ANNOTATED SEQUENCE RECORD

Molecular characterisation of two divergent variants of grapevineleafroll-associated virus 3 in New Zealand

Kar Mun Chooi • Daniel Cohen • Michael N. Pearson

Received: 4 September 2012 / Accepted: 28 December 2012 / Published online: 14 February 2013

� Springer-Verlag Wien 2013

Abstract Partial genomic sequences of two divergent

grapevine leafroll-associated virus 3 (GLRaV-3) variants,

NZ1-B and NZ2, from New Zealand were determined and

analysed (11,827 nt and 7,612 nt, respectively). At the

nucleotide level, both variants are more than 20 % differ-

ent from the previously published GLRaV-3 sequences,

from phylogenetic groups 1 to 5. Phylogenetic analysis

indicated that NZ1-B is a variant of the previously identi-

fied divergent NZ-1, while NZ2 is a novel sequence with

only 76 % nucleotide sequence identity to GLRaV-3

variants NZ-1, GH11, and GH30. Therefore, NZ2 is a new

variant of GLRaV-3. Amino acid sequence analysis of the

NZ1-B and NZ2 coat proteins indicated significant sub-

stitutions that are predicted to alter the coat protein struc-

ture, which potentially leads to the observed reduced

immunological reactivity of both variants to the Bioreba

anti-GLRaV-3 conjugated monoclonal antibody.

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

member of the genus Ampelovirus, family Closteroviridae

[19]. The virions are flexuous, filamentous particles,

approximately 1,800 nm in length. It has a positive-sense

single-stranded RNA genome that varies between 17,919

and 18,671 nt [2, 10, 14, 17, 18] and is organised into 13 open

reading frames (ORFs) [17]. The virus is restricted to the

phloem of grapevines and is only transmitted by mealybugs

(semi-persistent) and grafting [19]. There has been increas-

ing interest in the genetic variability of GLRaV-3 popula-

tions in various countries following the study by Turturo

et al. [26], with recent studies showing high genetic vari-

ability [2, 12, 15, 24, 27]. Most GLRaV-3 isolates identified

thus far fall within the phylogenetic groups 1 to 5 proposed

by Gouveia et al. [12]. However, an outlier isolate, NZ-1

(EF508151), from New Zealand is more than 20 % divergent

from group 1 variants at the nucleotide level and does not fall

into any of these five groups. Recently, isolates with 90 to

91 % nucleotide sequence identity to NZ-1 have been

described from South Africa (GH11, JQ655295; GH30,

JQ655296) [2] and the USA (GLRaV-3e cluster [24];

CA7246, JQ796828 [23]), and it has been proposed that these

divergent variants represent a new, sixth phylogenetic group

[2]. High genetic variability can affect the diagnostic

detection of virus, as was observed when ELISA, using

antibodies against GLRaV-3 (Bioreba), detected GLRaV-3

in samples testing negative by RT-PCR using common

diagnostic primers [4, 5]. Therefore, a genetic diversity study

was undertaken using RT-PCR with newly designed primers,

single-stranded conformation polymorphism analysis, and

sequence analysis [4]. From this study, two GLRaV-3 vari-

ants with low nucleotide sequence identity to the common

NY1 (AF037268) isolate were identified based on a 564 nt

region of ORF4. A BLAST search revealed one variant,

NZ1-B, that was highly similar to the divergent New Zealand

variant, NZ-1, and a second variant, NZ2, showed a maxi-

mum of 79 % nucleotide sequence identity to previously

described GLRaV-3 variants. Subsequent testing showed

that samples previously testing GLRaV-3 positive by ELISA

but negative by RT-PCR contained only NZ1-B and/or NZ2

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00705-013-1631-9) contains supplementarymaterial, which is available to authorized users.

K. M. Chooi (&) � M. N. Pearson

School of Biological Sciences, University of Auckland,

Private Bag 92019, Auckland, New Zealand

e-mail: kcho115@aucklanduni.ac.nz

D. Cohen

The New Zealand Institute for Plant and Food Research Limited,

Private Bag 92169, Auckland, New Zealand

123

Arch Virol (2013) 158:1597–1602

DOI 10.1007/s00705-013-1631-9

variants [6]. As a result, a study was conducted to confirm the

identity and better understand the genetic structure of these

divergent GLRaV-3 variants.

