Browsing by Author "Varsani, A"

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    Open Access
    Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context
    (Public Library of Science, 25) Candresse, T; Filloux, D; Muhire, B; Julian, C; Galzi, S; Fort, G; Bernardo, P; Daugrois, J-H; Fernandez, E; Martin, D P; Varsani, A; Roumagnac, P
    Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share .91% genomewide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.
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    Begomovirus 'melting pot' in the south-west Indian Ocean islands: molecular diversity and evolution through recombination
    (Microbiology Society, 2007) Lefeuvre, P; Martin, D P; Hoareau, M; Naze, F; Delatte, H; Thierry, M; Varsani, A; Becker, N; Reynaud, B; Lett, J-M
    During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.
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    Begomovirus 'melting pot' in the south-west Indian Ocean islands: molecular diversity and evolution through recombination
    (Microbiology Society, 2007) Lefeuvre, P; Martin, D P; Hoareau, M; Naze, F; Delatte, H; Thierry, M; Varsani, A; Becker, N; Reynaud, B; Lett, J-M
    During the last few decades, many virus species have emerged, often forming dynamic complexes within which viruses share common hosts and rampantly exchange genetic material through recombination. Begomovirus species complexes are common and represent serious agricultural threats. Characterization of species complex diversity has substantially contributed to our understanding of both begomovirus evolution, and the ecological and epidemiological processes involved in the emergence of new viral pathogens. To date, the only extensively studied emergent African begomovirus species complex is that responsible for cassava mosaic disease. Here we present a study of another emerging begomovirus species complex which is associated with serious disease outbreaks in bean, tobacco and tomato on the south-west Indian Ocean (SWIO) islands off the coast of Africa. On the basis of 14 new complete DNA-A sequences, we describe seven new island monopartite begomovirus species, suggesting the presence of an extraordinary diversity of begomovirus in the SWIO islands. Phylogenetic analyses of these sequences reveal a close relationship between monopartite and bipartite African begomoviruses, supporting the hypothesis that either bipartite African begomoviruses have captured B components from other bipartite viruses, or there have been multiple B-component losses amongst SWIO virus progenitors. Moreover, we present evidence that detectable recombination events amongst African, Mediterranean and SWIO begomoviruses, while substantially contributing to their diversity, have not occurred randomly throughout their genomes. We provide the first statistical support for three recombination hot-spots (V1/C3 interface, C1 centre and the entire IR) and two recombination cold-spots (the V2 and the third quarter of V1) in the genomes of begomoviruses.
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    Open Access
    Evidence of pervasive biologically functional secondary-structures within the genomes of eukaryotic single-stranded DNA viruses
    (American Society for Microbiology, 2013) Muhire, B M; Golden, M; Murrell, B; Lefeuvre, P; Lett, J-M; Gray, A; Poon, Art Y F; Ngandu, N K; Semegni, Y; Tanov, E P; Monjane, A L; Harkins, G W; Varsani, A; Shepherd, D N; Martin, D P
    Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary-structures through Watson-Crick base-pairing between their constituent nucleotides. A few of the structural elements formed by such base-pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. What is unknown, however, is (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist, and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae andGeminiviridae, and analysed these for evidence of natural selection favouring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base-paired than it is at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary-structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterised structural elements that likely have important functions within some of the ssDNA virus genomes analysed here.
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    A genome-wide pairwise-identity-based proposal for the classification of viruses in the genus Mastrevirus (family Geminiviridae)
    (Springer, 2013) Muhire, B; Martin, D P; Brown, J K; Navas-Castillo, J; Moriones, E; Zerbini, F M; Rivera-Bustamante, R; Malathi, V G; Briddon, R W; Varsani, A
    Recent advances in the ease with which the genomes of small circular single-stranded DNA viruses can be amplified, cloned, and sequenced have greatly accelerated the rate at which full genome sequences of mastreviruses (family Geminiviridae, genus Mastrevirus) are being deposited in public sequence databases. Although guidelines currently exist for species-level classification of newly determined, complete mastrevirus genome sequences, these are difficult to apply to large sequence datasets and are permissive enough that, effectively, a high degree of leeway exists for the proposal of new species and strains. The lack of a standardised and rigorous method for testing whether a new genome sequence deserves such a classification is resulting in increasing numbers of questionable mastrevirus species proposals. Importantly, the recommended sequence alignment and pairwise identity calculation protocols of the current guidelines could easily be modified to make the classification of newly determined mastrevirus genome sequences significantly more objective. Here, we propose modified versions of these protocols that should substantially minimise the degree of classification inconsistency that is permissible under the current system. To facilitate the objective application of these guidelines for mastrevirus species demarcation, we additionally present a user-friendly computer program, SDT (species demarcation tool), for calculating and graphically displaying pairwise genome identity scores. We apply SDT to the 939 full genome sequences of mastreviruses that were publically available in May 2012, and based on the distribution of pairwise identity scores yielded by our protocol, we propose mastrevirus species and strain demarcation thresholds of >78 % and >94 % identity, respectively.
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    Panicum streak virus diversity is similar to that observed for maize streak virus
    (Springer Verlag, 2008) Varsani, A; Oluwafemi, S; Windram, P; Shepherd, D N; Monjane, A L; Owor, B E; Rybicki, E P; Lefeuvre, P; Martin, D P
    Panicum streak virus (PanSV; genus Mastrevirus, family Geminiviridae) is, together with maize streak virus (MSV), sugarcane streak virus (SSV), sugarcane streak Reunion virus (SSRV) and sugarcane streak Egypt virus (SSEV), one of the currently described “African streak virus” (AfSV) species [6]. As with all the other AfSV species other than MSV, very little is known about PanSV genomic sequence diversity across Africa. Only two PanSV full genome sequences have ever been reported: one from Kenya [2], and the other from South Africa [17]. Both these genomes were isolated from Panicum maximum plants, but share only approximately 90% sequence identity. The reason this is noteworthy is that throughout mainland Africa all MSV genomes ever sampled from maize have been found to share >97% sequence identity. Although other MSV strains sharing between 78 and 90% identity with the maize-adapted strain (MSV-A) have been described, these have all been isolated from different host species, indicating that host adaptation is probably the main force driving MSV diversification. MSV and PanSV share common vector species (leafhoppers in the genus Cicadulina) and probably also share some host species. Although the host range of PanSV is currently unknown, the MSV host range is extensive and includes P. maximum [3]. One might therefore expect that similar evolutionary forces acting on both species might result in their sharing similar patterns of both geographical and host-associated diversity. Here we describe the full genome sequences of five new PanSV isolates (including two new strains) sampled from southern and western Africa, and report that PanSV and MSV do indeed have similar patterns of diversity. We find, however, that unlike with MSV, geographical separation rather than host adaptation is possibly the dominant force driving PanSV diversification.
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    Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8
    (Springer Verlag, 2010) Williamson, D S; Dent, K C; Weber, B W; Varsani, A; Frederick, J; Thuku, R; Cameron, R A; van Heerden, J H; Cowan, D A; Sewell, B T
    Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for wellconserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of ~600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, “c-shaped”, circular and “figure-8” shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.