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1 Tropical Crops and Biocommodities Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane 4001, Australia
2 Department of Plant Pathology, Hanoi Agriculture University, Gialam, Hanoi, Vietnam
Correspondence
Rob Harding
r.harding{at}qut.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn St, North Melbourne, Victoria 3051, Australia. 
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ641688–DQ641719 (see Table 1).
A supplementary table showing the full names and abbreviations of all reference sequences used in the DNA-A analysis is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
The largest genus, Begomovirus, currently contains 132 species (Fauquet & Stanley, 2005) that have either bipartite genomes (DNA-A and DNA-B) or monopartite genomes resembling DNA-A (hereafter called DNA-A). DNA-A typically has six open reading frames (ORFs): AV1/V1 (coat protein, CP) and AV2/V2 (AV2/V2 protein) on the virion-sense strand, and AC1/C1 (replication initiation protein, Rep), AC2/C2 (transcriptional activator, TrAP), AC3/C3 (replication enhancer, REn) and AC4/C4 (AC4/C4 protein) on the complementary-sense strand. DNA-B has two ORFs encoding movement proteins: BV1 (nuclear shuttle protein, NSP) on the virus-sense strand and BC1 (movement protein, MP) on the complementary-sense strand (Rojas et al., 2005; Seal et al., 2006).
The opposing transcription units of begomovirus DNA-A and -B molecules are separated by an intergenic region (IR) that generally shares a highly conserved region of approximately 200 nt, named the common region (CR) (Lazarowitz, 1992). The CR contains an origin of replication (ori) that includes a stem–loop structure containing an invariant nonanucleotide (TAATATTAC) sequence whose T7–A8 site is required for cleaving and joining viral DNA during replication, and conserved iterated sequences (iterons) required for specific recognition and binding by Rep during replication (Arguello-Astorga et al., 1994; Fontes et al., 1994a, b).
Based on phylogenetic studies and genome arrangement, begomoviruses have been divided broadly into two groups: the Old World (OW) viruses (eastern hemisphere, Europe, Africa, Asia) and the New World (NW) viruses (western hemisphere, the Americas) (Padidam et al., 1999; Paximadis et al., 1999; Rybicki, 1994). Begomovirus genomes have a number of characteristics that distinguish Old World and New World viruses. All indigenous New World begomoviruses are bipartite, whereas both bipartite and monopartite begomoviruses are present in the Old World. In addition, DNA-A of bipartite begomoviruses from the New World lacks an AV2 ORF (Rybicki, 1994; Stanley et al., 2005). New World begomoviruses also have an N-terminal PWRsMaGT motif in the CP that is absent from Old World viruses (Harrison et al., 2002). Until recently, it was thought that New World viruses arose more recently than Old World viruses, evolving after continental separation of the Americas from Gondwana (Rybicki, 1994). However, we recently identified a virus indigenous to Vietnam, Corchorus yellow vein virus (CoYVV), that resembles New World viruses more closely than Old World viruses (Ha et al., 2006). This was based primarily on phylogenetic analysis, the absence of an AV2 ORF and the presence of an N-terminal PWRLMAGT motif in the CP. The presence of CoYVV in Vietnam suggests that New World-like viruses were probably present in the Old World prior to the Gondwana separation although, to date, CoYVV is the only known New World geminivirus that is indigenous to the Old World.
Recently, two additional circular ssDNA molecules, known as DNA-β and nanovirus-like DNA-1 (DNA-1), have been identified in association with some Old World monopartite begomoviruses (Briddon & Stanley, 2006). These ‘satellite’ molecules are approximately half the size of the ‘helper’ begomovirus. DNA-β molecules contain one major ORF (βC1) on their complementary strand, depend on the helper virus for replication and are responsible for symptom induction (Briddon & Stanley, 2006). Begomovirus-associated DNA-1 molecules are similar to nanovirus-encoded DNA-1 molecules in that they contain one major ORF that encodes a Rep protein and they replicate autonomously. The role of the begomovirus-associated DNA-1-like molecules in disease progression is unclear (Briddon et al., 2004).
