Journal of General Virology (2009), 90, 1025–1034
DOI 10.1099/vir.0.006858-0
Genetic diversity and population structure of rice
stripe virus in China
Tai-Yun Wei,13 Jin-Guang Yang,13 Fu-Long Liao,1 Fang-Luan Gao,1
Lian-Ming Lu,1 Xiao-Ting Zhang,1 Fan Li,1,2 Zu-Jian Wu,1 Qi-Yin Lin,1
Lian-Hui Xie1 and Han-Xin Lin14
1
Correspondence
Institute of Plant Virology, Fujian Agricultural and Forestry University, Fuzhou, Fujian 350002,
PR China
Han-Xin Lin
[email protected]
2
Key Laboratory of Agricultural Biodiversity and Pest Management of Ministry of Education,
Yunnan Agricultural University, Kunming, PR China
or
[email protected]
Lian-Hui Xie
[email protected]
Received 26 August 2008
Accepted 29 December 2008
Rice stripe virus (RSV) is one of the most economically important pathogens of rice and is
repeatedly epidemic in China, Japan and Korea. The most recent outbreak of RSV in eastern
China in 2000 caused significant losses and raised serious concerns. In this paper, we provide a
genotyping profile of RSV field isolates and describe the population structure of RSV in China,
based on the nucleotide sequences of isolates collected from different geographical regions
during 1997–2004. RSV isolates could be divided into two or three subtypes, depending on
which gene was analysed. The genetic distances between subtypes range from 0.050 to 0.067.
The population from eastern China is composed only of subtype I/IB isolates. In contrast, the
population from Yunnan province (southwest China) is composed mainly of subtype II isolates, but
also contains a small proportion of subtype I/IB isolates and subtype IA isolates. However,
subpopulations collected from different districts in eastern China or Yunnan province are not
genetically differentiated and show frequent gene flow. RSV genes were found to be under strong
negative selection. Our data suggest that the most recent outbreak of RSV in eastern China was
not due to the invasion of new RSV subtype(s). The evolutionary processes contributing to the
observed genetic diversity and population structure are discussed.
INTRODUCTION
Due to their error-prone RNA replication, large population
size and short generation time, RNA viruses have high
mutation rates (Drake & Holland, 1999). Therefore, RNA
viruses exhibit high potential for genetic variation, and a
large number of nucleotide variations could exist in natural
populations. Analysing the polymorphic pattern of these
variations will help us to understand the phylogenetic
relationships, epidemiological routes, population structures and underlying evolutionary mechanisms of RNA
viruses. In turn, this information will facilitate the
development of effective control strategies for plant viral
diseases.
3These authors contributed equally to this paper.
4Present address: Department of Pathology and Molecular Medicine,
McMaster University, Hamilton, ON L8N 3Z5, Canada.
A supplementary table listing geographical location, collection year,
GenBank accession numbers and rice variety of RSV isolates, and
associated supplementary references are available with the online
version of this paper.
006858 G 2009 SGM
Rice stripe virus (RSV) is one of the most important plant
pathogens in China. The rice stripe disease induced by RSV
was first recorded in Jiangsu–Zhejiang–Shanghai (JZS)
district in 1963 and was later discovered in 16 provinces
(Lin et al., 1990). Outbreaks of this disease were reported
in JZS district in 1966, in Taiwan in 1969, in Yunnan
province in 1974, in Beijing in 1975, in Shandong in 1986
and in Liaoning in the early 1990s (Lin et al., 1990; Xie
et al., 2001). The disease is endemic in Yunnan, Jiangsu,
Shanghai, Shandong, Beijing and Liaoning provinces
(Fig. 1). Since 2000, the disease has circulated widely in
Jiangsu province and become more severe. For instance,
approximately 780 000 ha rice was infected in 2002 in
Jiangsu province. This increased to 957 000 ha in 2003 and
1 571 000 ha in 2004, accounting for 80 % of the rice fields
and a 30–40 % yield loss (http://www.hzag.gov.cn/bcjb/
200559154000.htm). The outbreak of RSV also spread to
adjacent provinces, such as Henan, Zhejiang, Anhui,
Shandong, Shanghai and Hebei. However, the reasons
behind this outbreak are not well understood. For example,
it is unclear whether the invasion was triggered by a new
RSV genotype. Outside China, RSV has been reported only
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T.-Y. Wei and others
Fig. 1. Maps of RSV sample-collection sites.
RSV samples used in this study were collected
from Liaoning, Beijing, Shandong, Henan,
Jiangsu, Zhejiang, Shanghai, Fujian and
Yunnan provinces in China. A more detailed
map of collection sites in Yunnan province is
also shown. Based on geographical proximity,
sites in Yunnan province were grouped into
five districts: BS (Baoshan–Shaba–Banqiao–
Xinjie–Hetu–Jiaguan),
DL
(Dali–Xizhou–
Fengyi–Weishan), CX (Chuxiong–Yongren–
Dayao–Yaoan),
KM
(Kunming–Luquan–
Wuding–Fumin–Yiliang–Shilin–Luliang) and
YX (Yuxi–Jiangchun).
in Japan, Korea and the Far East (ex-USSR) and has been
epidemic in Japan and Korea since the 1960s, causing
significant losses of rice yield (Hibino, 1996).
RSV is the type member of the genus Tenuivirus. It mainly
infects rice plants, but also infects some other species in the
family Poaceae, such as wheat and maize. RSV is
transmitted by a small brown planthopper, Laodelphax
striatellus Fallen (Hemiptera, Delphacidae), in circulative–
propagative and transovarial manners (Falk & Tsai, 1998).
The genome of RSV is composed of four negative-sense
single-stranded RNA segments, designated RNA 1, 2, 3 and
4 according to decreasing size (Fig. 2) (Takahashi et al.,
1993; Toriyama et al., 1994; Zhu et al., 1991, 1992). RNA 1
is of negative polarity, encoding the putative RNAdependent RNA polymerase (Toriyama et al., 1994). The
other three segments adopt an unusual ambisense coding
strategy, i.e. both the viral-sense RNA (vRNA) and viral
complementary-sense RNA (vcRNA) possess coding capacity, but the functions of the translated proteins are
unclear. What is known is that the vRNA 2 encodes a
membrane-associated protein and the vcRNA 2 encodes a
polyglycoprotein (Falk & Tsai, 1998; Takahashi et al.,
1993). The nucleocapsid (N) protein gene has been
mapped to vcRNA 3 (Kakutani et al., 1991; Zhu et al.,
1991), and the NS3 protein encoded by vRNA 3 could act
as an RNA-silencing suppressor based on the function of
the analogous NS3 of rice hoja blanca virus, another
member of the genus Tenuivirus (Bucher et al., 2003).
vRNA 4 encodes a protein known as major non-capsid
protein (NCP) that accumulates in infected plants and may
be involved in pathogenesis (Toriyama, 1986), whilst
vcRNA 4 encodes a protein that has recently been shown
to be involved in movement (Xiong et al., 2008).
Fig. 2. Schematic representation of the RSV genome. The lines
represent RNA segments and the empty boxes denote genes
encoded by the vRNAs (viral-sense RNAs) or vcRNAs (viral
complementary-sense RNAs). Oligonucleotide primers used for
RT-PCR of RSV genes are indicated by arrows. Pol, RNAdependent RNA polymerase.
Numerous studies have been performed in recent decades
on RSV aetiology, pathogenesis, ecology, molecular
biology, control strategies etc. (Falk & Tsai, 1998).
Nevertheless, there are still major gaps, especially in our
understanding of the genetic diversity and population
structure of RSV. Our previous studies have shown the
biological diversity and genetic variations of several RSV
isolates (Lin et al., 1999, 2001, 2002; Wei et al., 2003a, b).
In this report, we analysed five genes (NS2, N, NS3, NCP
and NSvc4) of 136 RSV isolates collected from different
areas in China during 1997–2004. Our data showed that
RSV isolates could be divided into two or three subtypes,
depending on which gene was analysed. The distribution of
these subtypes is correlated with their geographical
locations, but not with the collection years. All of the
isolates collected from eastern China and Japan belong to
subtype I/IB, whereas isolates from Yunnan province are
much more diverse, belonging to different subtypes with
subtype II being predominant.
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Journal of General Virology 90
Population structure of RSV
METHODS
Virus samples. RSV samples were collected in rice fields from eight
provinces (Fujian, Jiangsu, Shanghai, Zhejiang, Henan, Shandong,
Beijing and Liaoning) in eastern China and from Yunnan province in
southwest China during 1997–2004 (Fig. 1). A virus sample from an
individual rice plant was considered as one isolate. Infected rice plants
were either used for extraction of total RNA or stored at 280 uC for
future use. Some isolates were maintained on suitable rice varieties
(e.g. Hexi 28) via transmission by small brown planthoppers, L.
striatellus Fallen. The geographical locations, collection years and rice
varieties of RSV isolates used in this study are listed in Supplementary
Table S1 (available in JGV Online).
