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Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the “master” sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained by in silico analysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid’s accumulation by inducing the RNA silencing of the host’s defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid’s interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics.

Hop plant (Humulus lupulus L.) is an important industrial crop, grown for harvesting hop cones however, it is a host to four different viroids as well. The nature of viroid infections is not entirely clarified. In our work, we focused on analyzing viroid accumulation and their strands polarity through RNA sequencing and reverse transcription polymerase chain reaction in real time. RNA-seq data indicate that viroids amplify until saturation further demonstrating plant's biological capacity. Negative fold changes in accumulation of individual viroids between hop samples with single and multiple infections are suggesting an antagonistic relationship amongst viroids, where citrus bark cracking viroid seems to be the least and hop stunt viroid the most sensitive to the other two. RNA-seq data also show that on average (−) viroid strand is dominating over (+), especially for the citrus bark cracking viroid. Reverse transcription polymerase chain reaction in real time results from strand polarity analysis seem to be less consistent between different combinations of infection but are showing level of conformity with RNA-seq in the case of citrus bark cracking viroid.

Studies on the ways in which viroids are transmitted are important for understanding their epidemiology and for developing effective control measures for viroid diseases. Viroids may be spread via vegetative propagules, mechanical damage, seed, pollen, or biological vectors. Vegetative propagation is the most prevalent mode of spread at the global, national and local level while further dissemination can readily occur by mechanical transmission through crop handling with viroid-contaminated hands or pruning and harvesting tools. The current knowledge of seed and pollen transmission of viroids in different crops is described. Biological vectors shown to transmit viroids include certain insects, parasitic plants, and goats. Under laboratory conditions, viroids were also shown to replicate in and be transmitted by phytopathogenic ascomycete fungi; therefore, fungi possibly serve as biological vectors of viroids in nature. The term “mycoviroids or fungal viroids” has been introduced in order to denote these viroids. Experimentally, known sequence variants of viroids can be transmitted as recombinant infectious cDNA clones or transcripts. In this review, we endeavor to provide a comprehensive overview of the modes of viroid transmission under both natural and experimental situations. A special focus is the key findings which can be applied to the control of viroid diseases.

Viroids are a unique class of plant pathogens that consist of small circular RNA molecules, between 220 and 450 nucleotides in size. Viroids encode no proteins and are the smallest known infectious agents. Viroids replicate via the rolling circle mechanism, producing multimeric intermediates which are cleaved to unit length either by ribozymes formed from both polarities of the viroid genomic RNA or by coopted host RNAses. Many viroid-like small circular RNAs are satellites of plant RNA viruses. Ribozyviruses, represented by human hepatitis delta virus, are larger viroid-like circular RNAs that additionally encode the viral nucleocapsid protein. It has been proposed that viroids are direct descendants of primordial RNA replicons that were present in the hypothetical RNA world. We argue, however, that much later origin of viroids, possibly, from recently discovered mobile genetic elements known as retrozymes, is a far more parsimonious evolutionary scenario. Nevertheless, viroids and viroid-like circular RNAs are minimal replicators that are likely to be close to the theoretical lower limit of replicator size and arguably comprise the paradigm for replicator emergence. Thus, although viroid-like replicators are unlikely to be direct descendants of primordial RNA replicators, the study of the diversity and evolution of these ultimate genetic parasites can yield insights into the earliest stages of the evolution of life.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission. [ABSTRACT FROM AUTHOR]

Escherichia coli K1 is the leading cause of meningitis in newborns. Understanding the molecular basis of E. coli K1 pathogenicity will help develop treatment of meningitis and prevent neurological sequelae. E. coli K1 replicates in host blood and forms a high level of bacteremia to cause meningitis in human. However, the mechanisms that E. coli K1 employs to sense niche signals for survival in host blood are poorly understood. We identified one intergenic region in E. coli K1 genome that encodes a novel small RNA, sRNA-17. The expression of sRNA-17 was downregulated by ArcA in microaerophilic blood. The Δ sRNA-17 strain grew better in blood than did the wild-type strain and enhanced invasion frequency in human brain microvascular endothelial cells. Transcriptome analyses revealed that sRNA-17 regulates tens of differentially expressed genes. These data indicate that ArcA downregulates the sRNA-17 expression to benefit bacterial survival in blood and penetration of the blood–brain barrier. Our findings reveal a signaling mechanism in E. coli K1 for host adaptation. [ABSTRACT FROM AUTHOR]

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RVA and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RVA16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, norwas the C-terminal tail of STING that mediates IRF3 signaling. Despite coopting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus. [ABSTRACT FROM AUTHOR]