To date, the only begomoviruses reported in Vietnam have been CoYVV (Ha et al., 2006), tomato leaf curl Vietnam virus (ToLCVV) (Green et al., 2001), tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) (GenBank accession no. DQ169054
[GenBank]
) and two viruses infecting cucurbits, namely squash leaf curl China virus (SLCCNV) and Luffa yellow mosaic virus (LYMV) (Revill et al., 2003, 2004). In this paper, we have identified and characterized numerous additional geminiviruses and associated DNA molecules infecting crop and weed species throughout Vietnam, and provide further evidence that indigenous ‘New World’ viruses were present in the Old World.
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METHODS |
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Virus taxonomy.
New virus species names were assigned by using the rules proposed by the ICTV Geminiviridae Study Group (Fauquet et al., 2003; Fauquet & Stanley, 2005). Demarcation of new viral species was based on DNA-A sequence comparisons, using a threshold of 89 % nucleotide sequence identity (Fauquet et al., 2003). The sequence and arrangement of iterons, Rep N-terminal iterated-related domains (IRD) (Arguello-Astorga et al., 1994; Arguello-Astorga & Ruiz-Medrano, 2001) and recombinant regions were also considered in the taxonomic decisions. As a sanctioned taxonomic system was not available for naming the begomovirus-associated satellite molecules (DNA-β and DNA-1), names were assigned by using the protocol adopted by Zhou et al. (2003).
PCR
DNA-A.
The sequences of degenerate primers, BegoAFor1 and BegoARev1, and the PCR conditions used to detect DNA-A molecules have been described previously (Ha et al., 2006). These primers were used to detect DNA-A from all viruses except Corchorus golden mosaic virus (CoGMV). From the sequences amplified by degenerate primers, adjacent, outwardly extending, specific primers were designed to amplify the complete DNA-A molecules, using the Expand Long Template PCR system (Roche). For CoGMV, DNA-A was initially amplified from three different plants by using outwardly extending, CoYVV-specific primers 201For/201Rev1 (Ha et al., 2006). As these primers were derived from the CoYVV sequence, the sequence at the CoYVV priming sites was obtained by reamplification with the degenerate primers BegoAFor1 and BegoARev1 (Ha et al., 2006).
DNA-B.
With the exception of CoGMV, an antisense, specific primer located in the DNA-A CR was used in combination with a degenerate primer specific for DNA-B (BegoBFor, 5'-CCIDHIGCRTTRAWIGGIACYTG-3') to amplify a product of approximately 0.7 kbp. The PCR conditions for DNA-B amplification were as described previously (Ha et al., 2006). For CoGMV, DNA-B was initially obtained by using two primers specific for the BV1 gene of CoYVV (201BV1For, 5'-CGCTGATGATAAGATGACCAAACA-3'; 201BV1Rev, 5'-ACGCCCCATTACAACTATCAACAT-3'). The complete DNA-B molecules were subsequently amplified by using adjacent, outwardly extending, specific primers and the Expand Long Template PCR system.
DNA-β.
To detect DNA-β molecules, two consensus outwardly extending primers were designed in the satellite conserved region (SCR) (BetaFor2, 5'-TAGCTACGCCGGAGCTTAGCTCG-3'; BetaRev2, 5'-AAGGCTGCTGCGTAGCGTAGTGG-3'). The PCR conditions for DNA-β amplification were as described previously (Ha et al., 2006) except that an annealing temperature of 55 °C was used, due to a high G+C content. From the sequences amplified by using BetaFor2 and BetaRev2, adjacent, outwardly extending, specific primers were designed and used in the Expand Long Template PCR system to amplify the complete DNA-β molecules.
Nanovirus-like DNA-1.
To detect the nanovirus-like DNA-1 molecules, degenerate, outwardly extending primers (NLDNA1For, 5'-TGGTTYTATWCACGTGGHGG-3'; NLDNA1Rev, 5'-ARAWGATAGTKCKRTCATCTG-3') were designed from the conserved region of the DNA-1 Rep gene. The PCR conditions for DNA-1 amplification were as described previously (Ha et al., 2006) except that the annealing temperature used was 46 °C. Adjacent, outwardly extending, specific primers were subsequently designed from the NLDNA1For/NLDNA1Rev-primed amplicon sequences, and were used in the Expand Long Template PCR system to amplify the entire DNA-1 molecules.
