[Show abstract][Hide abstract] ABSTRACT: 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 and Geminiviridae, 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.
Journal of Virology 02/2014; 88:1972-1989. DOI:10.1128/JVI.03031-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Initially designed to infer evolutionary relationships based on morphological and physiological characters, phylogenetic reconstruction methods have greatly benefited from recent developments in molecular biology and sequencing technologies with a number of powerful methods having been developed specifically to infer phylogenies from macromolecular data. This chapter, while presenting an overview of basic concepts and methods used in phylogenetic reconstruction, is primarily intended as a simplified step-by-step guide to the construction of phylogenetic trees from nucleotide sequences using fairly up-to-date maximum likelihood methods implemented in freely available computer programs. While the analysis of chloroplast sequences from various Vanilla species is used as an illustrative example, the techniques covered here are relevant to the comparative analysis of homologous sequences datasets sampled from any group of organisms.
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.
PLoS ONE 11/2013; 8(11):e79704. DOI:10.1371/journal.pone.0079704 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens.
PLoS ONE 05/2013; 8(5):e63155. DOI:10.1371/journal.pone.0063155 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two complete nucleotide sequences of an alphasatellite isolated from a cassava plant with mosaic disease symptoms in Madagascar are described and analyzed. While the helper begomovirus was identified as an isolate of East African cassava mosaic Kenya virus (EACMKV), its associated alphasatellite was most closely related (80 % nucleotide sequence identity) to cotton leaf curl Gezira alphasatellite. These satellite molecules have typical features of alphasatellites, with a single gene in the virion sense, an A-rich region and a stem-loop structure. According to the proposed species demarcation threshold of alphasatellites (83 % nucleotide identity), they are isolates of a new species for which we propose the name "Cassava mosaic alphasatellite".
Archives of Virology 03/2013; 158(8). DOI:10.1007/s00705-013-1664-0 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cassava is a vital crop in Africa and represents the main food crop in Central African Republic (CAR). CAR has recently faced large reductions in cassava yields that have led to a surge in market prices. To better understand the causes of the reduction in yield, we identified biotic constraints to cassava production by means of a large-scale plant epidemiological survey conducted in 2007 and 2008. Standard protocols were used for the assessment of the major cassava pests and diseases. Cassava mosaic disease (CMD) was shown to be the most serious constraint to cassava in CAR, with symptoms observed at all localities surveyed. CMD is distributed throughout the country, with an average incidence of 85%. Importantly, 94% of diseased plants had cutting-derived CMD infection suggesting that farmers mostly use virus-infected cuttings for planting. PCR amplification and direct sequencing of partial fragments of the Rep ORF revealed that the causal agents of CMD in CAR are African cassava mosaic virus (ACMV) and the Uganda strain of East African cassava mosaic virus (EACMV-UG). We also demonstrated that 58% of CMD samples present mixed infections (ACMV and EACMV-UG) and that these samples had significantly higher symptom severities. Our results suggest that mixed infection and synergism between CMGs, could be an important feature in the yield reduction of cassava plants in CAR, similar to the other severe CMD epidemics reported in East Africa.
[Show abstract][Hide abstract] ABSTRACT: Background
Single-stranded (ss) DNA viruses in the family Geminiviridae are proving to be very useful in real-time evolution studies. The high mutation rate of geminiviruses and other ssDNA viruses is somewhat mysterious in that their DNA genomes are replicated in host nuclei by high fidelity host polymerases. Although strand specific mutation biases observed in virus species from the geminivirus genus Mastrevirus indicate that the high mutation rates in viruses in this genus may be due to mutational processes that operate specifically on ssDNA, it is currently unknown whether viruses from other genera display similar strand specific mutation biases. Also, geminivirus genomes frequently recombine with one another and an alternative cause of their high mutation rates could be that the recombination process is either directly mutagenic or produces a selective environment in which the survival of mutants is favoured. To investigate whether there is an association between recombination and increased basal mutation rates or increased degrees of selection favoring the survival of mutations, we compared the mutation dynamics of the MSV-MatA and MSV-VW field isolates of Maize streak virus (MSV; Mastrevirus), with both a laboratory constructed MSV recombinant, and MSV recombinants closely resembling MSV-MatA. To determine whether strand specific mutation biases are a general characteristic of geminivirus evolution we compared mutation spectra arising during these MSV experiments with those arising during similar experiments involving the geminivirus Tomato yellow leaf curl virus (Begomovirus genus).
