Arch Virol
DOI 10.1007/s00705-015-2566-0
REVIEW
Detection and genetic variation analysis of grapevine fanleaf virus
(GFLV) isolates in China
Jun Zhou1 Xudong Fan1 Yafeng Dong1 Zun ping Zhang1 Fang Ren1
Guojun Hu1
Received: 29 March 2015 / Accepted: 5 August 2015
Springer-Verlag Wien 2015
Abstract To investigate the prevalence and genetic
variation of grapevine fanleaf virus (GFLV) in China, 142
grapevine samples from 13 provinces and regions were
tested using DAS-ELISA, RT-PCR, and nested RT-PCR.
Of the samples, 38 % tested positive for GFLV by DAS-
ELISA, and 26.8 % tested positive by RT-PCR and nested
RT-PCR. Movement protein (MP) and coat protein (CP)
gene PCR products were cloned and sequenced. The MP or
CP nucleotide and protein sequences shared identities that
ranged from 94.9 % to 100 %. Phylogenetic analysis
revealed that Chinese GFLV isolates obtained in this study
were distinct from the isolates reported in GenBank.
Grapevine fanleaf virus (GFLV), a member of the genus
Nepovirus in the family Secoviridae [25], is the most
economically important virus that infects grapevine
worldwide [19]. It can reduce grape yield and quality, often
also reducing the productive life of grapevine canes [1].
The rooting ability of rootstocks and the graft take of
scions are both reduced substantially in GFLV-infected
material [1]. Infected grapevines show a range of foliar
symptoms including leaf deformation, yellow mosaic, vein
banding, ring and line patterns and flecks [18]. GFLV is
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-015-2566-0) contains supplementary
material, which is available to authorized users.
& Yafeng Dong
yfdong1234@163.com
1
National Center for Eliminating Viruses from Deciduous
Fruit Tree, Research Institute of Pomology, Chinese
Academy of Agriculture Sciences,
Xingcheng 125100, Liaoning, China
transmitted by the ectoparasitic root nematode Xiphinema
index [25] and by vegetative propagation and grafting.
GFLV is widespread in almost all viticultural regions,
including Asia, Africa, Europe, Oceania, and North and
South America [1]. Recently, GFLV has been found in
countries or areas where it had not previously been
observed, and 96 %, 50 %, 24 %, 9.6 %, and 1.4 % of
grapevines were reported to be infected in Alicante (Spain)
[4], La Cote (Switzerland) [23], Istria (Croatia) [21], Ata-
cama (Chile) [8], and Tunisia [16], respectively. The
results of this extensive GFLV survey in these viticulture
regions indicated an urgent need for an efficient virus
control strategy.
Understanding the genetic diversity of a virus popula-
tion can help in the development of reliable diagnostic
methods in clean-plant programs. The genetic diversity of
GFLV has been investigated in France [20], Iran [3],
Slovenia [22], South Africa [13], Tunisia [5], and Italy
[28]. These studies have focused mainly on the character-
ization of 2BMP (movement protein), and 2CCP (coat pro-
tein) genes. Phylogenetic analysis showed that the
sequenced GFLV isolates from Slovenia and Iran were
monophyletic, whereas the GFLV isolates from France,
Italy, and the United States formed several relatively
independent lineages, suggesting that differentiation had
occurred between geographical populations of GFLV [3].
Grapevine fanleaf virus diseases pose major threats to
the grapevine industry, and currently, the use of clean
propagation material is the only effective strategy for virus
disease control in viticulture. For this strategy to work
effectively, virus detection is essential. In addition to
enzyme-linked immunosorbent assay (ELISA), reverse
transcription-polymerase chain reaction (RT-PCR) assays
are the most commonly used technique for virus detection.
China is one of the worlds leading grape producers, with a
123

