ORIGINAL ARTICLE

Characterization of the complete genome of a novel citrivirusinfecting Actinidia chinensis

Ramesh R. Chavan • Arnaud G. Blouin •

Daniel Cohen • Michael N. Pearson

Received: 17 December 2012 / Accepted: 1 February 2013 / Published online: 15 March 2013

� Springer-Verlag Wien 2013

Abstract A ssRNA virus from kiwifruit (Actinidia spp.)

was identified as a member of the family Betaflexiviridae.

It was mechanically transmitted to the herbaceous indica-

tors Nicotiana benthamiana, N. clevelandii, N. glutinosa

and N. occidentalis. The complete genome was comprised

of three ORFs and a 3’poly (A) tail. Phylogenetic analysis

of the entire genome indicated it was a novel member of

the genus Citrivirus (family Betaflexiviridae). The com-

plete nucleotide sequence differed from that of citrus leaf

blotch virus (CLBV) by * 26 %. The movement protein

(ORF2) and coat protein (ORF3) shared 95-96 % and

90-92 % amino acid sequence identity, respectively, with

CLBV. The replicase polyprotein (ORF1) was distinctly

different from published CLBV sequences, with 78-79 %

amino acid sequence identity, while the 5’ UTR and 3’

UTR differed from CLBV by 28 % and 29 %, respectively.

The sequence differences indicate that the citrivirus from

Actinidia is either a divergent strain of CLBV or a member

of a new citrivirus species.

Introduction

The genus Actinidia, commonly known as kiwifruit or

Chinese gooseberry, is a native to temperate eastern Asia

and China, which represent the centre of genetic diversity

of the genus, with 52 of 55 species [15]. A. chinensis and

A. deliciosa are extensively cultivated in various parts of

the world for their nutritious berries [8]. In an endeavour to

improve the quality of kiwifruit to meet the demands of the

industry, Actinidia germplasm is imported into New Zea-

land from China and other countries for breeding new

cultivars. A new flexivirus was detected in germplasm from

China that was held in post-entry quarantine.

Kiwifruit losses due to bacterial and fungal diseases are

well documented, but so far there are no published reports

of similar losses due to viral diseases. However, a number

of viruses have been reported infecting members of the

genus Actinidia [19, 20], often associated with typical viral

foliar symptoms. These include apple stem grooving virus

[7], ribgrass mosaic virus [5, 6, 19], cucumber mosaic virus

and alfalfa mosaic virus [20], and most recently, two novel

vitiviruses (Actinidia virus A and Actinidia virus B)

infecting A. chinensis [4].

In addition to the above, a betaflexivirus was detected

from A. chinensis using carlavirus primers (PCR93000,

Agdia Inc., Elkhart, USA) that amplify a 274-nt sequence

from the replicase gene [20]. This sequence showed 81 %

nt and 92 % aa sequence identity to citrus leaf blotch virus

(CLBV), the type member of the genus Citrivirus (family

Betaflexiviridae). The family Betaflexiviridae consists of

the genera Capillovirus, Carlavirus, Citrivirus, Foveavirus,

Tepovirus, Trichovirus and Vitivirus [1], which include

viruses that infect numerous herbaceous and woody crops

from both monocotyledonous and dicotyledonous groups.

This paper reports the characterization of the complete

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00705-013-1654-2) contains supplementarymaterial, which is available to authorized users.

R. R. Chavan � M. N. Pearson (&)

School of Biological Sciences, The University of Auckland,

Private Bag 92019 Auckland, New Zealand

e-mail: m.pearson@auckland.ac.nz

A. G. Blouin � D. Cohen

The New Zealand Institute for Plant & Food Research Ltd,

Private Bag 92169 Auckland, New Zealand

123

Arch Virol (2013) 158:1679–1686

DOI 10.1007/s00705-013-1654-2

genome of a novel citrivirus from A. chinensis originating

from China.

Materials and methods

Source plants

Male and female plants of A. chinensis and A. deliciosa

from Shaanxi Province, China, were imported into New

Zealand as woody cuttings, whip grafted onto healthy

rootstocks of A. chinensis ‘Hort 16A’, and grown in *10-litre

containers in post-entry quarantine. Dormant plants were

chilled at 4 �C for a period of three weeks during winter to

hasten bud break. The spring and summer growth was

observed for viral symptoms for five years.

