Biological and Genetic Characterization of Physostegia Chlorotic Mottle Virus in Europe Based on Host Range, Location, and Time.

Application of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses.[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.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Route of a Multipartite Nanovirus across the Body of Its Aphid Vector.

Vector transmission plays a primary role in the life cycle of viruses, and insects are the most common vectors. An important mode of vector transmission, reported only for plant viruses, is circulative nonpropagative transmission whereby the virus cycles within the body of its insect vector, from gut to salivary glands and saliva, without replicating. This mode of transmission has been extensively studied in the viral families Luteoviridae and Geminiviridae and is also reported for Nanoviridae The biology of viruses within these three families is different, and whether the viruses have evolved similar molecular/cellular virus-vector interactions is unclear. In particular, nanoviruses have a multipartite genome organization, and how the distinct genome segments encapsidated individually transit through the insect body is unknown. Here, using a combination of fluorescent in situ hybridization and immunofluorescence, we monitor distinct proteins and genome segments of the nanovirus Faba bean necrotic stunt virus (FBNSV) during transcytosis through the gut and salivary gland cells of its aphid vector Acyrthosiphon pisum FBNSV specifically transits through cells of the anterior midgut and principal salivary gland cells, a route similar to that of geminiviruses but distinct from that of luteoviruses. Our results further demonstrate that a large number of virus particles enter every single susceptible cell so that distinct genome segments always remain together. Finally, we confirm that the success of nanovirus-vector interaction depends on a nonstructural helper component, the viral protein nuclear shuttle protein (NSP), which is shown to be mandatory for viral accumulation within gut cells.IMPORTANCE An intriguing mode of vector transmission described only for plant viruses is circulative nonpropagative transmission, whereby the virus passes through the gut and salivary glands of the insect vector without replicating. Three plant virus families are transmitted this way, but details of the molecular/cellular mechanisms of the virus-vector interaction are missing. This is striking for nanoviruses that are believed to interact with aphid vectors in ways similar to those of luteoviruses or geminiviruses but for which empirical evidence is scarce. We here confirm that nanoviruses follow a within-vector route similar to that of geminiviruses but distinct from that of luteoviruses. We show that they produce a nonstructural protein mandatory for viral entry into gut cells, a unique phenomenon for this mode of transmission. Finally, noting that nanoviruses are multipartite viruses, we demonstrate that a large number of viral particles penetrate susceptible cells of the vector, allowing distinct genome segments to remain together.

Aphid transmission of nanoviruses.

The genus Nanovirus consists of plant viruses that predominantly infect legumes leading to devastating crop losses. Nanoviruses are transmitted by various aphid species. The transmission occurs in a circulative nonpropagative manner. It was long suspected that a virus-encoded helper factor would be needed for successful transmission by aphids. Recently, a helper factor was identified as the nanovirus-encoded nuclear shuttle protein (NSP). The mode of action of NSP is currently unknown in contrast to helper factors from other plant viruses that, for example, facilitate binding of virus particles to receptors within the aphids' stylets. In this review, we are summarizing the current knowledge about nanovirus-aphid vector interactions.

Characterisation of a novel nucleorhabdovirus infecting alfalfa (Medicago sativa).