RT-PCR, cloning and sequencing. Extraction of total RNA from
infected rice leaves, cDNA synthesis and PCR amplification were
performed as described previously (Lin et al., 2001). Forward and
reverse primers were designed according to the nucleotide sequences
of RNAs 2, 3 and 4 of RSV isolate T (GenBank accession numbers
NC_003754, NC_003776 and NC_003753, respectively): 59ATGGCATTACTCCTTTTCAATG-39 (nt 81–102) and 59-CCAAATTCACATTAGAATAGG-39 (nt 666–686) for the NS2 gene; 59-GTTCAGTCTAGTCATCTGCAC-39 (nt 1437–1457) and 59-ACACAAAGTCTGGGT-39 (nt 2490–2504) for the N gene; 59-ACACAAAGTCCTGGGT-39 (nucleotides nt 1–15) and 59-CTACAGCACAGCTGGAGAGCTG-39 (nt 681–701) for the NS3 gene; 59-ACACAAAGTCCTGGG-39 (nt 1–15) and 59-GGTGGAAAATGTGATATGCAAT-39
(nt 597–616) for the NCP gene; and 59-TGGAACTGGTTATCTCACCT-39 (nt 1208–1228) and 59-ACACAAAGTCATGGC-39
(nt 2123–2137) for the NSvc4 gene.
RT-PCR products were purified by using a QIAquick PCR extraction
kit (Qiagen). The purified PCR products were inserted into the
pGEM-T vector (Promega) followed by transformation into
Escherichia coli DH5a. Nucleotide sequences were determined by
the Shanghai Jikang Biotech Company. For each gene of each isolate,
one or two clones were sequenced. The GenBank accession numbers
of these sequences are listed in Supplementary Table S1.
Phylogenetic analysis. Multiple nucleotide sequence alignments
were performed by using CLUSTAL W (Thompson et al., 1994).
Alignments were also adjusted manually to guarantee correct reading
frames. Non-coding sequences were removed before alignment. Eight
RSV isolates (T, M, O, C, Y, JS-YM, JSYD-05 and SD-JN2) that had
been sequenced by other laboratories were included as references
(Kakutani et al., 1990, 1991; Qu et al., 1997; Wang et al., 1992; Zhu et al.,
1991, 1992). Maize stripe virus (MStV), the most closely related virus to
RSV in the genus Tenuivirus, was used as outgroup for phylogenetic
analyses. The GenBank accession numbers for RNAs 2, 3 and 4 of MStV
are U53224, S40180 and AJ969410, respectively. All of the sequence
alignments used in this study are available upon request. Phylogenetic
trees were reconstructed by using the neighbour-joining (NJ) method
with the Kimura two-parameter model implemented in PAUP* 4.0b10.0
(Swofford, 2002). Gaps were treated as a fifth character state. Evaluation
of statistical confidence in nodes was based on 1000 bootstrap replicates.
Branches with ,50 % bootstrap value were collapsed.
Estimation of genetic distance and selection pressure. Genetic
distances (the average number of nucleotide substitutions between two
randomly selected sequences in a population) within and between
subtypes were calculated by MEGA 2.1 based on the Kimura twoparameter model (Kumar et al., 2001). The dn : ds ratio is used to
estimate selection pressure. dn (the average number of non-synonymous
substitutions per non-synonymous site) and ds (the average number of
synonymous substitutions per synonymous site) were estimated by
using the Pamilo–Bianchi–Li method implemented in MEGA 2.1. SEM
was computed by MEGA 2.1 using a bootstrap with 100 replicates.
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Statistical tests of genetic differentiation and measurement of
gene flow. Genetic differentiation between populations was
examined by three permutation-based statistical tests, Ks*, Z and
Snn, which represent the most powerful sequence-based statistical
tests for genetic differentiation and are recommended for use in cases
of high mutation rate and small sample size (Hudson, 2000; Hudson
et al., 1992). The extent of genetic differentiation or the level of gene
flow between populations was measured by estimating Fst (the
interpopulational component of genetic variation or the standardized
variance in allele frequencies across populations). Fst ranges from 0 to
1 for undifferentiated to fully differentiated populations, respectively.
Normally, an absolute value of Fst .0.33 suggests infrequent gene
flow. The statistical tests for genetic differentiation and estimation of
Fst were performed by DnaSP 4.0 (Rozas et al., 2003).
RESULTS
Genotype profile of RSV field isolates
To provide a genotype profile of RSV field isolates, we
performed phylogenetic analyses of RSV isolates collected
from different provinces in eastern and southwest China
during 1997–2004 (Fig. 1; Supplementary Table S1).
