Natural Spread and Molecular Analysis of Grapevine Leafroll-Associated Virus 3 in Australia

Natural spread of grapevine leafroll disease was observed in a Pinot Noir clonal evaluation trial in South Australia. The trial consisted of 13 clones with the spread apparently initiated from 3 leafroll-infected clones : Antav 543, Geisenheim 20, and Bourgogne H199A. The occurrence of grapevine leafroll-associated virus 3 (GLRaV-3) was suspected. All 104 vines in the trial were tested by enzyme-linked immunosorbent assay using antibody to GLRaV-3 and by slot blot hybridization analysis using double-stranded RNA as target and labeled GLRaV-3-specific cDNA as probe. Both tests linked GLRaV-3 with the disease spread and detected the infection prior to the onset of symptoms. A cDNA clone from an Italian isolate of GLRaV-3 hybridized in Northern blots with three major dsRNAs of 19.5, 1.9, and 0.9 kbp extracted from leafroll-infected vines. The cloned cDNA insert of approximately 1 kbp was sequenced, and a set of primers designed based on the sequence was used to obtain a corresponding polymerase chain reaction product from the Antav 543 isolate grown in Australia. The nucleotide sequence of the cDNA clones from the two isolates of GLRaV-3 showed 99.5% identity and contained an open reading frame (ORF) encoding a putative protein with a molecular mass of 20.4 kDa with no significant homology to known protein sequences. This ORF was mapped near the 3'-end of the plus strand viral genome and had a 3'-untranslated AU-rich region of approximately 347 nucleotide residues.