Twenty-four isolates of Chilli veinal mottle virus (ChiVMV) from China, India, Indonesia, Taiwan and Thailand were analysed to determine their genetic relatedness. Pathogenicity of virus isolates was confirmed by induction of systemic mosaic and/or necrotic ringspot symptoms on Capsicum annuum after mechanical inoculation. The 3′ terminal sequences of the viral genomic RNA were determined. The coat protein (CP) coding regions ranged from 858 to 864 nucleotides and the 3′ untranslated regions (3′UTR) from 275 to 289 nucleotides in length. All isolates had the inverted repeat sequence GUGGNNNCCAC in the 3′UTR. The DAG motif, conserved in aphid-transmitted potyviruses, was observed in all isolates. All 24 isolates were considered as belonging to ChiVMV because of their high CP amino acid and nucleotide identity (more than 94·8 and 89·5%, respectively) with the reported ChiVMV isolates including the pepper vein banding virus (PVBV), the chilli vein-banding mottle virus (CVbMV) and the CVbMV Chiengmai isolate (CVbMV-CM1). Based on phylogenetic analysis, ChiVMV isolates including all 24 isolates tested, PVBV, CVbMV and CVbMV-CM1 can be classified into three groups. In addition, a conserved region of 204 amino acids with more than 90·2% identity was identified in the C terminal of the CP gene of ChiVMV and Pepper veinal mottle virus (PVMV), and may explain the serological cross reaction between these two viruses. The conserved region may also provide useful information for developing transgenic resistance to both ChiVMV and PVMV.
ChiVMV contains a 9·7 kb single-strand genomic RNA which encodes a polyprotein (Anindya et al., 2004). The virus was previously identified by serological methods, such as ELISA (Green & Kim, 1991). Recently, degenerate primers based on the conserved regions of the nuclear inclusion b (NIb) and the coat protein (CP) gene of ChiVMV and other potyviruses have been developed (Hiskias, 1998; Chen et al., 2001; Moury et al., 2005). So far, however, only one complete genomic RNA sequence has been determined from an Indian isolate (Anindya et al., 2004), and three partial sequences, including one from Taiwan (Hiskias, 1998; Green et al., 1999) and two from Thailand (Chiemsombat et al., 1998), have been sequenced. All three partial sequences include the partial NIb, CP and 3′ untranslated region (3′UTR). The two Thai isolates were originally named ‘chilli vein-banding mottle virus’ (CVbMV, U72193; CVbMV-CM1, AB012221), and the Indian isolate was designated as ‘pepper vein-banding virus’ (PVBV, AJ237843) (Anindya et al., 2004).
Many criteria have been used in the taxonomy of potyviruses, including the vector, host range, symptomatology, cross-protection, morphology of cytoplasmic inclusion bodies and serology (Shukla et al., 1994; Fauquet et al., 2005). Recently, molecular criteria such as amino acid sequence identity of the CP gene and nucleotide sequence identity of the full-length viral RNA have become the accepted method for the taxonomy of potyviruses (Fauquet et al., 2005). Viruses having more than 85% nucleotide identity of the full-length viral RNA, or more than 80% amino acid identity of the CP gene should be considered as the same potyvirus species (Fauquet et al., 2005). Based on these criteria, the CVbMV, CVbMV-CM1 and PVBV isolates have recently been reclassified as strains of ChiVMV by the International Committee on Taxonomy of Viruses (ICTV) (Fauquet et al., 2005). The identity of the 3′UTR nucleotide sequence has also been used in potyvirus classification (Frenkel et al., 1989).
Knowledge of the virus epidemiology is important for disease management and resistance breeding. In this paper, molecular characterization including virus sequences and phylogenetic relationships of the ChiVMV isolates from South and Southeast Asia are reported. Other potyviruses have also been included in the sequence comparison and the phylogenetic analysis.