total production area of approximately 602,800 ha (FAO,
2012) mainly in the North, West, and East regions of
China, which share a major part of the grape production.
Although GFLV has been reported to be widespread in
other parts of the world, the prevalence of GFLV in China
has not yet been reported. In addition, sequence data of
GFLV isolates from China are limited. Therefore, in the
present study, we used ELISA, RT-PCR and nested RT-
PCR (RT-nPCR) to detect GFLV in grapevines from dif-
ferent regions of China and to characterize the genetic
variability of the GFLV isolates.
We collected 142 samples from 13 major grapevine-
growing areas, including the north-east (Liaoning Pro-
vince), north China (Hebei Province, and Beijing and
Tianjin regions), east China (Shangdong Province and
Shanghai region), west China (Xinjiang Uygur and Ningxia
Hui Autonomous Regions and Gansu and Sichuan Pro-
vinces), central China (Anhui Province) and south China
(Guangxi Zhuang Autonomous Region and Yunnan Pro-
vince). To preserve the virus isolates, these 142 samples
were planted in a greenhouse and protected with mesh
screening and were not transmitted by grafting. The dor-
mant canes from these samples were stored at 4 C until
used for double antibody sandwich ELISA (DAS-ELISA)
testing and RT-PCR detection. ELISA was done as
described previously [9] for detection of GFLV. An anti-
GFLV polyclonal serum (Agritest Srl, Valenzano, Italy)
and its alkaline phosphatase conjugate were used in the
DAS-ELISA, and the threshold for detection was twice the
average absorbance in three replicate tests of the healthy
controls.
Total RNA was extracted from the grapevines using the
adsorption column method described by MacKenzie et al.
[15] with some modifications. Briefly, approximately
50 mg of tissue samples was ground into fine powder and
homogenized in cracking buffer to release RNA. After
centrifugation of the homogenate, a half volume of anhy-
drous ethanol was added to the supernatant, and the mix-
ture was transferred to an adsorption column (Aidlab,
Beijing, China). The total RNA was washed, eluted from
the column with RNase-free water, and stored at -80 C
until used for RT-PCR.
To design degenerate primer sets that can be used to
amplify most of the isolates, RNA2 sequences of GFLVs
from Africa, Asia, Europe, and the United States were
retrieved from GenBank and aligned using MEGA 6.06
[27]. Four conserved sites in the GFLV MP (movement
protein) or CP (coat protein) domains were chosen for
primer design. The primers FL-MP1A (5-TCNACCAGA
GCCAATTACAC-3), FL-MP1B (5-GTGARCGRCTYC
TRATAGAG-3), FL-MPn1A (5-AGGCTYAATGGTAT
WCCGAC-3) and FL-MPn1B (5-KCKTGCYTCAGGV
GTTCCAG-3) corresponded to nucleotides 295-314,
123
J. Zhou et al.
951-970, 325-344 and 851-870, respectively, of the GFLV-
SACH44 RNA2 MP sequence. The primers FL-CP1A
(5-TAATYCGYTCTCCATATCACC-3), FL-CP1B (5-T
AGACTGGRAARCTGGTTCT-3), FL-CPn1A (5-CCW
GACYTMTCYYTRCCAAG-3) and FL-CPn1B (5-GGY
TTRCACAARACDCGGAG-3) corresponded to nucleo-
tides 846-865, 1495-1514, 1006-1025 and 1450-1469,
respectively, of the GFLV-SACH44 RNA2 CP sequence.
Viral isolates and accession numbers of the RNA2 MP- and
CP-encoding genes examined in this study are listed in
Tables S1 and S2.
The first-strand cDNAs were generated by reverse
transcription reactions with random hexamer primers.
Using the cDNA template, the first PCR amplification of
the MP and CP genes was carried out using rTaq DNA
polymerase (Takara, Dalian, China) with the newly
designed degenerate primers FL-MP1A/B and FL-CP1A/B.
The cycling profile was as follows: 94 C for 4 min, then
35 cycles of 94 C for 30 s, 54 C for 30 s, and 72 C for
50 s, followed by a final extension step at 72 C for
10 min. Amplification was conducted in a 96-well PCR
Thermal Cycler (Model PTC-200, Watertown, MA, USA).
Nested PCR reactions (25 lL) were performed using 1 lL
of the first PCR product with two primer pairs (FL-MPn1A/
B and FL-CPn1A/B) corresponding to GFLV MP and CP,
respectively. The expected lengths of the PCR products
were 546 bp (MP) and 464 bp (CP). All of the reaction
conditions for the nPCR were the same as those for the first
PCR, except that the number of cycles was reduced to 30.
The nPCRs products were gel-purified (Aidlab, Beijing,
China) and ligated into a pMD18-T vector (TaKaRa,
Dalian, China) according to the manufacturers instruc-
tions. The recombinant plasmids were introduced by
transformation into Escherichia coli DH5a cells, which
were made competent according to ref. [7]. At least three
independent clones in both orientations were then
sequenced, and a consensus sequence was derived when
the three independent clones showed C98 % identity, to
exclude in vitro RT-PCR errors. This consensus sequence
was termed the unique sequence. If the isolate showed
\98 % nucleotide sequence identity between the three
initially sequenced clones, additional clones were
sequenced to investigate the possible occurrence of mix-
tures of distinct sequence variants within individual
grapevines. A multiple nucleotide sequence alignment was
made from the obtained sequences and sequences in the
NCBI GenBank database (http://www.ncbi.nlm.nih.gov),
using BLAST.
Sequence identities were determined using ClustalW,
implemented in MegAlign (DNASTAR Lasergene v7.1.0
software package). Multiple sequence alignments were
performed using Clustal Omega (http://www.clustal.org/
omega/). Aligned sequences for MP and CP were scanned
Grapevine fanleaf virus in China