Cloning and sequencing.
Amplicons were purified from agarose gels by using standard protocols, ligated to the plasmid vector pGEM-T Easy (Promega) and transformed into Escherichia coli XL1-Blue competent cells (Stratagene). Cloned plasmids were purified by using a Wizard Miniprep kit (Promega) and three clones for each sample were sequenced in both orientations by using an ABI PRISM BigDye Terminator kit (PE Applied Biosystems) at the Australian Genomic Research Facility (University of Queensland).
Sequence analysis.
The genomic sequences of DNA-A, DNA-B, DNA-β and DNA-1 molecules were assembled from contiguous sequences by using the SEQMAN program (DNASTAR). ORFs were identified by using the Vector NTI Suite 7 program. To determine whether the molecules were similar to known viral/satellite sequences, they were initially analysed by using the BLAST program available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/).
Sequences were aligned with the CLUSTAL_X program (Thompson et al., 1997) and phylogenetic trees were constructed by using the neighbour-joining algorithm and viewed with the TreeView program (Page, 1996). Nucleotide identities were determined by using the MEGALIGN program (DNASTAR). Potential gene-recombination events were analysed by using the Recombination Detection Program version 2.0 (RDP2) (Martin et al., 2005), also available online (http://darwin.uvigo.es/rdp/rdp.html). The recombination analyses were implemented by using six automated methods: RDP (Martin & Rybicki, 2000; Martin et al., 2005), GENECONV (Padidam et al., 1999), BootScan (Salminen et al., 1995), SiScan (Gibbs et al., 2000; Salminen et al., 1995), MaxChi and CHIMAERA (Posada & Crandall, 2001; Smith, 1992), using default parameters except that the option ‘Reference sequence selection’ was set at ‘internal references only’.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These viruses were named Corchorus golden mosaic virus (CoGMV), infecting jute mallow (Corchorus capsularis); kudzu mosaic virus (KuMV), infecting kudzu (Pueraria montana); Clerodendrum golden mosaic virus (ClGMV), infecting glory bower (Clerodendrum philippinum); Spilanthes yellow vein virus (SpYVV), infecting paracress (Spilanthes paniculata); Mimosa yellow leaf curl virus (MiYLCV), infecting mimosa (Mimosa sp.); Sida yellow vein Vietnam virus (SiYVVNV), infecting sida (Sida rhombifolia); tomato yellow leaf curl Vietnam virus (TYLCVNV), infecting tomato; Erectites yellow mosaic virus (ErYMV), infecting fireweed (Erectites valerianifolia); and Ludwigia yellow vein Vietnam virus (LuYVVNV), infecting willow primrose (Ludwigia octovalvis). Three of the viruses (CoGMV, KuMV and ClGMV) were bipartite, whilst the other six viruses were apparently monopartite. A DNA-β molecule was found associated with four of the monopartite viruses (MiYLCV, SiYVVNV, TYLCVNV and ErYMV) and a nanovirus-like DNA-1 molecule was detected in association with two of these viruses (MiYLCV and SiYVVNV).
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). These were papaya leaf curl China virus (PaLCuCNV), infecting tobacco; Lindernia anagallis yellow vein virus (LaYVV), infecting false pimpernel (Lindernia procumbens); Alternanthera yellow vein virus (AlYVV), infecting zinnia (Zinnia elegans) and eclipta (Eclipta prostrata); Sida leaf curl virus (SiLCV), infecting abutilon (Abutilon indicum); and Ludwigia yellow vein virus (LuYVV), infecting willow primrose. A DNA-β molecule was identified in plants infected with PaLCuCNV, LaYVV, AlYVV and SiLCV, whilst plants infected with SiLCV also harboured a nanovirus-like DNA-1 molecule. In addition, two virus species previously reported in Vietnam were identified infecting tomato and eggplant, namely tomato leaf curl Vietnam virus (ToLCVV) and tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), respectively (Table 1).