Sap transmission and experimental host range

Leaf sap extracts from both young and mature leaves of

male line M3-A, and female lines F1-A, F1-N, F3-E4,

F3-N8, F3-O, F3-P, F3-QII and F4-J of A. chinensis were

used for sap transmission tests. Leaf tissue (1-2 g) was

homogenised in 4 mL 0.1 M phosphate buffer, pH 7.5 [23],

containing 5 % polyvinyl pyrrolidone and 0.012 % sodium

sulphite, using a pestle and mortar. The homogenate was

mixed with 400-mesh carborundum powder and mechani-

cally inoculated to the herbaceous indicators Chenopodium

amaranticolor, C. quinoa, Nicotiana benthamiana, N. clev-

elandii, N. glutinosa, N. occidentalis 37B, and Phaseolus

vulgaris ‘The Prince’. Buffer-inoculated plants were used as

negative controls. The inoculated plants were maintained in

the quarantine greenhouse at * 20-22 �C for up to six weeks

and observed for viral symptoms.

PCR and sequencing

In order to sequence the presumed citrivirus from Actinidia,

multiple PCR primers (Online resource 1) were designed

based on all available CLBV sequences and conserved

domains of closely related members of the family Betaflex-

iviridae, using Primer3 software (http://frodo.wi.mit.

edu/primer3) and Oligo Analyzer (http://molbiol-tools.ca/

molecular_biology_freeware.htm) to determine Tm, GC%,

primer loops, primer dimers and primer-primer

compatibility.

RNA was extracted from 100-mg leaf samples of

symptomatic Actinidia and indicators using a QIAgen

RNeasy� Plant Mini Kit (catalogue no. 74903, Germany)

according to the manufacturer’s protocol, with the fol-

lowing modification: the RLC lysis buffer containing

guanidine hydrochloride was modified by the addition of

2 M sodium acetate to a final concentration of 0.2 M and

polyvinyl pyrrolidone (MW 40,000) to a final concentra-

tion of 2.5 % (w/v), and the pH was adjusted to 5.0. The

RNA was eluted in molecular grade water or 1 mM sodium

citrate RNA storage solution at pH 6.4 (Ambion, UK).

The viral genomes were amplified as several fragments

using the primers listed in Online resource 1. For two-step

RT-PCR, reverse transcription was performed at 37 �C for

1 h using 3 ll of total RNA and 1 ll of reverse primer in a

25-ll reaction volume with either Maloney murine leu-

kaemia virus (M-MLV) reverse transcriptase (Promega,

Madison, WI, USA) or SuperScript III (Invitrogen Inc. UK)

according to the manufacturer’s protocols. PCR was per-

formed in a 50-ll reaction volume using AmpliTaq� DNA

Polymerase (Applied Biosystems, California) according to

the manufacturer’s protocol with 1 ll each of forward and

reverse primers. The amplification programme consisted of

94 �C for 5 min followed by 35 cycles of 94 �C for 15 s,

55-60 �C for 30 s, and 68 �C for 60-120 s, followed by

68 �C for 5 min. Some parts of the genome were amplified

using the SuperScriptTM One step RT-PCR System with

PlatinumR Taq DNA polymerase (Invitrogen, catalogue no.

12574-026). The reaction was performed according to the

manufacturer’s protocol in a total reaction volume of 25 ll

with 0.6 ll each of sense and antisense primers (20 pmole/ll).

Reverse transcription was carried out at 55 �C for 30 min

followed by 94 �C for 5 min. The PCR programme

consisted of 35 cycles of 94 �C for 15 s, 55-60 �C

(depending upon primers) for 30 s, and 68 �C for

60-120 s (depending upon amplicon length), followed by

68 �C for 5 min.

PCR products were analysed by agarose gel electro-

phoresis, stained with ethidium bromide and visualised

using a BIO-RAD transilluminator. For cloning, DNA was

purified from excised bands using a ‘Perfectprep Gel

Cleanup’ purification kit (Eppendorf, Hamburg, Germany),

ligated into pGEM-T Easy Vector (Promega, Madison, WI,

USA) and cloned in E. coli DH5a competent cells (Invit-

rogen Technologies). Plasmids were extracted using a

FastPlasmid�Mini Kit (Eppendorf, Hamburg, Germany),

and the inserts were sequenced using an ABI PRISM

automated DNA sequencer (University of Auckland, New

Zealand).

Sequence assembly

Prior to assembly, the individual sequences were edited to

remove the primer sequences and subjected to VecScreen

(http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html)

to identify sequence that could be of vector origin. Con-

sensus nucleotide sequences for the complete putative

replicase polyprotein, movement and coat protein genes,

and 5’ and 3’ UTR were created from forward and reverse

sequences using Sequencher 4.5 (Gene Codes Corporation,

1680 R. R. Chavan et al.

123

Michigan 48108, USA). The genes were translated into

amino acid sequences using the BioEdit biological

sequence alignment editor (Tom Hall, Ibis Therapeutics,

Carlsbad, CA 92008).

Phylogenetic analysis

The complete genome nucleotide sequence and amino acid

sequences of replicase polyprotein, movement and coat

proteins were compared with those of fourteen betaflexiv-

iruses, including members of two unassigned species

belonging to the family Betaflexiviridae [1]. Sequence

identity matrices were created using BioEdit, and phylo-

genetic analyses of amino acid sequences of the various

ORFs were conducted using MEGA 5 software [24]. The

neighbour joining (NJ) method was used to construct

phylogenetic trees with Poisson-corrected amino acid dis-

tances and pairwise gap deletion options. The node sig-

nificance was evaluated with 10,000 bootstrap random

addition replicates to create a consensus tree. Both uniform

and unequal rates of evolutions were tested to evaluate the

replicase polyprotein, movement and coat protein phylog-

enies. The complete genome phylogeny was constructed

using the NJ method (maximum composite likelihood

model) with pairwise gap deletion, substitution including

transitions ? transversions and uniform rate of evolution

options using the nucleotide sequences of representative

members of the family Betaflexiviridae. The nucleotide and

amino acid sequence similarities between Actinidia citri-

virus and other CLBV sequences were analysed using

SimPlot v.3.5.1 [16].

Results

Symptoms on virus-infected Actinidia and indicators

Vein clearing and mild mottling were observed on leaves

of the terminal and middle regions of shoots of the infected

A. chinensis male plant (M3-A) during spring, and inter-

veinal chlorosis during summer (Fig. 1a-b). Similar

symptoms were observed on female A. chinensis acces-

sions (F1-A and F3-N8), while five other accessions (F1-N,

F3-E4, F3-P, F3-QII and F4-J) remained symptomless for

four years.

Actinidia citrivirus isolates were sap transmitted to

N. benthamiana, N. clevelandii, N. occidentalis 37B and

N. glutinosa, and infection was confirmed by PCR. All of

the citrivirus-infected indicators were severely stunted

compared to the control plants and expressed the following

symptoms: N. occidentalis – localised necrotic ring spots

after c. 2 weeks followed by systemic leaf distortion and

mottling and /or vein chlorosis and significant vein

thickening 2-3 weeks post-inoculation; N. glutinosa – leaf

distortion developed on the inoculated leaves within

2 weeks post-inoculation followed by systemic mottle

(Fig. 1c) and vein clearing/banding; N. benthamiana

– distortion and leaf tip chlorosis 2-3 weeks post-inocula-

tion; N. clevelandii – leaf distortion, systemic necrotic ring

spots in older leaves and chlorotic vein banding and dis-

tortion and/or chlorotic spotting. Some of the original

Actinidia plants were subsequently found to be co-infected

with vitiviruses [4], and consequently, it is possible that

some of the symptomatic indicators may also have been

co-infected with a vitivirus. However, Actinidia citrivirus

isolates readily infected N. glutinosa and N. clevelandii,

whereas AcVA and AcVB did not.

PCR and sequencing

The complete genome (8782 nt) of the virus isolate from

A. chinensis, line M3-A, was amplified and sequenced

(GenBank accessions JN900477, JN983454, JN983455,

JN983456) using the primers listed in Online resource 1. In

addition, partial sequences were obtained for isolates from

other A. chinensis lines, as follows: F1-N (JN936275),

3683 nucleotides encompassing the 3’end of ORF 1, the

complete movement and coat protein genes and 3’UTR;

F1-A (JQ013961), F3-E4 (JQ013962) and F3-N8

(JQ013957), 1092 nt of the complete coat protein gene.

The primer sets used to amplify M3-A failed to amplify the

5’ end of the F1 and F3 viruses. Clonal variation between

the movement and coat protein sequences amplified from

the individual Actinidia lines was minimal, so only one

representative sequence from each line was chosen for

phylogenetic analysis.

Genome structure and organization

Multiple full genome sequences of M3-A (JN900477,

JN983454, JN983455, JN983456) were essentially identi-

cal, with just 16 point mutations (0.002 % variation) across

the genome. The following genome description is based on

the full genome sequence JN900477: The genome con-

sisted of a monopartite, linear, single-stranded, positive-

sense RNA, 8782 nucleotides long with 74 % nucleotide

sequence identity to CLBV (AJ318061.1). The genome

organisation is typical of members of the genus Citrivirus

(family Betaflexiviridae) with three non-overlapping open

reading frames and a 3’-terminal poly (A) tract.