using seven recombination detection programs (RDP,
BOOTSCAN, SISCAN, SEQ, GENECONV, LARD and
MAXCHI) implemented in the program RDP 4.22 [14] to
detect putative recombination events. Phylogenetic trees
were constructed in MEGA 6.06 [27], using the maximum-
likelihood (ML) method with 1000 bootstrap replicates
Genetic differentiation and gene flow between popula-
tions were examined using three permutation-based statis-
tical tests, Ks*, Z*, and Snn [11], and by estimating Fst
(the absolute value of the standardized variance in allele
frequencies across populations). The absolute value of Fst
ranges from 0 to 1 for undifferentiated to fully differenti-
ated populations, respectively. Generally, an absolute value
of Fst [ 0.33 suggests infrequent gene flow, while an
absolute value of Fst \ 0.33 suggests frequent gene flow
[6, 10]. The statistical tests for genetic differentiation and
Fst estimation were performed using DnaSP 5.10 [12].
GFLV was detected in 54 out of 142 samples (38 %) by
DAS-ELISA (Table 1). Although only two samples (LN-
SDHN1 and LN-SDHN2) had tested positive for GFLV by
the first PCR using primer sets FL-MP1A/B and FL-CP1A/
B (Online Resource: Table S4), we found that 35 samples
(24.6 %) and 23 samples (16.2 %) tested positive for
GFLV by nested PCR using primer sets FL-MPn1A/B and
FL-CPn1A/B, respectively (Table 1). These results showed
that 60 of 142 samples (42.3 %) tested positive for GFLV
using one or more of the above detection methods. We
found that GFLV was distributed widely in seven major
grapevine-growing areas in China: Tianjin area (75 %),
Liaoning Province (72.6 %), Xinjiang area (37.5 %), and
Hebei Province (20 %), with lowest incidences in Shan-
dong Province (15.2 %), Beijing area (15.4 %) and
Ningxia area (12.5 %) (Table 1). GFLV was not detected
in samples from other areas (Table 1). Because of the high
prevalence (42.3 %) and wide distribution of GFLV in
grapevine-growing regions, the establishment of quarantine
facilities and the implementation of rigorous certification
schemes may be warranted.
Among the GFLV-positive samples identified by nPCRs
using FL-MPn1A/B or FL-CPn1A/B, 29 of the 35 samples
(83 %) and all 23 samples (100 %), respectively, tested
positive using DAS-ELISA (Online Resource: Table S4).
Overall, 32 of the 54 ELISA-positive samples tested positive
for GFLV by RT-nPCR, and six ELISA-negative samples
also tested positive by RT-nPCR (Online Resource:
Table S4). Moreover, among the 23 GFLV-positive samples
identified by nPCR using the FL-CPn1A/B primer pair, 20
of them were also positive using the FL-MPn1A/B primer
pair (Online Resource: Table S4). These results suggest that
the nPCR assays had high detection accuracy. Some of the
DNA fragments amplified from infected grapevines by
nPCR using the primer pairs FL-MPn1A/B and FL-CPn1A/
B, respectively, were analyzed by agarose gel elec-
trophoresis (data not shown). The results showed that FL-
MPn1A/B had a higher detection rate than FL-CPn1A/B and
that the detection effect was better. To understand the
genetic diversity of GFLV in China, the 34 and 22 PCR
products obtained in the nPCR assays using the GFLV-
specific primer pairs FL-MPn1A/B and FL-CPn1A/B,
respectively, were cloned and sequenced The sequencing
results showed that 39 MP and 22 CP sequences were
obtained in this study as unique sequences, and these
were selected for subsequent analyses (Online Resource:
Table S5). After removing the primer sequences, all of the

Table 1 Prevalence and distribution of GFLV in grapevines in China

Province/ No. of samples No. positive for GFLV No. positive for GFLV No. positive for GFLV
region tested (using nPCR with one or (including nPCR and
Primer pair Primer pair DAS-ELISA two sets primers) (%) ELISA detection) (%)
FLMPn1A/B FLCPn1A/B

Liaoning 62 24 22 43 27/62 (43.5) 45/62 (72.6)

