Use of degenerate primers in a RT-PCR assay for the identification and analysis of some filamentous viruses, with special reference to clostero- and vitiviruses of the grapevine

RT-PCR with degenerate primers was used for the screening of the genome of some members of the Closterovirus, Vitivirus and Trichovirus genera. Two se ts of primers, targeted to conserved sequences of the heat shock protein 70 homologue of closteroviruses or to the RNA dependent RNA polymerase genes of tricho- and vitiviruses, amplified the expected fragments from total RNA extracts or double-stranded RNAs of infected plants. Amplified cDNAs were cloned, sequenced and phylogenetically analyzed. Results support the alloca tion of grapevine viruses A, B, D and heracleum latent virus (HLV) in the g enus Vitivirus, whereas, the detection of a HSP70 homologue in grapevine le afroll-associated viruses agrees with their assignment in the genus Closter ovirus. The use of degenerate primers for the identification of grapevine v iruses belonging to Vitivirus and Closterovirus genera is envisaged.