Genetic variability within RNA2 of Grapevine fanleaf virus

The genetic variability of Grapevine fanleaf virus (GFLV) was assessed within RNA2 of nine isolates from Vitis vinifera cv. Volovnik in a vineyard in Slovenia by immunocapture (IC)-reverse transcription (RT)-polymerase chain reaction (PCR)-restriction length fragment polymorphism (RFLP), followed by cloning and sequencing. Four, one, and nine distinct StyI restrictotypes were identified in the 2A(HP), 2B(MP), and 2C(CP) genes, respectively, by IC-RT-PCR-RFLP. Each isolate had a specific StyI RFLP profile across the three RNA2-encoded genes. Sequence analysis of cloned RNA2 ORF amplicons obtained by IC-RT-PCR showed mixed infection in four of the nine isolates and a slightly higher nucleotide variability in the 2A(HP) and 2C(CP) genes relative to the 2B(MP). Also, gene 2A(HP), unlike genes 2B(MP) and 2C(CP), had a variable size (765-774 nucleotides) and high amino acid diversity (up to 15%). In addition, a recombination event was identified at nucleotide position 220-225 of gene 2A(HP) in three of the nine isolates. No clear association was apparent between symptomatology and restrictotype composition, phylogenetic clustering, or occurrence of recombination. This study provides new insights into the genetic diversity of GFLV.