ORF1, the putative replicase polyprotein, includes

methyltransferase (M), AlkB (A), OTu-like peptidase (O),

papain-like protease (P), RNA helicase (H), and RNA-

dependent RNA polymerase (R) domains, typical of a

citrivirus [17]. It comprises 5964 nucleotides, spanning

from AUG at nucleotide position 72 to an ‘opal’

Genome sequence of a citrivirus from Actinidia chinensis 1681

123

termination codon (UGA) at position 6035 and codes for

1987 amino acids (229.8 kDa). ORF2, which codes for the

putative movement protein, extends from nucleotides 6035

to 7123 with an ‘ochre’ termination sequence and codes for

362 amino acids (40.2 kDa). A non-coding region of 55

nucleotides intercalates between ORF2 and ORF3, from

7124 to 7178. ORF3 codes for a putative coat protein and

spans nucleotides 7179 to 8255, terminating with an

‘amber’ codon, and codes for 358 amino acids (40.1 kDa).

The 5’ and 3’ UTRs are represented by 71 and 526

nucleotides, respectively. The 3’UTR terminates in a

poly(A) tail. The 5’UTR and 3’UTR nucleotide sequences

of M3-A differed from CLBV citrus isolates by 28 % and

29 %, respectively.

The complete ORF1 of M3-A (AFA 43527.1) shows an

overall 78-79 % amino acid identity to published citrus

CLBV sequences (Table 1), with many conserved blocks of

up to 83 amino acids, common to all sequences. However,

the level of similarity varies greatly across ORF1, with M3-A

being 25 amino acids longer than the citrus CLBV isolates,

24 of which are in the highly variable region between amino

acids 600 and 700 (Online resource 2). However, the 915

bases at the 3’ end of ORF1, of both M3-A and F1-N, show

much higher identity (93-96 %) to CLBV sequences (Table 1).

The polyprotein has a predicted molecular mass of 229.8 kDa

compared to 227 kDa for CLBV [25].

The movement proteins (ORF2) of M3-A and F1-N are

362 amino acids long (40.2 kDa), the same as CLBV, and

have 94-96 % amino acid sequence identity to CLBV

isolates (Table 2). An alignment of the movement protein

amino acid sequences of the Actinidia and citrus citrivi-

ruses shared nine conserved regions ranging from 10 to 88

amino acids, with two major conserved regions spanning

amino acids 77 to 131 and 133 to 220.

The coat proteins of the Actinidia isolates show 91-96 %

amino acid identity to CLBV isolates (Table 3). M3-A has

a five-amino-acid deletion at positions 140 to 144, resulting

in a coat protein of 358 amino acids with a predicted

molecular mass of 40.1 kDa, compared to 363 amino acids

(41 kDa) for CLBV and the other Actinidia isolates.

Clustal analysis of the coat protein amino acid sequences of

the Actinidia isolates and other citrivirus isolates revealed

seven highly conserved regions ranging from 10 to 66

amino acids, the largest between aa positions 240 and 305.

Phylogenetic analysis

A comparison of the complete genome sequences of

Actinidia isolate M3-A (JN900477) and 16 genome

sequences representing the six genera of the family Beta-

flexiviridae plus members of two unassigned species

(Fig. 2) clearly places M3-A in the genus Citrivirus, and

whole (M3-A) and partial (F1-A, F1-N, F3-E4 and F3-N8)

genome analysis consistently places the Actinidia isolates

into a clade that is separate from that of the citrus isolates.

Although the genome structure of Actinidia citrivirus

M3-A (JN900477) is similar to that of CLBV, the nucle-

otide sequences of 3’UTR, ORF1 and 5’UTR differed

significantly from all previously published CLBV sequen-

ces (AJ318061.1, EU857539.1, EU857540.1, FJ009367.1).

The Actinidia citrivirus isolates cluster together in a single

clade and are more variable than citrus CLBV isolates.

Citrus CLBV isolates show at least 97 % amino acid

sequence identity for all three ORFs, whereas the Actinidia

isolates showed 15-20 % nucleotide and 1-6 % amino acid

difference for the partial 3’ replicase (Table 1), 15 % nt

and 3 % aa difference for the movement protein (Table 2)

and 1-17 % nt and 1-7 % aa difference for the coat protein

(Table 3). The failure of primers that successfully ampli-

fied isolate M3-A to amplify a major portion of the 5’ end

of the replicase gene of F1-N suggests sequence differences

in this area.

Fig. 1 Symptoms associated with citrivirus infection in Actinidia chinensis and herbaceous indicators: a–b Actinidia chinensis male plant

(M3-A) showing vein chlorosis; c N. glutinosa with leaf mottle

1682 R. R. Chavan et al.

123

Table 1 Identity matrix of nucleotide (normal font) and amino acid

(bold font) sequences of the complete replicase polyprotein (ORF1) of

Actinidia citrivirus isolates M3-A (JN900477) and partial replicase of

Actinidia citrivirus isolate F1-N (JQ013958) with citrus leaf blotch

virus isolates from citrus

# Virus name/accession 1 2 3 4 5 6

1 JN900477 Actinidia citrivirus M3-A NA 73 72 72 72

85 81 81 81 81

2 JQ013958 Actinidia citrivirus F1-N NA NA NA NA NA

94 80 80 80 80

3 AJ318061 Citrus leaf blotch virus 79 NA 97 98 99

94 96 98 99 99

4 EU845539 Citrus leaf blotch virus NZ G18 79 NA 98 97 98

93 96 99 97 98

5 EU845540 Citrus leaf blotch virus NZ G78 78 NA 98 98 98

93 95 99 99 99

6 FJ009367 Dweet mottle virus 79 NA 99 99 99

94 96 100 99 99

1 2 3 4 5 6

NA = data not available, italicized values represent comparisons of 305 aa at the 3’ end, only

Table 2 Identity matrix of nucleotide (normal font) and amino acid (bold font) sequences of the complete movement protein (ORF 2) of

Actinidia citrivirus isolates M3-A and F1-N and citrus leaf blotch virus isolates from citrus

# Virus name / accession 1 2 3 4 5 6

1 JN900477 Actinidia citrivirus M3-A 85 78 78 78 78

2 JN936275 Actinidia citrivirus F1-N 97 78 78 78 78

3 AJ318061 Citrus leaf blotch virus 95 94 96 97 98

4 EU845539 Citrus leaf blotch virus NZ G18 95 94 98 97 98

5 EU845540 Citrus leaf blotch virus NZ G78 95 94 98 98 97

6 FJ009367 Dweet mottle virus 96 94 99 99 98

1 2 3 4 5 6

Table 3 Identity matrix of nucleotide (normal font) and amino acid (bold font) sequences of the complete coat protein (ORF3) of Actinidia

citrivirus isolates M3-A, F1-A, F1-N, F3-E4 and F3-N8 and citrus leaf blotch virus isolates from citrus

# Virus name/accession 1 2 3 4 5 6 7 8 9

1 JN900477 Actinidia citrivirus M3-A 83 83 83 83 84 83 84 84

2 JQ013961 Actinidia citrivirus F1-A 93 99 99 99 86 86 85 86

3 JQ013958 Actinidia citrivirus F1-N 93 99 99 99 85 85 85 86

4 JQ013957 Actinidia citrivirus F3-N8 93 99 99 99 86 86 85 86

5 JQ013962 Actinidia citrivirus F3-E4 93 99 99 98 85 85 85 85

6 AJ318061 Citrus leaf blotch virus 92 96 96 96 96 99 98 99

7 EU845539 Citrus leaf blotch virus NZ G18 91 96 96 95 95 99 97 98

8 EU845540 Citrus leaf blotch virus NZ G78 90 94 94 94 94 98 97 98

9 FJ009367 Dweet mottle virus 92 96 96 96 96 99 99 98

1 2 3 4 5 6 7 8 9

Genome sequence of a citrivirus from Actinidia chinensis 1683

123

Discussion

The closest published sequences to M3-A and other

Actinidia citrivirus isolates are those of isolates of citrus

leaf blotch virus (CLBV), first detected from Nagami

kumquat (Fortunella margirita (Lour.) Swing) in Spain

[10]. The characterization of the complete genome of

CLBV [25] led to the establishment of the genus Citrivirus

with Citrus leaf blotch virus as the type species [1]. Dweet

mottle virus [21], which produced symptoms similar to

those of CLBV [9, 27], was subsequently shown to

be * 98 % similar to CLBV [13] and is thus considered a

CLBV isolate [1]. A partial 3’-end sequence of the genome

of a betaflexivirus infecting Prunus persicae (3055 nt)

showed 73 % identity to CLBV, but further characteriza-

tion of the virus revealed a trichovirus-like genome orga-

nization [3]. Hence, Citrus leaf blotch virus is currently the

only recognised species in the genus Citrivirus.

CLBV is only known to produce symptomatic infections

in Citrus spp. through grafting [9, 18, 25], and dweet

mottle disease is transmitted from citrus to citrus by con-

taminated knife blades [13, 22, 28] and transmitted at a low

rate (c. 2.5 %) through seeds of citrange, kumquat and sour

orange [12]. CLBV has also been mechanically transmitted

to C. quinoa, Gomphrena globosa, N. benthamiana,

N. cavicola, N. clevelandii, N. glutinosa and N. occidentalis

[11, 26, 28], although all of these infections were symptom-

less. In contrast, Actinidia citrivirus isolates were readily

mechanically transmitted to several herbaceous species,

including N. benthamiana, N. clevelandii, N. glutinosa and N.

occidentalis, causing symptoms. Although possible co-

infection with vitiviruses may influence the symptoms

observed in Actinidia, the vitiviruses did not cause symptoms

in N. clevelandii and N. glutinosa, and in N. occidentalis, we

did not observe the vitivirus-associated necrotic local lesions

caused by vitiviruses [4].

Actinidia citrivirus isolate M3-A differs from CLBV

isolates in having a longer genome, largely due to an

additional 25 amino acids in ORF 1, although this is par-

tially offset by a deletion of 5 amino acids in ORF 3 and

slightly shortened non-coding regions (10 nt between the

movement and coat protein genes, 2 nt in the 5’UTR and

14 nt in the 3’UTR). In common with most viruses of the

family Betaflexiviridae, M3-A encodes an AlkB domain

that maintains the integrity of the viral RNA genome by

oxidative demethylation, through repair of deleterious

methylation damage [2, 17]. AlkB-containing viruses have

a remarkable capacity to infect woody or perennial plants,

and the long-term survival of viruses within a single

infected plant might be attributed to the functional

advantages provided by the AlkB protein [17]. This may

explain the successful establishment and survival of

Fig. 2 Phylogenetic relationship of the complete genome of Actinidia isolate M3-A (JN983454) with representative members of the genera of

the family Betaflexiviridae (neighbour-joining tree)

1684 R. R. Chavan et al.

123

citriviruses in citrus and Actinidia. The failure of M3-A

primers to amplify the 5’ end of isolates F1 and F3 suggests

significant variation in the replicase polyprotein of the

various Actinidia citirivirus isolates, which together with

the deletion of a five-amino-acid block in the coat protein,

is consistent with the plasticity of betaflexiviruses [17].

Based on the overall similarity of the genome organi-

sation, the common large conserved regions within the

replicase and coat protein genes, and grouping together

with CLBV strains in all of the phylogenetic analyses, we

conclude that virus isolate M3-A (JN900477), from

Actinidia, is a member of the genus Citrivirus. The

molecular criteria for species demarcation in the family

Betaflexiviridae [1] are that members of distinct species

have less than ca. 72 % nt identity or 80 % amino acid

identity between their coat protein or replicase genes.

Based on coat protein sequence, Actinidia citrivirus M3-A

could be considered a strain of CLBV, since it shares

83-84 % nt and 90-92 % aa sequence identity with CLBV

(Table 3). However there is a difference of 26 % across the

whole genome and the replicase polyprotein, with an

additional 25 amino acids, where it shows only 72-73 % nt

and 78-79 % aa sequence identity with CLBV, which is

below the threshold and represents a significant departure

from the CLBV isolates. Komatsu et al. [14] reported a

similar situation for Plantago asiatica mosaic virus

(PlAMV) (genus Potexvirus, family Alphaflexiviridae),

isolates of which share as little as 75-77 % nucleotide

sequence identity and 82-85 % amino acid sequence

identity in the polymerase gene, and about 68 % nt and

70-73 % aa sequence identity to the closely related Tulip

virus X (TVX). Based on these results, they suggest that

PlAMV and TVX are in the process of diverging from a

common ancestor and emerging as distinct virus.

In summary, we conclude that the citrivirus from

Actinidia line M3-A (JN900477) is either a diverging strain

of CLBV as the result of adaption to a different host or a

member of a new Citrivirus species.

Acknowledgments The authors wish to thank Zespri Innovation

and Plant and Food Research for funding this work, and Jane Lan-

caster and Peter Berry (Zespri Innovation) for their management of

the quarantine plants and associated testing programme.

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