate virus species within subgroup 2 of the Orthoreovirus genus.     

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).