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During cell entry, reovirus particles disassemble to generate ISVPs. ISVPs undergo conformational changes to form ISVP* and this conversion is required for membrane penetration. In tissues where ISVP formation occurs within endoso...
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During cell entry, reovirus particles disassemble to generate ISVPs. ISVPs undergo conformational changes to form ISVP* and this conversion is required for membrane penetration. In tissues where ISVP formation occurs within endosomes, ISVP-to-ISVP* conversion occurs at low pH. In contrast, in tissues where ISVP formation occurs extracellularly, ISVP-to-ISVP* transition occurs at neutral pH. Whether these two distinct pH environments influence the efficiency of cell entry is not known. In this study, we used Ouabain to lower the endosomal pH and determined its effect on reovirus infection. We found that Ouabain treatment blocks reovirus infection. In cells treated with Ouabain, virus attachment, internalization, and ISVP formation were unaffected but the efficiency of ISVP*s formation was diminished. Low pH also diminished the efficiency of ISVP-to-ISVP* conversion in vitro. Thus, the pH of the compartment where ISVP-to-ISVP* conversion takes place may dictate the efficiency of reovirus infection. (C) 2015 Elsevier Inc. All rights reserved.
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Blueberry latent virus (BBLV) was detected in 27 of 95 asymptomatic highbush blueberry trees in a blueberry field in Japan. In situ hybridization showed that the viral RNAs were detected in the palisade mesophyll, spongy mesophyll...
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Blueberry latent virus (BBLV) was detected in 27 of 95 asymptomatic highbush blueberry trees in a blueberry field in Japan. In situ hybridization showed that the viral RNAs were detected in the palisade mesophyll, spongy mesophyll and vascular bundle of leaves. In western blot analysis, an antibody to the viral protein encoded by open reading frame 1 (ORF1) reacted with a 48-kDa protein specific for blueberry trees in which BBLV was detected. Immunogold electron microscopy revealed that amorphous bodies in the cytoplasm of blueberry cells were labeled with antibodies to the ORF1 protein.
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Protists provide insights into the diversity and function of RNA viruses in marine systems. Among them, marine macroalgae are good targets for RNA virome analyses because they have a sufficient biomass in nature. However, RNA viru...
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Protists provide insights into the diversity and function of RNA viruses in marine systems. Among them, marine macroalgae are good targets for RNA virome analyses because they have a sufficient biomass in nature. However, RNA viruses in macroalgae have not yet been examined in detail, and only partial genome sequences have been reported for the majority of RNA viruses. Therefore, to obtain further insights into the distribution and diversity of RNA viruses associated with marine protists, we herein examined RNA viruses in macroalgae and a diatom. We report the putative complete genome sequences of six novel RNA viruses from two marine macroalgae and one diatom holobiont. Four viruses were not classified into established viral genera or families. Furthermore, a virus classified into Totiviridae showed a genome structure that has not yet been reported in this family. These results suggest that a number of distinct RNA viruses are widespread in a broad range of protists.
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Ebolavirus causes a severe hemorrhagic fever and is divided into five distinct species, of which Reston ebolavirus is uniquely nonpathogenic to humans. Disease caused by ebolavirus is marked by early immuno-suppression of innate i...
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Ebolavirus causes a severe hemorrhagic fever and is divided into five distinct species, of which Reston ebolavirus is uniquely nonpathogenic to humans. Disease caused by ebolavirus is marked by early immuno-suppression of innate immune signaling events, involving silencing and sequestration of double-stranded RNA (dsRNA) by the viral protein VP35. Here we present unbound and dsRNA-bound crystal structures of the dsRNA-binding domain of Reston ebolavirus VP35. The structures show that VP35 forms an unusual, asymmetric dimer on dsRNA binding, with each of the monomers binding dsRNA in a different way: one binds the backbone whereas the other caps the terminus. Additional SAXS, DXMS, and dsRNA-binding experiments presented here support a model of cooperative dsRNA recognition in which binding of the first monomer assists binding of the next monomer of the oligomeric VP35 protein. This work illustrates how ebolavirus VP35 could mask key recognition sites of molecules such as RIG-I, MDA-5, and Dicer to silence viral dsRNA in infection.
