Diagnosis and Characterization of Plant Viruses Using HTS to Support Virus Management and Tomato Breeding.

Viral diseases pose a significant threat to tomato crops (Solanum lycopersicum L.), one of the world's most economically important vegetable crops. The limited genetic diversity of cultivated tomatoes contributes to their high susceptibility to viral infections. To address this challenge, tomato breeding programs must harness the genetic resources found in native populations and wild relatives. Breeding efforts may aim to develop broad-spectrum resistance against the virome. To identify the viruses naturally infecting 19 advanced lines, derived from native tomatoes, high-throughput sequencing (HTS) of small RNAs and confirmation with PCR and RT-PCR were used. Single and mixed infections with tomato mosaic virus (ToMV), tomato golden mosaic virus (ToGMoV), and pepper huasteco yellow vein virus (PHYVV) were detected. The complete consensus genomes of three variants of Mexican ToMV isolates were reconstructed, potentially forming a new ToMV clade with a distinct 3' UTR. The absence of reported mutations associated with resistance-breaking to ToMV suggests that the Tm-1, Tm-2, and Tm-22 genes could theoretically be used to confer resistance. However, the high mutation rates and a 63 nucleotide insertion in the 3' UTR, as well as amino acid mutations in the ORFs encoding 126 KDa, 183 KDa, and MP of Mexican ToMV isolates, suggest that it is necessary to evaluate the capacity of these variants to overcome Tm-1, Tm-2, and Tm-22 resistance genes. This evaluation, along with the characterization of advanced lines using molecular markers linked to these resistant genes, will be addressed in future studies as part of the breeding strategy. This study emphasizes the importance of using HTS for accurate identification and characterization of plant viruses that naturally infect tomato germplasm based on the consensus genome sequences. This study provides crucial insights to select appropriate disease management strategies and resistance genes and guide breeding efforts toward the development of virus-resistant tomato varieties.

Co-Infection of Tobacco Rattle and Cycas Necrotic Stunt Viruses in Paeonia lactiflora: Detection Strategies, Potential Origins of Infection, and Implications for Paeonia Germplasm Conservation.

Increasing reports of tobacco rattle virus (TRV) and cycas necrotic stunt virus (CNSV) in herbaceous Paeonia worldwide highlight the importance of conserving the genetic resources of this economically important ornamental and medicinal crop. The unknown origin(s) of infection, differential susceptibility of peony cultivars to these viruses, and elusive disease phenotypes for CNSV in peonies make early detection and management challenging. Here, we report the presence of TRV and CNSV in plants of the University of Michigan living peony collection in the United States and a molecular characterization of their strains. Using sequences of the TRV 194 K RNA polymerase gene, we confirmed TRV infections in seven symptomatic plants (1.07% of all plants in the collection). Using newly developed primers, we recovered sequences of the CNSV RdRp gene and the polyprotein 1 gene region from nine out of twelve samples analyzed, including three from symptomless plants. Four of the nine plants had TRV and CNSV co-infections and showed more severe disease symptoms than plants only infected with TRV. Phylogenetic analyses of isolates from the University of Michigan living peony collection and publicly available isolates point to multiple origins of TRV and CNSV infections in this collection. This is the first report of TRV/CNSV co-infection and of a symptomatic detection of CNSV on cultivated P. lactiflora.

Exploring the viral landscape of saffron through metatranscriptomic analysis.

Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 52 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered 7 novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.

ICTV Virus Taxonomy Profile: Fimoviridae 2024.

Members of the family Fimoviridae are plant viruses with a multipartite negative-sense enveloped RNA genome (-ssRNA), composed of 4-10 segments comprising 12.3-18.5 kb in total, within quasi-spherical virions. Fimoviruses are transmitted to plants by eriophyid mites and induce characteristic cytopathologies in their host plants, including double membrane-bound bodies in the cytoplasm of virus-infected cells. Most fimoviruses infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the ICTV Report on the family Fimoviridae, which is available at ictv.global/report/fimoviridae.

Development and Application of Attenuated Plant Viruses as Biological Control Agents in Japan.