Materials and methods
Chilli (Capsicum sp.) plants showing symptoms such as yellow vein, mottle, mosaic, and/or leaf deformation were collected from fields in China, India, Indonesia, Taiwan and Thailand from 1994 to 2004 (Table 1). DAS-ELISA (Clark & Adams, 1977) was used to confirm the presence of viruses commonly found in Asia (Green & Kim, 1991), including Cucumber mosaic virus (CMV), Pepper mild mottle virus (PMMoV), PVY, Tomato mosaic virus (ToMV) and ChiVMV. All antisera were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Braunschweig, Germany). Samples positive for ChiVMV only were selected for virus isolation. The ChiVMV infected leaves were ground in 30 mm phosphate buffer (pH 7·0, containing 0·5% Na2SO3, 0·17% Na2-DIECA and 0·01% charcoal) and mechanically inoculated to Nicotiana tabacum cv. White Burley, a local lesion host. Necrotic spots indicated the presence of ChiVMV. One such spot was individually excised and used to inoculate a new tobacco plant. Following three single spot transfers, the virus was propagated and maintained in N. glutinosa, a systemic host. Infectivity of ChiVMV isolates was confirmed by mechanical inoculation onto pepper (C. annuum cv. Early Calwonder). Appearance of mosaic and/or necrotic ringspots indicated presence of ChiVMV. The presence of virus was further confirmed by DAS-ELISA.
Table 1. Chilli veinal mottle virus isolates used in this study
Viral cDNA synthesis, PCR amplification, cloning and sequencing
Total RNAs were extracted from N. glutinosa leaf tissue showing symptoms by RNeasy Plant Mini Kit (Qiagen), following the manufacturer's protocol. Viral cDNA was synthesized using Oligo(dT) (5′-GCGGGATCCTTTTTTTTTTTTTTTTTT-3′) as downstream primer (Hiskias, 1998). Three microlitres of extracted RNA was used for viral cDNA synthesis following a modified protocol for the Moloney murine leukemia virus reverse transcriptase (M-MLV-RT, Invitrogen), using 0·375 mm dNTP, 20 U RNase inhibitor, 100 U M-MLV-RT, and incubation at 42°C for 120 min.
PCR amplification was performed using CVMV1037Pol (the ChiVMV polymerase primer, 5′-AGCATGGAGAGAGCGACATTAGTC-3′) and Poty3 (degenerate primer for the coat protein of pepper and tomato potyviruses, 5′-TGAGGATCCTGGTG(C/T)AT(A/C)GA(A/G)AA (C/T)GG-3′) as upstream primers (Hiskias, 1998), and Oligo(dT) as the downstream primer. The primer pair CVMV1037Pol/Oligo(dT) was designed to amplify the 3′-end of ChiVMV genomic cDNA including the 3′ terminus of the polymerase (NIb) gene, the CP gene and the 3′UTR. The Poty3/Oligo(dT) is known to amplify the 3′UTR and part of the CP gene of the ChiVMV. Another primer pair, Pata-F1/R1 (Pata-F1: 5′-AACCTGAGCGTATAGTTTCA-3′; Pata-R1: 5′-TACGCTTCAGCAAGATTGCT-3′) was designed to amplify the 3′ terminus of the NIb gene and the 5′ terminus of the CP gene of the two Indonesian isolates Cikabayan2 and Pataruman. Viral cDNAs were amplified by PCR using Taq DNA polymerase (Invitrogen) with the first cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min, and a final cycle at 72°C for 5 min (Jan et al., 2000). The RT-PCR product was ligated with the pGEM-T easy vector (Promega), a TA-cloning vector for transformation into Escherichia coli DH5α. DNA from selected colonies were sequenced using BigDye Terminator v3·1 cycle sequencing Kit (Applied Biosystems). Sequencing reactions were analyzed using ABI Prism 3100 Genetic Analyzer (Applied Biosystems).
