Single-strand conformation polymorphism (SSCP), cloning and sequencing reveals two major groups of divergent molecular variants of grapevine leafroll-associated virus 3 (GLRaV-3)

Abstract

The SSCP and RE/SSCP analysis of ORF5, ORF6 and ORF7 of 25 local and overseas isolates of GLRaV-3 showed only two kinds of distinct SSCP profiles for each of these genomic regions. It suggested low molecular variability of the virus. Fragments of the 5’UTR + ORF1a, 3’ terminal part of ORF1a, ORF4, ORF5, ORF6 and ORF7 of three isolates, representing distinct SSCP profiles, were cloned and sequenced. Results revealed that although the 3’terminal half of the genome (ORF4-7) and the sequence located in the 3’ terminal part of ORF1a were relatively similar among isolates (91.3-96.2 % nt identity), their 5’terminal parts (88 nt of 5’UTR and adjacent first 329 nt of ORF1a) were clearly divergent (81.6-81.8 % nt identity). Analysis of this divergent part of GLRaV-3 for an additional 11 isolates showed that they cluster in two distinct molecular groups, sharing 94.7-99.7 % and 80.8-85.1 % nt identity within and between groups respectively. The correlation between the molecular groups and SSCP profiles of the 209 nt fragment of ORF5 of GLRaV-3 strongly suggests that SSCP analysis of this easily RT-PCR amplified region can be used for rapid identification of divergent molecular variants of the virus in field-collected grapevine samples.