First Report of Grapevine Virus Sequences Highly Similar to Grapevine Syrah virus-1 from Washington Vineyards

Abstract

Grapevine Syrah virus-1 (GSyV-1), a tentative member of the genus Marafivirus in the family Tymoviridae, has recently been found in a declining Syrah grapevine in California vineyards (1). To determine if GSyV-1 is present in grapevines grown in Washington State vineyards, extracts prepared from individual grapevines of six cultivars (Merlot, Chardonnay, Pinot Noir, Lemberger, Cabernet Sauvignon, and Syrah/Shiraz) were tested by single-tube reverse transcription (RT)-PCR using the primer pair GSyV-1 Det-F (5'-CAAGCCATCCGTGCATCTGG-3') and GSyV-1 Det-R (5'-GCCGATTTGGAACCCGATGG-3'). The primer GSyV-1 Det-F is identical to nucleotides (nt) 1125 to 1144 and GSyV-1 Det-R complementary to nt 1401 to 1420 of the GSyV-1 genome (GenBank Accession No. NC_012484) in the putative movement protein encoding gene (1). DNA fragment of approximately 296 base pairs (bp) was amplified only from 7 of 60 and 2 of 20 individual grapevines of cv. Syrah/Shiraz and Chardonnay, respectively, obtained from geographically separate vineyards. The 296-bp fragments from three Syrah/Shiraz and two Chardonnay grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones derived from each DNA fragment were sequenced from both orientations and the sequences edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparison of four of these sequences (Accession Nos. GU372349-52) showed 99 to 100% amino acid (aa) sequence identity among themselves and with corresponding sequences of GSyV-1. Because of the lack of antibodies, an additional 611-bp fragment specific to the capsid protein (CP) gene of GSyV-1 was amplified from six isolates (five from cv. Syrah/Shiraz, and one from cv. Chardonnay) (Accession Nos. GU372353-66) using primers GSyV-1-F (5'-TGTCGACGCTCCAATGTCTGA-3') and GSyV-1-R (5'-CATTGCTGCGCTTTGGAGGCTTTA-3'). GSyV-1-F is identical to nt 5775 to 5795 and GSyV-1-R is complementary to nt 6385 to 6408 of the GSyV-1 genome. The amplicons were cloned and sequenced as described above. Comparison of these sequences among themselves and with corresponding sequences of GSyV-1 showed 96 to 99% aa sequence identity, further complementing the results obtained above. To our knowledge, this is the first report of the occurrence of viral sequences closely related to GSyV-1 in Washington vineyards. Together with other reports (1,2), this study suggests that viruses similar to GSyV-1 could be widely distributed in wine grape cultivars across grape-growing regions.