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Cucumbers are susceptible to infections with many characterised virus species. In some cases, mixed virus infections occur and produce novel symptoms. In Greece, routine screening is carried out when virus infection is suspected, or novel symptoms are observed in the field. To identify the viruses associated with distinct symptoms observed in samples from commercial cucumber production areas on the island of Crete, Greece, we carried out high‐throughput sequencing (HTS) from a pool of six samples. Following assembly and BLAST analysis, we identified at least seven viruses based on similarity to published sequences. Two of these sequences represented novel, near‐complete genomes of a putative new nepovirus and of zucchini yellow fleck virus (ZYFV). To confirm the HTS results, the six samples were screened for all identified viruses, and their presence was confirmed through Sanger sequencing of PCR products. The full‐length genomes of both the nepovirus and ZYFV were amplified by PCR and confirmed by Sanger sequencing. We have generated the complete genome of a novel nepovirus from cucumber as well as the first complete genome sequence of a cucumber‐infecting ZYFV isolate from Crete. The nepovirus was mechanically transmissible to Nicotiana benthamiana and induced typical cytopathological modifications consistent with virus infection, as revealed by TEM studies. We propose to name this new virus Cucumber nepovirus A (CuNVA). [ABSTRACT FROM AUTHOR]

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium -mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8–13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, the transgenerational gene editing frequency of which ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals. [ABSTRACT FROM AUTHOR]

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]

Introduction More than 80 viral diseases of grapevines have been reported worldwide. The infectious degeneration disease complex causes growth reduction, stunting, shortening of internodes, bushy growth and decline in susceptible vines. Grapevine fanleaf viruses (GFLV), arabis mosaic virus (ArMV), grapevine deformation virus (GDefV), tomato ring spot virus (ToRSV) and tobacco ring spot virus (TRSV), which belong to the genus Nepovirus, are known to cause infectious degeneration. The genus Nepovirus has been divided into three subgroups A, B and C based on genome length, genomic organization, serological relationships, proteinase cleavage sites and the phylogenetic relationship of their coat protein (CP) gene. GDefV with the new name Nepovirus deformationis belongs to subgroup A of the genus Nepovirus and is closely related to ArMV and GFLV. Prevalence of GFLV in the vineyards of Iran raises the possibility that a mixed infection of GFLV and GDefV is also present in these areas. In Khorasan-Razavi Province, Iran's third-largest grape production center, research has addressed grape-affecting viruses with potential economic consequences, including reduced yield and product quality. Studying the distribution and genetic diversity of GDefV and other grape viruses is crucial for developing effective management and prevention strategies in the region's vineyards. In this study, GDefV was identified in the vineyards of Khorasan-Razavi province in Northeastern Iran and its complete genome was sequenced. In addition, the phylogenetic relationship of these isolates to other GDefV isolates deposited in GenBank was analyzed. Materials and Methods Samples from three grapevines were collected from a vineyard in Kashmer, Khorasan-Razavi Province; total RNA was extracted from the petiole of young leaves using the CTAB-PVPP method., GDefV and mixed infections of GDefV/GFLV were detected by PCR using specific primers. The PCR products were electrophoresed in a 1% agarose gel and sequenced. The miRNAs were extracted from grapevine petiole tissue using the modified CTAB method, and small RNA libraries were prepared using the TruSeq Small RNA Sample Prep Kit and sequenced on the Illumina Novaseq 6000 platform. After trimming the reads, contigs were generated using k-mer 15 in Velvet assembler 0.7.31. Contigs were verified using BLASTn and BLASTx in NCBI, and reconstruction of the GDefV genome from the NGS reads was performed using CLC Genomics Workbench (CLC Bio) software. Phylogenetic trees were generated in MEGA7 using the maximum likelihood (ML) method with 1000 replicates in the bootstrap test. The occurrence of possible recombinations in the GDefV genome was analyzed using the RDP v.6 package. Results and Discussion The samples showed leaf deformation, shortening of internodes, bushy growth, stunting and decline, but these symptoms are probably related to GFLV, and GDefV only causes leaf deformation in the infected vine. The PCR amplification and sequencing of a segment of the coat protein gene revealed a co-infection of GDefV and GFLV in the samples. GFLV is widely distributed in Iranian vineyards, GDefV has also been previously reported in the vineyards of northwestern Iran, but this is the first report of GDefV in the vineyards of Khorasan-Razavi Province. After refining the reads, approximately 15 million reads (92-99.7% of the original reads) remained for further analysis. The read sequences of each library were deposited in the Sequence Read Archive (SRA) under the accession number SAMN33747579-81. Three Iranian GDefV isolates obtained from three miRNA libraries were deposited in GenBank (accession numbers **-**). RNA1 and RNA2 of the three Iranian GDefV isolates had a length of 7386 and 3753 nucleotides, respectively. The RNA1 in GDefV had an open reading frame (ORF) of 6852 nucleotides in length, which started with the start codon AUG at position 288 and ended with the stop codons UAA or UAG at position 7142. The 5' end of the genome had 287 nucleotides long and contained two repeating sequences of 15 nucleotides that formed stem-loop structures. The length of the non-coding region at the 3' end had 244 nucleotides. Translation of RNA1 of the GDefV genome produces a polyprotein (p1) of 2285 amino acids (approximately 252 kda). The polyprotein p1 comprises the cofactor proteins proteinase, helicase, VPg, proteinase and polymerase with approximate weights of 45, 88, 3, 25 and 91 kDa, respectively. RNA2 also has one ORF, located between nucleotides 237 and 3560. This open reading frame also produces a 122 kDa polyprotein (P2) with 1108 amino acids. The second fragment of the GDeFV genome had 236 nucleotides as a 5'-noncoding region with three repeating sequences of 15 nucleotides that produced stem-loop structures with a 3-nucleotide loop and a 6bp stem. The 3'-noncoding region was also 193 nucleotides long. Polyprotein P2 comprised protein 2A, movement protein (MP) and coat protein (CP). The GDefV polyproteins had cysteine/alanine (C/A) and arginine/glycine (R/G) cleavage sites similar to those of the GFLV polyprotein. Comparison of the RNA1 sequences from three Iranian GDefV isolates with other GDefV isolates available in GenBank showed that the Iranian isolates had 88.1-92.2 % nucleotide identity with each other and 90.3-93.9 % with GenBank isolates at the nucleotide level. At the amino acid level, the Iranian isolates were 86.6-91.6 % identity with each other and 88.9-92.7 % with GenBank isolates. For RNA2, the Iranian isolates showed 89.4-92 % similar to each other and 89.6-94.2 % similar to GenBank isolates at the nucleotide level. The amino acid similarity between the Iranian GDefV isolates was 85-88.6 %. In the phylogenetic tree based on the nucleotide and amino acid sequences of RNA1 and RNA2 of the GDefV genome, the Iranian isolates of this study were clustered in a distinct clade than other GDefV isolates from Turkey (HE613269 and NC017939). GDefV was reported in 2003 and no further information is available on its distribution in vineyards around the world. GDefV has already been reported from Turkey and Iran, but the complete genome sequence of the Iranian GDefV isolate is being reported for the first time. Further studies on the population diversity of GDefV isolates in different regions of Iran are required to gain more insight into the mechanisms affecting the dynamics of GDefV populations. [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]

