Pauline Bernardo

Appearances Can Be Deceptive: Revealing a Hidden Viral Infection with Deep Sequencing in a Plant Quarantine Context

Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.

Identification and characterisation of a highly divergent geminivirus: Evolutionary and taxonomic implications

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 a wild spurge, Euphorbia caput-medusae. In addition to being infectious in E. caput-medusae, the cloned viral genome was also infectious in 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 tomato and N. benthamiana. The genome organisation 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 a set of highly divergent geminiviruses genomes, it is apparent that recombination has likely been a primary process in the genus-level diversification of geminiviruses. It is also demonstrated 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.

Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus

ABSTRACT 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.

Identification of novel Bromus- and Trifolium-associated circular DNA viruses

The genomes of a large number of highly diverse novel circular DNA viruses from a wide range of sources have been characterised in recent years, including circular single-stranded DNA (ssDNA) viruses that share similarities with plant-infecting ssDNA viruses of the family Geminiviridae. Here, we describe six novel circular DNA viral genomes that encode replication-associated (Rep) proteins that are most closely related to those of either geminiviruses or gemycircularviruses (a new group of ssDNA viruses that are closely related to geminiviruses). Four possible viral genomes were recovered from Bromushordeaceus sampled in New Zealand, and two were recovered from B. hordeaceus and Trifoliumresupinatum sampled in France. Two of the viral genomes from New Zealand (one from the North Island and one from the South Island each) share >99xa0% sequence identity, and two genomes recovered from B. hordeaceus and T. resupinatum sampled in France share 74xa0% identity. All of the viral genomes that were recovered were found to have a major open reading frame on both their complementary and virion-sense strands, one of which likely encodes a Rep and the other a capsid protein. Although future infectivity studies are needed to identify the host range of these viruses, this is the first report of circular DNA viruses associated with grasses in New Zealand.

Lepidopteran transcriptome analysis following infection by phylogenetically unrelated polydnaviruses highlights differential and common responses

The Polydnaviridae is a family of double-stranded DNA viruses that are symbionts of parasitoid wasps. The family is currently divided into two genera, the Ichnovirus (IV) and Bracovirus (BV), which are associated with wasps in the families Ichneumonidae and Braconidae, respectively. IVs and BVs have similar immunosuppressive and developmental effects on parasitized hosts but their encapsidated genomes largely encode different genes. To assess whether IV and BV infection has similar or disparate effects on the transcriptome of shared hosts, we characterized the effects of Hyposoter didymator Ichnovirus (HdIV) and Microplitis demolitor Bracovirus (MdBV) on the fat body and hemocyte transcriptome of Spodoptera frugiperda (Lepidoptera: Noctuidae). Our results indicated that HdIV and MdBV infection alters the abundance of a relatively low proportion of S.xa0frugiperda transcripts at 24xa0h post-infection. Axa0majority of the transcripts affected by infection also differed between MdBV and HdIV. However, we did identify some host transcripts that were similarly affected by both viruses. A majority of these genes were transcribed in the fat body and most belonged to functional classes with roles in immunity, detoxification, or cell structure. Particularly prominent in this suite of transcripts were genes encoding for predicted motor-related and collagen IV-like proteins. Overall, our data suggest that the broadly similar effects that HdIV and MdBV have on host growth and immunity are not due to these viruses inducing profound changes in host gene expression. Given though that IVs and BVs encode few shared genes, the host transcripts that are similarly affected by HdIV and MdBV could indicate convergence by each virus to target a few processes at the level of transcription that are important for successful parasitism of hosts by H.xa0didymator and M.xa0demolitor.

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.

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 × 4.5u2009km2 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.

Genome sequences of a capulavirus infecting Plantago lanceolata in the Åland archipelago of Finland

The discovery and full-genome sequences of two isolates of a fourth capulavirus species are reported. The viruses were discovered during a viral metagenomics survey of uncultivated Plantago lanceolata plants in the Åland archipelago of south western Finland. The newly discovered viruses apparently produce no symptoms in P. lanceolata. They have a genome organization that is very similar to that of the three known capulavirus species and additionally share between 62.9 and 67.1% genome-wide sequence identity with the isolates of these species. It is therefore proposed that these viruses be assigned to a new capulavirus species named “Plantago lanceolata latent virus”.

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.

A Novel Itera-Like Densovirus Isolated by Viral Metagenomics from the Sea Barley Hordeum marinum

ABSTRACT Densoviruses (DVs) infect arthropods and belong to the Parvoviridae family. Here, we report the complete coding sequence of a novel DV isolated from the plant Hordeum marinum (Poaceae) by viral metagenomics, and we confirmed reamplification by PCR. Phylogenetic analyses showed that this novel DV is related to the genus Iteradensovirus.