|Sequence analysis of Grapevine leafroll-associated virus 1 from Washington vineyards|
B. DONDA (1), N. Rayapati (1). (1) Washington State University, Prosser, WA, U.S.A.
<i>Grapevine leafroll-associated virus 1</i> (GLRaV-1, genus: <i>Ampelovirus</i>, family: <i>Closteroviridae</i>) has been reported in many grapevine-growing regions worldwide. However, only limited information is available on the molecular biology of GLRaV-1. In this study, we have isolated the genomic-length, double-stranded RNA (dsRNA) of GLRaV-1 from two wine grape (<i>Vitis vinifera</i>) cultivars that tested positive for the virus in reverse transcription-PCR (RT-PCR). The dsRNA was subsequently used as a template to amplify overlapping portions of the genome by RT-PCR using virus-specific primers. The 5’-end sequence was determined using a commercially available 5’RACE system. The resulting amplicons were cloned and nucleotide sequence determined. The genome sequences of two isolates of GLRaV-1 were determined to be ~18,730 (isolate WA-CH) and ~18,945 (isolate WA-PN) nucleotides (nt). WA-CH and WA-PN isolates encode nine open reading frames and the overall genome organization is similar with previously reported GLRaV-1 isolates. WA-CH and WA-PN isolates have ~857 nt and ~922 nt 5’nontranslated region (NTR), respectively, and contain a 130 nt tandem duplication. An additional 65 nt sequence was present only in the 5’NTR of the WA-PN isolate. The size of 3’NTR of WA-CH and WA-PN isolates is being determined. Using unique sites of a restriction enzyme in the 5’NTR, an RT-PCR-based RFLP assay was developed for molecular typing of GLRaV-1 isolates present in Washington vineyards.