Shongdong 33 4 1 4 4/33 (12.1) 5/33 (15.2)
Tianjin 4 3 0 2 3/4 (75) 3/4 (75)
Xingjiang 8 2 0 1 2/8 (25) 3/8 (37.5)
Beijing 13 1 0 2 1/13 (7.7) 2/13 (15.4)
Hebei, 5 1 0 1 1/5 (20) 1/5 (20)
Ningxia 8 0 0 1 0/8 (0) 1/8 (12.5)
Shanghai 4 0 0 0 0/4 (0) 0/4 (0)
Gansu 1 0 0 0 0/1 (0) 0/1 (0)
Anhui 1 0 0 0 0/1 (0) 0/1 (0)
Sichuan 1 0 0 0 0/1 (0) 0/1 (0)
Guangxi 1 0 0 0 0/1 (0) 0/1 (0)
Yunnan 1 0 0 0 0/1 (0) 0/1 (0)
Total 142 35 23 54 38/142 (26.8) 60/142 (42.3)

123
 J. Zhou et al.

LN-KSMS-1 LN-228-2
A LN-WMG B LN-SSMG-2
SD-GRX2-2 LN-ALHH1-3
LN-QM
HB-MSL LN-NX-3
LN-YBM LN-QB2-4
TJ-CXZ1 LN-QQWH-5
BJ-PLZ
LN-JFYN LN-SDHN2-12
SD-GRX1-1 LN-KSMS-2
LN-HFX-1 LN-ALHH2-5
LN-QQWH-3 LN-ZF-5
LN-GB-1
LN-LY LN-LN-BWSK
China
TJ-CXZ2 LN-HD-2
XJ-CXZ LN-DMN-2
LN-SSMG-1 LN-GB-2
LN-NX-1
LN-HD-1 LN-JZJ-3
LN-ZSX China LN-BJML-1
TJ-GRX LN-ZXWH-2
LN-QB2-2 100 LN-JMG2-1
LN-ZXWH-1
LN-HFS-1 SD-GRX2-4
100 SD-BBY-1 LN-HFX-2
LN-QB2-3 LN-SDHN1-3
LN-QQWH-2
94 LN-SDHN2-6 LN-HFS-2
81 LN-ALHH1-1 Bonab
82 LN-DMN-1 93 Urmia
LN-ALHH2-4
LN-ZF-1 100 Shir-Amin
84 XJ-ZXWH 100 KJ-18-2-2 Iran
LN-228-1 KJ-18-2-4
LN-JZJ-1 KH-12-2-3
LN-ZF-2
LN-SDHN2-9 91 KH-11-10-1
LN-SDHN2-7 C18c
LN-SDHN1-1 France
Tun1 Tunisia 98 C19a
99 AB21a 100 B10a
France
100 AB27a B6d
AB1a France 100 Ch FL162
AB12a Chile
Ch FL177
Sl1C 100 A40a
Vol51c3 90
100 Vol51c5 Slovenia 99
A18a
Sl1B France
A19a
Vol54c2 A16a
Hangzhou China 81 A6e
100 WAPN165
WACH911 American A2b
AB10a France B5a
AB8b 98
100
WAPN173 99 A32e France
WAPN8133 American A31b
99
100 SAPCS3 A14a
SACH44 South Africa 95 A17c
CAZINA5 American 96
CACSC2 American CACSC2
American
100 F13 99 CACSC1
94 Ch FLLA1 WACF2142 American
Ch FL1 Chile
SG1 Vol47c3
SG4 Italy Vol55c2 Slovenia
77 99 Ch FL177 99
99 Vol55c3
Ch FL162 Chile 100 B2a
Ch FL81 France
Ch FL1100 A22a
100 100 NP2 FA31
94 Italy
GFLV-NW Germany
SP2 100 NE185
95 Ch FLLA1
B19a
AB20a France Ch FL92
Chile
AB30a Ch FL4532
99
100 NE185
FA31 Italy 99 Ch FL4406
100 A17b C6d
France
France
A17a 99 C10b
Sl2 98 SAPCS3
Vol49c2 G2
Vol49c1 South Africa
Vol47c1 99 A1
100 Vol47c2 99 S2
Vol47c5 100 B5-7
Vol57c1 Slovenia Iran
Vol57c2 B5-5
Vol54c3 WAPN57
American
Sl1A
100 WAPN8133
87 Vol51c2 MOA4 393 Spain
Vol55c3
Vol55c2 C26b France
LGR12 100 A46b
99 LS2 France
34d
CACSB1 80 MMC 116
100
CAZINA3 99
95 La3-6-1 MMB34 116 Spain
100 La3-6-3 MMB2 116
X300-I1C1 CACSC4
La208-I1C3 American
97 Kh29-5 100 CACSC3
La208-I1C1 Hungary France
x300-I2C1 MOB4 393
100
99 Kj18-1 Iran Spain
Kj18-1-1 100 MOB5 393
100 KS-26
Kh4-2 Iran
100 Takestan KS-25
Bonab 100 JB3 429
94 Urmia Spain
Shir-Amin JB2 429
84 100 S33-2 99 Vol52c1
Slovenia
S32 Vol51c3
Kh12-7 D12
X400-I1C3 South Africa
98 X400-I2C1 100 D7
95

