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mRNA vaccines ( i.e. , COVID-19 vaccine) offer various advantages over traditional vaccines in preventing and reducing disease and shortening the time between pathogen discovery and vaccine creation. Production of mRNA vaccines results in several nucleic acid and enzymatic by-products, most of which can be detected and removed; however, double-stranded RNA (dsRNA) contaminants pose a particular challenge. Current purification and detection platforms for dsRNA vary in effectiveness, with problems in scalability for mass mRNA vaccine production. Effectively detecting dsRNA is crucial in ensuring the safety and efficacy of the vaccines, as these strands can cause autoimmune reactions with length-symptom dependency and enhance mRNA degradation. We present a new microfluidics method to rapidly identify and quantify dsRNA fragments in mRNA samples. Our innovation exploits the differences in the dynamic staining behavior between mRNA and dsRNA molecules to detect dsRNA contaminants in a high throughput approach. The limit of detection of the system for dsRNA was estimated to be between 17.7-76.6 pg μL -1 with a maximum loading capacity of mRNA of 12.99 ng μL -1 . Based on these estimated values, our method allows for the detection of dsRNA contaminants present in percentages as low as 0.14-0.59% compared to the total mRNA concentration. Here, we discuss the molecular mechanism of the dynamic staining behavior of dsRNA and mRNA for two different stains. We believe our method will accelerate the mRNA vaccine development from initial development to quality control workflows.

The psychrophilic (cold-loving) fungus Pseudogymnoascus destructans was discovered more than a decade ago to be the pathogen responsible for white-nose syndrome, an emerging disease of North American bats causing unprecedented population declines. The same species of fungus is found in Europe but without associated mortality in bats. We found P. destructans was infected with a mycovirus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)]. The virus is bipartite, containing two double-stranded RNA (dsRNA) segments designated as dsRNA1 and dsRNA2. The cDNA sequences revealed that dsRNA1 dsRNA is 1,683 bp in length with an open reading frame (ORF) that encodes 539 amino acids (molecular mass of 62.7 kDa); dsRNA2 dsRNA is 1,524 bp in length with an ORF that encodes 434 amino acids (molecular mass of 46.9 kDa). The dsRNA1 ORF contains motifs representative of RNA-dependent RNA polymerase (RdRp), whereas the dsRNA2 ORF sequence showed homology with the putative capsid proteins (CPs) of mycoviruses. Phylogenetic analyses with PdPV-1 RdRp and CP sequences indicated that both segments constitute the genome of a novel virus in the family Partitiviridae. The purified virions were isometric with an estimated diameter of 33 nm. Reverse transcription PCR (RT-PCR) and sequencing revealed that all US isolates and a subset of Czech Republic isolates of P. destructans were infected with PdPV-1. However, PdPV-1 appears to be not widely dispersed in the fungal genus Pseudogymnoascus, as non-pathogenic fungi P. appendiculatus (1 isolate) and P. roseus (6 isolates) tested negative. P. destructans PdPV-1 could be a valuable tool to investigate fungal biogeography and the host-pathogen interactions in bat WNS.

Background: Bacteriophages (phages) are the most numerous biological entities on Earth and play a crucial role in shaping microbial communities. Investigating the bacteriophage community from soil will shed light not only on the yet largely unknown phage diversity, but may also result in novel insights towards their functioning in the global biogeochemical nutrient cycle and their significance in earthbound ecosystems. Unfortunately, information about soil viromes is rather scarce compared to aquatic environments, due to the heterogeneous soil matrix, which rises major technical difficulties in the extraction process. Resolving these technical challenges and establishing a standardized extraction protocol is, therefore, a fundamental prerequisite for replicable results and comparative virome studies., Results: We here report the optimization of protocols for the extraction of phage DNA from agricultural soil preceding metagenomic analysis such that the protocol can equally be harnessed for phage isolation. As an optimization strategy, soil samples were spiked with Listeria phage A511 (Myovirus), Staphylococcus phage 2638AΔLCR (Siphovirus) and Escherichia phage T7 (Podovirus) (each 10 6 PFU/g soil). The efficacy of phage (i) elution, (ii) filtration, (iii) concentration and (iv) DNA extraction methods was tested. Successful extraction routes were selected based on spiked phage recovery and low bacterial 16S rRNA gene contaminants. Natural agricultural soil viromes were then extracted with the optimized methods and shotgun sequenced. Our approach yielded sufficient amounts of inhibitor-free viral DNA for shotgun sequencing devoid of amplification prior library preparation, and low 16S rRNA gene contamination levels (≤ 0.2‰). Compared to previously published protocols, the number of bacterial read contamination was decreased by 65%. In addition, 379 novel putative complete soil phage genomes (≤ 235 kb) were obtained from over 13,000 manually identified viral contigs, promising the discovery of a large, previously inaccessible viral diversity., Conclusion: We have shown a considerably enhanced extraction of the soil phage community by protocol optimization that has proven robust in both culture-dependent as well as through viromic analyses. Our huge data set of manually curated soil viral contigs substantially increases the amount of currently available soil virome data, and provides insights into the yet largely undescribed soil viral sequence space.

Plant RNA viruses are obligate intracellular parasites that hijack specific cellular membranes to replicate their genomes in what are commonly known as viral replication complexes (VRC). These contain host- and virus-encoded proteins and viral RNA. Double-stranded RNA (dsRNA) is a mandatory intermediate of RNA replication and a hallmark feature of VRCs. We have recently developed a method to isolate viral dsRNA and its associated proteins through pull-down of an ectopically expressed dsRNA-binding protein (B2:GFP) from infected Arabidopsis thaliana plants. After mass spectrometry analysis to identify the dsRNA-associated proteins, resulting candidate proteins of interest are tagged with a red fluorescent protein and their subcellular localization in relation to VRCs is assessed by transient expression within leaves of B2:GFP-transgenic Nicotiana benthamiana plants. In this chapter we describe in detail these experimental procedures to allow investigators to characterize the replication complexes of their plant RNA virus of interest.

Double-stranded RNA (dsRNA) plays an essential role in many biological processes and has a great potential for agronomic applications in disease and pest control. A simple and effective method to monitor dsRNA uptake by fungi is crucial for the use of dsRNA as alternative fungicide. The protocol reported in this chapter describes an efficient method to detect and localize labeled dsRNA in fungal hyphae. We use the fungal Verticillium longisporum, a fungal plant pathogen that commonly infects rapeseed and other Brassica species, to explain the procedure, though we have validated the method in a broad spectrum of fungi. Hereafter we elucidate step-by-step the production, fluorescence labeling, as well as detection of dsRNA via fluorescence microscopy in fungal mycelium.

Double-stranded RNA (dsRNA) is well characterized as an inducer of anti-viral interferon responses. We previously reported that dsRNA extracted from a specific edible plant possesses an immune-modulating capacity to confer, in mice, resistance against respiratory viruses, including the H1N1 strain of the influenza A virus (IAV). We report here that the systemic immune-activating capacity of the plant-derived dsRNA protected mice from infection by a highly virulent H5N1 strain of the IAV. In addition, subcutaneous inoculation of the dsRNA together with the inactivated virion of the H5N1 strain of the IAV suppressed the lethality of the viral infection as compared with individual inoculation of either dsRNA or HA protein, suggesting its potential usage as a vaccination adjuvant. Moreover, intra-peritoneal inoculation of the dsRNA limited the growth of B16-F10 melanoma cells through the activation of NK cells in murine models. Taken together, this study demonstrated the systemic immune-modulating capacity of a plant-derived dsRNA and its potential for nucleic acid-based clinical applications., (© The Japanese Society for Immunology. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

RNA viruses are a major source of emerging and re-emerging infectious diseases around the world. We developed a method to identify RNA viruses that is based on the fact that RNA viruses produce double-stranded RNA (dsRNA) while replicating. Purifying and sequencing dsRNA from the total RNA isolated from infected tissue allowed us to recover dsRNA virus sequences and replicated sequences from single-stranded RNA (ssRNA) viruses. We refer to this approach as dsRNA-Seq. By assembling dsRNA sequences into contigs we identified full length or partial RNA viral genomes of varying genome types infecting mammalian culture samples, identified a known viral disease agent in laboratory infected mice, and successfully detected naturally occurring RNA viral infections in reptiles. Here, we show that dsRNA-Seq is a preferable method for identifying viruses in organisms that don't have sequenced genomes and/or commercially available rRNA depletion reagents. In addition, a significant advantage of this method is the ability to identify replicated viral sequences of ssRNA viruses, which is useful for distinguishing infectious viral agents from potential noninfectious viral particles or contaminants.

The influenza A virus is a highly infectious respiratory pathogen that sickens many people with respiratory disease annually. To prevent outbreaks of this viral infection, an understanding of the characteristics of virus-host interaction and development of an anti-viral agent is urgently needed. The influenza A virus can infect mammalian species including humans, pigs, horses and seals. Furthermore, this virus can switch hosts and form a novel lineage. This so-called zoonotic infection provides an opportunity for virus adaptation to the new host and leads to pandemics. Most influenza A viruses express proteins that antagonize the antiviral defense of the host cell. The non-structural protein 1 (NS1) of the influenza A virus is the most important viral regulatory factor controlling cellular processes to modulate host cell gene expression and double-stranded RNA (dsRNA)-mediated antiviral response. This review focuses on the influenza A virus NS1 protein and outlines current issues including the life cycle of the influenza A virus, structural characterization of the influenza A virus NS1, interaction between NS1 and host immune response factor, and design of inhibitors resistant to the influenza A virus.

Trichomonas vaginalis (TV) is a parasitic protozoan responsible for the sexually transmitted infection trichomoniasis. Trichomonas vaginalis virus (TVV) is a nonsegmented, 4.5-5 kbp, double-stranded RNA virus, from the Totiviridae family, which inhabits TV. A capsid protein consisting of 120 subunits is covered in channels aiding in RNA release. TVV is closely associated with the Golgi complex and is transmitted vertically. TVV has four subspecies, TVV1, TVV2, TVV3, and TVV4. The clinical significance of TVV and its effect on the pathogenicity of TV is not well known. We performed a systematic review of the literature on TVV to better understand its clinical significance and its role in the pathogenesis of TV.

Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3' polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae . The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast.

The ability of the cytidine analog Çmf to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Çmf-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Çmf. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Çmf at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.

