Phylogenetic analysis of Melon chlorotic leaf curl virus from Guatemala: another emergent species in the Squash leaf curl virus clade

The genome of a new bipartite begomovirus Melon chlorotic leaf curl virus from Guatemala (MCLCuV-GT) was cloned and the genome sequence was determined. The virus causes distinct symptoms on melons that were not previously observed in melon crops in Guatemala or elsewhere. Phylogenetic analysis of MCLCuV-GT and begomoviruses infecting cucurbits and other host plant species indicated that its closest relative was MCLCuV from Costa Rica (MCLCuV-CR). The DNA-A components of two isolates shared 88.8% nucleotide identity, making them strains of the same species. Further, both MCLCuV-GT and MCLCuV-CR grouped with other Western Hemisphere cucurbit-infecting species in the SLCV-clade making them the most southerly cucurbit-infecting members of the clade to date. Although the common region of the cognate components of MCLCuV-GT and MCLCuV-CR, shared ～96.3% nucleotide identity. While DNA-A and DNA-B components of MCLCuV-GT were less than 86% nucleotide identity with the respective DNA-A and DNA-B common regions of MCLCuV-CR. The late viral genes of the two strains shared the least nt identity (<88%) while their early genes shared the highest nt identity (>90%). The collective evidence suggests that these two strains of MCLCuV are evolutionarily divergent owing in part to recombination, but also due to the accumulation of a substantial number of mutations. In addition they are differentially host-adapted, as has been documented for other cucurbit-infecting, bean-adapted, species in the SLCV clade.     

Molecular characterization of sweet potato leaf curl virus (SPLCV) isolates from Korea: phylogenetic relationship and recombination analysis

The complete DNA genome of sweet potato leaf curl virus (SPLCV) from samples obtained from eight regions was amplified by PCR and characterized in this study. The DNA genome of one group (SPLCV Korea group 1) consisted of 2828 nucleotides and that of the second group (SPLCV Korea group 2) consisted of 2829 nucleotides. Sequence comparisons showed that the genome sequences of SPLCV Korea isolates were closely related to those of SPLCV Brazil isolates (FJ969834, FJ969835, and FJ969836), SPLCV Japan isolate (AB433788), and SPLCV USA isolate (AF104036) with nucleotide sequence identity values ranging from 96-98%. Analysis of the phylogenetic relationship of SPLCV Korea isolates with other begomoviruses revealed that the majority of SPLCV Korea isolates were clustered with SPLCV Brazil isolates (FJ969834, FJ969835, and FJ969836). Recombination analysis results revealed three recombinations among SPLCV Korea isolates, SPLCV isolates from Brazil and Japan, and ipomoea yellow vein virus (IYVV) Italy isolate.     

Molecular characterization of Tobacco leaf curl Pusa virus, a new monopartite Begomovirus associated with tobacco leaf curl disease in India

Leaf curl disease of tobacco (TbLCD) is endemic in India. A monopartite Begomovirus, a betasatellite and an alphasatellite were found associated with the disease in Pusa, Bihar. The DNA-A of the Begomovirus associated with TbLCD in Pusa, Bihar was found to comprise of 2707 nt with a typical Old World begomovirus-like genome organization. The full-length sequence of DNA-A [HQ180391] showed that the Pusa isolate is a newly described member of the genus Begomovirus, as it had <89% sequence homology with DNA-A of all the known begomoviruses. The isolate is tentatively named as Tobacco leaf curl Pusa virus [India:Pusa:2010]. The betasatellite (HQ180395) associated with TbLCD in Pusa was identified as a variant of Tomato leaf curl Bangladesh betasatellite [IN:Raj:03], with which it shared 90.4% sequence identity. The alphasatellite (HQ180392) associated with the disease had highest 87% nucleotide sequence identity with Tomato leaf curl alphasatellite. The Begomovirus, betasatellite, and alphasatellite associated with TbLCD in Pusa, Bihar, India were found to be recombinants of extant begomoviruses, betasatellites and alphasatellites spreading in the Indian sub-continent and South-East Asia.     