Neighbour-joining (NJ) trees were constructed by using
datasets for five RSV genes: NS2 (600 bp, 55 sequences),
NS3 (636 bp, 44 sequences), N (969 bp, 87 sequences),
NCP (543 bp, 65 sequences) and NSvc4 (861 bp, 33
sequences). As illustrated in Figs 3 and 4, RSV isolates
fell into two monophyletic clades. As all of the isolates
collected from eastern China form one of the monophyletic
clades, we refer to it as subtype I and to the other as
subtype II. The mean genetic distance between these two
subtypes ranges from 0.054 to 0.067, and those within
subtypes range from 0.008 to 0.038 (Table 1). Unlike the
polytomic topology of subtype I in the NS2 and NS3 gene
trees, subtype I in the NCP and NSvc4 gene trees is
dichotomic (Fig. 3). Noticeably, one of the sister clades
only contains isolates that were collected from Yunnan
province. We thereby refer to this clade as subtype IA, and
to the other as subtype IB (Fig. 3c, d). Indeed, the genetic
distances between subtypes IA, IB and II fall into a range
(0.050–0.060) similar to those between subtypes I and II
(Table 2). Therefore, for the NCP and NSvc4 genes, we
divided RSV isolates into three subtypes.
Spatial and temporal distribution of RSV isolates
in nature
To better understand the spatial and temporal distribution
patterns, the collection years and geographical locations of
RSV isolates corresponding to taxa in the phylogenetic
trees were depicted (information for the N gene tree is
shown in Fig. 4). Considering the geographical separation/
proximity, the RSV population in China was first divided
into two subpopulations, YN and E, containing isolates
collected from Yunnan province and eastern China,
respectively. RSV isolates collected from Yunnan province
were further grouped into five districts: Baoshan–Shaba–
Banqiao–Xinjie–Hetu–Jiaguan (BS), Dali–Xizhou–Fengyi–
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Journal of General Virology 90
Fig. 3. Phylogenetic analysis of four RSV genes. Trees were constructed by the NJ algorithm with the Kimura two-parameter model implemented in PAUP* 4.0b10.0. Branches
were collapsed when the bootstrap value was ,50 %. Bootstrap values are given above the branches of each clade. The trees were rooted by using maize stripe virus (MStV)
as the outgroup. Eight RSV isolates (T, M, O, C, Y, JS-YM, JSYD-05 and SD-JN2) that were sequenced by other laboratories were included as references and are underlined.
Taxa on grey backgrounds indicate the subtype I, IB and IA isolates that were collected in Yunnan province. Bars, number of substitutions per site. (a) NS2 gene; (b) NS3
gene; (c) NCP gene; (d) NSvc4 gene.
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Population structure of RSV
Fig. 4. Phylogenetic analysis of RSV isolates based on the N gene and their spatial and temporal distributions. The left panel
shows the N gene tree, constructed by the NJ algorithm with the Kimura two-parameter model implemented in PAUP* 4.0b10.0.
Branches were collapsed when the bootstrap value was ,50 %. Bootstrap values are given above the branches of each clade.
The tree was rooted by using MStV as the outgroup. RSV isolates (T, M, C, Y, JS-YM and SD-JN2) that were sequenced by
other laboratories were included as references and are underlined. Taxa on grey backgrounds indicate the subtype I, IB and IA
isolates that were collected in Yunnan province. Bar, 0.005 substitutions per site. In the right panel, the collection year and site
for each isolate are shown. Collecting sites in China fall into two geographically large categories, Yunnan (YN) province and
eastern China. There are five districts [Baoshan–Shaba–Banqiao–Xinjie–Hetu–Jiaguan (BS), Dali–Xizhou–Fengyi–Weishan
(DL), Chuxiong–Yongren–Dayao–Yaoan (CX), Kunming–Luquan–Wuding–Fumin–Yiliang–Shilin–Luliang (KM) and Yuxi–
Jiangchun (YX)] in YN province and six districts, Fujian, Jiangsu–Zhejiang–Shanghai, Henan, Shandong and Liaoning province
(FJ, JZS, HN, SD, BJ and LN, respectively), in eastern China.
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T.-Y. Wei and others
Table 1. Genetic distances within and between subtypes I and II for five genes
Genetic distance refers to the average number of nucleotide substitutions between two randomly selected sequences
in a population and was estimated by the program MEGA2 based on the Kimura two-parameter model. SEM was
calculated by using a bootstrap of 100 replicates. Subtypes are designated based on the phylogenetic analyses of RSV
in Figs 3 and 4.
