WorldWideScience

Sample records for single-host rna viruses

  1. Sensing of RNA viruses

    DEFF Research Database (Denmark)

    Jensen, Søren; Thomsen, Allan Randrup

    2012-01-01

    Our knowledge regarding the contribution of the innate immune system in recognizing and subsequently initiating a host response to an invasion of RNA virus has been rapidly growing over the last decade. Descriptions of the receptors involved and the molecular mechanisms they employ to sense viral...... pathogen-associated molecular patterns have emerged in great detail. This review presents an overview of our current knowledge regarding the receptors used to detect RNA virus invasion, the molecular structures these receptors sense, and the involved downstream signaling pathways....

  2. RNA viruses in the sea.

    Science.gov (United States)

    Lang, Andrew S; Rise, Matthew L; Culley, Alexander I; Steward, Grieg F

    2009-03-01

    Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.

  3. RNA Viruses: ROS-Mediated Cell Death

    Science.gov (United States)

    Reshi, Mohammad Latif; Su, Yi-Che; Hong, Jiann-Ruey

    2014-01-01

    Reactive oxygen species (ROS) are well known for being both beneficial and deleterious. The main thrust of this review is to investigate the role of ROS in ribonucleic acid (RNA) virus pathogenesis. Much evidences has accumulated over the past decade, suggesting that patients infected with RNA viruses are under chronic oxidative stress. Changes to the body's antioxidant defense system, in relation to SOD, ascorbic acid, selenium, carotenoids, and glutathione, have been reported in various tissues of RNA-virus infected patients. This review focuses on RNA viruses and retroviruses, giving particular attention to the human influenza virus, Hepatitis c virus (HCV), human immunodeficiency virus (HIV), and the aquatic Betanodavirus. Oxidative stress via RNA virus infections can contribute to several aspects of viral disease pathogenesis including apoptosis, loss of immune function, viral replication, inflammatory response, and loss of body weight. We focus on how ROS production is correlated with host cell death. Moreover, ROS may play an important role as a signal molecule in the regulation of viral replication and organelle function, potentially providing new insights in the prevention and treatment of RNA viruses and retrovirus infections. PMID:24899897

  4. Newly discovered insect RNA viruses in China.

    Science.gov (United States)

    Qiu, Yang; Wang, ZhaoWei; Liu, YongXiang; Qi, Nan; Si, Jie; Xiang, Xue; Xia, XiaoLing; Hu, YuanYang; Zhou, Xi

    2013-08-01

    Insects are a group of arthropods and the largest group of animals on Earth, with over one million species described to date. Like other life forms, insects suffer from viruses that cause disease and death. Viruses that are pathogenic to beneficial insects cause dramatic economic losses on agriculture. In contrast, viruses that are pathogenic to insect pests can be exploited as attractive biological control agents. All of these factors have led to an explosion in the amount of research into insect viruses in recent years, generating impressive quantities of information on the molecular and cellular biology of these viruses. Due to the wide variety of insect viruses, a better understanding of these viruses will expand our overall knowledge of their virology. Here, we review studies of several newly discovered RNA insect viruses in China.

  5. Functional RNA during Zika virus infection

    NARCIS (Netherlands)

    Göertz, Giel P.; Abbo, Sandra R.; Fros, Jelke J.; Pijlman, Gorben P.

    2017-01-01

    Zika virus (ZIKV; family Flaviviridae; genus Flavivirus) is a pathogenic mosquito-borne RNA virus that currently threatens human health in the Americas, large parts of Asia and occasionally elsewhere in the world. ZIKV infection is often asymptomatic but can cause severe symptoms including

  6. The RNA synthesis machinery of negative-stranded RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

    2015-05-15

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

  7. The RNA synthesis machinery of negative-stranded RNA viruses

    International Nuclear Information System (INIS)

    Ortín, Juan; Martín-Benito, Jaime

    2015-01-01

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes

  8. How RNA viruses maintain their genome integrity.

    Science.gov (United States)

    Barr, John N; Fearns, Rachel

    2010-06-01

    RNA genomes are vulnerable to corruption by a range of activities, including inaccurate replication by the error-prone replicase, damage from environmental factors, and attack by nucleases and other RNA-modifying enzymes that comprise the cellular intrinsic or innate immune response. Damage to coding regions and loss of critical cis-acting signals inevitably impair genome fitness; as a consequence, RNA viruses have evolved a variety of mechanisms to protect their genome integrity. These include mechanisms to promote replicase fidelity, recombination activities that allow exchange of sequences between different RNA templates, and mechanisms to repair the genome termini. In this article, we review examples of these processes from a range of RNA viruses to showcase the diverse approaches that viruses have evolved to maintain their genome sequence integrity, focusing first on mechanisms that viruses use to protect their entire genome, and then concentrating on mechanisms that allow protection of the genome termini, which are especially vulnerable. In addition, we discuss examples in which it might be beneficial for a virus to 'lose' its genomic termini and reduce its replication efficiency.

  9. RNA polymerase activity of Ustilago maydis virus

    Energy Technology Data Exchange (ETDEWEB)

    Yie, S.W.

    1986-01-01

    Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

  10. Inhibition of virus replication by RNA interference

    NARCIS (Netherlands)

    Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

    2003-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

  11. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

    Science.gov (United States)

    Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

    2015-12-01

    Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

  12. Analysis of intermolecular RNA-RNA recombination by rubella virus

    International Nuclear Information System (INIS)

    Adams, Sandra D.; Tzeng, W.-P.; Chen, M.-H.; Frey, Teryl K.

    2003-01-01

    To investigate whether rubella virus (RUB) undergoes intermolecular RNA-RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5'-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3'-proximal structural protein (SP) ORF, and maintained the 3' end of the genome, including the putative 3' cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3' half of the genome including the SP-ORF and 3' CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA-RNA recombination. Using transcripts tagged with a 3'-terminal deletion, it was found that recombinants contained the 3' end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3' CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3' CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3' end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating

  13. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Lloyd, Richard E., E-mail: rlloyd@bcm.edu

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  14. ER stress, autophagy, and RNA viruses

    Directory of Open Access Journals (Sweden)

    Jia-Rong eJheng

    2014-08-01

    Full Text Available Endoplasmic reticulum (ER stress is a general term for representing the pathway by which various stimuli affect ER functions. ER stress induces the evolutionarily conserved signaling pathways, called the unfolded protein response (UPR, which compromises the stimulus and then determines whether the cell survives or dies. In recent years, ongoing research has suggested that these pathways may be linked to the autophagic response, which plays a key role in the cell’s response to various stressors. Autophagy performs a self-digestion function, and its activation protects cells against certain pathogens. However, the link between the UPR and autophagy may be more complicated. These two systems may act dependently, or the induction of one system may interfere with the other. Experimental studies have found that different viruses modulate these mechanisms to allow them to escape the host immune response or, worse, to exploit the host’s defense to their advantage; thus, this topic is a critical area in antiviral research. In this review, we summarize the current knowledge about how RNA viruses, including influenza virus, poliovirus, coxsackievirus, enterovirus 71, Japanese encephalitis virus, hepatitis C virus, and dengue virus, regulate these processes. We also discuss recent discoveries and how these will produce novel strategies for antiviral treatment.

  15. Purification and properties of cowpea mosaic virus RNA replicase

    NARCIS (Netherlands)

    Zabel, P.

    1978-01-01

    This thesis concerns the partial purification and properties of an RNA-dependent RNA polymerase (RNA replicase) produced upon infection of Vigna unguiculata plants with Cowpea Mosaic Virus (CPMV). The enzyme is believed to be coded, at least in part, by the virus genome and to

  16. RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

    NARCIS (Netherlands)

    Dorssers, L.

    1983-01-01

    The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.

    Previously, an RNA-dependent RNA polymerase produced upon infection of

  17. FLDS: A Comprehensive dsRNA Sequencing Method for Intracellular RNA Virus Surveillance.

    Science.gov (United States)

    Urayama, Syun-Ichi; Takaki, Yoshihiro; Nunoura, Takuro

    2016-01-01

    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.

  18. Single particle labeling of RNA virus in live cells.

    Science.gov (United States)

    Liu, Xiaohui; Ouyang, Ting; Ouyang, Hongsheng; Ren, Linzhu

    2017-06-02

    Real-time and visual tracking of viral infection is crucial for elucidating the infectious and pathogenesis mechanisms. To track the virus successfully, an efficient labeling method is necessary. In this review, we first discuss the practical labeling techniques for virus tracking in live cells. We then describe the current knowledge of interactions between RNA viruses (especially influenza viruses, immunodeficiency viruses, and Flaviviruses) and host cellular structures, obtained using single particle labeling techniques combined with real-time fluorescence microscopy. Single particle labeling provides an easy system for understanding the RNA virus life cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Highly activated RNA silencing via strong induction of dicer by one virus can interfere with the replication of an unrelated virus

    Science.gov (United States)

    Chiba, Sotaro; Suzuki, Nobuhiro

    2015-01-01

    Viruses often coinfect single host organisms in nature. Depending on the combination of viruses in such coinfections, the interplay between them may be synergistic, apparently neutral with no effect on each other, or antagonistic. RNA silencing is responsible for many cases of interference or cross-protection between viruses, but such antagonistic interactions are usually restricted to closely related strains of the same viral species. In this study, we present an unprecedented example of RNA silencing-mediated one-way interference between unrelated viruses in a filamentous model fungus, Cryphonectria parasitica. The replication of Rosellinia necatrix victorivirus 1 (RnVV1; Totiviridae) was strongly impaired by coinfection with the prototypic member of the genus Mycoreovirus (MyRV1) or a mutant of the prototype hypovirus (Cryphonectria hypovirus 1, CHV1) lacking the RNA silencing suppressor (CHV1-Δp69). This interference was associated with marked transcriptional induction of key genes in antiviral RNA silencing, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), following MyRV1 or CHV1-Δp69 infection. Interestingly, the inhibition of RnVV1 replication was reproduced when the levels of dcl2 and agl2 transcripts were elevated by transgenic expression of a hairpin construct of an endogenous C. parasitica gene. Disruption of dcl2 completely abolished the interference, whereas that of agl2 did not always lead to its abolishment, suggesting more crucial roles of dcl2 in antiviral defense. Taken altogether, these results demonstrated the susceptible nature of RnVV1 to the antiviral silencing in C. parasitica activated by distinct viruses or transgene-derived double-stranded RNAs and provide insight into the potential for broad-spectrum virus control mediated by RNA silencing. PMID:26283371

  20. Phomopsis longicolla RNA virus 1 - Novel virus at the edge of myco- and plant viruses.

    Science.gov (United States)

    Hrabáková, Lenka; Koloniuk, Igor; Petrzik, Karel

    2017-06-01

    The complete nucleotide sequence of a new RNA mycovirus in the KY isolate of Phomopsis longicolla Hobbs 1985 and its protoplasts subcultures p5, p9, and ME711 was discovered. The virus, provisionally named Phomopsis longicolla RNA virus 1 (PlRV1), was localized in mitochondria and was determined to have a genome 2822 nucleotides long. A single open reading frame could be translated in silico by both standard and mitochondrial genetic codes into a product featuring conservative domains for an RNA-dependent RNA polymerase (RdRp). The RdRp of PlRV1 has no counterpart among mycoviruses, but it is about 30% identical with the RdRp of plant ourmiaviruses. Recently, new mycoviruses related to plant ourmiaviruses and forming one clade with PlRV1 have been discovered. This separate clade could represent the crucial link between plant and fungal viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Viral counterdefense on RNA silencing : analysis of RNA silencing suppressors from arthropod-borne negative strand RNA plant viruses

    NARCIS (Netherlands)

    Schnettler, E.