Nucleotide sequence analysis
The DNAMAN Sequence Analysis Software (Lynnon Corporation) was used for assembly and analysis of viral sequences. Multiple sequence alignments and pairwise comparisons were done by the Multiple Sequence Alignment program provided by DNAMAN. The phylogenetic tree was generated by the neighbour-joining method provided by DNAMAN and bootstrap was tested in 1000 replications. The following sequences of potyviruses were retrieved from the NCBI-GenBank for comparison: Bean common mosaic virus (BCMV, L11890), Bean yellow mosaic virus (BYMV, D00490), Chilli vein-banding mottle virus (CVbMV, U72193), CVbMV Chiengmai isolate (CVbMV-CM1, AB012221), Clover yellow vein virus (CIYVV, D00605), Dasheen mosaic virus (DsMV, U00122), Johnsongrass mosaic virus (JGMV, Z26920), Lettuce mosaic virus (LMV, X65652), Leek yellow stripe virus (LYSV, X89711), Maize dwarf mosaic virus (MDMV, U07216), Papaya ringspot virus (PRSV, DQ374153), Pea seed-borne mosaic virus (PSbMV, D10930), Pepper severe mosaic virus (PepSMV, AM181350), Pepper mottle virus (PepMoV, M96425), Pepper vein banding virus (PVBV, AJ237843), Pepper veinal mottle virus-Cameroon (PVMV-Ca, AJ780967), PVMV-Ethiopia (PVMV-Et, AJ780970), PVMV-Ethiopia 1 (PVMV-Et1), PVMV-Ghana (PVMV-Gh, AJ780968), PVMV-Senegal (PVMV-Se, AJ780966), PVMV-Yemen (PVMV-Ye, AJ780969), Pepper yellow mosaic virus (PepYMV, AF348610), Peru tomato mosaic virus (PTV, AJ437280), Plum pox virus (PPV, D13751), Potato virus A (PVA, AJ296311), Potato virus V (PVV, AJ516024), Potato virus Y (PVY, AF237963), Soybean mosaic virus (SMV, D00507), Sugarcane mosaic virus (SCMV, AJ310105), Tobacco etch virus (TEV, M11458), Tobacco vein mottling virus (TVMV, X04083), Turnip mosaic virus (TuMV, AF169561), Wild potato mosaic virus (WPMV, AJ437279) and Zucchini yellow mosaic virus (ZYMV, AB188115).
Twenty-four ChiVMV isolates were obtained: eight from Taiwan (P1037, P3215, P3488, P3380, P3389, P3714, P3384 and P3525), eight from Thailand (T79, PJ, UB32, BP, CM1, K37, SKh5 and SRt8), four from India (BCV1, BeCV1, CCV3 and DCV3), two from China (China1 and China2) and two from Indonesia (Cikabayan2 and Pataruman) (Table 1). All isolates reacted positively with antiserum against ChiVMV in DAS-ELISA. All isolates induced systemic mosaic symptoms on C. annuum cv. Early Calwonder within 2 weeks of inoculation (Table 1; Fig. 2a) and seven isolates, five from Taiwan (P1037, P3215, P3380, P3389 and P3525) and two from China, also induced necrotic ringspots (Table 1; Fig. 2b).
Viral cDNA cloning, sequencing and analysis
RNAs isolated from virus-infected tobacco leaves were used as templates for viral cDNA synthesis. The viral cDNA synthesis was initiated by the primer-Oligo(dT). The predicted 0·8 kb DNA fragments were amplified from all virus isolates by RT-PCR using the primer pair Poty3 and Oligo(dT) (Fig. 1a). The predicted 1·2 kb DNA fragment was amplified with the primer pair CVMV1037Pol/Oligo(dT) from all isolates, except the two from Indonesia (Fig. 1b). The 1·2 kb DNA fragments were cloned and sequenced. The resulting sequences, which did not include the poly-A tail, ranged from 1237 to 1251 nucleotides (nt) in length (Table 1). For the Cikabayan2 and Pataruman isolates, the 3′ terminal genomic cDNAs were determined to be 1482 and 1476 nt, respectively, by assembling the sequences of the 0·8 kb fragment (Fig. 1a) and a 0·9 kb viral cDNA fragment amplified using the primer pair Pata-F1/R1 (Fig. 1c).
The full-length CP gene and 3′UTR sequences were determined (Table 1). The CP gene of all isolates, except Pataruman, contained 864 nt encoding 287 amino acids. The CP of Pataruman contained 858 nt, which encoded 285 amino acids. The cleavage site between NIb and CP was Q/S in all isolates, except BCV1 and DCV3 from India, which had cleavage site Q/A. The DAG motif, conserved in aphid transmitted potyviruses, was present in all isolates at amino acid positions 6–8 from the N-terminal of the CP gene. The length of 3′UTR ranged from 275 to 289 nt and had an inverted repeat sequence GUGGNNNCCAC present in all isolates.