Nucleic acid sensing is a central mechanism for innate immune defense against foreign molecules that culminates with an activation of interferon signaling pathways. This involves detection of molecular patterns associated with extracellular or intracellular pathogens by specialized receptors within the cell. In addition to foreign molecules, cells also sense endogenous molecules. One specific arm of nucleic acid sensors detects dsRNA structures. In this chapter, we discuss principles of dsRNA recognition and downstream activation of signaling pathways important in the process of antiviral responses. We also discuss various mechanisms by which endogenous dsRNA can form in a cell, in particular, through epigenetic regulation. Finally, we provide approaches for measuring and quantifying dsRNA accumulation and downstream activation in human colorectal cancer cells., (© 2019 Elsevier Inc. All rights reserved.)

A clinical isolate of Aspergillus thermomutatus (Teleomorph: Neosartorya pseudofischeri) was found to contain ~35 nm isometric virus-like particles associated with four double-stranded (ds) RNA segments, each of which coded for a single open reading frame. The longest dsRNA element (3589 nt) encodes a putative RNA-dependent RNA polymerase (1114 aa), the second longest dsRNA element (2772 nt) encodes a coat protein (825 aa), and the other two dsRNAs (2676 nt, 2514 nt) encode hypothetical proteins of 768 aa and 711 aa, respectively. Phylogenetic analysis of the amino acid sequences showed 41-60% similarity to the proteins coded by the dsRNAs of the most closely related virus, Penicillium janczewskii chrysovirus 2, indicating that it is a new species based on the International Committee on Taxonomy of Viruses criteria for the genus Chrysovirus. This is the first virus reported from A. thermomutatus and was tentatively named Aspergillus thermomutatus chrysovirus 1. A virus free line of the fungal isolate, cured by cycloheximide treatment, produced large numbers of conidia but no ascospores at both 20°C and 37°C, whereas the virus infected line produced ten-fold fewer conidia at 20°C and a large number of ascospores at both temperatures. The effects of the virus on fungal sporulation have interesting implications for the spread of the fungus and possible use of the virus as a biological control agent., Competing Interests: The authors have declared that no competing interests exist.

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen ( Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum , a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

Steric exclusion chromatography (SXC) is a method for separation of large target solutes based on their association with a hydrophilic stationary phase through mutual steric exclusion of polyethylene glycol (PEG). Selectivity in SXC is determined by the size or shape (or both) of the solutes alongside the size and concentration of PEG molecules. Elution is achieved by decreasing the PEG concentration. In this study, SXC applicability for the separation and purification of single-stranded (ss) and double-stranded (ds) RNA molecules was evaluated for the first time. The retention of ssRNA and dsRNA molecules of different lengths on convective interaction media (CIM) monolithic columns was systematically studied under variable PEG-6000 and NaCl concentrations. We determined that over 90% of long ssRNAs (700-6374 nucleotides) and long dsRNAs (500-6374 base pairs) are retained on the stationary phase in 15% PEG-6000 and ≥0.4 M NaCl. dsDNA and dsRNA molecules of the same length were partially separated by SXC. Separation of RNA molecules below 100 nucleotides from longer RNA species is easily achieved by SXC. Furthermore, SXC has the potential to separate dsRNAs from ssRNAs of the same length. We also demonstrated that SXC is suitable for the enrichment of ssRNA (PRR1 bacteriophage) and dsRNA (Phi6 bacteriophage) viral genomes from contaminating cellular RNA species. In summary, SXC on CIM monolithic columns is an appropriate tool for rapid RNA separation and concentration., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response., Methods: We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction., Results: HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication., Conclusions: Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Approaches based on next-generation sequencing (NGS) coupled with bioinformatics tools have been developed for detecting viruses and viroids infecting herbaceous and woody plants. Here we describe a protocol to extract nucleic acids from citrus bark and enrich them in double-stranded RNAs. These preparations can be efficiently used for generating cDNA libraries that, after pair-end sequencing and bioinformatics analyses, allow efficient identification of the viroids infecting the source plant.

High-throughput sequencing (or next-generation sequencing-NGS) is an emerging technology that allows the detection of plant viruses without any prior knowledge. Various sequencing techniques and various templates can be used as substrate for NGS. This chapter describes an optimized protocol for the extraction of double-stranded RNAs (dsRNAs) from a wide range of plants and for their random amplification prior to NGS sequencing.