Plant viruses of the families Potyviridae and Secoviridae encode 3C-like proteases (3CLpro) that are related to picornavirus 3C proteases. This review discusses recent advances in deciphering the multifunctional activities of plant virus 3CLpro. These proteases regulate viral polyprotein processing and facilitate virus replication. They are also determinants of host range, virulence, symptomatology and super-infection exclusion in some plant-virus interactions and facilitate aphid transmission. Potyvirid NIa-Pro proteases interact with host factors to interfere with a variety of defense mechanisms: salicylic acid-dependent signaling, ethylene-dependent signaling, transcriptional gene silencing and RNA decay. Potyvirid NIa-Pro also cleave host proteins at signature cleavage sites, although the biological impact of these cleavage remains to be determined. Recently, a plant defense mechanism was uncovered that inhibits the proteolytic activity of a comovirus 3CLpro. Future perspectives are discussed including using proteomic and degradomic techniques to elucidate the network of interactions of plant virus 3CLpro with the host proteome. • Potyvirids and secovirids encode proteases related to picornavirus 3C protease. • Potyvirid NIa-Pro counteract plant defenses targeted at the virus or their vector. • The NIa-Pro of three potyviruses cleave a variety of host proteins. • A plant antiviral mechanism inhibits the activity of a comovirus protease. [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]

Tomato black ring virus (TBRV) and beet ringspot virus (BRSV) are closely related but distinct members of subgroup B of the genus Nepovirus. Both viruses have broad host ranges and are transmitted by seed, pollen, and ectoparasitic nematodes. Although 13 TBRV and 3 BRSV genome sequences were already available, no attempt has been made to link sequence data from these recent sequences with those of historical isolates studied in the pre‐sequencing era. High‐throughput sequencing was used to generate eight new TBRV and BRSV genome sequences from three historical >60‐year‐old and two >30‐year‐old isolates, and three more recent isolates. These eight isolates were from the Czech Republic, Germany, and the UK. We compared these with all genomes sequenced previously. Intraspecies recombination (three of four TBRV and two of four BRSV isolates) was frequent amongst the eight new genomes. Interspecies recombination was also present within the RNA1 of TBRV isolates BRSV‐3393 SG GB and BRSV‐9888 ST GB. No satellite RNAs were associated with the eight new genomes. Two commercial enzyme‐linked immunosorbent assay (ELISA) kits used to detect TBRV during routine testing differed in that one detected only TBRV and the other only BRSV, so they are likely to provide incorrect but potentially complementary virus occurrence information. We suggest both ELISA kits, or appropriate molecular tests, be used by biosecurity authorities to avoid this problem. This study illustrates the value of sequencing historical isolates preserved from the pre‐sequencing era. [ABSTRACT FROM AUTHOR]

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.