Background: Cell released microvesicles specifically, exosomes, play an important role in mediating immunologic escape, treatment resistance, and disease persistence of Hepatitis C virus (HCV) infection. Reports on the molecular compositions of exosomes released by cells under diverse conditions, especially during viral infections, suggest that their cargo contents are not randomly loaded. However, the precise molecular mechanisms directing the selective cargo sorting and loading inside infectious viral exosomes remains elusive. Aim: To decipher the role of Reticulon 3 (RTN3) in the selective molecular cargo sorting and loading inside infectious viral exosomes during HCV infection. Methods: We used Huh7 cells–JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we analyzed human liver and serum exosome samples from healthy and treatment naïve HCV infected individuals. Our experiments made use of molecular biology and immunology techniques. Results: HCV infection (JFH1-Huh7 or HCV-FL replicon cells) was associated with increased RTN3L&S isoforms expression in cells and cell released exosomes. Accordingly, increased expression of RTN3L&S was observed in liver and serum exosome samples of HCV infected individuals compared to healthy controls. RNA-ChIP analysis revealed that RTN3L&S interacted with dsHCV RNA. Lentiviral CRISPR/Cas9 mediated knockdown (KD) of RTN3 and plasmid overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3L&S in HCV- infected Huh7 cells differentially impacted the cellular release of infectious viral exosomes. RTN3L&S KD significantly decreased, while RTN3S OE significantly increased the number of Huh7 cell-released infectious exosomes. The C-terminal domain of RTN3 interacted with and modulated the loading of dsHCV RNA inside infectious exosomes. Antiviral treatment of HCV infected Huh7 cells reduced virus-induced RTN3L&S expression and attenuated the release of infectious exosomes. Conclusion: RTN3 constitutes a novel regulator and a potential therapeutic target that mediates the specific loading of infectious viral exosomes. [ABSTRACT FROM AUTHOR]

Based on high-throughput sequencing (HTS) data, the existence of viroid-like RNAs (Vd-LRNAs) associated with fig trees grown in the Hawaiian Islands has been predicted. One of these RNAs has been characterized as a circular RNA ranging in size from 357 to 360 nucleotides. Structural and biochemical features of this RNA, tentatively named fig hammerhead viroid-like RNA (FHVd-LR), markedly resemble those previously reported for several viroids and viroid-like satellite RNAs (Vd-LsatRNAs), which are non-protein-coding RNAs infecting their hosts autonomously and in combination with a helper virus, respectively. The full-length sequence of FHVd-LR variants was determined by RT-PCR, cloning, and sequencing. Despite a low global sequence identity with known viroids and Vd-LsatRNAs, FHVd-LR contains a hammerhead ribozyme (HRz) in each polarity strand. Northern blot hybridization assays identified the circular and linear forms of both polarity strands of FHVd-LR and showed that one strand, assigned the (+) polarity, accumulates at higher levels than the (−) polarity strand in vivo. The (+) polarity RNA assumes a rod-like secondary structure of minimal free energy with the conserved domains of the HRzs located in opposition to each other, a feature typical of several viroids and Vd-LRNAs. The HRzs of both FHVd-LR polarity strands were shown to be active in vitro during transcription, self-cleaving the RNAs at the predicted sites. These data, together with the sequence variability observed in the cloned and sequenced full-length variants, indicate that FHVd-LR is a novel viroid or Vd-LsatRNA. According to HTS data, the coexistence of FHVd-LR of different sizes in the same host cannot be excluded. The relationships of FHVd-LR with previously reported viroids and Vd-LsatRNAs, and the need to perform bioassays to conclusively clarify the biological nature of this circular RNA, are discussed. [ABSTRACT FROM AUTHOR]

Apple russet ring and apple green crinkle are graft-transmitted diseases first reported more than 60 years ago, but at present, no association between a specific virus (variant) and the disease has been clearly demonstrated. In this study, we conducted the following series of experiments to identify the causal viruses (variants) of these apple diseases; (1) comprehensive analysis by next-generation sequencing of all viruses in each apple tree affected with russet ring or green crinkle disease, (2) amplification of full-length genomic cDNA of viruses using primers containing the T3 promoter and the in vitro transcription of infectious viral RNAs, (3) inoculation of viral RNA transcripts to both herbaceous and apple plants, (4) analysis of sequence variants of viruses present in infected plants, (5) back-inoculation of sequence variants of candidate viruses to apple seedlings combined with the virus-induced flowering technology using the apple latent spherical virus vector to reproduce the symptom on the fruit as soon as possible, and (6) reproduction of symptoms on the fruits of apple trees inoculated with sequence variants and the re-isolation of each virus variant from apples showing fruit symptoms. The results showed that one of the sequence variants of the apple chlorotic leaf spot virus causes a characteristic ring-shaped rust on the fruits of infected apple trees and that a sequence variant of the apple stem pitting virus probably causes green crinkle symptoms on an infected apple fruit. Thus, we were able to fulfill Koch's postulates to prove the viral etiology of both the apple russet ring and green crinkle diseases. We also propose an experimental system that can prove whether a virus found in diseased tissues is the pathogen responsible for the diseases when the etiology is undetermined. [ABSTRACT FROM AUTHOR]

Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the "master" sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained by in silico analysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid's accumulation by inducing the RNA silencing of the host's defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid's interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics. [ABSTRACT FROM AUTHOR]

Young pigeon disease syndrome (YPDS) is characterized as a seasonally occurring, acute and primarily enteric medical condition of mainly juvenile domestic pigeons (Columba livia) with highly variable mortality reaching more than 50%. Although the syndrome has been known in Europe for almost three decades, its aetiology remains largely obscure. Recently, a previously unknown pigeon‐associated clade of Rotavirus A (RVA) genotype G18P[17] was detected in Europe and Australia in association with fatal diseases resembling YPDS. Here we show for the first time, that peroral inoculation of healthy juvenile homing pigeons with two genetically different cell culture isolates of RVA G18P[17] (106.3 foci‐forming units per bird) induces an acute and self‐limiting YPDS‐like disease in all infected birds. Clinical signs included regurgitation, diarrhoea, congested crops, anorexia and weight loss, as described for naturally RVA‐infected pigeons. In agreement with the original outbreaks, RVA isolate DR‐7 induced more pronounced clinical signs as compared to isolate DR‐5, indicating strain‐dependent virulence factors to contribute to variable disease outcomes observed in the field. All inoculated birds developed rotavirus‐reactive antibodies starting at seven days after inoculation. High levels of viral RNA and infectious virus were detectable in cloacal swabs and faecal samples already three days after inoculation. While shedding of infectious virus subsided within few days, moderate viral RNA levels were still detectable in cloacal swabs, faeces, and tissue samples at the end of the experiment three weeks after inoculation. Histopathological analysis at this time point revealed inflammatory lesions in spleens and livers of pigeons from both infected groups. In summary, we fulfilled Henle‐Koch's postulates and confirmed RVA G18P[17] as a primary cause of YPDS‐like diseases in domestic pigeons. By establishing an infection model, we provide a crucial tool for future research, such as identification of transmission routes and establishing vaccination regimes. [ABSTRACT FROM AUTHOR]