From the initial genetic diversity study, a Syrah vine

from a germplasm collection in Auckland and a symp-

tomatic Pinot noir vine from a Hawke’s Bay vineyard,

singly infected with NZ1-B and NZ2, respectively, were

selected as a virus source for further molecular character-

isation. Total RNA was extracted from cane and leaf

material using a SpectrumTM Plant Total RNA Kit (Sigma-

Aldrich, St. Louis, MO) as described by the manufacturer

with minor modifications [5]. The genome sequences of the

two GLRaV-3 variants were obtained by amplifying seven

(NZ1-B) and five (NZ2) overlapping fragments of the

genome. All PCR primers were designed using the Primer3

program [22].

Amplicons of less than 2 kb were synthesised using the

SuperScriptTM III One-Step RT-PCR System with a Plati-

num� Taq DNA Polymerase Kit (Invitrogen, Carlsbad,

CA). Amplicons longer than 2 kb were synthesised using

two-step RT-PCR, with SuperscriptTM III Reverse Trans-

criptase (Invitrogen, Carlsbad, CA) used to synthesise the

first-strand cDNA, followed by treatment with RNase H

(Invitrogen, Carlsbad, CA) to remove RNA complementary

to cDNA. Long-extension PCR was conducted using a

Platinum� Taq DNA Polymerase High Fidelity Kit

(Invitrogen, Carlsbad, CA) with the addition of 1 % DMSO

for high GC content [1]. The 30 UTRs were confirmed

using yeast poly(A) polymerase (USB, Cleveland, OH),

and polyadenylated total RNA was then used as the tem-

plate for one-step RT-PCR using a SuperScriptTM III One-

Step RT-PCR System with Platinum� Taq DNA Poly-

merase Kit (Invitrogen, Carlsbad, CA). All PCR products

were analysed by gel electrophoresis, purified, and then

cloned using the pGEM�-T Easy Vector cloning system

(Promega, Madison, WI) and DH5a Escherichia coli cells

(Invitrogen, Carlsbad, CA). For each amplicon, at least two

clones were sequenced twice in both directions. With

longer fragments, additional internal primers were

designed to obtain the full-length sequence. Geneious v5.5

[9] was used to assemble all cloned sequences and con-

struct all nucleotide and amino acid alignments. Phyloge-

netic analysis of each respective ORF was conducted using

MEGA5 [25], and phylogenetic trees were constructed

using the neighbour-joining method with 1,000 bootstrap

replications.

Approximately 64 and 41 % of the NZ1-B and NZ2

genomes, respectively, were successfully assembled.

Analysis of the NZ1-B sequence confirmed that it is a

variant of the divergent NZ-1 isolate (99.6 % nucleotide

sequence identity based on 6,416 nt), and this extends the

known NZ-1 sequence by 5,410 nt. The NZ1-B sequence

starts within ORF1a (nt position 6,811 based on GP18,

EU259806), while the NZ2 sequence starts within ORF4

(nt position 10,897 based on GP18), and both terminate at

the 3’ UTR, resulting in a total of 11,827 nt and 7,612 nt

for NZ1-B and NZ2, respectively. The partial sequences

were deposited into the GenBank database as accessions

JX220900 and JX220899. Potential ORFs were identified

using Geneious v5.5 [9] and then compared to existing

GLRaV-3 full genomes.

