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International audience; A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.

Badnaviruses are double‐stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor., Banana‐related badnavirus sequences have undergone different coevolution processes with their host plant over the time, with some of them being either integrated or episomal while others are both integrated and episomal.

The number of plant viruses that are known likely remains only a vanishingly small fraction of all extant plant virus species. Consequently, the distribution and population dynamics of plant viruses within even the best-studied ecosystems have only ever been studied for small groups of virus species. Even for the best studied of these groups very little is known about virus diversity at spatial scales ranging from an individual host, through individual local host populations to global host populations. To date, metagenomics studies that have assessed the collective or metagenomes of viruses at the ecosystem scale have revealed many previously unrecognized viral species. More recently, novel georeferenced metagenomics approaches have been devised that can precisely link individual sequence reads to both the plant hosts from which they were obtained, and the spatial arrangements of these hosts. Besides illuminating the diversity and the distribution of plant viruses at the ecosystem scale, application of these "geometagenomics" approaches has enabled the direct testing of hypotheses relating to the impacts of host diversity, host spatial variations, and environmental conditions on plant virus diversity and prevalence. To exemplify how such top-down approaches can provide a far deeper understanding of host-virus associations, we provide a case-study focusing on geminiviruses within two complex ecosystems containing both cultivated and uncultivated areas. Geminiviruses are a highly relevant model for studying the evolutionary and ecological aspects of viral emergence because the family Geminiviridae includes many of the most important crop pathogens that have emerged over the past century. In addition to revealing unprecedented degrees of geminivirus diversity within the analyzed ecosystems, the geometagenomics-based approach enabled the focused in-depth analysis of the complex evolutionary dynamics of some of the highly divergent geminivirus species that were discovered.

International audience; Abstract The number of plant viruses that are known likely remains only a vanishingly small fraction of all extant plant virus species. Consequently, the distribution and population dynamics of plant viruses within even the best-studied ecosystems have only ever been studied for small groups of virus species. Even for the best studied of these groups very little is known about virus diversity at spatial scales ranging from an individual host, through individual local host populations to global host populations. To date, metagenomics studies that have assessed the collective or metagenomes of viruses at the ecosystem scale have revealed many previously unrecognized viral species. More recently, novel georeferenced metagenomics approaches have been devised that can precisely link individual sequence reads to both the plant hosts from which they were obtained, and the spatial arrangements of these hosts. Besides illuminating the diversity and the distribution of plant viruses at the ecosystem scale, application of these “geometagenomics” approaches has enabled the direct testing of hypotheses relating to the impacts of host diversity, host spatial variations, and environmental conditions on plant virus diversity and prevalence. To exemplify how such top-down approaches can provide a far deeper understanding of host–virus associations, we provide a case-study focusing on geminiviruses within two complex ecosystems containing both cultivated and uncultivated areas. Geminiviruses are a highly relevant model for studying the evolutionary and ecological aspects of viral emergence because the family Geminiviridae includes many of the most important crop pathogens that have emerged over the past century. In addition to revealing unprecedented degrees of geminivirus diversity within the analyzed ecosystems, the geometagenomics-based approach enabled the focused in-depth analysis of the complex evolutionary dynamics of some of the highly divergent geminivirus species that were discovered.

BGPI : équipe 2 / 7; International audience; Nanoviruses are multi-component plant-infecting single-stranded DNA viruses. Using a viral metagenomics-informed approach, a new nanovirus and two associated alphasatellite molecules have been identified in an uncultivated asymptomatic Vicia cracca plant in the Rhône region of France. This novel nanovirus genome includes eight genomic components (named DNA-R, DNA-S, DNA-M, DNA-C, DNA-N, DNA-U1, DNA-U2 and DNA-U4) and, across all components, shares < 66% pairwise sequence identity with other nanovirus genomes. The two associated alphasatellites share 62% identity with each other and < 81% identity will all other nanovirus-associated alphasatellites.

UMR BFP - Equipe Virologie; International audience; We here assessed the capability of the MinION sequencing approach to detect and characterize viruses infecting a water yam plant. This sequencing platform consistently revealed the presence of several plant virus species, including Dioscorea bacilliform virus, Yam mild mosaic virus and Yam chlorotic necrosis virus. A potentially novel ampelovirus was also detected by a complimentary Illumina sequencing approach. The full-length genome sequence of yam chlorotic necrosis virus was determined using Sanger sequencing, which enabled determination of the coverage and sequencing accuracy of the MinION technology. Whereas the total mean sequencing error rate of yam chlorotic necrosis virus-related MinION reads was 11.25%, we show that the consensus sequence obtained either by de novo assembly or after mapping the MinION reads on the virus genomic sequence was >99.8% identical with the Sanger-derived reference sequence. From the perspective of potential plant disease diagnostic applications of MinION sequencing, these degrees of sequencing accuracy demonstrate that the MinION approach can be used to both reliably detect and accurately sequence nearly full-length positive-sense single-strand polyadenylated RNA plant virus genomes.

The full-length genome sequences of two novel poleroviruses found infecting cowpea plants, cowpea polerovirus 1 (CPPV1) and cowpea polerovirus 2 (CPPV2), were determined using overlapping RT-PCR and RACE-PCR. Whereas the 5845-nt CPPV1 genome was most similar to chickpea chlorotic stunt virus (73% identity), the 5945-nt CPPV2 genome was most similar to phasey bean mild yellow virus (86% identity). The CPPV1 and CPPV2 genomes both have a typical polerovirus genome organization. Phylogenetic analysis of the inferred P1-P2 and P3 amino acid sequences confirmed that CPPV1 and CPPV2 are indeed poleroviruses. Four apparently unique recombination events were detected within a dataset of 12 full polerovirus genome sequences, including two events in the CPPV2 genome. Based on the current species demarcation criteria for the family Luteoviridae, we tentatively propose that CPPV1 and CPPV2 should be considered members of novel polerovirus species.

Several endogenous viral elements (EVEs) have been identified in plant genomes, including endogenous pararetroviruses (EPRVs). Here, we report the first characterization of EPRV sequences in the genome of African yam of the Dioscorea cayenensis-rotundata complex. We propose that these sequences should be termed 'endogenous Dioscorea bacilliform viruses' (eDBVs). Molecular characterization of eDBVs shows that they constitute sequences originating from various parts of badnavirus genomes, resulting in a mosaic structure that is typical of most EPRVs characterized to date. Using complementary molecular approaches, we show that eDBVs belong to at least four distinct Badnavirus species, indicating multiple, independent, endogenization events. Phylogenetic analyses of eDBVs support and enrich the current taxonomy of yam badnaviruses and lead to the characterization of a new Badnavirus species in yam. The impact of eDBVs on diagnosis, yam germplasm conservation and movement, and breeding is discussed.

BGPI : équipe 6/7; Little is known about the prevalence, diversity, evolutionary processes, genomic structures and population dynamics of viruses in the divergent geminivirus lineage known as the capulaviruses. We determined and analyzed full genome sequences of 13 Euphorbia caput-medusae latent virus (EcmLV) and 26 Alfalfa leaf curl virus (ALCV) isolates, and partial genome sequences of 23 EcmLV and 37 ALCV isolates. While EcmLV was asymptomatic in uncultivated southern African Euphorbia caput-medusae, severe alfalfa disease symptoms were associated with ALCV in southern France. The prevalence of both viruses exceeded 10% in their respective hosts. Besides using patterns of detectable negative selection to identify ORFs that are probably functionally expressed, we show that ALCV and EcmLV both display evidence of inter-species recombination and biologically functional genomic secondary structures. Finally, we show that whereas the EcmLV populations likely experience restricted geographical dispersion, ALCV is probably freely moving across the French Mediterranean region.

Banana bunchy top virus (BBTV), belonging to the genus Babuvirus, is the most devastating and widespread banana virus. Banana and plantain are major crops in terms of household income and food security in Democratic Republic of Congo (DRC). Despite the large area under banana and plantain cultivation in the country, before this study, the genetic characterization of BBTV isolates had only been undertaken for two provinces. In the study presented here, genetic variation in BBTV was assessed from 52 BBTV isolates collected in five out of 11 provinces in DRC (Bandundu, Bas-Congo, Katanga, Kinshasa and Kasai Oriental) and in two provinces using sequences previously described in databases. Full genome sequencing of DNA-R components was performed, revealing low genetic variation (98-100 % nucleotide identity) among the BBTV isolates detected. The phylogenetic analyses showed that all the DRC isolates were clustered in the South Pacific clade of BBTV. Based on the coding region for the replication initiator protein, haplotype diversity was estimated to be 0.944 ± 0.013, with 30 haplotypes from 68 isolates in DRC. Such diversity shows a haplotype distribution mainly at the sub-regional level in DRC. In addition, the sequence determination from the whole genome of selected isolates confirmed low genetic variation among isolates from seven DRC provinces (97-100 % nucleotide identity). This study strengthened the hypothesis of a single BBTV introduction some time ago, followed by the spread of the virus in the country.

The genome of Musa balbisiana spp. contains several infectious endogenous sequences of Banana streak virus (eBSV). We have shown previously that in vitro micropropagation triggers the activation of infectious eBSOLV (endogenous sequences of Banana streak Obino l'Ewai virus) in the synthetic tetraploid interspecific hybrid FHIA21 (AAAB). In this work, we show that another synthetic tetraploid (AAAB) hybrid and two natural triploid (AAB) plantains are equally prone to the activation of infectious eBSOLV during tissue culture. These results are a strong indication that such activation is a general phenomenon in interspecific Musa cultivars, whether synthetic or natural. We also report the first in-depth study of the correlation between the duration of tissue culture and the level of activation of infectious eBSOLV, and show that specific and common activation patterns exist in these banana plants. We hypothesize that these patterns result from the concomitant activation of infectious eBSOLV and a decrease in the virus titre in neoformed plantlets, resulting from cell multiplication outcompeting virus replication. We provide experimental data supporting this hypothesis. No activation of infectious eBSGFV (endogenous sequences of Banana streak Goldfinger virus) by tissue culture was observed in the two natural AAB plantain cultivars studied here, whereas such activation occurred in the AAAB synthetic hybrid studied. We demonstrate that this differential activation does not result from differences in the structure of eBSGFV, as all banana genomes harbour eaBSGFV-7.