Viroids are minute unencapsidated non-coding circular RNAs known to be present and to cause diseases only in plants. Infections were associated with the occurrence of specific single-stranded RNAs similar in size to miRNAs and endogenous small interfering RNAs, and viroid pathogenicity is suspected to occur through RNA interference. We looked for sequence similarities between viroids and the seed region of human microRNAs (hsa-miRNAs). Viroid genomes were retrieved from GenBank and mature hsa-mi-RNAs were retrieved from miRBase. Two hundred 300-nucleotide-long sequences were randomly generated as controls. BLAST searches were performed using viroids as queries and hsa-miRNAs as subjects with relaxed parameters, and matches involving hsa-miRNA seed regions were considered. A total of 81,021 matches were found, and 1,501 that showed 100% identity with whole hsa-miRNA seed regions were selected. The most frequent matches involved Chrysanthemum stunt viroid or Hop stunt viroidspecies with hsa-miR-4286, in 365 and 207 cases, respectively. Three hsa-mi-RNAs (miR-4286, miR-6808-5p, and miR-3622a-3p) were involved in 47% of all matches between viroids and hsa-mi-RNAs. Taken together, these findings warrant further investigation on the potential of viroids and their derived small RNAs to cross kingdoms and interact with nucleic acids in humans. [ABSTRACT FROM AUTHOR]

In about 5% of patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) no mutation in the SERPING1 gene is detected. C1-INH-HAE cases with no mutation in the coding region of SERPING1 after conventional genotyping were examined for defects in the intronic or untranslated regions of the gene. Using a next-generation sequencing (NGS) platform targeting the entire SERPING1 , 14 unrelated C1-INH-HAE patients with no detectable mutations in the coding region of the gene were sequenced. Detected variants with a global minor allele frequency lower than the frequency of C1-INH-HAE (0.002%), were submitted to in silico analysis using ten different bioinformatics tools. Pedigree analysis and examination of their pathogenic effect on the RNA level were performed for filtered in variants. In two unrelated patients, the novel mutation c.-22-155G > T was detected in intron 1 of the SERPING1 gene by the use NGS and confirmed by Sanger sequencing. All bioinformatics tools predicted that the variant causes a deleterious effect on the gene and pedigree analysis showed its co-segregation with the disease. Degradation of the mutated allele was demonstrated by the loss of heterozygosity on the cDNA level. According to the American College of Medical Genetics and Genomics 2015 guidelines the c.-22-155G > T was curated as pathogenic. For the first time, a deep intronic mutation that was detected by NGS in the SERPING1 gene, was proven pathogenic for C1-INH-HAE. Therefore, advanced DNA sequencing methods should be performed in cases of C1-INH-HAE where standard approaches fail to uncover the genetic alteration. Image 1 [ABSTRACT FROM AUTHOR]

All RNA viruses encode the RNA-dependent RNA polymerase (RdRp) which replicates and transcribes viral RNA. This essential viral enzyme does not exist in mammalian cells, thus presents a main target for the development of antiviral drugs with potential pan-antiviral activity. In this study, we take advantage of the structurally equivalent site in the dengue virus (DENV) RdRp, the N-pocket, and in the human norovirus (hNV) RdRp, the B-site, and performed a parallel structure-based virtual screening to discover compounds that can inhibit the RdRps of both hNV and DENV. We successfully identified a small molecule called Entrectinib (RAI-13) as a potent inhibitor of both hNV and DENV infection. Specifically, RAI-13 binds directly to hNV and DENV RdRps, effectively inhibits the polymerase activity in the in vitro biochemical assays, and exhibits does-responsive inhibition of murine norovirus (MNV) and DENV2 infection with IC50 values of 2.01 and 2.43 μM, respectively. Most promisingly, RAI-13 inhibits hepatitis C virus (HCV) infection by 95% at the 2 μM concentration. We have therefore discovered a small molecule compound that targets an allosteric site that is shared by different viral RdRps and strongly inhibits multiple pathogenic RNA viruses, thus holding the potential of being developed into a broad-spectrum antiviral drug. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) based virome analyses of mRNA and sRNA have recently become a routine approach for reliable detection of plant viruses and viroids. In the present study we identified the viral/viroidal spectrum of several Indian grapevine cultivars and reconstructed their whole genomes using the publically available mRNAome and sRNAome datasets. Twenty three viruses and viroids (including two variants of grapevine leafroll associated virus 4) were identified from two tissues (fruit peels and young leaves) of three cultivars among which nine unique grapevine viruses and viroids were identified for the first time in India. Irrespective of the assemblers and tissues used, the mRNA based approach identified more acellular pathogens than the sRNA based approach across cultivars. Further, the mRNAome was on par with the whole transcriptome in viral identification. Through de novo assembly of transcriptomes followed by mapping against reference genome, we reconstructed 19 complete/near complete genomes of identified viruses and viroids. The reconstructed viral genomes included four larger RNA genomes (>13 kb), a DNA genome (RG grapevine geminivirus A), a divergent genome (RG grapevine virus B) and a genome for which no reference is available (RG grapevine virus L). A large number of SNPs detected in this study ascertained the quasispecies nature of viruses. Detection of three recombination events and phylogenetic analyses using reconstructed genomes suggested the possible introduction of viruses and viroids into India from several continents through the planting material. The whole genome sequences generated in this study can serve as a resource for reliable indexing of grapevine viruses and viroids in quarantine stations and certification programs. [ABSTRACT FROM AUTHOR]