0.05 0.05

123
 Grapevine fanleaf virus in China
b Fig. 1 Maximum-likelihood phylogenetic trees based on nucleotide
sequences of MP (A) and CP (B) of Chinese and representative global
isolates of grapevine fanleaf virus. Bootstrap values were obtained
from 1,000 replications, and bootstrap values \75 % were collapsed.
Chinese isolates obtained in this study are indicated by their province
of origin (LN, Liaoning; HB, Hubei; TJ, Tianjin; SD, Shandong; BJ,
Beijing; XJ, Xinjiang) followed by the isolate name and a clone
number
cloned MP and CP sequences were 506 and 444 bp in
length, respectively. Sequence alignments showed that most
of the GFLV 2BMP sequences and all of GFLV 2CCP
sequences shared very high (up to 98.0 %) sequence simi-
larity. However, the cloned 2BMP sequences of isolate LN-
QQWH shared 96.499.8 % identity, the cloned sequences
of LN-ZF shared 96.699.8 % identity, and the cloned
sequences of LN-SDHN shared 95.899.8 % identity. These
results clearly show a slightly higher variability in the 2BMP
gene when compared with the 2CCP gene. We also found
that the nucleotide and amino acid sequence identities
between the different isolates from this study were relatively
high ([95 %) (data not shown), suggesting that they may be
variants. Species demarcation criteria in the genus Ne-
povirus, family Secoviridae are as follows: aa sequence of
CP with less than 75 % identity, aa sequence of conserved
Pro-Pol region (region between the conserved GC motif
in the protease and GDD motif in the polymerase) with
less than 80 % identity, differences in antigenic reactions,
host range and/or vector specificity [26]. When we aligned
the nucleotide and translated amino acid sequences from our
isolates and GFLV isolates downloaded from GenBank, we
found that the 2BMP nucleotide and amino acid sequences
shared 78.386.6 % and 89.396.4 % identity, respectively,
and the 2CCP nucleotide and amino acid sequences shared
74.883.3 % and 81.491.4 % identity, respectively.
Therefore, the Chinese GFLV isolates in this study were
considered different variants of these previously reported
isolates rather than belonging to a new species in the genus
Nepovirus. Because of the high degree of sequence identity
([95 %) of viral isolates in the analyzed samples, there are
no mixed infections of distinct sequence variants within
individual grapevines.
All virus-specific MP and CP sequences obtained in this
study and all previously reported GFLV MP and CP
sequences retrieved from GenBank (http://www.ncbi.nlm.
nih.gov) were used for recombination analysis, but no
recombinant sequences were found. Initially, two ML trees
were constructed using the obtained virus-specific MP and
CP sequences and all previously reported GFLV MP and
CP sequences retrieved from GenBank. To help make the
interpretation of the trees clearer, some closely related
sequences ([98 % nucleotide sequence identity) were
removed from each of the clusters for all subsequent
analyses. Finally, two ML trees were generated using 115
2BMP gene sequences (39 obtained in this study and 76
from GenBank) and 90 2CCP gene sequences (22 obtained
from this study and 68 from GenBank). In the 2BMP phy-
logenetic tree, the China GFLV isolates (from this study)
formed a separate subgroup, and all of the isolates from
Iran and most of the isolates from Slovenia formed distinct
clades, while the isolates from France, Italy, and the United
States apparently belonged to different lineages (Fig. 1A).
The phylogenetic tree based on the 2CCP nucleotide
sequences also showed that the China isolates clustered
together and formed a separate subgroup, like in the 2BMP
tree (Fig. 1B). However, in the 2CCP phylogenetic tree, the
isolates from Iran and Slovenia did not form a separate
cluster but belonged to different lineages, like the isolates
from France, Italy, and the United States (Fig. 1B). In any
case, the phylogenetic analysis showed a distinct evolu-
tionary relationship between the Chinese, Italian, French,
and United States populations. The sequenced GFLV iso-
lates from China were monophyletic, as described previ-
ously for the Slovenian isolates [3].
Ks*, Z*, and Snn tests were used to estimate the genetic
differentiation within and between GFLV populations from
China and other countries in the 2BMP and 2CCP genes. As
shown in Table 2, P-values of 0.0000 for all three tests
(Ks*, Z*, and Snn) and Fst values [0.33 were obtained
between the GFLV populations from China and other
countries, suggesting significant genetic differentiation and
infrequent gene flow among clusters. There appears to have
been infrequent gene flow between the GFLV populations
of China and other countries in the 2BMP gene and 2CCP
gene, possibly because of the large geographical distance.
The use of virus-free propagation material is the only
effective strategy for disease control in viticulture. Correct
diagnosis is essential for certified propagation material and
for the effective control of GFLV. ELISA and RT-PCR are
the most common and widely used techniques for routine
screening of pathogens. Serology-based detection systems
have to rely on the quality of the antisera available to detect
different strains that are constantly evolving. Therefore, the
detection accuracy of ELISA may be affected, so we have to
choose a more sensitive and reliable diagnostic RT-PCR
technique for detecting GFLV. However, we failed to
determine the prevalence and genetic variation of GFLV
using conventional RT-PCR assays as reported by Rowhani
et al. [24]. To improve GFLV detection, we established an
RT-nPCR protocol using degenerate primer pairs. The RT-
nPCR method uses the amplification products generated in
the first PCR as templates, which dramatically improved the
specificity and sensitivity of GFLV detection. However,
there were 22 ELISA-positive samples that gave no
amplification in the RT-nPCR. It is also worth noting that
all of the GFLV isolate sequences that were obtained
showed low variability and clustered in the same group. The
123
 J. Zhou et al.

