Browsing by Author "Shepherd, Dionne N"

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    Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of a recombinant variant.
    (Microbiology Society, 2007) Owor, Betty E; Martin, Darren P; Shepherd, Dionne N; Edema, Richard; Monjane, Ade´rito L; Rybicki, Edward P; Thomson, Jennifer A; Vasani, Arvind
    Maize streak virus (MSV) contributes significantly to the problem of extremely low African maize yields. Whilst a diverse range of MSV and MSV-like viruses are endemic in sub-Saharan Africa and neighbouring islands, only a single group of maize-adapted variants – MSV subtypes A1–A6 – causes severe enough disease in maize to influence yields substantially. In order to assist in designing effective strategies to control MSV in maize, a large survey covering 155 locations was conducted to assess the diversity, distribution and genetic characteristics of the Ugandan MSV-A population. PCR–restriction fragment-length polymorphism analyses of 391 virus isolates identified 49 genetic variants. Sixty-two full-genome sequences were determined, 52 of which were detectably recombinant. All but two recombinants contained predominantly MSV-A1-like sequences. Of the ten distinct recombination events observed, seven involved inter-MSV-A subtype recombination and three involved intra-MSV-A1 recombination. One of the intra-MSV-A1 recombinants, designated MSV-A1UgIII, accounted for .60 % of all MSV infections sampled throughout Uganda. Although recombination may be an important factor in the emergence of novel geminivirus variants, it is demonstrated that its characteristics in MSV are quite different from those observed in related African cassava-infecting geminivirus species.
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    Open Access
    A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family
    (BioMed Central, 2009) Varsani, Arvind; Shepherd, Dionne N; Dent, Kyle; Monjane, Aderito L; Rybicki, Edward P; Martin, Darren P
    Background: We have characterised a new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass. ECSV represents a new genus-level geminivirus lineage, and has a mixture of features normally associated with other specific geminivirus genera. Results: Whereas the ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses, curtoviruses and topocuviruses, its Rep also contains what is apparently a canonical retinoblastoma related protein interaction motif such as that found in mastreviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene found in begomoviruses, a mastrevirus-like coat protein gene and two intergenic regions. Conclusion: Although it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses. In addition to inferring how the ancestral geminivirus genome may have looked, we use the discovery of ECSV to refine various hypotheses regarding the recombinant origins of the major geminivirus lineages.
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    An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins
    (Springer Verlag (Germany), 2008) Windram, Oliver P; Weber, Brandon; Jaffer, Mohamed A; Rybicki, Edward P; Shepherd, Dionne N; Varsani, Arvind
    Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (*55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6–8 kb.
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    Maize streak virus: an old and complex 'emerging' pathogen
    (Wiley, 2010) Shepherd, Dionne N; Martin, Darren P; Van der Walt, Eric; Dent, Kyle; Varsani, Arvind; Rybicki, Edward P
    Maize streak virus (MSV; Genus Mastrevirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naudé and C. storeyi. In addition to maize, it can infect over 80 other species in the Family Poaceae.Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant streak disease in maize. Severe maize streak disease (MSD) manifests as pronounced, continuous parallel chlorotic streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in maize, and/or maize infections caused by non-maize-adapted MSV strains, can result in narrow, interrupted streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3–10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss.
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    Open Access
    A new African streak virus species from Nigeria
    (Springer Verlag, 2008) Oluwafemi, Sunday; Varsani, Arvind; Monjane, Ade´rito L; Shepherd, Dionne N; Owor, Betty E; Rybicki, Edward P; Martin, Darren P
    The African streak viruses (AfSVs) are a diverse group of mastrevirus species (family Geminiviridae) that infect a wide variety of annual and perennial grass species across the African continent and its nearby Indian Ocean islands. Six AfSV species (of which maize streak virus is the best known) have been described. Here we report the full genome sequences of eight isolates of a seventh AfSV species: Urochloa streak virus (USV), sampled from various locations in Nigeria. Despite there being good evidence of recombination in many other AfSV species, we found no convincing evidence that any of the USV sequences were either inter- or intra-species recombinants. The USV isolates, all of which appear to be variants of the same strain (their genome sequences are all more than 98% identical), share less than 69% nucleotide sequence identity with other currently described AfSV species.
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    Novel sugarcane streak and sugarcane streak Reunion mastreviruses from southern Africa and La Réunion.
    (Springer Verlag, 2008) Shepherd, Dionne N; Varsani, Arvind; Windram, Oliver P; Lefeuvre, Pierre; Monjane, Ade´rito L; Owor, Betty E; Martin, Darren P
    The sugarcane infecting streak viruses (SISVs) are mastreviruses (Family Geminiviridae) belonging to a group of ‘‘African streak viruses’’ (AfSVs) that includes the economically devastating Maize streak virus (MSV). Although there are three currently described SISV species (Sugarcane streak virus [SSV], Sugarcane streak Egypt virus [SSEV] and Sugarcane streak Re´union virus [SSRV]), only one strain variant has been fully sequenced for each of these species and as a result very little is known about the diversity and evolutionary origins of the SCISVs. Here we present annotated full genome sequences of four new SISV isolates, including a new strain of both SSRV and SSV, and one potentially new SISV species, sampled from wild grasses in La Re´union and Zimbabwe. For the first time, we report the finding of SSRV isolates in Zimbabwe and SSV isolates on the island of La Re´union. Phylogenetic and recombination analyses indicate continent-wide SSRV strain diversity and that our isolate potentially representing a new SISV species is a recombinant.