    2010-01-01

    This thesis describes that RNA silencing suppressor (RSS) proteins encoded by negative-stranded RNA plant viruses are able to interfere with different RNA silencing pathways in a variety of organisms by interacting with double stranded (ds)RNA molecules. These RSS proteins are able to counteract the

  2. Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

    Science.gov (United States)

    Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

    2015-09-01

    Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN

  3. RNA-virus proteases counteracting host innate immunity.

    Science.gov (United States)

    Lei, Jian; Hilgenfeld, Rolf

    2017-10-01

    Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen-associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern-recognition receptors of the host, and innate immune responses are induced. Through production of type-I and type-III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive-sense single-stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses. © 2017 Federation of European Biochemical Societies.

  4. Assembly of large icosahedral double-stranded RNA viruses.

    Science.gov (United States)

    Poranen, Minna M; Bamford, Dennis H

    2012-01-01

    Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.

  5. Sequence analysis of L RNA of Lassa virus

    International Nuclear Information System (INIS)

    Vieth, Simon; Torda, Andrew E.; Asper, Marcel; Schmitz, Herbert; Guenther, Stephan

    2004-01-01

    The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218-2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043-1546) could be assigned a function, namely that of an RNA polymerase. Secondary structure predictions suggest that this domain is very similar to RNA-dependent RNA polymerases of known structure encoded by plus-strand RNA viruses, permitting a model to be built. Outside the polymerase region, there is little structural data, except for regions of strong alpha-helical content and probably a coiled-coil domain at the N terminus. No evidence for reassortment or recombination during Lassa virus evolution was found. The secondary structure-assisted alignment of the RNA polymerase region permitted a reliable reconstruction of the phylogeny of all negative-strand RNA viruses, indicating that Arenaviridae are most closely related to Nairoviruses. In conclusion, the data provide a basis for structural and functional characterization of the Lassa virus L protein and reveal new insights into the phylogeny of negative-strand RNA viruses

  6. Arthropods as a source of new RNA viruses.

    Science.gov (United States)

    Bichaud, L; de Lamballerie, X; Alkan, C; Izri, A; Gould, E A; Charrel, R N

    2014-12-01

    The discovery and development of methods for isolation, characterisation and taxonomy of viruses represents an important milestone in the study, treatment and control of virus diseases during the 20th century. Indeed, by the late-1950s, it was becoming common belief that most human and veterinary pathogenic viruses had been discovered. However, at that time, knowledge of the impact of improved commercial transportation, urbanisation and deforestation, on disease emergence, was in its infancy. From the late 1960s onwards viruses, such as hepatitis virus (A, B and C) hantavirus, HIV, Marburg virus, Ebola virus and many others began to emerge and it became apparent that the world was changing, at least in terms of virus epidemiology, largely due to the influence of anthropological activities. Subsequently, with the improvement of molecular biotechnologies, for amplification of viral RNA, genome sequencing and proteomic analysis the arsenal of available tools for virus discovery and genetic characterization opened up new and exciting possibilities for virological discovery. Many recently identified but "unclassified" viruses are now being allocated to existing genera or families based on whole genome sequencing, bioinformatic and phylogenetic analysis. New species, genera and families are also being created following the guidelines of the International Committee for the Taxonomy of Viruses. Many of these newly discovered viruses are vectored by arthropods (arboviruses) and possess an RNA genome. This brief review will focus largely on the discovery of new arthropod-borne viruses. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. RNA Base Pairing Determines the Conformations of RNA Inside Spherical Viruses

    Science.gov (United States)

    Erdemci-Tandogan, Gonca; Orland, Henri; Zandi, Roya

    2017-11-01

    Many simple RNA viruses enclose their genetic material by a protein shell called the capsid. While the capsid structures are well characterized for most viruses, the structure of RNA inside the shells and the factors contributing to it remain poorly understood. We study the impact of base pairing on the conformations of RNA and find that it undergoes a swollen coil to globule continuous transition as a function of the strength of the pairing interaction. We also observe a first order transition and kink profile as a function of RNA length. All these transitions could explain the different RNA profiles observed inside viral shells.

  8. Evolution of mutational robustness in an RNA virus.

    Directory of Open Access Journals (Sweden)

    Rebecca Montville

    2005-11-01

    Full Text Available Mutational (genetic robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage phi6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.

  9. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses

    Science.gov (United States)

    Li, Ci-Xiu; Shi, Mang; Tian, Jun-Hua; Lin, Xian-Dan; Kang, Yan-Jun; Chen, Liang-Jun; Qin, Xin-Cheng; Xu, Jianguo; Holmes, Edward C; Zhang, Yong-Zhen

    2015-01-01

    Although arthropods are important viral vectors, the biodiversity of arthropod viruses, as well as the role that arthropods have played in viral origins and evolution, is unclear. Through RNA sequencing of 70 arthropod species we discovered 112 novel viruses that appear to be ancestral to much of the documented genetic diversity of negative-sense RNA viruses, a number of which are also present as endogenous genomic copies. With this greatly enriched diversity we revealed that arthropods contain viruses that fall basal to major virus groups, including the vertebrate-specific arenaviruses, filoviruses, hantaviruses, influenza viruses, lyssaviruses, and paramyxoviruses. We similarly documented a remarkable diversity of genome structures in arthropod viruses, including a putative circular form, that sheds new light on the evolution of genome organization. Hence, arthropods are a major reservoir of viral genetic diversity and have likely been central to viral evolution. DOI: http://dx.doi.org/10.7554/eLife.05378.001 PMID:25633976

  10. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses.

    Science.gov (United States)

    Li, Ci-Xiu; Shi, Mang; Tian, Jun-Hua; Lin, Xian-Dan; Kang, Yan-Jun; Chen, Liang-Jun; Qin, Xin-Cheng; Xu, Jianguo; Holmes, Edward C; Zhang, Yong-Zhen

    2015-01-29

    Although arthropods are important viral vectors, the biodiversity of arthropod viruses, as well as the role that arthropods have played in viral origins and evolution, is unclear. Through RNA sequencing of 70 arthropod species we discovered 112 novel viruses that appear to be ancestral to much of the documented genetic diversity of negative-sense RNA viruses, a number of which are also present as endogenous genomic copies. With this greatly enriched diversity we revealed that arthropods contain viruses that fall basal to major virus groups, including the vertebrate-specific arenaviruses, filoviruses, hantaviruses, influenza viruses, lyssaviruses, and paramyxoviruses. We similarly documented a remarkable diversity of genome structures in arthropod viruses, including a putative circular form, that sheds new light on the evolution of genome organization. Hence, arthropods are a major reservoir of viral genetic diversity and have likely been central to viral evolution.

  11. RNA Viruses and RNAi: Quasispecies Implications for Viral Escape

    Directory of Open Access Journals (Sweden)

    John B. Presloid

    2015-06-01

    Full Text Available Due to high mutation rates, populations of RNA viruses exist as a collection of closely related mutants known as a quasispecies. A consequence of error-prone replication is the potential for rapid adaptation of RNA viruses when a selective pressure is applied, including host immune systems and antiviral drugs. RNA interference (RNAi acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections. However, viruses containing mutations conferring resistance to RNAi were isolated in nearly all cases, underlining the problems of rapid viral evolution. Thus, while promising, the use of RNAi in treating or preventing viral diseases remains fraught with the typical complications that result from high specificity of the target, as seen in other antiviral regimens.

  12. Meta-transcriptomics and the evolutionary biology of RNA viruses.

    Science.gov (United States)

    Shi, Mang; Zhang, Yong-Zhen; Holmes, Edward C

    2018-01-02

    Metagenomics is transforming the study of virus evolution, allowing the full assemblage of virus genomes within a host sample to be determined rapidly and cheaply. The genomic analysis of complete transcriptomes, so-called meta-transcriptomics, is providing a particularly rich source of data on the global diversity of RNA viruses and their evolutionary history. Herein we review some of the insights that meta-transcriptomics has provided on the fundamental patterns and processes of virus evolution, with a focus on the recent discovery of a multitude of novel invertebrate viruses. In particular, meta-transcriptomics shows that the RNA virus world is more fluid than previously realized, with relatively frequent changes in genome length and structure. As well as having a transformative impact on studies of virus evolution, meta-transcriptomics presents major new challenges for virus classification, with the greater sampling of host taxa now filling many of the gaps on virus phylogenies that were previously used to define taxonomic groups. Given that most viruses in the future will likely be characterized using metagenomics approaches, and that we have evidently only sampled a tiny fraction of the total virosphere, we suggest that proposals for virus classification pay careful attention to the wonders unearthed in this new age of virus discovery. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

    Directory of Open Access Journals (Sweden)

    Brittney R Henderson

    Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

  14. RNA interference, arthropod-borne viruses, and mosquitoes.

    Science.gov (United States)

    Sanchez-Vargas, Irma; Travanty, Emily A; Keene, Kimberly M; Franz, Alexander W E; Beaty, Barry J; Blair, Carol D; Olson, Ken E

    2004-06-01

    RNA interference (RNAi) probably functions as an antiviral mechanism in most eukaryotic organisms. Variations in the activity of this antiviral pathway in mosquitoes could explain, in part, why some mosquitoes are competent vectors of medically important, arthropod-borne viruses (arboviruses) and others are not. There are three lines of evidence that show the RNAi pathway exists in Aedes species that transmit arboviruses. The first is that recombinant Sindbis viruses expressing a RNA fragment from a genetically unrelated dengue-2 virus (DENV-2) interfere with DENV-2 replication in Aedes aegypti mosquitoes by a mechanism similar to virus-induced gene silencing described in plants. The second is that transfection of C6/36 (Aedes albopictus) cells with either double-stranded RNA or synthetic small interfering RNAs derived from an arbovirus genome interferes with replication of the homologous virus. The third is that a hairpin DENV-2-specific RNA transcribed from a plasmid can generate virus-resistant C6/36 cells. We hypothesize that genetically modified mosquitoes can be generated that transcribe a flavivirus-specific dsRNA, triggering the RNAi response soon after ingestion of a blood meal. This could induce the RNAi pathway in the midgut prior to establishment of virus infection and profoundly change vector competence. Towards this goal, we are developing transgenic A. aegypti lines that are refractory to DENV by exploiting the RNAi pathway.

