American Journal of Plant Sciences, 2013, 4, 2006-2010
http://dx.doi.org/10.4236/ajps.2013.410250 Published Online October 2013 (http://www.scirp.org/journal/ajps)
Rt-PCR Analysis and Evolutionary Relationship of Some
Hungarian Grapevine leafroll associated virus 1 and 3
Isolates*
Eszter Cseh1#, András Péter Takács2, Richard Gáborjányi2, László Palkovics3, László Kocsis1
1Department of Horticulture, Georgikon Faculty, University of Pannonia, Keszthely, Hungary; 2Plant Protection Institute, Georgikon
Faculty, University of Pannonia, Keszthely, Hungary; 3Department of Plant Pathology, Faculty of Horticultural Science, Corvinus
University of Budapest, Budapest, Hungary.
Received January 30th, 2013; revised March 1st, 2013; accepted April 1st, 2013
Copyright © 2013 Eszter Cseh et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Hungarian isolates of Grapevine leafroll associated virus 1 and 3 (GLRaV-1, GLRaV-3) were tested using serological
(DAS-ELISA) and molecular (RT-PCR) methods. Five hundred bp long PCR products of the part of HSP70 gene of one
serologically positive GLRaV-1 and four GLRaV-3 isolates were sequenced. These sequences were applied for phy-
logenetic analysis and compared to foreign virus isolates of NCBI GenBank. Phylogenetic analysis of GLRaV-1 HSP70
gene supported the earlier results that it could be divided into two clusters: E and A. The Hungarian isolate 6.4.1 be-
longed to the group E. This isolate showed the highest homology with the AY754914 isolate from the Czech Republic.
GLRaV-3 sequence data could cluster five groups. Hungarian 2.2; 3.5 and 4.2 isolates were estimated belonging to the
group II. The 1.4 isolate from the same vineyard as 2.2 varied in sequence data so it belonged to the other, IV. variant
group with two South African, two Austrian and a Syrah isolate. According to the phylogenetic analysis, two variant
groups occurred in Hungary. These isolates related with each other, but showed higher similarity of foreign counties. In
some cases, they were similar to isolates of the neighbour countries such as Slovakia and Austria. It could be supposed
that mainly the exchange of virus infected propagation materials caused the dissemination of GLRaV isolates.
Keywords: Grapevine; Virus; GLRaV-1; GLRaV-3; HSP70; RT-PCR; Hungary
1. Introduction
Grapevine leafroll symptoms may be induced by a com-
plex of viruses, the majority of which belong to the
Closteroviridae family.
Members of GLRaV-1-4 have been distinguished in
Hungary by DAS-ELISA [1]. GLRaV-1 and -3 spread
via propagation material and grafting [2] and are transmit-
ted by the scale insect [3], and by mealybugs [4-11].
GLRaV-1 and -3 belong to the genus Ampelovirus. The
GLRaV-1 genome contains 9 open reading frames enco-
ding replication complex consisiting of the methyltrans-
ferase, helicase, RNA depending RNA polymerase do-
mains, a 70 kDa heat shock protein homologue, a HSP90-
like protein, the coat protein, two minor copies of coat
protein and other proteins of unknown function [12].
RNA genome of GLRaV-3 is positively single-stranded,
and contains 13 open reading frames [13]. It is most
closely related to GLRaV-1. Both viruses encode a 70
kDa heat shock protein homolog with conserved amino
acid sequence motives [14,15]. This region is used for
phylogenetic analysis in order to assess evolutionary re-
lationship among members of ampeloviruses [16].
2. Materials and Methods
2.1. Plant Material
Forty-eight grapevine leaf samples of cultivars Kékfran-
kos, Juhfark, Pinot noir, Kéknyelű, Cabernet sauvignon,
Olaszrizling and Tempranillo originated from different
geographical areas: North-West, Middle-West, South-
West and Middle-East part of Hungary (Kőszeg, Bala-
tonboglár, Badacsonytomaj, Cserszegtomaj, Kecskemét
*The nucleotide sequence data reported in this work have been submit-
ted in the Genbank database and assigned the accession numbers
HE794021, HE794022, HE794023, HE794024 and HE794025.
#Corresponding author.

Rt-PCR Analysis and Evolutionary Relationship of Some Hungarian Grapevine leafroll associated virus
1 and 3 Isolates 2007
and Pécs), showing typical leafroll symptoms, were col-
slected in autumn, 2010.
2.2. Das Elisa
Collected leaf samples were tested for GLRaV-1 and -3
by DAS-ELISA method [17] with antiserum of Bioreba
AG (Switzerland).