Table 2 Gene flow and genetic differentiation of GFLV populations

Gene Region (number of sequences) Parametera
Ks* (P-valueb) Z* (P-valueb) Snn (P-valueb) Fst

Between Chinese and other country populationsc

2BMP gene China (n = 39) vs. America (n = 8) 2.24607 5.77649 1.00000 0.58110
China (n = 39) vs. Chile (n = 6) 2.15608 5.72333 1.00000 0.61011
China (n = 39) vs. France (n = 12) 2.34286 5.82314 1.00000 0.62203
China (n = 39) vs. Germany (n = 3) 1.98007 5.62564 1.00000 0.81028
China (n = 39) vs. Iran (n = 19) 2.56298 6.00404 1.00000 0.58908
China (n = 39) vs. Italy (n = 6) 2.17994 5.72985 1.00000 0.55099
China (n = 39) vs. Slovenia (n = 18) 2.16467 5.84285 1.00000 0.76521
China (n = 39) vs. South Africa (n = 2) 1.97114 5.63125 1.00000 0.89035
2CCP gene China (n = 39) vs. America (n = 7) 1.79090 4.68106 1.00000 0.71204
China (n = 39) vs. Chile (n = 6) 1.70649 4.64984 1.00000 0.72259
China (n = 39) vs. France (n = 23) 2.48125 5.12523 1.00000 0.70155
China (n = 39) vs. Iran (n = 11) 2.04765 4.81748 1.00000 0.66714
China (n = 39) vs. Italy (n = 2) 1.32163 4.48522 1.00000 0.94844
China (n = 39) vs. Spain (n = 8) 1.83040 4.68139 1.00000 0.70636
China (n = 39) vs. Slovenia (n = 5) 1.55229 4.57417 1.00000 0.74970
China (n = 39) vs. South Africa (n = 6) 1.60421 4.60358 1.00000 0.79252
* 0.01 \ P \ 0.05; ** 0.001 \ P \ 0.01; *** P \ 0.001
a
Ks*, Z* and Snn are the sequence-based statistics described by Hudson [11]. Fst is the interpopulation component of genetic variation of the
standardized variance in allele frequencies across populations. An absolute value of Fst \ 0.33 suggests frequent gene flow
b
P \ 0.05 was considered the criterion for rejecting the null hypothesis that there is no genetic differentiation between two subpopulations. P-
values of 0.0000 were obtained in all three tests (Ks*, Z*, and Snn), so *** is omitted in Table 2

presence of other GFLV variants in those samples that were
not detected by PCR may be one reason that more GFLV
isolates were found in this study by ELISA than by PCR.
Similar results were obtained when ELISA-positive GFLV
field isolates in Iran were not amplified by RT-PCR [2].
Although 38 out of 142 grapevine samples were found
to be infected with GFLV using RT-nPCR with two primer
pairs, only two samples (LN-SDHN1 and LN-SDHN2) had
tested positive for GFLV by conventional RT-PCR. Other
nepoviruses have also been detected using nested PCR [17,
29], suggesting that those viruses may not be detectable
when using a single (not nested) PCR. Because we
obtained more positive isolates using nested PCR, we think
that the low virus concentration in the tested samples might
be one of the main reasons for the failure to detect GFLV
in a single (not nested) PCR. We also tested 11 different
primers (Online Resource: Table S3), including five pub-
lished primer sets and six new primer sets, to detect the
presence of GFLV in some samples that tested positive for
GFLV using ELISA (data not shown). However, only one
RNA sample (LN-SDHN1) tested positive using the FL-
F1/R1 primer pair. Sequence comparisons showed imper-
fect complementarity between the partial primer sets and
the nucleotide sequences generated in the present study,
while the FL-F1/R1 primers showed perfect
complementarity with these sequences (data not shown).
This may explain why only the FL-F1/R1 primers suc-
cessfully detected GFLV. Therefore, the establishment of
more-sensitive and reliable diagnostic molecular tech-
niques for detecting GFLV requires improved primer
specificity and an optimized RNA extraction method.
In this study, using the newly determined 2BMP and 2CCP
gene sequences, we investigated the genetic diversity of
GFLV from distinct geographical regions in China and other
parts of the world. The obtained sequence data showed
conservation of the 2BMP and 2CCP gene sequences among
the GFLV isolates from distinct geographical regions of
China, but the China isolates appeared to be distinct from the
previously published GFLV isolates. This finding was
confirmed by phylogenetic analysis (Fig. 1) and gene flow
analysis (Table 2). The phylogenetic analysis showed a
distinct evolutionary relationship between the Chinese,
Italian, French, and United States populations. The
sequenced GFLV isolates from China were monophyletic,
as described previously for the Slovenia isolates [3]. All of
the Chinese GFLV populations were differentiated from
other populations, because the Ks* values were well above
zero (Table 2). This differentiation was confirmed by the
Z*, Snn, and Fst tests (Table 2). Success in the amplifica-
tion of the 2BMP and 2CCP gene sequences is an important

