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.

How virulent are emerging maize-infecting mastreviruses?

Maize streak disease (MSD) is one of the most significant biotic constraints on the production of Africa's most important cereal crop. Until recently, the only virus known to cause severe MSD was the A-strain of maize streak virus (MSV/A), a member of the genus Mastrevirus, family Geminiviridae. However, over the past decade, two other mastreviruses, MSV/C and maize streak Réunion virus (MSRV), have been repeatedly found in the absence of MSV/A in maize plants displaying severe MSD symptoms. Here, we report on infectious clones of MSV/C and MSRV and test their ability to cause severe MSD symptoms. Although cloned MSV/C and MSRV genomes could cause systemic symptomatic infections in MSD-sensitive maize genotypes, these infections yielded substantially milder symptoms than those observed in the field. The MSV/C and MSRV isolates that we have examined are therefore unlikely to cause severe MSD on their own. Furthermore, mixed infections of MSRV and MSV/C with other mild MSV strains also consistently yielded mild MSD symptoms. It is noteworthy that MSRV produces distinctive striate symptoms in maize that are similar in pattern, albeit not in severity, to those seen in the field, showing that this virus may contribute to the severe MSD symptoms seen in the field. Therefore, despite not fulfilling Koch's postulates for MSV/C and MSRV as causal agents of severe MSD, we cannot exclude the possibility that these viruses could be contributing to currently emerging maize diseases.

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.

From Spatial Metagenomics to Molecular Characterization of Plant Viruses: A Geminivirus Case Study.

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.

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.

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.

Identification of an Australian-like dicot-infecting mastrevirus in Pakistan.

Although members of five distinct viral species in the genus Mastrevirus (family Geminiviridae) infect dicotyledonous plants in Australia, in the remainder of the world, only a single dicot-infecting mastrevirus, chickpea chlorotic dwarf virus (CpCDV) has ever been identified. This virus has been found infecting leguminous hosts in Africa, the Middle East and the Indian subcontinent. To further explore the diversity of CpCDV in Pakistan, ten full mastrevirus genome sequences from chickpea and lentil plants were determined. Eight of these genomes were from previously described CpCDV strains and included the first reported strain D and H isolates in Pakistan. Two other genomes derived from infected chickpea plants are more closely related to dicot-infecting mastreviruses found in Australia than they are to CpCDV. These two divergent genomes shared less than 75 % genome-wide nucleotide sequence identity with other characterised mastreviruses and therefore are likely to belong to a second species of dicot-infecting mastreviruses outside of Australia. We propose naming this species Chickpea yellow dwarf virus. We discuss how the presence of chickpea yellow dwarf virus (CpYDV) in Pakistan weakens the hypothesis that Australia is the geographical origin of the dicot-infecting mastreviruses.