In 1929, it was reported that yellowing symptoms caused by a tobacco mosaic virus (TMV) yellow mosaic isolate were suppressed in tobacco plants that were systemically infected with a TMV light green isolate. Similar to vaccination, the phenomenon of cross-protection involves a whole plant being infected with an attenuated virus and involves the same or a closely related virus species. Therefore, attenuated viruses function as biological control agents. In Japan, many studies have been performed on cross-protection. For example, the tomato mosaic virus (ToMV)-L11A strain is an attenuated isolate developed by researchers and shows high control efficiency against wild-type ToMV in commercial tomato crops. Recently, an attenuated isolate of zucchini yellow mosaic virus (ZYMV)-2002 was developed and registered as a biological pesticide to control cucumber mosaic disease. In addition, attenuated isolates of pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tobacco mild green mosaic virus (TMGMV), melon yellow spot virus (MYSV), and watermelon mosaic virus (WMV) have been developed in Japan. These attenuated viruses, sometimes called plant vaccines, can be used not only as single vaccines but also as multiple vaccines. In this review, we provide an overview of studies on attenuated plant viruses developed in Japan. We also discuss the application of the attenuated strains, including the production of vaccinated seedlings.

Transcriptome Analysis Reveals a Diverse Range of Novel Viruses in Australian Sugarcane Soldier Fly (Inopus flavus) Larvae.

In Australia, Soldier flies (Inopus spp.) are economically significant pests of sugarcane that currently lack a viable management strategy. Despite various research efforts, the mechanisms underlying the damage caused by soldier fly larvae remain poorly understood. Our study aims to explore whether this damage is associated with the transmission of plant viruses during larval feeding. We also explore the larval transcriptome to identify any entomopathogenic viruses with the potential to be used as biocontrol agents in future pest management programs. Seven novel virus sequences are identified and characterised using de novo assembly of RNA-Seq data obtained from salivary glands of larvae. The novel virus sequences belong to different virus families and are tentatively named SF-associated anphevirus (SFaAV), SF-associated orthomyxo-like virus (SFaOV), SF-associated narna-like virus (SFaNV), SF-associated partiti-like virus (SFaPV), SF-associated toti-like virus (SFaTV-1 and SFaTV-2) and SF-associated densovirus (SFaDV). These newly identified viruses are more likely insect-associated viruses, as phylogenetic analyses show that they cluster with other insect-specific viruses. Small RNA analysis indicates prominent peaks at both 21 nt and 26-29 nt, suggesting the activation of host siRNA and piwiRNA pathways. Our study helps to improve understanding of the virome of soldier flies and could identify insect viruses for deployment in novel pest management strategies.

Benchmarking of virome metagenomic analysis approaches using a large, 60+ members, viral synthetic community.

IMPORTANCE: We report here efforts to benchmark performance of two widespread approaches for virome analysis, which target either virion-associated nucleic acids (VANA) or highly purified double-stranded RNAs (dsRNAs). This was achieved using synthetic communities of varying complexity levels, up to a highly complex community of 72 viral agents (115 viral molecules) comprising isolates from 21 families and 61 genera of plant viruses. The results obtained confirm that the dsRNA-based approach provides a more complete representation of the RNA virome, in particular, for high complexity ones. However, for viromes of low to medium complexity, VANA appears a reasonable alternative and would be the preferred choice if analysis of DNA viruses is of importance. Several parameters impacting performance were identified as well as a direct relationship between the completeness of virome description and sample sequencing depth. The strategy, results, and tools used here should prove useful in a range of virome analysis efforts.

ICTV Virus Taxonomy Profile: Potyviridae 2022.

The family Potyviridae includes plant viruses with single-stranded, positive-sense RNA genomes of 8-11 kb and flexuous filamentous particles 650-950 nm long and 11-20 nm wide. Genera in the family are distinguished by the host range, genomic features and phylogeny of the member viruses. Most genomes are monopartite, but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Potyviridae, which is available at ictv.global/report/potyviridae.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Viruses Infecting Greenhood Orchids (Pterostylidinae) in Eastern Australia.

The Australasian biogeographic realm is a major centre of diversity for orchids, with every subfamily of the Orchidaceae represented and high levels of endemism at the species rank. It is hypothesised that there is a commensurate diversity of viruses infecting this group of plants. In this study, we have utilised high-throughput sequencing to survey for viruses infecting greenhood orchids (Pterostylidinae) in New South Wales and the Australian Capital Territory. The main aim of this study was to characterise Pterostylis blotch virus (PtBV), a previously reported but uncharacterised virus that had been tentatively classified in the genus Orthotospovirus. This classification was confirmed by genome sequencing, and phylogenetic analyses suggested that PtBV is representative of a new species that is possibly indigenous to Australia as it does not belong to either the American or Eurasian clades of orthotospoviruses. Apart from PtBV, putative new viruses in the genera Alphaendornavirus, Amalgavirus, Polerovirus and Totivirus were discovered, and complete genome sequences were obtained for each virus. It is concluded that the polerovirus is likely an example of an introduced virus infecting a native plant species in its natural habitat, as this virus is probably vectored by an aphid, and Australia has a depauperate native aphid fauna that does not include any species that are host-adapted to orchids.

Genomic characterization of silvergrass cryptic virus 1, a novel partitivirus infecting Miscanthus sinensis.

In the present study we report the identification of a novel partitivirus recovered from Miscanthus sinensis, for which the provisional name "silvergrass cryptic virus 1" (SgCV-1) is proposed. High-throughput sequencing (HTS) and rapid amplification of cDNA ends (RACE) allowed the assembly of the complete sequence of each double-stranded RNA genome segment of this novel virus. The largest dsRNA segment, dsRNA1 (1699 bp), was predicted to encode a viral RNA-dependent RNA polymerase protein (RdRp) with 478 aa, and dsRNA2 (1490 bp) and dsRNA3 (1508 bp) were predicted to encode putative capsid proteins (CPs) with 347 and 348 aa, respectively. SgCV-1 has the highest amino acid sequence identity (≤ 70.80% in RdPp and ≤ 34.5% in CPs) to members of the genus Deltapartitivirus, family Partitiviridae, especially to unclassified viruses related to members of this genus. Its genome segment and protein lengths are also within the range of those of deltapartitiviruses. Moreover, phylogenetic analysis based on RdRp amino acid sequences also showed clustering of this novel virus with the related unclassified deltapartitiviruses. An RT-PCR survey of 94 imported M. sinensis samples held in quarantine identified seven additional samples carrying SgCV-1. This new virus fulfils all ICTV criteria to be considered a new member of the genus Deltapartitivirus.

High-Throughput Sequencing of Small RNAs for Diagnostics of Grapevine Viruses and Viroids in Russia.

The use of high-throughput sequencing (HTS) technology has led to significant progress in the identification of many viruses and their genetic variants. In this study, we used the HTS platform to sequence small RNAs (sRNAs) of grapevine to study the virome. Isolation of RNA was performed using symptomatic grapevines collected from commercial vineyards in Krasnodar Krai in 2017-2018. To determine the viromes of vineyards, we used an integrated approach that included a bioinformatic analysis of the results of sRNA HTS and the molecular method RT-PCR, which made it possible to identify 13 viruses and 4 viroids. Grapevine leafroll-associated virus 4 (GLRaV-4), Grapevine Syrah Virus-1 (GSyV-1), Raspberry bushy dwarf virus (RBDV), Australian grapevine viroid (AGVd), and Grapevine yellow speckle viroid 2 (GYSVd-2) were identified for the first time in Russia. Out of 38 samples analyzed, 37 had mixed infections with 4-11 viruses, indicating a high viral load. Analysis of the obtained sequences of fragments of virus genomes made it possible to identify recombination events in GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GVT, GPGV, GRSPaV, GVA, and GFLV. The obtained results indicate a wide spread of the viruses and a high genetic diversity in the vineyards of Krasnodar Krai and emphasize the urgent need to develop and implement long-term strategies for the control of viral grapevine diseases.

ICTV Virus Taxonomy Profile: Solemoviridae 2021.

The family Solemoviridae includes viruses with icosahedral particles (26-34 nm in diameter) assembled on T=3 symmetry with a 4-6 kb positive-sense, monopartite, polycistronic RNA genome. Transmission of members of the genera Sobemovirus and Polemovirus occurs via mechanical wounding, vegetative propagation, insect vectors or abiotically through soil; members of the genera Polerovirus and Enamovirus are transmitted by specific aphids. Most solemoviruses have a narrow host range. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Solemoviridae, which is available at ictv.global/report/solemoviridae.

Identification of Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein-Banding Virus (CYVBV) in Cocoa (Theobroma cacao) Germplasm.

Cocoa, Theobroma cacao, is an important tropical perennial crop grown widely in the humid tropics. The exchange of cocoa germplasm between germplasm collections and breeding centres is vital for varietal development. Intermediate quarantine facilities, such as the International Cocoa Quarantine Centre, Reading UK (ICQC-R) play a vital role in ensuring the transfer of germplasm whilst minimising the risk of spreading pests and diseases. Current screening procedures combine visual inspection and molecular techniques, which are effective in detecting Cocoa swollen shoot virus (CSSV), a badnavirus, which causes severe losses but are restricted to West Africa. However, the detection of latent or mild virus infections that produce no visual symptoms has been a challenge. Recently two badnavirus species of cocoa producing mild symptoms, cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), have been sequenced. Here, we report new assays for the detection of these two species, for the first time in non-symptomatic accessions. Evolutionary and bioinformatic analyses of the viruses suggest their most recent source was from Trinidad, though there is historic evidence that these viruses may have their origin in South America and then become widespread globally over the last century. We also report a novel colorimetric Loop-mediated isothermal amplification (LAMP) assay for the detection of CYVBV. This simple and accurate method could be employed in field virus testing.

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Application of Plant Viruses in Biotechnology, Medicine, and Human Health.

Plant-based nanotechnology programs using virus-like particles (VLPs) and virus nanoparticles (VNPs) are emerging platforms that are increasingly used for a variety of applications in biotechnology and medicine. Tobacco mosaic virus (TMV) and potato virus X (PVX), by virtue of having high aspect ratios, make ideal platforms for drug delivery. TMV and PVX both possess rod-shaped structures and single-stranded RNA genomes encapsidated by their respective capsid proteins and have shown great promise as drug delivery systems. Cowpea mosaic virus (CPMV) has an icosahedral structure, and thus brings unique benefits as a nanoparticle. The uses of these three plant viruses as either nanostructures or expression vectors for high value pharmaceutical proteins such as vaccines and antibodies are discussed extensively in the following review. In addition, the potential uses of geminiviruses in medical biotechnology are explored. The uses of these expression vectors in plant biotechnology applications are also discussed. Finally, in this review, we project future prospects for plant viruses in the fields of medicine, human health, prophylaxis, and therapy of human diseases.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Characterisation and Distribution of Karaka Okahu Purepure Virus-A Novel Emaravirus Likely to Be Endemic to New Zealand.

We report the first emaravirus on an endemic plant of Aotearoa New Zealand that is, to the best of our knowledge, the country's first endemic virus characterised associated with an indigenous plant. The new-to-science virus was identified in the endemic karaka tree (Corynocarpus laevigatus), and is associated with chlorotic leaf spots, and possible feeding sites of the monophagous endemic karaka gall mite. Of the five negative-sense RNA genomic segments that were fully sequenced, four (RNA 1-4) had similarity to other emaraviruses while RNA 5 had no similarity with other viral proteins. A detection assay developed to amplify any of the five RNAs in a single assay was used to determine the distribution of the virus. The virus is widespread in the Auckland area, particularly in mature trees at Okahu Bay, with only occasional reports elsewhere in the North Island. Phylogenetic analysis revealed that its closest relatives are pear chlorotic leaf spot-associated virus and chrysanthemum mosaic-associated virus, which form a unique clade within the genus Emaravirus. Based on the genome structure, we propose this virus to be part of the family Emaravirus, but with less than 50% amino acid similarity to the closest relatives in the most conserved RNA 1, it clearly is a novel species. In consultation with mana whenua (indigenous Maori authority over a territory and its associated treasures), we propose the name Karaka Okahu purepure virus in te reo Maori (the Maori language) to reflect the tree from which it was isolated (karaka), a place where the virus is prevalent (Okahu), and the spotted symptom (purepure, pronounced pooray pooray) that this endemic virus appears to cause.

Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

Genomic characterization of two novel viruses infecting Barleria cristata L. from the genera Orthotospovirus and Polerovirus.

Barleria cristata L. has become naturalized in South Africa, where it is commonly used as an ornamental. In 2019, plants of B. cristata showing putative viral symptoms were collected from two locations in Gauteng, South Africa. RNAtag-seq libraries were prepared and sequenced using an Illumina HiSeq 2500 platform. De novo assembly of the resulting data revealed the presence of a novel member of the family Tospoviridae associated with the plants from both locations, and this virus was given the tentative name "barleria chlorosis-associated virus". Segments L, M, and S have lengths of 8752, 4760, and 2906 nt, respectively. Additionally, one of the samples was associated with a novel polerovirus, provisionally named "barleria polerovirus 1", with a complete genome length of 6096 nt. This is the first study to show the association of viruses with a member of the genus Barleria.