2.3. RT-PCR Detection
Total RNA was extracted and purified from grapevine
leaf tissues by SPEKTRUM Plant Total RNA Kit (Sig-
ma-Aldrich Chemie GmbH, Germany). The primer pairs
were designed based on the nucleic sequence of isolates
GLRaV-3 NY1 (GenBank accession number AF037268)
and GLRaV-1 according to the sequence data of Gen-
Bank Acc. No. AF 195822. The specific primers:
1FHSP70 5’-CAGGGCTCGTTTGTACTGG-3’, 1RHSP70
5’-TCGGACAGCGTTTAAGTT CC-3’ [4] and in the
case of GLRaV-3: LC1F 5’-CGCTAGGGCTGTGGAA-
GTATT-3’, LC2R 5’-GTTGTCCCGGGTACCAGAT-
AT-3’ [13] An 540 and an 546 bp fragments encompass
from the central part of HSP70 gene. The cDNA synthe-
sis was carried out by reverse transcription by M-MuLV
enzyme. PCR conditions in a PCR Applied Biosystems
GeneAmp PCR System were as follows: denaturation
94˚C/1 min, followed 40 cycles of 94˚C/75 s, 52˚C/30 s
and 72˚C/1 min. The final elongation step was at 72˚C/10
min. Aliquots of PCR products were run on 1.5% agarose
gel. PCR products were gel-purified using Roche High
Pure Purification Kit, cloned into pGEM-T Easy (Pro-
mega) cloning vector [18] and sequenced by BAY-GEN
(Hungary).
2.4. Phylogenetic Analysis
Phylogenetic studies were performed using alignments of
the HSP70h genes in both cases from several virus iso-
lates. Constructions of the evolutionary models were per-
formed using the CLC Sequence viewer 6.5.1. (CLC bio,
Denmark). The phylogenetic trees were also obtained
with CLC Sequence Viewer 6.5.1. using UPGMA me-
thod and 1000 bootstrap iterations as a confidence test. In
order to assess the relationship of the four Hungarian
GLRaV-3 isolates and one Hungarian GLRaV-1 isolate,
their HSP70h gene sequences were used in a phylogene-
tic analysis in which the HSP70h sequences of isolates
from elsewhere in the world were included.
3. Results
DAS-ELISA and RT-PCR gave positive results for
GLRaV-3 in four samples (1.4; 2.2; 3.5; 4.2) and one
sample (6.4.1) for GLRaV-1 (Table 1). Results of se-
quence analysis showed high homologies of Hungarian
GLRaV-3 virus isolates. However, this isolates showed
some differences. Alignment trees have been constructed
from the same items of sequence data from Gene bank of
500 nt part of HSP70 genes using UPGMA methods
compared of isolates from elsewhere in the world.
This phylogenetic analysis of GLRaV-1 HSP70 gene
is corresponded to the results of Kominek and his co-
worker’s [4]. According to their results this cluster can be
separated into two groups, an “E” and an “A”. Our results
suggest that Hungarian isolate from Badacsonytomaj
(6.4.1) belonged to the group “E” (Figure 1). This isolate
showed the highest homology to the (AY754914.1)
Table 1. Occurrence of Grapevine leafroll-associated virus 1 (GLRaV-1) and 3 (GLRaV-3) isolates in Hungary.
Number of
collected samples Cultivar Origin
Number of infected
samples by ELISA
Selected samples for
molecular analysis
7 Kékfrankos Kőszeg 2 GLRaV-3 1.4, 2.2
2 Pinot noir Balatonboglár 1 GLRaV-3 -
3 Juhfark 1 GLRaV-1 6.4.1
2 Kéknyelű - -
10 Olaszrizling 2 GLRaV-3 -
1 Pinot noir
Badacsonytomaj
1 GLRaV-1 3.5
3 Cabernet sauvignon 1 GLRaV-3 -
7 Unknown Kecskemét 2 GLRaV-1 -
3 Olaszrizling 1 GLRaV-3 4.2
3 Tempranillo
Cserszegtomaj 1 GLRaV-1 -
1 Pinot noir - -
6 Unknown Pécs - -
48
Abbreviations: Grapevine leafroll-associated virus 1 (GLRaV-1) Grapevine leafroll-associated 3 (GLRaV-3).

Rt-PCR Analysis and Evolutionary Relationship of Some Hungarian Grapevine leafroll associated virus
1 and 3 Isolates
2008
Figure 1. Phylogenetic tree reconstructed from 500 nt long fragment of HSP70 gene from GLRaV-1 isolates. Abbreviations:
AY754914.1 (Czech Republic), AY754912.1 (Czech Republic) AY754929.1 (Czech Republic), AY754931.1 (Czech Republic),
AY754920.1 (Czech Republic), AY754944.1 (Slovakia), AY754939.1 (Slovakia), FJ952150.1 (Iran), AF233935.1- (USA),
AY754924.1 (Czech Republic), AY644650.1 (Czech Republic), AY754933.1 (Czech Republic), AY754915.1 (Czech Republic),
AY754905.1 (Czech Republic), AF195822.1 (Australia), Hungarian isolate: 6.4.1 (Badacsonytomaj).
isolate from Czech Republic. The “E” group holds the
isolates (AY754912.1), (AY754929.1), (AY754931.1),
(AY754944.1) from Czech Republic; and the (AY754944.1)
and (AY754939.1) isolates from Slovakia. The group
“A” includes the Iranian (FJ952150.1), an Australian
(AF195822.1), an American (AF233935.1) and five
(AY754924.1), (AY754915.1), (AY754905.1), (AY754933.1)
and (AY644650.1) isolates from Czech Republic. Phy-
logenetic analysis showed that the GLRaV-1 Hungarian
isolate belonged to the European “E” group.