Recently developed technologies have revealed that the genomes of many organisms produce transcripts that do not encode proteins. These are called non-coding RNAs. Long non-coding RNAs (lncRNAs) are important regulators of the expression of their target genes at the levels of transcription, translation, and degradation. Multiple studies have demonstrated a role for lncRNAs in various biological responses, including pathogenic infection. Upon pathogenic infection, the expression levels of lncRNAs are dynamically altered, suggesting that lncRNAs are involved in the host immune response or propagation of pathogens. In this review, we focused on host lncRNAs that are involved in pathogenic infection. Some host lncRNAs act as host defense molecules to prevent pathogenic proliferation, while others are utilized by the pathogen to enhance the propagation of pathogens. [ABSTRACT FROM AUTHOR]

The initial molecular lesions through which viroids, satellite RNAs and viruses trigger signal cascades resulting in plant diseases are hotly debated. Since viroids are circular non-protein-coding RNAs of ∼250–430 nucleotides, they appear very convenient to address this issue. Viroids are targeted by their host RNA silencing defense, generating viroid-derived small RNAs (vd-sRNAs) that are presumed to direct Argonaute (AGO) proteins to inactivate messenger RNAs, thus initiating disease. Here, we review the existing evidence. Viroid-induced symptoms reveal a distinction. Those attributed to vd-sRNAs from potato spindle tuber viroid and members of the family Pospiviroidae (replicating in the nucleus) are late, non-specific and systemic. In contrast, those attributed to vd-sRNAs from peach latent mosaic viroid (PLMVd) and other members of the family Avsunviroidae (replicating in plastids) are early, specific and local. Remarkably, leaf sectors expressing different PLMVd-induced chloroses accumulate viroid variants with specific pathogenic determinants. Some vd-sRNAs containing such determinant guide AGO1-mediated cleavage of mRNAs that code for proteins regulating chloroplast biogenesis/development. Therefore, the initial lesions and the expected phenotypes are connected by short signal cascades, hence supporting a cause-effect relationship. Intriguingly, one virus satellite RNA initiates disease through a similar mechanism, whereas in the Pospiviroidae and in plant viruses the situation remains uncertain. [ABSTRACT FROM AUTHOR]

The innate immune system is normally programmed for immediate but transient upregulation in response to invading pathogens and interferon (IFN)-stimulated gene (ISG) activation is a central feature. In contrast, chronic innate immune system activation is typically associated with autoimmunity and a broad array of autoinflammatory diseases that include the interferonopathies. Here, we studied retroviral susceptibility in a transgenic mouse model with life-long innate immune system hyper-activation. The mice transgenically express low levels of a picornaviral RNA-dependent RNA polymerase (RdRP), which synthesizes double-stranded RNAs that are sensed by MDA5 to trigger constitutive upregulation of many ISGs. However, in striking counterpoint to the paradigm established by numerous human and murine examples of ISG hyperactivation, including constitutive MDA5 activation, it lacks auto-inflammatory sequelae. RdRP mice resist infection and disease caused by several pathogenic RNA and DNA viruses. However, retroviruses are sensed through other mechanisms, persist in the host, and have distinctive replication and immunity evading properties. We infected RdRP mice and wild type (WT) mice with varying doses of a pathogenic retrovirus (Friend virus) and assessed immune parameters and disease at 1, 4 and 8 weeks. Compared to WT mice, RdRP mice had significantly reduced splenomegaly, viral loads, and infection of multiple target cell types in the spleen and the bone marrow. During chronic infection, RdRP mice had 2.35 ± 0.66 log10 lower circulating viral RNA than WT. Protection required ongoing type I IFN signaling. The results show that the reconfigured RdRP mouse innate immune system substantially reduced retroviral replication, set point and pathogenesis. [ABSTRACT FROM AUTHOR]

A multiplex RT-PCR reaction was developed and validated for the simultaneous detection of two viruses and two viroids: grapevine fleck virus, grapevine Pinot gris virus, grapevine yellow speckle viroid 1, and hop stunt viroid. The multiplex reaction specificity was verified on a set of 210 grapevines from the Czech Republic. The results showed the occurrence of hop stunt viroid in 94.3% of the examined plants, while incidence of the other pathogens was lower: grapevine Pinot gris virus (64.8%), grapevine yellow speckle viroid 1 (52.4%), and grapevine fleck virus (15.2%). [ABSTRACT FROM AUTHOR]