BACKGROUND: Nucleorhabdoviruses possess bacilliform particles which contain a single-stranded negative-sense RNA genome. They replicate and mature in the nucleus of infected cells. Together with viruses of three other genera of the family Rhabdoviridae, they are known to infect plants and can be transmitted by arthropod vectors, during vegetative propagation, or by mechanical means. In 2010, an alfalfa (Medicago sativa) plant showing virus-like symptoms was collected from Stadl-Paura, Austria and sent to Julius Kühn Institute for analysis. METHODS: Electron microscopy (EM) of leaf extracts from infected plants revealed the presence of rhabdovirus-like particles and was further used for ultrastructural analyses of infected plant tissue. Partially-purified preparations of rhabdovirus nucleocapsids were used for raising an antiserum. To determine the virus genome sequence, high throughput sequencing (HTS) was performed. RT-PCR primers were designed to confirm virus infection and to be used as a diagnostic tool. RESULTS: EM revealed bacilliform virions resembling those of plant-infecting rhabdoviruses. HTS of ribosomal RNA-depleted total RNA extracts revealed a consensus sequence consisting of 13,875 nucleotides (nt) and containing seven open reading frames (ORFs). Homology and phylogenetic analyses suggest that this virus isolate represents a new species of the genus Nucleorhabdovirus (family Rhabdoviridae). Since the virus originated from an alfalfa plant in Austria, the name alfalfa-associated nucleorhabdovirus (AaNV) is proposed. Viroplasms (Vp) and budding virions were observed in the nuclei of infected cells by EM, thus confirming its taxonomic assignment based on sequence data. CONCLUSIONS: In this study, we identified and characterised a new nucleorhabdovirus from alfalfa. It shared only 39.8% nucleotide sequence identity with its closest known relative, black currant-associated rhabdovirus 1. The virus contains an additional open reading frame (accessory gene) with unknown function, located between the matrix protein and the glycoprotein genes. Serological and molecular diagnostic assays were designed for future screening of field samples. Further studies are needed to identify other natural hosts and potential vectors.

A divergent strain of melon chlorotic spot virus isolated from black medic (Medicago lupulina) in Austria.

A tenuivirus, referred to here as JKI 29327, was isolated from a black medic (Medicago lupulina) plant collected in Austria. The virus was mechanically transmitted to Nicotiana benthamiana, M. lupulina, M. sativa, Pisum sativum and Vicia faba. The complete genome was determined by high throughput sequencing. The genome of JKI 29327 consists of eight RNA segments closely related to those of melon chlorotic spot virus (MeCSV) isolate E11-018 from France. Since segments RNA 7 and 8 of JKI 29327 are shorter, its genome is slightly smaller (by 247 nts) than that of E11-018. Pairwise comparisons between the predicted virus proteins of JKI 29327 and their homologues in E11-018 showed aa identities ranging from 80.6 to 97.2%. Plants infected with E11-081 gave intermediate DAS-ELISA reactions with polyclonal antibodies to JKI 29327. Since JKI 29327 and E11-018 appear to be closely related both serologically and genetically, we propose to regard JKI 29327 as the black medic strain of MeCSV. To our knowledge, JKI 29327 represents the second tenuivirus identified from a dicotyledonous plant. Serological and molecular diagnostic methods were developed for future detection.

Caraway yellows virus, a novel nepovirus from Carum carvi.

A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus Nepovirus subgroup C.

Complete genome sequence of a highly divergent carrot torradovirus 1 strain from Apium graveolens.

A new virus was identified in a celery plant showing chlorotic rings, mosaic and strong yellowing symptoms, and its complete genome sequence was determined. The genomic organization of this novel virus is analogous to that of known members of the genus Torradovirus, consisting of two single-stranded RNAs of 6,823 (RNA1) and 4,263 nucleotides (RNA2), excluding the poly(A) tails. BLAST searches against the nucleotide and protein databases showed that this virus is closely related to but different from carrot torradovirus 1 (CaTV1). Comparisons between the two viruses demonstrated relatively low levels of nucleotide and amino acid similarity in different parts of their genomes, as well as considerable differences in the sizes of their two genomic RNAs. However, the protease-polymerase (Pro-Pol) and capsid protein (CP) regions of this virus share >80% amino acid identity with the corresponding regions of CaTV1. Therefore, based on the current ICTV species demarcation criteria for the family Secoviridae, the virus from celery is a divergent strain of CaTV1, named "CaTV1-celery". Nevertheless, differences between CaTV1 and CaTV1-celery in genome size, as well as in biological and epidemiological features, may warrant their separation into two distinct species in the future.

Red clover vein mosaic virus-A Novel Virus to New Zealand that is Widespread in Legumes.

Red clover vein mosaic virus (RCVMV) is an important virus of leguminous crops that can cause devastating losses. During a routine survey of legumes conducted on the South Island of New Zealand, RCVMV was found in mixed infections in clover plants with Alfalfa mosaic virus and White clover mosaic virus. The full-length sequence of the New Zealand isolate RCVMV-NZ from clover shared 96% nucleotide sequence identity with a chickpea isolate previously described from Washington (United States). Targeted surveys of pea, faba bean, and pasture crops showed that RCVMV-NZ is widespread on the South Island in New Zealand. This isolate is causing mild if any symptoms on experimental hosts and naturally infected plants.

Visual monitoring of Cucumber mosaic virus infection in Nicotiana benthamiana following transmission by the aphid vector Myzus persicae.

The single-stranded, positive-sense and tripartite RNA virus Cucumber mosaic virus (CMV) was used in this study as a method for monitoring the initial stages of virus infection following aphid transmission. The RNA2 of CMV was modified to incorporate, in a variety of arrangements, an open reading frame (ORF) encoding an enhanced green fluorescent protein (eGFP). The phenotypes of five engineered RNA2s were tested in Nicotiana tabacum, Nicotiana clevelandii and Nicotiana benthamiana. Only one construct (F4), in which the 2b ORF was truncated at the 3' end and fused in-frame with the eGFP ORF, was able to systemically infect N. benthamiana plants, express eGFP and be transmitted by the aphid Myzus persicae. The utility of this construct was demonstrated following infection as early as one day post-transmission (dpt) continuing through to systemic infection. Comparisons of the inoculation sites in different petiole sections one to three dpt clearly showed that the onset of infection and eGFP expression always occurred in the epidermal or collenchymatous tissue just below the epidermis; an observation consistent with the rapid time frame characteristic of the non-persistent mode of aphid transmission.

Genome diversity and evidence of recombination and reassortment in nanoviruses from Europe.

The recent identification of a new nanovirus, pea necrotic yellow dwarf virus, from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment and potential remnants of mixed nanovirus infections, as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogues) were observed, a type of genome complexity not described previously for any member of the family Nanoviridae. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.

Sequence-modified primers for the differential RT-PCR detection of Andean potato latent and Andean potato mild mosaic viruses in quarantine tests.

To enable the differential PCR detection of Andean potato latent virus (APLV) and Andean potato mild mosaic virus (APMMV) strains, sense primers were designed that correspond to regions directly upstream of the coat protein genes. Their differentiating power was increased by A->C or T->C replacements in their 3'-terminal parts. Together with the broad-specificity antisense primer EM3, primer AL-a-mod3C detected all APLV strains tested, but none of the APMMV strains. Primer AM-a-mod4C yielded PCR products with all APMMV preparations, but also with some APLV preparations. Sequence analysis revealed that this was not due to a lack of primer specificity, but to the sensitive detection of contaminating APMMV in some of our APLV preparations.

Correction: Haegeman et al. Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests. Plants 2023, 12, 2139.

In the original publication [...].

Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests.

High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.

To Be Seen or Not to Be Seen: Latent Infection by Tobamoviruses.

Tobamoviruses are among the most well-studied plant viruses and yet there is still a lot to uncover about them. On one side of the spectrum, there are damage-causing members of this genus: such as the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV) and cucumber green mottle mosaic virus (CGMMV), on the other side, there are members which cause latent infection in host plants. New technologies, such as high-throughput sequencing (HTS), have enabled us to discover viruses from asymptomatic plants, viruses in mixed infections where the disease etiology cannot be attributed to a single entity and more and more researchers a looking at non-crop plants to identify alternative virus reservoirs, leading to new virus discoveries. However, the diversity of these interactions in the virosphere and the involvement of multiple viruses in a single host is still relatively unclear. For such host-virus interactions in wild plants, symptoms are not always linked with the virus titer. In this review, we refer to latent infection as asymptomatic infection where plants do not suffer despite systemic infection. Molecular mechanisms related to latent behavior of tobamoviruses are unknown. We will review different studies which support different theories behind latency.

Molecular characterisation of a new tenuivirus from Festuca sp.