123
Grapevine fanleaf virus in China
achievement, because this opens up new avenues to design
new primers and identify more GFLV isolates from China.
The available nucleotide sequences of GFLV, also associ-
ated with the application of the latest developments in
molecular biology techniques, e.g., real-time RT-PCR,
loop-mediated isothermal amplification, etc., will be helpful
for detection of this virus. As more genome sequences are
determined in the future, it will be interesting to investigate
the extent of variation in viral genomic sequences and its
effects on variation in pathogenicity.
This study represents the first report of the prevalence
and distribution of GFLV in China and also provides a
sensitive RT-nPCR assay for detecting GFLV. In addition,
information about the genetic variability of Chinese iso-
lates is reported here for the first time. The results may help
improve regulatory measures, including quarantine, to
improve the sanitary status of planting material.
Acknowledgments This work was supported by the earmarked fund
of China Agriculture Research System (CARS-30-bc-3).
References
1. Andret-Link P, Laporte C, Valat L (2004) Grapevine fanleaf
virus: still a major threat to the grapevine industry. J Plant Pathol
86(3):183195
2. Bashir NS, Hajizadeh M (2007) Survey for Grapevine fanleaf
virus in vineyards of north-west Iran and genetic diversity of
isolates. Australas Plant Pathol 36:4652
3. Bashir NS, Melcher U (2012) Population genetic analysis of
Grapevine fanleaf virus. Arch Virol 157:19191929
4. Bertolini E, Garca J, Yuste A, Olmos A (2010) High prevalence
of viruses in table grape from Spain detected by real-time RT-
PCR. Eur J Plant Pathol 128:283287
5. Boulila M (2007) Phylogeny and genetic recombination of
Grapevine fanleaf virus isolates from naturally infected vineyards
in Tunisia. Phytopathol Mediterr 46:285294
6. Chen S, Zhou Y, Ye T, Hao L, Guo L, Fan Z, Li S, Zhou T (2014)
Genetic variation analysis of Apple chlorotic leaf spot virus coat
protein reveals a new phylogenetic type and two recombinants in
China. Arch Virol 159(6):14311438
7. Chung CT, Niemela SA, Miller RH (1989) One-step preparation
of competent Escherichia coli: transformation and storage of
bacterial cells in the same solution. Proc Natl Acad Sci USA
86:21722175
8. Fiore N, Zamorano A, Rivera L, Gonzalez F, Aballay E, Mon-
tealegre J, Pino AM (2011) Grapevine viruses in the Atacama
region of Chile. J Phytopathol 159:743750
9. Gambino G, Bondaz J, Gribaudo I (2006) Detection and elimi-
nation of viruses in callus, somatic embryos and regenerated
plantlets of grapevine. Eur J Plant Pathol 114:397404
10. Ge B, He Z, Zhang Z, Wang H, Li S (2014) Genetic variation in
potato virus M isolates infecting pepino (Solanum muricatum) in
China. Arch Virol 159(12):31973210
11. Hudson RR (2000) A new statistic for detecting genetic differ-
entiation. Genetics 155:20112014
12. Librado P, Rozas J (2009) DnaSP v5: a software for compre-
hensive analysis of DNA polymorphism data. Bioinformatics
25:14511452
13. Liebenberg A, Freeborough MJ, Visser CJ, Bellstedt DU, Burger
JT (2009) Genetic variability within the coat protein gene of
Grapevine fanleaf virus isolates from South Africa and the
evaluation of RT-PCR, DAS-ELISA and immunostrips as virus
diagnostic assays. Virus Res 142:2835
14. Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P
(2010) RDP3: a flexible and fast computer program for analysing
recombination. Bioinformatics 26:24622463
15. MacKenzie DJ, McLean MA, Mukerji S, Green M (1997)
Improved RNA extraction from woody plants for the detection of
viral pathogens by reverse transcription-polymerase chain reac-
tion. Plant Dis 81:222226
16. Mahfoudhi N, Harbi-Ben SM, Elair M, Selmi I, Ben Hamda H
(2014) Prevalence of viruses infecting autochthonous grapevines
in Tunisia. Tunisian J Plant Prot 9:111118
17. Maliogka V, Dovas CI, Efthimiou K, Katis NI (2004) Detection
and differentiation of Comoviridae species using a semi-nested
RT-PCR and a phylogenetic analysis based on the polymerase
protein. J Phytopathol 152:404409
18. Mekuria T, Martin RR, Naidu RA (2008) The occurrence of
Grapevine fanleaf Virus in Washington State Vineyards. In:
Proceedings of the 2nd annual national viticulture research con-
ference, July 911, University of California, Davis
19. Myles S, Boykob AR, Owens CL, Brown PJ, Grassi F, Aradhya
MK, Prins B, Reynolds A, Jer-Ming Chia, Ware D, Carlos D,
Bustamante, Buckler ES (2011) Genetic structure and domestica-
tion history of the grape. Proc Natl Acad Sci USA 108:35303535
20. Oliver JE, Vigne E, Fuchs M (2010) Genetic structure and
molecular variability of Grapevine fanleaf virus populations.
Virus Res 152:3040
21. Poljua D, Sladonja B, Bubola M (2010) Incidence of viruses
infecting grapevine varieties in Istria (Croatia). J Food Agric
Environ 8:166169
22. Pompe-Novak M, Gutierrez-Aguirre I, Vojvoda J, Blas M,
Tomazic I, Vigne E, Fuchs M, Ravnikar M, Petrovic N (2007)
Genetic variability within RNA2 of Grapevine fanleaf virus. Eur
J Plant Pathol 117:307312
23. Reynard JS, Gugerli P (2012) Current status of major grapevine
viruses in La Cote vineyards of Switzerland. In: 17th ICVG
Congress, Davis (US), pp 714
24. Rowhani A, Walker MA, Rokni S (1992) Sampling strategies for
the detection of Grapevine fanleaf virus and the grapevine strain
of Tomato ringspot virus. Vitis 31(1):3544
25. Sanfacon H, Wellink J, Le Gall O, Karasev A, van der Vlugt R,
Wetzel T (2009) Secoviridae: a proposed family of plant viruses
within the order Picornavirales that combines the families Se-
quiviridae and Comoviridae, the unassigned genera Cheravirus
and Sadwavirus, and the proposed genus Torradovirus. Arch
Virol 154:899907
26. Sanfacon H, Gorbalenya AR, Knowles NJ, Chen JP (2012) Order
Picornavirales. In: King AMQ, Adams MJ, Carstens EB,
Lefkowitz EJ (eds) Virus taxonomy. Classification and nomen-
clature of viruses. Ninth report of the international committee on
taxonomy of viruses. Elsevier Inc., Amsterdam, pp 835839
27. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013)
MEGA6: molecular evolutionary genetics analysis version 6.0.
Mol. Biol. Evol. 30(12):27252729
28. Terlizzi F, Pisi A, Beber R, Fiore N, Zamorano A, Credi R, Ratti
C (2015) Genetic variabitily of the movement and coat protein
genes of Grapevine fanleaf virus isolates from Italy. J Gen Plant
Pathol 81:6367
29. Wen WG, Cui JX, Sheng L (2007) Detection of Tobacco ringspot
virus and Tomato ringspot virus by semi-nested RT-PCR. ACTA
Phytophylacica Sinica 34(1):6165
123