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PubMed1991

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.

Antibodies↗
PubMed1991

Stability and extractability of double-stranded RNA of pangola stunt and sugarcane Fiji disease viruses in dried plant tissues.

When leaves infected with pangola stunt virus (PaSV) were dried at 23, 37, 50, 70 or 105 degrees C, the dsRNA was stable and could be extracted after aerobic storage at room temperature for 1 month, although at 105 degrees C the amount obtained was reduced. The dsRNA was also recovered after leaves were freeze dried and stored in vacuo at room temperature for 6 months, or were dried and stored aerobically at room temperature for 10.5 months. dsRNA of sugarcane Fiji disease virus (FDV) was also stable when infected leaves were dried at 23, 37, 50 or 105 degrees C and stored aerobically for 3 months or for at least 6 months when infected leaves were dried at 70 degrees C. The unexpected high stability and extractability of both PaSV and FDV dsRNA when dried in leaves at low or high temperatures and stored at room temperature indicate that these, and probably other plant-infecting reoviruses, can be transported readily in desiccated host tissue between different countries for later extraction and comparison of their dsRNAs.

Desiccation↗
PubMed1989

Identification of virus-like particles in Eimeria stiedae.

When nucleic acid samples purified from sporozoites of Eimeria stiedae were analyzed by agarose gel electrophoresis, an ethidium-stainable band with an apparent electrophoretic mobility of 6.5 kb was consistently observed. The band was readily degradable upon RNAse treatment, and its susceptibility towards ribonuclease A on a decreasing ionic strength was suggestive of double-stranded RNA (dsRNA). Electron microscopy revealed spherical, probably icosahedral, virus-like particles (VLP) with a diameter of 35 nm in sporozoite lysates. The VLP were purified by CsCl buoyant density gradient centrifugation. Upon extraction, these particles yielded dsRNA molecules of a uniform length of 1.63 microns. The presence of the VLP was investigated in different Eimeria strains. All E. stiedae isolates contained the RNA virus, whereas the Eimeria intestinalis and Eimeria magna isolates tested did not. RNA/RNA hybridization experiments where the E. stiedae VLP dsRNA was probed to the genomes of the dsRNA viruses of Trichomonas vaginalis and Giardia intestinalis revealed a strong relatedness of the E. stiedae virus to the G. intestinalis virus, in contrast with the T. vaginalis virus, where no homology could be detected.

Animals↗
PubMed1983

Reverse transcription of yeast double-stranded RNA and ribosomal RNA using synthetic oligonucleotide primers.

The ability of the four oligodeoxyribonucleotide primers oligo(dT)12-18, oligo(dA)12-18, oligo(dG)12-18 and oligo(dC)12-18 to act as primers for avian myeloblastosis virus reverse transcriptase on denatured yeast double-stranded (ds) RNA templates was investigated. Oligo(dT) and oligo(dA) were found to prime the synthesis of 1.1 and 1.0 kb reverse transcripts, respectively, using denatured M dsRNA as a template. The oligo(dT)- and oligo(dA)-primed cDNAs of M dsRNA hybridized to the region of the M dsRNA that encoded the killer toxin and to each other. Addition of oligo(dT) to reverse transcription reactions of denatured L dsRNA produced a 4.3 kb cDNA. During the course of this investigation oligo(dC) was observed to be a highly efficient primer for reverse transcription of yeast 18 S ribosomal RNA. Oligo(dC) primed the synthesis of a 1.0 kb transcript of 18 S rRNA which hybridized to the large Eco RI fragment of the 18 S rRNA gene. Reverse transcription of double-stranded RNA and 25 S ribosomal RNA was found to occur to some extent in the absence of added oligonucleotide primer.

Avian Myeloblastosis Virus↗
PubMed1985

In vitro translation of the major capsid polypeptide from Ustilago maydis virus stain P1.

Double-stranded RNA (dsRNA) from Ustilago maydis virus strain P1 was translated in vitro using a nuclease-treated rabbit reticulocyte lysate system. Following heat denaturation of the H2 double-stranded RNA segment in 90% dimethyl sulfoxide and incubation in the cell free extract, a primary translation product was observed which showed the same molecular weight and co-migrated with viral coat protein on 10% SDS-polyacrylamide gels. The in vitro product of the H2 dsRNA segment could also be immunoprecipitated with antibodies prepared against viral coat protein. Limited proteolysis of the in vitro product and authentic viral coat protein using Staphylococcus aureus V8 protease produced similar peptide patterns on SDS gels. In vitro translation products from other dsRNA segments that make up the P1 viral genome could not be precipitated by antibody to viral coat protein. These results complement the genetic data that indicated that information for coat formation and maintenance was contained within the H segments of dsRNA.

