USE OF DEGENERATE PRIMERS FOR PARTIAL SEQUENCING AND RT-PCR-BASED ASSAYS OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUS-4 AND VIRUS-5

Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), tw o putative closteroviruses. Reverse-transcriptase polymerase chain rea ction (RT-PCR) was performed on this dsRNA using degenerate oligonucle otides designed to amplify an approximately 550- to 650-nucleotide fra gment from the heat shock protein 70 homolog (HSP70) of the known clos teroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-P CR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated fr om a plant infected with GLRaV-5. These clones were also sequenced. Th e two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate tha t degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detectio n of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR det ection of multiple GLRaV serotypes. Lastly, the presence of closterovi rus-like HSP70 sequences in these putative closteroviruses implies tha t they are indeed members of this taxonomic group.