Metagenomics reveals the structure of Mastrevirus-host interaction network within an agro-ecosystem.

As highly pervasive parasites that sometimes cause disease, viruses are likely major components of all natural ecosystems. An important step towards both understanding the precise ecological roles of viruses and determining how natural communities of viral species are assembled and evolve is obtaining full descriptions of viral diversity and distributions at ecosystem scales. Here, we focused on obtaining such 'community-scale' data for viruses in a single genus. We chose the genus Mastrevirus (family Geminiviridae), members of which have predominantly been found infecting uncultivated grasses (family Poaceae) throughout the tropical and sub-tropical regions of the world. We sampled over 3 years, 2,884 individual Poaceae plants belonging to thirty different species within a 2-ha plot which included cultivated and uncultivated areas on the island of Reunion. Mastreviruses were found in ∼8 per cent of the samples, of which 96 per cent did not have any discernible disease symptoms. The multitude of host-virus associations that we uncovered reveals both the plant species that most commonly host mastreviruses and the mastrevirus species (such as maize streak virus and maize streak Reunion virus) that have especially large host ranges. Our findings are consistent with the hypothesis that perennial plant species capable of hosting years-long mixed mastrevirus infections likely play a disproportionately important role in the generation of inter-species and inter-strain mastrevirus recombinants.

Synergy between an emerging monopartite begomovirus and a DNA-B component.

In recent decades, a legion of monopartite begomoviruses transmitted by the whitefly Bemisia tabaci has emerged as serious threats to vegetable crops in Africa. Recent studies in Burkina Faso (West Africa) reported the predominance of pepper yellow vein Mali virus (PepYVMLV) and its frequent association with a previously unknown DNA-B component. To understand the role of this DNA-B component in the emergence of PepYVMLV, we assessed biological traits related to virulence, virus accumulation, location in the tissue and transmission. We demonstrate that the DNA-B component is not required for systemic movement and symptom development of PepYVMLV (non-strict association), but that its association produces more severe symptoms including growth arrest and plant death. The increased virulence is associated with a higher viral DNA accumulation in plant tissues, an increase in the number of contaminated nuclei of the phloem parenchyma and in the transmission rate by B. tabaci. Our results suggest that the association of a DNA-B component with the otherwise monopartite PepYVMLV is a key factor of its emergence.

Nanopore Sequencing Is a Credible Alternative to Recover Complete Genomes of Geminiviruses.

Next-generation sequencing (NGS), through the implementation of metagenomic protocols, has led to the discovery of thousands of new viruses in the last decade. Nevertheless, these protocols are still laborious and costly to implement, and the technique has not yet become routine for everyday virus characterization. Within the context of CRESS DNA virus studies, we implemented two alternative long-read NGS protocols, one that is agnostic to the sequence (without a priori knowledge of the viral genome) and the other that use specific primers to target a virus (with a priori). Agnostic and specific long read NGS-based assembled genomes of two capulavirus strains were compared to those obtained using the gold standard technique of Sanger sequencing. Both protocols allowed the detection and accurate full genome characterization of both strains. Globally, the assembled genomes were very similar (99.5-99.7% identity) to the Sanger sequences consensus, but differences in the homopolymeric tracks of these sequences indicated a specific lack of accuracy of the long reads NGS approach that has yet to be improved. Nevertheless, the use of the bench-top sequencer has proven to be a credible alternative in the context of CRESS DNA virus study and could offer a new range of applications not previously accessible.

Tomato interveinal yellowing virus: a novel tomato-infecting monopartite begomovirus from Côte d'Ivoire.

In this report, we present the first description of the complete genome sequences of a new monopartite begomovirus isolated from tomato with symptoms of interveinal yellowing of leaves collected in the region of Worodougou in the northwest of Côte d'Ivoire and provisionally named "tomato interveinal yellowing virus" (ToIYV). The DNA-A-like nucleotide sequences of ToIYV share the highest nucleotide sequence identity (83%) with tobacco leaf curl Zimbabwe virus (ToLCZWV). Phylogenetic analysis confirmed that ToIYV is related to Old World monopartite begomoviruses. The discovery of a member of a new virus species on diseased tomato plants confirms the high genetic diversity in monopartite begomoviruses in West Africa and stresses the importance of maintaining epidemiological crop surveillance.

Sorghum mastrevirus-associated alphasatellites: new geminialphasatellites associated with an African streak mastrevirus infecting wild Poaceae plants on Reunion Island.

