Journal of Integrative Agriculture 2016, 15(7): 16671671

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Complete nucleotide sequences of two isolates of Cherry virus A

from sweet cherry in China

GAO Rui, LI Shi-fang, LU Mei-guang

State Key Laboratory of Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural
Sciences, Beijing 100193, P.R.China

Abstract
Cherry virus A (CVA) is a member of the genus Capillovirus, in the family Betaflexiviridae. The infection rate of CVA was
high in sweet cherry in China. We determined the complete nucleotide sequences of two isolates of CVA from Taian, Shan-
dong Province, China using high fidelity PCR enzymes and specific primer pairs for amplifying long fragments in RT-PCR
and RACE. The full-length sequences from isolates ChTA11 and ChTA12 are both 7 382 nucleotide (nt) long, excluding
the poly(A) tail, encode two open reading frames (ORFs) and have similar genome organization to the two isolates in Gen-
Bank. The complete nucleotide sequence of ChTA11 is 98.2 and 81.2% nt identity to the isolates from Germany and India
in GenBank, respectively, and the ChTA12 isolate is 98.2 and 81.0% similar. Analysis of the nucleotide and amino acid
sequences showed that the domain of unknown function (DUF1717) is more variable compared with other domains. This
is the first report of the complete nucleotide sequences of CVA isolates infecting sweet cherry in China.