Viruses are normally defined as pathogens and have a bad reputation because of pandemics such as Influenza, HIV/AIDS, Ebola, and SARS. Most viruses are, however, not enemies or killers but play important roles in the origin, development and maintenance of life of all species on our planet. This is new information we learnt by new technologies such as sequencing. Viruses are the most successful species on Earth, they are ubiquitous, in the oceans, in our environment, in animals, plants, bacteria, up in the air, perhaps even in the universe, within our body and even as part of our genomes. They influence our health, our well‐being, mental properties, our gut microbiota including obesity, and may help to cope with multi‐drug‐resistant bacteria. There the phages, viruses of bacteria, raise hopes. Viruses built our immunity: viruses protect against viruses. We do not have to lay eggs – thanks to viruses! They are the drivers of evolution and adaptation to environmental changes, also e. g. in plankton. The success story of viruses started about 3.5 billion years ago when life began. Newly discovered giant viruses are almost bacteria in their composition, suggesting that the borderline between dead matter and life is continuous. There are many open questions – how did life begin, is there life on exoplanets, how to find it? Are virus‐like elements, viroids, important for the origin of life? Will viruses eliminate mankind [1]? [ABSTRACT FROM AUTHOR]

A list of plant species, in alphabetical order by their scientific name, and the viruses found naturally infecting them in Brazilian territory, with some comments, was prepared. The production of such a list was based on a yearly catalog of publications on plant viruses collected by the author, from 1926 to 2018. Listed species of viruses were those recognized by the International Committee on Taxonomy of Viruses (ICTV), but also those characterized and still waiting official recognition, were included. Several cases of putative viral diseases were listed for historical reasons expecting to raise interest for their clarification. This list includes 345 plants species belonging to 74 families naturally infected by plant viruses in Brazil. Fabaceae and Asteraceae had most virusinfected species, respectively 49 and 36. Until 2018, a total of 213 plant virus and 6 viroid species belonging to 57 genera and 22 families and 6 orders, officially recognized by ICTV, were found naturally infecting these plants. Begomovirus and Potyvirus genera have most representatives, with 45 and 42 species, respectively. There are 59 characterized plant viruses, up to species level, described in Brazil waiting for the inclusion in the ICTV Master Species List. One hundred and thirteen viruses were identified up to genus level but still uncharacterized, while four putative isometric viruses and eleven presumptive viral diseases ("unidentified") are included in the list. A reverse catalog, listing viruses and the plant species in which they were found is also included. [ABSTRACT FROM AUTHOR]

Context • RNA viruses exhibit an extraordinary ability to evolve in a changing environment and to switch from animal hosts to humans. The ongoing COVID-19 pandemic, recognized as a respiratory disease, is an example of zoonotic transmission of the RNA virus known as SARS-CoV-2. The development and regulatory approval of a vaccine against SARS-CoV-2 pose multiple preventive and therapeutic challenges, especially during an ongoing pandemic. Objective • The review intended to examine the challenges and recent achievements in the development of vaccine candidates against COVID-19. Design • The research team performed a literature review, searching relevant and up to date information from the literature. The sources of data included Google Scholar, PubMed, NCBI, and Yahoo. The search terms used were COVID-19 challenges, SARS-CoV-2 prospective challenges, RNA viruses adoptability, host switching by RNA viruses, COVID-19 vaccines. Setting • The study took place at the digital libraries of contributing institutions. The data was combined, selected for further analysis and manuscript preparation at King Abdulaziz University. Results • RNA viruses with high rate of genome alterations and evolution have better chances to survive in the adverse environmental conditions by adopting the alternate host species. The recent epidemics such as SARS, MERS, and COVID-19 are examples of zoonotic transmission of RNA viruses from animal species to the humans. However, the mechanisms involved in the switching-on to new host species need further investigations to control the zoonotic transmissions in near future. As of April 2020, 115 candidate vaccines were being evaluated; 78 of them had been found to be active, and a few of them are in Phase I trials. In the development of different types of vaccine candidates against COVID- 19, multiple international pharmaceutical and biotechnology companies are involved. Conclusions • Emerging and re-emerging pathogenic RNA viruses pose a serious threat to human health. Little is known about the human-host adoptive mechanism for zoonotic transmission. Deep insights into the molecular mechanism responsible for the switching of animal or bird viruses to humans could provide target molecules or events to prevent such transmissions in the near future. Fast development and approval of efficacious and safe vaccines is key to the effort to provide preventive measures against COVID-19 and future viruses. However, the development and availability of a vaccine candidate is a time-consuming process and often can’t be completed during an epidemic. Currently, several types of vaccines are under development, and most of them won’t realistically be available in time for the present COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

Background: Wolbachia pipientis are bacterial endosymbionts of arthropods currently being implemented as biocontrol agents to reduce the global burden of arboviral diseases. Some strains of Wolbachia, when introduced into Aedes aegypti mosquitoes, reduce or block the replication of RNA viruses pathogenic to humans. The wAlbB strain of Wolbachia was originally isolated from Aedes albopictus, and when transinfected into Ae. aegypti, persists in mosquitoes under high temperature conditions longer than other strains. The utility of wAlbB to block a broad spectrum of RNA viruses has received limited attention. Here we test the ability of wAlbB to reduce or block the replication of a range of Flavivirus and Alphavirus species in cell culture. Methods: The C6/36 mosquito cell line was stably infected with the wAlbB strain using the shell-vial technique. The replication of dengue, West Nile and three strains of Zika (genus Flavivirus), and Ross River, Barmah Forest and Sindbis (genus Alphavirus) viruses was compared in wAlbB-infected cells with Wolbachia-free controls. Infectious virus titres were determined using either immunofocus or plaque assays. A general linear model was used to test for significant differences in replication between flaviviruses and alphaviruses. Results: Titres of all viruses were significantly reduced in cell cultures infected with wAlbB versus Wolbachia-free controls. The magnitude of reduction in virus yields varied among virus species and, within species, also among the strains utilized. Conclusion: Our results suggest that wAlbB infection of arthropods could be used to reduce transmission of a wide range of pathogenic RNA viruses. [ABSTRACT FROM AUTHOR]

Hand, foot, and mouth disease (HFMD) is a febrile exanthematous disease with typical or atypical symptoms. Typical HFMD is usually caused by enterovirus 71 (EV71) or coxsackievirus A16, while atypical HFMD is usually caused by coxsackievirus A6 (CA6). In recent years, worldwide outbreaks of CA6-associated HFMD have dramatically increased, although the pathogenic mechanism of CA6 is still unclear. EV71 has been established to induce caspase-dependent apoptosis, but in this study, we demonstrate that CA6 infection promotes a distinct pathway of cell death that involves loss of cell membrane integrity. Necrostatin-1, an inhibitor of necroptosis, blocks the cell death induced by CA6 infection, but Z-DEVD-FMK, an inhibitor of caspase-3, has no effect on CA6-induced cell death. Furthermore, CA6 infection up-regulates the expression of the necroptosis signaling molecule RIPK3. Importantly, necrostatin-1 inhibits CA6 viral production, as assessed by its ability to inhibit levels of VP1 protein and genomic RNA and infectious particles. CA6-induced necroptosis is not dependent on the generation of reactive oxygen species; however, viral 3D protein can directly bind RIPK3, which is suggestive of a direct mechanism of necroptosis induction. Therefore, these results indicate that CA6 induces a mechanism of RIPK3-dependent necroptosis for viral production that is distinct from the mechanism of apoptosis induced by typical HFMD viruses. [ABSTRACT FROM AUTHOR]

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of those viruses and viroids (hereafter referred to as viruses) of Solanum tuberosum and other tuber‐forming Solanum spp. (hereafter referred to as potato) which are considered to be either non‐EU or of undetermined standing based on a previous EFSA opinion. These viruses belong to different families and genera and either have an established identity or produce consistent symptoms. Plants for planting is the main pathway for entry for all categorised viruses as they can all be transmitted by vegetative propagation. Several categorised viruses have a relatively wide host range and/or are vector‐transmitted, increasing the potential for entry. The information currently available on geographical distribution, biology, epidemiology, impact and potential entry pathways has been evaluated with regard to the criteria to qualify as potential Union quarantine pest or as Union regulated non‐quarantine pest (RNQP). Since this opinion addresses specifically the non‐EU potato viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non‐quarantine pests. The following viruses meet the criteria to qualify as potential Union quarantine pest: APLV, APMMV, APMoV, ChiLCV, CYSDV, PAMV, PBRSV, PVH, PVP, PVT, PYDV, PYMV, PYV, PYVV, RCVMV, SALCV, SB26/29, ToCV, ToLCNDV, ToMHaV, ToMoTV, ToSRV and ToYVSV. With the exception of the criterion regarding the potential for consequences in the EU territory, for which the Panel is unable to conclude because of lack of information, AVB, CPSbV, PaLCrV, PapMV, PVB, PVU, SB41 and TVBMV meet all the other criteria to qualify as potential Union quarantine pest. PotLV and WPMV do not qualify as potential Union quarantine pest, since they are not reported to have any impact. For most of the categorised viruses, the conclusions of the Panel have inherent uncertainties, due to the lack of quantitative data on their impact and/or absence or limited availability of information on the biology, epidemiology and geographical distribution. [ABSTRACT FROM AUTHOR]

Viroids, the smallest plant pathogenes, are single-stranded, covalently closed, circular, highly structured noncoding RNAs that cause disease in several economically important crop plants. They replicate autonomously and move systemically in host plants with the aid of the host cell machinery. Based on structure and location of replication, viroids are taxonomically divided into two families: Avsunviroidae and Pospiviroidae. Avsunviroidae replicate in the chloroplast, while Pospiviroidae reproduce in the nucleus. Rod-like structures are typical for the Pospiviroidae, whereas more branched structures are typical for Avsunviroidae. Viroids and viroid diseases are distributed globally. Viroids are transmitted with vegetative parts of plants, seeds, pollen and indirectly also with insects. Insects transmit viroids mostly mechanically, with infected plant sap that remains on different body parts during feeding or pollination. Indirectly viruses are also involved in viroids transmission with insects, in which viruses are encapsulated. The host symptoms of all these viroid diseases are similar, and include stunting, apical proliferation, apical leaf narrowing and yellowing, leaf crinkling, tissue brittleness and necrosis. [ABSTRACT FROM AUTHOR]

RT-PCR and RT-qPCR (real-time RT-PCR) methods are routinely used to determine viroids in hops, allowing rapid, reliable, and sensitive detection. The main limitation is RNA isolation, which can result in false negative results, due to low viroid concentration or the presence of PCR inhibitors. Six commercial RNA extraction reagents (Sigma-Aldrich TRIzol, SpectrumTM Plant Total kit, New England Biolabs (NEB) Monarch Total RNA Miniprep kit, Macherey-Nagel (MN) NucleoSpin® RNA Plus kit, Bioline ISOLATE II RNA Plant Kit, Qiagen RNeasy® Plant Mini Kit) and total nucleic acids isolation method using CTAB reagent were compared for their ability to extract hop viroids. Infected (CBCVd + HLVd) and healthy hop plants, different tissue (leaves and cones) and homogenization methods (use of press and mortar) were tested. The concentration of the samples was measured and analyzed by RT-PCR and RT-qPCR. With RTPCR, the strongest signals were detected after CTAB isolation. In the case of the use of commercial extraction kits, only RNA was isolated, and samples isolated with Qiagen, NEB and MN kits, were amplified by RT-qPCR without non-specific signals. For less sensitive determination of viroids by RT-PCR, the use of CTAB isolation is sufficient. While in the case of testing samples with lower viroid concentrations, the use of commercially available reagents is recommended. [ABSTRACT FROM AUTHOR]

Scientists at Stanford University have discovered a new type of virus-like entity called "obelisks" that inhabit bacteria in the human mouth and gut. These obelisks have genomes composed of loops of RNA and have been found worldwide. It is not yet known if obelisks affect human health, but they could potentially alter the genetic activity of their bacterial hosts, which could in turn impact human genes. This discovery adds to the ongoing exploration of the viral universe and raises questions about the evolution of viruses. [Extracted from the article]

SARS-CoV-2, a type of respiratory virus, has exerted a great impact on global health and economy over the past three years. Antibody-based therapy was initially successful but later failed due to the accumulation of mutations in the spike protein of the virus. Strategies that enable antibodies to resist virus escape are therefore of great significance. Here, we engineer a bispecific SARS-CoV-2 neutralizing nanobody in secretory Immunoglobulin A (SIgA) format, named S2-3-IgA2m2, which shows broad and potent neutralization against SARS-CoV-1, SARS-CoV-2 and its variants of concern (VOCs) including XBB and BQ.1.1. S2-3-IgA2m2 is ∼1800-fold more potent than its parental IgG counterpart in neutralizing XBB. S2-3-IgA2m2 is stable in mouse lungs at least for three days when administrated by nasal delivery. In hamsters infected with BA.5, three intranasal doses of S2-3-IgA2m2 at 1 mg/kg significantly reduce viral RNA loads and completely eliminate infectious particles in the trachea and lungs. Notably, even at single dose of 1 mg/kg, S2-3-IgA2m2 prophylactically administered through the intranasal route drastically reduces airway viral RNA loads and infectious particles. This study provides an effective weapon combating SARS-CoV-2, proposes a new strategy overcoming the virus escape, and lays strategic reserves for rapid response to potential future outbreaks of "SARS-CoV-3". [ABSTRACT FROM AUTHOR]

Rab1, a GTPase, is present in all eukaryotes, and is mainly involved in vesicle trafficking between the endoplasmic reticulum and Golgi, thereby regulating many cellular activities and pathogenic infections. However, little is known of how Rab1 functions in fish during virus infection. Groupers (Epinephelus spp.) are high in economic value and widely cultivated in China and Southeast Asia, although they often suffer from diseases. Red-spotted grouper nervous necrosis virus (RGNNV), a highly pathogenic RNA virus, is a major pathogen in cultured groupers, and causes huge economic losses. A series of host cellular proteins involved in RGNNV infection was identified. However, the impact of Rab1 on RGNNV infection has not yet been reported. In this study, a novel Rab1 homolog (EcRab1) from Epinephelus coioides was cloned, and its roles during virus infection and host immune responses were investigated. EcRab1 encoded a 202 amino acid polypeptide, showing 98% and 78% identity to Epinephelus lanceolatus and Homo sapiens , respectively. After challenge with RGNNV or poly(I:C), the transcription of EcRab1 was altered both in vitro and in vivo , implying that EcRab1 was involved in virus infection. Subcellular localization showed that EcRab1 was displayed as punctate structures in the cytoplasm, which was affected by EcRab1 mutants. The dominant negative (DN) EcRab1, enabling EcRab1 to remain in the GDP-binding state, caused EcRab1 to be diffusely distributed in the cytoplasm. Constitutively active (CA) EcRab1, enabling EcRab1 to remain in the GTP-binding state, induced larger cluster structures of EcRab1. During the late stage of RGNNV infection, some EcRab1 co-localized with RGNNV, and the size of EcRab1 clusters was enlarged. Importantly, overexpression of EcRab1 significantly inhibited RGNNV infection, and knockdown of EcRab1 promoted RGNNV infection. Furthermore, EcRab1 inhibited the entry of RGNNV to host cells. Compared with EcRab1, overexpression of DN EcRab1 or CA EcRab1 also promoted RGNNV infection, suggesting that EcRab1 regulated RGNNV infection, depending on the cycles of GTP- and GDP-binding states. In addition, EcRab1 positively regulated interferon (IFN) immune and inflammatory responses. Taken together, these results suggest that EcRab1 affects RGNNV infection, possibly by regulating host immunity. Our study furthers the understanding of Rab1 function during virus infection, thus helping to design new antiviral strategies. • EcRab1 was obviously downregulated after RGNNV infection. • EcRab1 significantly inhibited RGNNV infection. • EcRab1 inhibits RGNNV entry, which was the first report of the effect of Rab1 on virus entry. • EcRab1 has a positive regulatory effect on host immunity. [ABSTRACT FROM AUTHOR]