Complete nucleotide sequence of Iranian tomato yellow leaf curl virus isolate: further evidence for natural recombination amongst begomoviruses

Summary. Complete nucleotide sequence of the Iranian strain of tomato yellow leaf curl virus (TYLCV-IR) was determined and compared with some begomoviruses. The complete sequence of TYLCV-IR clustered together with TYLCV and TYLCV-MId from Israel. A similar relationship holds when the deduced amino acid sequences of V1, V2, C2 and C3 and nucleotide sequences of IR, and RIR were compared. In contrast, phylogenetic analyses of amino acid sequences of C4, C1, and nucleotide sequences of LIR revealed that TYLCV-IR clustered with TLCIRV and two Indian species: ToLCBV- [Ban4], and ToLCKV. The phylogenetic analyses, Recombination Detection Program analyses, and sequence alignment survey provided evidence of the occurrence of recombination between an Israeli TYLCV-MId, as major parent, and TLCIRV, as minor parent. In this recombination event, a region (from nt 2149 to 2766) of TYLCV-MId genome were replaced with corresponding genome sequences of TLCIRV (RDP P-value = 5.976 × 10^–72), which include LIR, C4, and N-terminal of C1. Infectivity of the cloned TYLCV-IR genome was demonstrated by successful agroinoculation of tomato (Lycopersicon esculentum) and other plant species. The disease was transmitted by the natural vector Bemisia tabaci from agroinoculated plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-IR consists of only one circular single-stranded DNA molecule.     

Transreplication of a Tomato yellow leaf curl Thailand virus DNA-B and replication of a DNAbeta component by Tomato leaf curl Vietnam virus and Tomato yellow leaf curl Vietnam virus

The genomes of two tomato-infecting begomoviruses from Vietnam were cloned and sequenced. A new variant of Tomato leaf curl Vietnam virus (ToLCVV) consisting of a DNA-A component and associated with a DNAbeta molecule as well as an additional begomovirus tentatively named Tomato yellow leaf curl Vietnam virus (TYLCVV) consisting also of a DNA-A component were identified. To verify if monopartite viruses occurring in Vietnam and Thailand are able to transreplicate the DNA-B component of Tomato yellow leaf curl Thailand virus-[Asian Institute of Technology] (TYLCTHV-[AIT]) infectivity assays were performed via agroinoculation and mechanically. As result, the DNA-B component of TYLCTHV-[AIT] was transreplicated by different DNA-A components of viruses from Vietnam and Thailand in Nicotiana benthamiana and Solanum lycopersicum. Moreover, the TYLCTHV-[AIT] DNA-B component facilitated the mechanical transmission of monopartite viruses by rub-inoculation as well as by particle bombardment in N. benthamiana and tomato plants. Finally, defective DNAs ranging from 735 to 1457 nucleotides were generated in N. benthamiana from those combinations containing TYLCTHV-[AIT] DNA-B component.     

Genetic diversity of tomato-infecting Tomato yellow leaf curl virus (TYLCV) isolates in Korea

Epidemic outbreaks of Tomato yellow leaf curl virus (TYLCV) diseases occurred in greenhouse grown tomato (Solanum lycopersicum) plants of Busan (TYLCV-Bus), Boseong (TYLCV-Bos), Hwaseong (TYLCV-Hwas), Jeju Island (TYLCV-Jeju), and Nonsan (TYLCV-Nons) in Korea during 2008-2009. Tomato disease by TYLCV has never occurred in Korea before. We synthesized the full-length genomes of each TYLCV isolate from the tomato plants collected at each area and determined their nucleotides (nt) sequences and deduced the amino acids of six open reading frames in the genomes. TYLCV-Bus and -Bos genomes shared higher nt identities with four Japanese isolates -Ng, -Omu, -Mis, and -Miy. On the other hand, TYLCV-Hwas, -Jeju, and -Nons genomes shared higher nt identities with five Chinese isolates TYLCV-AH1, -ZJ3, -ZJHZ12, -SH2, -Sh10, and two Japanese isolates -Han and -Tosa. On the basis of a neighbor-joining tree, five Korean TYLCV isolates were separated into three clades. TYLCV-Bus and -Bos formed the first clade, clustering with four Japanese isolates TYLCV-Mis, -Omu, -Ng, and -Miy. TYLCV-Jeju and -Nons formed the second clade, clustering with two Chinese isolates -ZJHZ212 and -Sh10. TYLCV-Hwas was clustered with two Japanese isolates -Han and -Tosa and three Chinese isolates -AH1, -ZJ3, and -SH2. Two fragments that had a potentially recombinant origin were identified using the RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, and 3Seq methods implemented in RDP3.41. On the basis of RDP analysis, all TYLCV isolates could originated from the interspecies recombination between TYLCV-Mld[PT] isolated from Portugal as a major parent and TYLCTHV-MM isolated from Myanmar as a minor parent.     

