title: Molecular analysis of double-stranded RNAs reveals complex infection of grapevines with multiple viruses.
abstract: The table grape variety "Waltham Cross" was infected with Leafroll and Shiraz Disease. To reveal specific viruses that are associated with the diseased plants, we used an RT-PCR-based strategy to determine partial genome sequences of these viruses. Upon cloning and sequencing of the RT-PCR products, we detected seven groups of viral variants that are related to four species of the Closteroviridae: Grapevine leafroll-associated virus 1, 2, 3, and 5, in addition to Rupestris stem pitting-associated virus. The population composition of GLRaV-2 and GLRaV-3-like viruses is complex and consists of two or three distinct groups of viral variants. Based on the consensus sequence of several GLRaV-2 strains, we designed a pair of broad-spectrum primers (GLR2-4 and GLR2-5) and used them to detect a range of GLRaV-2 variants from "Waltham Cross". Moreover, we identified a novel group of viral variants from the diseased grapevines, which possess a stretch of 19 nucleotides inserted in the 3' non-coding region as compared to strain "PN" and "93/955" for which the complete genomes have been sequenced. In contrast, the population composition of GLRaV-1 and GLRaV-5-like virus seems to be more uniform and each consists of a single viral variant. Furthermore, the central 5.7kb genomic region encompassing ORF1b-ORF4 of the GLRaV-1 isolate detected in "Waltham Cross" was sequenced. The new isolate is designated GLRaV-1 "WC", which differs from GLRaV-1 "Type" by 16% in nucleotide sequence. The taxonomic standing of the GLRaV-5-like and GLRaV-3-like viruses detected in "Waltham Cross" is discussed.
authors.affiliation: Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Sean W
authors.initials: SW
authors.lastname: Prosser
authors.forename: Dariusz E
authors.initials: DE
authors.lastname: Goszczynski
authors.forename: Baozhong
authors.initials: B
authors.lastname: Meng
mesh_terms: D044822:Biodiversity; D029381:Closteroviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D052677:Flexiviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014644:Genetic Variation; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology*; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D027843:Vitis / Q000821:virology*
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral