SHORT COMMUNICATION

Brief Report of a New Highly Divergent Variant of Grapevineleafroll-associated virus 3 (GLRaV-3)Dariusz E. Goszczynski

Plant Protection Research Institute, Agricultural Research Council, Private Bag X134, Queenswood 0121, Pretoria, South Africa

Keywords

Grapevine leafroll-associated virus 3, highly

divergent variants, RT-PCR, sequencing, South

Africa, SSCP

Correspondence

D. E. Goszczynski, Plant Protection Research

Institute, Agricultural Research Council,

Pretoria, South Africa.

E-mail: GoszczynskiD@arc.agric.za

Received: April 10, 2013; accepted: May 23,

2013.

doi: 10.1111/jph.12139

Abstract

The alignment of the complete genomes of genetic variants of Grapevine

leafroll-associated virus 3 (GLRaV-3) representing phylogenetic groups I, II,

III and VI revealed numerous regions with exceptionally high divergence

between group I to III and group VI variants. Oligonucleotide primers uni-

versal for all the above groups of the virus were designed in conserved

short stretches of sequences flanking the divergent regions in the helicase

(Hel) and RNA-dependent RNA polymerase (RdRP) domains of the repli-

case gene and the divergent copy of the capsid protein (dCP) gene. Clon-

ing and sequencing of the 549-bp RT-PCR amplicon of the helicase

domain from grapevine cv. Shiraz lead to the detection of a variant of

GLRaV-3, which shared only 69.6–74.1% nt similarity with other vari-

ants, including the recently reported, new, highly divergent variant, iso-

late 139. This was confirmed by the results of the analysis of 517-bp

amplicon of the HSP70 gene of GLRaV-3 generated in RT-nested PCR

based on degenerate primers for the simultaneous amplification of mem-

bers of the Closteroviridae family designed by Dovas and Katis (J Virol

Methods, 109, 2003, 217). In this genomic region, the variant shares

72.3–78.7% nt similarity with other variants of GLRaV-3. This previously

unreported, new, highly divergent variant was provisionally named

GTG10. From the alignment of the HSP70 sequences primers for the spe-

cific RT-nested PCR amplification of the variant GTG10 and members of

group VI, and specific simultaneous amplification of variants of groups I,

II and III, were designed. The results obtained from brief testing of various

grapevines using all these primers suggest a relatively limited presence of

GTG10 variant in vineyards.

Introduction

Grapevine leafroll-associated virus 3 (GLRaV-3), the type

member of the genus Ampelovirus of the family Closte-

roviridae (Martelli et al. 2011), is regarded as the

main contributor to the problem of grapevine leafroll

disease (GLD) of wine grapevine, Vitis vinifera L, in

South Africa and worldwide (Pietersen 2010; Martelli

et al. 2011; Almeida et al. 2013). The disease is of

great concern to grapevine industries because it

delays maturation, decreases the sugar content of

berries and ultimately negatively influences the qual-

ity of produced wine (Martelli et al. 2011). Multipli-

cation of the virus in grapevines, limited to phloem,

leads to degeneration of this tissue, which prevents

translocation of synthesized carbohydrates from

leaves (Hoefert and Gifford 1967). This causes an

accumulation of starch, which makes laminae of

leaves thick and brittle. In the advanced stage of

GLD, in white- and red-berried cultivars, leaf margins

roll downwards and, in red-berried cultivars, the col-

our of the leaves changes characteristically to red –except for a narrow strip along the main veins that

remains green. The intensity of the GLD symptoms in

GLRaV-3-infected grapevines differs from mild to

severe. Although it depends on many factors such as

� 2013 Blackwell Verlag GmbH 1

J Phytopathol

grapevine cultivar, environmental stress, as well as

co-infection of plants with other pathogens, some

researchers postulate the existence of mild variants of

this virus (Habili et al. 2009). In American Vitis spe-

cies, used as rootstocks, the virus does not induce

any symptoms of GLD. The virus has positive-sense

ssRNA genome of 18 433–18 671 nucleotides, which

is organized into 12–13 protein-encoding open read-

ings frames (ORF1-13) (Ling et al. 2004; Bester et al.

2012a). Since the first report of the near complete

genome sequence of GLRaV-3 (Ling et al. 2004)

sequence data on this virus has increased substan-

tially. To date, 10 complete genome sequences of

GLRaV-3 were deposited in the GenBank/EMBL

database by various laboratories worldwide. The data

revealed that the virus is highly genetically variable.

Genetic variants of GLRaV-3 reported to date, cluster

into six (I-VI) phylogenetic groups (Bester et al.

2012a; Maree et al. 2013). The genomes of variants

of groups I–V are relatively closely related when

compared with variants of group VI (Bester et al.

2012a). These highly divergent variants share only

68.4–68.8% nt similarity with variants of other

groups. Recent results revealed that these variants

are common in vineyards (Bester et al. 2012b).

Although the high genome divergence suggests that

GLRaV-3 variants differ in pathogenicity to grape-

vines, nothing is known about the biological proper-

ties of variants. The study is hampered by the

relatively extended period required for GLD symp-

toms to develop, and grapevine is the only known

host of GLRaV-3 (Klassen et al. 2011). Also, mixed

infections of grapevine plants by various variants of

GLRaV-3 (Jooste et al. 2010; Bester et al. 2012b),

and other virus species (Prosser et al. 2007) are com-

mon. The recent construction of a DNA clone of

GLRaV-3 (Jarugula et al. 2012) has created new pos-

sibilities in the study of this virus. Knowledge on

existing divergence among genetic variants of

GLRaV-3 may contribute significantly to progress in

the study of the molecular basis of pathogenicity of

this virus and may, ultimately, lead to its control in

vineyards. Prosser et al. (2007) and Chooi et al.

(2013) reported 603 and 7612 nt sequences of

GLRaV-3, named WC-HSP-10 and NZ2, sharing

respectively only 70.6–71.6% and 70.5–76.2% nt

similarity with other variants of this virus. Recently,

after this investigation was completed, the full-length

genome sequence of a highly divergent variant of the

virus, isolate 139, was deposited in the GenBank/

EMBL database by Rast et al. (2012). The variant

shares 98.5 nt% nt similarity with the sequence

WC-HSP-10 mentioned above.

Results presented in this study are part of the inves-

tigation of genetic variants of GLRaV-3 present in

grapevines cv. Shiraz affected by Shiraz disease (SD)

(D.E. Goszczynski, unpublished data). The virus,

along with Grapevine virus A (GVA), is always present

in diseased plants in South Africa (Goszczynski and

Habili 2012). The initial aim was to design oligonu-

cleotide primers universal to the genetic variants of all

six phylogenetic groups of GLRaV-3, flanking 500–600 nt regions clearly divergent between variants.

The amplicons of this size deliver substantial amount

of sequence data and are usually very useful in the

rapid analysis of heterogeneity of amplified sequences

using the single-strand conformation polymorphism

(SSCP) technique (Koenig et al. 1995). RT-PCR

amplification of GLRaV-3 from various grapevines

using designed primers and SSCP analysis of ampli-

cons followed by cloning and sequencing described in

this article, led to the detection of a new, highly diver-

gent variant of this virus.

Materials and Methods

Plant material and isolation of dsRNA

Grapevines cvs Waltham Cross (JP98) and Shiraz

(GTG10), primarily used in this study, and grapevines

cvs Shiraz, Black Spanish, Ohanez, Barlinka and

hybrid LN33 were all from the ARC-PPRI grapevine

collection. Grapevines of cv. Shiraz with various SD

statuses were collected from a vineyard in Stel-

lenbosch, Western Cape. dsRNA was isolated from

these grapevines using the ‘butch’ procedure

described by Goszczynski (2010).

Primers design and Reverse Transcription–Polymerase

Chain Reaction (RT-PCR)

Three genomic regions flanked by conserved stretches

of sequences were selected from the alignment of

South African GLRaV-3 genetic variants 621, 623,

PL20, H11 (GenBank/EMBL accession numbers

GQ352631, GQ352632, GQ352633, JQ655295), rep-

resenting respectively groups I, II, III and VI (the

alignment not shown). The fragments, 548, 575 and

614 nt, are located in the helicase (Hel) (3173–3721 nt) and RNA-dependent RNA polymerase

(RdRP) (7717–8293) domains of the replicase gene

and the divergent copy of the capsid protein (dCP)

(15 335–15 949) of the virus genome. Nucleotide

sequence similarities between the above variants in

these three regions are respectively 66.1–91.0%,

77.3–93.9% and 64.2–88.9%. The sequences of the

� 2013 Blackwell Verlag GmbH2

GLRaV-3, highly divergent variant D. E. Goszczynski

oligonucleotide primer sets were as follows: Hel2F/

Hel2R GGCGAAGAGTATTCGCTC/CCAGAAAAGGC-

CTTCGTC; RdRP1.F/RdRP1R GCGCAACACCTTGAA

GTG/GGCACTCTGAGATTTGTC; dCP1F/dCP1R CGA

ATGCGGCGTGTGTC/CGTTCATCGTAGATATCC. The

primers were tested in a standard two-step RT-PCR

(Goszczynski 2010), using dsRNA isolated from grape-

vine cv. Waltham Cross (JP98). The plant in addition

to GLRaV-3 is also infected with GLRaV-1, -2, -10,

GVA and GRSPaV (D.E. Goszczynski, unpublished

data). Thermal cycling parameters of RT-PCR were as

follows: reverse transcription (RT) at 42°C for 1 h;

PCR, 1 cycle of 94°C for 4 min; 35 cycles of 94°C for

30 s, 52°C for 30 s, 72°C for 1 min; and a final elon-

gation at 72°C for 5 min. RT-nested PCR based on

degenerate primers for the simultaneous amplification

of members of the Closteroviridae family, also used in

this study, was essentially as described by Dovas and

Katis (2003), except that the technique was carried

out in three separate steps. The technique targets

500–535 nt fragment of the 5′ terminal half of the

HSP70 gene of closteroviruses.

Oligonucleotide primers for the specific simulta-

neous amplification of variants of groups I, II, and

III, and specific amplification of variants of group VI

and the variant GTG10 were designed manually from

the alignments of overlapping fragments of HSP70

gene of various GLRaV-3 variants. In the alignment,

in addition to the variants described above and the

517 nt sequence of the variant reported in this study,

the sequence, WC-HSP-10, deposited in GenBank/

EMBL database, accession number AF037268, by

Prosser et al. (2007), was also included. The name

and sequences of respective sets of primers were as

follows: GL3-I,II,III.F/GL3.Gen.R (TTATCGCGACGG

TGTAGAG/TAACGACGCCTCTAACCG); GL3VI.F/GL

3.Gen.R (GTTTCGTGAAGGTACGTCC/see above); GL

3.GTG10.F/GL3.Gen.R (CTATCGTGAGGGCGTAGTA/

see above). The primers were used in a three-step

RT-nested PCR. Reverse Transcription (RT) of dsRNA

(see above), performed at 42°C for 1 h, was followed

by the first round of PCR, the thermal cycling param-

eters of which were described by Dovas and Katis

(2003), for the simultaneous amplification of mem-

bers of the Closteroviridae family. In the RT step and

the first round of the PCR, the closterovirus-specific

degenerate primers of Dovas and Katis (2003) were

used. The GLRaV-3-specific primers designed in this

study were used in the second round of the nested

PCR, the thermal cycling parameters of which were

described above for the standard two-step RT-PCR,

except that the primer annealing temperature was

60°C.

