Detection Of Double-stranded RNA Research Articles

Activation of PKR by a short-hairpin RNA

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54’s potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

Double-stranded RNA immunohistochemistry as a screening tool for viral encephalitis.

Viral infections of the central nervous system can be challenging to diagnose because of the wide range of causative agents and nonspecific histologic features. We sought to determine whether detection of double-stranded RNA (dsRNA), produced during active RNA and DNA viral infections, could be used to select cases for metagenomic next-generation sequencing (mNGS) from formalin-fixed, paraffin-embedded brain tissue. Eight commercially available anti-dsRNA antibodies were optimized for immunohistochemistry (IHC) and the top antibody tested in a series of cases with confirmed viral infections (n = 34) and cases with inflammatory brain lesions of unclear etiology (n = 62). Among known positives, anti-dsRNA IHC produced a strong cytoplasmic or nuclear staining pattern for Powassan virus, West Nile virus, rabies virus, JC polyoma virus, and adenovirus while failing to detect Eastern equine encephalitis virus, Jamestown Canyon virus, or any herpesvirus. All the unknown cases were negative by anti-dsRNA IHC, while mNGS detected rare viral reads (0.3-1.3 reads per million total reads) in 2 cases (3%), with only 1 having potential clinical significance. Anti-dsRNA IHC can effectively identify a subset of clinically relevant viral infections but not all. The absence of staining should not exclude cases from mNGS if sufficient clinical and histologic suspicion exists.

Viral sensing by RNA helicases.

A key aspect of antiviral immunity is the distinction between "self" and "non-self" components. This distinction can be established through the detection of double-stranded RNA (dsRNA), a common sign of viral infection, by cytosolic RNA helicases. Depending on the organism, two major antiviral pathways can be induced by dsRNA helicases: RNA interference (RNAi) and interferon (IFN) signaling. In the RNAi pathway, dsRNAs are recognized by a Dicer protein, and are then used for the sequence-dependent recognition and subsequent degradation of the complementary viral RNAs. In the IFN signaling pathway, dsRNAs are recognized by a RIG-like receptor (RLR), which induces a signaling cascade in order to induce the expression of IFNs, cytokines and chemokines. In this review, we discuss the RNA features that can be used by the cell to detect a viral infection, the two aforementioned types of helicase-mediated sensing, as well as some viral escape mechanisms developed to avoid recognition.

Viral sensing by RNA helicases

A key aspect of antiviral immunity is the distinction between "self" and "non-self" components. This distinction can be established through the detection of double-stranded RNA (dsRNA), a common sign of viral infection, by cytosolic RNA helicases. Depending on the organism, two major antiviral pathways can be induced by dsRNA helicases: RNA interference (RNAi) and interferon (IFN) signaling. In the RNAi pathway, dsRNAs are recognized by a Dicer protein, and are then used for the sequence-dependent recognition and subsequent degradation of the complementary viral RNAs. In the IFN signaling pathway, dsRNAs are recognized by a RIG-like receptor (RLR), which induces a signaling cascade in order to induce the expression of IFNs, cytokines and chemokines. In this review, we discuss the RNA features that can be used by the cell to detect a viral infection, the two aforementioned types of helicase-mediated sensing, as well as some viral escape mechanisms developed to avoid recognition.

Discovery and Characterisation of Castlerea Virus, a New Species of Negevirus Isolated in Australia.

With advances in sequencing technologies, there has been an increase in the discovery of viruses that do not group with any currently described virus families. The newly described taxon Negevirus encompasses a group of viruses displaying an insect-specific phenotype which have been isolated from multiple host species on numerous continents. Using a broad-spectrum virus screening assay based on the detection of double-stranded RNA and next-generation sequencing, we have detected a novel species of negevirus, from Anopheles, Culex, and Aedes mosquitoes collected in 4 geographically separate regions of Australia. Bioinformatic analysis of the virus, tentatively named Castlerea virus, revealed that it is genetically distinct from previously described negeviruses but clusters in the newly proposed Nelorpivirus clade within this taxon. Analysis of virions confirmed the presence of 2 proteins of 24 and 40 kDa which support previous bioinformatic predictions of negevirus structural proteins.

Toll-Like Receptor Tag Team

Cell Signaling How many receptors does it take to initiate an antiviral response? Yamashita et al. say it's two, plus an additional tyrosine kinase. First, you need Toll-like Receptor 3 (TLR3), which has a well-characterized role in the detection of double-stranded RNA (dsRNA) and initiation of protective cellular responses. Earlier findings showed that TLR3 differs from other TLRs in requiring phosphorylation of two tyrosine residues in its cytoplasmic tail for activation. Here, another receptor is implicated in the activation of antiviral responses by TLR3—the epidermal growth factor receptor (EGFR), which is itself a tyrosine kinase. The EGFR was found to associate with dsRNA-activated TLR3 in endosomal membranes along with another tyrosine kinase, Src. Both EGFR and Src appeared to phosphorylate a particular tyrosine residue in TLR3, and inhibition of the kinases inhibited TLR3-dependent antiviral responses. Thus, antiviral responses may involve interactions between signaling systems controlling cell growth and those mediating innate immune responses. Sci. Signal. 5 , ra50 (2012).