Keywords: Cherry virus A (CVA), genome sequence, Capillovirus

many, India, Italy, Japan, Canada, Poland, France and

1. Introduction
Cherry virus A (CVA) is a member of the genus Capillovi-
rus, in the family Betaflexiviridae. It was first described in
Germany from a sample of sweet cherry (Prunus avium)
(Jelkmann 1995). CVA has now been reported from many
different cherry-growing areas of the world, including Ger-
Received 2 September, 2015 Accepted 9 March, 2016
GAO Rui, E-mail: jiaoxiaoleng@163.com; Correspondence LU
Mei-guang, Tel: +86-10-62815615, E-mail: mglu@ippcaas.cn
2016, CAAS. Published by Elsevier Ltd. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/)
doi: 10.1016/S2095-3119(16)61343-6
China (Jelkmann 1995; James and Jelkmann 1998; Isogai
et al. 2004; Komorowska and Cieslinska 2004; Barone et al.
2006; Marais et al. 2008; Rao et al. 2009; Noorani et al.
2010; Marais et al. 2012). The virus has been detected in
sweet cherry (P. avium) (Marais et al. 2012), sour cherry
(Prunus cerasus) (Sabanadzovic et al. 2005), Japanese
flowering Kwanzan cherry (Prunus serrulata) (Eastwell and
Bernardy 1998), and also in noncherry hosts such as apricot
and peach (James and Jelkmann1998; Barone et al. 2006),
plum (Svanella-Dumas et al. 2005) and Japanese apricot
(Prunus mume) (Marais et al. 2008).
At present, there is no clear evidence for any association
between CVA infection and specific disease symptoms in
any of its hosts, but the situation is often complicated by
the frequent occurrence in woody hosts of mixed infections
with other viruses such as Little cherry virus 1 (LChV-1) or
Little cherry virus 2 (LChV-2), Prune dwarf virus (PDV),
1668 GAO Rui et al. Journal of Integrative Agriculture 2016, 15(7): 16671671
Plum bark necrosis and stem pitting-associated virus
(PBNSPaV), Apple chlorotic leaf spot virus (ACLSV), Apricot
pseudo chlorotic leaf spot virus (APCLSV), Cherry green
ring mottle virus (CGRMV) (Jelkmann 1995; Eastwell and
Bernardy 1998; Isogai et al. 2004; Sabanadzovic et al. 2005;
Svanella-Dumas et al. 2005; Barone et al. 2006). It is still not
known whether CVA can produce severe disease symptoms
when presents in mixed infections; rather in some cases of
mixed infections where CVA is a co-infecting virus, severe
disease symptoms are observed (Eastwell and Bernardy
1998; James and Jelkmann 1998; Marais et al. 2012).
The genome of the reference CVA isolate is a single
stranded, positive-sense RNA molecule of 7 383 nt, ex-
cluding the poly(A) tail (Jelkmann 1995), with two open
reading frames (ORFs). Presently, only two complete nu-
cleotide sequences of CVA are deposited in GenBank. One
(X82547.1) was determined from P. avium variety Sam in
Germany (Jelkmann 1995), while the second (FN691959)
was determined from P. avium variety Manigam in India
(Noorani et al. 2010). CVA was initially detected in P. mume
and P. avium in China (Marais et al. 2008; Rao et al. 2009).
The infection rate of CVA in China was found to be 60.8%
(Wang et al. 2013) and 60.0% (Lu et al. 2015), respectively,
similar to other countries, such as India (58%) (Noorani
et al. 2015), Germany (40%) (James and Jelkmann 1998)
and Japan (92%) (Isogai et al. 2004). This study provides
the first complete nucleotide sequences of CVA isolates
from P. avium variety Hongdeng and Tieton in China. This
information will provide the basis for further studies on the
molecular evolution of CVA.
2. Materials and methods
2.1. Virus isolate and RNA extraction
In this study, complete nucleotide sequences were de-
termined for CVA isolates recovered from sweet cherry
in Taian, Shandong Province, China; these are isolate
ChTA11, from sweet cherry variety Hongdeng, and isolate
ChTA12, from sweet cherry variety Tieton. Total RNA was
extracted using the RNAprep Pure Plant Kit (Polysac-
charide- & Polyphenolic-rich) (Tiangen, Beijing, China),
from 60 sweet cherry leaf samples either with or without
symptoms that were collected from Taian and Yantai cities
in Shandong Province, Dalian in Liaoning Province, and
Beijing. RT-PCR was used for the detection of CVA using
primer pair CVA-F (5-ATGCTTCGCAGGTGACGATA-3)
and CVA-R (5-GCTTGTTTGTGGAGGGAGAC-3) (Lu
et al. 2015). Two leaf samples (ChTA11 and ChTA12) that
were infected with CVA were selected for amplification of
the entire CVA genome.
2.2. Genome cloning
Complementary DNA (cDNA) was synthesized using a Re-
verse Transcription (RT) Kit (Promega, Madison, WI USA).
Two pairs of PCR primers (Appendix A) were designed
based on the reference sequence of a CVA isolate from Gen-
Bank (accession no. X82547.1). Sequences of the 3-ter-
minal region were amplified using oligo(dT) and specific
primers, and sequences from the 5-terminal region were
amplified using the 5-Full RACE Kit with TAP (TaKaRa, Bei-
jing, China) (Appendix A). The concentration of all primers
was 20 mol. Reverse transcription was performed at 42C
for 1 h using 1 L of total RNA and 1 L of oligo(dT) primer in
a 10 L reaction volume with Maloney murine leukemia virus
(M-MLV) reverse transcriptase (Promega, Madison, WI,
USA), according to the manufacturers protocol. PCR was
performed in a 50 L volume reaction mixture containing 25
L of 2Phanta buffer, 2 L of each primer (20 mol L1), 3 L
cDNA and 2 L Phanta Max Super-Fidelity DNA polymerase
(Vazyme, Nanjing, China). The PCR profile employed for all
primer sets consisted of an initial denaturation at 95C for
5 min followed by 35 cycles of 95C for 15 s, 54C or 65C
for 30 s, 72C for 4 min, and a final extension for 10 min at
72C. Amplified products were purified using the Wizard SV
Gel and PCR Clean-Up System (Promega, Beijing, China),
and the resulting fragments were cloned into the CE entry
vector (Vazyme, Nanjing, China). The recombinant DNA
was used to transform Escherichia coli DH5 cells.
2.3. Sequence analysis
The nucleotide (nt) and deduced amino acid (aa) sequenc-
es were compared to other CVA sequences published in
GenBank using DNAMAN 6.0 (Lynnon Biosoft, Quebec,
Canada). Phylogenetic trees were constructed using the
maximum likelihood method with 1 000 bootstrap replicates,
as implemented in the molecular evolutionary genetics
analysis (MEGA) software (ver. 5.1).
3. Results
The full-length sequences of CVA from the ChTA11 and
ChTA12 isolates are both 7 382 nt in length, excluding the
poly(A) tail. The two sequences were submitted to GenBank
with accession nos. KT285841 and KT310083. As shown in
Table 1, the ChTA11 and ChTA12 genomes share 99.5% se-
quence identity with each other. The ChTA11 isolate is 98.2
and 81.2% homologous at the nucleotide level to the CVA
isolates from Germany and India, respectively; the ChTA12
isolate is 98.2 and 81.0% similar to the CVA isolates from
Germany and India at the nucleotide level. The genome
 GAO Rui et al. Journal of Integrative Agriculture 2016, 15(7): 16671671 1669