Figure 2 represents the relationship of 500 bp frag-
ments of HSP70 gene of GLRaV-3 from twenty five iso-
lates from different countries, included four Hungarian
ones. These sequences clustered into five groups similar
in Fuchs’ study [19].
Only one virus isolate (ef508151) from New-Zealand
belongs to the first group.
The largest second group contained fifteen isolates as
Asian isolates: one (aj748524) from Israel; two Chinese
(aj748514) and (dq780887); and one (aj748517) from
Syria. Other members of this group were from America
(North and South part), two from USA (dq780891) and
(af037268) and one (eu344893) from Chile. South Africa
was represented by one isolate (gq352631). Some se-
quence data of GLRaV from Europe showed high ho-
mology with the second group, as one (aj748521) Italian;
two Austrian (aj748512) and (aj748511); one Tunisian
(aj748522) and three Hungarian (3.5 from Badacsony-
tomaj; 2.2 from Kőszeg and 4.2 from Cserszegtomaj).
Only one isolate from China (dq780889) represented the
third group.
Six isolates, (aj748513) and (aj748510) from Austria;
(gq352632) and (eu259806) from South Africa; (aj748516)
from Syria and one Hungarian isolate 1.4 from Kőszeg
formed the fourth numerous group. 1.4. showed the hi-
ghest homogeneity to the South African isolates. The two
members of the fifth group are from Italy (aj748519) and
South Africa (gq352633). Hungarian isolates (2.2; 4.2)
showed homology to the isolates of China (dq780887)
and Chile (eu344893). It is noteworthy that the Hungar-
ian (3.5); the Israeli (aj748524); the Italian (aj748521)
and the Austrian (aj748512) isolates showed no sequence
differences with each other.
4. Discussion
HSP70 gene was used as a basic target to initial molecu-
lar analysis of the members of Closteroviridae and eva-
luation of their genetic diversity. In the most cases this
sequenced part is available in the GenBank. In this ma-
nuscript could be used HSP70 sequence data of GLRaV-
1 isolates from Australia, Iran, America and some intra-
isolate sequence variant from Slovakia and Czech Re-
public. The isolate of Hungary show the highest homoge-
neity to the sequence data of isolate “sv12-5” from Czech
Republic.
Phylogenetic analysis of partial gene sequence of
HSP70 of Grapevine leafroll-associated virus 3 showed,
that four Hungarian isolate grouped into two biggest
clades. Sequence identities were found among the
GLRaV-3 isolates from four European isolates, including
the Hungarian 3.5 isolates. The 2.2, 3.5, 4.2 isolates are
related to the NY1 isolate in the group II. The isolate 1.4
riginated from the same vineyard in Kőszeg, as the o

Rt-PCR Analysis and Evolutionary Relationship of Some Hungarian Grapevine leafroll associated virus
1 and 3 Isolates 2009
Figure 2. Phylogenetic tree of the genomic region HSP70 GLRaV-3 constructed by UPGMA method. Abbreviations: ef508151
(New-Zealand), aj748524 (Israel), aj748521 (Italy), aj748512 (Austria), aj748514 (China), dq780887 (China), dq780891 (USA),
gq352631 (South Africa), aj748511 (Austria), aj748517 (Syria), eu344893 (Chile), af037268 (USA), aj748522 (Tunisia), dq
780889 (China), aj748513 (Austria), gq352632 (South Africa), eu259806 (South Africa), aj748516 (Syria), aj748510 (Austria),
aj748519 (Italy), gq352633 (South Africa), Hungarian isolates: 3.5 (Badacsonytomaj), 2.2 (Kőszeg), 4.2 (Cserszegtomaj) and
1.4 (Kőszeg).
isolate 2.2, but this sequence data show higher homoge-
neity to the members of the group IV.
Some authors have recognised five variant groups.
Three major, one isolate from New-Zealand and one from
China represent two other groups [13,19-22]. Our results
confirm these clustering and the Hungarian isolates could
insert in these groups.
5. Conclusion
According to the phylogenetic analysis, two variant groups
of GLRaV-3 occurred in Hungary. There is a lack of cor-
relation of sequence data with geographical origin if the
isolates were consistent with the view that the infected
propagation material plays the most important role in the
spreading. The results are not suitable for the justification
of origin and spread of Hungarian virus isolates. Further
studies are needed to gain information of the biological
significance and role in the transmission of these sequence
diversity of grapevine leafroll associated viruses.
6. Acknowledgements
This work was partly supported by the OTKA K67658,
TÁMOP-4.2.1./B-09/1-KMR-2010-0005, TÁMOP-4.2.2./
B-10/1-2010-0023 and TÁMOP-4.2.1./B-09/1/KONV-
2010-0003 grants.
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