Characterization of Six Ampeloviruses Infecting Pineapple in Reunion Island Using a Combination of High-Throughput Sequencing Approaches.

The cultivation of pineapple (Ananas comosus) is threatened worldwide by mealybug wilt disease of pineapple (MWP), whose etiology is not yet fully elucidated. In this study, we characterized pineapple mealybug wilt-associated ampeloviruses (PMWaVs, family Closteroviridae) from a diseased pineapple plant collected from Reunion Island, using a high-throughput sequencing approach combining Illumina short reads and Nanopore long reads. Reads co-assembly resulted in complete or near-complete genomes for six distinct ampeloviruses, including the first complete genome of pineapple mealybug wilt-associated virus 5 (PMWaV5) and that of a new species tentatively named pineapple mealybug wilt-associated virus 7 (PMWaV7). Short reads data provided high genome coverage and sequencing depths for all six viral genomes, contrary to long reads data. The 5' and 3' ends of the genome for most of the six ampeloviruses could be recovered from long reads, providing an alternative to RACE-PCRs. Phylogenetic analyses did not unveil any geographic structuring of the diversity of PMWaV1, PMWaV2 and PMWaV3 isolates, supporting the current hypothesis that PMWaVs were mainly spread by human activity and vegetative propagation.

Virome release of an invasive exotic plant species in southern France.

The increase in human-mediated introduction of plant species to new regions has resulted in a rise of invasive exotic plant species (IEPS) that has had significant effects on biodiversity and ecosystem processes. One commonly accepted mechanism of invasions is that proposed by the enemy release hypothesis (ERH), which states that IEPS free from their native herbivores and natural enemies in new environments can outcompete indigenous species and become invasive. We here propose the virome release hypothesis (VRH) as a virus-centered variant of the conventional ERH that is only focused on enemies. The VRH predicts that vertically transmitted plant-associated viruses (PAV, encompassing phytoviruses and mycoviruses) should be co-introduced during the dissemination of the IEPS, while horizontally transmitted PAV of IEPS should be left behind or should not be locally transmitted in the introduced area due to a maladaptation of local vectors. To document the VRH, virome richness and composition as well as PAV prevalence, co-infection, host range, and transmission modes were compared between indigenous plant species and an invasive grass, cane bluestem (Bothriochloa barbinodis), in both its introduced range (southern France) and one area of its native range (Sonoran Desert, Arizona, USA). Contrary to the VRH, we show that invasive populations of B. barbinodis in France were not associated with a lower PAV prevalence or richness than native populations of B. barbinodis from the USA. However, comparison of virome compositions and network analyses further revealed more diverse and complex plant-virus interactions in the French ecosystem, with a significant richness of mycoviruses. Setting mycoviruses apart, only one putatively vertically transmitted phytovirus (belonging to the Amalgaviridae family) and one putatively horizontally transmitted phytovirus (belonging to the Geminiviridae family) were identified from B. barbinodis plants in the introduced area. Collectively, these characteristics of the B. barbinodis-associated PAV community in southern France suggest that a virome release phase may have immediately followed the introduction of B. barbinodis to France in the 1960s or 1970s, and that, since then, the invasive populations of this IEPS have already transitioned out of this virome release phase, and have started interacting with several local mycoviruses and a few local plant viruses.

Historical Review on Sugarcane Streak Mosaic Virus That Has Recently Emerged in Africa.

Sugarcane streak mosaic virus (SCSMV), now assigned to the genus Poacevirus of the family Potyviridae, was reported for the first time in 1932 in Louisiana and was believed to be strain F of sugarcane mosaic virus (SCMV) for more than six decades. SCMV-F was renamed SCSMV in 1998 after partial sequencing of its genome and phylogenetic investigations. Following the development of specific molecular diagnostic methods in the 2000s, SCSMV was recurrently found in sugarcane exhibiting streak mosaic symptoms in numerous Asian countries, but not in the Western hemisphere or in Africa. In this review, we give an overview of the current knowledge on this disease and the progression in research on SCSMV. This includes symptoms, geographical distribution and incidence, diagnosis and genetic diversity of the virus, epidemiology, as well as control. Finally, we highlight future challenges as sugarcane streak mosaic has recently been found in Africa where this disease represents a new threat to sugarcane production.

The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

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.

Molecular diversity of yam virus Y and identification of banana mild mosaic virus isolates infecting yam (Dioscorea spp.).

