Further studies on olive leaf yellowing-associated virus

Recent surveys of commercial olive orchards in several Mediterranean countries have shown that Olive leaf yellowing-associated virus (OLYaV) is one of the most widespread viruses in the region. OLYaV, which is currently classified as unassigned virus to the family Closteroviridae, is little known at the molecular (only a sequence of 611 nt is available) and biological level, and its particles have never been observed in the electron microscope. To secure more information on the nucleotide sequence and organization of the viral genome, double stranded RNA (dsRNA) was extracted from a OLYaV-infected plant and used as template for cloning. Rapid amplification of cDNA ends (RACE-PCR) and degenerate oligonucleotide primer (DOP-PCR) protocols were successfully applied to amplify unknown regions of the viral genome. Resulting cDNA clones consisted of a total of 4,605 nucleotides (including the 611 nt previously sequenced), comprising the 3’ terminus of the RdRp gene (39 kDa), the whole 21 kDa, 6 kDa and HSP70 homologue (65 kDa) proteins, and the 5’ terminus of the HSP90-like protein. RdRp and HSP70 sequences were clearly related to (ca 39% and 30% homology, respectively) but phylogenetically different from comparable genes of other members of the family Closteroviridae. Preliminary data on genomic variability using single strand conformation polymorphism (SSCP) were also obtained. Filamentous particles, morphologically similar to those of members of the family Closteroviridae, were observed by electron microscopy in dip preparations from petioles of young leaves of greenhouse-grown OLYaV-infected plants.