A novel virus with a quadruple genome of negative-sense, single-stranded RNA was identified by high-throughput sequencing (HTS) in a grass sample from Saxony-Anhalt, Germany, and tentatively called Festuca stripe-associated virus (FSaV). The genome of FSaV consists of four segments and a total of 16,535 nucleotides (nt) which encode seven open reading frames (ORF). FSaV shares highest nt identity (between 72.84% to 80.74%) to Iranian wheat stripe virus (IWSV) and rice hoja blanca virus (RHBV). Additionally, pairwise comparisons between the amino acid sequences of the ORFs on the genome of FSaV and the corresponding ones on the genomes of the members of the Tenuvirus genus showed that FSaV shared 83.17% and 90.85% (amino acid) aa identity to IWSV. Moreover, the non-coding intergenic regions (ncIR) shared only between 49.5% to 60.87% nt identity to the corresponding regions on the IWSV genome. Based on the ICTV species demarcation, the results suggest that FSaV may represent a new species of the genus Tenuivirus. Plastid sequence analysis of the HTS data showed that the original host is a member of the genus Festuca most likely the species Festuca pratensis.

Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence.

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data.

Comparative study on three viral enrichment approaches based on RNA extraction for plant virus/viroid detection using high-throughput sequencing.

High-throughput sequencing (HTS) has become increasingly popular as virus diagnostic tool. It has been used to detect and identify plant viruses and viroids in different types of matrices and tissues. A viral sequence enrichment method prior to HTS is required to increase the viral reads in the generated data to ease the bioinformatic analysis of generated sequences. In this study, we compared the sensitivity of three viral enrichment approaches, i.e. double stranded RNA (dsRNA), ribosomal RNA depleted total RNA (ribo-depleted totRNA) and small RNA (sRNA) for plant virus/viroid detection, followed by sequencing on MiSeq and NextSeq Illumina platforms. The three viral enrichment approaches used here enabled the detection of all viruses/viroid used in this study. When the data was normalised, the recovered viral/viroid nucleotides and depths were depending on the viral genome and the enrichment method used. Both dsRNA and ribo-depleted totRNA approaches detected a divergent strain of Wuhan aphid virus 2 that was not expected in this sample. Additionally, Vicia cryptic virus was detected in the data of dsRNA and sRNA approaches only. The results suggest that dsRNA enrichment has the highest potential to detect and identify plant viruses and viroids. The dsRNA approach used here detected all viruses/viroid, consumed less time, was lower in cost, and required less starting material. Therefore, this approach appears to be suitable for diagnostics laboratories.

Complete Genome Sequence of a Soybean Dwarf Virus Isolate from White Clover in Germany.

In this study, we present the complete genome of a new isolate of soybean dwarf virus (SbDV) (genus Luteovirus, family Luteoviridae) from white clover in Germany. The complete genome of the isolate (JKI ID 23556) consists of 5,858 nucleotides and displays 94.98% nucleotide identity to its most similar SbDV relative (GenBank accession number MN412736).

Novel targets for engineering Physostegia chlorotic mottle and tomato brown rugose fruit virus-resistant tomatoes: in silico prediction of tomato microRNA targets.

BACKGROUND: Physostegia chlorotic mottle virus (PhCMoV; genus: Alphanucleorhabdovirus, family: Rhabdoviridae) and tomato brown rugose fruit virus (ToBRFV; genus: Tobamovirus, family: Virgaviridae) are newly emerging plant viruses that have a dramatic effect on tomato production. Among various known virus-control strategies, RNAi-mediated defence has shown the potential to protect plants against various pathogens including viral infections. Micro(mi)RNAs play a major role in RNAi-mediated defence. METHODS: Using in silico analyses, we investigated the possibility of tomato-encoded miRNAs (TomiRNA) to target PhCMoV and ToBRFV genomes using five different algorithms, i.e., miRanda, RNAhybrid, RNA22, Tapirhybrid and psRNATarget. RESULTS: The results revealed that 14 loci on PhCMoV and 10 loci on ToBRFV can be targeted by the TomiRNAs based on the prediction of at least three algorithms. Interestingly, one TomiRNA, miR6026, can target open reading frames from both viruses, i.e., the phosphoprotein encoding gene of PhCMoV, and the two replicase components of ToBRFV. There are currently no commercially available PhCMoV- or ToBRFV-resistant tomato varieties, therefore the predicted data provide useful information for the development of PhCMoV- and ToBFRV-resistant tomato plants.