ORIGINAL ARTICLE Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5 0 UTR A. E. C. Jooste • H. J. Maree • D. U. Bellstedt • D. E. Goszczynski • G. Pietersen • J. T. Burger Received: 29 April 2010 / Accepted: 27 August 2010 / Published online: 10 September 2010 Ó Springer-Verlag 2010 Abstract Three genetic variants of grapevine leafroll- associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymor- phism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5 0 ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5 0 UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the pres- ence of two or three additional variant groups elsewhere in the world. Introduction Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causative agent of grapevine leafroll disease (leafroll) in South Africa and spreads rapidly in local vineyards [18]. As GLRaV-3 is consistently associated with leafroll, it is viewed to be an important etiological agent of this economically important disease [2]. Grapevine leafroll- associated virus 3 is the type species of the genus Ampe- lovirus in the family Closteroviridae [17]. Virions are flexuous, filamentous particles, about 1,800 nm long, with a positive-sense single-stranded RNA genome organised into 13 open reading frames [12]. Research worldwide has shown the existence of several molecular variants of GLRaV-3. The population structure and genetic variability of 45 GLRaV-3 isolates, from dif- ferent grapevine varieties and 14 different countries, by single-stranded conformation polymorphism (SSCP) and sequence analysis of three genomic regions, RdRp, Hsp70h and coat protein (CP), was investigated [22]. The results for the RdRp and Hsp70h regions showed that 10% of the isolates analysed had mixed-variant infections, whilst 15% of the isolates had mixed infections when the CP region was analysed [22]. Multiple alignment of sequences deposited in GenBank revealed that the sequences used in the Italian study had nucleotide identities of above 90% The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are GQ352631, GQ352632 and GQ352633. Electronic supplementary material The online version of this article (doi:10.1007/s00705-010-0793-y) contains supplementary material, which is available to authorized users. A. E. C. Jooste (&) Á D. E. Goszczynski Á G. Pietersen ARC-Plant Protection Research Institute, Queenswood, Private Bag X134, Pretoria 0121, South Africa e-mail: JoosteE@arc.agric.za H. J. Maree Á J. T. Burger Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch 7602, South Africa D. U. Bellstedt Department of Biochemistry, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch 7602, South Africa 123 Arch Virol (2010) 155:1997–2006 DOI 10.1007/s00705-010-0793-y