The immunogenetics of wildlife populations influence the epidemiology and evolutionary dynamic of the host-pathogen system. Profiling immune gene diversity present in wildlife may be especially important for those species that, while not at risk of disease or extinction themselves, are host to diseases that are a threat to humans, other wildlife, or livestock. Hantaviruses (genus: Orthohantavirus) are globally distributed zoonotic RNA viruses with pathogenic strains carried by a diverse group of rodent hosts. The marsh rice rat (Oryzomys palustris) is the reservoir host of Orthohantavirus bayoui , a hantavirus that causes fatal cases of hantavirus cardiopulmonary syndrome in humans. We performed a genome wide association study (GWAS) using the rice rat "immunome" (i.e., all exons related to the immune response) to identify genetic variants associated with infection status in wild-caught rice rats naturally infected with their endemic strain of hantavirus. First, we created an annotated reference genome using 10× Chromium Linked Reads sequencing technology. This reference genome was used to create custom baits which were then used to target enrich prepared rice rat libraries (n = 128) and isolate their immunomes prior to sequencing. Top SNPs in the association test were present in four genes (Socs5 , Eprs , Mrc1 , and Il1f8) which have not been previously implicated in hantavirus infections. However, these genes correspond with other loci or pathways with established importance in hantavirus susceptibility or infection tolerance in reservoir hosts: the JAK/STAT, MHC, and NFκB. These results serve as informative markers for future exploration and highlight the importance of immune pathways that repeatedly emerge across hantavirus systems. Our work aids in creating cross-species comparisons for better understanding mechanisms of genetic susceptibility and host-pathogen coevolution in hantavirus systems. • We identify genetic variants (SNPs) associated with infection in a hantavirus host. • SNPs affected new genes in known pathways linked to hantavirus infections. • Identified SNPs in novel genes were present in the JAK/STAT, MHC, NFκB, GAIT pathways. • Our results highlight immune pathways that regularly emerge across hantavirus systems. [ABSTRACT FROM AUTHOR]

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.

Tomato (Solanum lycopersicum L.), family Solanaceae, has become in the past fifty years one of the most important and extensively grown horticultural crops in the Mediterranean region and throughout the world. In 2019, more than 180 million tonnes of tomato have been produced worldwide, out of which around 42 million tonnes in Mediterranean countries. Due to its genetic properties, tomato is afflicted by numerous plant diseases induced by fungal, bacterial, phytoplasma, virus, and viroid pathogens. Not only is its genetic inheritance of great importance to the management of the numerous tomato pathogens, but equally as important are also the present climate changes, the recently revised phytopathological control measures, and the globalization of the seed industry. Thus, the recognition of symptoms and the knowledge of the distribution and spread of the disease and of the methods for early detection of the pathogens are the major prerequisites for a successful management of the disease. In this review, we will describe the main tomato pathogens in the Mediterranean area that impact mostly the tomato yield and provide the current and perspective measures necessary for their successful management.

Kolmioviridae is a family for negative-sense RNA viruses with circular, viroid-like genomes of about 1.5-1.7 kb that are maintained in mammals, amphibians, birds, fish, insects and reptiles. Deltaviruses, for instance, can cause severe hepatitis in humans. Kolmiovirids encode delta antigen (DAg) and replicate using host-cell DNA-directed RNA polymerase II and ribozymes encoded in their genome and antigenome. They require evolutionary unrelated helper viruses to provide envelopes and incorporate helper virus proteins for infectious particle formation. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Kolmioviridae , which is available at ictv.global/report/kolmioviridae.

Bacteriophages, or phages, are viruses that infect and replicate within bacterial hosts, playing a significant role in regulating microbial populations and ecosystem dynamics. However, phages from extreme environments such as polar regions remain relatively understudied due to challenges such as restricted ecosystem access and low biomass. Understanding the diversity, structure, and functions of polar phages is crucial for advancing our knowledge of the microbial ecology and biogeochemistry of these environments. In this review, we will explore the current state of knowledge on phages from the Arctic and Antarctic, focusing on insights gained from -omic studies, phage isolation, and virus-like particle abundance data. Metagenomic studies of polar environments have revealed a high diversity of phages with unique genetic characteristics, providing insights into their evolutionary and ecological roles. Phage isolation studies have identified novel phage-host interactions and contributed to the discovery of new phage species. Virus-like particle abundance and lysis rate data, on the other hand, have highlighted the importance of phages in regulating bacterial populations and nutrient cycling in polar environments. Overall, this review aims to provide a comprehensive overview of the current state of knowledge about polar phages, and by synthesizing these different sources of information, we can better understand the diversity, dynamics, and functions of polar phages in the context of ongoing climate change, which will help to predict how polar ecosystems and residing phages may respond to future environmental perturbations., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, an analysis of the virome of 51 grapevines from the Don ampelographic collection named after Ya. I. Potapenko (Russia) was performed using high-throughput sequencing of total RNA. A total of 20 previously described grapevine viruses and 4 viroids were identified. The most detected were grapevine rupestris stem pitting-associated virus (98%), hop stunt viroid (98%), grapevine Pinot gris virus (96%), grapevine yellow speckle viroid 1 (94%), and grapevine fleck virus (GFkV, 80%). Among the economically significant viruses, the most present were grapevine leafroll-associated virus 3 (37%), grapevine virus A (24%), and grapevine leafroll-associated virus 1 (16%). For the first time in Russia, a grapevine-associated tymo-like virus (78%) was detected. After a bioinformatics analysis, 123 complete or nearly complete viral genomes and 64 complete viroid genomes were assembled. An analysis of the phylogenetic relationships with reported global isolates was performed. We discovered and characterized the genomes of five novel grapevine viruses: bipartite dsRNA grapevine alphapartitivirus (genus Alphapartitivirus , family Partitiviridae ), bipartite (+) ssRNA grapevine secovirus (genus Fabavirus , family Secoviridae ) and three (+) ssRNA grapevine umbra-like viruses 2, -3, -4 (which phylogenetically occupy an intermediate position between representatives of the genus Umbravirus and umbravirus-like associated RNAs).