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Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (Vitis spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1BHel, as well as their fused form (1ABHel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophanglycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1BHel in their systemic RNA silencing suppression ability by co-infiltrating Nicotiana benthamiana 16c line plants with a GFP silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity in planta, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1ABHel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate NbAGO2 expression by 1ABHel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of NbDCL2, NbDCL4,, and NbRDR6 expression by 1BHel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection in planta. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate NbDCL2 and NbRDR6 expression. Finally, in silico protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology. [ABSTRACT FROM AUTHOR]

Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant. [ABSTRACT FROM AUTHOR]

Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9-100% and 91.8-100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6-100% and 71.3-99.3%, respectively). This study is the first to determine the virome of pawpaw.

Grapevine fanleaf virus (GFLV) (genus Nepovirus, family Secoviridae) causes fanleaf degeneration, one of the most damaging viral diseases of grapevines. Despite substantial advances at deciphering GFLV-host interactions, how this virus overcomes the host antiviral pathways of RNA silencing is poorly understood. In this study, we identified viral suppressors of RNA silencing (VSRs) encoded by GFLV, using fluorescence assays, and tested their capacity at modifying host gene expression in transgenic Nicotiana benthamiana expressing the enhanced green fluorescent protein gene (EGFP). Results revealed that GFLV RNA1-encoded protein 1A, for which a function had yet to be assigned, and protein 1BHel, a putative helicase, reverse systemic RNA silencing either individually or as a fused form (1ABHel) predicted as an intermediary product of RNA1 polyprotein proteolytic processing. The GFLV VSRs differentially altered the expression of plant host genes involved in RNA silencing, as shown by reverse transcription-quantitative PCR. In a co-infiltration assay with an EGFP hairpin construct, protein 1A upregulated NbDCL2, NbDCL4, and NbRDR6, and proteins 1BHel and 1A+1BHel upregulated NbDCL2, NbDCL4, NbAGO1, NbAGO2, and NbRDR6, while protein 1ABHel upregulated NbAGO1 and NbRDR6. In a reversal of systemic silencing assay, protein 1A upregulated NbDCL2 and NbAGO2 and protein 1ABHel upregulated NbDCL2, NbDCL4, and NbAGO1. This is the first report of VSRs encoded by a nepovirus RNA1 and of two VSRs that act either individually or as a predicted fused form to counteract the systemic antiviral host defense, suggesting that GFLV might devise a unique counterdefense strategy to interfere with various steps of the plant antiviral RNA silencing pathways during infection. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plant virus genomes encode proteins that are involved in replication, encapsidation, cell-to-cell, and long-distance movement, avoidance of host detection, counter-defense, and transmission from host to host, among other functions. Even though the multifunctionality of plant viral proteins is well documented, contemporary functional repertoires of individual proteins are incomplete. However, these can be enhanced by modeling tools. Here, predictive modeling of proteins encoded by the two genomic RNAs, i.e., RNA1 and RNA2, of grapevine fanleaf virus (GFLV) and their satellite RNAs by a suite of protein prediction software confirmed not only previously validated functions (suppressor of RNA silencing [VSR], viral genome-linked protein [VPg], protease [Pro], symptom determinant [Sd], homing protein [HP], movement protein [MP], coat protein [CP], and transmission determinant [Td]) and previously identified putative functions (helicase [Hel] and RNA-dependent RNA polymerase [Pol]), but also predicted novel functions with varying levels of confidence. These include a T3/T7-like RNA polymerase domain for protein 1AVSR, a short-chain reductase for protein 1BHel/VSR, a parathyroid hormone family domain for protein 1EPol/Sd, overlapping domains of unknown function and an ABC transporter domain for protein 2BMP, and DNA topoisomerase domains, transcription factor FBXO25 domain, or DNA Pol subunit cdc27 domain for the satellite RNA protein. Structural predictions for proteins 2AHP/Sd, 2BMP, and 3A? had low confidence, while predictions for proteins 1AVSR, 1BHel*/VSR, 1CVPg, 1DPro, 1EPol*/Sd, and 2CCP/Td retained higher confidence in at least one prediction. This research provided new insights into the structure and functions of GFLV proteins and their satellite protein. Future work is needed to validate these findings. [ABSTRACT FROM AUTHOR]