Gene
NS2
NS3
N
NCP
NSvc4
Genetic distance
Within subtype I
Within subtype II
Between subtypes I and II
0.022±0.003
0.021±0.002
0.017±0.002
0.036±0.004
0.038±0.004
0.025±0.004
0.029±0.004
0.023±0.002
0.008±0.001
0.021±0.003
0.054±0.008
0.067±0.009
0.057±0.009
0.055±0.007
0.058±0.006
Weishan (DL), Chuxiong–Yongren–Dayao–Yaoan (CX),
Kunming–Luquan–Wuding–Fumin–Yiliang–Shilin–Luliang
(KM) and Yuxi–Jiangchun (YX). Those from eastern China
were further grouped into six districts: Fujian (FJ), Jiangsu–
Zhejiang–Shanghai (JZS), Henan (HN), Shandong (SD),
Beijing (BJ) and Liaoning (LN) (Figs 1, 4).
Two interesting patterns are seen in Fig. 4. Firstly, all of the
isolates collected from eastern China and Japan belong to
subtype I. Secondly, all of the subtype II isolates were
collected from Yunnan province. However, two isolates, Y
and CX2 (shown on grey backgrounds in Fig. 4), collected
from Yunnan province also belong to subtype I. Therefore,
isolates collected from Yunnan province are more diverse,
belonging to different subtypes. A similar genetic structure
was observed in the trees for the other four genes (Fig. 3).
We also examined the spatial distribution pattern of isolates
collected from different districts of eastern China and
Yunnan province, but failed to see any obvious pattern.
Despite examining the phylogenetic grouping of RSV
isolates and their collection years closely, we are unable to
find a general pattern as clear as that for spatial
distribution. However, we discovered that most of the
subtype IA isolates in the NCP and NSvc4 gene trees were
collected in 2004 (Fig. 3c, d).
Genetic differentiation between subpopulations
To test the genetic differentiation between and within
subpopulations E and YN, three statistical tests, Ks*, Z and
Snn, were used (Hudson, 2000; Hudson et al., 1992). As
shown in Table 3, all of the tests strongly support the
hypothesis that these two subpopulations are genetically
differentiated (P50). Although, for some genes, significant
genetic differentiation could also be observed within
subpopulations YN and E, it was not supported by all three
statistical tests. To estimate the extent of genetic differentiation, we measured the coefficient Fst, which is also an
estimate of gene flow. Except for the NS2 gene, the values of
Fst between subpopulations are all .0.33 (Table 3), an
indication of infrequent gene flow. The absolute values of Fst
for within subpopulations are all ,0.33, suggesting frequent
gene flow. This was also seen when Fst between any two
districts within YN or E was measured (data not shown).
Strong selective pressures acting on RSV genes
We also estimated the negative selection pressure acting on
RSV genes. Overall, the values of the dn : ds ratio for five
genes were markedly low (0.046–0.123; Table 4), implying
that all of these genes are under strong negative selection.
The value of the dn : ds ratio for the NS2 gene is slightly
higher than that for the N and NS3 genes, but is 2.6 times
higher than that of the NCP and NSvc4 genes. Therefore,
the NCP and NSvc4 genes are subject to stricter selective
constraints. Interestingly, the values of the dn : ds ratio for
genes on the same RNA segment, i.e. the NS3 and N genes
on RNA 3, and the NCP and NSvc4 genes on RNA 4, are
almost identical (Table 4). This suggests that the single
Table 2. Genetic distances within and between subtypes IA, IB and II of the NCP and NSvc4 genes
Subtypes are designated based on the phylogenetic analyses of RSV in Figs 3 and 4.
Gene
NCP
NSvc4
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Genetic distance
Within subtype IA
Within subtype IB
Within subtype II
0.025±0.004
0.012±0.002
0.025±0.004
0.024±0.003
0.008±0.001
0.021±0.003
Between IA and IB Between IA and II Between IB and II
0.051±0.008
0.055±0.006
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0.057±0.009
0.060±0.007
0.050±0.007
0.056±0.006
Journal of General Virology 90
Population structure of RSV
Table 3. Genetic differentiation between and within subpopulations YN and E
Subpopulation division is based on the geographical sites of origin of
RSV samples. The RSV population in China was first divided into two
subpopulations, Yunnan province (YN) and eastern China (E).
Subpopulation YN was further divided into five districts: Baoshan
(BS), Dali (DL), Chuxiong (CX), Kunming (KM) and Yuxi (YX) (Fig.
