Annex I
(Fac-simile)
GRAPEVINE FLECK VIRUS AS THE TYPE SPECIES OF A POSSIBLE NEW GENUS OF PLANT VIRUSES
S. Sabanadzovicl, N. Abou-Ghaneml, P. Saldarelli2 and G.P. Martelli2
lIstituto
Agronomico Mediterraneo, Via Ceglie 9,70010 Valenzano (Bari), Italy
di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Centro di Studio del CNR sui
Virus e le Virosi delle Colture Mediterranee, Via Amendola 165/A, 70126 Bari, Italy.
2Dipartimento
Grapevine fleck is a graft-transmissible disease with a worldwide distribution. Its causal agent, Grapevine fleck
virus (GFkV), is a non-mechanically transrnissible phloem-restricted isometric virus c. 30 nm in diameter, exhibiting surface
structure resembling that of tymovirus and marafivirus particles. GFkV has a positive sense RNA genome estimated to be
7500 nucleotide in size and a single coat protein of c. 28 kDa (1).
Using viral RNA as template, cDNA clones were generated and cloned, and complete re-sequencing on both
strands was done. Two missing nucleotides were discovered at positions 1983 and 5402, which resulted in a modified
genomic organization as compared with that previously reported (4). A fragment of 7099 nt, representing more than 95% of
the viral genome, was sequenced and its base content was determined to be 14.4%A, 19.6%T, 16.6%G and 49.4%C.
Typically, members of the genera Tymovirus and Marafivirus have a high citosine content whose level, however, ranges
between 40 and 43%, i.e. lower than that found in GFkV. In the sequenced GFkV genome fragment two main open reading
frames (ORF) were detected. ORF 1, which starts at position 291 and ends position 6140, encodes a high molecular weight
protein of c. 215 kDa (p215) containing, in the order, the methyltransferase, protease, helicase and polymerase motifs
conserved in the tymo-lineage of Alpha- like positive-strand RNA viruses. Computer-assisted analysis of these motifs
showed that GFkV has a phylogenetic relationship with both marafiviruses and tymoviruses, but is closer to Oat blue dwarf
virus (genus Marafivirus) than to sequenced members of the genus Tymovirus. ORF 2 was found in frame with ORF 1, from
which it is separated by a double stop codon (amber and opal, in succession) nucletodes apart. ORF 2 starts at position 6366,
ends at position 7058 and encodes a protein of c. 25 kDa (p25) identified as the viraI coat protein (CP). GFkV CP proved to
be related to CPs of marafiviruses and tymoviruses. No ORF was found comparable to ORF OP (putative movement protein)
overlapping the replicase cistron of tymoviruses (3)
GFkV has intriguing morphological, molecular, ultrastructural and biological similarities with two other grapevine
viruses, Grapevine asteroid mosaic-associated virus (GAMaV) and Grapevine red globe virus (GRGV), which have been
identified as different species (5), and constitute a coherent cluster of viruses. On the other hand, the sequence, organization
and, perhaps, expression strategy of GFkV genome resemble those of members of the genera Marafivirus and Tymovirus.
However, the biological, physico-chemical, cytopathological and some of the molecular properties of GFkV differ enough
from those of tymoviruses and marafviruses to warrant the establishment of a different genus.
References
1. Boulila M., Boscia D., Di Terlizzi B., Castellano M.A., Minafra A., Savino V. and Martelli G.P., 1990. Some properties
of a phloem-lirnited non mechanically-transmissibile grapevine virus. J. Phytopathol. 129: 151-158.
2. Edwards M.C., Zhang Z., Weiland J.J., 1997. Oat blue dwarf marafivirus resembles the tymoviruses in sequence,
genome organization and expression strategy. Virology 232, 217-229.
3. Morch M.D., Boyer J.C. and Henni A.L., 1988. Overlapping open reading frames revealed by complete nucleotide
sequencing of turnip yellow mosaic virus genomic RNA. Nucleic Acids Research 16,6157-6173.
4. Sabanadzovic S., Abou-Ghanem N., Saldarelli P. and Martelli G.P., 1997. Physico-chemical and molecular
characterization of grapevine fleck virus. Extended Abstracts 12th Meeting ICVG, Lisbon, 1997: 25-26.
5. Sabanadzovic S., Abou-Ghanem N., Castellano M.A., Digiaro M. and Martelli G.P., 2000. Grapevines host a family of
grapevine fleck virus-like viruses.
