Determination of the complete genome sequence of European mountain ash ringspot-associated emaravirus from Sorbus intermedia reveals two additional genome segments.

We report the complete genome sequence, comprising six single-stranded RNAs of negative orientation, of a European mountain ash ringspot-associated virus (EMARaV) isolate identified in a diseased Sorbus intermedia (Swedish whitebeam) tree exhibiting prominent chlorotic ringspots, mottle and line pattern on leaves. Since the first observation of EMARaV-like symptoms and detection of the virus in whitebeam in 2012, the tree displayed leaf symptoms every year in at least one third of its canopy, developed dieback symptoms, and showed signs of decline. Two previously unrecorded genome segments of the virus were identified, each encoding a single protein in a negative orientation. RNA5 is 1629 nucleotides long and encodes the putative movement protein (MP) of EMARaV with a molecular mass of 42.4 kDa. RNA6 (1362 nucleotides) encodes a small protein (26.8 kDa) exhibiting some sequence similarity to the P4 protein encoded by EMARaV RNA4. However, its biological function remains to be elucidated. Both novel genome segments are systematically present in EMARaV-infected Sorbus spp., and no additional genome segments could be identified by two independent methods. It is concluded that the six RNAs represent the complete genome of EMARaV.

Detection of European mountain ash ringspot-associated virus-specific RNA and protein P3 in the pear leaf blister mite Phytoptus pyri (Eriophyidae).

The means by which European mountain ash ringspot-associated virus (EMARaV), a minus-strand ssRNA virus and the type member of the genus Emaravirus, is naturally spread, is unknown. In attempts to identify an EMARaV vector, galls induced by the eriophyid mite Phytoptus pyri were frequently found on infected leaves. By immunofluorescence microscopy, the presence of EMARaV nucleocapsid protein P3 was demonstrated in P. pyri individuals collected from diseased plants. Furthermore, RT-PCR analysis of entire P. pyri individuals revealed the presence of both viral genomic ss(-)RNAs and antigenomic ss(+)RNAs, suggesting that P. pyri might be a candidate vector of EMARaV.

Detection, distribution, and genetic variability of European mountain ash ringspot-associated virus.

European mountain ash ringspot-associated virus (EMARAV) was recently characterized from mountain ash (rowan) (Sorbus aucuparia) in Germany. The virus belongs tentatively to family Bunyaviridae but is not closely related to any classified virus. How commonly EMARAV occurs in ringspot disease (EMARSD) affected mountain ash trees was not reported and was investigated here. Virus-specific detection tools such as reverse transcription-polymerase chain reaction and dot blot hybridization using digoxigenin-labeled RNA probes were developed to test 73 mountain ash trees including 16 trees with no virus-like symptoms from 16 districts in Finland and Viipuri, Russia. All trees were infected with EMARAV. Hence, EMARAV is associated with EMARSD and can also cause latent infections in mountain ash. Symptom expression and the variable relative concentrations of viral RNA detected in leaves showed no correlation. Infectious EMARAV was detected also in dormant branches of trees in winter. Subsequently, genetic variability, geographical differentiation, and evolutionary selection pressures were investigated by analyzing RNA3 sequences from 17 isolates. The putative nucleocapsid (NP) gene sequence (944 nucleotides) showed little variability (identities 97 to 99%) and was under strong purifying selection. Amino acid substitutions were detected in two positions at the N terminus and one position at the C terminus of NP in four isolates. The 3' untranslated region (442 nucleotides) was more variable (identities 94 to 99%). Six isolates from a single sampling site exhibited as wide a genetic variability as isolates from sites that were hundreds of kilometers apart and no spatial differentiation of populations of EMARAV was observed.

A novel, multipartite, negative-strand RNA virus is associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.).

Four RNAs from a new plant-pathogenic virus, which we have tentatively named European mountain ash ringspot-associated virus (EMARAV), were identified and sequenced completely. All four viral RNAs could be detected in previous double-stranded RNA preparations. RNA 1 (7040 nt) encodes a protein with similarity to the RNA-dependent RNA polymerase of different members of the Bunyaviridae, a family containing five genera with viruses infecting invertebrates, vertebrates and plants. RNA 2 (2335 nt) encodes a 75 kDa protein containing a conserved motif of the glycoprotein precursor of the genus Phlebovirus. Immunological detection indicated the presence of proteins with the expected size of the precursor and one of its processing products. The amino acid sequence of protein p3 (35 kDa) encoded by RNA 3 shows similarities to a putative nucleocapsid protein of two still unclassified plant viruses. The fourth viral RNA encodes a 27 kDa protein that has no significant homology to any known protein. As is typical for members of the family Bunyaviridae, the 5' and 3' ends of all viral RNAs are complementary, which allows the RNA to form a panhandle structure. Comparison of these sequences demonstrates a conserved terminal part of 13 nt, similar to that of the bunyaviral genus Orthobunyavirus. Despite the high agreement of the EMARAV genome with several characteristics of the family Bunyaviridae, there are a few features that make it difficult to allocate the virus to this group. It is therefore more likely that this plant pathogen belongs to a novel virus genus.

Double-stranded RNA pattern and partial sequence data indicate plant virus infection associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.).

Double-stranded RNA (dsRNA) has been extracted from tissue of European mountain ash trees (Sorbus aucuparia L.) showing typical ringspot and mottling symptoms on leaves and a gradual decay in general. A characteristic dsRNA pattern was found in leaf samples of symptomatic mountain ash trees from various stands in Germany. Bands of dsRNA molecules of approximately 7 kb, 2.3 kb, 1.5 kb, and 1.3 kb, respectively, were repeatedly detected. By random primed reverse transcription cDNA was synthesised from dsRNA and amplified by degenerate oligonucleotide primed PCR. After TA cloning, the cDNA clones obtained were screened with an enhanced-chemiluminescence-labelled dsRNA probe. Positive clones were further analysed by using them as hybridisation probes in Northern blots of total plant RNA and in Southern hybridisation with genomic DNA from Sorbus aucuparia leaves. From cDNA clones that were found to be specific for dsRNA in Northern analysis, primers were deduced for 5'-RACE analyses and further cloning. Finally, a cDNA fragment of 3,737 bp was obtained, which showed homology to viral proteins, particularly to the RNA-dependent RNA polymerase of members of the family Bunyaviridae, but without high similarity to a known genus. The dsRNA pattern and the sequence information strongly indicate a virus associated with the mountain ash ringspot disease. The putative virus remains still unidentified.