  15. The reverse genetics applied to fish RNA viruses

    Directory of Open Access Journals (Sweden)

    Biacchesi Stéphane

    2011-01-01

    Full Text Available Abstract Aquaculture has expanded rapidly to become a major economic and food-producing sector worldwide these last 30 years. In parallel, viral diseases have emerged and rapidly spread from farm to farm causing enormous economic losses. The most problematic viruses encountered in the field are mainly, but not exclusively, RNA viruses belonging to the Novirhabdovirus, Aquabirnavirus, Alphavirus and Betanodavirus genera. The recent establishment of reverse genetics systems to recover infectious fish RNA viruses entirely from cDNA has made possible to genetically manipulate the viral genome. These systems have provided powerful tools to study all aspects of the virus biology and virus-host interactions but also gave the opportunity to use these viruses as live vaccines or as gene vectors. This review provides an overview on the recent breakthroughs achieved by using these reverse genetics systems in terms of viral protein function, virulence and host-specificity factor, vaccine development and vector design.

  16. Identification of a Novel RNA Virus Lethal to Tilapia

    OpenAIRE

    Eyngor, Marina; Zamostiano, Rachel; Kembou Tsofack, Japhette Esther; Berkowitz, Asaf; Bercovier, Hillel; Tinman, Simon; Lev, Menachem; Hurvitz, Avshalom; Galeotti, Marco; Bacharach, Eran; Eldar, Avi

    2014-01-01

    Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and ...

  17. A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

    Directory of Open Access Journals (Sweden)

    Baodong Wu

    2009-03-01

    Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

  18. Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

    NARCIS (Netherlands)

    Holm, Christian K; Rahbek, Stine H; Gad, Hans Henrik; Bak, Rasmus O; Jakobsen, Martin R; Jiang, Zhaozaho; Hansen, Anne Louise; Jensen, Simon K; Sun, Chenglong; Thomsen, Martin K; Laustsen, Anders; Nielsen, Camilla G; Severinsen, Kasper; Xiong, Yingluo; Burdette, Dara L; Hornung, Veit; Lebbink, Robert Jan; Duch, Mogens; Fitzgerald, Katherine A; Bahrami, Shervin; Mikkelsen, Jakob Giehm; Hartmann, Rune; Paludan, Søren R

    2016-01-01

    Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent,

  19. Pathogenesis of Noroviruses, Emerging RNA Viruses

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    Stephanie M. Karst

    2010-03-01

    Full Text Available Human noroviruses in the family Caliciviridae are a major cause of epidemic gastroenteritis. They are responsible for at least 95% of viral outbreaks and over 50% of all outbreaks worldwide. Transmission of these highly infectious plus-stranded RNA viruses occurs primarily through contaminated food or water, but also through person-to-person contact and exposure to fomites. Norovirus infections are typically acute and self-limited. However, disease can be much more severe and prolonged in infants, elderly, and immunocompromised individuals. Norovirus outbreaks frequently occur in semi-closed communities such as nursing homes, military settings, schools, hospitals, cruise ships, and disaster relief situations. Noroviruses are classified as Category B biodefense agents because they are highly contagious, extremely stable in the environment, resistant to common disinfectants, and associated with debilitating illness. The number of reported norovirus outbreaks has risen sharply since 2002 suggesting the emergence of more infectious strains. There has also been increased recognition that noroviruses are important causes of childhood hospitalization. Moreover, noroviruses have recently been associated with multiple clinical outcomes other than gastroenteritis. It is unclear whether these new observations are due to improved norovirus diagnostics or to the emergence of more virulent norovirus strains. Regardless, it is clear that human noroviruses cause considerable morbidity worldwide, have significant economic impact, and are clinically important emerging pathogens. Despite the impact of human norovirus-induced disease and the potential for emergence of highly virulent strains, the pathogenic features of infection are not well understood due to the lack of a cell culture system and previous lack of animal models. This review summarizes the current understanding of norovirus pathogenesis from the histological to the molecular level, including

  20. The Battle of RNA Synthesis: Virus versus Host

    Science.gov (United States)

    Landick, Robert; Berkhout, Ben

    2017-01-01

    Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage. PMID:29065472

  1. The evolution of RNA viruses: A population genetics view

    Science.gov (United States)

    Moya, Andrés; Elena, Santiago F.; Bracho, Alma; Miralles, Rosario; Barrio, Eladio

    2000-01-01

    RNA viruses are excellent experimental models for studying evolution under the theoretical framework of population genetics. For a proper justification of this thesis we have introduced some properties of RNA viruses that are relevant for studying evolution. On the other hand, population genetics is a reductionistic theory of evolution. It does not consider or make simplistic assumptions on the transformation laws within and between genotypic and phenotypic spaces. However, such laws are minimized in the case of RNA viruses because the phenotypic space maps onto the genotypic space in a much more linear way than on higher DNA-based organisms. Under experimental conditions, we have tested the role of deleterious and beneficial mutations in the degree of adaptation of vesicular stomatitis virus (VSV), a nonsegmented virus of negative strand. We also have studied how effective population size, initial genetic variability in populations, and environmental heterogeneity shapes the impact of mutations in the evolution of vesicular stomatitis virus. Finally, in an integrative attempt, we discuss pros and cons of the quasispecies theory compared with classic population genetics models for haploid organisms to explain the evolution of RNA viruses. PMID:10860958

  2. The evolution of RNA viruses: A population genetics view.

    Science.gov (United States)

    Moya, A; Elena, S F; Bracho, A; Miralles, R; Barrio, E

    2000-06-20

    RNA viruses are excellent experimental models for studying evolution under the theoretical framework of population genetics. For a proper justification of this thesis we have introduced some properties of RNA viruses that are relevant for studying evolution. On the other hand, population genetics is a reductionistic theory of evolution. It does not consider or make simplistic assumptions on the transformation laws within and between genotypic and phenotypic spaces. However, such laws are minimized in the case of RNA viruses because the phenotypic space maps onto the genotypic space in a much more linear way than on higher DNA-based organisms. Under experimental conditions, we have tested the role of deleterious and beneficial mutations in the degree of adaptation of vesicular stomatitis virus (VSV), a nonsegmented virus of negative strand. We also have studied how effective population size, initial genetic variability in populations, and environmental heterogeneity shapes the impact of mutations in the evolution of vesicular stomatitis virus. Finally, in an integrative attempt, we discuss pros and cons of the quasispecies theory compared with classic population genetics models for haploid organisms to explain the evolution of RNA viruses.

  3. Mechanisms of human immunodeficiency virus type 2 RNA packaging

    DEFF Research Database (Denmark)

    Ni, Na; Nikolaitchik, Olga A; Dilley, Kari A

    2011-01-01

    Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2...... proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results...... do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins...

  4. Hepatitis C virus RNA functionally sequesters miR-122

    DEFF Research Database (Denmark)

    Luna, Joseph M; Scheel, Troels K H; Danino, Tal

    2015-01-01

    Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during...... HCV infection showed robust AGO binding on the HCV 5'UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 "sponge" effect was relieved and redirected to miR-15...... targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration...

  5. Metagenomic analysis of RNA viruses in a fresh water lake.

    Directory of Open Access Journals (Sweden)

    Appolinaire Djikeng

    Full Text Available Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic and possibly be involved in the spread of emerging and pandemic diseases.

  6. Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay

    International Nuclear Information System (INIS)

    Panet, A.; Baltimore, D.; Hanafusa, T.

    1975-01-01

    A radioimmunoassay was developed that can detect and quantitate 3 ng or more of the avian RNA tumor virus reverse transcriptase. The assay detected no antigenic sites in Rous sarcoma virus α virions or in virions of a murine RNA tumor virus. About 70 molecules of reverse transcriptase were found per virion of avian myeloblastosis virus with this assay or with an assay based on antibody inhibition of enzymatic activity. The assay detected about 270 ng of enzyme per mg of cell protein in virus-producing cells; uninfected cells had much less antigenic material but contained some determinants able to displace radioactive antigen. No additional antigenic determinants on reverse transcriptase could be detected that were not found on the separated α subunit of the enzyme. Although sevenfold less sensitive than enzymatic activity as a measure of reverse transcriptase, the radioimmunoassay can detect antigen using small amounts of protein and in the presence of inhibitors

  7. RNA interference against animal viruses: how morbilliviruses generate extended diversity to escape small interfering RNA control.

    Science.gov (United States)

    Holz, Carine L; Albina, Emmanuel; Minet, Cécile; Lancelot, Renaud; Kwiatek, Olivier; Libeau, Geneviève; Servan de Almeida, Renata

    2012-01-01

    Viruses are serious threats to human and animal health. Vaccines can prevent viral diseases, but few antiviral treatments are available to control evolving infections. Among new antiviral therapies, RNA interference (RNAi) has been the focus of intensive research. However, along with the development of efficient RNAi-based therapeutics comes the risk of emergence of resistant viruses. In this study, we challenged the in vitro propensity of a morbillivirus (peste des petits ruminants virus), a stable RNA virus, to escape the inhibition conferred by single or multiple small interfering RNAs (siRNAs) against conserved regions of the N gene. Except with the combination of three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The genetic modifications involved consisted of single or multiple point nucleotide mutations and a deletion of a stretch of six nucleotides, illustrating that this virus has an unusual genomic malleability.

  8. Hepatitis C virus translation preferentially depends on active RNA replication.

    Directory of Open Access Journals (Sweden)

    Helene Minyi Liu

    Full Text Available Hepatitis C virus (HCV RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.