Two members of the family Betaflexiviridae associated with yam (Dioscorea spp.) have been described so far: yam latent virus (YLV) and yam virus Y (YVY). However, their geographical distribution and molecular diversity remain poorly documented. Using a nested RT-PCR assay, we detected YVY in D. alata, D. bulbifera, D. cayenensis, D. rotundata, and D. trifida in Guadeloupe, and in D. rotundata in Côte d'Ivoire, thus extending the known host range of this virus and geographical distribution. Using amplicon sequencing, we determined that the molecular diversity of YVY in the yam samples analyzed in this work ranged between 0.0 and 29.1% and that this diversity is partially geographically structured. We also identified three isolates of banana mild mosaic virus (BanMMV) infecting D. alata in Guadeloupe, providing the first evidence for BanMMV infection in yam.

Long-Term Anthropogenic Management and Associated Loss of Plant Diversity Deeply Impact Virome Richness and Composition of Poaceae Communities.

Modern agriculture has influenced plant virus emergence through ecosystem simplification, introduction of new host species, and reduction in crop genetic diversity. Therefore, it is crucial to better understand virus distributions across cultivated and uncultivated communities in agro-ecological interfaces, as well as virus exchange among them. Here, we advance fundamental understanding in this area by characterizing the virome of three co-occurring replicated Poaceae community types that represent a gradient of grass species richness and management intensity, from highly managed crop monocultures to little-managed, species-rich grasslands. We performed a large-scale study on 950 wild and cultivated Poaceae over 2 years, combining untargeted virome analysis down to the virus species level with targeted detection of three plant viruses. Deep sequencing revealed (i) a diversified and largely unknown Poaceae virome (at least 51 virus species or taxa), with an abundance of so-called persistent viruses; (ii) an increase of virome richness with grass species richness within the community; (iii) stability of virome richness over time but a large viral intraspecific variability; and (iv) contrasting patterns of virus prevalence, coinfections, and spatial distribution among plant communities and species. Our findings highlight the complex structure of plant virus communities in nature and suggest the influence of anthropogenic management on viral distribution and prevalence. IMPORTANCE Because viruses have been mostly studied in cultivated plants, little is known about virus diversity and ecology in less-managed vegetation or about the influence of human management and agriculture on virome composition. Poaceae (grass family)-dominated communities provide invaluable opportunities to examine these ecological issues, as they are distributed worldwide across agro-ecological gradients, are essential for food security and conservation, and can be infected by numerous viruses. Here, we used multiple levels of analysis that considered plant communities, individual plants, virus species, and haplotypes to broaden understanding of the Poaceae virome and to evaluate host-parasite richness relationships within agro-ecological landscapes in our study area. We emphasized the influence of grass diversity and land use on the composition of viral communities and their life history strategies, and we demonstrated the complexity of plant-virus interactions in less-managed grass communities, such as the higher virus prevalence and overrepresentation of mixed virus infection compared to theoretical predictions.

Molecular characterization of Cordyline virus 1 isolates infecting yam (Dioscorea spp).

Cordyline virus 1 (CoV1) is a velarivirus that has so far only been reported in ornamental Ti plants (Cordyline fruticosa). Using high-throughput sequencing, we identified CoV1 infection in yam accessions from Vanuatu. Using a specific RT-PCR assay, we found that CoV1 is also present and highly prevalent in Dioscorea alata, D. cayenensis, and D. trifida in Guadeloupe. Phylogenetic analysis showed that CoV1 isolates infecting yam in Guadeloupe display a low level of molecular diversity. These data provide insights into the transmission of CoV1 in yam in Guadeloupe.

Identification of a novel vitivirus from pineapple in Reunion Island.

We report the complete genome sequence of a novel member of the genus Vitivirus (family Betaflexiviridae, subfamily Trivirinae) infecting pineapple. The complete genome sequence of this virus was obtained from total RNA extracted from pineapple leaf samples collected in Reunion Island, using a combination of high-throughput sequencing technologies. The viral genome is 6,757 nt long, excluding the poly(A) tail, and shares all the hallmarks of vitiviruses. Phylogenetic analysis performed on the replication-associated protein and capsid protein gene sequences unambiguously place this new virus, for which we propose the name "pineapple virus A", in the genus Vitivirus.

Virion-Associated Nucleic Acid-Based Metagenomics: A Decade of Advances in Molecular Characterization of Plant Viruses.