Despite its first description in 1977 and numerous reports of its presence in various plant species in many countries, the molecular information available in GenBank for artichoke Italian latent virus (AILV) is still limited to a single complete genome sequence (RNA1 and 2) of a grapevine isolate (AILV-V) and a partial portion of the RNA2 sequence from an isolate of unknown origin and host. Here, we report the results of molecular analyses conducted on the RNA2 of some AILV isolates, sequenced for the first time in this study, together with the first-time identification of AILV in a new host plant species, namely chard (Beta vulgaris subsp. vulgaris), associated with vein clearing and mottling symptoms on leaves. The different AILV isolates sequenced were from artichoke (AILV-C), gladiolus (AILV-G), Sonchus (AILV-S), and chard (AILV-B). At the molecular level, the sequencing results of the RNA2 segments showed that AILV-C, AILV-G, AILV-S, and AILV-B had a length of 4629 nt (excluding the 3′ terminal polyA tail), which is one nt shorter than that of the AILV-V reported in GenBank. A comparison of the RNA2 coding region sequences of all the isolates showed that AILV-V was the most divergent isolate, with the lowest sequence identities of 83.2% at the nucleotide level and 84.7% at the amino acid level. Putative intra-species sequence recombination sites were predicted among the AILV isolates, mainly involving the genomes of AILV-V, AILV-C, and AILV-B. This study adds insights into the variability of AILV and the occurrence of recombination that may condition plant infection. [ABSTRACT FROM AUTHOR]

Introduction: Soybean is a plant belonging to the Fabaceae family and is one of the most important oil seed in the world. The northern provinces of Iran are the main soybean cultivation regions in the country, but over the years, soybean podding disorder has reduced yield in this regions and caused up to 100% damage. The disorder is more severe in late planting farms, and symptoms can be observed as plant greening, accumulation of flowers and pods, abnormal pods, bud blight, and lack of seed in pod. The results of previous studies have shown the possible role of viruses in occurrence of soybean disorder. This research was conducted to investigate the Nepovirus effect on soybean yield and cultivars, and possible role on soybean podding disorder. Materials and Methods: 770 soybean sample from Golestan provinces were collected befor and after disorder appeared, and tested by DAS-ELISA and RT-PCR test for investigate the possible role of Nepovirus in disorder. The response of soybean cultivars to Nepovirus and soybean podding disorder, was evaluated in greenhouse and natural conditions. In greenhouse, 2 soybean cultivars (Katol, Saman) were inoculated mechanically at the 4-6 leaf stage. For comparison of soybean cultivars response to studied viruses and evaluation of possible role of Nepovirus on soybean podding disorder in natural condition, the experiment was performed in Golestan Agricuitural Research center as a split plot design with three factors: using net, virus inoculation and soybean cultivars with 3 replications in two years. Nepoviruses inoculated on specific plots on soybean plants at 4-6 leaf stages. ELISA and RT-PCR tests were used to ensure the plants infection with inoculated viruses, and growth indices were measured at physiological growth stage. Data were analyzed by two-way ANOVA, and the means were compared with LSD test at 5% confidence level, using SPSS software version 16.0. Results and Discussion: Comparing of viruse frequency in not disorder plants with podding disorder plants, did not show significant relationship between viruses and disorder incidence. In greenhouse, virus inoculation on soybean plants, caused chlorosis, stunt and systemic necrosis, but the typical symptoms of disorder such as severe falling of flowers, re-flowering, and plant greening did not show. In natural conditions, disorder was observed in all soybean cultivars, but disorder incidence was different among cultivars, and Williams variety showed less disorder in four treatments. The highest and lowest plant growth indices were observed in non-inoculation virus with use of net treatment and, virus inoculation with no use of net, respectively. Mechanical virus inoculation on different soybean cultivars, although reduced growth indices and soybeans yield, but had no significant effect on the podding disorder. In treatments with not controlling of sucker pests, the incidence of disorder was significantly higher than other treatments. Conclusion: Virus inoculation on soybean plants in greenhouse and natural conditions did not cause podding disorder syndrome. Viruses in soybean plants, reduce growth indices and aggravate disorder due to the stress they inflict on the soybean plant, but are not probably the cause of the disorder alone. In treatments with not controlling of sucker pests, disorder incidence was significantly higher, so the role of sucker pests was estimated effective and important. [ABSTRACT FROM AUTHOR]