  9. Infectious Bursal disease virus: ribonucleoprotein complexes of a double-stranded RNA virus.

    Science.gov (United States)

    Luque, Daniel; Saugar, Irene; Rejas, María Teresa; Carrascosa, José L; Rodríguez, José F; Castón, José R

    2009-02-27

    Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some double-stranded DNA (dsDNA) viruses, and the nucleocapsid proteins of single-stranded RNA viruses. dsRNA viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T=2 core involved in the metabolism and insulation of the dsRNA genome. The birnaviruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T=13 capsid only. Moreover, as in many negative single-stranded RNA viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease virus, the best characterized member of the Birnaviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at approximately 450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg-dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNase III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA viruses.

  10. Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

    Directory of Open Access Journals (Sweden)

    Kiwamu Hyodo

    2015-05-01

    Full Text Available Eukaryotic positive-strand RNA [(+RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA, a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids, but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+RNA virus, Red clover necrotic mosaic virus (RCNMV. We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

  11. [Diverse double-stranded RNA viruses infecting fungi].

    Science.gov (United States)

    Chiba, Sotaro; Suzuki, Nobuhiro

    2014-01-01

    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.

  12. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis

    Science.gov (United States)

    te Velthuis, Aartjan J.W.; Fodor, Ervin

    2016-01-01

    The genome of influenza viruses consists of multiple segments of single stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, forming viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, how it carries out transcription and replication, and how its activities are modulated by viral and host factors. Furthermore, we discuss how advances in our understanding of polymerase function could help identifying new antiviral targets. PMID:27396566

  13. RNA Editing and its Control in Hepatitis Delta Virus Replication

    Directory of Open Access Journals (Sweden)

    John L. Casey

    2010-01-01

    Full Text Available The hepatitis delta virus genome is a small circular RNA, similar to viroids. Although HDV contains a gene, the protein produced (HDAg is encoded by less than half the genome and possesses no RNA polymerase activity. Because of this limited coding capacity, HDV relies heavily on host functions and on structural features of the viral RNA—very much like viroids. The virus’ use of host RNA editing activity to produce two functionally distinct forms of HDAg is a particularly good example of this reliance. This review covers the mechanisms and control of RNA editing in the HDV replication cycle.

  14. RNA interference against viruses: strike and counterstrike

    NARCIS (Netherlands)

    Haasnoot, Joost; Westerhout, Ellen M.; Berkhout, Ben

    2007-01-01

    RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA. RNAi holds great promise as a novel nucleic acid-based therapeutic against a wide variety of diseases, including cancer, infectious diseases and genetic disorders. Antiviral

  15. Infectious bursal disease virus capsid protein VP3 interacts both with VP1, the RNA-dependent RNA polymerase and with viral double-stranded RNA

    NARCIS (Netherlands)

    Tacken, M.G.J.; Peeters, B.P.H.; Thomas, A.A.M.; Rottier, P.J.M.; Boot, H.J.

    2002-01-01

    Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus of the Birnaviridae family. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shell capsid that is composed of VP2 and VP3. In this study we

  16. RNA folding inside a virus capsid and dimensional reduction.

    Science.gov (United States)

    Ghafouri, Rouzbeh; Bruinsma, Robijn; Rudnick, Joseph

    2006-03-01

    As RNA folds on itself , in certain conditions, it takes the form of a branched polymer. So the problem of RNA folding in a virus capsid is essentially the problem of a branched polymer in a confined environment. In this paper we attack the problem using the technique of dimensional reduction which relates a branched polymer with self interation in D dimension to a hardcore classical gas in (D-2) dimension. We look for phase transitions and intersting physical quantities such as pressure.

  17. Evidence of pestivirus RNA in human virus vaccines.

    Science.gov (United States)

    Harasawa, R; Tomiyama, T

    1994-01-01

    We examined live virus vaccines against measles, mumps, and rubella for the presence of pestivirus RNA or of pestiviruses by reverse transcription PCR. Pestivirus RNA was detected in two measles-mumps-rubella combined vaccines and in two monovalent vaccines against mumps and rubella. Nucleotide sequence analysis of the PCR products indicated that a modified live vaccine strain used for immunization of cattle against bovine viral diarrhea is not responsible for the contamination of the vaccines. Images PMID:8077414

  18. Assessment of the RNASound RNA Sampling Card for the preservation of influenza virus RNA

    Directory of Open Access Journals (Sweden)

    Hilda Lau

    2016-11-01

    Full Text Available Shipping influenza virus specimens, isolates or purified RNA is normally conducted at ultra-low temperatures using dry ice to ensure minimal degradation of the samples but this is expensive and requires special packaging and shipping conditions. Therefore, alternative methods for shipping influenza viruses or RNA at ambient temperatures would be desirable.The RNASound RNA Sampling Card (FortiusBio LLC, CA, USA is a device that enables specimens or isolates to be applied to a card, whereby viruses are inactivated, while RNA is preserved and purified RNA can also easily be eluted. To evaluate this card, we applied influenza virus cell culture isolate supernatants to either the RNASound card or Whatman Grade No. 1 filter paper (GE Healthcare, NSW, Australia and compared the preservation to that of material stored in liquid form. Preservation was tested using influenza A and B viruses at two different storage temperatures (cool 2-8oC or room temperature 18-22oC and these were compared with control material stored at -80°C, for 7, 14 or 28 days. The quality of the RNA recovered was assessed using real time RT-PCR and Sanger sequencing. The RNASound card was effective in preserving influenza RNA at room temperature for up to 28 days, with only a minor change in real-time RT-PCR cycle threshold values for selected gene targets when comparing between viruses applied to the card or stored at -80°C. Similar results were obtained with filter paper, whilst virus in liquid form performed the worst. Nevertheless, as the RNASound card also has the capability to inactivate viruses in addition to preserving RNA at room temperature for many weeks, this makes it feasible to send samples to laboratories using regular mail, and thus avoid the need for expensive shipping conditions requiring biohazard containers and dry ice. Moreover, the quick and simple RNA recovery from the RNASound card allows recipient labs to obtain RNA without the need for special reagents

  19. A comparative analysis of measles virus RNA by oligonucleotide fingerprinting

    International Nuclear Information System (INIS)

    Stephenson, J.R.; Meulen, V. ter

    1982-01-01

    Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T 1 oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin. (Author)

  20. RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication

    NARCIS (Netherlands)

    Feenstra, F.; Gennip, van H.G.P.; Water, van de S.G.P.; Rijn, van P.A.

    2014-01-01

    Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges.

  1. dsRNA interference on expression of a RNA-dependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus.

    Science.gov (United States)

    Pan, Zhong-Hua; Gao, Kun; Hou, Cheng-Xiang; Wu, Ping; Qin, Guang-Xing; Geng, Tao; Guo, Xi-Jie

    2015-07-01

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens in silkworm. Its infection often results in significant losses to sericulture. Studies have demonstrated that RNAi is one of the important anti-viral mechanisms in organisms. In this study, three dsRNAs targeting the RNA-dependent RNA polymerase (RDRP) gene of BmCPV were designed and synthesized with 2'-F modification to explore their interference effects on BmCPV replication in silkworm larvae. The results showed that injecting dsRNA in the dosage of 4-6 ng per mg body weight into the 5th instar larvae can interfere with the BmCPV-RDRP expression by 93% after virus infection and by 99.9% before virus infection. In addition, the expression of two viral structural protein genes (genome RNA segments 1 and 5) was also decreased with the decrease of RDRP expression, suggesting that RNAi interference of BmCPV-RDRP expression could affect viral replication. The study provides an effective method for investigating virus replication as well as the virus-host interactions in the silkworm larvae using dsRNA. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. A Broad RNA Virus Survey Reveals Both miRNA Dependence and Functional Sequestration

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Luna, Joseph M; Liniger, Matthias

    2016-01-01

    , critically depended on the interaction of cellular miR-17 and let-7 with the viral 3' UTR. Unlike canonical miRNA interactions, miR-17 and let-7 binding enhanced pestivirus translation and RNA stability. miR-17 sequestration by pestiviruses conferred reduced AGO binding and functional de...... immunoprecipitation (CLIP) of the Argonaute (AGO) proteins to characterize strengths and specificities of miRNA interactions in the context of 15 different RNA virus infections, including several clinically relevant pathogens. Notably, replication of pestiviruses, a major threat to milk and meat industries...

  3. Genome rearrangement affects RNA virus adaptability on prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Kendra ePesko

    2015-04-01

    Full Text Available Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wildtype gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense nonsegmented RNA viruses (order Mononegavirales have specific genome architecture: 3′ UTR – core protein genes – envelope protein genes – RNA-dependent RNA-polymerase gene – 5′ UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV variants with the nucleocapsid (N gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N-gene translocation towards the 5’ end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function, especially on PC3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective

  4. Inhibition of Monkeypox virus replication by RNA interference

    Directory of Open Access Journals (Sweden)

    Jahrling Peter B

    2009-11-01

    Full Text Available Abstract The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV, Vaccinia (VACV, monkeypox (MPV and Variola (VARV viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA. Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R or an important gene in viral entry (E8L, inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses.

  5. An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments

    Science.gov (United States)

    Amplification and sequencing of the complete M- and S-RNA segments of Tomato spotted wilt virus and Impatiens necrotic spot virus as a single fragment is useful for whole genome sequencing of tospoviruses co-infecting a single host plant. It avoids issues associated with overlapping amplicon-based ...

  6. Beet yellows virus replicase and replicative compartments: parallels with other RNA viruses.

    Science.gov (United States)

    Gushchin, Vladimir A; Solovyev, Andrey G; Erokhina, Tatyana N; Morozov, Sergey Y; Agranovsky, Alexey A

    2013-01-01

    In eukaryotic virus systems, infection leads to induction of membranous compartments in which replication occurs. Virus-encoded subunits of the replication complex mediate its interaction with membranes. As replication platforms, RNA viruses use the cytoplasmic surfaces of different membrane compartments, e.g., endoplasmic reticulum (ER), Golgi, endo/lysosomes, mitochondria, chloroplasts, and peroxisomes. Closterovirus infections are accompanied by formation of multivesicular complexes from cell membranes of ER or mitochondrial origin. So far the mechanisms for vesicles formation have been obscure. In the replication-associated 1a polyprotein of Beet yellows virus (BYV) and other closteroviruses, the region between the methyltransferase and helicase domains (1a central region (CR), 1a CR) is marginally conserved. Computer-assisted analysis predicts several putative membrane-binding domains in the BYV 1a CR. Transient expression of a hydrophobic segment (referred to here as CR-2) of the BYV 1a in Nicotiana benthamiana led to reorganization of the ER and formation of ~1-μm mobile globules. We propose that the CR-2 may be involved in the formation of multivesicular complexes in BYV-infected cells. This provides analogy with membrane-associated proteins mediating the build-up of "virus factories" in cells infected with diverse positive-strand RNA viruses (alpha-like viruses, picorna-like viruses, flaviviruses, and nidoviruses) and negative-strand RNA viruses (bunyaviruses).

  7. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species.

    Science.gov (United States)

    Di Giallonardo, Francesca; Schlub, Timothy E; Shi, Mang; Holmes, Edward C

    2017-04-15

    Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies and

  8. The RNA of turnip yellow mosaic virus exhibits icosahedral order

    International Nuclear Information System (INIS)

    Larson, Steven B.; Lucas, Robert W.; Greenwood, Aaron; McPherson, Alexander

    2005-01-01

    Difference electron density maps, based on structure factor amplitudes and experimental phases from crystals of wild-type turnip yellow mosaic virus and those of empty capsids prepared by freeze-thawing, show a large portion of the encapsidated RNA to have an icosahedral distribution. Four unique segments of base-paired, double-helical RNA, one to two turns in length, lie between 33-A and 101-A radius and are organized about either 2-fold or 5-fold icosahedral axes. In addition, single-stranded loops of RNA invade the pentameric and hexameric capsomeres where they contact the interior capsid surface. The remaining RNA, not seen in electron density maps, must serve as connecting links between these secondary structural elements and is likely icosahedrally disordered. The distribution of RNA observed crystallographically appears to be in agreement with models based on biochemical data and secondary structural analyses

  9. Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

    OpenAIRE

    Certo, Jeanine L.; Shook, Betsy F.; Yin, Philip D.; Snider, John T.; Hu, Wei-Shau

    1998-01-01

    It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Ψ) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-base...

  10. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  11. Quasispecies theory and the behavior of RNA viruses.

    Directory of Open Access Journals (Sweden)

    Adam S Lauring

    2010-07-01

    Full Text Available A large number of medically important viruses, including HIV, hepatitis C virus, and influenza, have RNA genomes. These viruses replicate with extremely high mutation rates and exhibit significant genetic diversity. This diversity allows a viral population to rapidly adapt to dynamic environments and evolve resistance to vaccines and antiviral drugs. For the last 30 years, quasispecies theory has provided a population-based framework for understanding RNA viral evolution. A quasispecies is a cloud of diverse variants that are genetically linked through mutation, interact cooperatively on a functional level, and collectively contribute to the characteristics of the population. Many predictions of quasispecies theory run counter to traditional views of microbial behavior and evolution and have profound implications for our understanding of viral disease. Here, we discuss basic principles of quasispecies theory and describe its relevance for our understanding of viral fitness, virulence, and antiviral therapeutic strategy.

  12. Interferon Induction by RNA Viruses and Antagonism by Viral Pathogens

    Directory of Open Access Journals (Sweden)

    Yuchen Nan

    2014-12-01

    Full Text Available Interferons are a group of small proteins that play key roles in host antiviral innate immunity. Their induction mainly relies on host pattern recognition receptors (PRR. Host PRR for RNA viruses include Toll-like receptors (TLR and retinoic acid-inducible gene I (RIG-I like receptors (RLR. Activation of both TLR and RLR pathways can eventually lead to the secretion of type I IFNs, which can modulate both innate and adaptive immune responses against viral pathogens. Because of the important roles of interferons, viruses have evolved multiple strategies to evade host TLR and RLR mediated signaling. This review focuses on the mechanisms of interferon induction and antagonism of the antiviral strategy by RNA viruses.

  13. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus.

    Directory of Open Access Journals (Sweden)

    Aaron M Collier

    2016-04-01

    Full Text Available During the replication cycle of double-stranded (ds RNA viruses, the viral RNA-dependent RNA polymerase (RdRP replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV. In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1 the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2 the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3 RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4 the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.

  14. Mapping the active site of vaccinia virus RNA triphosphatase

    International Nuclear Information System (INIS)

    Gong Chunling; Shuman, Stewart

    2003-01-01

    The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called ''triphosphate tunnel''). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery

  15. Specificity in the association of tomato black ring virus satellite RNA with helper virus.

    Science.gov (United States)

    Oncino, C; Hemmer, O; Fritsch, C

    1995-10-20

    The satellite RNAs (sat-RNAs) associated with some isolates of tomato black ring virus (TBRV) consist of single-stranded molecules of about 1375 nucleotides, encoding a nonstructural protein of 48K which has been shown to be involved in the replication of the sat-RNA. The TBRV sat-RNAs are also dependent for their replication and for their encapsidation on the helper virus. To characterize the nature of the association between sat-RNA and helper virus, transcripts of sat-RNA from TBRV isolates C and L (respectively, of serotypes G and S) have been prepared and inoculated onto Chenopodium quinoa leaves or protoplasts. Transcript of the TBRV sat-RNA C is efficiently multiplied when coinoculated with the genomic RNAs of TBRV isolate G (used instead of TBRV isolate C, because isolate G was depleted of sat-RNA), but does not multiply with TBRV isolate L. On the other hand, transcript of the sat-RNA L is able to multiply with the cognate helper virus and, less efficiently, with grapevine chrome mosaic virus (another nepovirus, 80% similar to TBRV), but does not multiply with TBRV G. The specificity of the association resides at the level of sat-RNA replication. Analysis of the multiplication of chimeric sat-RNAs, obtained by exchanging different regions between the two sat-RNAs C and L, showed that the 5' and the 3' noncoding regions of the sat-RNA, although important for replication, are not implicated in specificity. The results suggest that the determinants of the specificity are contained in the 48K sat-RNA-encoded protein.

  16. Small RNA profiles from virus-infected fresh market vegetables.

    Science.gov (United States)

    Frizzi, Alessandra; Zhang, Yuanji; Kao, John; Hagen, Charles; Huang, Shihshieh

    2014-12-10

    Functional small RNAs, such as short interfering RNAs (siRNAs) and microRNAs (miRNAs), exist in freshly consumed fruits and vegetables. These siRNAs can be derived either from endogenous sequences or from viruses that infect them. Symptomatic tomatoes, watermelons, zucchini, and onions were purchased from grocery stores and investigated by small RNA sequencing. By aligning the obtained small RNA sequences to sequences of known viruses, four different viruses were identified as infecting these fruits and vegetables. Many of these virally derived small RNAs along with endogenous small RNAs were found to be highly complementary to human genes. However, the established history of safe consumption of these vegetables suggests that this sequence homology has little biological relevance. By extension, these results provide evidence for the safe use by humans and animals of genetically engineered crops using RNA-based suppression technologies, especially vegetable crops with virus resistance conferred by expression of siRNAs or miRNAs derived from viral sequences.

  17. RNA viruses and microRNAs: challenging discoveries for the 21st century

    Science.gov (United States)

    Swaminathan, Gokul; Martin-Garcia, Julio

    2013-01-01

    RNA viruses represent the predominant cause of many clinically relevant viral diseases in humans. Among several evolutionary advantages acquired by RNA viruses, the ability to usurp host cellular machinery and evade antiviral immune responses is imperative. During the past decade, RNA interference mechanisms, especially microRNA (miRNA)-mediated regulation of cellular protein expression, have revolutionized our understanding of host-viral interactions. Although it is well established that several DNA viruses express miRNAs that play crucial roles in their pathogenesis, expression of miRNAs by RNA viruses remains controversial. However, modulation of the miRNA machinery by RNA viruses may confer multiple benefits for enhanced viral replication and survival in host cells. In this review, we discuss the current literature on RNA viruses that may encode miRNAs and the varied advantages of engineering RNA viruses to express miRNAs as potential vectors for gene therapy. In addition, we review how different families of RNA viruses can alter miRNA machinery for productive replication, evasion of antiviral immune responses, and prolonged survival. We underscore the need to further explore the complex interactions of RNA viruses with host miRNAs to augment our understanding of host-virus interplay. PMID:24046280

  18. RNA viruses and microRNAs: challenging discoveries for the 21st century.

    Science.gov (United States)

    Swaminathan, Gokul; Martin-Garcia, Julio; Navas-Martin, Sonia

    2013-11-15

    RNA viruses represent the predominant cause of many clinically relevant viral diseases in humans. Among several evolutionary advantages acquired by RNA viruses, the ability to usurp host cellular machinery and evade antiviral immune responses is imperative. During the past decade, RNA interference mechanisms, especially microRNA (miRNA)-mediated regulation of cellular protein expression, have revolutionized our understanding of host-viral interactions. Although it is well established that several DNA viruses express miRNAs that play crucial roles in their pathogenesis, expression of miRNAs by RNA viruses remains controversial. However, modulation of the miRNA machinery by RNA viruses may confer multiple benefits for enhanced viral replication and survival in host cells. In this review, we discuss the current literature on RNA viruses that may encode miRNAs and the varied advantages of engineering RNA viruses to express miRNAs as potential vectors for gene therapy. In addition, we review how different families of RNA viruses can alter miRNA machinery for productive replication, evasion of antiviral immune responses, and prolonged survival. We underscore the need to further explore the complex interactions of RNA viruses with host miRNAs to augment our understanding of host-virus interplay.

  19. A stable RNA virus-based vector for citrus trees

    International Nuclear Information System (INIS)

    Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe; Dawson, William O.

    2007-01-01

    Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter. These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees

  20. Structural and functional characterisation of Aichi virus RNA dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Šilhán, Jan; Bäumlová, Adriana; Chalupská, Dominika; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 15, č. 1 (2017), s. 7-8 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : Aichi virus * RNA replication Subject RIV: CE - Biochemistry

  1. Yeast double-stranded RNA virus L-A deliberately synthesizes RNA transcripts with 5'-diphosphate.

    Science.gov (United States)

    Fujimura, Tsutomu; Esteban, Rosa

    2010-07-23

    L-A is a persistent double-stranded RNA virus commonly found in the yeast Saccharomyces cerevisiae. Isolated L-A virus synthesizes positive strand transcripts in vitro. We found that the 5' termini of the transcripts are diphosphorylated. The 5'-terminal nucleotide is G, and GDP was the best substrate among those examined to prime the reaction. When GTP was used, the triphosphate of GTP incorporated into the 5'-end was converted to diphosphate. This activity was not dependent on host CTL1 RNA triphosphatase. The 5'-end of the GMP-primed transcript also was converted to diphosphate, the beta-phosphate of which was derived from the gamma-phosphate of ATP present in the polymerization reaction. These results demonstrate that L-A virus commands elaborate enzymatic systems to ensure its transcript to be 5'-diphosphorylated. Transcripts of M1, a satellite RNA of L-A virus, also had diphosphate at the 5' termini. Because viral transcripts are released from the virion into the cytoplasm to be translated and encapsidated into a new viral particle, a stage most vulnerable to degradation in the virus replication cycle, our results suggest that the 5'-diphosphate status is important for transcript stability. Consistent with this, L-A transcripts made in vitro are resistant to the affinity-purified Ski1p 5'-exonuclease. We also discuss the implication of these findings on translation of viral RNA. Because the viral transcript has no conventional 5'-cap structure, this work may shed light on the metabolism of non-self-RNA in yeast.

  2. RNA polymerase of the killer virus of yeast

    International Nuclear Information System (INIS)

    Georgopoulos, D.E.; Leibowitz, M.J.

    1984-01-01

    The L/sub A/ and M double-stranded (ds) RNA segments of the cytoplasmically inherited killer virus of Saccharomyces cerevisiae are encapsidated in virions that contain a DNA-independent transcriptase activity. This enzyme catalyzes the synthesis of full-length (+) stranded copies of the genomic dsRNA segments, denoted l/sub A/ and m. The L/sub A/ dsRNA segment appears to encode the major capsid protein in which both dsRNA molecules are encapsidated, while M dsRNA encodes products responsible for the two killer phenotypes of toxin production and resistance to toxin. Proteins extracted from transcriptionally active virions fail to cross-react with antibody to yeast DNA-dependent RNA polymerases, suggesting that none of the subunits of the host cell polymerases are active in viral transcription. Sequence analysis of the in vitro transcripts reveals neither to be 3'-terminally polyadenylated, although m contains an apparent internal polyA-like tract. In the presence of any three ribonucleoside triphosphates (0.5 mM), the fourth ribonucleoside triphosphate shows an optimal rate of incorporation into transcript at a concentration of 20 μM. However, in a 3-hour reaction, the yield of a product RNA increases with the concentration of the limiting ribonucleotide up to 0.5 mM. Gel electrophoresis of the reaction products reveals that increasing the substrate concentration accelerates the appearance of radioactivity in full-length l/sub A/ and m transcripts

  3. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, C.G.; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...

  4. Inhibition of RNA recruitment and replication of an RNA virus by acridine derivatives with known anti-prion activities.

    Directory of Open Access Journals (Sweden)

    Zsuzsanna Sasvari

    Full Text Available BACKGROUND: Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV, a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ and quinacrine (QC, which are active against prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii reduction of minus-strand synthesis by the tombusvirus replicase; and (iii inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. CONCLUSION/SIGNIFICANCE: Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.

  5. Inhibition of RNA Recruitment and Replication of an RNA Virus by Acridine Derivatives with Known Anti-Prion Activities

    Science.gov (United States)

    Sasvari, Zsuzsanna; Bach, Stéphane; Blondel, Marc; Nagy, Peter D.

    2009-01-01

    Background Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases. Methodology/Principal Findings Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication. Conclusion/Significance Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses. PMID:19823675

  6. Simple genomes, complex interactions: Epistasis in RNA virus

    Science.gov (United States)

    Elena, Santiago F.; Solé, Ricard V.; Sardanyés, Josep

    2010-06-01

    Owed to their reduced size and low number of proteins encoded, RNA viruses and other subviral pathogens are often considered as being genetically too simple. However, this structural simplicity also creates the necessity for viral RNA sequences to encode for more than one protein and for proteins to carry out multiple functions, all together resulting in complex patterns of genetic interactions. In this work we will first review the experimental studies revealing that the architecture of viral genomes is dominated by antagonistic interactions among loci. Second, we will also review mathematical models and provide a description of computational tools for the study of RNA virus dynamics and evolution. As an application of these tools, we will finish this review article by analyzing a stochastic bit-string model of in silico virus replication. This model analyzes the interplay between epistasis and the mode of replication on determining the population load of deleterious mutations. The model suggests that, for a given mutation rate, the deleterious mutational load is always larger when epistasis is predominantly antagonistic than when synergism is the rule. However, the magnitude of this effect is larger if replication occurs geometrically than if it proceeds linearly.

  7. Innate immune evasion strategies of DNA and RNA viruses.

    Science.gov (United States)

    Beachboard, Dia C; Horner, Stacy M

    2016-08-01

    Upon infection, both DNA and RNA viruses can be sensed by pattern recognition receptors (PRRs) in the cytoplasm or the nucleus to activate antiviral innate immunity. Sensing of viral products leads to the activation of a signaling cascade that ultimately results in transcriptional activation of type I and III interferons, as well as other antiviral genes that together mediate viral clearance and inhibit viral spread. Therefore, in order for viruses to replicate and spread efficiently, they must inhibit the host signaling pathways that induce the innate antiviral immune response. In this review, we will highlight recent advances in the understanding of the mechanisms by which viruses evade PRR detection, intermediate signaling molecule activation, transcription factor activation, and the actions of antiviral proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    Science.gov (United States)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  9. Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements

    Science.gov (United States)

    Watters, Kyle E; Choudhary, Krishna; Aviran, Sharon; Perry, Keith L

    2018-01-01

    Abstract In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites. PMID:29294088

  10. Global organization of a positive-strand RNA virus genome.

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    Baodong Wu

    Full Text Available The genomes of plus-strand RNA viruses contain many regulatory sequences and structures that direct different viral processes. The traditional view of these RNA elements are as local structures present in non-coding regions. However, this view is changing due to the discovery of regulatory elements in coding regions and functional long-range intra-genomic base pairing interactions. The ∼4.8 kb long RNA genome of the tombusvirus tomato bushy stunt virus (TBSV contains these types of structural features, including six different functional long-distance interactions. We hypothesized that to achieve these multiple interactions this viral genome must utilize a large-scale organizational strategy and, accordingly, we sought to assess the global conformation of the entire TBSV genome. Atomic force micrographs of the genome indicated a mostly condensed structure composed of interconnected protrusions extending from a central hub. This configuration was consistent with the genomic secondary structure model generated using high-throughput selective 2'-hydroxyl acylation analysed by primer extension (i.e. SHAPE, which predicted different sized RNA domains originating from a central region. Known RNA elements were identified in both domain and inter-domain regions, and novel structural features were predicted and functionally confirmed. Interestingly, only two of the six long-range interactions known to form were present in the structural model. However, for those interactions that did not form, complementary partner sequences were positioned relatively close to each other in the structure, suggesting that the secondary structure level of viral genome structure could provide a basic scaffold for the formation of different long-range interactions. The higher-order structural model for the TBSV RNA genome provides a snapshot of the complex framework that allows multiple functional components to operate in concert within a confined context.

  11. Use of Cellular Decapping Activators by Positive-Strand RNA Viruses

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    Jennifer Jungfleisch

    2016-12-01

    Full Text Available Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5′-3′ mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus.

  12. Analysis of RNA biosynthesis in tobacco cell infected by pimiento ring virus

    International Nuclear Information System (INIS)

    Silva, D.M.; Nogueira, N.L.; Lage, G.

    1984-01-01

    The results about the RNA biosynthesis of pimiento ring virus are analysed. The possible virus influence on the nucleolus, mitochondria and chloroplast of the cellular RNA is studied by electron microscopy radio-authogram. The presence of the virus in the cell seems to modify 3 H-uridine transport to hostess cell interior. (M.A.C.) [pt

  13. RNA binding specificity of Ebola virus transcription factor VP30.

    Science.gov (United States)

    Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

    2016-09-01

    The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

  14. RNA viruses and microRNAs: challenging discoveries for the 21st century

    OpenAIRE

    Swaminathan, Gokul; Martin-Garcia, Julio; Navas-Martin, Sonia

    2013-01-01

    RNA viruses represent the predominant cause of many clinically relevant viral diseases in humans. Among several evolutionary advantages acquired by RNA viruses, the ability to usurp host cellular machinery and evade antiviral immune responses is imperative. During the past decade, RNA interference mechanisms, especially microRNA (miRNA)-mediated regulation of cellular protein expression, have revolutionized our understanding of host-viral interactions. Although it is well established that sev...

  15. Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

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    Liu Yong

    2011-01-01

    Full Text Available Abstract Background An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV movement protein (MP gene and the partial Cucumber mosaic virus (CMV replication protein (Rep gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58 immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32 immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃ did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.

  16. Stem-Loop RNA Hairpins in Giant Viruses: Invading rRNA-Like Repeats and a Template Free RNA

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    Hervé Seligmann

    2018-02-01

    Full Text Available We examine the hypothesis that de novo template-free RNAs still form spontaneously, as they did at the origins of life, invade modern genomes, contribute new genetic material. Previously, analyses of RNA secondary structures suggested that some RNAs resembling ancestral (tRNAs formed recently de novo, other parasitic sequences cluster with rRNAs. Here positive control analyses of additional RNA secondary structures confirm ancestral and de novo statuses of RNA grouped according to secondary structure. Viroids with branched stems resemble de novo RNAs, rod-shaped viroids resemble rRNA secondary structures, independently of GC contents. 5′ UTR leading regions of West Nile and Dengue flavivirid viruses resemble de novo and rRNA structures, respectively. An RNA homologous with Megavirus, Dengue and West Nile genomes, copperhead snake microsatellites and levant cotton repeats, not templated by Mimivirus' genome, persists throughout Mimivirus' infection. Its secondary structure clusters with candidate de novo RNAs. The saltatory phyletic distribution and secondary structure of Mimivirus' peculiar RNA suggest occasional template-free polymerization of this sequence, rather than noncanonical transcriptions (swinger polymerization, posttranscriptional editing.

  17. Promotion of Hendra virus replication by microRNA 146a.

    Science.gov (United States)

    Stewart, Cameron R; Marsh, Glenn A; Jenkins, Kristie A; Gantier, Michael P; Tizard, Mark L; Middleton, Deborah; Lowenthal, John W; Haining, Jessica; Izzard, Leonard; Gough, Tamara J; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G; Pallister, Jackie A; Foord, Adam J; Bean, Andrew G; Wang, Lin-Fa

    2013-04-01

    Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease.