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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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    Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen.
    (Microbiology Society, 2008) Varsani, Arvind; Shepherd, Dionne N; Monjane, Ade´rito L; Owor, Betty E; Erdmann, Julia B; Rybicki, Edward P; Peterschmitt, Michel; Briddon, Rob W; Markham, Peter G; Oluwafemi, Sunday; Windram, Oliver P; Lefeuvre, Pierre; Lett, Jean-Michel; Martin, Darren P
    Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus), the causal agent of maize streak disease, ranks amongst the most serious biological threats to food security in subSaharan Africa. Although five distinct MSV strains have been currently described, only one of these – MSV-A – causes severe disease in maize. Due primarily to their not being an obvious threat to agriculture, very little is known about the ‘grass-adapted’ MSV strains, MSV-B, -C, -D and -E. Since comparing the genetic diversities, geographical distributions and natural host ranges of MSV-A with the other MSV strains could provide valuable information on the epidemiology, evolution and emergence of MSV-A, we carried out a phylogeographical analysis of MSVs found in uncultivated indigenous African grasses. Amongst the 83 new MSV genomes presented here, we report the discovery of six new MSV strains (MSV-F to -K). The non-random recombination breakpoint distributions detectable with these and other available mastrevirus sequences partially mirror those seen in begomoviruses, implying that the forces shaping these breakpoint patterns have been largely conserved since the earliest geminivirus ancestors. We present evidence that the ancestor of all MSV-A variants was the recombinant progeny of ancestral MSV-B and MSV-G/-F variants. While it remains unknown whether recombination influenced the emergence of MSV-A in maize, our discovery that MSV-A variants may both move between and become established in different regions of Africa with greater ease, and infect more grass species than other MSV strains, goes some way towards explaining why MSV-A is such a successful maize pathogen.
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    Replicative intermediates of maize streak virus found during leaf development
    (Microbiology Society, 2010) Erdmann, Julia B; Shepherd, Dionne N; Martin, Darren P; Varsani, Arvind; Rybicki, Edward P; Jeske, Holger
    Geminiviruses of the genera Begomovirus and Curtovirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombinationdependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that maize streak virus (MSV), the type member of the most divergent geminivirus genus, Mastrevirus, does the same. Although mastreviruses have fewer regulatory genes than other geminiviruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiviruses, MSV also apparently uses dimeric open circular DNA as a template for RCR.
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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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    Open Access
    The use of maize streak virus (MSV) replication-associated protein mutants in the development of MSV-resistant plants
    (2003) Shepherd, Dionne N; Thomson, Jennifer Ann; Rybicki, Edward P
    Maize streak virus (MSV) is the type member of the Mastrevirus genus of the Geminiviridae. As the causal agent of maize streak disease (MSD), MSV is the most significant pathogen of maize in Africa, resulting in crop yield losses of up to 100%. Transmitted by leafhoppers (Cicadulina spp.), MSV is indigenous to Africa and neighbouring Indian Ocean Islands. Despite maize being a crucial staple food crop in Africa, the average maize yield per hectare in Africa is the lowest in the world, leading to food shortages and famine. A major contributing factor to these low yields is MSD. To genetically engineer MSV-resistant maize using the pathogen-derived resistance (PDR) strategy, the viral replication-associated (Rep) protein gene was targeted, whose multifunctional products Rep and RepA are the only viral proteins essential for replication. Rep constructs had previously been made containing deleterious mutations in several conserved amino acid motifs. In this study, these mutants and the wild type Rep gene were truncated to remove key motifs involved in viral replication. A quantitative PCR assay was developed to determine the effects of the mutant and truncated Reps on viral replication in black Mexican sweetcorn (BMS) suspension cells. The MSVsensitive grass Digitaria sanguinalis was then transformed with Rep constructs that inhibited MSV replication in BMS, and transgenic lines were tested for virus resistance. Several plants of a D. sanguinalis line transgenic for a mutated full-length Rep gene showed excellent resistance (immunity) to MSV, but the transgene had negative effects on aspects of plant growth and development. Transformation with a mutated/truncated Rep gene resulted in healthy fertile transgenic D. sanguinalis plants, many of which showed good MSV resistance. Fertile maize (Hi-II) T 1 transgenic plants expressing the truncated/mutated Rep gene have been obtained, the offspring of which will be tested for resistance to MSV. Considering the success in achieving MSV-resistant D. sanguinalis, there is good reason to believe that the transgenic maize will too be resistant to MSV.