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Keywords:

  • genome sequence;
  • phylogenetic relationship;
  • tospovirus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Calla lily chlorotic spot virus (CCSV) collected from calla lily plants showing symptoms in Taiwan has been identified as a tentative species of the genus Tospovirus based on the comparison of S RNA sequences. In this investigation, the complete sequences of its L and M RNAs were determined. The L RNA contains 8911 nucleotides (nt) and encodes a putative RNA-dependent RNA polymerase of 2882 amino acids (aa) (332 kDa) in the viral complementary (vc) sense. The M RNA was shown to be 4704 nt in length, encoding a nonstructural NSm protein of 309 aa (35 kDa) in the viral sense and a Gn/Gc glycoprotein precursor (GP) of 1123 aa (128 kDa) in the vc sense. Phylogenetic analysis of the individual tospoviral proteins indicated that CCSV is closely related to Tomato zonate spot virus, a new tentative tospovirus isolated from tomato in southern China. Two degenerate primer pairs gL2740/gL3920c and gM410/gM870c, designed, after sequence comparison, from the highly conserved regions of the L and NSm genes, respectively, were successfully used to detect 12 greenhouse-cultured tospoviruses, including six formal species and six tentative species, by reverse transcription-polymerase chain reaction. They were also used to detect Melon yellow spot virus and Watermelon silver mottle virus from field cucurbit samples in Taiwan. The results indicate that the two degenerate primer pairs can be used for prompt detection of most tospoviruses and exploration of unknown tospovirus species.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Virus species in the genus Tospovirus of the family Bunyaviridae have quasi-spherical enveloped particles with diameters of 80–120 nm, containing a tripartite single-stranded (ss) RNA genome denoted as L, M and S RNAs. Each of the three RNAs contains a consensus sequence of 5′-AGAGCAAU-3′ at the 5′ end and a complementary 5′-AUUGCUCU-3′ at the 3′ end to form a pan-handle pseudocircular structure (Fauquet et al., 2005). The L RNA is of negative sense, encoding an RNA-dependent RNA polymerase (RdRp) from the viral complementary (vc) strand for virus replication (de Haan et al., 1991). Both M and S RNAs are ambisense, each containing two open reading frames (ORFs). The viral (v) sense of M RNA encodes a nonstructural NSm protein associated with viral cell-to-cell movement in infected cells (Kormelink et al., 1994; Lewandowski & Adkins, 2005) and its vc sense encodes a membrane-bound Gn/Gc glycoprotein precursor (GP) composing spikes on the viral envelope, responsible for virus–thrips interactions (Kikkert et al., 2001). The v strand of S RNA encodes a nonstructural NSs protein as the suppressor of gene silencing to counteract plant innate defence (Takeda et al., 2002). The NSs protein behaves as an avirulence gene in the interaction between Tomato spotted wilt virus (TSWV) and the Tsw resistance gene in pepper (Margaria et al., 2007). This protein also has a bifunction as ATPase and phosphatase as recently shown from Peanut bud necrosis virus (PBNV) (Lokesh et al., 2010). The vc strand of S RNA encodes a nucleocapsid protein (NP) for binding viral RNAs (de Haan et al., 1990).

Viruses classified within the genus Tospovirus have similar virion morphology, genome organization and a vector relationship with thrips. A threshold of 90% amino acid (aa) identity based on NPs is one of the important criteria for demarcation of a tospovirus at the species level (Fauquet et al., 2005). In addition, tospoviruses are also clustered as a serogroup or a serotype according to their serological relationships of NPs (Adam et al., 1993). So far 20 formal and tentative tospovirus species have been characterized (Hassani-Mehraban et al., 2010). Based on the serological and phylogenetic analysis of NPs, most species are clustered into three major serogroups, with TSWV, Watermelon silver mottle virus (WSMoV) and Iris yellow spot virus (IYSV) as the respective type members. Species prevailing in Europe or America, including Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV), Chrysanthemum stem necrosis virus (CSNV), Zucchini lethal chlorosis virus (ZLCV), Melon severe mosaic virus (MeSMV) and Alstroemeria necrotic streak virus (ANSV), are classified as members of the TSWV serogroup. Species occurring in Asia, including Capsicum chlorosis virus (CaCV), Calla lily chlorotic spot virus (CCSV), Melon yellow spot virus (MYSV), Peanut bud necrosis virus (PBNV), Watermelon bud necrosis virus (WBNV) and Tomato zonate spot virus (TZSV), are clustered in the WSMoV serogroup. Tomato yellow ring virus (TYRV) and Polygonum ringspot virus (PolRSV) are considered as the IYSV serogroup. Three tospovirus species, Impatiens necrotic spot virus (INSV), Peanut yellow spot virus (PYSV) and Peanut chlorotic fan-spot virus (PCFV), have no serological relationships with other tospoviruses and are regarded as distinct serotypes.