  18. Nucleoproteins of Negative Strand RNA Viruses; RNA Binding, Oligomerisation and Binding to Polymerase Co-Factor

    Directory of Open Access Journals (Sweden)

    Thibaut Crépin

    2010-01-01

    Full Text Available Commentary on Tawar, R.G.; Duquerroy, S.; Vonrhein, C.; Varela, P.F.; Damier-Piolle, L.; Castagné, N.; MacLellan, K.; Bedouelle, H.; Bricogne, G.; Bhella, D.; Eléouët, J.-F.; Rey, F.A. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 2009, 326, 1279-1283.

  19. Synthesis and methylation of ribosomal RNA in HeLa cells infected with the herpes virus pseudorabies virus

    International Nuclear Information System (INIS)

    Furlong, J.C.; Kyriakidis, S.; Stevely, W.S.

    1982-01-01

    The effects of infection with the herpes virus pseudorabies virus on the metabolism of HeLa cell ribosomal RNA were examined. There is a decline both in the synthesis of nucleolar 45S ribosomal precursor RNA and in its processing to mature cytoplasmic RNA. The methylated oligonucleotides in the ribosomal RNA species were studied. The methylation of cytoplasmic ribosomal RNA was essentially unchanged. However there was some undermethylation of the nucleolar precursor. If undermethylated RNA does not mature then this may partly explain the reduced processing in the infected cells. (Author)

  20. Emetine inhibits replication of RNA and DNA viruses without generating drug-resistant virus variants.

    Science.gov (United States)

    Khandelwal, Nitin; Chander, Yogesh; Rawat, Krishan Dutt; Riyesh, Thachamvally; Nishanth, Chikkahonnaiah; Sharma, Shalini; Jindal, Naresh; Tripathi, Bhupendra N; Barua, Sanjay; Kumar, Naveen

    2017-08-01

    At a noncytotoxic concentration, emetine was found to inhibit replication of DNA viruses [buffalopoxvirus (BPXV) and bovine herpesvirus 1 (BHV-1)] as well as RNA viruses [peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)]. Using the time-of-addition and virus step-specific assays, we showed that emetine treatment resulted in reduced synthesis of viral RNA (PPRV and NDV) and DNA (BPXV and BHV-1) as well as inhibiting viral entry (NDV and BHV-1). In addition, emetine treatment also resulted in decreased synthesis of viral proteins. In a cell free endogenous viral polymerase assay, emetine was found to significantly inhibit replication of NDV, but not BPXV genome, suggesting that besides directly inhibiting specific viral polymerases, emetine may also target other factors essentially required for efficient replication of the viral genome. Moreover, emetine was found to significantly inhibit BPXV-induced pock lesions on chorioallantoic membrane (CAM) along with associated mortality of embryonated chicken eggs. At a lethal dose 50 (LD 50 ) of 126.49 ng/egg and at an effective concentration 50 (EC 50 ) of 3.03 ng/egg, the therapeutic index of the emetine against BPXV was determined to be 41.74. Emetine was also found to significantly delay NDV-induced mortality in chicken embryos associated with reduced viral titers. Further, emetine-resistant mutants were not observed upon long-term (P = 25) sequential passage of BPXV and NDV in cell culture. Collectively, we have extended the effective antiviral activity of emetine against diverse groups of DNA and RNA viruses and propose that emetine could provide significant therapeutic value against some of these viruses without inducing an antiviral drug-resistant phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

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    Kim Chan-Mi

    2007-07-01

    Full Text Available Abstract Background Japanese encephalitis virus (JEV NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the

  2. Ebola Virus RNA in Semen from an HIV-Positive Survivor of Ebola.

    Science.gov (United States)

    Purpura, Lawrence J; Rogers, Emerson; Baller, April; White, Stephen; Soka, Moses; Choi, Mary J; Mahmoud, Nuha; Wasunna, Christine; Massaquoi, Moses; Kollie, Jomah; Dweh, Straker; Bemah, Philip; Ladele, Victor; Kpaka, Jonathan; Jawara, Mary; Mugisha, Margaret; Subah, Onyekachi; Faikai, Mylene; Bailey, Jeff A; Rollin, Pierre; Marston, Barbara; Nyenswah, Tolbert; Gasasira, Alex; Knust, Barbara; Nichol, Stuart; Williams, Desmond

    2017-04-01

    Ebola virus is known to persist in semen of male survivors of Ebola virus disease (EVD). However, maximum duration of, or risk factors for, virus persistence are unknown. We report an EVD survivor with preexisting HIV infection, whose semen was positive for Ebola virus RNA 565 days after recovery from EVD.

  3. Ebola Virus RNA in Semen from an HIV-Positive Survivor of Ebola

    OpenAIRE

    Purpura, Lawrence J.; Rogers, Emerson; Baller, April; White, Stephen; Soka, Moses; Choi, Mary J.; Mahmoud, Nuha; Wasunna, Christine; Massaquoi, Moses; Kollie, Jomah; Dweh, Straker; Bemah, Philip; Ladele, Victor; Kpaka, Jonathan; Jawara, Mary

    2017-01-01

    Ebola virus is known to persist in semen of male survivors of Ebola virus disease (EVD). However, maximum duration of, or risk factors for, virus persistence are unknown. We report an EVD survivor with preexisting HIV infection, whose semen was positive for Ebola virus RNA 565 days after recovery from EVD.

  4. Variation in RNA virus mutation rates across host cells.

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    Marine Combe

    2014-01-01

    Full Text Available It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6 to 10(-4 substitutions per nucleotide per round of copying (s/n/r and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV, which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5 s/n/r. Cell immortalization through p53 inactivation and oxygen levels (1-21% did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

  5. Convergent evolution of argonaute-2 slicer antagonism in two distinct insect RNA viruses.

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    Joël T van Mierlo

    Full Text Available RNA interference (RNAi is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A antagonized Argonaute-2 (AGO2 Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.

  6. Exclusion of West Nile Virus Superinfection through RNA Replication▿

    Science.gov (United States)

    Zou, Gang; Zhang, Bo; Lim, Pei-Yin; Yuan, Zhiming; Bernard, Kristen A.; Shi, Pei-Yong

    2009-01-01

    Superinfection exclusion is the ability of an established viral infection to interfere with a second viral infection. Using West Nile virus (WNV) as a model, we show that replicating replicons in BHK-21 cells suppress subsequent WNV infection. The WNV replicon also suppresses superinfections of other flaviviruses but not nonflaviviruses. Mode-of-action analysis indicates that the exclusion of WNV superinfection occurs at the step of RNA synthesis. The continuous culturing of WNV in the replicon-containing cells generated variants that could overcome the superinfection exclusion. The sequencing of the selected viruses revealed mutations in structural (prM S90R or envelope E138K) and nonstructural genes (NS4a K124R and peptide 2K V9M). Mutagenesis analysis showed that the mutations in structural genes nonselectively enhance viral infection in both naïve and replicon-containing BHK-21 cells; in contrast, the mutations in nonstructural genes more selectively enhance viral replication in the replicon-containing cells than in the naïve cells. Mechanistic analysis showed that the envelope mutation functions through the enhancement of virion attachment to BHK-21 cells, whereas the 2K mutation (and, to a lesser extent, the NS4a mutation) functions through the enhancement of viral RNA synthesis. Furthermore, we show that WNV superinfection exclusion is reversible by the treatment of the replicon cells with a flavivirus inhibitor. The preestablished replication of the replicon could be suppressed by infecting the cells with the 2K mutant WNV but not with the wild-type virus. These results suggest that WNV superinfection exclusion is a result of competition for intracellular host factors that are required for viral RNA synthesis. PMID:19726510

  7. Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

    OpenAIRE

    Magden, Julia; Takeda, Naokazu; Li, Tiancheng; Auvinen, Petri; Ahola, Tero; Miyamura, Tatsuo; Merits, Andres; Kääriäinen, Leevi

    2001-01-01

    Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group f...

  8. A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

    Energy Technology Data Exchange (ETDEWEB)

    Gumpper, Ryan H.; Li, Weike; Castañeda, Carlos H.; Scuderi, M. José; Bashkin, James K.; Luo, Ming; Dutch, Rebecca Ellis

    2018-02-07

    Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.

    IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit

  9. Preparation and characterization of high-specific activity radiolabeled 50 S measles virus RNA

    International Nuclear Information System (INIS)

    Spruance, S.L.; Ashton, B.N.; Smith, C.B.

    1980-01-01

    A method is described to radiolabeled measles virus RNA for hybridization studies. Tritiated nucleosides were added to the media of measles virus infected Vero cells and negative-strand (genome) RNA with a specific activity of 6X10 5 c.p.m./μg was purified from viral nucleocapsids. 50 S RNA was the sole RNA present in nucleocapsids and self-annealed to 50% due to the presence of 25% 50 S plus-strands (anti-genomes). (Auth.)

  10. Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

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    Jozef Julian Bujarski

    2013-03-01

    Full Text Available RNA recombination is one of the driving forces of genetic variability in (+-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings along with nonreplicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (i How various factors modulate the ability of viral replicase to switch templates, (ii What is the intracellular location of RNA-RNA template switchings, (iii Mechanisms and factors responsible for non-replicative RNA recombination, (iv Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (v What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

  11. Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

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    Kiran Zahid

    2015-01-01

    Full Text Available Satellite RNAs (satRNAs are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS transgene fused with a Cucumber mosaic virus (CMV Y satellite RNA (Y-Sat sequence (35S-GUS:Sat was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

  12. Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

    Science.gov (United States)

    Zahid, Kiran; Zhao, Jian-Hua; Smith, Neil A; Schumann, Ulrike; Fang, Yuan-Yuan; Dennis, Elizabeth S; Zhang, Ren; Guo, Hui-Shan; Wang, Ming-Bo

    2015-01-01

    Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

  13. Genotype phenotype mapping in RNA viruses - disjunctive normal form learning.

    Science.gov (United States)

    Wu, Chuang; Walsh, Andrew S; Rosenfeld, Roni

    2011-01-01

    RNA virus phenotypic changes often result from multiple alternative molecular mechanisms, where each mechanism involves changes to a small number of key residues. Accordingly, we propose to learn genotype-phenotype functions, using Disjunctive Normal Form (DNF) as the assumed functional form. In this study we develop DNF learning algorithms that attempt to construct predictors as Boolean combinations of covariates. We demonstrate the learning algorithm's consistency and efficiency on simulated sequences, and establish their biological relevance using a variety of real RNA virus datasets representing different viral phenotypes, including drug resistance, antigenicity, and pathogenicity. We compare our algorithms with previously published machine learning algorithms in terms of prediction quality: leave-one-out performance shows superior accuracy to other machine learning algorithms on the HIV drug resistance dataset and the UCIs promoter gene dataset. The algorithms are powerful in inferring the genotype-phenotype mapping from a moderate number of labeled sequences, as are typically produced in mutagenesis experiments. They can also greedily learn DNFs from large datasets. The Java implementation of our algorithms will be made publicly available.