Animals↗
PubMed1990

Evidence for equimolar synthesis of double-strand RNA and minus-strand RNA in rotavirus-infected cells.

The genome of the rotaviruses consists of eleven segments of double-strand RNA (dsRNA). Each segment is replicated asymmetrically with viral plus-strand RNA, i.e. messenger (m)RNA, serving as the template for the synthesis of minus-strand RNA to produce dsRNA. To examine the relative frequency of replication of each of the eleven genome segments, MA104 cells were infected with low (3rd) and high (12th) passage stocks of simian rotavirus SA11. The total cytoplasmic RNA of the infected cell was radiolabeled either by maintaining the infected cells in the presence [3H]uridine prior to harvest or by 3'-endlabeling the purified RNA with [32P]pCp and T4 RNA ligase. The RNA was then analyzed for the presence of 3H- and 32P-labeled dsRNA by electrophoresis on 10% polyacrylamide gels. Total cytoplasmic RNA from infected cells was also 3'-end-labeled with [32P]pCp and T4 RNA ligase and examined for the presence of minus-strand RNA by electrophoresis on low pH agarose-urea gels. Bands representing dsRNAs and minus-strand RNAs on autoradiographs of the gels were analyzed for intensity by densitometry. The results showed that the eleven segments of viral dsRNA were present in equimolar concentrations in cells either infected with low or high passage stocks of virus. Like intracellular dsRNAs, full-length minus-strand RNAs were also present in equimolar concentration in cells either infected with low or high passage rotavirus. These data indicate that, despite the non-equimolar levels of viral RNAs in the cell, the eleven genome segments of rotavirus are replicated with equal frequencies in vivo.

Animals↗
PubMed1995

Sequences at four termini of the giardiavirus double-stranded RNA.

The linear double-stranded RNA (dsRNA) genome of giardiavirus (GLV) was estimated to be 6100 nucleotides (nt) [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 8585-8599]. As the 5'- and 3'-untranslated regions (UTR) of viral genomes are known to contain critical information for viral replication, we reexamine the sequences at all four termini of GLV dsRNA by (i) direct RNA sequencing with RT, (ii) tailing GLV dsRNA with UTP or CTP in addition to ATP in 3' rapid amplification of cDNA ends (RACE) and (iii) adding poly(dG) to the products of primer extension in 5'-RACE. The results confirmed the reported sequence for the 5'-terminus of the GLV sense strand RNA, but uncovered an additional 177 nt at the 3'-terminus. The new study also showed conclusively that there are no protruding overhangs at either terminus of GLV dsRNA.

Base Sequence↗
PubMed2004

Small interfering RNA molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism.

RNA silencing or interference (RNAi) is a sequence-specific, post-transcriptional process of mRNA degradation. The degradation of target gene mRNA can be induced by short dsRNA molecules (21-25-nt) corresponding to the sequence of the target gene to be silenced. Short dsRNA molecules have been shown to be very effective in inducing RNA silencing in several human cell lines. In this study, we have shown that short dsRNA molecules corresponding to the human rhinovirus-16 (HRV-16) genome induce effective inhibition of the viral replication in cell culture. This inhibition is sequence-specific and dose-dependent. A single or double nucleotide sequence change in an effective dsRNA molecule can significantly reduce the ability of the molecule to induce RNA silencing. Reducing the length of siRNA molecules to 19-nt or shorter abolishes their activity. Therefore, the results of this study demonstrate certain siRNA molecules are inhibitory for the replication of HRV-16 when transfected into human cells; further studies are warranted to explore the potential clinical value of these siRNA molecules as anti-human rhinovirus agents.

Antiviral Agents↗
PubMed2006

Double-stranded RNA and antiviral immunity in marine shrimp: inducible host mechanisms and evidence for the evolution of viral counter-responses.

