Browsing by Subject "Geminivirus"

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    Molecular diversity of Chickpea chlorotic dwarf virus in Sudan: High rates of intra-species recombination – a driving force in the emergence of new strains
    (Elsevier, 2015) Kraberger, S; Kumari, S G; Hamed, A A; Gronenborn, B; Thomas, J E; Sharman, M; Harkins, G W; Muhire, B M; Martin, D P; Varsan, A
    Sudan Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae) is an important pathogen of pulses that are grown both for local consumption, and for export. Although a few studies have characterised CpCDV genomes from countries in the Middle East, Africa and the Indian subcontinent, little is known about CpCDV diversity in any of the major chickpea production areas in these regions. Here we analyse the diversity of 146 CpCDV isolates characterised from pulses collected across the chickpea growing regions of Sudan. Although we find that seven of the twelve known CpCDV strains are present within the country, strain CpCDV-H alone accounted for 73% of the infections analysed. Additionally we identified four new strains (CpCDV-M, -N, -O and -P) and show that recombination has played a significant role in the diversification of CpCDV, at least in this region. Accounting for observed recombination events, we use the large amounts of data generated here to compare patterns of natural selection within protein coding regions of CpCDV and other dicot-infecting mastrevirus species.
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    A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue
    (Elsevier, 2008) Shepherd, Dionne N; Martin, Darren P; Lefeuvre, Pierre; Monjane, Aderito L; Owor, Betty E; Rybicki, Edward P; Varsani, Arvind
    A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-AmpTM-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA.
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    Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism
    (Elsevier, 2006) Shepherd, Dionne N; Martin, Darren P; Varsani, Arvind; Thomson, Jennifer A; Rybicki, Edward P; Klump, Horst H
    We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of singlestranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely aVected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold diVerently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic eVect, which might have broad implications in the evolution of plants and their viruses.
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    Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes
    (Elsevier, 2007) Owor, Betty E; Shepherd, Dionne N; Taylor, Nigel J; Edema, Richard; Monjane, Aderito L; Thomson, Jennifer A; Martin, Darren P; Varsani, Arvind
    Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers’ fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA® Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage 29 DNA polymerase using the TempliPhiTM system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the 29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.
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    Turnip curly top virus, a highly divergent geminivirus infecting turnip in Iran
    (Elsevier, 2010) Briddon, Rob W; Heydarnejad, Jahangir; Khosrowfar, Fakhrosadat; Massumi, Hossain; Martin, Darren P; Varsani, Arvin
    From 2006 onwards turnip crops in Fars province, Iran, have been noted with unusual leaf curling and vein swelling symptoms which are characteristic of the leafhopper-transmitted viruses of the genus Curtovirus (family Geminiviridae). Rolling circle amplification was used to clone viruses from five turnip isolates exhibiting leaf curl symptoms. Analysis of the sequences showed them to have >93% sequence identity and to be distinct from all other geminiviruses previously characterised. Analysis of the sequence of this virus, for which we propose the name Turnip curly top virus (TCTV), showed it to have a genome arrangement in the complementary-sense similar to that of curtoviruses (consisting of four overlapping genes) but only two open reading frames in the virion-sense (the curtoviruses encode three). The complementarysense genes are homologous to those of curtoviruses but show little sequence identity to their curtovirus homologs, with the exception of the product of the C4 open reading frame (ORF) which shows ∼70.6% amino acid sequence identity to the C4 of the North American curtoviruses, Pepper curly top virus and Beet mild curly top virus. For curtoviruses the C4 protein is a symptom determinant, which likely explains the similarity of TCTV symptoms to those of curtoviruses. In the virion-sense the predicted product of the V2 ORF of TCTV shows no significant similarity with any proteins in the databases whereas the product of the V1 ORF (encoding the coat protein [CP] of geminiviruses) shows low levels of sequence identity to the CPs of curtoviruses. These findings show TCTV to be a highly divergent geminivirus with similarities to viruses of the genus curtovirus. The significance of these findings, particularly the taxonomic implications are discussed.