FURTHER CHARACTERIZATION OF OLIVE LEAF YELLOWINGASSOCIATED VIRUS
T. El Beaino, M. Saponari, A. Minafra, M.A. Castellano, V. Savino, G.P. Martelli
RACE-PCR using a denatured dsRNA extract from an olive plant infected by Olive leaf yellowing-associated virus (OLYaV), extended a previously sequenced large fragment of the viral genome by 854 nt downstream in the heat shock protein 90 (HSP90). This sequence showed from 45 to 53% identity at the nucleotide level with that of putative HSP90s of different members of the family Closteroviridae. In phylogenetic trees constructed with published HSP90 and RdRp sequences, OLYaV was well separated from other viruses, as was Little cherry virus 1 (LChV-1), another unassigned species in the family Closteroviridae. A polyclonal antiserum was raised to a recombinant protein encoded by the HSP70 gene in fusion with sequence encoding the maltose binding protein. In Western blots of extracts from infected olive tissues, IgGs purified from this antiserum reacted with a polypeptide with a Mr of ca. 65 kDa, which was identified as the putative viral HSP70. Closterovirus-like flexuous particles with distinct cross banding and a maximum length of ca. 2000 nm were seen in extracts from infected roots and leaves of rooted olive cuttings. The cytopathology of infected leaves was similar to that induced by other members of the family Closteroviridae. However, the new information is still insufficient for the ultimate classification of OLYaV.