Table 1 Comparison of the complete genome sequences of ChTA11 and ChTA12 and the different genomic regions with the
Cherry virus A (CVA) isolates from Germany (X82547.1) and India (FN691959) deposited in GenBank
Genome 5 UTR1) MT2) DUF17173) Hel4) RdRp5) CP6) MP7) 3 UTR1)
nt aa nt aa nt aa nt aa nt aa nt aa
(nt, %) (nt, %) (nt, %)
(%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)
ChTA11/Germany 98.2 98.2 98.8 99.4 96.7 100 96.7 100 97.3 97.0 99.2 100 98.3 100 95.6
ChTA12/Germany 98.2 98.2 98.6 99.0 97.0 100 97.0 100 97.3 97.0 99.2 98.5 98.2 100 99.3
ChTA11/India 81.2 90.7 81.7 92.2 70.6 77.2 81.1 91.7 81.4 93.7 88.1 99.0 91.9 98.5 92.3
ChTA12/India 81.0 90.7 81.3 91.9 70.3 77.2 81.1 91.7 81.4 93.7 88.0 98.5 91.5 98.5 92.3
ChTA11/ChTA12 99.5 100 99.4 99.7 99.0 100 99.5 100 100 100 99.8 99.5 99.7 100 95.7
India/Germany 81.1 92.6 82.0 91.9 74.5 77.2 80.9 90.0 79.8 93.7 88.5 95.3 91.0 98.5 91.3
1)
UTR, untranslated region.
2)
MT, viral methyl transferase.
3)
DUF1717, a domain of unknown function.
4)
Hel, helicase.
5)
RdRp, RNA-dependent RNA polymerase.
6)
CP, coat protein.
7)
MP, movement protein.

A ORF 1

MT DUF1717 Hel RdRp CP

Poly(A)
MP
5 RACE 872
ORF 2
511 3 372
2 892 7 182

7 124 Oligo(dT)

B
CVA (FN691959.1)
100
CVA (X82547.1)
CVA
100 CVA (ChTA11)
100 Capillovirus
98 CVA (ChTA12)

CTLV (JQ765412.1)
100 ASGV (KJ579253.1)

100 ACLSV (KM207212.1)

100 CMLV (NC_002500.1)

CLBV (JN983456.1)

CRMaV (KF030870.2)
100
CNRMV (KF030832.2)
100
93 CTLaV (NC_024449.1)

CGRMV (NC_001946.1)
100
ASPV (KF321966.1)

Fig. 1 A, diagram of the genomic structure of the two Cherry virus A (CVA) isolates from China. Numbers are the nucleotide
positons of the PCR primers used in amplification. B, phylogenetic analysis based on the complete genome sequences of CVA
isolates ChTA11 and ChTA12 from sweet cherry in China and two previously reported CVA isolates from sweet cherry. Sequences
of Apple chlorotic leaf spot virus (ACLSV) and Cherry mottle leaf virus (CMLV) in the genus Trichovirus; Apple stem grooving virus
(ASGV) and Citrus tatter leaf virus (CTLV) in the genus Capillovirus; Apple stem pitting virus (ASPV) in the genus Foveavirus; Cherry
green ring mottle virus (CGRMV); Cherry rusty mottle associated virus (CRMaV); Cherry necrotic rusty mottle virus (CNRMV) and
Cherry twisted leaf associated virus (CTLaV) in the genus unassigned Betaflexiviridae; and Citrus leaf blotch virus (CLBV) in the
genus Citrivirus were also used to construct the phylogenetic tree. The two CVA genome sequences from this study are indicated
by . Bootstrap values below 60% are not shown.
1670 GAO Rui et al. Journal of Integrative Agriculture 2016, 15(7): 16671671
structures of the two isolates are identical to those of the
German and Indian CVA isolates in GenBank, and consist-
ed of two ORFs, except for 3 untranslated region (UTR)
(Fig. 1-A). The lengths of 3 UTR are 300 nt in German
isloate, 296 nt in Indian isolate and 299 nt in two Chinese
isolates. ORF1 (557 083 nt) encodes a polyprotein. Five
domains were identified: the viral methyl transferase (MT),
a domain of unknown function (DUF1717), helicase (Hel),
the RNA-dependent RNA polymerase (RdRp), and the coat
protein (CP). ORF2 (5 4006 791 nt) encodes a putative
movement protein (MP) (Noorani et al. 2010). In general,
the DUF1717 domain is more variable compared with other
domains, and the three isolates (Germany, ChTA11 and
ChTA12) have 70.374.5% nt and 77.2% aa sequence
identities overall to the India isolate. The CP and MP
genes appear to the more conserved, sharing 98.5100%
aa identity among the four CVA isolates.
Phylogenetic analysis was conducted on the complete
genomic sequences of four CVA isolates, including those
from this study; ACLSV and Cherry mottle leaf virus (CMLV)
in the genus Trichovirus, Apple stem grooving virus (ASGV)
and Citrus tatter leaf virus (CTLV) in the genus Capillovirus;
Apple stem pitting virus (ASPV) in the genus Foveavirus;
CGRMV; Cherry rusty mottle associated virus (CRMaV),
Cherry twisted leaf associated virus (CTLaV) and Cherry ne-
crotic rusty mottle virus (CNRMV) in the genus unassigned
Betaflexiviridae; and Citrus leaf blotch virus (CLBV) in the
genus Citrivirus. The analysis showed that all isolates of
CVA group into a single, well-supported cluster, which in-
cludes the two CVA isolates from Germany and India. The
two CVA isolates from this study are more closely related to
the isolate from Germany than they are to the isolate from
India (Fig. 1-B). For analysis of the relationship between
TA11, TA12 and other CVA isolates, phylogenetic analysis
based on partial CP gene sequences of two CVA isolates in
this study and 19 previously reported CVA isolates in Gen-
Bank was performed. This analysis revealed that four CVA
isolates (TA11, TA12, X82547.1 and AB181355) were in a
single cluster and far from the isolate from India (FN691959)
(Appendix B). The result of phylogenetic analysis based on
partial CP gene sequences was similar to the result based
on the full genomic sequences.
4. Discussion
CVA is frequent in sweet and sour cherry and it could have
a worldwide distribution in these hosts (Marais et al. 2011).
In China, CVA was firstly detected respectively in P. mume
in Jiangsu Province and in P. avium in Yunnan Province
(Marais et al. 2008; Rao et al. 2009). Later on, leaf samples
of sweet cherry variety Hongdeng were collected and de-
tected from 11 orchards around Bohai Bay and the infection
rate of CVA in China was found to be 60.8% (Wang et al.
2013). Recently, Lu et al. (2015) investigated virus diseases
in eight orchards or cultivated areas in Shandong, Liaoning
provinces and Beijing of China and found that the infection
rate of CVA in sweet cherry was 60% and some symptoms
of shrivel, deformation, mosaic, yellow and green mottle
and necrosis ringspot were observed when mixed infections
with other viruses. However, the genomic sequence of CVA
was only reported in Germany and India. It is necessary to
understand the genomic features of CVA from China. The
results showed the ChTA11 and ChTA12 isolates shared
high nucleotide sequence and amino acid sequence iden-
tity with the isolate from Germany. The complete genome
sequences reported here provides additional information on
genome sequence information of CVA from different geo-
