Reovirus genomes from plant-feeding insects represent a newly discovered lineage within the family Reoviridae

A complex set of double-stranded RNAs (dsRNAs) was isolated from threecornered alfalfa hopper (Spissistilus festinus), a plant-feeding hemipteran pest. A subset of these dsRNAs constitute the genome of a new reovirus, provisionally designated Spissistilus festinus reovirus (SpFRV). SpFRV was present in threecornered alfalfa hopper populations in the San Joaquin Valley of California, with incidence ranging from 10% to 60% in 24 of 25 sample sets analyzed. The 10 dsRNA segments of SpFRV were completely sequenced and shown to share conserved terminal sequences (5′-AGAGA and CGAUGUUGU-3′) of the positive-sense strand that are distinct from known species of the family Reoviridae. Comparisons of the RNA directed RNA polymerase (RdRp) indicated SpFRV is most closely related (39.1% amino acid identity) to another new reovirus infecting the angulate leafhopper (Acinopterus angulatus) and provisionally designated Acinopterus angulatus reovirus (AcARV). The RdRp of both viruses was distantly related to Raspberry latent virus RdRp at 27.0% (SpFRV) and 30.0% (AcARV) or Rice ragged stunt virus RdRp at 26.2% (SpFRV) and 29.0% (AcARV) amino acid identity. RdRp phylogeny confirmed that SpFRV and AcARV are sister taxa sharing a most recent common ancestor. SpFRV segment 6 encodes a protein containing two NTP binding motifs that are conserved in homologs of reoviruses in the subfamily Spinareovirinae. The protein encoded by SpFRV segment 4 was identified as a guanylyltransferase homolog. SpFRV segments 1, 3, and 10 encode homologs of reovirus structural proteins. No homologs were identified for proteins encoded by SpFRV segments 5, 7, 8, and 9. Collectively, the low level of sequence identity with other reoviruses, similar segment terminal sequences, RdRp phylogeny, and host taxa indicate that SpFRV and AcARV may be considered members of a proposed new genus of the family Reoviridae (subfamily Spinareovirinae), with SpFRV assigned as the type species.     

Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta. Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584), comprised of 10,386 nucleotides, and polyadenylated at the 3′ terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5′ proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3′ proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.     

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

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

Genomic analysis of grapevine leafroll associated virus-5 and related viruses

The grapevine leafroll-associated viruses (GLRaVs) (Closteroviridae) represent an emerging threat to world grape production. One group of GLRaVs within the genus Ampelovirus, the GLRaV-4-like viruses (GLRaV-4LVs), contains a fragmented collection of seven viruses only two of which (GLRaV-Pr and GLRaCV) are fully sequenced. Here in reporting the sequence of GLRaV-5, a member of GLRaV-4LVs, we identify genomic elements common to the GLRaV-4LV group. Exclusive properties include a highly conserved p5 gene product and phylogenies for complete genes that, except for the p23 gene, are reliably monophyletic. In comparison with other members of the genus Ampelovirus, GLRaV-4LVs form a tight cluster for all genes analyzed. In addition, they all possess a conserved AlkB domain which is most similar to the more distantly related GLRaV-3, suggesting recombination. In silico RNA structural analyses revealed a conserved five stem-loop structure at the 3′ untranslated region that extends to all GLRaV-4LVs, and the ampeloviruses Pineapple mealybug wilt-associated virus 1 and Pineapple mealybug wilt-associated virus 3. A conserved G-U rich stem loop was also found upstream of the ORF1a stop and 1b start codons. Taken together, this work allows for a more thorough contextualization of GLRaV-5 and the GLRaV-4LVs as a group within the genus Ampelovirus.     

Three unrelated viruses occur in a single isolate of Gremmeniella abietina var. abietina type A

Five enclosed double-stranded RNA (dsRNA) bands in electrophoresis, probably of viral origin, were found from a single isolate (SurS4) of Gremmeniella abietina var. abietina type A. Analysis of the dsRNAs revealed that they represented three different viruses, named as Gremmeniella abietina mitochondrial RNA virus S2 (GaMRV-S2), Gremmeniella abietina RNA virus MS2 (GaRV-MS2) and Gremmeniella abietina RNA virus L2 (GaRV-L2). The genome of GaMRV-S2 was 2587 base pairs (bp) long and had a very low GC content (31%). Sequence variations occurred at both ends. The genome coded for a putative RNA-dependent RNA polymerase (RdRp) under a mitochondrial translation code. The GaRV-MS2 genome was composed of three dsRNA molecules (1781 bp, 1586 bp and 1186 bp). They coded for a putative RdRp, a coat protein (CP) and a protein with an unknown function, respectively. The GaRV-L2 genome was 5129 bp long and contained two ORFs. The 5′-proximal ORF coded for a putative CP, whereas the 3′-proximal ORF encoded for a putative RdRp. The buoyant density of GaRV-MS2 and GaRV-L2 were 1.37 and 1.42 g/ml, respectively. GaMRV-S2, GaRV-MS2 and GaRV-L2 were closely related to the previously described viruses GaMRV-S1, GaRV-MS1 and GaRV-L1, respectively, and are putative members of the genera Mitovirus, Partitivirus and Totivirus, respectively. This is the first report on the occurrence of viruses of all these different genera in a single fungal isolate.     

Complete nucleotide sequence of rose yellow leaf virus,a new member of the family Tombusviridae

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.     

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 novel mycovirus identified from the rice false smut fungus <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis>

The complete sequence of a novel mycovirus infecting Ustilaginoidea virens, the causal agent of false smut of rice, is reported here and designated as Ustilaginoidea virens unassigned RNA virus HNND-1 (UvURV-HNND-1). This virus has an undivided dsRNA genome of 2903 nt in length and contains two non-overlapping open reading frames (ORF1 and 2), with the small ORF1 encoding a protein of unknown function that showed sequence similarity to the comparable protein in virus Alternaria longipes dsRNA virus 1(AlRV1) and a larger ORF2 encoded the protein showing identities to the RNA-dependent RNA polymerases of AlRV1 and some other unassigned dsRNA viruses. Phylogenetic analysis showed that UvURV-HNND-1 is more closely related to unclassified viruses such as AlRV1 and distinct from distantly related members of the family Partitiviridae. Here, we propose in accordance with previous reports that UvURV-HNND-1 might belong to a new mycovirus genus together with AlRV1 and other similar viruses.     

Genome organization, serology and phylogeny of Grapevine leafroll-associated viruses 4 and 6: taxonomic implications

Complete nucleotide sequences of the type isolate of Grapevine leafroll-associated virus 4 (GLRaV-4) and of an isolate of GLRaV-6 from cv ‘Estellat’ (GLRaV-6Est) were generated and compared mutually and with related viruses. The genome organization of both viruses resembled that of members of Subgroup I in the genus Ampelovirus (fam. Closteroviridae). The availability of these sequences, along with previously existing data on related GLRaVs, allowed critical review of the taxonomy and nomenclature of these viruses. In phylogenetic analyses, GLRaV-4 and -6Est consistently grouped with GLRaV-5, -9, and -Pr forming a poorly resolved sub-cluster (“GLRaV-4 group”) within the genus Ampelovirus. In-depth study showed that genetic distances between these viruses do not exceed the intra-species diversity observed in other closteroviruses. In Western blots, partially purified preparations of GLRaVs -4, -5, -6 and -9 reacted only with homologous monoclonal antibodies, but were all recognized by polyclonal antisera to GLRaV-5 and GLRaV-9. Serological relatedness among these viruses was further confirmed in DAS-ELISA. In immuno-electron microscopy, GLRaV-6 particles appeared uniformly decorated with homologous monoclonal antibodies, whereas GLRaV-2, used as a control, showed “bipolar” morphology of the virion. Results of this study challenge taxonomy and nomenclature of several GLRaVs suggesting that they are divergent isolates of Grapevine leafroll-associated virus 4 and not, as has been assumed, distinct species (definitive and/or putative) in the genus Ampelovirus.     

Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV)

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82 °C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication.     