Infectivity of Euphorbia leaf curl virus and interaction with Tomato yellow leaf curl China betasatellite

To investigate the infectivity of Euphorbia leaf curl virus (EuLCV), an infectious clone was constructed and tested by agroinoculation and whitefly inoculation. EuLCV infected Nicotiana benthamiana, N. glutinosa, Solanum lycopersicum, Petunia hybrida efficiently upon agroinoculation and induced leaf curling, vein swelling and stunting in these plants but no symptoms in N. tabacum. Co-inoculation of EuLCV with a betasatellite DNA from an unrelated begomovirus enhanced symptoms in N. benthamiana, N. glutinosa, N. tabacum, S. lycopersicum and P. hybrida plants but had no effect on the accumulation of EuLCV DNA. Euphorbia pulcherrima plants were only infectable by insect transmission from agro-infected P. hybrida as a source. This is the first report about a monopartite begomovirus that has been reintroduced into a plant of the genus Euphorbia.     

Molecular characterization of squash leaf curl Yunnan virus,a new begomovirus and evidence for recombination

Summary. Virus isolate Y23V, obtained from squash showing leaf curl symptoms in Yunnan, China, was readily differentiated from four studied Chinese begomovirus isolates in reactions with a set of monoclonal antibodies raised against begomoviruses. The complete nucleotide sequence (2714nts) of the DNA-A-like molecule of Y23V was determined. The DNA-A of Y23V is most closely related to that of tomato yellow leaf curl Thailand virus-[1] (TYLCTHV-[1]) (84% sequence identity). However, the AC1 and AC4 gene of Y23V DNA-A resembled to Pepper leaf curl virus from Bangladesh (PepLCBDV). The DNA-A of Y23V has three distinct regions: the region from 74-2071nts is 95% identical to TYLCTHV-[1] excluding a 27nt deletion; the following 386nts are 91% identical to PepLCBDV and the rest of the DNA-A is not closely related to any reported begomovirus. Y23V, therefore, is considered to have arisen by recombination. The 84% sequence identity of Y23V with TYLCTHV-[1] allows Y23V to be considered as a distinct begomovirus species, for which the name squash leaf curl Yunnan virus (SLCYNV) is proposed.Received August 5, 2003; accepted May 14, 2003     

A new strain of tomato severe leaf curl virus and a unique variant of tomato yellow leaf curl virus from Mexico

The complete genome sequence of a distinct variant of tomato yellow leaf curl virus-Israel (TYLCV-IL) and the DNA-A sequence of a new strain of tomato severe leaf curl virus (ToSLCV) isolated in San Luis Potosi, Mexico, are described and analyzed. The TYLCV-IL[MX:SLP:11] variant differs from all TYLCV-IL isolates described so far by a unique 42-nt duplicated sequence comprising a part of the conserved stem-loop element of the virion-strand replication origin and adjacent regulatory sequences. TYLCV-IL[MX:SLP:11] was associated with tomato chino La Paz virus (ToChLPV-B[MX:SLP:11]) in a Solanum pimpinellifolium plant, and with pepper huasteco yellow vein virus (PHYVV-[MX:SLP:11]) and ToSLCV-GT[MX:SLP:11] in a Solanum lycopersicum plant. In addition, a distinct ToSLCV exhibiting low sequence identity (<89?%) to other ToSLCV isolates from Mexico was found in a tomato plant collected in the same field. Sequence analysis of this new ToSLCV strain indicates that it is a recombinant of close relatives of ToSLCV-GT[MX:SLP:11] and ToChLPV-B[MX:SLP:11] found in mixed infections with TYLCV-IL[MX:SLP:11].     

Complete nucleotide sequence of chili leaf curl virus and its associated satellites naturally infecting potato in Pakistan

Archives of Virology -     

Molecular characterization of Chilli leaf curl virus and satellite molecules associated with leaf curl disease of Amaranthus spp

Amaranthus, collectively known as amaranth, is an annual or short-lived perennial plant used as leafy vegetables, cereals and for ornamental purposes in many countries including India. During 2011, leaf samples of Amaranthus plants displaying leaf curling, leaf distortion, leaf crinkling and yellow leaf margins were collected from Banswara district, Rajasthan in India. Full-length clones of a monopartite begomovirus, a betasatellite and an alphasatellite were characterized. The complete nucleotide sequence of the isolated begomovirus features as a typical ‘Old World’ begomovirus with the highest nucleotide per cent identity with Chilli leaf curl virus and hence, considered as an isolate of Chilli leaf curl virus. The complete nucleotide sequences of betasatellite and alphasatellite possess maximum nucleotide identity with Tomato yellow leaf curl Thailand betasatellite and Chilli leaf curl alphasatellite, respectively. This is the first report of the association of chilli-infecting begomovirus and satellite molecules infecting a new host, Amaranthus, causing leaf curl disease.     

The complete nucleotide sequence of an isolate of Tomato yellow leaf curl Sardinia virus found in Sicily

Partial sequences of Tomato yellow leaf curl Sardinia virus (TYLCSV) derived from tomato samples collected in Sicily in 1999, 2002 and 2004 indicated the presence of a TYLCSV different from the one previously described as the Sic strain. Here, we report a complete DNA sequence that is classified as belonging to the TYLCSV type strain (Sar strain), confirming the co-existence in Sicily of virus populations of both strains. Moreover, comparisons between this new sequence and those of the two recombinants recently described in Sicily revealed unequivocally (99% identity) that their TYLCSV-derived portion originated from the Sar strain.     

Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus

Summary. Tomato plants grown in greenhouses in Thailand developed typical symptoms of a tomato yellow leaf curl Thailand virus (TYLCTHV) infection. After confirmation by ELISA, a Phi29 DNA polymerase approach was chosen for further molecular analysis of TYLCTHV. Total DNA purified from infected tomato leaves was subjected to rolling-circle amplification (RCA) of DNA-A and DNA-B of TYLCVTHV. In addition, a new monopartite geminivirus with a putative recombinant background was identified by RCA and tentatively named tobacco leaf curl Thailand virus (TbLCTHV). To confirm the composition of both geminiviruses, full-length clones were established and used for inoculation of Nicotiana benthamiana by particle bombardment or agroinfection. When TYLCTHV DNA-A and DNA-B were applied together by particle bombardment or agroinfection, severe stunting, yellowing, and leaf curling were observed. Whereas TYLCTHV DNA-A and TbLCTHV revealed no infection after'particle bombardment, similar symptoms in N. benthamiana, like leaf upward curling and yellowing were observed following agroinfection. DNA components of TYLCTHV DNA-A and DNA-B were excised from their respective plasmids, ligated, and amplified by Phi29 DNA polymerase. The ability of viral concatamere inoculation was evaluated in particle co-bombardment experiments on N. benthamiana. Thus, particle bombardment of RCA-derived multimeric products proved to be at least as effective as inoculation with a partial repeat construct and tenfold as effective as inoculation with excised unit-lengths of DNA-A and DNA-B of TYLCVTHV when using each DNA component in an amount of 5 ng.     

Phylogenetic and recombination analysis of genomic sequences of PCV2 isolated in Korea

The complete genomic sequences of 13 PCV2 viruses obtained between 2005 and 2007 were analyzed in order to determine their phylogenetic relationship and identify possible recombination events between PCV2a and PCV2b. Twelve PCV2b viruses and one PCV2a virus were identified by phylogenetic analysis. Notably, two PCV2b viruses (PF163 and C7201-1) were shown to belong to the 1B subgroup of PCV2b, which had not been previously reported in Korea. Theses two viruses were also predicted to be possible recombinants between PCV2a (the minor parent) and PCV2b (the major parent) by the RDP program (P < 0.01). A recombination site was predicted to exist in ORF1 of both viruses. This additional evidence of PCV2 recombination in Korea further supports the important role of recombination in genetic evolution.     