Single-strand conformation polymorphism (SSCP),

cloning, sequencing and sequence analysis

For SSCP, the amplicons were extracted from agarose

using the GeneJet DNA extraction kit (Thermo Scien-

tific Cat. #K0691), according to the manufacturer’s

instructions. Five microlitre of the amplicon DNA was

mixed with 5 ll of bromophenol blue loading solu-

tion (Promega Cat. #DV4371), incubated at 99°C for

10 min, cooled on ice for 2 min and analyzed using

SSCP technique. All SSCP analyses in this study were

carried out in 8% acrylamide/bis-acrylamide (29.2/

0.8), 0.75-mm gels in 0.59 TBE buffer at 5°C for 1 h,

using Mini-protean II dual slab cell (Bio-Rad,

Hercules, CA, USA). To identify the amplified

sequences, the amplicons were cloned using pGEM-T

Easy cloning kit (Promega Cat# A1380). SSCP analysis

of clones and sequencing was as described by Gos-

zczynski (2010). Nucleotide sequence alignments and

analyses of the homology of the sequences (percent-

age identity) were carried out using the DNAMAN ver-

sion 5.2.9 (Lynnon Biosoft, 1994–2001, QC, Canada)

software package. The sequences of the alignments

were scanned for recombinants using the GDP software

package version 4.2 developed by Martin et al.

(2010). Phylogenetic analysis was conducted using

MEGA version 4 (Tamura et al. 2007). Phylogenetic

tree was constructed with the neighbour-joining

method (Saitou and Nei 1987) using evolutionary dis-

tances calculated using maximum composite likeli-

hood method of the MEGA4 package. Bootstrap

analysis of the data, based on 1000 permutations,

was used to assess the statistical confidence of the

topologies of phylogenetic tree. In the analysis, in

addition to genome sequence data for GLRaV-3 iso-

lates 621, 623, PL20 and H11 (see above for Gen-

Bank/EMBL accession numbers), sequence data for

isolates: NY1 (NC004667), 3138-07 (JX559465),

Clone 3 (JQ796828), WA-MR (GU983863), CI-766

(EU344893), GP18 (EU259806) and 139 (JX266782)

were also used. In the analysis of the HSP70 gene,

the sequences WC-HSP-10 (see above) and NZ2

(JX220899) were included.

Results and Discussion

Although GLRaV-3 was successfully RT-PCR ampli-

fied from cv. Waltham Cross (JP98) using all three

sets of primers (Hel2F/Hel2R, RdRP1.F/RdRP1R and

dCP1F/dCP1R), SSCP analysis of amplicons revealed

the highest number of DNA bands for the 548-bp

amplicon of the helicase (Fig. 1). The amplicons using

Hel2F/Hel2R primers were obtained for seven

� 2013 Blackwell Verlag GmbH 3

D. E. Goszczynski GLRaV-3, highly divergent variant

additional GLD-affected and GLRaV-3-infected grape-

vines of various cultivars from the ARC-PPRI collec-

tion. Results of SSCP analysis of these amplicons

revealed that the individual plants are infected with

one or more GLRaV-3 variants (Fig. 2). The virus

from two of these plants, cvs Waltham Cross (JP98)

and Shiraz (GTG10), showed a relatively high number

of strongly EtBr-stained DNA bands in SSCP (Fig. 2).

To determine the identity of amplified GLRaV-3

sequences from these two grapevines the amplicons

were cloned, randomly selected clones were subjected

to SSCP analysis, and clones with a distinct SSCP pat-

tern were sequenced. Seventy-eight clones for each of

the Hel2F/Hel2R amplicon were analyzed using SSCP,

and 26 and 22 of them, respectively, were sequenced.

In studying the GLRaV-3 variant status of grapevines

cv. Waltham Cross (JP98) and Shiraz (GTG10),

a RT-nested PCR based on the degenerate primers for

the simultaneous amplification of members of the

Closteroviridae family, designed by Dovas and Katis

(2003), was also used. The same work plan described

above was used: the amplicons were cloned, and 52

and 39 clones, respectively, were analyzed using the

SSCP technique. Of these, 22 and 9 clones were

sequenced. The sequence data for amplicons gener-

ated in both RT-PCR’s revealed that Waltham Cross

(JP98) is infected with GLRaV-3 genetic variants of

groups I, II, VI, and the Shiraz (GTG10) grapevine is

infected with variants of groups II, VI and, unexpect-

edly, another highly divergent variant of this virus

(Table 1). As shown in Table 1, the divergent

sequences of helicase domain and HSP70 genes of

GLRaV-3 amplified from cv. Shiraz (GTG10) grape-

vine share only 69.6–74.1% nt and 72.3–78.7% nt

similarity with other variants of the virus. This also

includes the respective sequences of highly divergent

variants of group VI, isolates 139 and NZ2. In the phy-

logenetic tree, which was constructed using the

HSP70 gene fragments of GLRaV-3 variants, the

sequence of the variant amplified from cv. Shiraz

(GTG10) created a new, clearly separated and well-

supported branch (Fig. 3). A similar result was

obtained for 549 nt fragments of helicase domain (not

shown). The analysis of the sequence data sets of phy-

logenetic trees using the GDP software package version

4.2 (Martin et al. 2010) did not reveal any recombina-

tion events in the sequences of the GTG10 variant.

Therefore, the sequences represent a new, currently

unreported, highly divergent variant of GLRaV-3. The

variant has been named GTG10 provisionally.

The presence of divergent fragments in the 5′ and 3′terminal parts of the HSP70 sequences alignment

allowed the design of primers for specific RT-nested

PCR amplification of variant GTG10 and variants of

group VI and specific simultaneous amplification of

variants of groups I, II and III (see Materials and

Methods). To investigate the specificity of primers

designed for amplification of the variant GTG10 and

variants of group VI, the respective amplicons

(354 bp) of GLRaV-3 from cv. Shiraz (GTG10) were

cloned, and eight randomly selected clones per each

primers set were sequenced. Results confirmed spe-

cific amplification of the GTG10 variant. All eight

cloned sequences of the amplicon generated in PCR

for the specific amplification of this variant shared

99.2–99.4% of nt similarity with the sequence used to

design GTG10-specific primers. For the amplicon gen-

erated in PCR using primers designed for amplifica-

tion of variants of group VI, 7 cloned sequences

shared 90.1–99.4% of nt similarity with the GH11

variant of this group, and one shared 96.6% nt simi-

larity with sequences of the GTG10 variant. In further

tests, Waltham Cross (JP98) grapevine, as expected,

was strongly positive in PCR for the simultaneous

amplification of variants of groups I-III and the vari-

ant of group VI, and consistently negative for variant

GTG10 (not shown). Surprisingly, the variant GTG10

along with a variant of group II was detected in the

grapevine cv. Cinsaut Blanc clone P163/12, which

was used by the South African grapevine industry as a

control source of SD in woody indexing (not shown).

The results of brief testing of various grapevines with

different SD statuses, however, ruled out the possible

1 2 3

Fig. 1 Single-strand conformation polymorphism (SSCP) DNA bands

patterns of RT-PCR amplicons of (1) RNA-dependent RNA polymerase

(RdRP), (2) Helicase (Hel) domains of the RNA replicase and (3) the diver-

gent copy of capsid protein (dCP) genes of Grapevine leafroll-associated

virus-3 (GLRaV-3) from cv. Waltham Cross (JP98) grapevine.

1 2 3 4 5 6 7 8

Fig. 2 SSCP DNA band patterns of RT-PCR amplicons of the Helicase

gene of GLRaV-3 from grapevines (1) cv. Waltham Cross (JP98), (2) cv.

Shiraz (GTG10), (3) cv. Black Spanish (93/1053), (4) cv. Shiraz (93/1035),

(5) cv. Barlinka (92/1027), (6) cv. Ohanez (92/1023), (7) cv. Pinot Noir

(93/944) and (8) LN33 (93/955).

� 2013 Blackwell Verlag GmbH4

GLRaV-3, highly divergent variant D. E. Goszczynski

association of the variant with SD (Fig. 4). In testing,

dsRNA isolated from six GLD-affected Shiraz plants, of

which three (plants 4–6) were also showing symp-

toms of SD, was used. The dsRNA isolated from the

original Shiraz (GTG10) and a sibling plant propa-

gated from cane cuttings of Shiraz (GTG10), which

was infected with SD (Goszczynski and Habili 2012)

was also included. As expected, all samples were

strongly GLRaV-3 positive in PCR using primers for

the simultaneous amplification of members of groups

I, II and III (Fig. 4a). Most samples were also positive

(b)

(a)

M 1 2 3 4 5 6 7 8

M 1 2 3 4 5 6 7 8

M 1 2 3 4 5 6 7 8(c)

Fig. 4 Detection of Grapevine leafroll-associated virus 3 (GLRaV-3) in

various GLD-affected cv. Shiraz grapevines using RT-nested PCR based

on Closterovirus-specific degenerate primers of Dovas and Katis (2003)

and designed in this study primers specific for genetic variants of

groups (a) I, II and III, (b) VI, and (c) variant GTG10. Numbers 1–6 refer to

field-collected plants and 7 and 8 to two plants of Shiraz (GTG10) (see

above).

Is. 621

Is. 3138-07

Is. NY1

Is. CI-766

Is. WA-MR

Is. 623

Is. GP18

Is. PL20

Is. NZ2

Is. NZ-1

Is. GH11

Is. Clone3

Cl.GTG10

Is. 139

Cl.WC-HSP-10

GLRaV-9

57

45

98

8875

69

94

85

75

58

8851

42

0.5

I

II

III

VI

Fig. 3 Phylogenetic tree constructed with

partial 305 nt sequence of HSP70 gene of vari-

ous genetic variants of GLRaV-3 to illustrate

the position of the sequence GTG10 (boxed)

among members of groups I, II, III, VI and, the

recently reported, new, highly divergent iso-

lates NZ2 and 139 (arrows). The sequence data

for GLRaV-9 (AY297819) were used as an out-

group.

Table 1 Molecular divergences of Grapevine leafroll-associated virus-3

(GLRaV-3) isolate GTG10 identified in this study

Gene Amplicona

Nucleotide similarity between isolate GTG10

and members of group I, II, III, VI, and the

isolates NZ2 and 139

I, II, III VI Is. NZ2b Is. 139

Helicase 549 bp 69.6–70.1% 73.0–74.1% – 73.0%

HSP70 517 bp 75.8–78.7% 73.5–75.2% 73.8% 72.3%

a549 and 517 nt sequences, clones G10.3.3 and 18C.C6.6, respectively,

are deposited in GenBank/EMBL database under accession numbers

KC731553 and KC731554.bNucleotide similarity in 305 nt NZ2 fragment overlapping with the

sequence of GTG10 clone 18C.C6.6.

� 2013 Blackwell Verlag GmbH 5

D. E. Goszczynski GLRaV-3, highly divergent variant

for the variant of group VI (Fig. 4b), which confirms

the report by Bester et al. (2012b) of the wide pres-

ence of this highly divergent variant in South African

vineyards. The new, highly divergent variant GTG10

was present only in Shiraz (GTG10) grapevines

(Fig. 4c), suggesting its relatively limited presence in

vineyards.

Acknowledgements

The author thanks Winetech, South Africa, for

partially funding this study.

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