Over the last decade, viral metagenomic studies have resulted in the discovery of thousands of previously unknown viruses. These studies are likely to play a pivotal role in obtaining an accurate and robust understanding of how viruses affect the stability and productivity of ecosystems. Among the metagenomics-based approaches that have been developed since the beginning of the 21st century, shotgun metagenomics applied specifically to virion-associated nucleic acids (VANA) has been used to disentangle the diversity of the viral world. We summarize herein the results of 24 VANA-based studies, focusing on plant and insect samples conducted over the last decade (2010 to 2020). Collectively, viruses from 85 different families were reliably detected in these studies, including capsidless RNA viruses that replicate in fungi, oomycetes, and plants. Finally, strengths and weaknesses of the VANA approach are summarized and perspectives of applications in detection, epidemiological surveillance, environmental monitoring, and ecology of plant viruses are provided. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Host range and molecular variability of the sadwavirus dioscorea mosaic associated virus.

Dioscorea mosaic associated virus (DMaV) is a member of the genus Sadwavirus, family Secoviridae, that is associated with mosaic symptoms in Dioscorea rotundata in Brazil. The genome of a DMaV isolate detected in D. trifida in Guadeloupe was sequenced by high-throughput sequencing. Using an RT-PCR-based detection assay, we found that DMaV infects D. alata, D. bulbifera, D. cayenensis-rotundata, D. esculenta, and D. trifida accessions conserved in Guadeloupe and Côte d'Ivoire and displays a very high level of molecular diversity in a relatively small region of the genome targeted by the assay. We also provide evidence that DMaV is also present in D. rotundata in Benin and in D. alata in Nigeria.

Complete genome sequence of a novel marafivirus infecting pearl millet in Burkina Faso.

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a staple food that is widely cultivated in sub-Saharan Africa. In August 2018, a survey was conducted in the main producing regions of Burkina Faso, and leaf samples were analyzed using virion-associated nucleic acid (VANA)-based metagenomic approach and Illumina sequencing. A new virus, tentatively named "Pennisetum glaucum marafivirus" (PGMV), was detected, and its complete nucleotide sequence of 6364 nucleotides was determined. The sequence contains a large open reading frame (ORF) encoding a polyprotein of 224.2 kDa with five domains (methyltransferase, papain-like protease, helicase, RNA-dependent RNA polymerase, and coat proteins), typical of marafiviruses. Additionally, a characteristic conserved marafibox domain was detected in the genome. The nucleotide sequence of the complete PGMV genome shares 68.5% identity with that of sorghum bicolor marafivirus, and its coat protein shares 58.5% identity with that of oat blue dwarf virus. Phylogenetic analysis confirmed that the pearl millet virus is unambiguously grouped with members of the genus Marafivirus in the family Tymoviridae. This is the first report on the occurrence of a marafivirus in pearl millet.

Novel Ampeloviruses Infecting Cassava in Central Africa and the South-West Indian Ocean Islands.

Cassava is one of the most important staple crops in Africa and its production is seriously damaged by viral diseases. In this study, we identify for the first time and characterize the genome organization of novel ampeloviruses infecting cassava plants in diverse geographical locations using three high-throughput sequencing protocols [Virion-Associated Nucleotide Acid (VANA), dsRNA and total RNA], and we provide a first analysis of the diversity of these agents and of the evolutionary forces acting on them. Thirteen new Closteroviridae isolates were characterized in field-grown cassava plants from the Democratic Republic of Congo (DR Congo), Madagascar, Mayotte, and Reunion islands. The analysis of the sequences of the corresponding contigs (ranging between 10,417 and 13,752 nucleotides in length) revealed seven open reading frames. The replication-associated polyproteins have three expected functional domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). Additional open reading frames code for a small transmembrane protein, a heat-shock protein 70 homolog (HSP70h), a heat shock protein 90 homolog (HSP90h), and a major and a minor coat protein (CP and CPd respectively). Defective genomic variants were also identified in some cassava accessions originating from Madagascar and Reunion. The isolates were found to belong to two species tentatively named Manihot esculenta-associated virus 1 and 2 (MEaV-1 and MEaV-2). Phylogenetic analyses showed that MEaV-1 and MEaV-2 belong to the genus Ampelovirus, in particular to its subgroup II. MEaV-1 was found in all of the countries of study, while MEaV-2 was only detected in Madagascar and Mayotte. Recombination analysis provided evidence of intraspecies recombination occurring between the isolates from Madagascar and Mayotte. No clear association with visual symptoms in the cassava host could be identified.

Genome characterization and diversity of trifolium virus 1: identification of a novel legume-infecting capulavirus.

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.

Comparison of the Virome of Quarantined Sugarcane Varieties and the Virome of Grasses Growing near the Quarantine Station.

Visacane is a sugarcane quarantine station located in the South of France, far away from sugarcane growing areas. Visacane imports up to 100 sugarcane varieties per year, using safe control and confinement measures of plants and their wastes to prevent any risk of pathogen spread outside of the facilities. Viruses hosted by the imported material are either known or unknown to cause disease in cultivated sugarcane. Poaceae viruses occurring in plants surrounding the quarantine glasshouse are currently unknown. These viruses could be considered as a source of new sugarcane infections and potentially cause new sugarcane diseases in cases of confinement barrier failure. The aim of this study was to compare the plant virome inside and outside of the quarantine station to identify potential confinement failures and risks of cross infections. Leaves from quarantined sugarcane varieties and from wild Poaceae growing near the quarantine were collected and processed by a metagenomics approach based on virion-associated nucleic acids extraction and library preparation for Illumina sequencing. While viruses belonging to the same virus genus or family were identified in the sugarcane quarantine and its surroundings, no virus species was detected in both environments. Based on the data obtained in this study, no virus movement between quarantined sugarcane and nearby grassland has occurred so far, and the confinement procedures of Visacane appear to be properly implemented.

New World Cactaceae Plants Harbor Diverse Geminiviruses.

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.

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.

Genetic Diversity of Rice stripe necrosis virus and New Insights into Evolution of the Genus Benyvirus.

The rice stripe necrosis virus (RSNV) has been reported to infect rice in several countries in Africa and South America, but limited genomic data are currently publicly available. Here, eleven RSNV genomes were entirely sequenced, including the first corpus of RSNV genomes of African isolates. The genetic variability was differently distributed along the two genomic segments. The segment RNA1, within which clusters of polymorphisms were identified, showed a higher nucleotidic variability than did the beet necrotic yellow vein virus (BNYVV) RNA1 segment. The diversity patterns of both viruses were similar in the RNA2 segment, except for an in-frame insertion of 243 nucleotides located in the RSNV tgbp1 gene. Recombination events were detected into RNA1 and RNA2 segments, in particular in the two most divergent RSNV isolates from Colombia and Sierra Leone. In contrast to BNYVV, the RSNV molecular diversity had a geographical structure with two main RSNV lineages distributed in America and in Africa. Our data on the genetic diversity of RSNV revealed unexpected differences with BNYVV suggesting a complex evolutionary history of the genus Benyvirus.

Characterisation of the Viral Community Associated with the Alfalfa Weevil (Hypera postica) and Its Host Plant, Alfalfa (Medicago sativa).

Advances in viral metagenomics have paved the way of virus discovery by making the exploration of viruses in any ecosystem possible. Applied to agroecosystems, such an approach opens new possibilities to explore how viruses circulate between insects and plants, which may help to optimise their management. It could also lead to identifying novel entomopathogenic viral resources potentially suitable for biocontrol strategies. We sampled the larvae of a natural population of alfalfa weevils (Hypera postica), a major herbivorous pest feeding on legumes, and its host plant alfalfa (Medicago sativa). Insect and plant samples were collected from a crop field and an adjacent meadow. We characterised the diversity and abundance of viruses associated with weevils and alfalfa, and described nine putative new virus species, including four associated with alfalfa and five with weevils. In addition, we found that trophic accumulation may result in a higher diversity of plant viruses in phytophagous pests compared to host plants.

[From boots on the ground to nucleotides in the sequencer: a century of advances in the study of the plant virus ecology]. / De la paire de bottes à la paire de bases : de l'intérêt d'étudier l'écologie des viromes de plantes.

Plant virus ecology began to be explored at the end of the 19th century. Since then, major advances have revealed complex virus-host-vector interactions in a variety of environments. These advances have been accelerated by development of new technologies for virus detection and characterization, the latest of which being high-throughput sequencing (HTS). HTS technologies have proved to be effective for non-targeted characterization of all or nearly all viruses present in a sample without requiring prior information about virus identity, as would be needed for virus-targeted tests. Phytoviromic studies have thus made important advances, including characterization of the complex interactions between phytovirus dynamics and the structure of multi-species host communities, and documentation of the effects of anthropogenic ecosystem simplification on plant virus emergence and diversity. However, such studies must overcome challenges at every stage, from plant sampling to bioinformatics analysis. This review summarizes major advances in plant virus ecology, in association with technological developments, and presents key considerations for use of HTS in the study of the ecology of phytovirus communities.