Similar to group 6 variants NZ-1, GH11, GH30, and

CA7246 variants, NZ1-B does not contain an ORF2, but

the intergenomic region between ORF1b and ORF3 is

1,596 nt long, 34 nt shorter than GH11 and GH30. NZ1-B,

GH11, GH30, and CA7246 also share the same frameshift,

which leads to a premature stop codon and shortens the

ORF12 polypepetide by six amino acids [2]. In contrast,

ORF11 of NZ2 is translated in the same frame as in other

GLRaV-3 isolates; however, transcription starts 3 nt

upstream and terminates 15 nt downstream of the corre-

sponding positions in NZ1-B, GH11, and GH30. Thus, the

NZ2 ORF11 is 18 nt longer than those of all known

GLRaV-3 isolates, resulting in an ORF11 polypeptide that

is six amino acids longer. Variation in the translation of

ORF11 was found by Wang et al. [27] in GLRaV-3 vari-

ants from group 4, which requires the use of an alternative

start codon (ACG). The longer NZ2 ORF11 does not affect

the positioning of ORF12, as it is predicted to start in the

same position as other GLRaV-3 variants; however, a

frameshift within ORF12 leads to a premature stop codon

and an ORF12 polypeptide that is four amino acids shorter.

The 3’ UTR of NZ1-B and NZ2 are 263 nt and 289 nt long,

respectively.

Based on the partial genome sequence, NZ1-B is closely

related to GH11 and GH30, with 91.5 % nucleotide

sequence identity, while the NZ2 variant is most closely

related to the NZ1-B, GH11, and GH30 variants with 76 %

nucleotide sequence identity. To understand the distribu-

tion of the nucleotide sequence similarity between GLRaV-

3 isolates over the partial genome, similarity plots were

made using the Kimura 2-parameter model [16] (Supple-

mentary Fig. S1), and the nucleotide and amino acid

sequence identities for the individual ORFs and the 3’ UTR

of NZ1-B and NZ2 variants compared to NY1, GH11, and

to each other, are displayed in Table 1. Sequence com-

parisons indicated that the ORF6 (which encodes the coat

protein [CP]) of NZ1-B and NZ2 had the highest sequence

similarity to that of NY1, with 78.5 % and 78.6 % nucle-

otide and 90.1 % and 91.4 % amino acid sequence identity,

respectively. Other regions of high similarity to NY1 were

ORF1b and ORF4, with at least 74.0 % and 84.5 %

nucleotide and amino acid sequence identity, respectively.

Sequence variation increased towards the 3’ UTR, with

nucleotide and amino acid sequence differences of more

than 34.5 and 38.0 % respectively, for ORF9, ORF10, and

1598 K. M. Chooi et al.

123

ORF12 when compared to NY1. ORF11, which codes for

the p4 protein (function unknown), is unique to GLRaV-3,

and it displayed the greatest variation. At the amino acid

level, NZ1-B and NZ2 are 72.3 % and 86.1 % different

from NY1 and 16.7 % and 69.4 % different from GH11.

Recombination analysis using RDP3 [20] based on partial

genome sequence alignments of NZ1-B and NZ2 and

corresponding GLRaV-3 sequences revealed no recombi-

nation events between NZ1-B and NZ2 and other GLRaV-

3 variants. Sequencing of the remaining parts of the gen-

ome would also be advantageous for further comparisons,

particularly in the highly variable 5’ UTR, ORF1a, and

intergenomic region [2, 18].

Phylogenetic analysis based on sequences from a 428 nt

region of the ORF6 from this study and GenBank placed

most of the GLRaV-3 isolates in the previously proposed

groups 1 to 5 [12]. However, the isolate 43-15 (JF421951)

is in a separate clade, which is referred to as GLRaV-3f by

Sharma et al. [24], NZ1-B is in a separate clade with

variants from South Africa and the USA (the proposed

group 6 of Bester et al. [2]), and NZ2 is positioned sepa-

rately (99 % confidence) from all other variants (Fig. 1).

Noticeably, the branch lengths within group 6 are signifi-

cantly longer than for the other five groups. The average

genetic distance between the seven sequences within the

proposed sixth group (6.3 %) is considerably higher than in

the other five groups (0.6 to 0.9 %). Therefore, to ensure

that the phylogenetic classification is consistent for all

GLRaV-3 variants, further sequence information is

required to determine the integrity of group 6.

