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.