Detection of Double-Stranded RNA in Macrophomina phaseolina

Detection of Double-Stranded RNA in Macrophomina phaseolina

Detection of double-stranded RNA in Macrophomina phaseolina

One hundred ten isolates of Macrophomina phaseolina, the causal agent of charcoal rot of many different plant species, were obtained from Mexico, Somalia, and several locations in the United States, and analyzed for the presence of double-stranded RNA (dsRNA). Twenty-one isolates from the United States and Mexico contained dsRNA elements. The sizes of the dsRNAs ranged from ca 0.4 kbp to 10 kbp and the number of dsRNAs present among the isolates ranged from one to ten. Attempts to detect virus particles by sucrose gradient centrifugation were unsuccessful. Three isolates having dsRNA and three dsRNA-free isolates were evaluated for virulence on sorghum at two different field locations and for mycelial growth in PDA and V8 agar. For both locations, the isolates that had dsRNA were significantly less virulent (P < 0.05) than the dsRNA-free isolates. Differences between locations were not significant (P < 0.05). The contrasts between means of isolates with and without dsRNA suggest a strong association between the presence of dsRNA and a reduction in both mycelial growth and the capacity to cause disease. These results suggest an association between the presence of dsRNA and hypovirulence in the fungus.

Abstracts, The Canadian Phytopathological Society, Annual Meeting, Toronto, Ontario, 25-28 June 1995

Abstracts, The Canadian Phytopathological Society, Annual Meeting, Toronto, Ontario, 25-28 June 1995

Detection of Double‐Stranded RNA (DSRNA) in Crude Extracts of Virus‐Infected Plants by Indirect ELISA

AbstractAn antiserum against polyinosinic‐polycytidylic acid (In‐Cn) was used to detect double‐stranded RNA (dsRNA) by indirect ELISA (ELISA‐I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)‐infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA‐I 50 days after inoculation. ELISA values then obtained with the In‐Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.

Detection of double-stranded RNA in Mucor ramannianus

Double-stranded RNA (dsRNA) molecules have been found in numerous fungal species and are considered to be indicators of the presence of virus-like particles (VLPs) in the cells (Hollings 1978 Adv. Virus Res. 22:1-53; Buck ed., 1986 Fungal Virology, CRC Press, Boca Raton, FL). Such mycoviruses have been found mainly from studies of the nucleic acids of different strains. In this respect, fungal species belonging to the Zygomycetes are rarely screened. We have investigated the nucleic acids of 30 Mucor strains representing ten different species. The presence of dsRNA molecules was detected in only one of the two Mucor ramannianus strains examined.

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

Abstracts of Papers Presented at the Annual Meeting of the Canadian Phytopathological Society Banff, Alberta June 1991

Detection of double-stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by several serological techniques. DsRNA was readily detected by indirect ELISA (ELISA-I) and dot immunobinding assay (DIA). Addition of the antigen to poly- l-lysine-precoated plates and blocking with uncreamed milk powder allowed detection levels of 100 pg · ml −1 In-Cn by ELISA-I. Concentrations as low as 1 ng · ml −1 were detected by DIA using polyvinyliden difluoride (PVDF) membranes. Detection capacity with nitrocellulose membranes was 1000 times lower than with PVDF. ELISA-I and DIA enabled detection of dsRNA in enriched fractions from cucumber mosaic virus (CMV)- and citrus tristeza virus (CTV)-infected plants and from virus-infected Penicillium chrysogenum mycelium. These techniques showed similar or higher sensitivity for detection of dsRNA than separation by polyacrylamide gel electrophoresis and silver staining.

Detection of double-stranded RNA in shake-cultures of Agaricus bisporus affected by ‘La France’ disease

Double-stranded ribonucleic acid (dsRNA) was detected in sporophores, and in mycelial shake-cultures derived from sporophores, produced in a tray inoculated with mushroom material affected by ‘La France’ disease. Cultures from ‘diseased’ sporophores had lower growth rates, in vitro, than cultures from ‘apparently normal’ sporophores, and contained higher concentrations of dsRNA. Up to nine ‘major’ bands of dsRNA were resolved, by gel electrophoresis, in extracts of ‘diseased’ mycelia. No dsRNA was detected in extracts of shake-cultures derived from sporophores produced in uninoculated trays.

Detection of double-stranded RNA by serologically specific electron microscopy

Details of a procedure for detecting double-stranded RNA (dsRNA) in virus and viroid infected tissue extracts using serologically specific electron microscopy are given. A method for staining dsRNA, based on in situ formation of uranyl phosphate, that consistently permits the examination of dsRNA by electron microscopy without shadowing with heavy metals, is described. The method provides for routine assays for dsRNA in crude extracts without the variable results associated with shadowing procedures. DsRNA was found to accumulate in older leaves of sorghum systemically infected with sugarcane mosaic virus. In contrast, dsRNA was not detected in older cowpea leaves systemically infected with cowpea mosaic virus but was readily found in inoculated leaves 4 days post inoculation and young systemically infected leaves.

Detection of double-stranded RNA in Semliki Forest virus-infected cells by an indirect solid phase radioimmunoassay: an assay for interferon.

An indirect solid phase radioimmunoassay using rabbit antibodies to the synthetic double-stranded RNA poly rI-rC and 125I-labelled sheep anti-rabbit IgG could detect 1 ng of poly rI-rC. This assay could also detect the presence of double-stranded RNA in Semliki Forest virus-infected chick fibroblasts and can be used as an assay for interferon activity.

Detection of Double-Stranded RNA from Polyhedral Virus-like Particles in Lentinus edodes (Berk.) Sing

Detection of Double-Stranded RNA from Polyhedral Virus-like Particles in Lentinus edodes (Berk.) Sing