In 2001, a tospovirus, designated as CCSV, was isolated from a calla lily plant showing symptoms in central Taiwan. This virus reacted with the antiserum against the NP of WSMoV (As-WSMoV) (Chu et al., 2001b) in indirect enzyme-linked immunosorbent assays (ELISA) and immunoblotting, but could not be amplified by reverse transcription–polymerase chain reaction (RT-PCR) when the WSMoV N gene-specific primers were used (Chen et al., 2005). The complete sequence of the S RNA of this virus has been determined as 3172 nt, encoding a 51·8 kDa NSs protein and a 30·4 kDa NP in an ambisense organization (Lin et al., 2005). Phylogenetic analysis of the S RNA and NP indicated that CCSV is a new tentative species in the genus Tospovirus (Lin et al., 2005). The reciprocal serological analysis using antisera and MAbs to CCSV NP or WSMoV NP in indirect ELISA and immunoblotting indicated that CCSV is serologically related to WSMoV and thus the virus has been clustered as a member of the WSMoV serogroup. CCSV is transmitted by Thrips palmi and has become a potential threat for the floral industry in Taiwan (Chen et al., 2005).

In this investigation, the complete sequences of the M and L RNAs of CCSV were determined. Individual coding sequences of the CCSV genome were compared with those of other tospoviruses. Through sequence comparison, degenerate primer pairs were designed from the conserved regions of L genes and NSm genes and used to amplify the corresponding fragments from total RNA samples of WSMoV-serogroup species, including CaCV, CCSV, MYSV, PBNV, WBNV and WSMoV; TSWV-serogroup species, including GRSV, TCSV and TSWV; IYSV-serogroup species, including IYSV and TYRV; and the INSV serotype by RT-PCR. These degenerate primer pairs were also used on field cucurbit samples in Taiwan to see if they could detect MYSV and WSMoV.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Virus sources

The calla lily isolate of CCSV (Chen et al., 2005), the watermelon isolates of WSMoV (Yeh et al., 1992) and MYSV (Chen et al., 2008), and the peanut isolate of PCFV (Chu et al., 2001b) were collected from Taiwan. A high temperature-recovered isolate (HT-1) of CaCV collected from gloxinia in the United States was provided by Dr H.-T. Hsu (Hsu et al., 2000). PBNV and WBNV collected from tomato and watermelon, respectively, in India were provided by Dr P. A. Rajagopalan (Mahyco Co., Jalna, India). A New York isolate of TSWV isolated from tomato was provided by Dr R. Provvidenti (New York Agricultural Experiment Station, Geneva, NY, USA). TCSV originating from tomato (de Avila et al., 1993) was obtained from the DSMZ Plant Virus Collection, Germany. GRSV isolated from tomato in Brazil was given by Dr D. Gonsalves (Pang et al., 1993). INSV isolated from impatiens in the United States was provided by Dr J. Moyer (Law & Moyer, 1990). The iris isolate of IYSV from the Netherlands (Cortes et al., 1998) and the tomato isolate of TYRV from Iran (Hassani-Mehraban et al., 2005) were supplied by Dr R. Kormelink (Wageningen University, Wageningen, The Netherlands). Different taxonomic plant viruses, including a calla lily isolate (YC5) of Turnip mosaic virus (TuMV) (Chen et al., 2003), a sponge gourd isolate (TW-TN3) of Zucchini yellow mosaic virus (ZYMV) (Lin et al., 1998) and a melon isolate of Cucumber mosaic virus (CMV) (collected by this laboratory), were also used in this investigation. Viruses were maintained in the systemic host Nicotiana benthamiana or local-lesion host Chenopodium quinoa by mechanical inoculation and cultured in a temperature-controlled (25–28°C) greenhouse.

Reverse transcription–polymerase chain reaction (RT-PCR) and sequencing of L and M RNAs

Leaves of CCSV-infected C. quinoa were harvested 3 days post-inoculation (dpi) for extraction of total RNAs as described (Verwoerd et al., 1989). The primers used for amplification of L and M RNAs of CCSV by RT-PCR are listed in Table 1. The degenerate primers L1 and L9143, designed from the conserved regions of L RNAs of WSMoV-serogroup species, and the specific primers L3922 and L4499, designed from the previously determined sequence of the conserved region of CCSV L RNA (Chen et al., 2005), were used in RT-PCR for amplification of CCSV L RNA. The primer pairs M1/M1750, M1809/M2770 and M3821/M4920, designed from the conserved regions of M RNAs, were used in RT-PCR for amplification of CCSV M RNA. RT was performed with 2 μg of total RNAs mixed with individual primers and SuperScript™ III reverse transcriptase (Invitrogen). Mixtures were incubated at 55°C for 1 h to synthesize the first strand cDNA, and then the reaction was inactivated by heating at 70°C for 15 min. RT products were heated at 94°C for 5 min and amplification performed with 35 cycles of 1 min for strand separation at 94°C, 1 min for primer annealing at 55°C and 1 min for synthesis at 72°C, and 10 min at 72°C for final extension. PCR products shorter than 3 kb were cloned into pCR2.1-TOPO vector by TOPO TA cloning kit (Invitrogen), and PCR products longer than 3 kb were cloned into pCR-XL-TOPO vector by TOPO XL PCR cloning kit (Invitrogen), according to the manufacturer’s instructions. DNA sequences were determined by an automatic DNA sequencing system (ABI377-19; Perkin-Elmer Applied Biosystems). Specific primer pairs M788/M2392 and M2440/M4132 were designed from the known sequences and used for amplification of the remaining parts of the M RNA (Table 1).

Table 1.   Primers used for cloning of L and M RNAs of Calla lily chlorotic spot virus
PrimerDirectionSequence
  1. F: forward primers; R: reverse primers.

L1F5′-AGAGCAATCC(A/T)GCAACAA-3′
L3922R5′-CCAAGGACTCTAAATCTATCTCTG-3′
L4499F5′-GCCAGACACAGTACGGATCA-3′
L9143R5′-AGAGCAATCC(A/T)GCAAC-3′
L5′RACERTR5′-TTTTATTTGTTTGGCTACACCGA-3′
L5′RACEnestR5′-CTTTAATGAATTTTTGACAGGG-3′
L3′RACERTF5′-GGGGTTAAGTGCTGAAACACTAGTT-3′
L3′RACEnestF5′-TCATGTTTTCGATCTTGTCTAGAC-3′
gL3637F5′-CCTTTAACAGT(A/T/G)GAAACAT-3′
gL4435cR5′-CAT(A/T/G)GC(A/G)CAAGA(A/G)TG(A/G)TA(A/G)ACAGA-3′
M1F5′-AGAGCAATCGGTGC(A/G)CCAA-3′
M788F5′-CTTGTGATGTTATACCAATCAATAGAG-3′
M1750R5′-TGGGA(C/T)TGG(A/G)T(A/C/T)AAAGCACC-3′
M1809F5′-TCATC(A/G)TGTGCACTTTT(A/G)TCATC-3′
M2392R5′-CCTCAGATCACTATTGATGG-3′
M2440F5′-GGCACAGATAATGGTGTTC-3′
M2770R5′-CCAAC(C/T)TCTTGGTGGGG(A/T)TG-3′
M3821F5′-GGGAAATA(C/T)(G/T)(G/T)CCA(C/T)AACCA-3′
M4132R5′-CCTCTGTGGAGATCGTTC-3′
M4920R5′-GAGCAATCAGTGCAACAATT-3′
M5′RACERTR5′-ATGAAATATAAAACAAGCTGGGTTCG-3′
M5′RACEnestR5′-TTCCATTACCAACTAAAGAATCATTTTT-3′
M3′RACERTF5′-TGATTTATCGACAAAGAAGAATTTTGT-3′
M3′RACEnestF5′-CTAAGGATTCGAGAAAGTACTTTTGTAGTT-3′

Rapid amplification of cDNA ends (RACE)

Specific primers designed for amplification of cDNAs corresponding to the 5′- and 3′-ends of CCSV L and M RNAs are also listed in Table 1. Total RNAs extracted from the CCSV-infected plant tissues using Trizol reagent (Invitrogen) were denatured by heating at 65°C for 5 min and then mixed with the first primers, L5′RACERT and L3′RACERT for L RNA, and M5′RACERT and M3′RACERT for M RNA. The first strand cDNA fragments were synthesized by MMLV reverse transcriptase (Epicentre). After removal of template RNAs by RNaseH digestion, the first strand cDNAs were tailed with 0·4 mm dCTP and 10–15 U terminal deoxynucleotidyl transferase (Takara). PCR amplification of the polyC-tailed cDNAs was performed with Ex Taq DNA polymerase (Takara) using a primer polyG18 and a second primer L5′RACEnest or L3′RACEnest for L RNA, and M5′RACEnest or M3′RACEnest for M RNA. The amplified DNA fragments were cloned by the TOPO TA cloning kit (Invitrogen) for sequencing.

Sequence assembly and alignment

Sequence analysis was carried out using programs within the software vector nti v. 10 (Invitrogen). Contigs of CCSV L and M RNAs were assembled using the Assemble Selected program and analysed by the ContigExpress program. Prediction of ORFs in the CCSV L and M RNAs was conducted using the ORF program. Multiple sequence alignments were carried out with the alignment program ClustalW. The nucleotide sequences were translated into deduced amino acid sequences using the Translate Selection program. All reported sequences used for comparison were obtained from GenBank through the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). The accession codes corresponding to individual sequences are shown in Table 2. Sequences were analysed by multiple alignments using ClustalW. The amino acid sequence alignment was performed by the AlignX program and exported as MSF format with vector nti.

Table 2.   Accession codes corresponding to the individual tospoviral sequences used in this study
SpeciesAbbreviationM RNAL RNA
  1. –: no sequence available.

Calla lily chlorotic spot virusCCSVFJ822961FJ822962
Capsicum chlorosis virusCaCVNC_008303NC_008302
Chrysanthemum stem necrosis virusCSNVAF213675 for NSm; AB274026 for GP
Groundnut ringspot virusGRSVAF513220 for NSm; AY574055 for GP
Impatiens necrotic spot virusINSVGQ336990NC_003625
Iris yellow spot virusIYSVAF214014FJ623474
Melon severe mosaic virusMeSMV
Melon yellow spot virusMYSVNC_008307NC_008306
Peanut bud necrosis virusPBNVNC_003620AF025538
Peanut chlorotic fan-spot virusPCFV
Peanut yellow spot virusPYSV
Polygonum ringspot virusPolRSVEU271753
Tomato chlorotic spot virusTCSVAF213674 for NSm; AY574054 for GP
Tomato spotted wilt virusTSWVAF208497NC_002052
Tomato yellow ring virusTYRV
Tomato zonate spot virusTZSVNC_010490EF552435
Watermelon bud necrosis virusWBNVGU584185
Watermelon silver mottle virusWSMoVNC_003841NC_003832
Zucchini lethal chlorosis virusZLCVAF213676 for NSm; AB274027 for GP

Phylogenetic analysis

Multiple amino acid sequence identities were obtained by the Similarity Tables program of vector nti. Input alignment data were changed to the phylip format by the readseq program (University of Indiana). Phylogenetic analysis was conducted using phylip 3·66 (University of Washington). Bootstrapping produced 1000 repeats to generate multiple resampled data sets by the Seqboot program of phylip. A distance matrix of amino acids was produced by the Protdist program of phylip under PAM matrices of the Dayhoff model. Phylogenetic branching was set using the Neighbour–Joining method. Finally, a consensus tree was produced by the Consense program of phylip. Sequences of tospoviruses used for comparison were obtained from the GenBank database (Table 1).

Design of degenerate primers for detection of tospoviruses by RT-PCR

The degenerate primer pair gL2740 [5′-ATGGG(A/G/T)AT(A/T/G/C)TTTGATTTCATG(A/G)TATGC-3′] (forward primer) and gL3920c [5′-TCATGCTCAT(C/G)AG(A/G)TAAAT(T/C)TCTCT-3′] (reverse primer) were designed from the conserved regions of L RNAs of WSMoV, PBNV, CaCV, CCSV, MYSV, TSWV and INSV, after multiple sequence alignments using ClustalW. The degenerate primer pair gM410 [5′-AACTGGAAAAATGATT(T/C)(A/T/C/G)(T/C)TTGTTGG-3′] (forward primer) and gM870c [5′-ATTAG(C/T)TTGCA(T/G)GCTTCAAT(A/T/G/C)AA(A/G)GC-3′] (reverse primer) were designed from the conserved regions of NSm sequences of WSMoV, PBNV, CaCV, CCSV, MYSV, TSWV, TCSV, GRSV and INSV. Total RNAs extracted from leaves of C. quinoa or N. benthamiana plants infected with distinct tospovirus species, including CCSV, CaCV, PBNV, WBNV, WSMoV, MYSV, IYSV, TYRV, TSWV, TCSV, GRSV, PCFV and INSV, were used for tests. In addition, total RNAs from leaf tissues of plants uninoculated or infected with individual viruses of ZYMV, TuMV and CMV were used as controls.

Detection of WSMoV and MYSV in cucurbit field samples using the degenerate and species-specific primers

Because WSMoV and MYSV are two important tospoviruses infecting cucurbits in Taiwan (Chen et al., 2010), the degenerate primer pairs gL2740/gL3920c and gM410/gM870c were used to test their feasibility for detecting these tospoviruses in cucurbit samples collected from the field. Tissues of watermelon, melon and cucumber collected from fields in central Taiwan were used for analysis. Total RNAs from plant tissues were extracted using Plant Total RNA Miniprep Purification kit (Hopegen) and one tube-based RT-PCR was performed using One-Step RT-PCR kit (Hopegen) following the manufacturer’s instructions. The first strand cDNAs were synthesized at 50°C for 30 min and terminated at 94°C for 2 min. PCR for the degenerate primer pairs was conducted by 35 cycles of 94°C for 30 sec, 52°C for 30 sec and 72°C for 30 sec, and a final cycle for 10 min at 72°C. To verify the infection by the two tospoviruses, the N gene primer pairs, WN2963 (5′-AATAATCGGTGCCAGTCCCCTT-3′)/WN3469c (5′-ATGTCTAACGTTAAGCAGCTCACA-3′) specific for WSMoV and MYSV-N-f (5′-GCCATGGCATGCATGTCTACCGTTACTAAGCTGACA-3′)/MYSV-N-r (5′-GTCTAGAGGTACCAACTTCAATGGACTTAGCTCTGGA-3′) specific for MYSV (Chen et al., 2010), were used. The annealing was modified to 58°C for 30 sec for the N gene-specific primer sets.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Completion of full-length genome sequencing of CCSV

For CCSV L RNA, three overlapping PCR fragments and two RACE products were cloned and sequenced. The complete CCSV L RNA sequence assembled from these five clones was determined as 8911 nt in length (GenBank accession code FJ822962). Five overlapping PCR fragments covering most of the CCSV M RNA and two RACE products corresponding to the 5′- and 3′-ends were cloned and sequenced. The complete sequence of CCSV M RNA assembled from these seven clones was determined as 4704 nt in length (GenBank accession code FJ822961). The typical 5′-terminal consensus sequence (5′-AGAGCAAU-3′) and its complementary 3′-terminal sequence (5′-AUUGCUCU-3′) were determined by RACE to verify the ends of both L and M RNAs. These results, combined with the previously reported S RNA (Lin et al., 2005), completed the full-length genomic sequence of CCSV.

Sequence analysis of CCSV L RNA

The 5′- and 3′-untranslatable terminal regions (UTRs) of the CCSV L RNA have 230 and 29 nt, respectively. Due to the negative polarity of L RNA, an ORF (8652 nt) starting from nt 30 with an AUG start codon to nt 8681 with a UGA stop codon was found in the vc strand, encoding a deduced RdRp of 2883 aa (332 kDa). The RdRp of CCSV shows the highest degree of aa homology, 89·0% identity and 92·5% similarity, with that of TZSV. It also shares higher identities and similarities of 66·9–77·3% and 75·9–83·9%, respectively, with those of CaCV, MYSV, PBNV, WSMoV and IYSV, but lower homologies with TSWV (45·0% identity and 56·3% similarity) and INSV (46·4% identity and 57·3% similarity) (Table 3). Although most tospoviral L RNA sequences are still not available, the phylogenetic analysis of RdRps among the reported tospoviruses showed that CCSV is clustered in the WSMoV serogroup and closely related to TZSV (Fig. 1a). The five conserved motifs, DXXKW in motif A, QGXXXXXSS in motif B, SDD in motif C, K in motif D, and EXXS in motif E, were also found within the RdRp of CCSV (Fig. 1b).

Table 3.   Sequence identities and similarities (%) of individual proteins of Calla lily chlorotic spot virus (CCSV) compared with those of other tospoviruses
SpeciesM RNAL RNA
NSmGPRdRp
IdentitySimilarityIdentitySimilarityIdentitySimilarity
  1. –: no sequence available.

WSMoV serogroup
 TZSV87·791·691·795·289·092·5
 WBNV69·178·872·780·7
 PBNV73·380·873·881·777·083·9
 WSMoV71·079·272·480·477·383·8
 CaCV75·282·472·579·872·579·8
 MYSV58·569·066·274·674·181·4
IYSV serogroup
 IYSV67·377·161·772·666·975·9
 PolRSV66·174·061·372·8
TSWV serogroup
 TSWV41·850·236·448·345·056·3
 GRSV43·252·238·150·4
 CSNV41·949·537·449·1
 ZLCV37·246·237·348·1
 TCSV42·551·237·950·1
Distinct serotype
 INSV41·849·837·449·146·457·3
image

Figure 1.  Sequence analysis of the reported L RNAs of tospoviruses. (a) A phylogenetic tree based on the amino acid sequences of RNA-dependent RNA polymerases (RdRp). The dendrogram was produced using the Neighbour–Joining algorithm with 1000 bootstrap replicates. (b) The RdRp conserved motifs in viruses of the genus Tospovirus. The consensus amino acid residues in each RdRp motif of the family Bunyaviridae are shown in bold. The positions of the conserved motifs in RdRp are indicated. For virus abbreviations see Table 1.

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Sequence analysis of CCSV M RNA

The results indicated that CCSV M RNA contains two ORFs, the NSm and the glycoprotein precursor (GP), in an ambisense coding strategy and separated by an AU-rich intergenic region (IGR). The 5′- and 3′-UTRs of CCSV M RNA contain 54 and 45 nt, respectively. The IGR of CCSV M RNA has 303 nt, ranging from nt 985 to 1287 in the v sense. The NSm coding sequence of CCSV contains 930 nt, starting from nt 55 with an AUG start codon to nt 984 with a UAA stop codon in the v strand of M RNA, encoding a protein of 309 aa (35 kDa). The NSm protein of CCSV shares the highest degree of aa homology, 87·7% identity and 91·6% similarity, with that of TZSV. It also shares high identities and similarities of 58·5–75·2% and 69·0–82·4%, respectively, with those of CaCV, MYSV, PBNV, PolRSV, WBNV, WSMoV and IYSV, but lower degrees of homology, 37·2–43·2% identities and 46·2–52·2% similarities, with those of CSNV, GRSV, INSV, TCSV, TSWV and ZLCV (Table 3).