1). Subpopulation E was further divided into six districts: Liaoning
(LN), Beijing (BJ), Henan (HN), Shandong (SD), Jiangsu–Zhejiang–
Shanghai (JZS) and Fujian (FJ) (Fig. 1).
Gene
Test
Subpopulation
Between
YN and E
NS2
P-value
P-value
P-value
Fstd
NS3
P-value
P-value
P-value
Fst
N
P-value
P-value
P-value
Fst
NCP
P-value
P-value
P-value
Fst
NSvc4 P-value
P-value
P-value
Fst
of Ks*
of Z
of Snn
of Ks*
of Z
of Snn
of Ks*
of Z
of Snn
of Ks*
of Z
of Snn
of Ks*
of Z
of Snn
0.000D
0.000D
0.000D
0.310
0.000D
0.000D
0.000D
0.476
0.000D
0.000D
0.000D
0.604
0.000D
0.000D
0.000D
0.415
0.000D
0.000D
0.000D
0.435
Within YN Within E
0.510
0.767
0.353
20.048
0.098
0.189
0.030D
0.133
0.385
0.197
0.206
0.069
0.648
0.863
0.087
20.043
0.007D
0.008D
0.090
0.282
0.126
0.403
0.032D
20.010
0.232
0.066
0.033D
0.281
0.436
0.344
0.281
0.042
0.514
0.686
0.831
20.059
0.318
0.334
0.858
20.095
DP,0.05, which was considered as significantly rejecting the null
hypothesis that there is no genetic differentiation between two
subpopulations.
dFst is a coefficient of the extent of genetic differentiation and
provides an estimate of the extent of gene flow. An absolute value of
Fst.0.33 suggests infrequent gene flow.
RNA segments of the divided genome may be the
evolutionary unit of selection. With regard to selection
pressure acting on different subtypes, there is no general
pattern of which subtype is under stronger or weaker
selection pressure.
DISCUSSION
A number of methods have been used to differentiate virus
isolates in order to provide a genotyping basis for
examining the genetic composition of viral populations.
These include restriction fragment-length polymorphism
(RFLP; Arboleda & Azzam, 2000), PCR–single-strand
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conformation polymorphism (PCR-SSCP; Lin et al.,
2004), RNase-protection assays (Fraile et al., 1997) and
molecular phylogeny (Abubakar et al., 2003). In this paper,
we applied a distance-based NJ method to construct the
phylogenetic trees of RSV isolates collected from different
areas in China during 1997–2004. Overall, these isolates fell
into two monophyletic clades, namely subtypes I and II
(Figs 3, 4). However, the subtype I clade in the NSvc4 gene
tree is only poorly supported (51 % bootstrap value,
Fig. 3d). Furthermore, some subtype clades collapsed when
the character-based maximum-parsimony method was
used (data not shown). The low resolution of the
phylogenetic trees of RSV isolates could be due to the
low genetic diversity (0.05–0.067 between subtypes and up
to 0.092 between isolates) and insufficient informative sites
regarding evolution of the genome. Nevertheless, our
analyses provided a genotyping profile of RSV field isolates,
and this allowed us to investigate the genetic structure of
RSV populations in China.
Our data show that the population collected from eastern
China is composed only of subtype I or IB isolates. In
contrast, the population from Yunnan province is
composed of different subtypes, with subtype II predominating (Fig. 4). Subpopulations collected from different
districts in eastern China or Yunnan province are not
genetically differentiated and show frequent gene flow
(Table 3). Particularly, subtype I/IB isolates prevailing in
the outbreak sites (Jiangsu–Zhejiang–Shangha and Henan
districts) show high sequence identities to isolates collected
from other provinces in eastern China (Fig. 4). These data
indicate that the most recent outbreak of RSV in these
provinces was not due to the invasion of a new subtype(s)
from Yunnan province.
Although the prevailing genotype in Yunnan province is
subtype II, we noticed a recent expansion of a ‘new’
lineage, subtype IA. Subtype IA isolates were collected from
different districts in Yunnan province and mainly in the
year 2004 (Fig. 3c, d; Supplementary Table S1). In fact,
some of them already existed in nature before 2004. For
example, isolate CHUX was collected in 2001, and the
collection year of isolate Y was 1995 (Qu et al., 1997). It
would be interesting to see whether this subtype expands
further in the field and to compare the biological
properties of isolates of subtype IA with those of subtypes
IB and II.