Double-stranded RNA (dsRNA) is a common virus-associated molecular pattern and a potent inducer of antiviral responses in many organisms. While it is clear that the specific RNA interference (RNAi) response, a phenomenon triggered by dsRNA, serves antiviral functions in invertebrates, innate (non-specific) antiviral immune reactions induced by dsRNA (e.g. the Interferon response) have long been thought to be restricted to vertebrates. Recent work in an underappreciated experimental model, the penaeid shrimp, is challenging these traditional distinctions, by demonstrating the existence of both innate (non sequence-specific) and RNAi-related (sequence-specific) antiviral phenomena in crustacea. Here we discuss the evidence for this bivalent role of dsRNA in the initiation of antiviral responses in shrimp, and present new data that suggest that the antiviral functions of the shrimp RNAi machinery have imposed selective pressures on an evolving viral pathogen. These findings open the door for the discovery of novel mechanisms of innate immunity, and provide a basis for the future development of strategies to control viral diseases in the commercially important penaeid shrimp.

Animals↗
PubMed2005

Comparison between Rosellinia necatrix isolates from soil and diseased roots in terms of hypovirulence.

The white root rot fungus, Rosellinia necatrix, is a devastating soil-borne pathogen of many plant species. Biocontrol with the hypovirulence factor is promising, but disease symptoms, signs or culture morphology of the pathogen cannot be reliably used as markers for hypovirulence in this fungus. We attempted to obtain hypovirulent isolates from soil rather than from diseased roots, based on the hypothesis that hypovirulent isolates were more likely to persist in soil as saprobes. Sixteen isolates, belonging to eight mycelial compatibility groups (MCGs), were obtained from soil in two active and one abandoned Japanese pear orchards. Comparison of these isolates based on clonality revealed that six MCGs were commonly recovered from both diseased roots and soil and two MCGs exclusively from soil. No MCG was found in more than one orchard. With two exceptions, isolates within the same MCG were similar in virulence, competitive saprophytic ability (CSA) and mycelial growth rate whether or not they carried dsRNA. The two exceptional isolates recovered from soil had multiple dsRNA segments that caused hypovirulence, weakened CSA and restricted mycelial growth on nutrient-rich media. They belonged to different MCGs, each including dsRNA-free isolates. Isolates from soil contained various dsRNAs (44%), including the hypovirulence factor, more frequently than isolates from diseased roots in the same fields (25%), which is much higher than the proportion of isolates with dsRNA from diseased roots (19%) in a total of 424 isolates from Japan examined so far. These results suggest that isolation of R. necatrix from soil is an effective method to obtain isolates with dsRNAs, including the hypovirulence factor.

Ascomycota↗
PubMed2006

Role of cytosolic malate dehydrogenase in oocyte maturation and embryo development.

OBJECTIVE: To elucidate the function of cytosolic malate dehydrogenase (Mor2) in oocyte maturation and embryo development using RNA interference (RNAi). DESIGN: Experimental animal study. SETTING: Research unit of university. ANIMAL(S): Female 4-week-old (C57/BL6) mice. INTERVENTION(S): Isolation of immature germinal vesicle (GV) oocytes or fertilized pronucleus (PN) embryos, microinjection of Mor2 double-stranded RNA (dsRNA), and reverse transcription and polymerase chain reaction (RT-PCR) analysis to investigate Mor2-specific messenger RNA (mRNA) knockdown. MAIN OUTCOME MEASURE(S): Relative changes in mRNA levels after microinjection of Mor2 dsRNA and in rates of oocyte maturation and preimplantation embryo development. RESULT(S): Mor2 mRNA mostly was knocked down in germinal vesicle- and metaphase I (MI)-arrested oocytes, compared with metaphase II (MII)-developed oocytes, after microinjection of Mor2 dsRNA and in vitro culture for 16 hours. In vitro oocyte maturation was significantly decreased (34%), compared with noninjected (73.4%) and buffer-injected (67.5%) control groups. The rate of blastocyst development (48.1%) was lower in the Mor2 dsRNA-injected group than in buffer-injected control (88.2%). CONCLUSION(S): In the present study, the function of Mor2 was analyzed with the aid of RNAi. On the basis of the data obtained, we propose that Mor2 is an essential factor for oocyte maturation and embryo development in mouse.

Animals↗
PubMed2004

Using RNA interference to develop dengue virus resistance in genetically modified Aedes aegypti.