The begomoviruses Honeysuckle yellow vein mosaic virus and Tobacco leaf curl Japan virus with DNAbeta satellites cause yellow dwarf disease of tomato

The complete nucleotide sequences of two begomoviruses (Nara virus-1 and Nara virus-2), a satellite DNA (DNAbeta-Nara) and defective DNAs were obtained from honeysuckle (Lonicera japonica) showing characteristic yellow vein mosaic symptoms in Nara Prefecture, Japan. One begomovirus (Ibaraki virus) and a satellite DNA (DNAbeta-Ibaraki) was isolated and cloned from honeysuckle plants exhibited typical yellowing of veins and small elliptical shaped enations along veins on the under side of the leaves in Ibaraki Prefecture, Japan. The genome organization of the three viruses is the same as those of other Old World monopartite begomoviruses. Nara virus-1 had overall nucleotide sequence identity with Nara virus-2 of 94% and Ibaraki virus of 90%. DNAbeta-Nara had overall nucleotide sequence identity with DNAbeta-Ibaraki of 83%. Comparison of the nucleotide sequences with other begomoviruses revealed that Nara virus-1 and Nara virus-2 are strains of Honeysuckle yellow vein mosaic virus (HYVMV), hence named as HYVMV-Nara1 and HYVMV-Nara2, whereas Ibaraki virus was a strain of Tobacco leaf curl Japan virus (TbLCJV), designated as TbLCJV-Hs[Iba]. HYVMV-Nara1 and HYVMV-Nara2 have hybrid genomes, which are likely to have formed recombination between HYVMV and TbLCJV. TbLCJV-Hs[Iba] or HYVMV-Nara2 could infect and cause yellowing, leaf crinkling and stunting symptoms when partial tandem dimeric constructs were agroinoculated on tomato plants. However, in the presence of DNAbeta, both TbLCJV-Hs[Iba] or HYVMV-Nara2 produced more severe stunting symptoms in tomato plants. Therefore, these viruses along with their satellites are causal agents of tomato yellow dwarf disease in Japan, and honeysuckle acts as a potential reservoir host. Previously available evidence indicated that DNAbeta elements do not contain iteron sequences of their helper viruses; hence this is the first evidence that DNAbeta satellites have the iteron of their helper virus.     

Molecular characterization and pathogenicity of tomato yellow leaf curl virus in China

Several tomato production regions in China were surveyed for tomato yellow leaf curl disease (TYLCD), and 31 tomato leaf samples showing TYLCD-like symptoms were collected. The partial or full-length genomes of these isolates were sequenced and tomato yellow leaf curl virus (TYLCV) was detected in Shanghai, Zhejiang, Jiangsu Shandong and Hebei provinces of China. The TYLCV isolates found in China share high sequence identity (>98%) and have more than 97% sequence identity with TYLCV-IL[IL:Reo] (X15656). Phylogenetic relationship analysis reveals that although with little genetic variability, they can form two groups and all the TYLCV isolates in China belong to the group I. An infectious clone of TYLCV-[CN:SH2] (AM282874) was constructed and agro-inoculated into Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Solanum lycopersicum, Petunia hybrida, Cucumis sativus, Gossypium hirsutum, S. melongena, and Capsicum annuum. TYLCV-[CN:SH2] can induce severe leaf curling and stunting symptoms in these plants except C. sativus, G. hirsutum, S. melongena and C. annuum. We verified that TYLCV can trans-replicate tomato yellow leaf curl China virus DNA-β in N. benthamiana and S. lycopersicum and induced more severe symptoms with distortion and yellow vein.     

Characterization and transient replication of tomato leaf curl virus defective DNAs

Summary. Distinct subgenomic DNA species known as defective (df) DNA molecules were found in plants infected with tomato leaf curl virus (TLCV). Four df DNAs derived from TLCV Type and Darwin 1 strains were found to contain large deletions that disrupt all of the viral genes required for viral replication, encapsidation and spread. However, the viral origin of replication (ori), including the replication-associated protein (Rep) binding domains, was present in all four df DNAs. Co-agroinfection of leaf strips with tandem repeat constructs of the viral and df DNAs resulted in their replication in the presence of the respective TLCV strain. However, the df DNAs failed to move in whole plants when co-inoculated with TLCV. The df DNAs were shown to be associated with TLCV coat protein, which may indicate encapsidation. Mutational analysis showed the minimum sequence requirements for df DNA replication by TLCV to be the intergenic region containing the Rep-binding domains.     

Replicative intermediates of Tomato leaf curl virus and its satellite DNAs

Several plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA beta satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates.