Differentiation of Cherry leaf roll virus isolates from various host plants by immunocapture-reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations

A restriction fragment length polymorphism assay (RFLP) was developed to differentiate Cherry leaf roll virus (CLRV) isolates according to phylogenetic clades by examining restriction patterns from partial 3′ non-coding region (NCR) genomic fragments (approx. 420 bp). The 3′ NCR fragment from 43 CLRV isolates belonging to different phylogenetic groups were compared after restriction analysis with the endonucleases Bsp143I, AluI, RsaI, EcoRI and Eco130I, and another 23 isolates were analyzed by computer assisted restriction analysis. The restriction endonucleases Bsp143I, AluI and RsaI enabled the differentiation of isolates from group B and all but two isolates belonging to group A. A major proportion of group E isolates could also be discriminated. The remainder of the group E isolates were indistinguishable from isolates belonging to phylogenetic group C or D2. Isolates belonging to group D1 could not be differentiated from two group A isolates. The method was applied successfully in an IC-RT-PCR-RFLP assay to differentiate samples from walnut, black elderberry and birch and determine their phylogenetic relationships. In future, this method will facilitate rapid phylogenetic classification of CLRV isolates detected in certain host plants by the universal immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR), and will be suitable for studying CLRV population diversity as well as genetic drift within virus populations.