Comparison and phylogenetic relationship of the viral CP gene and 3′UTR of the ChiVMV
The nucleotide sequences of the CP gene plus 3′UTR of the 24 new isolates shared 88·6 to 99·9% identity among themselves, and showed 87·4 to 97·4% identity with those of previously reported ChiVMV isolates (including PVBV, CVbMV and CVbMV-CM1), and less than 56·9% identity with that of other potyvirus species. The CP nucleotide and amino acid sequences of new isolates shared 89·9 to 99·9% and 95·1 to 100% identity, respectively. The CP genes of the new ChiVMV isolates shared 89·5 to 97·8% of nucleotide and 94·4 to 99·3% of amino acid identity with those of the reported ChiVMV isolates (including PVBV, CVbMV and CVbMV-CM1), but less than 79·4% of nucleotide and 83·6% of amino acid identity with that of other potyviruses as mentioned in materials and methods. The 3′UTR of the newly collected ChiVMV isolates showed nucleotide identity ranging from 82·1 to 100% among themselves. They also showed 77 to 97·9% identity with previously reported ChiVMV isolates (CVbMV, CVbMV-CM1 and PVBV), but less than 51·9% with other potyvirus species, as mentioned in materials and methods, in the 3′UTR nucleotide sequences.
Based on the phylogenetic analyses of the CP plus 3′UTR, the ChiVMV isolates in this study, PVBV, CVbMV and CVbMV-CM1 can be grouped into three groups (Fig. 3). Group 1 includes all isolates from China and Taiwan, three from Thailand (UB32, PJ and T97) and CVbMV-CM1. Group 2 includes the remaining five Thai isolates, two Indonesian isolates and CVbMV. Group 3 includes all Indian isolates and the PVBV. The nucleotide identity of the CP plus 3′UTR ranged from 93·3 to 99·9%, 95·2 to 99·7%, and 90·4 to 99·9% of the viruses within groups 1, 2 and 3, respectively (Table 2). The viruses within each group showed more than 92·2% and 96·9% identity of the CP nucleotide and CP amino acid sequences, respectively, and more than 90·7% identity of the 3′UTR nucleotide sequences (Table 2).
Table 2. Percentage sequence identities of CP genes and the 3′UTRs among isolates of Chilli veinal mottle virus (ChiVMV) and Pepper veinal mottle virus (PVMV)
Alignment of the viral CP gene and 3′UTR between the ChiVMV and PVMV isolates
Sequences of six CP genes and one 3′UTR (which is the only one available in GenBank) of the PVMV and all of the ChiVMV isolates, including the new isolates and three previously reported isolates (CVbMV, CVbMV-CM1 and PVBV), were analyzed. The CP genes between ChiVMV and PVMV isolates showed 72·1 to 76·3% and 81 to 84% identity of the nucleotide and the amino acid sequences, respectively (Table 2). When the CP amino acid sequences of the ChiVMV and PVMV isolates were aligned, a conserved region was identified which showed more than 90·2% amino acid identity (Table 2) and contained 204 amino acid residues from the C terminal. The remaining region was shown to be highly diverse (less than 61·9% amino acid identity) (Table 2). The nucleotide identity of the conserved and diverse regions were more than 79·2% and less than 68·3%, respectively (Table 2). The conserved region showed 64 to 82·4% and 63·5 to 83·8% amino acid identity of ChiVMV and PVMV with those of other potyviruses (Table 2). The nucleotide identity of the CP plus 3′UTR and the 3′UTR alone between ChiVMV and PVMV isolates were less than 71·9% and 57·4%, respectively (Table 2).
High CP nucleotide and amino acid sequence identities of > 89·5 and > 94·4%, respectively, were observed among all 24 ChiVMV isolates, PVBV, CVbMV and CVbMV-CM1. Only low CP gene homology (< 79·4% in nucleotide and < 83·6% in amino acid sequence identity) was found with other potyviruses included in this study. Based on the taxonomic criteria for potyvirus, a virus should be considered as a distinct species if it possesses less than 80% CP amino acid sequence identity (Fauquet et al., 2005). The results here provide strong evidence that all ChiVMV isolates from China, India, Indonesia, Taiwan and Thailand belong to the species Chilli veinal mottle virus. Moreover, based on one of the criteria for potyvirus species classification, namely 83–99% identity of the 3′UTR sequence (Frenkel et al., 1989), all isolates in this study should be considered strains of ChiVMV because their 3′UTR nucleotide sequence had > 83% identity. Exceptions were found when the 3′UTR of the PVBV was compared with those viruses in group 2 (77 to 79·2% identity), the CVbMV was compared with the BeCV1 (82·8%), CCV3 (82·8%), and UB32 (82·6%) isolates, and the SKH5 was compared with the BeCV1 (82·1%), CCV3 (82·1%) and UB32 (82·2%) isolates. However, the CP amino acids of the PVBV, CVbMV and SKH5 showed more than 94·1% identity in their sequence with those of other ChiVMV isolates. These results further confirm that PVBV, CVbMV and ChiVMV belong to the same virus species.
Based on the phylogenetic analysis of the CP and 3′UTR nucleotide sequences, all Chilli veinal mottle virus isolates can be classified into three groups (Fig. 3). Based on the high nucleotide identities (> 90·4% of the CP plus 3′UTR, > 92·2% of the CP and > 90·7% of the 3′UTR) among the isolates within each group, the viruses within the three groups can be considered strains of Chilli veinal mottle virus. Two types of symptoms, mosaic only or with necrotic ringspot, were induced in pepper plants (C. annuum cv. Early Calwonder) infected by the viruses of group 1. The sequence information of other viral genomic regions, such as P1, HC-Pro and P3 gene (Urcuqui-Inchima et al., 2001) and the gene function study should be helpful to the understanding of the genes or sequence elements related to the symptomatology.
Alignment of the CP gene of the ChiVMV with PVMV, a potyvirus widespread throughout Africa (Moury et al., 2005) showed 72·1 to 76·3% nucleotide sequence identity (Table 2), thus confirming that these two viruses are distinct (Moury et al., 2005). The ‘conserved’ CP gene region (204 amino acid residues from the C terminal) of the ChiVMV and PVMV isolates and their high homology (> 90·2% amino acid sequence identity) may explain the serological cross reactivity observed in other studies (Moury et al., 2005; unpublished data). The serological cross reaction was weak compared with the reaction of the virus with the antiserum against the virus itself (Moury et al., 2005; unpublished data). This may be due to the diverse N-terminal region (< 61·9% amino acid identity) of the CP of the ChiVMV and PVMV. The related region is exposed on the surface of the virus particle and is the major virus-specific epitope of potyviruses (Shukla & Ward, 1989; Urcuqui-Inchima et al., 2001). Because of the serological cross reaction, the recently detected ChiVMV in Tanzania by DAS-ELISA (Nono-Womdim et al., 2001) should be confirmed by the virus sequence identification.
The present study provides information on the molecular diversity of ChiVMV in Asia based on the CP and 3′UTR regions. More sequence data need to be collected from other areas to ascertain the population profile of ChiVMV worldwide. Recently, virus resistances have been generated through the post-transcriptional gene silencing (PTGS) mechanism (Tenllado et al., 2004). Broad-spectrum virus resistance has been developed through the production of transgene-specific siRNAs against cassava-infecting geminiviruses (Chellappan et al., 2004). A similar approach may be used to obtain transgenic plants resistant to both ChiVMV and PVMV based on the ‘conserved’ CP gene region. A total of 167 siRNAs were predicted to be generated in the conserved CP region of ChiVMV-China2 (group1) by an siRNA design program provided by the Whitehead Institute, MA, USA. Among them, three siRNA with perfect identity were also found in the related region of PVMV-Et1. If a tolerance of two or less mismatched nucleotides is considered, 33 additional siRNAs (11 with one mismatched and 22 with two mismatched) are predicted in the related region of PVMV. Based on the siRNA prediction, it is possible to use this conserved CP region for generating transgenic plants with broad resistance to both viruses via PTGS.
We are grateful to Dr Wen-Hsiug Ko, Emeritus Professor, University of Hawaii at Manoa, and V. Panwar for reviewing and editing the manuscript. This work was supported by the Gesellschaft für Technische Zusammenarbeit (German Agency for Technical Cooperation), Germany (International Agricultural Research Project No. 2001·7860·8-001·00), and the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan (Project No. 89-ST-6·2-BQ-90).