The initial detection of NZ-1 and NZ2 was not an iso-

lated occurrence. Both occur frequently within the New

Zealand GLRaV-3 population and have been detected in

both commercial vineyards and older germplasm collec-

tions, individually and in mixed infection with other

GLRaV-3 variants [5]. Cabernet Sauvignon and Pinot noir

vines infected with NZ-1 and NZ2 express characteristic

leafroll symptoms, and both variants are graft transmissible

to cultivars Syrah, Cabernet Sauvignon, Sauvignon blanc,

and rootstocks 3309 (Vitis riparia x V. rupestris), 101-14

(V. berlandieri x V. rupestris), SO4 (V. berlandieri x V.

riparia), Riparia Gloire (V. riparia), and Schwarzmann (V.

rupestris x V. riparia). In addition, natural spread of NZ2

was also recently observed within a commercial Hawke’s

Bay block [5].

Testing of samples by ELISA showed that both NZ2

and, to greater extent, NZ-1, had reduced immunological

reactivity to a monoclonal antibody (Bioreba) prepared

against NY1 compared to a polyclonal antiserum [7]. Thus,

in an attempt to explain the difference in immunological

reactivity, the complete CP amino acid sequences of GH11,

NZ1-B, and NZ2 were compared to NY1. There are 38

amino acid differences between these four variants, gen-

erally positioned closer to the 5’-terminal end (Fig. 2). Of

Table 1 Comparison of nucleotide (nt) and amino acid (aa) sequence identities (%) between available open reading frames of NZ1-B and NZ2

with corresponding sequences from virus variants NY1 and GH11, and between each other

NZ1-B versus NY1 NZ1-B versus GH11 NZ2 versus NY1 NZ2 versus GH11 NZ2 versus NZ1-B

nt aa nt aa nt aa nt aa nt aa

Overalla 70.7 - 91.5 70.3 - 76.3 - 76.2 -

5’UTR - - - - - - - - - -

ORF1ab 74.4 87.3 93.8 98.6 - - - - - -

ORF1b 78.7 88.7 92.4 97.0 - - - - - -

ORF2 - - - - - - - - - -

ORF3 72.5 75.6 94.9 95.6 - - - - - -

ORF4 (HSP70 h)c 74.4 84.9 92.8 95.4 74.0 84.5 79.5 90.2 79.1 89.8

ORF5 (HSP90 h) 68.5 72.7 90.4 94.4 68.7 72.1 77.4 86.3 77.1 86.7

ORF6 (CP) 78.5 90.1 92.9 96.5 78.6 91.4 82.3 94.9 81.3 94.6

ORF7 (dCP) 70.5 77.1 90.7 93.1 70.9 77.1 75.2 81.8 75.1 82.0

ORF8 75.4 77.8 91.8 95.7 76.0 78.4 75.6 81.6 76.7 83.2

ORF9 61.6 55.9 89.7 88.1 61.8 59.3 68.5 66.1 68.9 65.5

ORF10 64.3 62.0 86.7 90.5 63.3 63.7 73.0 78.2 72.8 78.8

ORF11 43.6 27.7 89.1 83.3 40.9 13.9 52.7 30.6 55.4 38.8

ORF12 65.5 61.1 92.7 88.9 64.8 61.1 70.9 72.2 72.1 75.9

3’ UTR 79.4 - 96.5 - 80.9 - 87.2 - 87.9 -

a The overall identities based on the entire partial genome sequence of NZ1-B (11,827 nt) and NZ2 (7,612 nt)b NZ1-B identities are based on the partial ORF1a sequence (641 nt)c NZ2 identities are based on the partial ORF4 sequence (1,418 nt)

Grapevine leafroll-associated virus 3 in New Zealand 1599

123

the 38 amino acid changes, 24 are conservative, i.e., amino

acid changes with similar physiochemical properties, and 8

out of the 14 non-conservative changes are considered

neutral substitutions [3]. Five out of the six remaining non-

conservative amino acid changes are located between

amino acid positions 70 and 84. In NY1, all five of these

amino acids are polar and would be expected to be found

on the protein surface or at active site(s), whereas in the

divergent GLRaV-3 genetic variants, these amino acids

have been substituted by small hydrophobic amino acids

with non-reactive side chains that are rarely directly

involved in protein function [3]. In addition, the secondary

structure predicted using the Garnier Osguthorpe Robson

algorithm [11] showed significant differences between

NY1 and the divergent variants, particularly at the 5’-ter-

minal end (Fig. 2).

Amino acid substitutions that alter the CP protein

structure and, in turn, the epitope recognised by the Bio-

reba conjugated monoclonal antibody may lead to reduced

immunoreactivity. At present, there is limited information

on the identity, types, and distribution of epitopes on the

GLRaV-3 CP. However, Zhou et al. [28, 29] and Orecchia

et al. [21] have identified a similar potential epitope near

the N’-terminus, between amino acids 61 and 148 and

between amino acids 59 and 78, respectively. This coin-

cides with the region that contains most of the non-con-

servative substitutions between NY1 and divergent

variants, further supporting the possibility of structural

changes to the CP of both divergent variants. The CP is

part of the quintuple gene block and forms the helical body

for the virion, encapsidating approximately 95 % of the

viral RNA, which protects the viral RNA during transport

[8]. Future work is required to understand the effect of the

amino acid substitutions on virion assembly, replication,

and movement within grapevines.

Similarly, Gouveia et al. [13] proposed critical amino

acid substitutions within ORF10 that alter the p19.7 protein

structure and are potentially linked to differences in the

RNA silencing suppressor activity between variants from

each of the five common groups. The overall average

amino sequence acid identity of ORF10 between the five

variants used in that study was 84.9 % (JQ763393–

JQ763397). Therefore, future work should be conducted on

the activity of the NZ1-B and NZ2 variant p19.7 protein, as

these genetic variants have only 62.0 and 63.7 % amino

acid sequence identity to NY1 (group 1), respectively, and

the predicted secondary structures are considerably

different.

In conclusion, this paper describes two divergent

GLRaV-3 variants: NZ1-B, which is a variant of NZ-1 and

is closely related to South African GH11 and GH30 vari-

ants, and NZ2, which at the nucleotide level is more than

Group 1

Group 2

Group 5

Group 4

Group 3

43-15 [USA] (JF421951)

22-15 [USA] (JF421827)

21-12 [USA] (JF421818)

44-2 [USA] (JF421958)

CA7246 [USA] (JQ796828)

GH30 [South Africa] (JQ655296)

GH11 [South Africa] (JQ655295)

NZ1-B [New Zealand] (JX220900)

Group 6

NZ2 [New Zealand] (JX220899)

99

9999

99

99

99

99

99

99

0.05

Fig. 1 Phylogenetic analysis of grapevine leafroll-associated virus

3 (GLRaV-3) isolates from this study and GenBank, based on a

428 nt region within open reading frame 6, conducted in MEGA5

[25]. For GLRaV-3 isolate names and accession numbers, see

Supplementary Table S1. The NZ1-B and NZ2 GLRaV-3 isolates

are highlighted in bold. Evolutionary history was inferred using the

neighbour-joining method, and the Kimura 2-parameter method was

used to compute evolutionary distances. Elongated triangles

represent the compressed subtrees of the phylogenetic groupings

based on Gouveia et al. [12] and Bester et al. [2]. The length of

the triangle corresponds to the respective intra-group diversity, and

the thickness is proportional to the number of taxa. The percent-

ages of bootstrap support (C 90 %) from 1,000 replicates are

shown at nodes. The scale represents 0.05 nucleotide substitutions

per site

1600 K. M. Chooi et al.

123

20 % different from the next most closely related GLRaV-

3 variant. The high genetic difference between NZ2 and

other GLRaV-3 variants suggests that NZ2 is a novel

variant of GLRaV-3 and potentially represents a new

phylogroup, though more sequence data are required to

confirm the new grouping. Furthermore, the classification

of the GLRaV-3 phylogenetic groupings should be revis-

ited because of significantly higher genetic variability

within group 6 compared to the other groups. The high

GLRaV-3 sequence variability described in this paper also

highlights the need for further work to be undertaken to

determine any differences in vector transmission effi-

ciency, virus virulence, and symptom severity between the

different GLRaV-3 variants.

Acknowledgments This research was supported by Corbans Viti-

culture Ltd, the University of Auckland, New Zealand Winegrowers,

Plant and Food Research, and the Tertiary Education Commission.

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