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Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported pre...
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Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.
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Since the 1970s, several dsRNA viruses, including Radish yellow edge virus, Raphanus sativus virus 1, Raphanus sativus virus 2, and Raphanus sativus virus 3, have been identified and reported as infecting radish. In the present st...
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Since the 1970s, several dsRNA viruses, including Radish yellow edge virus, Raphanus sativus virus 1, Raphanus sativus virus 2, and Raphanus sativus virus 3, have been identified and reported as infecting radish. In the present study, in conjunction with a survey of seed-borne viruses in cultivated Brassica and Raphanus using the dsRNA diagnostic method, we discovered 3 novel cryptoviruses that infect Brassica and Raphanus: Raphanus sativus partitivirus 1, which infects radish (Raphanus sativus); Sinapis alba cryptic virus 1, which infects Sinapis alba; and Brassica rapa cryptic virus 1 (BrCV1), which infects Brassica rapa. The genomic organization of these cryptoviruses was analyzed and characterized. BrCV1 might represent the first plant partitivirus found in Gammapartitivirus. Additionally, the evolutionary relationships among all of the partitiviruses reported in Raphanus and Brassica were analyzed.
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Invertebrates are a source of previously unknown RNA viruses that fill gaps in the viral phylogenetic tree. Although limited information is currently available on RNA viral diversity in the marine sponge, a primordial multicellula...
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Invertebrates are a source of previously unknown RNA viruses that fill gaps in the viral phylogenetic tree. Although limited information is currently available on RNA viral diversity in the marine sponge, a primordial multicellular animal that belongs to the phylum Porifera, the marine sponge is one of the well-studied holobiont systems. In the present study, we elucidated the putative complete genomc sequences of five novel RNA viruses from Hymeniacidon sponge using a combination of double-stranded RNA sequencing. called fragmented and primer ligated dsRNA sequencing, and a conventional transcriptome method targeting single-stranded RNA. We identified highly diverged RNA-dependent RNA polymerase sequences. including a potential novel RNA viral lineage, in the sponge and three viruses presumed to infect sponge cells.
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Knowledge of the distribution and diversity of RNA viruses is still limited in spite of their possible environmental and epidemiological impacts because RNA virus-specific metagenomic methods have not yet been developed. We herein...
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Knowledge of the distribution and diversity of RNA viruses is still limited in spite of their possible environmental and epidemiological impacts because RNA virus-specific metagenomic methods have not yet been developed. We herein constructed an effective metagenomic method for RNA viruses by targeting long double-stranded (ds) RNA in cellular organisms, which is a hallmark of infection, or the replication of dsRNA and single-stranded (ss) RNA viruses, except for retroviruses. This novel dsRNA targeting metagenomic method is characterized by an extremely high recovery rate of viral RNA sequences, the retrieval of terminal sequences, and uniform read coverage, which has not previously been reported in other metagenomic methods targeting RNA viruses. This method revealed a previously unidentified viral RNA diversity of more than 20 complete RNA viral genomes including dsRNA and ssRNA viruses associated with an environmental diatom colony. Our approach will be a powerful tool for cataloging RNA viruses associated with organisms of interest.
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RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limite...
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RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi
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The presence of virus associated with 'La France' disease was confirmed in the mushroom sporophores collected in 2000, on the farm located in Central Poland. Two different types of isometric virus-like particles (diameter 25 and 3...
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The presence of virus associated with 'La France' disease was confirmed in the mushroom sporophores collected in 2000, on the farm located in Central Poland. Two different types of isometric virus-like particles (diameter 25 and 34-35 nm) were observed in electron microscope. After electrophoretic separation of dsRNA isolated from suspected material revealed five characteristic bands. After RT-PCR amplification, a specific PCR product of expected size (about 480 bp) was obtained from samples of infected sporophores. In few cases, a positive RT-PCR reaction was also found for apparently healthy mushrooms, suggesting the high incidence of latent infections with LFD.
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