graphical locations and will form the basis for development
of molecular diagnostics and development a more effective
disease control strategy of CVA in China.
Whats more, we used six primers to amplify short
fragments in order to obtain the full-length sequences in
the beginning (data not shown). However, we found many
mutations in the overlapping regions of two neighbored frag-
ments. To ensure the accuracy of the genome sequences,
in this study, we designed two pair new primers to amplify
longer fragments and the final full-length CVA genomic se-
quences were assembled from two long fragments amplified
with Phanta MAX Super-Fidelity DNA polymerase. It is
important to ensure the accuracy of the genome sequenc-
es using a high fidelity PCR enzymes and primer pairs to
amplify long fragments.
5. Conclusion
This study provides the first complete nucleotide sequences
of CVA isolates from sweet cherry in China. The CVA iso-
lates from China were found to be phylogenetically closest to
the isolate from Germany. This information will provide the
basis for further studies on the molecular evolution of CVA.
Acknowledgements
This work was supported by the Special Fund for Agro-sci-
entific Research in the Public Interest, China (201203076)
and the National Natural Science Foundation of China
(31471752).
Appendix associated with this paper can be available on
http://www.ChinaAgriSci.com/V2/En/appendix.htm
References
Barone M, Alioto D, Marais A, Candresse T, Ragozzino A. 2006.
 GAO Rui et al. Journal of Integrative Agriculture 2016, 15(7): 16671671
First report and high prevalence in noncherry host of Cherry
virus A in Italy. Plant Disease, 90, 1459.
Eastwell K C, Bernardy M G. 1998. Relationship of Cherry virus
A to little cherry disease in British Columbia. In: Proceedings
of the 17th International Symposium on Virus and Virus-Like
Diseases of Temperate Fruit Crops: Fruit Tree Diseases,
472, 305313.
Isogai M, Aoyagi J, Nakagawa M, Kubodera Y, Satoh K, Katoh
T, Inamori M, Yamashita K, Yoshikawa N. 2004. Molecular
detection of five cherry viruses from sweet cherry trees in
Japan. Journal of General Plant Pathology, 70, 288291.
James D, Jelkmann W. 1998. Detection of Cherry virus A
in Canada and Germany. In: Proceedings of the 17th
International Symposium on Virus and Virus-Like Diseases
of Temperate Fruit Crops: Fruit Tree Diseases, 472,
299303.
Jelkmann W, 1995. Cherry virus A - cDNA cloning of dsRNA,
nucleotide sequence analysis and serology reveal a new
plant capillovirus in sweet cherry. Journal of General
Virology, 76, 20152024.
Komorowska B, Cieslinska M. 2004. First report of Cherry virus
A and Little Cherry virus-1 in Poland. Plant Disease, 88, 909.
Lu M G, Wu B, Gao R, Zhang Z X, Xiao H, Chen R R, Li S F.
2015. Preliminary investigation on cherry virus diseases and
detection the pathogens in some regions of China. Plant
Protection, 41, 98103. (in Chinese)
Marais A, Faure C, Svanella-Dumas L, Candresse T. 2008.
First report of Cherry virus A in Prunus mume in China.
Plant Disease, 92, 1589.
Marais A, Candresse T, Svanella-Dumas L, Jelkmann W.
2011. Cherry virus A. In: Hadidi A, Barba M, Candresse T,
1671
Jelkmann W, eds., Virus and Virus-Like Diseases of Pome
and Stone Fruits. APS Press, USA. pp. 147150.
Marais A, Svanella-Dumas L, Barone M, Gentit P, Faure C,
Charlot G, Ragozzino A, Candresse T. 2012. Development
of a polyvalent RT-PCR detection assay covering the
genetic diversity of Cherry capillovirus A. Plant Pathology,
61, 195204.
Noorani M S, Awasthi P, Singh R M, Ram R, Sharma M P,
Singh S R, Ahmed N, Hallan V, Zaidi A A. 2010. Complete
nucleotide sequence of Cherry virus A (CVA) infecting
sweet cherry in India. Archives of Virology, 155, 20792082.
Noorani M S, Awasthi P, Sukapaka M, Singh L, Ram R, Sharma
M P, Zaidi A A, Hallan V. 2015. Immunodiagnostics for
Cherry virus A and Cherry necrotic rusty mottle virus.
Journal of Plant Biochemistry and Biotechnology, 24,
93104.
Rao W L, Zhang Z K, Li R. 2009. First report of Cherry virus
A in sweet cherry trees in China. Plant Disease, 93, 425.
Sabanadzovic S, Abou-Ghanem-Sabanadzovic N, Rowhani A,
Grant J A, Uyemoto J K. 2005. Detection of Cherry virus
A, Cherry necrotic rusty mottle virus and Little cherry virus
1 in California orchards. Journal of Plant Pathology, 87,
173177.
Svanella-Dumas L, Marais A, Gentit P, Lamorte J, Candresse
T. 2005. First report on the natural occurrence of Cherry
virus A in mirabelle plum (Prunus domestica var. insititia).
Plant Disease, 89, 433.
Wang W W, Zong X J, Wang J W, Wei H R, Xu L, Chen X, Liu
J W, Liu Q Z. 2013. Identification and investigation of Little
cherry virus and Cherry virus A on sweet cherry around
Bohai bay. Plant Protection, 39, 128133. (in Chinese)
(Managing editor ZHANG Juan)