Virus Genes (2009) 39:120–125 DOI 10.1007/s11262-009-0361-2
A new potyvirus first isolated and identified from Angelica sinensis Yu Zhang Æ Ruoyu Wang Æ Jianhui Wang Æ Jiangfeng Chang Æ Xinfang Zhang Æ Tuo Chen Æ Lizhe An Æ Shijian Xu
Received: 4 February 2009 / Accepted: 15 April 2009 / Published online: 5 May 2009 Ó Springer Science+Business Media, LLC 2009
Abstract A filamentous virus was isolated in Angelica sinensis (Angelica sinensis (Oliv.) Diels) which shows mosaic symptoms on leaves in Minxian, Gansu province, China. According to morphology and molecular biology properties, this virus, which has a flexuous rod-shaped particle about 750 nm in length and 12 nm in width, was assigned to the genus Potyvirus, family Potyviridae. Its coat protein (CP) shows high similarity with six other potyviruses by analysis of peptide mass fingerprinting (PMF). The 919 bp nucleotides of 30 terminal covering partial CP gene and 30 -untranslated region was amplified by RT-PCR using degenerate primers which were designed according to the result of PMF. In sequence comparisons and phylogenetic analysis, the new isolate was found to be closely related to Japanese hornwort mosaic virus (JHMV), Konjak mosaic virus (KoMV), and Zantedeschia mosaic virus (ZaMV). The most closely related virus is JHMV03 (AB251346), with 96.59% aa and 87.60% nt identity to the isolate. All results suggest the presence of a new member of potyvirus, tentatively named Dang Gui strain of Japanese hornwort mosaic virus (JHMV-DG*). In our research the antiserum against the CP of JHMV-DG had also been prepared. To our
*Nucleotide sequence date reported appear in GenBank with the accession number EF157843. Y. Zhang J. Chang X. Zhang L. An S. Xu (&) Key Laboratory of Arid and Grassland Ecology of Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou 730000, People’s Republic of China e-mail:
[email protected];
[email protected] R. Wang J. Wang T. Chen L. An S. Xu Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China
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knowledge, it is the first time that a potyvirus has been isolated and identified in Angelica sinensis. Keywords Angelica sinensis Potyvirus Peptide mass fingerprinting (PMF) RT-PCR JHMV
Introduction Angelica sinensis (Angelica sinensis (Oliv.) Diels, family Umbelliferae), a well-known Chinese herbal medicine, has been used for the treatment of various diseases and a tonic medicine for thousands of years [1, 2]. Sometimes it is referred to its Chinese name as Dong Quai or Dang Gui [3, 4]. As the major producing area of angelica in China, Minxian County in Gansu province has more than 1700 years planting history. In recent years, lots of works have been focused on plant disease and insect pests in Angelica sinensis [5–7]. However, there is no study on virus disease in this plant. Potyvirus, the major genus in the Potyviridae family, infect a broad range of host plants, both monocot and dicot in different climatic regions. They are transmitted predominantly by aphids in a non-persistent manner, and can cause a range of symptoms including leaf mosaic and necrosis, flower breaking or mottling [8]. As a result, these potyviruses lead to severe economic damage to crops [9, 10]. The members of this genus have filamentous particles 650–900 nm in length and 11–13 nm in width, and these dimensions vary among species [11]. Their single-stranded positive-sense RNA genome of 8,500–12,000 nucleotides possesses a poly (A) tail at the 30 -terminus [12] and a viral protein genome-linked (VPg) at the 50 -end. During the infection process, the RNA is translated into a large precursor polyprotein that is cleaved co- and post-translationally into 10 mature proteins [13]. KoMV, ZaMV, and
Virus Genes (2009) 39:120–125
JHMV are members of the same species, a distinct species in Potyvirus genus according to the 8th ICTV report [14– 16]. Isolated from konjak plants, the complete nucleotide sequence of KoMV F, which can infect a relatively wide range of plants, had been determined by Nishiguchi et al. [16]. The genome is 9,544 nucleotides long excluding the 3-terminal polyA tail and encodes a typical potyviral 350 kDa polyprotein of 3,087 amino acids [16]. Some researchers had found that ZaMV could infect calla lily plants in Taiwan, Korea, and Typhomium flagelliforme in China [16–18]. Okuno et al. [19] had isolated JHMV from Japanese hornwort plants (Cryptotaenia japonica) with mosaic disease symptoms. Because of its narrow-ranged host plants, JHMV could systemically infect only plants belonging to Umbelliferae. Considering KoMV was the first of these to be described [20, 21], Nishiguchi et al. [16] suggested that it would be reasonable to state that ZaMV and JHMV are isolates of KoMV. In this research, leaves of Angelica sinensis with typical mosaic symptoms were collected to be studied on plant virus, including preliminary electron microscopic observation of the purified virus, peptide mass fingerprinting (PMF) analysis of virus CP, and sequencing the product of RT-PCR. The comparison and phylogenetic analyses have also been done among several other potyviruses, while the antiserum has also been prepared against the CP protein in this study.
Materials and methods Plant material Leaves of Angelica sinensis with typical mosaic symptom were collected from the fields in Minxian County, Gansu province, and stored at -80°C (Fig. 1a).
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Virus purification Virus was purified from the leaves following the protocol. Leaves of Angelica sinensis (30 g) were homogenized with grinder in liquid nitrogen, adding 60 ml of extraction buffer (0.05 M Tris–HCl pH 7.4, 0.1% b-mercaptoethanol, 0.1% pvp, 0.01 M EDTA-Na2, and 1 mM PMSF) and 60 ml chloroform quickly, and then centrifuged at 50009g for 10 min at 4°C. The supernatant which had been filtered by filter paper was added with polyethyleneglycol (PEG) 6000 to 10% (w/v) and NaCl to 2% (w/v), and then stored for 12–16 h at 4°C. After centrifugation at 50009g for 20 min at 4°C, the pellet was resuspended by 5 ml of elution buffer (0.05 M Tris–HCl pH 7.4, 0.1% b-mercaptoethanol, and 1 mM PMSF). Then the crude preparation of virus particles was passed through a Sephadex G-200 column (35 cm 9 1.0 cm). The eluant solution was mon¨ KTA basic 100 Purification System itored at 254 nm by A (GE Healthcare, USA). Outflows with the expected peak were collected and then concentrated by centrifugation. By this means, satisfactorily purified products containing viral particles were obtained. Electron microscopy Purified virus particles were placed onto formvar-coated copper grids and stained with 2% uranyl acetate. The specimens were examined with a transmission electron microscope (Hitachi-8100, Japan) at 80 kV. SDS-PAGE and peptide mass fingerprinting (PMF) analysis Purified preparations were concentrated and mixed with 59 SDS-PAGE loading buffer, then boiled for 5 min, and
Fig. 1 Leaves of Angelica sinensis with typical mosaic symptom (a). Electron micrograph of purified virus particles (b1, b2)
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Virus Genes (2009) 39:120–125
subjected to SDS-PAGE in a 15% gel. Protein of purified virus band was cut from the gel, and then digested with trypsin (Promega, USA). Peptide mass mapping was performed by matrix assisted laser desorption-ionization/timeof-flight mass spectrometry (MALDI-TOF-MS) using a Proteomics System I (ABI, USA). PMF and MS/MS data from MALDI-TOF-MS were analyzed by searching against an NCBInr database using GPS (Matrix Science, London) search software. Primer design and RT-PCR PMF shows that the highest homologous proteins are six other potyviruses coat proteins. Alignment of those six CP coding sequences and 30 -UTR sequences were used to design the degenerate primers (Fig. 2). P1_50 -CARCCDCCAAAGAAGGATAA-30 and P2_50 -CY CCYRYTTAWRARACAYRACT-30 (G/A = R, T/C = Y, A/T = W, G/A/T = D). Viral genomic RNA was isolated from the purified virus preparations by TRIZOL Reagent (Sangon, Shanghai). The first strand of cDNA was synthesized by MMLV reverse transcriptase (promega) with the Oligo (dT). Primers (P1, P2) were used to amplify fragment about 900 bp covering partial CP and 30 -UTR gene of virus. Cloning and sequencing After verified by agarose gel electrophoresis and purified using the Gel Extraction Kit (QIAGEN), the PCR product was cloned into the pUCm-T vector (Sangon, Shanghai). The ligated plasmid was transformed into DH5a cells and the recombinant plasmids were auto-sequenced using the ABI PRISMTM377 DNA Sequencer. Sequence analysis Comparisons of the sequences between the isolate and the other potyviruses retrieved from the GenBank databases (Table 2) were performed with complete alignment using DNAMAN package (version 5.1, Lynnon Biosoft, Canada). Multiple sequence alignments were taken by CLUSTALW algorithm in the Mega 3.0. Using the neighbor-joining (NJ) algorithm, phylogenetic analysis was performed in Mega 3.0 to address the relationship between
3'UTR
5'
NIb
CP
...... PolyA
P1 P2
Fig. 2 Position of primes on potyvirus genome
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this virus and other potyviruses after bootstrapping in 1000 replicates according to Saitou and Nei [22]. Preparation of antiserum and serological test Based on the virus sequence data, primers were designed to amplify the part of CP about 792 bp coding 264 amino acids. Restriction sites for Nco I and Hind III (underlined) were created at the ends of the primers: CP-up 50 -GGCCATGGAACCGCCAAAGAAGGATA A-30 and CP-down 50 -ATAAGCTTGATGGCGCGA ACA CCCAT-3. The PCR product of the CP gene was purified and inserted into Pet28 vector, and then transformed into BL21 cells for expression. The protocol of prokaryotic overexpression was taken as described by Schenk et al. [23]. Antiserum against the expressed protein was raised in rabbits. Protein of the purified virus was prepared on 15% SDS-PAGE and then transferred onto PVDF membranes for western blot analysis [24].
Results Using gel filtration chromatograph with Sephadex G-200, the crude sap of virus was purified. The fractions of the virus at first peak with white opalescence were eluted with approx. 6 ml Elution Buffer. It was collected and concentrated for the next step. Then the impurity fractions that had a slight yellow color and no opalescence were eluted in another absorbance peak. Under electron microscope, purified virus has a notably flexuous, filamentous shape about 750 nm in length and 12 nm in width (Fig. 1b1 and b2) with a morphological similarity to viruses in family Potyviridae. The purified virus was analyzed in SDS-PAGE and an estimated 30 kDa protein band was observed in gel (Fig. 3a1). According to the PMF analysis of the virus protein band (data not shown), six potyviruses proteins, which received high scores in the PMF results (Table 1), show great conformability with the purified virus. Using degenerate primers designed according to the result of PMF, a fragment about 900 nt was obtained in PCR. The sequence was determined and it showed to contain the partial CP (795 nt) gene and the 30 -UTR (124 nt). GenBank accession number: EF157843. Percentage identities at the nucleotide sequence, amino acid of the partial CP gene, and 30 -UTR levels between new isolate and other potyviruses are shown in Table 2. The alignment results showed that the amino acid (aa) sequence of the partial CP and the nucleotide(nt) sequence have 96.59% and 87.60% identities to the corresponding gene of Japanese hornwort mosaic virus isolate (JHMV03,
Virus Genes (2009) 39:120–125
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Discussion
Fig. 3 SDS-PAGE of coat protein (a1. CP) and western blot analysis (a2). Expression of the partial CP gene in BL21 (b). M is a molecular weight maker (kDa), lane 1 is total proteins of BL21 which has recombinant vector, ep in lane 2 is the purified expression of protein. Lane 3 is a control of BL21 which has unrecombinant vector Pet28
Table 1 Scores of other six potyviruses by PMF analysis Virus name
Accession number
Mass
Expect
Queries matched
Score
JHMV-FK*
AB181353
55768
1.8e-06
19
112
JHMV-IW*
AB181352
55770
3.1e-05
18
100
JHMV-FK2*
AB181354
55771
3.5e-05
18
99
ZaMV NNI*
AJ628757
56338
0.00030
16
90
JHMV*
AB081518
56646
0.00077
16
86
ZaMV-KR*
AB081519
56551
0.00180
16
82
Score is probability based on Mowse Score. Protein scores greater than 67 are significant (P \ 0.05) * Virus used to design primers and compared with the isolate in sequence analysis
AB251346). Phylogenetic trees (Fig. 4a–c) were performed to show the relationships between the isolate and other potyviruses at the levels of nt, aa, and 30 -UTR, respectively. The result revealed that the virus clusters with Japanese hornwort mosaic virus (JHMV), Konjak mosaic virus (KoMV), and Zantedeschia mosaic virus (ZaMV). In the antiserum preparation and serological test, the partial CP gene inserted into Pet28 has been expressed in BL21 (Fig. 3b). And the expressed protein was used to prepare antiserum in rabbit. The antiserum (dilution 1/200) against the expressed protein reacts strongly with the coat protein of the purified virus in western-blot (Fig. 3a2).
This Virus was easily purified from the collected leaf tissues by PEG 6000 sedimentation and gel filtration chromatograph without density centrifugation and ultra centrifugation. It was observed that fractions of virus and fractions of impurity were separated completely according to absorbance curve (data not shown). The result indicates that it is feasible to obtain preparations of these viruses in Angelica sinensis by GFC technique. Under electron microscope, purified virus particles appear to have the typical morphology of members in the genus Potyvirus. Then the hypothesis was confirmed by analysis of virus CP using PMF, which showed that the most homologous proteins to the identified CP are six other potyviruses (JHMV and ZaMV, in Table 1). Based on the above result, primers have been designed for PCR and an accordant result with the PMF analysis was obtained. Sequence analysis and alignment of the PCR product show that the identified virus has the highest identity to JHMV03, 87.60% and 96.59% at nucleotide and amino acid sequences, respectively, and 91.13% identity at the 30 -untranslated region (Table 2). Multiple alignments of sequences (nt, aa, and 30 -UTR) between the virus and other potyviruses show that it is closely related to JHMV, ZaMV, and KoMV. However, \57.00, \57.20, and \49.64% identities to the other four potyviruses (PVY-LYE84.2, PPV SoC, PeMV, PVY in Table 2) at nt, aa, and 30 -UTR levels had been found in the above results. All these results indicate that it is a new distinct potyvirus. In additional phylogenetic analysis using NJ algorithm at those three levels, the identified virus shows a significant grouping with JHMV, ZaMV, and KoMV, and a wider group including Plum pox virus SoC (Fig. 4). They are located in a different group with the other three potyviruses. Based on the aa sequence of partial CP gene, the phylogenetic tree (Fig. 4, tree (c)) indicates that the identified virus is more closely related to JHMV than ZaMV and KoMV. All results indicated that the isolate from Angelica sinensis is a new member of the same potyvirus species as JHMV, KoMV, and ZaMV. According to the 8th ICTV report, JHMV, ZaMV, and KoMV are members of the same species, belonging to distinct potyvirus species of the genus Potyvirus [14, 15]. Here we have tentatively named it Dang Gui strain of Japanese hornwort mosaic virus (JHMV-DG) because of the closer relationship to JHMV than ZaMV and KoMV and the highest identity with JHMV03. In our study, we also prepared the antiserum against CP of JHMV-DG, which can be used for detection of this virus. This is the first report that a new potyvirus was isolated and identified in Angelica sinensis. The complete
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Virus Genes (2009) 39:120–125
Table 2 Potyviruses used in multiple alignments and phylogenetic analysis. Percentage sequence identities between the nucleotide, amino acid of partial CP gene, and 30 -UTR of JHMV-DG and other potyviruses Virus names
Acronym
Accession number
% Identities Nucleotide
Amino acid
30 -UTR
Japanese hornwort mosaic virus 03
JHMV03
AB251346
87.60
96.59
91.13
Japanese hornwort mosaic virus-IW
JHMV-IW*
AB181352
87.05
96.59
90.32
Japanese hornwort mosaic virus-FK2 Japanese hornwort mosaic virus FK1
JHMV-FK2* JHMV-FK1*
AB181354 AB181353
86.83 87.38
96.59 96.21
90.32 88.71
Japanese hornwort mosaic virus*
JHMV*
AB081518
86.29
95.83
87.90
Zantedeschia mosaic virus-KR
ZaMV-KR*
AB081519
86.77
92.08
90.32
Zantedeschia mosaic virus NN1
ZaMV NN1*
AJ628757
85.42
92.05
88.71
Konjak mosaic virus F
KoMV-F
AB219545
84.94
90.57
87.20
Zantedeschia mosaic virus R7
ZaMV R7
AF470620
85.95
89.85
90.32
Zantedeschia mosaic virus BG
ZaMV BG
AY026463
85.95
89.47
90.32
Zantedeschia mosaic virus ZAN
ZaMV ZAN
AF332872
85.84
89.47
90.32
Zantedeschia mosaic virus DB
ZaMV DB
AY026464
85.95
89.10
90.32
Potato virus Y LYE84.2
PVY LYE84.2
AJ439545
56.36
58.71
42.54
Pepper mottle virus
PeMV
M11598
57.00
57.20
41.73
Potato virus Y
PVY
AF255659
56.42
56.44
44.44
Plum pox virus SoC
PPV SoC
AY184478
53.05
55.10
49.64
* Virus used to design primers
Fig. 4 Phylogenetic relationships between the isolate (JHMV-DG) and members of the genus Potyvirus. The trees are constructed by the neighbor-joining algorithm. Numbers shown at branch point indicate
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the bootstrap values. The dataset is subjected to 1,000 bootstrap replicates. Tree (a) based on nucleotide sequence from PCR. Tree (b) based on 30 -UTR. Tree (c) based on amino acid of the partial CP gene
Virus Genes (2009) 39:120–125
nucleotide sequence of the genomic RNA is underway, and the infective mechanism of this virus still remains to be researched furtherly in our work. Acknowledgment We gratefully acknowledge the technical assistance of Yu Bai, Daqun Yang, Qi Zhao, and Xin Yang of Lanzhou University. The work was funded by the Gansu Provincial Natural Science Foundation of China (3ZS061-A25-063), Gansu Provincial Agricultural Science Foundation of China, and the National Natural Science Foundation of China (30870378).
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