Sequence and recombination analysis
DNA-A and DNA-B.
The genomes of CoGMV, KuMV and ClGMV were bipartite. All DNA-A molecules contained four complementary-sense ORFs (AC1, AC2, AC3 and AC4) and two virion-sense ORFs (AV1 and AV2), with the exception of CoGMV, which did not contain an AV2 ORF. The stem–loop sequence in the putative intergenic region of all DNA-A molecules contained the nonanucleotide sequence TAATATTAC, with the exception of CoGMV, which contained the sequence TATTATTAC.
The CoGMV, KuMV and ClGMV DNA-B molecules each possessed two major ORFs: BC1 on the complementary-sense strand and BV1 on the virion-sense strand, separated by an IR that contained a CR shared with DNA-A. The CR sequences between the cognate DNA components of CoGMV, KuMV and ClGMV shared 66.2, 58.7 and 87.4 % identity, respectively. The unusually low identities of the CoGMV and KuMV CR sequences were due to differences in the region between the TATA box and stem–loop, as well as almost 50 % variability in the putative stem sequences that spanned the conserved TAATATTAC sequence in KuMV. For all three viruses, however, the sequence and arrangement of the iterons in the CRs of both components were identical (Fig. 1).
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). In addition, the sequence and arrangement of the iterons and IRD differed for the two viruses (Fig. 1), providing further evidence that they were two distinct species. The N-terminal region of the CoGMV CP encoded a putative PWRsMaGT motif, identical to that encoded by CoYVV and other New World begomoviruses (Table 3). In addition, the CP N-terminal region of all Old World begomoviruses, with the exception of CoGMV and CoYVV, encoded either two or three basic domains, typically KR, KVRRR and K/RRRR, that formed part of the nuclear-localization signal (NLS). In contrast, the CP N-terminal sequence of CoGMV, CoYVV and all New World begomoviruses encoded only the first basic domain, KR (Table 3).
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). DNA-A of ClGMV was most similar to the genome of a putative novel monopartite virus identified in the current study (TYLCVNV, 68.8 %), whereas ClGMV DNA-B showed most similarity to DNA-B from tomato yellow leaf curl Thailand virus (TYLCTHV, 38.7 %), a bipartite virus (Table 2).
Most of the new monopartite viruses identified in this study showed significant sequence identity to known virus sequences (Table 2), with the exception of SpYVV, which shared only 65.7 % identity with the most closely related virus, tobacco leaf curl Yunnan virus (TbLCYNV). Over the entire nucleotide sequence, MiYLCV was most similar (82.3 %) to TYLCVNV, whilst SiYVVNV was most similar (84.7 %) to Sida yellow mosaic China virus (SiYMCNV). Although close to the 89 % threshold for classification as the same virus species, marked differences in both the sequences and arrangement of the iterons and the IRD of SiYVVNV and SiYMCNV (Fig. 1) suggested that they warrant classification as distinct species.
Although the complete nucleotide sequence of TYLCVNV was most similar (85.2 %) to that of PalCuCNV, the nucleotide sequence of the TYLCVNV V1 ORF was almost identical to that of ToLCVV (97.7 %), whilst the C1 nucleotide sequence showed most similarity to that of Ageratum yellow vein virus (AYVV, 88.5 %) (Table 2). Recombination analysis of the TYLCVNV genome (Fig. 2) identified a 929 nt region in V1 that shared 97.4 % identity with the cognate region of ToLCVV. This putative recombinant fragment encompassed 31 nt of the IR 3' terminus, the complete V2 ORF and most of V1, with the exception of 33 nt at the V1 3' terminus. The overall nucleotide identity of TYLCVNV and ToLCVV was 83.2 % and the TYLCVNV IR was most similar to that of ToLCVV (82.5 %) (Table 2). However, the arrangement and sequence of the TYLCVNV iterons and IRD were almost identical to those of AYVV (Fig. 1), suggesting that TYLCVNV may have emerged through two recombination events.