Nucleic acids carrying pathogen-associated molecular patterns trigger innate immune responses and are used to activate host immunity. Although synthetic nucleic acids have been used for that purpose, they have shown limitations for in vivo and clinical applications. To address this issue, we tested a naturally occurring dsRNA extracted from rice bran (rb-dsRNA) and characterized it as a potent ligand of TLR3 and MDA5. In this study, intranasal administration of rb-dsRNA induced production of type I IFNs by alveolar macrophages and protected mice from morbidity and mortality resulting from respiratory virus infection, such as influenza A virus. This protection was completely absent in mice lacking both TRIF and MDA5, indicating the essential role of TLR3- and MDA5-dependent pathways. Interestingly, IFNAR1-deficient mice retained residual antiviral protection, which was abolished by pharmacological inhibition of caspase 1, but not IL-1β signaling. In fact, rb-dsRNA activated caspase 1 via TRIF, resulting in the release of IL-1β and LDH. In addition to the direct antiviral activity, rb-dsRNA modulated the immune cell population in the lungs by repopulating virus-depleted alveolar macrophages. Our data demonstrate that rb-dsRNA orchestrates IFN-dependent and -independent direct antiviral protection and that it is a potent immune stimulator modulating antiviral immunity in the lungs. These findings open doors to a range of precise immune-modulating studies and therapeutic options., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The Dicer family of ribonucleases plays a key role in small RNA-based regulatory pathways by generating short dsRNA fragments that modulate expression of endogenous genes, or protect the host from invasive nucleic acids. Beginning with its initial discovery, biochemical characterization of Dicer has provided insight about its catalytic properties. However, a comprehensive understanding of how Dicer's domains contribute to substrate-specific recognition and catalysis is lacking. One reason for this void is the lack of high-resolution structural information for a metazoan Dicer in the apo- or substrate-bound state. Both biochemical and structural studies are facilitated by large amounts of highly purified, active protein, and Dicer enzymes have historically been recalcitrant to overexpression and purification. Here we describe optimized procedures for the large-scale expression of Dicer in baculovirus-infected insect cells. We then outline a three-step protocol for the purification of large amounts (3-4mg of Dicer per liter of insect cell culture) of highly purified and active Dicer protein, suitable for biochemical and structural studies. Our methods are general and are extended to enable overexpression, purification and biochemical characterization of accessory dsRNA binding proteins that interact with Dicer and modulate its catalytic activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Neofusicoccum luteum species belongs to the Botryosphaeriaceae family and is involved in grapevine wood decay diseases. The present study reports the discovery and the molecular characterization of a novel mitovirus infecting this fungus. Double-stranded RNAs were purified from cultivated N. luteum and analysed by next generation sequencing. Using contigs showing BlastX homology with the RNA-dependent RNA polymerase (RdRp) gene of various members of the family Narnaviridae, a single contig of approximately 1.2 kb was constructed. The genomic sequence was completed and phylogenetic analyses indicated that this virus represents a new member of the genus Mitovirus, for which the name of "Neofusicoccum luteum mitovirus 1" is proposed. The genome is 2,389 nucleotides long and, based on the fungal mitochondrial genetic code, it encodes a putative protein of 710 amino acids, homologous to the RdRps of members of the Narnaviridae family. The neofusicoccum luteus mitovirus 1 (NLMV1) RdRp contains the six conserved motifs previously reported for mitoviral RdRps. Our findings represent the first evidence that a mycovirus can infect N. luteum, an important pathogenic fungus of grapevine.

The Totiviridae family includes a number of viruses that can infect protozoan parasites such as Leishmania and Giardia and fungi like Saccharomyces cerevisiae. Some isolates of Trichomonas vaginalis are also infected with one or more double-stranded RNA (dsRNA) viruses. In this study, the frequency of Trichomonas vaginalis virus (TVV1) was evaluated in Iranian isolates of T. vaginalis in Tehran, Iran. One thousand five hundred vaginal samples were collected from patients attending obstetrics and gynaecology hospitals associated with Iran University of Medical Sciences in Tehran, Iran from October 2015 to September 2016. Trichomonas vaginalis isolates were cultured in Diamond's modified medium. Nucleic acids were extracted using a DNA/RNA extraction kit and RT-PCR was performed. Among 1500 collected vaginal samples, 8 (0.53%) cases of T. vaginalis infection were found. Half (4/8) of the T. vaginalis positive cases were infected with TVV1. Phylogenetic mapping indicated that the Iranian isolates were most closely related to TVV1-OC5, TVV1-UR1. Iranian isolates of T. vaginalis were infected with TVV1. The frequency of viral infection (TVV1) in T. vaginalis isolates found in this study is higher than previously reported in Iran., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ustilaginoidea virens , the causal agent of rice false smut, is one of the most devastating grain diseases that causes loss of yield in most rice-growing areas worldwide. In this study, we performed a dsRNA screen to isolate mycoviruses from 35 U. virens strains. The results revealed that 34 of the tested isolates were infected by various dsRNA elements, displaying highly viral diversity and mixed infections. We characterized a 5.3 kbp dsRNA from a typical isolate containing dsRNA segments with sizes ranging from 0.5 to 5.3 kbp. Sequence analysis of its genomic properties indicated that it is a novel victorivirus, named Ustilaginoidea virens RNA virus 5 (UvRV5), that belongs to the family Totiviridae . RT-PCR detection was performed and indicated that not all the dsRNA bands that were 5.3 kbp in size contained UvRV5. Moreover, the genetic relatedness of all the U. virens strains was estimated according to phylogenetic analysis of the partial intergenic spacer region (IGS) sequences. However, concordance was not found between the dsRNA profiles and the IGS-based genetic relatedness of their host fungi.

RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Bacteriophages (phages) are widely distributed in the biosphere and play a key role in modulating microbial ecology in the soil, ocean, and humans. Although the role of DNA bacteriophages is well described, the biology of RNA bacteriophages is poorly understood. More than 1900 phage genomes are currently deposited in NCBI, but only 6 dsRNA bacteriophages and 12 ssRNA bacteriophages genome sequences are reported. The 6 dsRNA bacteriophages were isolated from legume samples or lakes with Pseudomonas syringae as the host. Here, we report the first Pseudomonas aeruginosa phage phiYY with a three-segmented dsRNA genome. phiYY was isolated from hospital sewage in China with the clinical P. aeruginosa strain, PAO38, as a host. Moreover, the dsRNA phage phiYY has a broad host range, which infects 99 out of 233 clinical P. aeruginosa strains isolated from four provinces in China. This work presented a detailed characterization of the dsRNA bacteriophage infecting P. aeruginosa.

The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS)., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Two dsRNAs of approximately 6.0-kb and 3.0-kb in length were detected in strain SsSn-1 of Sclerotinia nivalis. Genomic analysis showed that the 6.0-kb dsRNA was a victorivirus, named as Sclerotinia nivalis victorivirus 1 (SnVV1). The genome of SnVV1 is 5162bp in length containing two large open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to CPs of viruses belonging to the family Totiviridae. The stop codon of ORF1 overlaps the start codon of ORF2 in the tetranucleotide sequence AUGA. ORF2 was predicted to encode for a RNA-dependent RNA polymerase (RdRp) that was very similar to the RdRps of victoriviruses. The 3.0-kb dsRNA was consisted of two species of mitoviruses, named as Sclerotinia nivalis mitovirus 1/SsSn-1 (SnMV1/SsSn-1) and Sclerotinia sclerotiorum mitovirus 3/SsSn-1 (SsMV3/SsSn-1). The genomes of SnMV1/SsSn-1 and SsMV3/SsSn-1 were 2720nt and 2583nt in length, respectively. Both mitoviruses were AU-rich and deduced to contain a major large ORF encoding a mitoviral RdRp with the fungal mitochondrial codon usages. SnMV1/SsSn-1 was most closely related to Sclerotinia sclerotiorum mitovirus 4 (SsMV4/NZ1) and shared 76.5% and 80.1% identity with SsMV4/NZ1 for nucleotide and RdRp sequences, respectively. In addition, the nucleotide and RdRp sequences of SsMV3/SsSn-1 were 90.6% and 95.9% identical to the nucleotide and RdRp sequences of SsMV3/NZ1, respectively. Considering their nucleotide and RdRp sequence identities with other mitoviruses, SnMV1/SsSn-1 may belong to the genus Mitovirus, whereas SsMV3/SsSn-1 is possibly a strain of SsMV3. Both SnMV1/SsSn-1 and SsMV3/SsSn-1 were transmitted to a recipient virus-free colony faster than was SnVV1 through hyphal anastomosis. Co-infection by these mycoviruses had no apparent effects on growth and pathogenicity of S. nivalis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Next-generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to justify their use in large-scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double-stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time-consuming and labour-intensive. We present an alternative method to enrich dsRNA from plant extracts using anti-dsRNA monoclonal antibodies in a pull-down assay. The extracted dsRNA can be amplified by reverse transcriptase-polymerase chain reaction and sequenced by next-generation sequencing. In our study, we have selected three distinct plant hosts: Māori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand-native ornamental plant and a weed, respectively. Of the sequence data obtained, 31-74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples., (© 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.)

Background: The known plant viruses mostly infect angiosperm hosts and have RNA or small DNA genomes. The only other lineage of green plants with a relatively well-studied virome, unicellular chlorophyte algae, is mostly infected by viruses with large DNA genomes. Thus RNA viruses and small DNA viruses seem to completely displace large DNA virus genomes in late branching angiosperms. To understand better the expansion of RNA viruses in the taxonomic span between algae and angiosperms, we analyzed the transcriptomes of 66 non-angiosperm plants characterized by the 1000 Plants Genomes Project., Results: We found homologs of virus RNA-dependent RNA polymerases in 28 non-angiosperm plant species, including algae, mosses, liverworts (Marchantiophyta), hornworts (Anthocerotophyta), lycophytes, a horsetail Equisetum, and gymnosperms. Polymerase genes in algae were most closely related to homologs from double-stranded RNA viruses leading latent or persistent lifestyles. Land plants, in addition, contained polymerases close to the homologs from single-stranded RNA viruses of angiosperms, capable of productive infection and systemic spread. For several polymerases, a cognate capsid protein was found in the same library. Another virus hallmark gene family, encoding the 30 K movement proteins, was found in lycophytes and monilophytes but not in mosses or algae., Conclusions: The broadened repertoire of RNA viruses suggests that colonization of land and growth in anatomical complexity in land plants coincided with the acquisition of novel sets of viruses with different strategies of infection and reproduction.

The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as "DECS-C," is a powerful method for detecting novel plant viruses.

The aim of this study was to investigate the functions of Trichinella spiralis Nudix hydrolase (TsNd) during the larval invasion of intestinal epithelial cells (IECs), development and survival in host by RNAi. The TsNd-specific double-stranded RNA (dsRNA) was designed to silence the expression of TsNd in T. spiralis larvae. DsRNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with dsRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. After being soaked with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 65.8% and 56.4%, respectively. After being electroporated with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 74.2% and 58.2%, respectively. Silencing TsNd expression by both soaking and electroporation inhibited significantly the larval invasion of IECs in a dose-dependent manner (r1 = -0.96798, r2 = -0.98707). Compared with the mice inoculated with untreated larvae, mice inoculated with larvae soaked with TsNd dsRNA displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larvae, while mice inoculated with larvae electroporated with TsNd dsRNA displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larvae, indicating that electroporation has a higher efficiency than soaking in inhibiting the larval development and survival in mice. Our results showed that silencing TsNd expression in T. spiralis inhibited significantly the larval invasion and survival in host., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The identification of mycoviruses contributes greatly to understanding of the diversity and evolutionary aspects of viruses. Powdery mildew fungi are important and widely studied obligate phytopathogenic agents, but there has been no report on mycoviruses infecting these fungi. In this study, we used a deep sequencing approach to analyze the double-stranded RNA (dsRNA) segments isolated from field-collected samples of powdery mildew fungus-infected red clover plants in Japan. Database searches identified the presence of at least ten totivirus (genus Totivirus)-like sequences, termed red clover powdery mildew-associated totiviruses (RPaTVs). The majority of these sequences shared moderate amino acid sequence identity with each other (<44%) and with other known totiviruses (<59%). Nine of these identified sequences (RPaTV1a, 1b and 2-8) resembled the genome of the prototype totivirus, Saccharomyces cerevisiae virus-L-A (ScV-L-A) in that they contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA dependent RNA polymerase (RdRp), while one sequence (RPaTV9) showed similarity to another totivirus, Ustilago maydis virus H1 (UmV-H1) that encodes a single polyprotein (CP-RdRp fusion). Similar to yeast totiviruses, each ScV-L-A-like RPaTV contains a -1 ribosomal frameshift site downstream of a predicted pseudoknot structure in the overlapping region of these ORFs, suggesting that the RdRp is translated as a CP-RdRp fusion. Moreover, several ScV-L-A-like sequences were also found by searches of the transcriptome shotgun assembly (TSA) libraries from rust fungi, plants and insects. Phylogenetic analyses show that nine ScV-L-A-like RPaTVs along with ScV-L-A-like sequences derived from TSA libraries are clustered with most established members of the genus Totivirus, while one RPaTV forms a new distinct clade with UmV-H1, possibly establishing an additional genus in the family. Taken together, our results indicate the presence of diverse, novel totiviruses in the powdery mildew fungus populations infecting red clover plants in the field., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus). Virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. Double-stranded RNA (dsRNA) is produced during the replication of many of these viruses as either an intermediate in RNA replication (e.g., flaviviruses, togaviruses) or the double-stranded RNA genome (e.g., reoviruses). Detection and discovery of novel viruses from field and clinical samples usually relies on recognition of antigens or nucleotide sequences conserved within a virus genus or family. However, due to the wide antigenic and genetic variation within and between viral families, many novel or divergent species can be overlooked by these approaches. We have developed two monoclonal antibodies (mAbs) which show co-localised staining with proteins involved in viral RNA replication in immunofluorescence assay (IFA), suggesting specific reactivity to viral dsRNA. By assessing binding against a panel of synthetic dsRNA molecules, we have shown that these mAbs recognise dsRNA greater than 30 base pairs in length in a sequence-independent manner. IFA and enzyme-linked immunosorbent assay (ELISA) were employed to demonstrate detection of a panel of RNA viruses from several families, in a range of cell types. These mAbs, termed monoclonal antibodies to viral RNA intermediates in cells (MAVRIC), have now been incorporated into a high-throughput, economical ELISA-based screening system for the detection and discovery of viruses from mosquito populations. Our results have demonstrated that this simple system enables the efficient detection and isolation of a range of known and novel viruses in cells inoculated with field-caught mosquito samples, and represents a rapid, sequence-independent, and cost-effective approach to virus discovery.

Recent evidence has suggested the existence of sense-antisense transcription in mammals, but the existence of double-stranded RNAs endowed with biological function has remained elusive. Herein we show that hundreds of putative natural double-stranded RNAs (ndsRNAs) are expressed from interspersed genomic locations and respond to cellular cues. We demonstrate that a subset of ndsRNAs localize in the nucleus and, in their double-stranded form, interact with nuclear proteins. Detailed characterization of an ndsRNA (nds-2a) revealed that this molecule displays differential localization throughout the cell cycle and directly interacts with RCC1 and RAN and, through the latter, with the mitotic RANGAP1-SUMO1-RANBP2 complex. Notably, altering nds-2a levels led to postmitotic abnormalities, mitotic catastrophe and cell death, thus supporting a mitosis-related role. Altogether, our study reveals a hitherto-unrecognized class of RNAs that potentially participate in major biological processes in human cells.

Fungal viruses (mycoviruses) often have a significant impact not only on phenotypic expression of the host fungus but also on higher order biological interactions, e.g., conferring plant stress tolerance via an endophytic host fungus. Arbuscular mycorrhizal (AM) fungi in the phylum Glomeromycota associate with most land plants and supply mineral nutrients to the host plants. So far, little information about mycoviruses has been obtained in the fungi due to their obligate biotrophic nature. Here we provide a technical breakthrough, "two-step strategy" in combination with deep-sequencing, for virological study in AM fungi; dsRNA is first extracted and sequenced using material obtained from highly productive open pot culture, and then the presence of viruses is verified using pure material produced in the in vitro monoxenic culture. This approach enabled us to demonstrate the presence of several viruses for the first time from a glomeromycotan fungus.

When a diseased plant is suspected to be infected with unknown viruses, the approach of isolating double-stranded RNA (dsRNA) from diseased tissues and analyzing the sequence has been useful for detecting the viruses. This procedure owes its success to the majority of plant pathogenic viruses being RNA viruses, which accumulate dsRNAs as copies of their genome or as a replicative intermediate in infected cells. Conventional dsRNA isolation methods (e.g., chromatography using CF-11 cellulose) require a significant amount of plant material and are laborious and time consuming. Therefore, it has been impractical to isolate dsRNA from many samples at the same time. To overcome these problems, we developed a novel dsRNA isolation method involving a recombinant dsRNA-binding protein. Using this method, we can readily isolate viral dsRNA from a small amount of plant material, and can process numerous samples simultaneously. Purified dsRNA can be used as a template for cDNA synthesis and sequencing, enabling detection of both known and unknown viruses.

