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Complete nucleotide sequences of two isolates of Cherry virus A from sweet cherry in China
Journal of Integrative Agriculture 2016, 15(7): 1667–1671 Available online at www.sciencedirect.com
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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 Tai’an, Shandong 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 GenBank. 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
1. Introduction Cherry virus A (CVA) is a member of the genus Capillovirus, 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: [email protected]; Correspondence LU Mei-guang, Tel: +86-10-62815615, E-mail: [email protected] © 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
many, India, Italy, Japan, Canada, Poland, France and 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),
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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, excluding the poly(A) tail (Jelkmann 1995), with two open reading frames (ORFs). Presently, only two complete nucleotide 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.
2.2. Genome cloning
2. Materials and methods
The nucleotide (nt) and deduced amino acid (aa) sequences 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).
2.1. Virus isolate and RNA extraction In this study, complete nucleotide sequences were determined for CVA isolates recovered from sweet cherry in Tai’an, 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 (Polysaccharide- & Polyphenolic-rich) (Tiangen, Beijing, China), from 60 sweet cherry leaf samples either with or without symptoms that were collected from Tai’an 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.
Complementary DNA (cDNA) was synthesized using a Reverse 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 GenBank (accession no. X82547.1). Sequences of the 3´-terminal 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, Beijing, China) (Appendix A). The concentration of all primers was 20 μmol. Reverse transcription was performed at 42°C for 1 h using 1 μL of total RNA and 1 μL of oligo(dT) primer in a 10 μL reaction volume with Maloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA), according to the manufacturer’s protocol. PCR was performed in a 50 μL volume reaction mixture containing 25 μL of 2×Phanta buffer, 2 μL of each primer (20 μmol L–1), 3 μL cDNA and 2 μL Phanta Max Super-Fidelity DNA polymerase (Vazyme, Nanjing, China). The PCR profile employed for all primer sets consisted of an initial denaturation at 95°C for 5 min followed by 35 cycles of 95°C for 15 s, 54°C or 65°C for 30 s, 72°C for 4 min, and a final extension for 10 min at 72°C. 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 DH5α cells.
2.3. Sequence analysis
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% sequence 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
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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)
(nt, %)
(nt, %)
ChTA11/Germany ChTA12/Germany ChTA11/India ChTA12/India ChTA11/ChTA12 India/Germany
98.2 98.2 81.2 81.0 99.5 81.1
98.2 98.2 90.7 90.7 100 92.6
MT2) nt aa (%) (%) 98.8 99.4 98.6 99.0 81.7 92.2 81.3 91.9 99.4 99.7 82.0 91.9
DUF17173) nt aa (%) (%) 96.7 100 97.0 100 70.6 77.2 70.3 77.2 99.0 100 74.5 77.2
Hel4) RdRp5) nt aa nt aa (%) (%) (%) (%) 96.7 100 97.3 97.0 97.0 100 97.3 97.0 81.1 91.7 81.4 93.7 81.1 91.7 81.4 93.7 99.5 100 100 100 80.9 90.0 79.8 93.7
CP6) nt aa (%) (%) 99.2 100 99.2 98.5 88.1 99.0 88.0 98.5 99.8 99.5 88.5 95.3
MP7) 3´ UTR1) nt aa (nt, %) (%) (%) 98.3 100 95.6 98.2 100 99.3 91.9 98.5 92.3 91.5 98.5 92.3 99.7 100 95.7 91.0 98.5 91.3
1)
UTR, untranslated region. 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. 2)
ORF 1
A MT 5´ RACE
DUF1717
Hel
CP
RdRp
Poly(A)
MP
872
ORF 2 3 372
511
7 182
2 892 7 124 B
CVA (FN691959.1)
100
CVA (X82547.1) CVA (ChTA11)
100
100
Oligo(dT)
98
CVA (ChTA12)
CVA Capillovirus
CTLV (JQ765412.1) 100
ASGV (KJ579253.1)
100
ACLSV (KM207212.1)
100
CMLV (NC_002500.1) CLBV (JN983456.1) CRMaV (KF030870.2)
100 100 100
CNRMV (KF030832.2) 93
CTLaV (NC_024449.1) CGRMV (NC_001946.1) 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.
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structures of the two isolates are identical to those of the German and Indian CVA isolates in GenBank, and consisted 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 (55–7 083 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 (5 400–6 791 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 70.3–74.5% nt and 77.2% aa sequence identities overall to the India isolate. The CP and MP genes appear to the more conserved, sharing 98.5–100% 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 necrotic 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 includes 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 GenBank 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 detected 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 identity with the isolate from Germany. The complete genome sequences reported here provides additional information on genome sequence information of CVA from different geographical locations and will form the basis for development of molecular diagnostics and development a more effective disease control strategy of CVA in China. What’s 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 fragments. 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 sequences were assembled from two long fragments amplified with Phanta® MAX Super-Fidelity DNA polymerase. It is important to ensure the accuracy of the genome sequences 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 isolates 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-scientific 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.
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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, 305–313. 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, 288–291. 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, 299–303. 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, 2015–2024. 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, 98–103. (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,
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Jelkmann W, eds., Virus and Virus-Like Diseases of Pome and Stone Fruits. APS Press, USA. pp. 147–150. 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, 195–204. 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, 2079–2082. 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, 93–104. 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, 173–177. 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, 128–133. (in Chinese) (Managing editor ZHANG Juan)
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SHORT COMMUNICATION
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 Tai’an, Shandong 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 GenBank. 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
1. Introduction Cherry virus A (CVA) is a member of the genus Capillovirus, 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: [email protected]; Correspondence LU Mei-guang, Tel: +86-10-62815615, E-mail: [email protected] © 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
many, India, Italy, Japan, Canada, Poland, France and 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),
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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, excluding the poly(A) tail (Jelkmann 1995), with two open reading frames (ORFs). Presently, only two complete nucleotide 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.
2.2. Genome cloning
2. Materials and methods
The nucleotide (nt) and deduced amino acid (aa) sequences 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).
2.1. Virus isolate and RNA extraction In this study, complete nucleotide sequences were determined for CVA isolates recovered from sweet cherry in Tai’an, 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 (Polysaccharide- & Polyphenolic-rich) (Tiangen, Beijing, China), from 60 sweet cherry leaf samples either with or without symptoms that were collected from Tai’an 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.
Complementary DNA (cDNA) was synthesized using a Reverse 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 GenBank (accession no. X82547.1). Sequences of the 3´-terminal 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, Beijing, China) (Appendix A). The concentration of all primers was 20 μmol. Reverse transcription was performed at 42°C for 1 h using 1 μL of total RNA and 1 μL of oligo(dT) primer in a 10 μL reaction volume with Maloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA), according to the manufacturer’s protocol. PCR was performed in a 50 μL volume reaction mixture containing 25 μL of 2×Phanta buffer, 2 μL of each primer (20 μmol L–1), 3 μL cDNA and 2 μL Phanta Max Super-Fidelity DNA polymerase (Vazyme, Nanjing, China). The PCR profile employed for all primer sets consisted of an initial denaturation at 95°C for 5 min followed by 35 cycles of 95°C for 15 s, 54°C or 65°C for 30 s, 72°C for 4 min, and a final extension for 10 min at 72°C. 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 DH5α cells.
2.3. Sequence analysis
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% sequence 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
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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)
(nt, %)
(nt, %)
ChTA11/Germany ChTA12/Germany ChTA11/India ChTA12/India ChTA11/ChTA12 India/Germany
98.2 98.2 81.2 81.0 99.5 81.1
98.2 98.2 90.7 90.7 100 92.6
MT2) nt aa (%) (%) 98.8 99.4 98.6 99.0 81.7 92.2 81.3 91.9 99.4 99.7 82.0 91.9
DUF17173) nt aa (%) (%) 96.7 100 97.0 100 70.6 77.2 70.3 77.2 99.0 100 74.5 77.2
Hel4) RdRp5) nt aa nt aa (%) (%) (%) (%) 96.7 100 97.3 97.0 97.0 100 97.3 97.0 81.1 91.7 81.4 93.7 81.1 91.7 81.4 93.7 99.5 100 100 100 80.9 90.0 79.8 93.7
CP6) nt aa (%) (%) 99.2 100 99.2 98.5 88.1 99.0 88.0 98.5 99.8 99.5 88.5 95.3
MP7) 3´ UTR1) nt aa (nt, %) (%) (%) 98.3 100 95.6 98.2 100 99.3 91.9 98.5 92.3 91.5 98.5 92.3 99.7 100 95.7 91.0 98.5 91.3
1)
UTR, untranslated region. 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. 2)
ORF 1
A MT 5´ RACE
DUF1717
Hel
CP
RdRp
Poly(A)
MP
872
ORF 2 3 372
511
7 182
2 892 7 124 B
CVA (FN691959.1)
100
CVA (X82547.1) CVA (ChTA11)
100
100
Oligo(dT)
98
CVA (ChTA12)
CVA Capillovirus
CTLV (JQ765412.1) 100
ASGV (KJ579253.1)
100
ACLSV (KM207212.1)
100
CMLV (NC_002500.1) CLBV (JN983456.1) CRMaV (KF030870.2)
100 100 100
CNRMV (KF030832.2) 93
CTLaV (NC_024449.1) CGRMV (NC_001946.1) 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.
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structures of the two isolates are identical to those of the German and Indian CVA isolates in GenBank, and consisted 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 (55–7 083 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 (5 400–6 791 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 70.3–74.5% nt and 77.2% aa sequence identities overall to the India isolate. The CP and MP genes appear to the more conserved, sharing 98.5–100% 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 necrotic 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 includes 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 GenBank 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 detected 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 identity with the isolate from Germany. The complete genome sequences reported here provides additional information on genome sequence information of CVA from different geographical locations and will form the basis for development of molecular diagnostics and development a more effective disease control strategy of CVA in China. What’s 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 fragments. 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 sequences were assembled from two long fragments amplified with Phanta® MAX Super-Fidelity DNA polymerase. It is important to ensure the accuracy of the genome sequences 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 isolates 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-scientific 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
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