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). However, the nucleotide sequences of the ErYMV C1 ORF and IR were most similar to those of tomato yellow leaf curl China virus (TYLCCNV; 88.9 and 77.9 %, respectively) (Table 2). Recombination analysis (Fig. 2) identified a 451 nt region in the C1 ORF that shared 96.9 % identity with the cognate region in TYLCCNV, although the sequence identity of the complete genomes was only 81.4 %. This region spanned 364 nt downstream of the 5' end of the C1 ORF (covering 208 nt of the 5' end of the C4 ORF), to position 2680 (22 nt upstream of the TATA box, 71 nt downstream of the nicking site). The nucleotide sequence identity of ErYMV and PepLCV in this region was only 82.5 %. This putative recombinant region contained the iterons and the IRD and, whereas the virion-sense iteron sequences of ErYMV, TYLCCNV and PepLCV were all identical, the ErYMV IRD and complementary iteron sequences were more similar to those of TYLCCNV than to those of PepLCV (Fig. 1).
The nucleotide sequence of LuYVVNV was most similar to that of LuYVV from China, with nucleotide identities of 87.1 and 97 % for the complete genome and V1 ORF, respectively (Table 2). The high level of sequence identity over the entire genome was very close to the 89 % threshold for delineation of different viral species, suggesting that LuYVVNV and LuYVV may be two distal isolates of the same virus. However, sequence comparisons between LuYVVNV and LuYVV identified a highly disparate region of 586 nt in the LuYVVNV C1 ORF that extended to the 5' end of the stem–loop in the IR; the nucleotide identity of LuYVVNV and LuYVV in this region was only 55.6 %. Although no putative recombination events were detected by computing analysis, the LuYVVNV sequence in this region had higher identity with the corresponding fragment of tomato leaf curl Laos virus (ToLCLV; 86 %) than that of LuYVV, suggesting that this region was a putative recombinant fragment (Fig. 2). Indeed, the iterons and IRD of LuYVVNV were more similar to those of ToLCLV than to those of LuYVV (Fig. 1).
The isolates of the seven previously characterized begomoviruses identified in this study (TYLCKaV, ToLCVV, PaLCuCNV, LaYVV, AlYVV, SiLCV and LuYVV) all shared >93 % identity over the entire nucleotide sequence with the corresponding virus sequences in databases (Table 2). A putative recombinant region of 431 nt, located near the 5' end of the genome, was detected between SiLCV-[Vietnam : Thanhhoa : Abutilon : 2000 : 61] and Stachytarphyta leaf curl virus (StaLCuV) (Fig. 2).
DNA-β and DNA-1.
The DNA-β molecules associated with AlYVV, ErYMV, LaYVV, MiYLCV, PaLCuCNV, SiYVVNV, SiLCV and TYLCVNV ranged in size from 1322 to 1367 nt (Table 1) and each encoded one major complementary-sense ORF (βC1). Each DNA-β molecule contained (i) a putative stem–loop structure with the loop sequence, TAATATTAC, (ii) an SCR immediately upstream of the putative stem–loop sequence, and (iii) an adenosine (A)-rich region upstream of the SCR. Sequence comparisons of the complete DNA-β genomes (Table 4) revealed that they could be divided into two distinct groups. One group consisted of DNA-β molecules associated with MiYLCV, SiLCV and PaLCuCNV; these all had high sequence identities with the respective DNA-β sequences in GenBank (80.3 % for MiYLCVβ and GenBank accession no. AJ54249180; 92.5 % for SiLCVβ and AM050732
[GenBank]
; and 96 % for PaLCCNVβ and AJ971257
[GenBank]
). The second group, containing DNA-β molecules associated with AlYVV, ErYMV, LaYVV, SiYVVNV and TYLCVNV, shared <70 % sequence identity with known DNA-β molecules and each other, suggesting that they were all novel satellite molecules.