The majority of picornaviral 3C proteinases (3Cpro) cleavage sites possess glutamine at the P1 position. Plant nepovirus 3C-like proteinases (3CLpro) show however much broader specificity, cleaving not only after glutamine, but also after several basic and hydrophobic residues. To investigate this difference, we employed AlphaFold to generate structural models of twelve selected 3CLpro, representing six substrate specificities. Generally, we observed favorable correlations between the architecture and charge of nepovirus proteinase S1 subsites and their ability to accept or restrict larger residues. The models identified a conserved aspartate residue close to the P1 residue in the S1 subsites of all nepovirus proteinases examined, consistent with the observed strong bias against negatively-charged residues at the P1 position of nepovirus cleavage sites. Finally, a cramped S4 subsite along with the presence of two unique histidine and serine residues explains the strict requirement of the grapevine fanleaf virus proteinase for serine at the P4 position. • We used AlphaFold to generate models of twelve nepovirus proteinases. • All models show close relationships to picornavirus 3C proteinases. • The residues found in the S1 pockets of the nepovirus proteinases differ. • These differences can explain the varied specificities of the nepovirus proteinases. • Comparison of the models illuminates the evolution of the proteinases. [ABSTRACT FROM AUTHOR]

Grapevine fanleaf virus (GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by Xiphinema index. This paper aims to isolate Xiphinema species from Tunisian vineyard soil samples and assess their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as X. index and Xiphinema italiae. These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of Xiphinema and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that X. index could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in X. italiae , under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.

Tomato necrotic ringspot virus (TNRV) was first reported in Thailand in 2011, where it continues to reduce the yield and quality of pepper and tomato crops. Here, we report the complete genome sequence of TNRV isolate chilli-CR derived from next-generation sequencing. The TNRV genome comprises 16,595 nucleotides (nt) on three RNA segments. The L RNA is 8,858 nt, the M RNA is 4,724 nt, and the S RNA is 3,013 nt in length. The genome structure and organization are typical of orthotospoviruses, encoding five proteins, named L, NSm, G N G C , NSs, and N. Pairwise comparison of each genomic RNA segment and its deduced amino acid (aa) sequence showed that TNRV chilli-CR shares 73.6-82.3% nt sequence identity and 81.1-91.9% aa sequence identity with pepper chlorotic spot virus (PCSV). Similar phylogenetic groupings were observed based on each genomic RNA or deduced aa sequence, and with concatenated genomic RNA sequences. The clustering of TNRV and PCSV in all phylogenetic analyses, and the 78.9% overall nt sequence identity observed using the concatenated genomic RNAs suggest that TNRV is a distinct orthotospovirus and that analysis of concatenated orthotospovirus genome sequences will be of value in future phylogenetic studies of this virus group., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Cotton is one of the most salient cash crops globally and in the United States. Lately, several virus-like diseases have been reported from cotton in the United States such as the tobacco ringspot virus (TRSV) in Oklahoma. TRSV has been reported from various hosts worldwide with minimal phylogenetic examination. In this study, complete genome sequences of four TRSV isolates from cotton were isolated, and the genetic diversity was investigated along with additional available TRSV isolates retrieved from GenBank. Phylogenetic analysis based on the complete RNA1 and RNA2 sequences distributed all TRSV isolates into three major phylogenetic clades exhibiting a differential clade composition depending on the segment. The TRSV cotton isolates exhibited differential grouping between the RNA1 and RNA2 analyses. Additionally, monophyletic subclades of isolates appeared to be conserved between both segments. Thirty-five recombination events in RNA1 and 23 in RNA2 were identified with implications in the variation of the phylogenetic analyses. Furthermore, multiple hypotheses of TRSV evolution were generated based on the phylogenetic analyses, but to test them, more complete genomes of TRSV will be needed. This study provides the first complete genome analysis of TRSV isolates infecting cotton in the United States and a detailed analysis of global TRSV isolates., Competing Interests: The author(s) declare no conflict of interest.

Icosahedral, 30 nm diameter, grapevine fanleaf virus (GFLV) virus particles are adsorbed onto electrodes and used as nanoscaffolds for the assembly of an integrated glucose oxidizing system, comprising the enzyme pyrroloquinoline quinone-glucose dehydrogenase (PQQ-GDH) and ferrocenylated polyethylene glycol chains (Fc-PEG) as a redox co-substrate. Two different GFLV-specific nanobodies, either fused to the enzyme, or chemically conjugated to Fc-PEG, are used for the regio-selective immunodecoration of the viral particles. A comprehensive kinetic characterization of the enzymatic function of the particles, initially decorated with the enzyme alone shows that simple immobilization on the GFLV capsid has no effect on the kinetic scheme of the enzyme, nor on its catalytic activity. However, we find that co-immobilization of the enzyme and the Fc-PEG co-substrate on GFLV does induce enzymatic enhancement, by promoting cooperativity between the two subunits of the homodimeric enzyme, via "synchronization" of their redox state. A decrease in inhibition of the enzyme by its substrate (glucose) is also observed., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Vianney Poignavent and Christophe Ritzenthaler have patent EP3596219A pending, which applies to the use of Nanobodies to functionalize GFLV., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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

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.)

Viruses can elicit varying types and severities of symptoms during plant host infection. We investigated changes in the proteome and transcriptome of Nicotiana benthamiana plants infected by grapevine fanleaf virus (GFLV) with an emphasis on vein clearing symptom development. Comparative, time-course liquid chromatography tandem mass spectrometry and 3' ribonucleic acid sequencing analyses of plants infected by two wildtype GFLV strains, one symptomatic and one asymptomatic, and their asymptomatic mutant strains carrying a single amino acid change in the RNA-dependent RNA polymerase (RdRP) were conducted to identify host biochemical pathways involved in viral symptom development. During peak vein clearing symptom display at 7 days post-inoculation (dpi), protein and gene ontologies related to immune response, gene regulation, and secondary metabolite production were overrepresented when contrasting wildtype GFLV strain GHu and mutant GHu-1E K802G Pol . Prior to the onset of symptom development at 4 dpi and when symptoms faded away at 12 dpi, protein and gene ontologies related to chitinase activity, hypersensitive response, and transcriptional regulation were identified. This systems biology approach highlighted how a single amino acid of a plant viral RdRP mediates changes to the host proteome (∼1%) and transcriptome (∼8.5%) related to transient vein clearing symptoms and the network of pathways involved in the virus-host arms race.

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.

Two common bean leaf samples from Ethiopia that had shown chlorotic fleck and veinal mosaic symptoms but tested ELISA-negative for known viruses were mechanically transmitted to herbaceous hosts to obtain virus isolates ET-773/4 and ET-779. Virus purification from Chenopodium quinoa systemically infected with ET-773/4 yielded icosahedral particles measuring ~ 30 nm in diameter and containing a single capsid protein of ~ 58 kDa, suggesting a nepovirus infection. Analysis of nucleotide sequences generated from RNA1 and RNA2 of the isolates indicated that they represent a distinct virus species in the genus Nepovirus. Surprisingly, the most closely related sequence in the GenBank database was that of Hobart nepovirus 3, an incompletely described metagenomic sequence obtained from honey bees in Tasmania. This new nepovirus from Ethiopia is provisionally named "bean chlorotic fleck virus"., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates., Competing Interests: The authors declare no conflict of interest.

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. [ABSTRACT FROM AUTHOR]

The Ampelovirus Grapevine leafroll-associated virus 3 (GLRaV-3) and the Nepovirus Grapevine fanleaf virus (GFLV) are pathogens reported in many grapevine-growing areas all over the world, main causal agents of grapevine leafroll disease and grapevine fanleaf disease, respectively. Prevention of virus spread thanks to rapid diagnosis of infected plants is a key factor for control of both diseases. Although serological (e.g., enzyme-linked immunosorbent assay-ELISA test) and molecular methods are available to reveal the presence of the viruses, they turn out to be quite expensive, time-consuming and laborious, especially for large-scale health screening. Here we report the optimization of a lab-on-a-chip (LOC) for GLRaV-3 and GFLV detection, based on an electrochemical transduction and a microfluidic multichamber design for measurements in quadruplicate and simultaneous detection of both targets. The LOC detect GLRaV-3 and GFLV at dilution factors more than 15 times higher than ELISA, providing a higher sensitivity in the detection of both viruses. Furthermore, the platform offers several advantages as easy-to-use, rapid-test, portability and low costs, favoring its potential application for large-scale monitoring programs. Compared to other grapevine virus biosensors, our sensing platform is the first one to provide a dose-dependent calibration curve combined with a microfluidic module for sample analysis and a portable electronics providing an operator-independent read-out scheme.