Replication of biotinylated human immunodeficiency viruses

Previous work demonstrated recently the adaptation of the Escherichia coli biotin ligase BirA - biotin acceptor sequence (BAS) labeling system to produce human immunodeficiency virus type 1 viruses with biotinylated integrase (NLXIN_B) and matrix (NLXMA_B) proteins (Belshan et al., 2009). This report describes the construction of an HIV permissive cell line stably expressing BirA (SupT1.BirA). Consistent with the results in the previous report, NLXMA_B replicated similar to wild-type levels and expressed biotinylated Gag and MA proteins in the SupT1.BirA cells, whereas the replication of NLXIN_B was reduced severely. Three additional HIV type 2 (HIV-2) viruses were constructed with the BAS inserted into the vpx and vpr accessory genes. Two BAS insertions were made into the C-terminal half of the Vpx, including one internal insertion, and one at the N-terminus of Vpr. All three viruses were replication competent in the SupT1.BirA cells and their target proteins biotinylated efficiently and incorporated into virions. These results demonstrate the potential utility of the biotinylation system to label and capture HIV protein complexes in the context of replicating virus.     

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.     

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.     

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.     

Genetic and phenotypic characterization of a 2006 United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field

The objective of this study was to characterize a porcine reproductive and respiratory syndrome virus (PRRSV) isolated from United States pigs experiencing high morbidity (50%) and mortality (20%). The PRRSV isolate, designated NC16845b, was characterized through phenotypic analysis and genomic sequencing and compared to Type 2 PRRSV isolates VR-2332, MN184 and VR-2385. NC16845b demonstrated slower replication in vitro compared to the three other isolates and grew to a peak titer of 5.4 × 10⁵ plaque forming units (PFU) per ml at 60 h post inoculation, which was 4- to 13-fold less than the peak titer of the other three viruses. NC16845b plaques were intermediate size averaging 3.3 mm in diameter that was larger than MN184 plaques and smaller than VR-2385 and VR-2332. Using Northern blot analysis, viral and subgenomic RNA were detected that demonstrated variable levels of hybridization in some open reading frames (ORF) compared to the other viruses. NC16845b is 15,389 nucleotides in length and ORF 5 restriction fragment length polymorphism (RFLP) analysis demonstrated a 1-18-2 pattern. Among all available Type 2 complete genome sequences, NC16845b showed the highest nucleotide homology (91.2%) to atypical PRRSV strain JA142. Compared to prototype VR-2332, NC16845b demonstrated marked nucleotide variability within non-structural protein (nsp) 1β and nsp2, and a nucleotide deletion of 24 bases in nsp2. Sequence homology with VR-2332 and MN184 was 88.4% and 82.9%, respectively; homology with the ORF2-7 of VR-2385 was 90.4%. Collectively, these data indicate that, compared to prototype Type 2 PRRSV isolates, NC16845b exhibited slower in vitro growth properties, had regions of heterogeneity within ORF1a that corresponded to at least two individual virus quasispecies, and also contained a continuous 8 amino acid deletion in the nsp2 protein.     

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.     

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.     

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.     

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.     

Complete nucleotide sequence of four RNA segments of fig mosaic virus

The complete sequence of four viral RNA segments of fig mosaic virus (FMV) was determined. Each of the four RNAs comprises a single open reading frame (ORF) 7,093, 2,252, 1,490 and 1,472 nucleotides in size, respectively. These ORFs encode the following proteins in the order: RNA-dependent RNA polymerase (p1 264 kDa), a putative glycoprotein (p2 73 kDa), a putative nucleocapsid protein (p3 35 kDa) and a protein with unknown function (p4 40.5 kDa). All RNA segments possess untranslated regions containing at the 5′ and 3′ termini a 13-nt complementary sequence. A conserved motif denoted premotif A was found to be present in addition to the five RdRp motifs A–F in RNA-1. In phylogenetic trees constructed with the amino acid sequences of RNA-1 and RNA-2, FMV clustered consistently with European mountain ash ringspot-associated virus (EMARaV) in a clade close to those comprising members of the genera Hantavirus, Orthobunyavirus and Tospovirus. The amino acid sequence of the putative FMV nucleocapsid protein encoded by RNA-3 shared identity with comparable sequences of EMARaV and the unclassified viruses pigeonpea sterility mosaic virus (PPSMV) and maize red stripe virus (MRSV). The nucleocapsid sequences rooted the four viruses in a clade close to the genus Tospovirus. Based on molecular, morphological and epidemiological features, FMV appears to be very closely related to PPSMV and MRSV. All these viruses are phylogenetically related to EMARaV and therefore seem to be eligible for classification in the proposed genus Emaravirus, which, in turn, may find a taxonomic allocation in the family Bunyaviridae.