Viral diseases affecting the essential oil rose, which is a valuable object of agricultural production, may have a significant negative impact on the economic value of this crop. Hence, the study and control of potentially dangerous viruses is essential to improving the quality of cultivars of this raw plant material, to enable production of valuable derivatives. The diversity of viruses affecting Rosa L. plants manifests itself in their conditional division into those that are specific to this crop, and those that are hosted by other plants. Representatives of both groups are found in different countries, however, a low number of viruses identified have been thoroughly studied through the use of experimental methods. In particular, with regard to many viruses, the issue of their spread remains open. The viruses infecting Rosa L. plants along with other crops are described in the literature in detail, as the range of hosts they affect is rather wide and well-studied. It is also possible to single out the three most significant viruses affecting this host—Prunus necrotic ringspot virus, Apple mosaic virus and Arabis mosaic virus which individually, or collectively, cause viral diseases that manifest themselves in mosaic symptoms. The most likely mechanisms for the spread of the Rosa L. species viruses are vegetative propagation procedures and transmission by various pests. These presumptions underlie viral infection control methods, including a well-thought-out planting scheme and provision of accurate plant care, which considers plant disinfection, disease monitoring associated with diagnostics and obtaining virus-free material through biotechnology techniques. [ABSTRACT FROM AUTHOR]

Papa criolla is one of the most important agricultural products in the Colombian Andean region. Viral diseases, mostly caused by PYVV, PVS, PLRV and PVV, are one of the most limiting factors that constraint the productivity of this crop. In order to gain new knowledge of the S. phureja virome in Colombia, in this study using high-throughput sequencing (HTS) and quantitative RT-PCR (RT-qPCR) we present the first report of a natural infection with Potato virus B (PVB) in this country. PVB was detected in leaf samples from different S. phureja plots in Antioquia. Using bioinformatics analysis, it was possible to get the genome assembly of PVB, which comprised two segments of 7.126 nt (RNA1) and 4.298 nt (RNA2) coding for polyproteins P1 and P2. Genome sequences were used to design PVB-specific primers for RT-PCR and RT-qPCR. PVB was found in 35 % of S. phureja samples but was not detected in 20 samples of S. tuberosum var. Diacol-Capiro. The methodology used in this study could be useful in a PVB epidemiology surveillance program in Colombia and other Andean countries. [ABSTRACT FROM AUTHOR]

BackgroundGenome-wide association study (GWAS) is a useful tool for detecting and characterizing traits of interest including those associated with disease resistance in soybean. The availability of 50,000 single nucleotide polymorphism (SNP) markers (SoySNP50K iSelect BeadChip; www.soybase.org ) on 19,652 soybean and wild soybean plant introductions (PIs) in the USDA Soybean Germplasm Collection allows for fast and robust identification of loci associated with a desired phenotype. By using a genome-wide marker set to predict phenotypic values, genomic prediction for phenotype-unknown but genotype-determined PIs has become possible. The goal of this study was to describe the genetic architecture associated with sensitivity to Tobacco ringspot virus (TRSV) infection in the USDA Soybean Germplasm Collection.ResultsTRSV-induced disease sensitivities of the 697 soybean PIs were rated on a one to five scale with plants rated as one exhibiting mild symptoms and plants rated as five displaying terminal bud necrosis (i.e., bud blight). The GWAS identified a single locus on soybean chromosome 2 strongly associated with TRSV sensitivity. Cross-validation showed a correlation of 0.55 (P < 0.01) to TRSV sensitivity without including the most significant SNP marker from the GWAS as a covariate, which was a better estimation compared to the mean separation by using significant SNPs. The genomic estimated breeding values for the remaining 18,955 unscreened soybean PIs in the USDA Soybean Germplasm Collection were obtained using the GAPIT R package. To evaluate the prediction accuracy, an additional 55 soybean accessions were evaluated for sensitivity to TRSV, which resulted in a correlation of 0.67 (P < 0.01) between actual and predicted severities.ConclusionA single locus responsible for TRSV sensitivity in soybean was identified on chromosome 2. Two leucine-rich repeat receptor-like kinase genes were located near the locus and may control sensitivity of soybean to TRSV infection. Furthermore, a comprehensive genomic prediction for TRSV sensitivity for all accessions in the USDA Soybean Germplasm Collection was completed.

Aim. The aim of the current work is to analyse the epiphytotic situation in the south of the Russian Far East in connection with viral diseases of legumes (Fabaceae Lindl., 1836).Discussion contains a description of 18 viruses that infect legumes in this region: Alfalfa mosaic (Martellivirales: Bromoviridae, Alfamovirus); Vicia unijuga mosaic (Martellivirales: Bromoviridae, Bromovirus); Cucumber mosaic (Martellivirales: Bromoviridae, Cucumovirus); Vicia unijuga ringspot virus (Martellivirales: Closteroviridae, Unidentified); Trifolium hybridum yellow mosaic virus, Bean common mosaic virus, Bean yellow mosaic virus, Trifolium repens mottle virus, Mountain clover mosaic virus, Red clover mosaic virus, Soybean chlorotic deformation virus, Soybean chlorotic mottle virus, Soybean mosaic virus, Soybean weak mosaic virus (Patatavirales: Genus, Potyvirus); Tobacco ringspot virus (Picornavirales: Secoviridae, Nepovirus); Pea enation mosaic virus (Tolivirales: Luteoviridae, Enamovirus); White clover mosaic virus (Tymovirales: Alphaflexiviridae, Potexvirus); Vicia pseudorobus necrotic mosaic virus (Tymovirales: Betaflexiviridae, Carlavirus). The description of the established natural reservoirs and the main vectors of these viruses is given.Conclusion. A list of measures are recommended for the prevention of viral diseases of legumes and a thesis is provided on the need to continue the planned monitoring of the phytovirological situation in the Russian Far East.

Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9–100% and 91.8–100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6–100% and 71.3–99.3%, respectively). This study is the first to determine the virome of pawpaw.

Abstract Background Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. Results The epitope sequence was genetically inserted in the αB-αB” domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. Conclusions The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

In 2015 and 2016, lettuce (Lactuca sativa) plants showing yellow ringspots and line patterns were observed in Argolida (Peloponnese, Greece). Symptoms resembled those caused by nepoviruses, especially artichoke yellow ringspot virus (AYRSV), an endemic virus in artichoke (Cynara scolymus) and onion (Allium cepa var. cepa) crops in this area. The aim of this work was to identify and characterize the viral agent associated with the observed disease. We carried out mechanical inoculations onto Nicotiana benthamiana and lettuce plants and also virus‐specific reverse transcription (RT)‐PCR. Results confirmed the presence of AYRSV in all lettuce plants with symptoms tested, and the natural and experimental host range of the virus was studied. The species Heliotropium europaeum, Solanum nigrum, Origanum vulgare, Lactuca seriola, and Sinapis arvensis, are recorded as hosts of AYRSV for the first time. Also, AYRSV was detected by RT‐PCR in seeds and in the produced seedlings of lettuce, N. benthamiana, N. glutinosa, N. tabacum, and S. nigrum. Sequencing of a partial fragment of the gene encoding the RNA‐dependent RNA polymerase was performed for 29 isolates of AYRSV originating from different hosts (lettuce, artichoke, onion, and arable weeds). Phylogenetic analysis of AYRSV isolates classified them in diverse clusters that exhibited no host grouping. All groups included isolates from cultivated and wild plant species. In this study, AYRSV was associated with a new lettuce disease for the first time, thus increasing the known natural host range of the virus. [ABSTRACT FROM AUTHOR]

We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.

Abstract. The aim of the presented work consists in the analysis of results of long-term monitoring of the members of one of the most important and interesting phytovirus taxons – genus Nepovirus (Picornavirales, Secoviridae, Comovirinae) – which is carried out since 1962 on the planned basis in the south of the Far East (Primorsky krai, Khabarovsk krai, the Sakhalin region) by the Laboratory of Virology of Federal Scientific Center of the East Asia terrestrial biodiversity of Far Eastern Branch of Russian Academy of Sciences (Vladivostok, Russia). Discussion. Many (it is possible – all) nepoviruses are capable to cause epiphytoties with significant economic damage for world economy. In the current article the organization of nepovirus genome, the principles of their division into three genetic groups (A, B, and C), virion morphology, the principles of division into three beyond density fractions (T, M, and B), the characteristics of phytopathology connected with nepoviruses, their ecology in the south of the Far East (including sources of isolation and vectors) are discussed as well as physical-chemical properties of some the most interesting strains from this region which apparently could represent new species of Nepovirus genus: Phryma asiatica mosaic virus (PhAMV); dayflower mosaic virus (DFMV); foenugreek necrotic spot virus (FoNSV); Capsicum annuum necrotic spot virus (CaNSV). Conclusion. Nepovirus genus is one of the most adapted for circulation in the phytocenoses of midlatitudes having natural reservoir among wild plants and pose serious threat for crops. On the basis of ecological features of nepoviruses the scientifically based list of preventive and protective actions against epiphytoties with nepovirus nature is provided. One of the most important elements of such actions is their planned monitoring at the regional level with obligatory comparison of the received results to universal data.

Tomato black ring virus (TBRV) is a member of the Nepovirus genus in the Secoviridae family, which infects a wide range of important crop species worldwide. In this work, we constructed four cDNA infectious clones of the TBRV tagged with the green fluorescent protein (TBRV-GFP), which varied in (i) the length of the sequences flanking the GFP insert, (ii) the position of the GFP insert within the RNA2 polyprotein, and (iii) the addition of a self-cutting 2A protein. The presence of the GFP coding sequence in infected plants was verified by RT-PCR, while the infectivity and stability of the constructs were verified by mechanical inoculation of the host plants. The systemic spread of TBRV-GFP within plants was observed under UV light at a macroscopic level, monitoring GFP-derived fluorescence in leaves, and at a microscopic level using confocal microscopy. The obtained clones are a valuable tool for future studies of TBRV-host interactions, virus biology, and the long-term monitoring of its distribution in infected plants.

Abstract ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21–27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions. [ABSTRACT FROM AUTHOR]

Summary: Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs. [ABSTRACT FROM AUTHOR]

An emerging virus-like flower yellowing disease (FYD) of green Sichuan pepper (Zanthoxylum armatum v. novemfolius) has been recently reported. Four new RNA viruses were discovered in the FYD-affected plant by the virome analysis using high-throughput sequencing of transcriptome and small RNAs. The complete genomes were determined, and based on the sequence and phylogenetic analysis, they are considered to be new members of the genera Nepovirus (Secoviridae), Idaeovirus (unassigned), Enamovirus (Luteoviridae), and Nucleorhabdovirus (Rhabdoviridae), respectively. Therefore, the tentative names corresponding to these viruses are green Sichuan pepper-nepovirus (GSPNeV), -idaeovirus (GSPIV), -enamovirus (GSPEV), and -nucleorhabdovirus (GSPNuV). The viral population analysis showed that GSPNeV and GSPIV were dominant in the virome. The small RNA profiles of these viruses are in accordance with the typical virus-plant interaction model for Arabidopsis thaliana. Rapid and sensitive RT-PCR assays were developed for viral detection, and used to access the geographical distributions. The results revealed a correlation between GSPNeV and the FYD. The viruses pose potential threats to the normal production of green Sichuan pepper in the affected areas due to their natural transmission and wide spread in fields. Collectively, our results provide useful information regarding taxonomy, transmission and pathogenicity of the viruses as well as management of the FYD.

Competing Interests: The author(s) declare no conflict of interest.

For quarantine purpose, we developed the RT- and nested PCR module of Tomato black ring virus (TBRV), Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV) and Grapevine fanleaf virus (GFLV). The PCR modules, developed in this study make diagnosis more convenient and speedy because of same PCR condition. And also, the methods are more accurate because it can check whether the result is contamination or not using the mutation-positive control. We discard or return the 27 cases of Nepovirus infection seed by employing the module past 3 years. This study provides a rapid and useful method for detection of four quarantine plant viruses.

In this work, the infection of Grapevine fanleaf virus (GFLV) disease was described on the base of symptomatic differences within eight grapevine plants of six grape ciltivars with positive tests on GFLV. Among them, cultivars Kodrjanka, Pamjati Negrula, Kišmiš Lučistyj were planted in wine region of the South Moravia (Czech Republic), three infected grapevine cultivars (URS, Cinsaut, Dimrit) included in this study originating from Italy. Except symptomatic evaluation, the differences between isolates were emphasized at the genetic level too, exactly in the frame of RNA2 genomic region coding movement protein. The variability of the tested isolates within the eight plants was in the range from 86.59 to 97.61% at the nucleotide level. The results confirmed very high degree of similarity between virus isolates of GFLV within studied RNA2 region. This fact was assessed by the phylogenetic analysis of obtained sequencing data too.

Viruses are frequently a microbial biocontaminant of healthy plants. The occurrence of the infection can be also due to environmental stress, like urbanisation, air pollution and increased air temperature, especially under the ongoing climate change. The aim of the present study was to investigate the hypothesis that worsened air quality and fewer green areas may favour the higher frequency of common viral infections, particularly in a common tree in temperate and continental climates, Betula pendula ROTH. We examined 18 trees, during the years 2015-2017, the same always for each year, in the region of Augsburg, Germany. By specific PCR, the frequency of two viruses, Cherry leaf roll virus (CLRV, genus Nepovirus, family Secoviridae), which is frequent in birch trees, and a novel virus tentatively named birch idaeovirus (BIV), which has been only recently described, were determined in pollen samples. The occurrence of the viruses was examined against the variables of urban index, air pollution (O 3 and NO 2 ), air temperature, and tree morphometrics (trunk perimeter, tree height, crown height and diameter). Generalized Non-linear models (binomial logit with backward stepwise removal of independent variables) were employed. During the study period, both CLRV and BIV were distributed widely throughout the investigated birch individuals. CLRV seemed to be rather cosmopolitan and was present independent of any abiotic factor. BIV's occurrence was mostly determined by higher values of the urban index and of NO 2 . Urban birch trees, located next to high-traffic roads with higher NO 2 levels, are more likely to be infected by BIV. Increased environmental stress may lead to more plant viral infections. Here we suggest that this is particularly true for urban spaces, near high-traffic roads, where plants may be more stressed, and we recommend taking mitigation measures for controlling negative human interventions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Virus-like particles ( VLPs) derived from nonenveloped viruses result from the self-assembly of capsid proteins ( CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self-assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus ( GFLV). We found that the N- and C-terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant-based production of nucleic acid-free VLPs. Remarkably, expression of N- or C-terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules. [ABSTRACT FROM AUTHOR]

Myrobalan latent ringspot virus (MLRSV) was purified from the extract of infected Chenopodiumquinoa Willd., using n-octanol, differential centrifugation, and saccharose gradient centrifugation. Electron microscopy showed 28 nm large isometric particles in a sample of purified virus. Antiserum to MLRSV was prepared by immunisation of rabbits with intravenous injections of antigen in combination with intramuscular injections of antigen with Freund's adjuvant. The antibody titer of the obtained antiserum was determined by drop-precipitation method to be 1:1024. Immunoglobulins against MLRSV were isolated from antiserum with ammonium acetate, caprylic acid, and precipitation with ammonium sulphate. The isolated immunoglobulins (IgG) were conjugated with alkaline phosphatase. The optimal dilution of IgG for detection of MLRSV using ELISA was 1×10-3μg/ml which was also the optimal dilution of conjugated IgG. Using this dilution of antibodies, the absorbance of samples from MLRSV-infected plants of C. quinoa varied between 0.71 and 1.45, while absorbance of samples from healthy plants (control) was 0.01 to 0.07.

Grapevine is a widely grown fruit crop that is seriously affected by different viruses, reducing grape yield and quality, as well as threatening profitability. Vineyard disease management requires accurate identification of viral infections. This study aimed to survey the presence of ten grapevine viruses in four geographic sites in the Mendoza province of Argentina. Two hundred twenty-three composite cane samples from 1060 plants of six cultivars were collected from 26 blocks distributed across 11 vineyards. The cane samples were screened by RT-PCR for the following viruses: grapevine leafroll-associated viruses 1-4 (GLRaV 1, 2, 3, and 4), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine virus A (GVA) and B (GVB), grapevine rupestris stem pitting associated virus (GRSPaV), and arabis mosaic virus (ArMV). The results showed an uneven occurrence of viruses through the sampled regions, with GRSPaV being prevalent (71.1%), followed by GFLV (28.9%), GFkV (20.6%), and GLRaV-2 (14.7%). GVB was not detected. This study revealed a moderate prevalence of viruses associated with economically impactful diseases in the vineyards surveyed.

Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain trans-membrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase) proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C and 3A membrane proteins. The identification of membrane-association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted.

The Panel on Plant Health performed a pest categorisation of Cherry leafroll virus (CLRV) for the European Union (EU) territory. CLRV is a well-defined virus species of the genus Nepovirus, for which the entire genome sequence and molecular detection assays are available. CLRV is transmitted by vegetative multiplication of infected hosts and is also seed- and pollen-transmitted (both horizontally and vertically) in many of its hosts. There is, however, evidence that interspecific transmission by the pollen-mediated mechanism is very limited. CLRV has a wide host range, which contains both woody and herbaceous hosts, many of which are largely present in natural environments. It is listed on plants of Rubus spp. for planting in Annex IIAI of Directive 2000/29/EC. CLRV is widely present in the EU but there are very few records on its regulated hosts. It is not expected to be affected by ecoclimatic conditions wherever its hosts are present and has the potential to establish in large parts of the EU territory. CLRV can spread through efficient seed- and pollen-mediated transmission mechanisms and through the movement of vegetatively propagated plants for planting of numerous hosts. However, the existence of efficient and widely adopted certification systems for some of these hosts constitutes a limitation to CLRV spread. Although the virus alone or when in mixed infection can cause significant disease in some hosts, the actual impact of CLRV is very difficult to evaluate but generally appears to be limited. [ABSTRACT FROM AUTHOR]

Background: The relationship between allopolyploidy and plant virus resistance is poorly understood. To determine the relationship of plant evolutionary history and basal virus resistance, a panel of Nicotiana species from diverse geographic regions and ploidy levels was assessed for resistance to non-coevolved viruses from the genus Nepovirus, family Secoviridae. The heritability of resistance was tested in a panel of synthetic allopolyploids. Leaves of different positions on each inoculated plant were tested for virus presence and a subset of plants was re-inoculated and assessed for systemic recovery. Results: Depending on the host-virus combination, plants displayed immunity, susceptibility or intermediate levels of resistance. Synthetic allopolyploids showed an incompletely dominant resistance phenotype and manifested systemic recovery. Plant ploidy was weakly negatively correlated with virus resistance in Nicotiana species, but this trend did not hold when synthetic allopolyploids were taken into account. Furthermore, a relationship between resistance and geographical origin was observed. Conclusion: The gradients of resistance and virulence corresponded to a modified matching allele model of resistance. Intermediate resistance responses of allopolyploids corresponded with a model of multi-allelic additive resistance. The variable virus resistance of extant allopolyploids suggested that selection-based mechanisms surpass ploidy with respect to evolution of basal resistance to viruses. [ABSTRACT FROM AUTHOR]

Abstract: The N-terminal domains of the RNA 2-encoded 2AHP proteins of the arabis mosaic (ArMV) and grapevine fanleaf (GFLV) nepoviruses were shown to be highly variable and a hotspot for intra- and inter-species recombination events. Chimeric ArMV-NW clones in which the N-terminal domain of 2AHP or the entire 2AHP of GFLV isolates replaced the corresponding domains of ArMV retained their infectivity, showing that the 2AHP proteins of ArMV-NW and GFLV are exchangeable. ArMN-NW clones with deletions of the N-terminal, core, or C-terminal domains of the ArMV-NW 2AHP were infectious in Chenopodium quinoa although viral RNA (especially RNA 2) accumulated at reduced levels. In contrast, deletion of the entire 2AHP protein or of the C-terminal two thirds of the protein abolished infectivity of the ArMV-NW clones. These results suggest that multiple functional domains are distributed throughout the 2AHP protein and are essential for the accumulation of viral RNA 2. [Copyright &y& Elsevier]

Abstract: Symptom recovery in nepovirus-infected plants has been attributed to the induction of RNA silencing. However, recovery is not always accompanied with viral RNA clearance. In this study, we show that recovery of Nicotiana benthamiana plants infected with the tomato ringspot virus (ToRSV) is associated with a reduction of the steady-state levels of RNA2-encoded coat protein (CP) and movement protein but not of RNA2. In vivo labeling experiments revealed efficient synthesis of the CP early in infection, but reduced RNA2 translation later in infection. Silencing of Argonaute1-like (Ago1) genes prevented both symptom recovery and RNA2 translation repression. Similarly, growing the plants at lower temperature (21°C rather than 27°C) alleviated the recovery and the translation repression. Taken together, our results suggest that recovery of ToRSV-infected plants is associated with an Ago1-dependent mechanism that represses the translation of viral RNA2. [Copyright &y& Elsevier]

A survey of nepoviruses in small fruits and fruit trees in thirteen localities of Slovakia was undertaken during the last three years. The samples of spontaneously infected small fruits and fruit trees with symptoms such as leaf yellowing, mottling, chlorotic spotting, vein clearing, chlorosis, dwarfing, and in some cases even plant dying, were analysed. Virus identification in collected samples from affected crops was performed by ELISA using polyclonal antibodies. The following quarantine nepoviruses were detected: Tobacco ringspot nepovirus, Tomato ringspot nepovirus, Tomato black ring nepovirus, Raspberry ringspot nepovirus, Cherry leafroll nepovirus and Arabis mosaic nepovirus. Screening of virus vector nematodes in the rhizosphere of infected plants was also undertaken. Three Longidorus (L. elongatus, L. picenus and L. leptocephalus) and four Xiphinema phytonematode species (X. vuittenezi, X. diversicaudatum, X. taylori and X. pachtaicum) were recorded in localities tested.