The population structure described here for RSV in China
is distinct from that of cucumber mosaic virus (CMV), but
is somewhat similar to those of two insect vector-borne rice
viruses, rice tungro bacilliform virus (RTBV) and rice
yellow mottle virus (RYMV). The genetic composition of
11 CMV populations collected in Spain was not correlated
with their geographical locations and collection years, and
was described as metapopulation with local colonization,
extinction and recolonization (Fraile et al., 1997). In
contrast, RTBV populations also showed a spatial distribution pattern with a greater genetic diversity in the
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T.-Y. Wei and others
Table 4. Estimation of nucleotide diversity of five RSV genes
Nucleotide diversity, defined here as the average number of nucleotide substitutions per site, was computed separately for
non-synonymous sites (dn) and synonymous sites (ds). The dn : ds ratio gives an estimate of selection pressure; if
dn : ds,1.0, negative selection is implied.
Gene
NS2
NS3
N
NCP
NSvc4
Nucleotide diversity
dn
ds
dn : ds
dn
ds
dn : ds
dn
ds
dn : ds
dn
ds
dn : ds
dn
ds
dn : ds
Subtype IA*
Subtype I/IB
Subtype II
AllD
NA
0.006±0.001
0.058±0.008
0.103
0.010±0.002
0.052±0.006
0.192
0.004±0.001
0.047±0.006
0.085
0.008±0.002
0.065±0.012
0.123
0.007±0.002
0.067±0.012
0.104
0.009±0.002
0.073±0.014
0.123
0.006±0.001
0.083±0.013
0.072
0.008±0.002
0.064±0.008
0.125
0.004±0.001
0.021±0.006
0.190
0.004±0.001
0.061±0.009
0.066
0.013±0.003
0.105±0.015
0.123
0.014±0.003
0.139±0.019
0.100
0.011±0.003
0.111±0.012
0.099
0.007±0.002
0.151±0.025
0.046
0.007±0.001
0.149±0.015
0.047
NA
NA
NA
NA
NA
NA
NA
NA
0.003±0.002
0.090±0.017
0.033
0.004±0.001
0.032±0.006
0.125
*NA, Not applied. The subdivisions of subtype IA, IB and II were applied only to the NCP and NSvc4 genes.
DAll isolates were included in the analysis.
Indonesian population than in Philippine or Vietnamese
populations (Azzam et al., 2000). Similarly, the genetic
diversity of the RYMV population is highest in east Africa,
especially in eastern Tanzania, and decreases progressively
from the east to the west of Africa (Abubakar et al., 2003).
The geographical origin of a virus can be inferred from the
extent of its genetic diversity. If a viral population shows
higher genetic diversity, it is normally considered more
ancient (Azzam et al., 2000; Fargette et al., 2006; Gessain
et al., 1992; Giri et al., 1997; Koralnik et al., 1994; Moya &
Garcia-Arenal, 1995). The historical record of disease may
or may not be consistent with the extent of genetic
diversity. For example, the higher genetic diversity of the
RTBV Indonesian population is consistent with historic
records of rice tungro disease in 1840 in Indonesia, but this
disease was not reported in the Philippines until 1940 and
has been described in Vietnam only recently (Azzam et al.,
2000). Likewise, eastern Tanzania is believed to be the
centre of origin for RYMV, as the most divergent isolates
were found in this area (Abubakar et al., 2003; Fargette
et al., 2006). The virus was first reported in Kenya;
however, Kenya is located directly north-east of Tanzania
(Fargette et al., 2006). According to the population
structure described in our report, Yunnan province could
be the geographical origin of RSV in China. Although the
report of rice stripe disease in this province occurred
almost a decade after its discovery in Jiangsu–Zhejiang–
Shanghai district (Lin et al., 1990; Xie et al., 2001), the
1032
disease could have been unnoticed in Yunnan province for
many years. This is likely considering the fact that plant
virology research in Yunnan province greatly lagged behind
that in eastern China. The scenario of a Yunnan origin is
reinforced by the fact that this province is well known as
the ‘kingdom of plants and animals’ that may harbour
primary indigenous hosts and efficient insect vectors of
RSV. Analogously, Tanzania is also reputed to be a
biodiversity hotspot for plants and animals, and this rich
biodiversity has been proposed to account for the origin of
RYMV as well as another insect-borne plant virus, cassava
mosaic virus, in Africa (Fargette et al., 2006). Despite this
evidence, we cannot exclude the possibility that different
subtypes might have been present in eastern China a long
time ago, giving rise to subtype I/IB only in recent years.
Interestingly, although the occurrence of rice stripe disease
dates back to the early 1990s in Japan (Hibino, 1996), the
only three Japanese isolates (T, M and O) with available
nucleotide sequences belong to subtype I/IB (Figs 3, 4).