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). Each DNA contained one large ORF that encoded a putative protein of 315 aa; analyses of these sequences revealed high levels of similarity to the Rep proteins of nanoviruses. Each molecule also contained a putative stem–loop structure, containing the loop sequence TAGTATTAC and an A-rich region immediately downstream of the ORF. The DNA-1 molecules associated with MiYLCV, SiLCV and SiYVVNV had highest sequence identities with DNA-1 molecules associated with tobacco curly shoot virus (83.8 %), SiLCV (84.8 %) and TYLCCNV (69.9 %), respectively (Table 4).
Phylogenetic analyses
DNA-A and DNA-B.
Phylogenetic analyses based on the complete nucleotide sequences of DNA-A (Fig. 3a) showed that CoGMV and CoYVV formed a distinct clade that was related more closely to New World begomoviruses than to viruses from the Old World. KuMV grouped tightly with three Old World legume-infecting bipartite begomoviruses: HgYMV, mungbean yellow mosaic virus (MYMV) and mungbean yellow mosaic india virus (MYMIV), to form an intermediate clade. The remaining viruses isolated from Vietnam grouped into one major clade that included both bipartite and monopartite Old World begomoviruses. Nearly identical topologies were observed in phylogenetic trees constructed by using both the amino acid and nucleotide sequences of REn, TrAP and CP (data not shown). Similarly, phylogenetic analysis of DNA-B molecules using either the complete sequence or the BV1 and BC1 nucleotide and amino acid sequences showed that CoGMV and CoYVV clustered closer to New World viruses than to viruses from the Old World. Unlike the tree topology obtained by using DNA-A sequences, however, the legume-infecting viruses clustered more closely with cassava-infecting viruses [African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and South African cassava mosaic virus (SACMV)] from the Old World (data not shown). The separation of New World and Old World begomoviruses was less distinct using the AC1 sequence or the IR, and there was no separation between Old World and New World viruses using the AC4 sequences (data not shown).
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). All of the DNA-β molecules isolated from Vietnam fell within the non-Malvaceae clade. With the exception of LaYVV DNA-β, which was related more closely to sequences isolated from Japan and the UK, all DNA-β molecules from Vietnam grouped closely with molecules previously isolated from China or Laos. Interestingly, the tree topologies for many of the DNA-β molecules differed from that of their cognate DNA-A molecules, with the exception of viruses/DNA-β molecules isolated from sida in Vietnam and China and abutilon from Vietnam. In general, the DNA-β molecules isolated from Vietnam showed high sequence diversity, as can be seen from their distal positions on the trees.
Phylogenetic analyses of the nanovirus-like DNA-1 molecules also identified a high degree of sequence diversity (Fig. 3c). Similar to DNA-β, the DNA-1 molecule isolated from abutilon was related most closely to a DNA-1 molecule isolated from sida in China. The cognate DNA-A components associated with these satellite molecules from Vietnam and China were both associated with isolates of SiLCV. The DNA-1 molecule from mimosa formed a distinct branch between DNA-1 sequences originating from China. Although the DNA-1 sequence from sida formed a separate branch, its exact position in the tree was not well supported by bootstrap values. Similar to that observed for DNA-β, the DNA-1 sequences were positioned distally on the tree, indicating a high level of sequence diversity in Vietnam and suggesting that the sequences had been present in the region for a considerable period.
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DISCUSSION |
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). In addition, differences in the CoGMV and CoYVV iteron sequences and the results of phylogenetic analyses showed that CoGMV and CoYVV are distinct viruses. The genomes of CoGMV, CoYVV and other New World viruses shared many common features, including (i) they are all bipartite, (ii) all have a CP N-terminal 7-PWRsMaGT motif, (iii) all lack the second and third basic domains in the CP N-terminal region, and (iv) all lack an AV2 ORF. In addition, the identification of SiLCV infecting abutilon, PaLCuCNV infecting tobacco and AlYVV infecting zinnia and eclipta suggested that these viruses had a wider natural host range than reported previously (Guo & Zhou, 2005, 2006; Wang et al., 2004).
The basic domains in the CP N-terminal region form an essential part of the NLS in Old World viruses and their role in nuclear targeting has been demonstrated for TYLCV (Kunik et al., 1998), ACMV (Unseld et al., 2001) and MYMV (Guerra-Peraza et al., 2005). The reason for the absence of these domains in New World viruses is unclear. Bipartite viruses were thought to have evolved from monopartite viruses by gene duplication and/or DNA acquisition, with gene products encoded by DNA-B providing enhanced viral movement within the host (Rojas et al., 2005). The evolution of bipartite viruses is thought to have occurred before continental separation, due to the presence of bipartite viruses in both the Old and New Worlds (Rojas et al., 2005). All New World viruses lack the AV2 ORF, and it has been proposed that they evolved from a common ancestor that had lost the AV2 ORF after the Gondwana continental separation (Rybicki, 1994). However, the occurrence of both CoYVV (Ha et al., 2006) and CoGMV, bearing features similar to New World viruses, in Vietnam suggests that viruses with characteristics of New World viruses were present in the Old World prior to continental separation.
The mechanisms by which bipartite viruses evolved into distinct Old World and New World populations is unclear, although it is possible that this process involves the region encoding the AV2 ORF and the CP N-terminal region. Harrison et al. (2002) reported variability in the N-terminal 50 residues from 27 begomovirus CP sequences originating from six continents. Similarly, in a comparison of the CP N-terminal region of six Old World and four New World CP sequences, Sharma et al. (2005) observed that the first 50 aa were highly variable when comparing all viruses together, but were much more conserved when the two groups were compared separately (approx. 62 and 68 % identity, respectively). In the current study, comparison of the deduced CP sequences from a large number of New World and Old World viruses showed that their CPs were clearly divided into distinct N-terminal and C-terminal regions. The N-terminal region consisted of approximately 39 aa for the New World viruses and approximately 45 aa for the Old World viruses (Table 3). This region was relatively conserved within the two groups (mean 62.9 and 66.8 % identity, respectively), but differed markedly between them (mean 28.8 % identity) (Table 5). In contrast, the C-terminal CP region was conserved in all begomoviruses, irrespective of whether they were from the Old or New World (mean 80.5 % identity between the two groups; Table 5). The higher conservation (mean 92.7 % identity) in the C-terminal region of the New World viruses compared with the Old World viruses (mean 81.6 % identity) (Table 5) supports the hypothesis that New World viruses emerged more recently (Rybicki, 1994).
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; Rigden et al., 1993). The AV2 ORF (approx. 115 aa), which is lacking in New World viruses, completely overlaps the above-mentioned CP N-terminal region present in Old World viruses, suggesting that functions normally attributed to this CP region and AV2 are encoded by the DNA-B component present in all New World viruses. The presence of the N-terminal CP region and the AV2 gene in Old World viruses may explain why many of them are monopartite and also why DNA-B is not required for symptom expression in viruses such as TYLCTHV (Rochester et al., 1990) or Sri Lankan cassava mosaic virus (SLCMV) (Saunders et al., 2002).
Among the nine newly identified viruses in this current study, we had difficulties determining the taxonomic status of ErYMV and LuYVVNV. Both viruses had overall nucleotide identities close to the 89 % threshold (87.5 and 87.1 % identity with PepLCV and LuYVV, respectively). Fauquet (2002) reported that most viruses with approximately 87 % identity may be recombinants. Indeed, computer analysis detected a putative recombinant fragment in ErYMV that covered the Rep N-terminal region and Rep-binding site, which contains species-specific factors essential for replication (Arguello-Astorga et al., 1994; Arguello-Astorga & Ruiz-Medrano, 2001; Fontes et al., 1994a, b; Hanley-Bowdoin et al., 2000; Jupin et al., 1995). A putative recombinant fragment, at a similar position, was also detected in LuYVVNV by sequence comparison. Sequence analyses of these fragments suggested that ErYMV and LuYVVNV would have higher affinity, in terms of trans-replication, with TYLCCNV and ToLCLV, respectively, than with their most closely related viruses based on overall nucleotide sequence. This needs to be demonstrated experimentally, however, as the ability of viruses to trans-replicate is considered an important criterion in begomovirus classification (Fauquet et al., 2003). Strict application of the 89 % taxonomy rule and phylogenetic analysis also supported the classification of ErYMV and LuYVVNV as distinct virus species.