Diseases caused by arthropod-borne viruses are significant public health problems, and novel methods are needed to control pathogen transmission. We hypothesize that genetic manipulation of Aedes aegypti mosquitoes can profoundly and permanently reduce vector competence and subsequent transmission of dengue viruses (DENV) to human hosts. We have identified RNA interference (RNAi) as a potential anti-viral, intracellular pathway in the vector that can be triggered by expression of virus-specific, double stranded RNAs (dsRNAs) to reduce vector competence to DENV. We identified DENV-derived RNA segments using recombinant Sindbis viruses to trigger RNAi, that when expressed in mosquitoes ablate homologous DENV replication and transmission. We also demonstrated that heritable expression of DENV-derived dsRNA in cultured mosquito cells can silence virus replication. We now have developed a number of transgenic mosquito lines that transcribe the effector dsRNA from constitutive promoters such as immediate early 1 (baculovirus) and polyubiquitin (Drosophila melanogaster). We have detected DENV-specific small interfering RNAs, the hallmark of RNAi, in at least one of these lines. Surprisingly, none of these lines expressed dsRNA in relevant tissues (e.g., midguts) that will ultimately affect transmission. A major challenge now is to express the effector dsRNA from tissue-specific promoters to allow RNAi to silence virus replication at critical sites in the vector such as midguts and salivary glands. If successful, this strategy has the advantage of harnessing a naturally occurring vector response to block DENV infection in a mosquito vector and profoundly affect virus transmission.

Aedes↗
PubMed2006

Functional analysis of the allatostatin-A type gene in the cricket Gryllus bimaculatus and the armyworm Spodoptera frugiperda.

Double-stranded RNA (dsRNA) gene interference is an efficient method to silence gene expression in a sequence specific manner. Here we show, that dsRNA targeting the allatostatin (AS)-A type (FGL/I/V-amide) gene of Gryllus bimaculatus (Ensifera, Gryllidae) and Spodoptera frugiperda (Lepidoptera, Noctuidae) injected into freshly moulted larvae or adult crickets and moths produced a rapid and long-lasting reduction in the mRNA levels in various tissues. The effect lasted up to 7 days. Following dsRNA injection, the juvenile hormone (JH) titers in the hemolymph were clearly raised in both species. AS-dsRNA injection induced a reduced body weight in larval and adult crickets and the imaginal moult was incomplete. Silencing allatostatin type-A expression also reduced the egg and testes development in crickets, and the oviposition rate was drastically diminished in both species.

Animals↗
PubMed2006

Toll-like receptor 2, 3, and 4 expression and function in human airway smooth muscle.

BACKGROUND: Host defense against microbial pathogens is elicited through the innate immune system by means of Toll-like receptors (TLRs). Airway smooth muscle cells (ASMCs) display proinflammatory and immunomodulatory functions. ASMCs might participate in airway inflammatory responses associated with innate immune activation. OBJECTIVES: We determined the effects of cytokines, TLR ligands, and corticosteroids on TLR expression and function in human ASMCs. METHODS: Real-time PCR and flow cytometry were used to assess TLR mRNA and protein expression, respectively. ASMCs were stimulated with TLR ligands, and chemokine release was measured by means of ELISA. RESULTS: ASMCs expressed TLR1 to TLR10 mRNA, and TLR2 and TLR3 protein expression was demonstrated. TNF-alpha and double-stranded RNA (dsRNA; TLR3 ligand) were potent inducers of TLR2 and TLR3 mRNA expression, and both stimuli had additive or synergistic effects with IFN-gamma on TLR2 and TLR4, but not TLR3, mRNA expression. Peptidoglycan (TLR2 ligand) and LPS (TLR4 ligand) weakly enhanced TLR2 mRNA expression. Peptidoglycan, dsRNA, and LPS induced IL-8 and eotaxin release, with dsRNA being most potent. dsRNA also modulated cytokine-induced chemokine release in a differential manner. Dexamethasone inhibited cytokine- and ligand-induced TLR2, TLR3, and TLR4 expression and chemokine release. However, dexamethasone potentiated TLR2 expression induced by combined IFN-gamma and TNF-alpha stimulation. CONCLUSION: Expression of TLR2, TLR3, and TLR4 is regulated by cytokines and TLR ligands, and their activation mediates chemokine release in ASMCs. CLINICAL IMPLICATIONS: Proinflammatory responses mediated by activation of pathogen-recognition receptors in ASMCs might contribute to infectious exacerbations of airway inflammatory conditions, such as asthma and chronic obstructive pulmonary disease.