Apparently, more sequences, especially for Japanese and
Korean isolates, are needed to fully disclose the secrets of
the origin of RSV.
Why is the RSV Yunnan population composed of different
subtypes, whereas the eastern China population is only
composed of a single subtype? This can be explained simply
by the founder effect, which is often invoked to explain the
low genetic diversity of certain populations of various plant
viruses, including the RYMV population in western Africa
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Journal of General Virology 90
Population structure of RSV
(Fargette et al., 2006; Garcia-Arenal et al., 2001). However,
we believe that the interplay between RSV and its insect
vector, L. striatellus, could explain the observed population
structure of RSV in China more specifically. Unlike CMV
or RYMV, which can be transmitted by contact and several
different vectors (Fargette et al., 2006; Palukaitis et al.,
1992), RSV is strictly transmitted by L. striatellus in
persistent propagative and transovarial manners
(Toriyama, 1986). Therefore, the RSV–vector interaction
probably played a critical role in shaping the population
structure. It is generally believed that L. striatellus lacks the
ability to migrate long distances unless helped by a
monsoon and therefore has evolved to adapt to the local
climate (Hoshizaki, 1997). Therefore, it is possible that L.
striatellus populations in Yunnan and eastern China are
genetically differentiated and have different affinities for
RSV subtypes. The vector for the Yunnan population may
be favourable for the transmission of the predominant
subtype II isolates, whereas the vector for the eastern China
population is favourable for the subtype I/IB isolates. Such
biased transmissibility has been reported for other plant
virus–vector systems. For example, whitefly Bemisia tabaci
populations from various geographical locations had
different transmission efficiencies with different isolates
of tomato leaf curl geminivirus or other geminiviruses
(Bedford et al., 1994; McGrath & Harrison, 1995). This
scenario is supported by our unpublished data indicating
that four L. striatellus populations collected from Yunnan
province grouped together with similar random-amplified
polymorphic DNA (RAPD) patterns, and were distinct
from another group containing nine insect populations
collected from eastern China. The specific interaction
between RSV subtypes and insect vectors is being
investigated further in our laboratory.
The strict manner of insect-vector transmission, together
with the narrow host range of RSV, may explain the
observed low genetic diversity. Compared with the highly
variable CMV isolates (genetic distance up to 0.4 between
subgroup I and II isolates), RSV isolates show very low
genetic diversity (0.05–0.067 between subtypes and up to
0.092 between isolates). Similarly, compared with a list of
plant viruses (Chare & Holmes, 2004; Garcia-Arenal et al.,
2001), RSV genes are subjected to very strong negative
selection (Table 4). It has been shown that the genetic
diversities of several plant viruses were correlated with their
host ranges and controlled by virus–host interactions
(Schneider & Roossinck, 2000, 2001). In addition, plant
virus variability is believed to be constrained more by the
specificity of virus–vector interaction than by the specificity of virus–host plant interaction (Chare & Holmes, 2004;
Power, 2000). Considering the facts that (i) RSV only
infects rice and some species in the family Poaceae, whereas
CMV is able to infect more than 1000 species in 85 plant
families (Palukaitis et al., 1992), and (ii) RSV is transmitted
by L. striatellus in a persistent propagative manner, whereas
CMV can be transmitted by sap and different species of
aphids in a non-persistent manner, it is understandable
http://vir.sgmjournals.org
that RSV has low genetic diversity and is under strong
negative selection.
ACKNOWLEDGEMENTS
We are grateful to Bashan Huang, Jun Zheng, Kexian Zong, Yushan
Wang, Weimin Li, Dechun Fang, Lingeng Gong, Xiaomin Li, Wenwu
Shi, Yexiu Huang, Dunfan Fang, Yun Xu, Yunkun He, Yijun Zhou,
Zhaoban Chen, Maosheng Li, Xuelian Lai, Honglian Li, Dalin Shen,
Chongguan Pan and Guanghe Zhou for their help in collecting RSV
samples. We also thank Dr Bryce Falk and Dr Dustin Johnson for
their help with the writing. This work was supported by projects from
the National Natural Scientific and Technological Foundation of
China (items 39900091 and 30000002 to L.-H. X. and H.-X. L.,
respectively), Natural Scientific and Technological Foundation of
Fujian province (item B0110014 to H.-X. L.), the Ministry of
Education of China (item 020401 to H.-X. L.) and the Natural
Scientific and Technological Foundation of Yunnan province (item
2003C0042M to F. L.)
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