Complete nucleotide sequence and genome organization of Grapevine fleck virus

The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3'-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5' and 3' ends, respectively. ORF 1 potentially encodes a 215.4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24.3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3' end of the viral genome and encode proline-rich proteins of 31.4 kDa (p31) and 15.9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus.