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