Vitellogenin (Vg) is a precursor of the major yolk protein, an essential nutrient for the embryonic development of oviparous animals including insects. Here, the gene(CceVg [Corcyra cephalonica Vg] ) encoding the Vg (CceVg of moth, C. cephalonica, was cloned and sequenced. The gene sequence was 6,721-bp long and contained 5five introns and six exons that together formed a 5,382-bp open reading frame. The deduced protein (CceVg) consisted of 1,793 amino acid residues, including a 16-amino-acid signal peptide. The putative molecular weight of the primary Vg protein was 202.46 kDa. The CceVg contained all conserved domains and motifs that were commonly found in most insect Vgs except the presence of a polyserine tract at the C-terminal region, which had not been reported in other lepidopteran Vgs. The expression pattern showed that CceVg was first transcribed at a very low level in the early larval stage but disappeared in later stage larva. In female, the CceVg mRNA was detected in early pupal stage and throughout adult stage. Interestingly, the CceVg mRNA was detected only in mated males at low levels, not in the virgin ones. Injection of CceVg double-stranded RNA into early-emergent females caused severely abnormal ovaries., (© 2014 Wiley Periodicals, Inc.)

Unlabelled: To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae. A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1β, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses., Importance: Virus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vector-based vaccination by immune modulators.

Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88 %), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs.

An infectious agent was transmitted mechanically from samples of Viola spp. showing white mosaic and leaf deformation to Nicotiana benthamiana. dsRNA extracted from the N. benthamiana plants migrated as four specific bands that were absent in non-inoculated plants. Sequence analysis of cDNA clones generated from the second-smallest dsRNA showed the greatest similarity to the RNA3 of prune dwarf virus (PDV) (genus Ilarvirus, family Bromoviridae). However, because of differences in molecular, biological, and serological properties between this virus isolate and PDV, a new ilarvirus species, named "Viola white distortion associated virus" (VWDaV) is proposed. Specific oligonucleotides and a TaqMan(®) probe were designed for diagnostic purposes. The possible association between the virus and the original white distortion symptoms is discussed.

Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88%), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs.

p19 is an RNA binding protein originally isolated from the Carnation Italian ring-spot virus (CIRV). It has been shown that p19 is a plant RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. A bifunctional p19 fusion protein, with an N-terminal maltose binding protein (MBP) and a C-terminal chitin binding domain (CBD) allows protein purification and binding of p19 to chitin magnetic beads via the chitin binding domain. The fusion p19 protein recognizes and binds double-stranded RNAs (dsRNA) in the size range of 20-23 nucleotides, but does not bind single strand RNA (ssRNA) or dsDNA. Furthermore, p19 can also bind mRNA, if there is a 19 bp blunt RNA duplex at the exact end of the RNA. Binding specificity of the p19 fusion protein for small dsRNA allows for detection of siRNAs derived either from exogenous or endogenous long dsRNA or microRNAs when hybridized to a complementary RNA. Here we describe a robust method using p19 and radioactive RNA probes to detect siRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. This chapter describes the potential of p19 fusion protein to detect miRNAs, isolate exogenous or endogenous siRNAs, and purify longer RNAs that contain a 19-bp terminal RNA duplex.

Most of reported fungal viruses (mycoviruses) have double-stranded RNA (dsRNA) genomes. This may reflect the simple, easy method for mycovirus hunting that entails detection of dsRNAs as a sign of viral infections. There are an increasing number of screens of various fungi, particularly phytopathogenic fungi for viruses pathogenic to host fungi or able to confer hypovirulence to them. This bases on an attractive research field of biological control of fungal plant diseases using viruses (virocontrol), mainly targeting important phytopathogenic fungi. While isolated viruses usually induce asymptomatic symptoms, they show a considerably high level of diversity. As of 2014, fungal dsRNA viruses are classified into six families: Reoviridae, Totiviridae, Chrysoviridae, Partitiviridae, Megabirnaviridae and Quadriviridae. These exclude unassigned mycoviruses which will definitely be placed into distinct families and/or genera. In this review article, dsRNA viruses isolated from the kingdom Fungi including as-yet-unclassified taxa are overviewed. Some recent achievements in the related field are briefly introduced as well.

The etiology of viruses in osteoarthritis remains controversial because the prevalence of viral nucleic acid sequences in peripheral blood or synovial fluid from osteoarthritis patients and that in healthy control subjects are similar. Until now the presence of virus has not been analyzed in cartilage. We screened cartilage and chondrocytes from advanced and non-/early osteoarthritis patients for parvovirus B19, herpes simplex virus-1, Epstein Barr virus, cytomegalovirus, human herpes virus-6, hepatitis C virus, and human endogenous retroviruses transcripts. Endogenous retroviruses transcripts, but none of the other viruses, were detected in 15 out the 17 patients. Sequencing identified the virus as HERV-WE1 and E2. HERV-W activity was confirmed by high expression levels of syncytin, dsRNA, virus budding, and the presence of virus-like particles in all advanced osteoarthritis cartilages examined. Low levels of HERV-WE1, but not E2 envelope RNA, were observed in 3 out of 8 non-/early osteoarthritis patients, while only 3 out of 7 chondrocytes cultures displayed low levels of syncytin, and just one was positive for virus-like particles. This study demonstrates for the first time activation of HERV-W in cartilage of osteoarthritis patients; however, a causative role for HERV-W in development or deterioration of the disease remains to be proven.

Aspergillus foetidus virus (AfV) contains at least two icosahedral particle types named AfV-fast (-F) and AfV-slow (-S), based on relative electrophoretic mobility. AfV-F is a quadripartite double-stranded RNA (dsRNA) virus, and AfV-S contains AfV-S1, which is a member of the genus Victorivirus in the family Totiviridae, and AfV-S2, which may be a satellite RNA or satellite virus and is described here. Analysis of the complete AfV-S2 nucleotide sequence reveals it to be significantly similar to an unclassified RNA from the fungus Rosellinia necatrix and distantly related to the RNA-dependent RNA polymerases of several single-stranded RNA genomes.

The family Partitiviridae includes plant (Alphacryptovirus and Betacryptovirus), fungal (Partitivirus) and protozoan (Cryspovirus) viruses with bisegmented dsRNA genomes and isometric virions. Cryptic viruses commonly occur in different plant species without causing any symptoms. So far, numerous sequences have been determined for viruses of the genus Alphacryptovirus, but no sequence is available for any assigned member of the genus Betacryptovirus. Following extraction, cloning and sequence analysis of double-stranded RNA in this study, we report the molecular properties of three assigned members of the genus Betacryptovirus, white clover cryptic virus 2, red clover cryptic virus 2 and hop trefoil cryptic virus 2, and two new putative betacryptoviruses found in crimson clover and dill. Betacryptoviruses share sequence motifs with members of the genus Partitivirus. In phylogenetic analyses, members of the genus Betacryptovirus formed a new sub-cluster within the clusters containing members of the genus Partitivirus. Our results provide evidence for a distinct evolutionary lineage of dsRNA viruses of plants and fungi.

Recent advances in the field of RNA interference and new cost-effective approaches for large-scale double-stranded RNA (dsRNA) synthesis have fuelled the demand for robust high-performance purification techniques suitable for dsRNA molecules of various lengths. To address this issue, we developed an improved dsRNA purification method based on anion exchange chromatography utilizing convective interaction media (CIM) monolithic columns. To evaluate column performance we synthesized a selection of dsRNA molecules (58-1810 bp) in a one-step enzymatic reaction involving bacteriophage T7 DNA-dependent RNA polymerase and phi6 RNA-dependent RNA polymerase. In addition, small interfering RNAs (siRNAs) of 25-27 bp were generated by Dicer digestion of the genomic dsRNA of bacteriophage phi6. We demonstrated that linearly scalable CIM monolithic quaternary amine (QA) columns can be used as a fast and superior alternative to standard purification methods (e.g. LiCl precipitation) to obtain highly pure dsRNA preparations. The impurities following Dicer treatment were quickly and efficiently removed with the QA CIM monolithic column, yielding siRNA molecules of high purity suitable for potential therapeutic applications. Moreover, baseline separation of dsRNA molecules up to 1 kb in non-denaturing conditions was achieved., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In general, mycoviruses are transmitted through hyphal anastomosis between vegetatively compatible strains of the same fungi, and their entire intracellular life cycle within host fungi limits transmission to separate species and even to incompatible strains belonging to the same species. Based on field observations of the white root rot fungus, Rosellinia necatrix, we found two interesting phenomena concerning mycovirus epidemiology. Specifically, apple trees in an orchard were inoculated with one or two R. necatrix strains that belonged to different mycelial compatibility groups (MCGs), strains W563 (virus-free, MCG139) and NW10 (carrying a mycovirus-like double-stranded (ds) RNA element (N10), MCG442). Forty-two sub-isolates of R. necatrix, which were retrieved 2-3 years later, were all genetically identical to W563 or NW10: however, 22 of the sub-isolates contained novel dsRNAs. Six novel dsRNAs (S1-S6) were isolated: S1 was a new victorivirus; S2, S3, and S4 were new partitiviruses; and S5 and S6 were novel viruses that could not be assigned to any known mycovirus family. N10 dsRNA was detected in three W563 sub-isolates. These findings indicated that novel mycoviruses, from an unknown source, were infecting strains W563 and NW10 of R. necatrix in the soil, and that N10 dsRNA was being transmitted between incompatible strains, NW10 to W563., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Background: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence., Methodology/principal Findings: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice., Conclusions/significance: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

RNA interference (RNAi) is an evolutionary conserved gene-silencing pathway that can be efficiently utilized as a tool to study gene function. RNAi is initiated by long double-stranded RNAs (dsRNAs), which are processed into small duplexes called small-interfering RNAs (siRNAs). In turn, these duplexes target mRNAs for degradation in a sequence-specific manner. Mouse oocytes, unlike most mammalian cell types, lack an interferon response to long dsRNA. Moreover, they are a rare example of a mammalian cell type with a robust endogenous RNAi pathway. For these reasons microinjection of either long dsRNAs or siRNAs results in efficient, sequence-specific gene silencing. Here, we describe a protocol for preparation and microinjection of long dsRNA into mouse oocytes.