Although both the genomic distribution of mutations and the occurrence of various convergent mutations at specific genomic sites indicated that either mutation hotspots or selection for adaptive mutations might elevate observed mutation rates in MSV, we found no association between recombination and mutation rates. Importantly, when comparing the mutation spectra of MSV and TYLCV we observed similar strand specific mutation biases arising predominantly from imbalances in the complementary mutations G → T: C → A.
While our results suggest that recombination does not strongly influence mutation rates in MSV, they indicate that high geminivirus mutation rates are at least partially attributable to increased susceptibility of all geminivirus genomes to oxidative damage while in a single stranded state.
[Show abstract][Hide abstract] ABSTRACT: In the last 20 years, molecular ecology approaches have proven to be extremely useful to identify and assess factors associated with viral emerging diseases, particularly in economically and socially important tropical crops such as maize (maize streak disease) and cassava (cassava mosaic disease). Molecular ecology approaches were applied in Reunion Island to analyze the epidemic of tomato yellow leaf curl disease, which has been affecting the island since the end of the 1990s. Before the invasive biotype B (currently known as Middle East-Asia Minor 1 cryptic species) of Bemisia tabaci spread across the world, Reunion Island (South West Indian Ocean) only hosted an indigenous biotype of B. tabaci, Ms (currently known as Indian Ocean cryptic species). Wild hybrids between invasive and indigenous species were subsequently characterized over multiple generations. Endosymbiont analysis of the hybrid population indicated that matings were non-random. Similarly, while no indigenous begomoviruses have ever been reported on Reunion Island, the two main strains of one of the most damaging and emerging plant viruses in the world, the Mild and Israel strains of the Tomato yellow leaf curl virus (TYLCV-Mld and TYLCV-IL), were introduced in 1997 and 2004 respectively. While these introductions extensively modified the agricultural landscape of Reunion Island, they also provided an invaluable opportunity to study the ecological and genetic mechanisms involved in biological invasion and competition.
[Show abstract][Hide abstract] ABSTRACT: Background
Cassava (Manihot esculenta) is a major food source for over 200 million sub-Saharan Africans. Unfortunately, its cultivation is severely hampered by cassava mosaic disease (CMD). Caused by a complex of bipartite cassava mosaic geminiviruses (CMG) species (Family: Geminivirideae; Genus: Begomovirus) CMD has been widely described throughout Africa and it is apparent that CMG's are expanding their geographical distribution. Determining where and when CMG movements have occurred could help curtail its spread and reveal the ecological and anthropic factors associated with similar viral invasions. We applied Bayesian phylogeographic inference and recombination analyses to available and newly described CMG sequences to reconstruct a plausible history of CMG diversification and migration between Africa and South West Indian Ocean (SWIO) islands.
The isolation and analysis of 114 DNA-A and 41 DNA-B sequences demonstrated the presence of three CMG species circulating in the Comoros and Seychelles archipelagos (East African cassava mosaic virus, EACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV). Phylogeographic analyses suggest that CMG’s presence on these SWIO islands is probably the result of at least four independent introduction events from mainland Africa occurring between 1988 and 2009. Amongst the islands of the Comoros archipelago, two major migration pathways were inferred: One from Grande Comore to Mohéli and the second from Mayotte to Anjouan. While only two recombination events characteristic of SWIO islands isolates were identified, numerous re-assortments events were detected between EACMV and EACMKV, which seem to almost freely interchange their genome components.
Rapid and extensive virus spread within the SWIO islands was demonstrated for three CMG complex species. Strong evolutionary or ecological interaction between CMG species may explain both their propensity to exchange components and the absence of recombination with non-CMG begomoviruses. Our results suggest an important role of anthropic factors in CMGs spread as the principal axes of viral migration correspond with major routes of human movement and commercial trade. Finer-scale temporal analyses of CMGs to precisely scale the relative contributions of human and insect transmission to their movement dynamics will require further extensive sampling in the SWIO region.
[Show abstract][Hide abstract] ABSTRACT: Cassava mosaic geminiviruses (CMGs) are implicated in cassava mosaic disease (CMD), the main constraint to cassava production in Africa. Here, we report the complete nucleotide sequences of the DNA-A and DNA-B of a newly characterized CMG found infecting cassava in Madagascar, for which we propose the tentative name cassava mosaic Madagascar virus. With the exception of two recombinant regions that resembled a CMG, we determined that the non-recombinant part of the DNA-A component is distantly related to the other CMGs. Whereas the DNA-B component possesses one recombinant region originating from an unidentified virus, the rest of the genome was seen to be closely related to members of the species East African cassava mosaic Zanzibar virus (EACMZV). Phylogenetic analysis based on complete genome sequences demonstrated that DNA-A and DNA-B components are outliers related to the clade of EACMV-like viruses and that DNA-A is related to the monopartite tomato leaf curl begomoviruses described in islands in the south-west Indian Ocean.
Archives of Virology 07/2012; 157(10):2027-30. DOI:10.1007/s00705-012-1399-3 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 4 Groupement de Défense des Cultures sous Abri (GDCSA), 98848 Nouméa, Nouvelle-Calédonie; 5 IRD, CBGP, Campus international de Baillarguet, 34988 Montferrier sur Lez, France; 6 Chambre dAgriculture de la Nouvelle Calédonie, GDS-V, In 2007, severe symptoms of leaf curling and yellowing resembling those of tomato yellow leaf curl disease were observed for the first time on tomato plants (Solanum lycopersicum) with a high incidence in fields and greenhouses in the south western region (Nouméa) of the Pacific island of New Caledonia. Tomato samples with leaf curling and yellowing symptoms were collected in the south west and west of New Caledonia and also Ouvéa (Loyalty Islands), respectively, in November and December 2010 (Table 1). Samples were tested for the presence of begomoviruses using a polymerase chain reaction (PCR) assay with a set of degenerate primers designed to amplify genomic regions of the Old World begomovirus DNA-A component (Delatte et al., 2005). PCR products of the expected sizes were obtained for all the five samples from New Caledonia and for four of the six samples from Ouvéa suggesting the presence of an Old World monopartite begomovirus. PCR positive samples were processed further and full-length viral genomes were successfully amplified from six samples (Table 1) by rolling-circle amplification, cloned using XmnI restriction enzyme and sequenced (Shepherd et al., 2008). The complete DNA-A genome sequences obtained (EMBL-GenBank-DDBJ Accession Nos. HE603241-HE603246), showed the highest pairwise sequence identity of 97.6 to 99.4% (BLAST, NCBI) with isolates of the Israel strain of Tomato yellow leaf curl virus (TYLCV-IL) from Spain ([SP:Alm], AJ489258) and Reunion ([RE:SGi:RE4:04], AM409201). The new sequences were aligned with representative sequences of TYLCV strains using MUSCLE (using default settings) in MEGA5 (Tamura et al., 2011). A maximum-likelihood (ML) phylogenetic tree (Fig. 1) was constructed from the full alignment using PHYML with GTR+G4 selected as the best model of sequence evolution by RDP3 (Martin et al., 2010). The ML phylogenetic tree confirmed the relationship of New Caledonia and Ouvéa isolates of TYLCV-IL with the isolates of Spain [SP:Alm] and Reunion [RE:SGi:RE4:04] (Fig. 1). Surprisingly despite the proximity of New Caledonia and Ouvéa to Australia, the New Caledonian TYLCV isolates seem to have a different origin/source of introduction to those recently described from Australia [AU:Bri1:06] and [AU:Bun1:06] (Van Brunschot et al., 2010). This divergence suggests that the epidemic of TYLCD in New Caledonia and Loyalty Islands seems not directly associated with the introduction of TYLCV-IL in Australia where the first symptoms of TYLCD were described in 2006. To our knowledge, this is the first report of the Old World TYLCV implicated in yellow leaf curl disease on tomato in New Caledonia and Loyalty Islands. This description confirms the invasion and the dissemination of the Israel strain, also called "severe" strain, of TYLCV in the Pacific region, and represents a new record of first importance for the regional management of emerging crop diseases and regulatory institutions.
[Show abstract][Hide abstract] ABSTRACT: Despite extensive sampling, only one virus belonging to the genus Mastrevirus of the family Geminiviridae, maize streak virus (MSV), has until now been detected in maize with maize streak disease (MSD) symptoms. Here, we report for the first time a second, highly divergent, mastrevirus isolated from two maize plants displaying characteristic MSD-like symptoms, sampled on the South-west Indian Ocean Island, La Réunion. The two isolates shared <57 % genome-wide identity with all other known mastreviruses. We propose calling the new species Maize streak Réunion virus.
Archives of Virology 04/2012; 157(8):1617-21. DOI:10.1007/s00705-012-1314-y · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cassava mosaic disease (CMD) is a major constraint on cassava cultivation in Africa. The disease is endemic and is caused by seven distinct cassava mosaic geminiviruses (CMGs), some of them including several variants.
From cassava leaf samples presenting CMD symptoms collected in Burkina Faso, four DNA-A begomovirus components were cloned and sequenced, showing 99.9% nucleotide identity among them. These isolates are most closely related to African cassava mosaic virus (ACMV) but share less than 89% nucleotide identity (taxonomic threshold) with any previously described begomovirus. A DNA-B genomic component, sharing 93% nucleotide identity with DNA-B of ACMV, was also characterized. Since all genomic components have a typical genome organization of Old World bipartite begomoviruses, this new species was provisionally named African cassava mosaic Burkina Faso virus (ACMBFV). Recombination analysis of the new virus demonstrated an interspecies recombinant origin, with major parents related to West African isolates of ACMV, and minor parents related to Tomato leaf curl Cameroon virus and Cotton leaf curl Gezira virus.
This is the first report of an ACMV-like recombinant begomovirus arisen by interspecific recombination between bipartite and monopartite African begomoviruses.
[Show abstract][Hide abstract] ABSTRACT: MultiLocus Sequence Analysis (MLSA) and Amplified Fragment Length Polymorphism (AFLP) were used to measure the genetic relatedness of a comprehensive collection of xanthomonads pathogenic to solaneous hosts to Xanthomonas species. The MLSA scheme was based on partial sequences of four housekeeping genes (atpD, dnaK, efp and gyrB). Globally, MLSA data unambiguously identified strains causing bacterial spot of tomato and pepper at the species level and was consistent with AFLP data. Genetic distances derived from both techniques showed a close relatedness of (i) X. euvesicatoria, X. perforans and X. alfalfae and (ii) X. gardneri and X. cynarae. Maximum likelihood tree topologies derived from each gene portion and the concatenated data set for species in the X. campestris 16S rRNA core (i.e. the species cluster comprising all strains causing bacterial spot of tomato and pepper) were not congruent, consistent with the detection of several putative recombination events in our data sets by several recombination search algorithms. One recombinant region in atpD was identified in most strains of X. euvesicatoria including the type strain.
[Show abstract][Hide abstract] ABSTRACT: The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools.