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.

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.

Genetic Diversity, Evolution, and Diagnosis of Sugarcane Yellow Leaf Virus from 19 Sugarcane-Producing Locations Worldwide.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf, has been reported in an increasing number of sugarcane-growing locations since its first report in the 1990s in Brazil, Florida, and Hawaii. In this study, the genetic diversity of SCYLV was investigated using the genome coding sequence (5,561 to 5,612 nt) of 109 virus isolates from 19 geographical locations, including 65 new isolates from 16 geographical regions worldwide. These isolates were distributed in three major phylogenetic lineages (BRA, CUB, and REU), except for one isolate from Guatemala. Twenty-two recombination events were identified among the 109 isolates of SCYLV, thus confirming that recombination was a significant driving force in the genetic diversity and evolution of this virus. No temporal signal was found in the genomic sequence dataset, most likely because of the short temporal window of the 109 SCYLV isolates (1998 to 2020). Among 27 primers reported in the literature for the detection of the virus by RT-PCR, none matched 100% with all 109 SCYLV sequences, suggesting that the use of some primer pairs may not result in the detection of all virus isolates. Primers YLS111/YLS462, which were the first primer pair used by numerous research organizations to detect the virus by RT-PCR, failed to detect isolates belonging to the CUB lineage. In contrast, primer pair ScYLVf1/ScYLVr1 efficiently detected isolates of all three lineages. Continuous pursuit of knowledge of SCYLV genetic variability is therefore critical for effective diagnosis of yellow leaf, especially in virus-infected and mainly asymptomatic sugarcane plants.

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.

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.

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.

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.

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.

Emergence of Southern Rice Black-Streaked Dwarf Virus in the Centuries-Old Chinese Yuanyang Agrosystem of Rice Landraces.

Southern rice black-streaked dwarf virus (SRBSDV), which causes severe disease symptoms in rice (Oriza sativa L.) has been emerging in the last decade throughout northern Vietnam, southern Japan and southern, central and eastern China. Here we attempt to quantify the prevalence of SRBSDV in the Honghe Hani rice terraces system (HHRTS)-a Chinese 1300-year-old traditional rice production system. We first confirm that genetically diverse rice varieties are still being cultivated in the HHRTS and categorize these varieties into three main genetic clusters, including the modern hybrid varieties group (MH), the Hongyang improved modern variety group (HY) and the traditional indica landraces group (TIL). We also show over a 2-year period that SRBSDV remains prevalent in the HHRTS (20.1% prevalence) and that both the TIL (17.9% prevalence) and the MH varieties (5.1% prevalence) were less affected by SRBSDV than were the HY varieties (30.2% prevalence). Collectively we suggest that SRBSDV isolates are freely moving within the HHRTS and that TIL, HY and MH rice genetic clusters are not being preferentially infected by particular SRBSDV lineages. Given that SRBSDV can cause 30-50% rice yield losses, our study emphasizes both the need to better monitor the disease in the HHRTS, and the need to start considering ways to reduce its burden on rice production.

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.

A New Prevalent Densovirus Discovered in Acari. Insight from Metagenomics in Viral Communities Associated with Two-Spotted Mite (Tetranychus urticae) Populations.

Viral metagenomics and high throughput sequence mining have revealed unexpected diversity, and the potential presence, of parvoviruses in animals from all phyla. Among arthropods, this diversity highlights the poor knowledge that we have regarding the evolutionary history of densoviruses. The aim of this study was to explore densovirus diversity in a small arthropod pest belonging to Acari, the two-spotted spider mite Tetranychus urticae, while using viral metagenomics based on virus-enrichment. Here, we present the viromes obtained from T. urticae laboratory populations made of contigs that are attributed to nine new potential viral species, including the complete sequence of a novel densovirus. The genome of this densovirus has an ambisens genomic organization and an unusually compact size with particularly small non-structural proteins and a predicted major capsid protein that lacks the typical PLA2 motif that is common to all ambidensoviruses described so far. In addition, we showed that this new densovirus had a wide prevalence across populations of mite species tested and a genomic diversity that likely correlates with the host phylogeny. In particular, we observed a low densovirus genomic diversity between the laboratory and natural populations, which suggests that virus within-species evolution is probably slower than initially thought. Lastly, we showed that this novel densovirus can be inoculated to the host plant following feeding by infected mites, and circulate through the plant vascular system. These findings offer new insights into densovirus prevalence, evolution, and ecology.

