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International audience; 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.

The Mastrevirus genus (Geminiviridae family) contains circular single-stranded DNA viruses transmitted by leafhoppers (in the Cicadellidae family) to a wide range of either monocotyledonous or dicotyledonous host species. Most of the known monocot-infecting mastreviruses have been identified either in Africa or surrounding islands. This group of mastreviruses have collectively been called the “African streak viruses” (AfSV). Of the 13 AfSV species infecting cultivated and wild Poaceae species, six have been identified on Reunion Island. Interestingly, these species were probably introduced at different time and they present with putatively distinct host ranges. Understanding how this virus community operates remains an essential question in the understanding of virus ecology, evolution and emergence. Therefore, to elucidate the diversity, host ranges and structure of mastrevirus communities on Reunion Island, we undertook an extensive survey in a single sampling site of one acre including crop fields, orchards and uncultivated areas. After four sampling campaigns, 2917 samples of 30 cultivated and uncultivated Poaceae species were randomly collected, regardless of their health status. Total plant DNA was isolated and circular viral genomes were amplified by a sequence-independent amplification procedure combining rolling circle amplification, a random amplification tagging (RCA-RA) and high-throughput sequencing (Illumina HiSeq). For every sample, mastrevirus reads were classified using phylogenetic placement within species and strains. Mastrevirus species were confirmed by cloning and Sanger sequencing. Besides uncovering previously undescribed mastrevirus species, our results provide an exhaustive view of the mastrevirus-host association network within an agroecosystem. The topology of this network suggests (1) the co-existence of viruses ranging from generalist to specialist and (2) that certain hosts may act as hubs of virus infection and transmission.

Mastreviruses are single stranded DNA viruses belonging to the family Geminiviridae. These viruses are transmitted by leafhoppers mostly to monocotyledonous plants species. As for most plant viruses, research have been traditionnally focused on the fraction of the viruses that causes disease on crops despite uncultivated Poaceae plants being well established as mastreviruses hosts. Nevertheless, the demonstration of the emergence of the maize streak virus-strain A (MSV-A, the most devasting mastrevirus) through recombination between strains originating from uncultivated grasses shed lights on the importance to obtain a better understanding of the diversity and structure of virus communities at the scale of the agro-ecosystems. In our preliminary study, 144 samples of uncultivated species of Poaceae were collected from the agro-ecosystems interfaces in Reunion. A sequence-independent amplification procedure, that combined rolling circle amplification with a random amplification tagging (RCA-RA) and high-throughput sequencing (Illumina HiSeq), was then applied on full DNA extracts of plants. Several viruses reads belonging to the Mastrevirus genus were reported from twelve of the 144 samples (~8%). Beside multiple MSV variants and the maize streak Reunion virus strains, at least two species of viruses related to the Axonopus compressus streak virus and the Switchgrass mosaic streak virus were identified on multiple plant species. Cloning and Sanger sequencing procedure were carried out to obtain full viral genomes and further confirm the assignation of these isolates. Our preliminary results demonstrate that mastreviruses diversity in Reunion is larger than previsouly suspected and confirm the importance to characterize their diversity at the scale of the full agro-ecosystems. They strongly suggest that the unique mastrevirus community present in the volcanic Island of Reunion results from multiple introduction events. The study of the evolution imprints within the virus genomes will certainly be informative on the process of adaptation that operates in the evolutionary history of this viral community.

Background and objectives begomoviruses (family geminiviridea) are transmitted by Bemisia tabaci and are responsible for serious diseases in the world. African begomoviruses described on crops are mainly monopartite (dna-a component), except cassava-infecting bipartite begomoviruses (dna-a and dna-b components). In recent years, a complex of more than 10 monopartite begomoviruses responsible for tomato (yellow) leaf curl diseases (tylcd-tolcd) has been described on tomato in sub-Saharan Africa [1]. Faced with the upsurge of vegetable virus diseases in Burkina Faso, we have undertaken (1) to identify the causal agents and their molecular diversity, and (2) to investigate their main epidemiological parameters in the field and under controlled conditions. Material and methods forty-five localities from the main vegetable-growing areas of Burkina Faso were surveyed from 2013 to 2016. In total, 2065 leaf samples were collected from cultivated and uncultivated plants. Some of them were subjected to begomovirus diagnosis (sequence-dependent methods) by pcr, rolling circle amplification, cloning and sequencing and others were used for more accurate and complete description of virus communities using a metagenomic approach (sequence-independent methods) based on rca, pcr-random amplification tagging and illumina sequencing. Agroinfectious clones were constructed in order to assess the pathogenicity of some characterized viruses. The transmission rate of these viruses was estimated after acquisition of the virus by B. tabaci. Results we have cloned and sequenced 143 dna sequences (109 dna-a and 34 dna-b). Nucleotide similarity analyses confirmed the presence of at least four known African monopartite begomoviruses (clcugv, pepyvmv, tolmlv and tolcghv). It also revealed for the first time in West Africa the mastrevirus cpcdv on tomato and a new species named “tomato leaf curl Burkina Faso virus”. We characterized a dna-b molecule on cultivated and uncultivated plants, unexpectedly always associated with the dna-a of pepyvmv, which is distantly related to the known dna-b diversity. In addition to the previous five geminiviruses, the metagenomic approach suggested the presence of at least five other viruses. Our agroinoculation tests demonstrate that the sole pepyvmv dna-a does not induce tylcd-tolcd symptoms on tomato in experimental conditions. However, in coinfection dna-b is an activator of the virulence of the cognate pepyvmv dna-a. Conclusions our work suggest that tylcd-tolcd in Burkina Faso is associated with a complex of geminiviruses and that sequence-independent deep sequencing based approaches are interesting and complementary to further study the diversity of plant viruses. We demonstrate experimentally that the newly characterized dna-b component represents a major pathogenity activator for pepyvmv dna-a. Our first investigations may suggest that this dna-b component recruited by pepyvmv dna-a could be the main epidemiological factor for the emergence of pepyvmv as the most prevalent and severe plant virus disease on tomato and pepper in Burkina Faso.

BACKGROUND and OBJECTIVES Begomoviruses (family Geminiviridea) are transmitted by Bemisia tabaci and are responsible for serious diseases in the world. Except cassava-infecting bipartite begomoviruses (DNA-A and DNA-B components), all African begomoviruses described on crops are monopartite (DNA-A component). In recent years, a complex of more than 10 monopartite begomoviruses responsible for tomato (yellow) leaf curl diseases (TYLCDToLCD) has been described on tomato in sub-Saharan Africa [1]. Faced with the upsurge of vegetable virus diseases in Burkina Faso, we have undertaken (1) to identify the causal agents and their molecular diversity, and (2) to investigate their main epidemiological parameters in the field and under controlled conditions. MATERIAL and METHODS Thirty-nine localities from the main vegetable-growing areas of Burkina Faso were surveyed during 2013 to 2015. Altogether 939 leaf samples from cultivated and uncultivated crops were collected. Samples were subjected to begomovirus diagnosis by PCR, RCA, cloning and sequencing. Agroinfectious clones were constructed in order to assess the pathogenicity of some characterized viruses. RESULTS We have cloned and sequenced 143 DNA sequences (109 DNA-A and 34 DNA-B). Whereas, nucleotide similarity analyses confirmed the presence of at least four known African monopartite begomoviruses (CLCuGV, PepYVMV, ToLMLV and ToLCGHV), it also revealed for the first time in West Africa the mastrevirus CpCDV on tomato and a new species named “tomato leaf curl Burkina Faso virus”. Unexpectedly, we characterized a DNA-B molecule on cultivated and uncultivated plants, always associated with the DNA-A of PepYVMV, which is distantly related to the known DNA-B. Our agroinoculation tests demonstrate that the sole PepYVMV DNA-A doesn't induce TYLCD-ToLCD symptoms on tomato in experimental conditions. However, in mixed infection the DNA-B is an activator of the virulence of the cognate PepYVMV DNA-A. CONCLUSIONS A complex of four known begomoviruses (CLCuGV, PepYVMV, ToLMLV and ToLCGHV) and a new begomovirus species on tomato (ToLCBFV) were described on diseased vegetable crops in Burkina Faso. Interestingly, a mastrevirus (CpCDV) was described for the first time in West Africa on tomato. Finally, we demonstrate experimentally that the newly characterized DNA-B component, naturally associated with PepYVMV DNA-A component in the field, represents a major pathogenicity activator. Our first investigations may suggest that this DNA-B component recruited by PepYVMV DNA-A could be the main epidemiological factor for the emergence of PepYVMV as the most prevalent and severe plant virus disease on tomato and pepper in Burkina Faso.

International audience; In the last decades, begomoviruses emerged as probably the most economically important crop pathogens. Despites the hypothesis that uncultivated hosts, such as wild plants or weeds, provide alternative hosts during intercropping periods, few studies effectively identified the plant species involved. Here, we used multiple parallel-sequencing of a conserved region of begomovirus genome, using newly designed primers, to identify the presence of begomoviruses in a large number of wild plants and crops in Madagascar and sub-Saharan African countries. Whereas our primers set successfully amplified begomoviruses in a wide range of plant species and families, we focused our analyses on Madagascan samples, and identified four distinct Operational Taxonomic Units (OTUs), that infect both crop and weeds. While one of these OTUs likely represents an example of a malvaceous-adapted virus which acquired the ability to infect bean, three closely related OTUs, largely prevalent on tomato, were found infecting weeds. One of these tomato-associated OTU in particular, related to variants of tomato leaf curl Madagascar virus, was found in eight distinct plant species, suggesting its evolution as a generalist pathogen. Despites several biases associated to the use of primers designed from known sequences, and the need to confirm our results with the cloning of complete sequences, our study has unravelled the complexity of the relationships between begomoviruses and their hosts and raised some exciting questions on the niche partitioning of begomoviruses in natural and cultivated ecosystems. (Résumé d'auteur)

Cassava is the staple food for hundreds of millions of people in Africa but its cultivation is seriously constrained by cassava mosaic disease (CMD) in Madagascar and in Africa in general. In this study, we first identified the cassava mosaic geminiviruses (CMGs) involved in CMD in Madagascar and their associated epidemiological characteristics from countrywide surveys. Molecular diagnosis of CMGs in Madagascar revealed an unprecedented diversity and co-occurrence of six CMGs. Distinct geographical distributions were observed for the six viruses. While EACMCV and SACMV were widespread, ACMV was more prevalent in the central highlands, and EACMV and EACMKV were prevalent in lowlands and coastal regions. PCR diagnosis revealed that mixed infection (up to four co-infected viruses) occurred in 21% of the samples and were associated with higher symptom severity scores. Pairwise comparisons of virus associations showed that EACMCV was found in mixed infections more often than expected while ACMV and SACMV were mostly found in single infections. Even if the whitefly vector Bemisia tabaci was more abundant in lowland and coastal areas, infected cuttings remain the primary source of CMD propagation (95%) in Madagascar. Secondly, the genetic and spatial structure of CMGs populations was studied and we applied Bayesian phylogeographic modelling 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 islands. 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 (EACMV, 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. Our study highlights both the complexity of CMD on Madagascar, and the distinct evolutionary and spatial dynamics of the different viral species that collectively are associated with this disease.

International audience; Cassava is the staple food for hundreds of millions of people in Africa but its cultivation is seriously constrained by cassava mosaic disease (CMD) in Madagascar and in Africa in general. In this study, we first identified the cassava mosaic geminiviruses (CMGs) involved in CMD in Madagascar and their associated epidemiological characteristics from countrywide surveys. Molecular diagnosis of CMGs in Madagascar revealed an unprecedented diversity and co-occurrence of six CMGs. Distinct geographical distributions were observed for the six viruses. While EACMCV and SACMV were widespread, ACMV was more prevalent in the central highlands, and EACMV and EACMKV were prevalent in lowlands and coastal regions. PCR diagnosis revealed that mixed infection (up to four co-infected viruses) occurred in 21% of the samples and were associated with higher symptom severity scores. Pairwise comparisons of virus associations showed that EACMCV was found in mixed infections more often than expected while ACMV and SACMV were mostly found in single infections. Even if the whitefly vector Bemisia tabaci was more abundant in lowland and coastal areas, infected cuttings remain the primary source of CMD propagation (95%) in Madagascar. Secondly, the genetic and spatial structure of CMGs populations was studied and we applied Bayesian phylogeographic modelling 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 islands. 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 (EACMV, 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. Our study highlights both the complexity of CMD on Madagascar, and the distinct evolutionary and spatial dynamics of the different viral species that collectively are associated with this disease. (Résumé d'auteur)

International audience

International audience; Biological invasions are major threats to biodiversity and the main causes of emerging viral diseases. The ongoing spread of Tomato yellow leaf curl virus is a major concern to the sustainable tomato production throughout the world. The two main strains of TYLCV have been successively introduced in Reunion Island providing a fortuitous field experiment to study the invasion and competition of these two emerging strains in a tropical and insular environment. In this study, a seven-year field survey was performed following the introduction of the Israel strain of TYLCV (TYLCV-IL) into a niche occupied by the Mild strain of TYLCV (TYLCV-Mld). A displacement of the resident TYLCV-Mld by the newcomer TYLCV-IL was observed in this short period. To understand the factors associated with this displacement, biological traits related to fitness were measured to compare these strains. Besides demonstrating a better ecological aptitude of TYLCV-IL, which explains its rapid spread and increasing prevalence, the first estimate of the number of viral particles efficiently transmitted by an insect vector for a circulative virus was obtained. However, TYLCV-Mld persistence in the field (especially in mixed infections with TYLCV-IL) spurred further experiments regarding the effects of the mixed infections on these biological traits. Our study revealed complex interplay between these two strains of one of the most emergent plant virus following their successive introductions in the insular and tropical environment of Reunion Island. This rare case of unilateral facilitation between two pathogens led to the epidemiological rescue and maintenance of the less fit strain. (Texte intégral)

Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs, Geminiviridae) is a major threat on cassava production throughout Africa. In Madagascar, severe symptoms with high prevalence of CMD were observed. From 700 mostly symptomatic cassava leaves, we undertook molecular characterization of the CMGs involved. The PCR diagnosis confirmed the presence of CMGs in Madagascar and six species were detected with the frequent occurrence of mixed infections. Based on rolling circle amplification, cloning and sequencing, we obtained full genome sequences of 281 DNA-A and 119 DNA-B of CMGs. DNA-A sequences were most closely related to those of African cassava mosaic virus (ACMV), South African cassava mosaic virus (SACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Kenya virus (EACMKV) and a new species named cassava mosaic Madagascar virus (CMMGV). DNAB sequences were most closely related to those of ACMV, EACMKV, EACMCV and CMMGV. Phylogenetic reconstruction allowed to assess the geographical structure of CMG populations and to determine the recent history of CMG migrations in Madagascar. Taken together, our results point to Madagascar as a potential major center of begomovirus diversity.