ANNOTATED SEQUENCE RECORD The first complete nucleotide sequence of a grapevine virus E variant Beatrix Coetzee • Hans J. Maree • Dirk Stephan • Michael-John Freeborough • Johan T. Burger Received: 4 March 2010 / Accepted: 29 April 2010 / Published online: 16 May 2010 Ó Springer-Verlag 2010 Grapevine virus E (GVE) was first isolated from a Japanese table grape cultivar (Vitis labrusca cv. Aki Queen and Pione) and classified as a new member of the genus Viti- virus in 2008 [1]. Although one of the plants used as a source of virus had stem pitting disease, no relationship between GVE and any disease symptoms could be estab- lished. Grapevine virus E is a positive-sense single-stran- ded RNA virus with a genome organization resembling that of grapevine virus A (GVA). Currently, the only sequence data available are the incomplete sequences of GVE iso- lates TvAQ7 (AB432910) and TvP15 (AB432911). Both isolates are transmissible by the mealybug Pseudococcus comstocki [1]. Recently, a metagenomic sequencing study reported the presence of GVE in South African vineyards for the first time [2]. The virus was detected in a severely diseased vineyard (cv. Merlot) in the Stellenbosch region of South Africa. Sequence data obtained using an Illumina Genome Analyzer II were subjected to de novo assembly, and two scaffolds were found to align preferentially to GVE when doing BLAST searches against the NCBI’s non-redundant nucleotide databases. The GVE variant present in the meta- genomic sample was homologous to the incomplete GVE TvP15 sequence [1]. A scaffold assembled from metage- nomic sequencing data [2], GVE Node 3404, was edited to 4,216 nt to contain only high-quality sequence data and submitted to GenBank (accession number GU903011). This scaffold aligned to TvAQ7 at nt positions 373–4,589. Based on the available sequence information (GVE Node 3404, TvP15 and TvAQ7 sequence data), the following set of diagnostic primers was designed: GVE-1-For 5 0 -AA TGGAGTCAAAAGCGATCC-3 0 and GVE-Rev 5 0 -GTAG GGTCAATCAACCAACA-3 0 . To identify a GVE-infected plant, total RNA was isolated from individual plants using a CTAB method adapted for grapevine [3]. The extracted RNA was used as a template in RT-PCR using the diag- nostic primers. A single grapevine plant, SA94 (Vitis vinifera cv. Shiraz), from a different vineyard and cultivar than the metagenomic study, was identified. The vine dis- played typical Shiraz disease symptoms (e.g., canes with a lack of lignification, delayed leaf fall and reduced vigor) and tested positive for GVE. Sequencing of the PCR product confirmed the GVE infection status. The vine also tested positive for grapevine rupestris stem pitting-associ- ated virus (GRSPaV), grapevine leafroll-associated virus 3 (GLRaV-3) and GVA infections. RNA extracted from this vine was used to determine the complete nucleotide sequence of the South African GVE isolate SA94. A set of nine primer pairs was designed to amplify overlapping regions spanning the GVE genome. Primer pairs were used to synthesize cDNA from total RNA using AMV reverse transcriptase. The cDNA was then used as a template in a PCR with a high-fidelity DNA polymerase. Annealing temperatures and extension times were adapted according to the primer pairs and the expected sizes of the amplified fragments. PCR amplicons of the expected sizes were excised from agarose gels, purified and sequenced directly using the primers used for amplification. Sequence data from the 9 overlapping amplicons generated a contig of 7,042 nt using BioEdit [4]. The 3 0 -terminal nucleotide sequence of the virus genome was determined by cDNA synthesis from total RNA with the oligo(dT) primer 5 0 -TACGATGGCTGCAG(T) 17 -3 0 [5]. B. Coetzee Á H. J. Maree Á D. Stephan Á M.-J. Freeborough Á J. T. Burger (&) Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa e-mail: jtb@sun.ac.za 123 Arch Virol (2010) 155:1357–1360 DOI 10.1007/s00705-010-0685-1