Bronchi↗
PubMed2005

Myostatin gene silenced by RNAi show a zebrafish giant phenotype.

Myostatin is a member of the transforming growth factor-beta (TGF-beta) family that functions as a negative regulator of skeletal muscle development and growth. Recently, it has been reported that the transgenic zebrafish expressing myostatin prodomain exhibited an increased number of fiber in skeletal muscle. Other novel results suggest that myostatin plays a mayor role during myogenesis, apart from inhibition of proliferation as well as differentiation. We have investigated the ability of double-stranded RNA (dsRNA) to inhibit myostatin function in the zebrafish. By microinjection dsRNA, corresponding to biologically active C-terminal domain from aminoacid 268 to end codon of tilapia myostatin protein, we produced an increased body mass in treated fish. The dsRNA injection in early development stage in zebrafish produced hyperplasia or hypertrophy. In addition, the interference of gene function showed a strong dependence on the amount of dsRNA.

Animals↗
PubMed2005

Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth.

Schistosoma mansoni is an important flatworm parasite of man that has remained intractable to experimental analyses of gene function. We have developed an approach for using dsRNA to target schistosome transcripts for RNA interference, and used it to address the role of cathepsin B (SmCB1), a cysteine protease that has been proposed to play a central role in hemoglobin digestion in the schistosome gut. Electroporation of 3 h old larval schistosomes with SmCB1-specific dsRNA (SmCB1-dsRNA) resulted in a greater than 10-fold reduction in SmCB1 transcript levels that persisted for >20 days. RNAi mediated reductions in transcript levels led to associated reductions in SmCB1 enzyme activity. Schistosomes treated with SmCB1-dsRNA were viable and developed intestinal heme pigmentation indicative of hemoglobin digestion, but showed significant growth retardation when compared to control parasites, indicating that SmCB1 function is not essential for hemoglobin digestion but is necessary for normal parasite growth. This effect on growth was apparent when parasites were maintained in culture or introduced into mammalian hosts. The report sheds new light on the role of SmCB1 and provides a template for using RNAi to examine gene function in the mammal-parasitic stages of schistosomes during early development in vitro and in vivo.

Animals↗
PubMed2006

Activation of PKR: an open and shut case?

The double-stranded (ds) RNA-activated protein kinase, PKR, has a key role in the innate immunity response to viral infection in higher eukaryotes. PKR contains an N-terminal dsRNA-binding domain and a C-terminal kinase domain. In the prevalent autoinhibition model for PKR activation, dsRNA binding induces a conformational change that leads to the release of the dsRNA-binding domain from the kinase, thus relieving the inhibition of the latent enzyme. Structural and biophysical data now favor a model whereby dsRNA principally functions to induce dimerization of PKR via the kinase domain. This dimerization model has implications for other PKR regulatory mechanisms and represents a new structural paradigm for control of protein kinase activity.

Animals↗
PubMed2006

Intergeneric relationship between the Aspergillus ochraceous virus F and the Penicillium stoloniferum virus S.

It was reported that the "slow" component (PsV-S) of Penicillium stoloniferum virus complex also occurred in a second genus, Aspergillus ochraceous. The responsible virus for this intergeneric occurrence was considered to be the "fast" component (AoV-F) of A. ochraceous virus complex. In this investigation, AoV dsRNA 1, that was previously shown to cross-hybridize with PsV-S dsRNA, has been cloned. It was 1754 bp in length and contained one open reading frame of 539 amino acids (p63), had the same genome organization as PsV-S dsRNA S1 and also had the conserved sequence motif of the PsV-S dsRNAs (5'-GCGCAAAA-3') at the 5' terminus. A BLAST search indicated that p63 was a putative dsRNA-dependent RNA polymerase (RdRp), had 81% of sequence homology to members of the genus Partitivirus, and grouped together with PsV-S in phylogenetic analysis. But immunoblot analysis showed that the capsid protein (P3) of AoV-F virus component did not reacted against PsV-S antiserum. These evidences suggest that the cross serological relationship between AoV-F and PsV-S previously observed may have been due to the RdRps of the respective viruses rather than between their respective capsid proteins as was assumed in 1985.

Aspergillus ochraceus↗