Nanopore-based detection and characterization of yam viruses.

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.

Viral Metagenomics Approaches for High-Resolution Screening of Multiplexed Arthropod and Plant Viral Communities.

Viral metagenomic approaches have become essential for culture-independent and sequence-independent viral detection and characterization. This chapter describes an accurate and efficient approach to (1) concentrate viral particles from arthropods and plants, (2) remove contaminating non-encapsidated nucleic acids, (3) extract and amplify both viral DNA and RNA, and (4) analyze high-throughput sequencing (HTS) data by bioinformatics. Using this approach, up to 96 arthropod or plant samples can be multiplexed in a single HTS library.

Geometagenomics illuminates the impact of agriculture on the distribution and prevalence of plant viruses at the ecosystem scale.

Disease emergence events regularly result from human activities such as agriculture, which frequently brings large populations of genetically uniform hosts into contact with potential pathogens. Although viruses cause nearly 50% of emerging plant diseases, there is little systematic information about virus distribution across agro-ecological interfaces and large gaps in understanding of virus diversity in nature. Here we applied a novel landscape-scale geometagenomics approach to examine relationships between agricultural land use and distributions of plant-associated viruses in two Mediterranean-climate biodiversity hotspots (Western Cape region of South Africa and Rhône river delta region of France). In total, we analysed 1725 geo-referenced plant samples collected over two years from 4.5 × 4.5 km2 grids spanning farmlands and adjacent uncultivated vegetation. We found substantial virus prevalence (25.8-35.7%) in all ecosystems, but prevalence and identified family-level virus diversity were greatest in cultivated areas, with some virus families displaying strong agricultural associations. Our survey revealed 94 previously unknown virus species, primarily from uncultivated plants. This is the first effort to systematically evaluate plant-associated viromes across broad agro-ecological interfaces. Our findings indicate that agriculture substantially influences plant virus distributions and highlight the extent of current ignorance about the diversity and roles of viruses in nature.

Occurrence of a novel mastrevirus in sugarcane germplasm collections in Florida, Guadeloupe and Réunion.

BACKGROUND: In Africa and Asia, sugarcane is the host of at least seven different virus species in the genus Mastrevirus of the family Geminiviridae. However, with the exception of Sugarcane white streak virus in Barbados, no other sugarcane-infecting mastrevirus has been reported in the New World. Conservation and exchange of sugarcane germplasm using stalk cuttings facilitates the spread of sugarcane-infecting viruses. METHODS: A virion-associated nucleic acids (VANA)-based metagenomics approach was used to detect mastrevirus sequences in 717 sugarcane samples from Florida (USA), Guadeloupe (French West Indies), and Réunion (Mascarene Islands). Contig assembly was performed using CAP3 and sequence searches using BLASTn and BLASTx. Mastrevirus full genomes were enriched from total DNA by rolling circle amplification, cloned and sequenced. Nucleotide and amino acid sequence identities were determined using SDT v1.2. Phylogenetic analyses were conducted using MEGA6 and PHYML3. RESULTS: We identified a new sugarcane-infecting mastrevirus in six plants sampled from germplasm collections in Florida and Guadeloupe. Full genome sequences were determined and analyzed for three virus isolates from Florida, and three from Guadeloupe. These six genomes share >88% genome-wide pairwise identity with one another and between 89 and 97% identity with a recently identified mastrevirus (KR150789) from a sugarcane plant sampled in China. Sequences similar to these were also identified in sugarcane plants in Réunion. CONCLUSIONS: As these virus isolates share <64% genome-wide identity with all other known mastreviruses, we propose classifying them within a new mastrevirus species named Sugarcane striate virus. This is the first report of sugarcane striate virus (SCStV) in the Western Hemisphere, a virus that most likely originated in Asia. The distribution, vector, and impact of SCStV on sugarcane production remains to be determined.