The GP coding sequence of CCSV is 3372 nt in length, ranging from nt 4659 with an AUG start codon to nt 1288 with a UAG stop codon in the v strand of CCSV M RNA, encoding a GP of 1123 aa (128 kDa) from the antisense polarity. The GP of CCSV shares the highest degree of aa homology, 91·7% identity and 95·2% similarity, with that of TZSV; it also shares high identities, 61·3–73·8%, and similarities, 72·6–81·7%, with those of CaCV, MYSV, PBNV, PolRSV, WBNV, WSMoV and IYSV. Lower degrees of homology, of 36·4–38·1% identity and 48·1–50·4% similarity, were observed when the CCSV GP was compared with those of CSNV, GRSV, INSV, TCSV, TSWV and ZLCV (Table 3). Phylogenetic trees of NSm proteins and GPs among tospoviruses are shown in Figure 2, indicating that CCSV is clustered within the WSMoV serogroup and closest to TZSV.

image

Figure 2.  Phylogenetic trees of tospoviruses based on the amino acid sequences of NSm proteins (a) and Gn/Gc glycoprotein precursors (GP) (b). The dendrograms were produced using the Neighbour–Joining algorithm with 1000 bootstrap replicates. For virus abbreviations see Table 1.

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Broad-spectrum detection of tospovirus species by RT-PCR using the degenerate primers

After sequence analysis, the degenerate primer pairs gL2740/gL3920c (Fig. 3a) and gM410/gM870c (Fig. 4a) were designed from the conserved regions within the reported sequences of L and M RNAs, respectively. Using the degenerate primer pair gL2740/gL3920c in RT-PCR amplification, an expected DNA fragment of 1·2 kb was obtained from most tested tospovirus samples except PCFV, including six formal species: GRSV, INSV, PBNV, TCSV, TSWV and WSMoV, and six tentative species: CCSV, CaCV, IYSV, MYSV, TYRV and WBNV (Fig. 3b). No DNA fragments were obtained from the uninoculated and non-tospovirus samples, such as CMV, TuMV and ZYMV. Similarly, the results also indicated that the degenerate primer pair gM410/gM870c can be used to amplify a 0·5 kb DNA fragment from all the tested tospoviruses except PCFV, and no signals were observed from the samples of healthy control and non-tospoviruses (Fig. 4b).

image

Figure 3.  Detection of tospoviruses by reverse transcription-PCR (RT-PCR) using the degenerate primers designed from the L RNAs of tospoviruses. (a) Sequences of the degenerate forward primer gL2740 and the degenerate reverse primer gL3920c designed from multiple alignments of the conserved regions within tospoviral L RNAs. The positions of the conserved nucleotides within each tospoviral RNA-dependent RNA polymerase reading frame are indicated. The nucleotides in black boxes were degenerate in primer design. (b) The degenerate primers gL2740 and gL3920c were used to detect individual tospovirus species in RT-PCR. A DNA fragment of 1·2 kb was amplified from total RNA extracted from each tested tospovirus-infected sample. Tospoviruses belonging to the same serogroups or distinct serotypes are indicated. No signals were detected from the plant samples infected with Turnip mosaic virus (TuMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV) used as outgroup controls. Healthy Nicotiana benthamiana sample (H) was used as a negative control. For tospovirus abbreviations see Table 1.

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image

Figure 4.  Detection of tospoviruses by reverse transcription-PCR (RT-PCR) using the degenerate primers designed from the NSm genes of tospoviruses. (a) Sequences of the degenerate forward primer gM410 and the degenerate reverse primer gM870c designed from multiple alignments of the conserved regions within tospoviral NSm genes. The positions of the conserved nucleotides in each tospoviral NSm reading frame are indicated. The nucleotides in black boxes were degenerate in primer design. (b) The degenerate primers gM410 and gM870c were used to detect individual tospovirus species in RT-PCR. A DNA fragment of 0·5 kb was amplified from total RNA extracted from each tested tospovirus-infected sample. Tospoviruses belonging to the same serogroups or distinct serotypes are indicated. No signals were detected from the plant samples infected with Turnip mosaic virus (TuMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV) used as outgroup controls. Healthy Nicotiana benthamiana sample (H) was used as a negative control. For tospovirus abbreviations see Table 1.

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Detection of tospoviruses from field cucurbit samples by RT-PCR with the designed degenerate primers and species-specific primers

The two designed degenerate primer pairs, gL2740/gL3920c and gM410/gM870c, were further used to detect tospoviruses in field samples. Forty total RNA samples of watermelon and melon individually infected with WSMoV or MYSV, 20 samples for each crop, randomly selected from a previously reported survey in 2008 (Chen et al., 2010) were used for the test. Total RNAs extracted from two cucumber samples showing chlorotic and necrotic spots on leaves newly collected from the field were also used for virus detection. Parts of the results are shown in Figure 5. The predicted DNA fragments of 1·2 and 0·5 kb were amplified from all tested samples using gL2740/gL3920c and gM410/gM870c, respectively (Fig. 5a,b). When the N gene-specific primer pairs, WN2963/WN3469c for WSMoV and MYSV-N-f/MYSV-N-r for MYSV, were further used for verification of the distinct virus species, DNA fragments of 0·5 and 0·8 kb were amplified from the WSMoV- and MYSV-infected tissues, respectively, as shown in Figure 5c,d. The two tested cucumber samples were infected with MYSV, and the results were further confirmed by serological assays using a monoclonal antibody against the NP of MYSV (Chen et al., 2010) (data not shown).

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Figure 5.  Detection of tospoviruses from field samples of cucurbits using the genus-specific and species-specific primer pairs in reverse transcription-PCR. The degenerate primer pairs gL2740/gL3920c (a) and gM410/gM870c (b) were used to detect tospoviruses using total RNAs of the tissues of melon, watermelon and cucumber plants showing symptoms, collected from fields. The species-specific primer pair WN2963/WN3469c designed from the N gene of Watermelon silver mottle virus (WSMoV) (c) and the Melon yellow spot virus (MYSV) N gene-specific primer pair MYSV-N-f/MYSV-N-r (d) were used to identify WSMoV and MYSV, respectively. The samples of Nicotiana benthamiana infected with WSMoV (W) or MYSV (M) were used as positive controls. The healthy N. benthamiana sample (H) was used as a negative control.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Calla lily chlorotic spot virus is a tentative tospovirus species as indicated by sequence comparison of the complete S RNA sequence (Lin et al., 2005). Here the sequences of the CCSV L and M RNAs are reported to complete the whole genomic sequence. CCSV is the ninth species for which the entire genome sequence has been completely determined in the genus Tospovirus, following CaCV (DQ256123 for S RNA, DQ256125 for M RNA and DQ256124 for L RNA), INSV (NC_003624 for S RNA, NC_003616 for M RNA and NC_003625 for L RNA), IYSV (AF001387 for S RNA, AF214014 for M RNA and FJ623474 for L RNA), MYSV (AB038343 for S RNA, NC_008307 for M RNA and NC_008306 for L RNA), PBNV (U27809 for S RNA, NC_003620 for M RNA and AF025538 for L RNA), TSWV (D13926 for S RNA, AF208497 for M RNA and NC_002052 for L RNA), TZSV (NC_010489 for S RNA, NC_010490 for M RNA and EF552435 for L RNA) and WSMoV (U78734 for S RNA, NC_003841 for M RNA and NC_003832 for L RNA). These tospoviral sequences provide important information for understanding the molecular characteristics and phylogenetic relationships of tospoviruses.

Different methods can be applied for extracting the viral RNAs, for example, from purified virions of tospoviruses to construct a cDNA library or from double-stranded (ds) RNAs of tospoviruses to be used for cDNA cloning. In this study, the total RNAs were extracted from CCSV-infected C. quinoa plants. In addition, degenerate primers were designed, using the published M RNA and L RNA sequences of WSMoV-related tospoviruses from the database for the alignment. The RT-PCR resulted in clear products for cloning and sequencing, indicating that this approach is feasible. In RACE experiments, the sequences of the 5′- and 3′-ends of CCSV L and M RNAs were also determined using total RNAs. The total RNAs extracted from the leaves of CCSV-infected N. benthamiana contain viral ssRNAs, dsRNAs and subgenomic mRNAs. Degenerate primers of 5′- and 3′-conserved regions were used for 5′ RACE and the clones with the terminal sequences of L and M RNAs were selected for sequencing. In addition to the exact sequence reflecting the terminal conserved eight nucleotides, some clones with additional sequences (13–15 bp) were obtained. These 5′ extensions are apparently derived from viral mRNAs that are generated by cap-snatching in host cells, through which the 5′-ends of viral mRNA possess a 5′ extension with 12 and 17 nt in addition to the genomic template (Duijsings et al., 2001).

Although the N gene sequence of S RNA is the most important criterion for classification of a tospovirus species, determination of full-length genomic sequences is an important step to further demarcate different tospoviruses. CCSV has been classified as a member of the WSMoV serogroup based on serological relationships of its NP (Lin et al., 2005). Results of this study further indicated that other CCSV-encoded proteins are phylogenetically related to those of WSMoV-serogroup tospoviruses. It is notable that all CCSV-encoded proteins share higher aa identities of 80·9–91·7% with those of TZSV, a newly identified WSMoV-serogroup tospovirus species collected from tomato in the Yunnan province of Southern China (Dong et al., 2008). The high aa identities imply that CCSV and TZSV may be difficult to distinguish by serological assays with polyclonal antibodies or even monoclonal antibodies. The close relationship between CCSV and TZSV can be verified with the phylogenetic analysis of other viral genes, and the economic importance of these two viruses in southern China and Taiwan need to be further investigated.

Only nine tospoviruses sequences have been determined so far for the L RNA. Five conserved RdRp motifs are observed in all analysed tospoviruses, including CCSV (Fig. 1b). These conserved motifs are found in the middle portion of the RdRp of tospoviruses and may play critical functions in the RdRp for replication and transcription. Motifs A, B, C and D are conserved among RNA-dependent DNA polymerases (reverse transcriptases) and RdRp encoded by viruses (Porch et al., 1989). These four motifs are also conserved within all RNA-dependent polymerases of eukaryotes and transcriptional elements, the so-called ‘polymerase module’ (Delarue et al., 1990). The particular types of short consensus sequences in the conserved motifs imply their essential roles for the function of RdRp (Muller et al., 1994). Motif E has been found in segmented negative-stranded viruses, and it is considered to be involved in the mechanism of cap-snatching (Kolakofsky & Hacker, 1991).

In the M RNA, a predicted signal peptide at the N-terminal region of CCSV GP was found in this study. The signal peptide length of CCSV GP is 24 aa, the same as those of CaCV, WSMoV, PBNV, MYSV and TZSV. In addition, IYSV and PolRSV have a similar signal peptide with a length of 28 aa, and TSWV, GRSV, TCSV, CSNV, ZLCV and INSV share a similar signal peptide length of 35 aa (data not shown). This observation indicates that tospoviruses classified into a distinct serogroup share a similar signal peptide length and sequence in their GPs. The signal peptide may play a role for leading the migration of GP from the endoplasmic reticulum to the Golgi body (Ribeiro et al., 2008, 2009). Moreover, other properties in GP were also found to demonstrate the relationship of tospoviruses. The RGD motif that is known to interact with β-integrins on cell surfaces is present in the viral GPs of some animal-infecting viruses, such as coxsackievirus A9 (Roivainen et al., 1994) and herpesvirus (KSHV/HHV-8) (Akula et al., 2002). In this study, RGD was found to be absent from the GPs of CCSV, WSMoV, PBNV, CaCV, MYSV and TZSV that belong to the WSMoV serogroup, and IYSV and PolRSV that belong to the IYSV serogroup. These tospoviruses are also clustered as Asian type because of their predominant distribution in Asia (Pappu et al., 2009). In contrast, RGD is only present in the GPs of TSWV, TCSV, GRSV, CSNV and ZLCV that belong to the TSWV serogroup, and the distinct serotype INSV (data not shown). These tospoviruses occurring worldwide, but predominantly in European and American areas, are clustered as Euro-American type (Pappu et al., 2009). These results further reflect that tospoviruses belonging to the same group have a similar evolutionary ancestor, and their proteins should play similar functions in vector specificity.

In this report, based on the phylogenetic analysis of the genomic products of CCSV, the virus is verified as a distinct species of the genus Tospovirus, and is closely related to TZSV, a member of the WSMoV serogroup. CCSV and TZSV have probably evolved from the same ancestor and have diverged through geographic separation. The current classification status of tospoviruses is proposed as three major serogroups and three serotypes based on NP relationships (Chen et al., 2010). Although the major criterion for demarcation of tospoviruses at the species level is the homology of NP (Adam et al., 1993), the phylogenetic results here support the previous reports that the other tospoviral proteins may also provide a sufficient basis for classification of tospoviruses (Hassani-Mehraban et al., 2007; Dong et al., 2008). Both WSMoV and IYSV serogroup tospoviruses, belonging to the Asian type, have biologically parallel properties to support their evolutionary relationship, such as being mainly transmitted by vectors Thrips palmi and Thrips tabaci, respectively, which distinguish them from the Euro-American TSWV serogroup tospoviruses and INSV serotype, that are mainly transmitted by Franklinella spp. (Pappu et al., 2009).

In this study, the highly conserved regions within tospoviral genomes were used to develop degenerate primer pairs for detection of tospoviruses. Previously, the degenerate primer pair gL3637/gL4435c designed from the conserved regions of tospoviral L RNAs was used to amplify a 0·8 kb DNA fragment by RT-PCR from total RNAs extracted from WSMoV, CaCV, CCSV, PCFV, TSWV, GRSV, and INSV-infected plants (Chu et al., 2001a). In this study, two other degenerate primer pairs, gL2740/gL3920c and gM410/gM870c, were designed from L and NSm genes, respectively. These two degenerate primer pairs were successfully used to amplify the corresponding DNA fragments from 12 of the 13 tested tospovirus species, except PCFV which was isolated from peanut in Taiwan. The molecular and serological relationships of PCFV and PYSV, both peanut-infecting tospoviruses, indicate that they are phylogenetically distant from other tospoviruses (Satyanarayana et al., 1998; Chu et al., 2001b) and are not related to other Asia-type tospoviruses. Completion of the unknown genomic sequences of these two tospoviruses is necessary for designing suitable degenerate primer pairs for detection and identification of all species in the genus Tospovirus.

Furthermore, gL2740/gL3920c and gM410/gM870c were used in a field survey to detect WSMoV and MYSV, two serious threats of cucurbits in Taiwan. The results indicated that gL2740/gL3920c and gM410/gM870c are broad-spectrum, and a combination of gL2740/gL3920c, gM410/gM870c and gL3637/gL4435c can be used for detection and diagnosis of known or unknown tospoviruses emerging in fields.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported in part by the projects NSC96-3111-P-225-001-Y and NSC99-2324-B-468-003-MY2 from the National Science Council and 97AS-14.1.2-BQ-B1 from the Council of Agriculture of Taiwan.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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