Although CoYVV and CoGMV share many features in common with New World viruses, their position on phylogenetic trees basal to the New World viruses suggests that they were distinct from New World viruses, but may share a common ancestor. This was further supported by the observations that they originated from the Old World and they shared very low overall sequence identity with the New World viruses [maximum 60.2 % for CoYVV (Ha et al., 2006); 58.4 % for CoGMV]. This scenario is very similar to that of the sweet potato viruses [sweet potato leaf curl virus (SPLCV), sweet potato leaf curl Georgia virus (SPLCGV) and Ipomoea yellow vein virus (IYVV)], which (i) are present in both the New World (Lotrakul et al., 1998) and the Old World (Briddon et al., 2006), (ii) have genome organizations typical of Old World monopartite viruses (Lotrakul & Valverde, 1999), and (iii) formed an evolutionary lineage independent of both Old World and New World viruses (Fauquet & Stanley, 2003). Our phylogenetic analysis, based on the complete DNA-A sequence, identified two geographically defined major clusters (Old World and New World viruses) and three other distinct clusters that were distinguished on the basis of host (legume, sweet potato and jute). The relatively intermediate positions of the sweet potato and jute viruses between the Old World and New World populations suggests that (i) geographical separation appears to play a less important role than proposed previously in evolution of the genus Begomovirus, and (ii) host factors appear to be the major selection force driving the evolution of the sweet potato and jute viruses.
Two unexpected observations from this study were (i) that the nonanucleotide sequence of CoGMV was TATTATTAC rather than TAATATTAC and (ii) that the putative stem sequences of DNA-A and DNA-B differed in KuMV. Although the third residue of the TATTATTAC sequence is variable among nanoviruses (TAT/GTATTAC) and animal circoviruses (T/C/AAT/GTATTAC) (Hattermann et al., 2003), this is the first report of such variability in geminiviruses. Similarly, KuMV is the first geminivirus identified with variability in the putative stem sequence. In all other bipartite begomoviruses characterized to date, the putative stem sequence is almost identical in both components, even in viruses exhibiting low identities in the remainder of the CR sequences (Chakraborty et al., 2003; Ha et al., 2006; Hill et al., 1998; Idris & Brown, 2004). These differences are unlikely to affect replication, however, as the presence of the stem–loop structure, and not the sequence of the stem itself, is important for DNA replication (Orozco & Hanley-Bowdoin, 1996). However, this remains to be demonstrated experimentally.
The majority of the viruses characterized during the current study were isolated from weeds that can serve as important virus reservoirs (Gilbertson et al., 1993; Stonor et al., 2003). Viruses in mixed infections may recombine, resulting in a novel virus that can cross host barriers. Indeed, we were able to detect a number of putative recombination events between SiLCV and StaLCuV, ErYMV and TYLCCNV, and TYLCVNV and ToLCVV, with the TYLCVNV iterons probably originating from an AYVV-like weed-infecting virus. In addition, the conserved nature of iteron sequences observed between distinct viruses such as ErYMV/TYLCCNV, LuYVVNV/ToLCLV and TYLCVNV/AYVV suggests that they could replicate in a trans-acting manner (Fontes et al., 1994a); this may facilitate gene transfer through recombination-dependent replication (Jeske et al., 2001).
The identification of CoGMV in this study, together with our previous characterization of CoYVV (Ha et al., 2006), provides strong evidence of a New World virus present in the Old World prior to Gondwana separation. This, together with a high degree of virus diversity that includes putative recombinant viruses, satellite molecules and viruses with previously undescribed variability in the putative stem–loop sequences, suggests that South-East Asia, and Vietnam in particular, is one of the origins of begomovirus diversity.
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ACKNOWLEDGEMENTS |
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Received 14 June 2007;
accepted 13 September 2007.
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