Metagenomic-Based Screening and Molecular Characterization of Cowpea-Infecting Viruses in Burkina Faso.

Cowpea, (Vigna unguiculata L. (Walp)) is an annual tropical grain legume. Often referred to as "poor man's meat", cowpea is one of the most important subsistence legumes cultivated in West Africa due to the high protein content of its seeds. However, African cowpea production can be seriously constrained by viral diseases that reduce yields. While twelve cowpea-infecting viruses have been reported from Africa, only three of these have so-far been reported from Burkina Faso. Here we use a virion-associated nucleic acids (VANA)-based metagenomics method to screen for the presence of cowpea viruses from plants collected from the three agro-climatic zones of Burkina Faso. Besides the three cowpea-infecting virus species which have previously been reported from Burkina Faso (Cowpea aphid borne mosaic virus [Family Potyviridae], the Blackeye cowpea mosaic virus-a strain of Bean common mosaic virus-[Family Potyviridae] and Cowpea mottle virus [Family Tombusviridae]) five additional viruses were identified: Southern cowpea mosaic virus (Sobemovirus genus), two previously uncharacterised polerovirus-like species (Family Luteoviridae), a previously uncharacterised tombusvirus-like species (Family Tombusviridae) and a previously uncharacterised mycotymovirus-like species (Family Tymoviridae). Overall, potyviruses were the most prevalent cowpea viruses (detected in 65.5% of samples) and the Southern Sudan zone of Burkina Faso was found to harbour the greatest degrees of viral diversity and viral prevalence. Partial genome sequences of the two novel polerovirus-like and tombusvirus-like species were determined and RT-PCR primers were designed for use in Burkina Faso to routinely detect all of these cowpea-associated viruses.

Molecular characterization and prevalence of two capulaviruses: Alfalfa leaf curl virus from France and Euphorbia caput-medusae latent virus from South Africa.

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.

Prevalence of Sugarcane yellow leaf virus in Sugarcane-Producing Regions in Kenya Revealed by Reverse-Transcription Loop-Mediated Isothermal Amplification Method.

Yellow leaf (YL) is a disease of sugarcane that is currently widespread throughout most American and Asian sugarcane-producing countries. However, its actual distribution in Africa remains largely unknown. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to facilitate and improve the detection of Sugarcane yellow leaf virus (SCYLV), the causal agent of YL. The RT-LAMP assay was found to be comparable with or superior to conventional RT-polymerase chain reaction (PCR) for the detection of SCYLV genotypes CUB and BRA in infected sugarcane 'C132-81' and 'SP71-6163', respectively. Additionally, 68 sugarcane samples that tested negative by RT-PCR were found positive by RT-LAMP, whereas the RT-LAMP assay failed to detect SCYLV in only 5 samples that tested positive by RT-PCR. Combining RT-PCR and RT-LAMP data enabled the detection of SCYLV in 86 of 183 Kenyan sugarcane plants, indicating high SCYLV prevalence throughout the country (ranging from 36 to 64% in individual counties). Seminested PCR assays were developed that enabled the amplification of a fragment encompassing the capsid protein coding region gene and its flanking 5' and 3' genomic regions. Sequences of this fragment for four Kenyan SCYLV isolates indicated that they shared 99.2 to 99.6% pairwise identity with one another and clearly clustered phylogenetically with SCYLV-BRA genotype isolates. To our knowledge, this is the first report of SCYLV in Kenya.

Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus.

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.