Molecular characterization of Euphorbia caput-medusae stunt virus: Evidence for the existence of a new genus within the family Geminiviridae

Studies focusing on phytoviruses isolated from the wild are rare. Nevertheless, even if those studies remain scarce, it is increasingly accepted that viruses coming from wild plants might play a role on disease emergence and in the functioning of ecosystems. Geminivirus are a major cause of disease on plants of agronomic interest. We hypothesize that strengthening our knowledge of the geminivirus diversity coming from the wild could help reconstructing the evolutionary history of the Geminiviridae family but also could help us understanding and predicting future epidemics. Over the past two decades, rolling circle amplification (RCA) has been more and more employed for the detection of small circular single-stranded DNA viruses, including geminivirus coming from the wild 1. We have used this method for detecting the presence of ssDNA from 236 plants collected in the South African fynbos. We have obtained amplified DNAs from 36% of the plants (85 out of 236 plants). Using classical cloning and sequencing methods, we have obtained ten sequences of which one was identified as a plant virus. This viral sequence corresponds to a new geminivirus, which infects a wild spurge (Euphorbia caput-medusae). This geminivirus is highly divergent from the current known members of the family Geminiviridae and is likely to represent a new previously unknown genus of this agriculturally highly relevant family of viruses. The virus, which we have named Euphorbia caput-medusae stunt virus (EcmSV) is not obviously a recombinant of viruses in the known geminivirus genera, has features most similar to viruses in the genus Mastrevirus (the presence of a repA gene and the production transcripts that are almost certainly spliced), but it also has unique features among geminiviruses (potential product of spliced V2-V3 ORFs). Besides EcmSV providing new information on the evolutionary history of geminiviruses, its discovery stresses the need to better assess viral diversity at the interface between wild and cultivated areas (by in situ sampling) and to study viruses isolated from wild hosts for their potential to infect crop species and vice-versa (by in vitro experimentation). 1. Varsani,A. et al. A highly divergent South African geminivirus species (Texte intégral

New insights into the evolutionary history of geminiviruses derived through the discovery of divergent viruses isolated from wild plants

During a large scale "non a priori" survey in 2010 of South African plant-infecting single stranded DNA viruses, a highly divergent geminivirus genome was isolated from an uncultivated spurge, Euphorbia caput-medusae. In addition to being infectious in E. caputmedusae, the cloned viral genome was also infectious in the cultivated hosts, tomato and Nicotiana benthamiana. The virus, named Euphorbia caput-medusae Latent virus (EcmLV) due to the absence of infection symptoms displayed by its natural host, caused severe symptoms in both of the cultivated plant species. The genome organization of EcmLV is unique amongst geminiviruses and it likely expresses at least two proteins without any detectable homologues within public sequence databases. Although clearly a geminivirus, EcmLV is so divergent that we propose its placement within a new genus that we have tentatively named Capulavirus. Using the most divergent set of geminivirus genomes ever assembled, we detect strong evidence that recombination has likely been a primary process in the genus-level diversification of geminiviruses. We demonstrate how this insight, taken together with phylogenetic analyses of predicted coat protein and replication associated protein (Rep) amino acid sequences indicate that the most recent common ancestor of the geminiviruses was likely a dicot-infecting virus that, like modern day mastreviruses and becurtoviruses, expressed its Rep from a spliced complementary strand transcript. (Résumé d'auteur

Night at the museum: Contribution of small RNA from historical herbarium specimens in the reconstruction of evolutionary histories of geminiviruses

Emerging infectious diseases of plants, almost half of which are caused by viruses, are recognized as a growing threat to global food security. However, little is known about the evolutionary processes and ecological factors that underlie the emergence and success of viruses that have caused past epidemics. With technological advances in the field of ancient DNA and RNA, it is now possible to sequence historical viral genomes, which provides us direct access to the dimension of time in evolutionary studies. Herbarium collections are an enormous source of dated, identified and well-preserved material that can be used to elucidate the emergence and evolutionary history of viral plant pathogens. Geminiviruses are responsible for many of the emerging plant diseases worldwide with a major economic impact on food crops such as cassava, which are a vital source of dietary calories in many sub-Saharan African countries. Their high potential for evolution, with high rates of mutation and recombination, makes such viruses an ideal model for understanding the epidemiological and evolutionary processes associated with viral emergence. Our proof of concept study investigated whether small interfering RNA (siRNA) can be used to reconstruct a complete geminivirus DNA genome from a herbarium sample despite the existence of post-mortem nucleic acid damage. Using a metagenomics approach based on the high-throughput sequencing of siRNA, we obtained a siRNA database from cassava leaf samples presenting typical symptoms of cassava mosaic disease that were collected in 1928 from Madagascar and 1968 from Cameroon, and then stored in the National Museum of Natural History herbarium in Paris. Our preliminary results demonstrate our ability to reconstruct the almost complete sequence of bipartite begomoviruses in particular from a 90-year-old herbarium specimen. These sequences are now used in phylogenetic, comparative genomic and phylogeographic studies to elucidate the emergence and evolutionary history of this important crop pathogen

Diversity and structure of Poaceae-infecting mastreviruses communities on Reunion Island using a viral metagenomics-based approach

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

Hidden diversity of endogenous geminiviral sequences across plant genomes and transcriptomes

Endogenous viral elements (EVE) can be used as 'fossil records' to 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. Most of the EVEs that have been characterized so far belong to family Caulimoviridae. However the first plant EVEs to be discovered were geminivirus derived sequences in the nuclear genomes of various Nicotiana species. Since then, endogenous geminivirus-like elements (EGV) have also been identified in the genomes of several plants, including yam (several Dioscorea species), apple (Malus domestica), lettuce (Lactuca sativa), cottonwood (Populus trichocarpa) and coffee (Coffea canephora). We therefore search for evidence of EGVs within 134 plant genome sequences and 797 plant transcriptome sequences. We detected homologues of geminivirus replication-associated protein (rep) genes from 17 genomes and 39 transcriptomes from angiosperms. Copy numbers of EGVs within these genomes varied widely with the highest copy numbers, approximately 1000, being found in two varieties of tea (Camellia sinensis). Phylogenetic and similarity-based analyses revealed multiple taxonomically novel geminivirus lineages, including two in Camellia species which might represent novel genera. We found that some of the Camellia and Dioscorea EGVs are transcriptionally active, and display evidence of purifying selection, suggesting that expressed geminivirus proteins were, and may still be, functionally active in certain host plants. Collectively our analysis expands the known breadth of past geminivirus diversity, provides a first large-scale view of EGV prevalence, and strengthens support for the hypothesis that EGVs impact the biology of their hosts

Geo-metagenomics: deciphering the spatial biodiversity of ssDNA viruses associated with Western Cape and Camargue agroecosystems

Over the past three years we have developed a geometagenomics approach which, based on the sampling design and the depth of sampling, enables the quantitative ecosystem-scale evaluation of spatial variations in, viral demographics, host distributions, and gene-flow. The approach is particularly well suited to analysing viral dynamics in ecosystem perturbations. The geometagenomics approach can precisely link individual sequence reads from bulked mixed sequencing reactions to information on abiotic and biotic conditions of the samples from which the sequences were obtained, the plant hosts from which samples were collected and the spatial arrangement of the samples. Our experimental design sampling locations are systematically placed within a predefined grid; the location of which is placed according to available geographic information systems data. This a priori choice of the sampling points allows the identification of reference ecosystems that should be appropriate for determining, for example, the impacts of agriculture on viral demographics and evolution within natural endangered ecosystems or the transmission rates of viruses between wild and cultivated plants. Our study was conducted in the Western Cape (South Africa) and Camargue (France) regions, which include wild areas, including renosterveld shrubland and strandveld shrubland besides wide fertile plains under introduced crops such as barley, wheat and wine in South Africa and sansouires, marshes and meadows surrounded by areas under intensive agriculture such as rice and wheat in Camargue. Besides determining the spatial and host distributions of various groups of both known and previously unknown ssDNA viruses, we compare the ssDNA species richness of the various wild and cultivated sampling locations. Amongst a large number of apparently novel single stranded DNA viruses, was one in South Africa belonging to a new Geminivirus genus that we have tentatively named, Capulavirus. (Texte intégral