Nine complete nucleotide sequences of geminialphasatellites (subfamily Geminialphasatellitinae, family Alphasatellitidae) recovered from the wild Poaceae Sorghum arundinaceum collected in Reunion are described and analyzed. While the helper geminivirus was identified as an isolate of maize streak virus (genus Mastrevirus, family Geminiviridae), the geminialphasatellite genomes were most closely related to, and shared ~63% identity with, clecrusatellites. Even though the geminialphasatellite molecules lack an adenine rich-region, they have the typical size of geminialphasatellites, encode a replication-associated protein in the virion sense, and have probable stem-loop structures at their virion-strand origins of replication. According to the proposed geminialphasatellite species and genus demarcation thresholds (88% and 70% nucleotide identity, respectively), the genomes identified here represent a new species (within a new genus) for which we propose the name "Sorghum mastrevirus-associated alphasatellite" (genus "Sorgasalphasatellite").

Tomato leaf curl Kunene virus: a novel tomato-infecting monopartite begomovirus from Namibia.

This is the first description of the complete genome sequence of a new monopartite begomovirus isolated from tomato with symptoms of tomato (yellow) leaf curl disease collected in northwestern Namibia, which we provisionally name "tomato leaf curl Kunene virus". The DNA-A-like nucleotide sequence shares the highest nucleotide sequence identity (82.8%) with tobacco leaf curl Zimbabwe virus (ToLCZWV). Phylogenetic analysis confirmed its affiliation with Old World monopartite begomoviruses. The discovery of a new begomovirus in a vegetable plant in Namibia highlights the importance of maintaining epidemiological surveillance in southern Africa.

Exploring the diversity of Poaceae-infecting mastreviruses on Reunion Island using a viral metagenomics-based approach.

Mostly found in Africa and its surrounding islands, African streak viruses (AfSV) represent the largest group of known mastreviruses. Of the thirteen AfSV species that are known to infect either cultivated or wild Poaceae plant species, six have been identified on Reunion Island. To better characterize AfSV diversity on this island, we undertook a survey of a small agroecosystem using a new metagenomics-based approach involving rolling circle amplification with random PCR amplification tagging (RCA-RA-PCR), high-throughput sequencing (Illumina HiSeq) and the mastrevirus reads classification using phylogenetic placement. Mastreviruses that likely belong to three new species were discovered and full genome sequences of these were determined by Sanger sequencing. The geminivirus-focused metagenomics approach we applied in this study was useful in both the detection of known and novel mastreviruses. The results confirm that Reunion Island is indeed a hotspot of AfSV diversity and that many of the mastrevirus species have likely been introduced multiple times. Applying a similar approach in other natural and agricultural environments should yield sufficient detail on the composition and diversity of geminivirus communities to precipitate major advances in our understanding of the ecology and the evolutionary history of this important group of viruses.

High-throughput sequencing of complete genomes of ipomoviruses associated with an epidemic of cassava brown streak disease in the Comoros Archipelago.

Using high-throughput sequencing of small interfering RNAs (siRNAs), virion-associated nucleic acid (VANA), and double stranded RNAs (dsRNAs), we have determined the complete genome sequences of Comorian isolates of two ipomoviruses, cassava brown streak virus (CBSV) and a divergent isolate of Ugandan cassava brown streak virus (UCBSV-KM) representing a new strain of this virus. While the large ORF of CBSV shares the highest nucleotide sequence identity (95.9%) with a Tanzanian isolate of CBSV, the large UCBSV-KM ORF shares the highest nucleotide sequence identity (77.5%) with a Malawian isolate of UCBSV. This low value is near the species demarcation threshold for the family Potyviridae (<76%). Phylogenetic analysis confirms that UCBSV-KM represents a new lineage that is genetically distinct from the currently described UCBSV strains.

Tomato leaf curl Mahé virus: a novel tomato-infecting monopartite begomovirus from the Seychelles.

This is the first description of the complete genome sequence of a new monopartite begomovirus isolated from tomato with symptoms of tomato (yellow) leaf curl disease collected in the Seychelles. The DNA-A-like nucleotide sequences share the highest nucleotide sequence identity (84%) with tomato leaf curl Anjouan virus (ToLCAnjV) from the Comoros islands. Phylogenetic analysis confirmed its recombinant nature and its relationship to Old World monopartite and bipartite begomoviruses. This discovery of a new member of a species confirms the high genetic diversity of begomoviruses in the south-western Indian Ocean islands.

New strains of chickpea chlorotic dwarf virus discovered on diseased papaya and tomato plants in Burkina Faso.

This is the first description of full genome sequences of chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus; family Geminiviridae) identified in papaya and tomato plants sampled in Burkina Faso. The CpCDV full genome sequences from papaya and tomato share the highest pairwise sequence identity (84% and 93.5%) with Sudanese isolates of the CpCDV-K and CpCDV-M strains, respectively. Based on the strain demarcation threshold (>94% identity) for mastreviruses, we propose two new strains, CpCDV-Q and CpCDV-R, identified in papaya and tomato, respectively. Phylogenetic analysis confirmed that the sequences belong to a distinct clade of the highly diverse population of CpCDVs. Evidence of inter-strain recombination provided more support for the important role of recombination in CpCDV evolution. The discovery of CpCDV on papaya, a previously unsuspected host, raises many questions about the natural and potential host range of this dicot-infecting mastrevirus species that is reported to be emerging worldwide.

Tomato leaf curl Burkina Faso virus: a novel tomato-infecting monopartite begomovirus from Burkina Faso.

In this report, we present the first description of the complete genome sequence of a new monopartite begomovirus isolated from tomatoes collected in Burkina Faso and presenting with symptoms of tomato leaf curl disease. We propose the tentative name "tomato leaf curl Burkina Faso virus'' (ToLCBFV). DNA-A-like nucleotide sequence of ToLCBFV shares the highest nucleotide sequence identity (85%) with the pepper yellow vein Mali virus (PepYVMLV). Phylogenetic analysis confirmed the affiliation of ToLCBFV to Old World monopartite begomoviruses. This discovery of a new species confirms the existence of high genetic diversity in monopartite begomoviruses in sub-Saharan Africa and particularly in West Africa.

Divergent evolutionary and epidemiological dynamics of cassava mosaic geminiviruses in Madagascar.

BACKGROUND: Cassava mosaic disease (CMD) in Madagascar is caused by a complex of at least six African cassava mosaic geminivirus (CMG) species. This provides a rare opportunity for a comparative study of the evolutionary and epidemiological dynamics of distinct pathogenic crop-infecting viral species that coexist within the same environment. The genetic and spatial structure of CMG populations in Madagascar was studied and Bayesian phylogeographic modelling was applied to infer the origins of Madagascan CMG populations within the epidemiological context of related populations situated on mainland Africa and other south western Indian Ocean (SWIO) islands. RESULTS: The isolation and analysis of 279 DNA-A and 117 DNA-B sequences revealed the presence in Madagascar of four prevalent CMG species (South African cassava mosaic virus, SACMV; African cassava mosaic virus, ACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV), and of numerous CMG recombinants that have, to date, only ever been detected on this island. SACMV and ACMV, the two most prevalent viruses, displayed low degrees of genetic diversity and have most likely been introduced to the island only once. By contrast, EACMV-like CMG populations (consisting of East African cassava mosaic virus, EAMCKV, EACMCV and complex recombinants of these) were more diverse, more spatially structured, and displayed evidence of at least three independent introductions from mainland Africa. Although there were no statistically supported virus movement events between Madagascar and the other SWIO islands, at least one mainland African ACMV variant likely originated in Madagascar. CONCLUSIONS: Our study highlights both the complexity of CMD in Madagascar, and the distinct evolutionary and spatial dynamics of the different viral species that collectively are associated with this disease. Given that more distinct CMG species and recombinants have been found in Madagascar than any other similarly sized region of the world, the risks of recombinant CMG variants emerging on this island are likely to be higher than elsewhere. Evidence of an epidemiological link between Madagascan and mainland African CMGs suggests that the consequences of such emergence events could reach far beyond the shores of this island.

Tomato chlorotic mottle Guyane virus: a novel tomato-infecting bipartite begomovirus from French Guiana.

This is the first description of the complete genome sequence of a new bipartite begomovirus isolated from tomato (Solanum lycopersicum) in French Guiana, for which we propose the tentative name "tomato chlorotic mottle Guyane virus" (ToCMoGFV). DNA-A and -B nucleotide sequences of ToCMoGFV are only distantly related to known New World begomoviruses. They share the highest nucleotide sequence identity of 80% with the Brazilian isolates of macroptilium yellow spot virus (MacYSV) and 73% with soybean chlorotic spot virus (SBCSV). Phylogenetic analysis demonstrated that this novel virus belongs to a new lineage of New World bipartite begomoviruses. The discovery of this new virus confirms the high genetic diversity of begomoviruses in Latin America.

Asystasia mosaic Madagascar virus: a novel bipartite begomovirus infecting the weed Asystasia gangetica in Madagascar.

Here, we describe for the first time the complete genome sequence of a new bipartite begomovirus in Madagascar isolated from the weed Asystasia gangetica (Acanthaceae), for which we propose the tentative name asystasia mosaic Madagascar virus (AMMGV). DNA-A and -B nucleotide sequences of AMMGV were only distantly related to known begomovirus sequence and shared highest nucleotide sequence identity of 72.9 % (DNA-A) and 66.9 % (DNA-B) with a recently described bipartite begomovirus infecting Asystasia sp. in West Africa. Phylogenetic analysis demonstrated that this novel virus from Madagascar belongs to a new lineage of Old World bipartite begomoviruses.

Frequency-dependent assistance as a way out of competitive exclusion between two strains of an emerging virus.

Biological invasions are the main causes of emerging viral diseases and they favour the co-occurrence of multiple species or strains in the same environment. Depending on the nature of the interaction, co-occurrence can lead to competitive exclusion or coexistence. The successive fortuitous introductions of two strains of Tomato yellow leaf curl virus (TYLCV-Mld and TYLCV-IL) in Réunion Island provided an ideal opportunity to study the invasion of, and competition between, these worldwide emerging pathogens. During a 7-year field survey, we observed a displacement of the resident TYLCV-Mld by the newcomer TYLCV-IL, with TYLCV-Mld remaining mostly in co-infected plants. To understand the factors associated with this partial displacement, biological traits related to fitness were measured. The better ecological aptitude of TYLCV-IL in single infections was demonstrated, which explains its rapid spread. However, we demonstrate that the relative fitness of virus strains can drastically change between single infections and co-infections. An epidemiological model parametrized with our experimental data predicts that the two strains will coexist in the long run through assistance by the fitter strain. This rare case of unilateral facilitation between two pathogens leads to frequency-dependent selection and maintenance of the less fit strain.

Molecular characterization of a new alphasatellite associated with a cassava mosaic geminivirus in Madagascar.

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".

East African cassava mosaic-like viruses from Africa to Indian ocean islands: molecular diversity, evolutionary history and geographical dissemination of a bipartite begomovirus.

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. RESULTS: 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. CONCLUSIONS: 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.

A novel cassava-infecting begomovirus from Madagascar: cassava mosaic Madagascar virus.

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.

Evolution of African cassava mosaic virus by recombination between bipartite and monopartite begomoviruses.

BACKGROUND: 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. FINDINGS: 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. CONCLUSION: This is the first report of an ACMV-like recombinant begomovirus arisen by interspecific recombination between bipartite and monopartite African begomoviruses.

Complex recombination patterns arising during geminivirus coinfections preserve and demarcate biologically important intra-genome interaction networks.

Genetic recombination is an important process during the evolution of many virus species and occurs particularly frequently amongst begomoviruses in the single stranded DNA virus family, Geminiviridae. As in many other recombining viruses it is apparent that non-random recombination breakpoint distributions observable within begomovirus genomes sampled from nature are the product of variations both in basal recombination rates across genomes and in the over-all viability of different recombinant genomes. Whereas factors influencing basal recombination rates might include local degrees of sequence similarity between recombining genomes, nucleic acid secondary structures and genomic sensitivity to nuclease attack or breakage, the viability of recombinant genomes could be influenced by the degree to which their co-evolved protein-protein and protein-nucleotide and nucleotide-nucleotide interactions are disreputable by recombination. Here we investigate patterns of recombination that occur over 120 day long experimental infections of tomato plants with the begomoviruses Tomato yellow leaf curl virus and Tomato leaf curl Comoros virus. We show that patterns of sequence exchange between these viruses can be extraordinarily complex and present clear evidence that factors such as local degrees of sequence similarity but not genomic secondary structure strongly influence where recombination breakpoints occur. It is also apparent from our experiment that over-all patterns of recombination are strongly influenced by selection against individual recombinants displaying disrupted intra-genomic interactions such as those required for proper protein and nucleic acid folding. Crucially, we find that selection favoring the preservation of co-evolved longer-range protein-protein and protein DNA interactions is so strong that its imprint can even be used to identify the exact sequence tracts involved in these interactions.