Banana streak viruses are among the most widely distributed viruses of banana and are responsible for banana streak disease. Natural field spread occurs by either mealybugs or use of infected planting material, such as suckers. Banana streak viruses are pararetroviruses belonging to the genus Badnavirus, in the family Caulimoviridae. Like all members of the Badnavirus genus, they have bacilliform virions, 30 x 150 nm in size and a circular dsDNA genome of 7.4kbp. Fifteen years ago, an increasing number of outbreaks of banana streak disease were reported worldwide. Many occured in banana breeding lines and micropropagated inter-specific banana hybrids. The origin of infections in new hybrids and tissue-cultured plants was linked to the presence of viral DNA sequences integrated into the Musa balbisiana genome. Although integration is not an essential step in the replication cycle of pararetroviruses, it is assumed that under stress conditions some endogenous banana streak viruses could become infectious by reconstituting a complete replication-competent viral genome. Several serological and molecular tools have been developed to detect either virions or endogenous banana streak viruses. They specificity and potential to prevent and control outbreaks of banana streak disease is discussed. (Resume d'auteur)

An immunocapture (IC) One-step RT-PCR assay was developed to improve the detection of Banana bract mosaic virus (BBrMV) in single and bulked samples of banana plants. In this paper, an atypical strain of BBrMV was described, the BBrMV "Ref" strain, and we showed that detection with available BBrMV tools using ELISA and RT-PCR approaches was not reliable. Primer sets Bract N1/NR and N2/NR specific to BBrMV were designed and used in RT-PCR and IC-RT-PCR assays with two commercial kits that allow the RT and the PCR reactions to take place simultaneously in the same tube. The new assay enabled detection of BBrMV in leaf extract diluted up to 1 x 10(-10) and in bulked samples of 10 plants, and was proposed as a new international standard to index BBrMV.

The family Geminiviridae comprises seven genera differentiated by genome organization, sequence similarity, and insect vector. Capulavirus, an eighth genus, has been proposed to accommodate two newly discovered highly divergent geminiviruses that presently have no known vector. Alfalfa leaf curl virus, identified here as a third capulavirus, is shown to be transmitted by Aphis craccivora. This is the first report of an aphid-transmitted geminivirus.

This chapter describes an efficient approach that combines quality and yield extraction of viral nucleic acids from plants containing high levels of secondary metabolites and a sequence-independent amplification procedure for both the inventory of known plant viruses and the discovery of unknown ones. This approach turns out to be a useful tool for assessing the virome (the genome of all the viruses that inhabit a particular organism) of plants of interest. We here show that this approach enables the identification of a novel Potyvirus member within a single plant already known to be infected by two other Potyvirus species.

Equipe BGPI : 6 / 7; International audience; This chapter describes an efficient approach that combines quality and yield extraction of viral nucleic acids from plants containing high levels of secondary metabolites and a sequence-independent amplification procedure for both the inventory of known plant viruses and the discovery of unknown ones. This approach turns out to be a useful tool for assessing the virome (the genome of all the viruses that inhabit a particular organism) of plants of interest. We here show that this approach enables the identification of a novel Potyvirus member within a single plant already known to be infected by two other Potyvirus species.

BGPI : équipe 2/7; International audience; Endogenous viral sequences are essentially ‘fossil records’ that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21–24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species.

Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of badnaviruses, and determine if badnavirus genomes are integrated in the yam genome. Leaf samples (n = 58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n = 112) and landraces (n = 15) at IITA, Nigeria, were screened using generic badnavirus PCR primers. Positive amplification of an expected ca. 579 bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from badnavirus particles or putative endogenous badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.

Filamentous viral particles were observed by electron microscopy in several banana plants without symptoms. The same type of particles were observed in mixed infections with cucumber mosaic virus (CMV) in banana plants developing unusual symptoms of necrosis. Some of them were serologically related to one of two filamentous particles that are encountered in plants affected by the Banana Bract Mosaic Disease (BBrMD). Samples were collected from several banana cultivars in the banana collection of CIRAD-FLHOR at NeufChdteau (Guadeloupe) and during a survey of the banana growing areas in Guadeloupe. A procedure based on western blots combining three antibodies for detection (two reacting with BBrMD and one with potyviruses) and PCR amplification with potyvirus degenerate primers was used to classify these particles with regards to BBrMD and potyviruses. At least three types of filamentous viral particles were encountered in banana plants. Two were serologically related to those involved in the BBrMD: one was in mixed infections with CMV correlated with an unusual necrosis symptom and seemed to be aphid transmitted; the other one appeared to be associated with the 'AAB' banana genome. The third particle seemed to be a virus different from a potyvirus. (Resume d'auteur)