  14. Robustness promotes evolvability of thermotolerance in an RNA virus

    Directory of Open Access Journals (Sweden)

    Turner Paul E

    2008-08-01

    Full Text Available Abstract Background The ability for an evolving population to adapt to a novel environment is achieved through a balance of robustness and evolvability. Robustness is the invariance of phenotype in the face of perturbation and evolvability is the capacity to adapt in response to selection. Genetic robustness has been posited, depending on the underlying mechanism, to either decrease the efficacy of selection, or increase the possibility of future adaptation. However, the true effect of genetic robustness on evolvability in biological systems remains uncertain. Results Here we demonstrate that genetic robustness increases evolvability of thermotolerance in laboratory populations of the RNA virus φ6. We observed that populations founded by robust clones evolved greater resistance to heat shock, relative to populations founded by brittle (less-robust clones. Thus, we provide empirical evidence for the idea that robustness can promote evolvability in this environment, and further suggest that evolvability can arise indirectly via selection for robustness, rather than through direct selective action. Conclusion Our data imply that greater tolerance of mutational change is associated with virus adaptability in a new niche, a finding generally relevant to evolutionary biology, and informative for elucidating how viruses might evolve to emerge in new habitats and/or overcome novel therapies.

  15. NF90 Binds the Dengue Virus RNA 3′ Terminus and Is a Positive Regulator of Dengue Virus Replication

    Science.gov (United States)

    Gehrke, Lee

    2011-01-01

    Background Viral RNA translation and replication are regulated by sequence and structural elements in the 5′ and 3′ untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5′ m7GpppG cap, and a conserved 3′-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3′ terminus. Methodology/Principal Findings Proteins eluted from a dengue 3′ SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny. Conclusions/Significance The results indicate that NF90 interacts with the 3′ SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide. PMID:21386893

  16. Induction of virus resistance by exogenous application of double-stranded RNA.

    Science.gov (United States)

    Mitter, Neena; Worrall, Elizabeth A; Robinson, Karl E; Xu, Zhi Ping; Carroll, Bernard J

    2017-10-01

    Exogenous application of double-stranded RNA (dsRNA) for virus resistance in plants represents a very attractive alternative to virus resistant transgenic crops or pesticides targeting virus vectors. However, the instability of dsRNA sprayed onto plants is a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only 5 days. Innovative approaches, such as the use of nanoparticles as carriers of dsRNA for improved stability and sustained release, are emerging as key disruptive technologies. Knowledge is still limited about the mechanism of entry, transport and processing of exogenously applied dsRNA in plants. Cost of dsRNA and regulatory framework will be key influencers towards practical adoption of this technology. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Transfection of RNA from organ samples of infected animals represents a highly sensitive method for virus detection and recovery of classical swine fever virus.

    Science.gov (United States)

    Meyer, Denise; Schmeiser, Stefanie; Postel, Alexander; Becher, Paul

    2015-01-01

    Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.

  18. Allosteric inhibitors of Coxsackie virus A24 RNA polymerase.

    Science.gov (United States)

    Schein, Catherine H; Rowold, Diane; Choi, Kyung H

    2016-02-15

    Coxsackie virus A24 (CVA24), a causative agent of acute hemorrhagic conjunctivitis, is a prototype of enterovirus (EV) species C. The RNA polymerase (3D(pol)) of CVA24 can uridylylate the viral peptide linked to the genome (VPg) from distantly related EV and is thus, a good model for studying this reaction. Once UMP is bound, VPgpU primes RNA elongation. Structural and mutation data have identified a conserved binding surface for VPg on the RNA polymerase (3D(pol)), located about 20Å from the active site. Here, computational docking of over 60,000 small compounds was used to select those with the lowest (best) specific binding energies (BE) for this allosteric site. Compounds with varying structures and low BE were assayed for their effect on formation of VPgU by CVA24-3D(pol). Two compounds with the lowest specific BE for the site inhibited both uridylylation and formation of VPgpolyU at 10-20μM. These small molecules can be used to probe the role of this allosteric site in polymerase function, and may be the basis for novel antiviral compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus.

    Science.gov (United States)

    Wang, Minghong; Wang, Yong; Sun, Xiangzhong; Cheng, Jiasen; Fu, Yanping; Liu, Huiquan; Jiang, Daohong; Ghabrial, Said A; Xie, Jiatao

    2015-08-01

    Mycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus, Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1), in an apparently hypovirulent strain (SX466) of Sclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related to Rosellinia necatrix megabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5'-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) of Cryphonectria hypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1. Mycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus

  20. Birds shed RNA-viruses according to the pareto principle.

    Science.gov (United States)

    Jankowski, Mark D; Williams, Christopher J; Fair, Jeanne M; Owen, Jennifer C

    2013-01-01

    A major challenge in disease ecology is to understand the role of individual variation of infection load on disease transmission dynamics and how this influences the evolution of resistance or tolerance mechanisms. Such information will improve our capacity to understand, predict, and mitigate pathogen-associated disease in all organisms. In many host-pathogen systems, particularly macroparasites and sexually transmitted diseases, it has been found that approximately 20% of the population is responsible for approximately 80% of the transmission events. Although host contact rates can account for some of this pattern, pathogen transmission dynamics also depend upon host infectiousness, an area that has received relatively little attention. Therefore, we conducted a meta-analysis of pathogen shedding rates of 24 host (avian) - pathogen (RNA-virus) studies, including 17 bird species and five important zoonotic viruses. We determined that viral count data followed the Weibull distribution, the mean Gini coefficient (an index of inequality) was 0.687 (0.036 SEM), and that 22.0% (0.90 SEM) of the birds shed 80% of the virus across all studies, suggesting an adherence of viral shedding counts to the Pareto Principle. The relative position of a bird in a distribution of viral counts was affected by factors extrinsic to the host, such as exposure to corticosterone and to a lesser extent reduced food availability, but not to intrinsic host factors including age, sex, and migratory status. These data provide a quantitative view of heterogeneous virus shedding in birds that may be used to better parameterize epidemiological models and understand transmission dynamics.

  1. Birds shed RNA-viruses according to the pareto principle.

    Directory of Open Access Journals (Sweden)

    Mark D Jankowski

    Full Text Available A major challenge in disease ecology is to understand the role of individual variation of infection load on disease transmission dynamics and how this influences the evolution of resistance or tolerance mechanisms. Such information will improve our capacity to understand, predict, and mitigate pathogen-associated disease in all organisms. In many host-pathogen systems, particularly macroparasites and sexually transmitted diseases, it has been found that approximately 20% of the population is responsible for approximately 80% of the transmission events. Although host contact rates can account for some of this pattern, pathogen transmission dynamics also depend upon host infectiousness, an area that has received relatively little attention. Therefore, we conducted a meta-analysis of pathogen shedding rates of 24 host (avian - pathogen (RNA-virus studies, including 17 bird species and five important zoonotic viruses. We determined that viral count data followed the Weibull distribution, the mean Gini coefficient (an index of inequality was 0.687 (0.036 SEM, and that 22.0% (0.90 SEM of the birds shed 80% of the virus across all studies, suggesting an adherence of viral shedding counts to the Pareto Principle. The relative position of a bird in a distribution of viral counts was affected by factors extrinsic to the host, such as exposure to corticosterone and to a lesser extent reduced food availability, but not to intrinsic host factors including age, sex, and migratory status. These data provide a quantitative view of heterogeneous virus shedding in birds that may be used to better parameterize epidemiological models and understand transmission dynamics.

  2. Identification of a Novel RNA Virus Lethal to Tilapia

    Science.gov (United States)

    Eyngor, Marina; Zamostiano, Rachel; Kembou Tsofack, Japhette Esther; Berkowitz, Asaf; Bercovier, Hillel; Tinman, Simon; Lev, Menachem; Hurvitz, Avshalom; Galeotti, Marco; Eldar, Avi

    2014-01-01

    Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide. PMID:25232154

  3. Identification of a novel RNA virus lethal to tilapia.

    Science.gov (United States)

    Eyngor, Marina; Zamostiano, Rachel; Kembou Tsofack, Japhette Esther; Berkowitz, Asaf; Bercovier, Hillel; Tinman, Simon; Lev, Menachem; Hurvitz, Avshalom; Galeotti, Marco; Bacharach, Eran; Eldar, Avi

    2014-12-01

    Tilapines are important for the sustainability of ecological systems and serve as the second most important group of farmed fish worldwide. Significant mortality of wild and cultured tilapia has been observed recently in Israel. The etiological agent of this disease, a novel RNA virus, is described here, and procedures allowing its isolation and detection are revealed. The virus, denominated tilapia lake virus (TiLV), was propagated in primary tilapia brain cells or in an E-11 cell line, and it induced a cytopathic effect at 5 to 10 days postinfection. Electron microscopy revealed enveloped icosahedral particles of 55 to 75 nm. Low-passage TiLV, injected intraperitoneally in tilapia, induced a disease resembling the natural disease, which typically presents with lethargy, ocular alterations, and skin erosions, with >80% mortality. Histological changes included congestion of the internal organs (kidneys and brain) with foci of gliosis and perivascular cuffing of lymphocytes in the brain cortex; ocular inflammation included endophthalmitis and cataractous changes of the lens. The cohabitation of healthy and diseased fish demonstrated that the disease is contagious and that mortalities (80 to 100%) occur within a few days. Fish surviving the initial mortality were immune to further TiLV infections, suggesting the mounting of a protective immune response. Screening cDNA libraries identified a TiLV-specific sequence, allowing the design of a PCR-based diagnostic test. This test enables the specific identification of TiLV in tilapines and should help control the spread of this virus worldwide. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Noncoding Subgenomic Flavivirus RNA: Multiple Functions in West Nile Virus Pathogenesis and Modulation of Host Responses

    Directory of Open Access Journals (Sweden)

    Justin A. Roby

    2014-01-01

    Full Text Available Flaviviruses are a large group of positive strand RNA viruses transmitted by arthropods that include many human pathogens such as West Nile virus (WNV, Japanese encephalitis virus (JEV, yellow fever virus, dengue virus, and tick-borne encephalitis virus. All members in this genus tested so far are shown to produce a unique subgenomic flavivirus RNA (sfRNA derived from the 3' untranslated region (UTR. sfRNA is a product of incomplete degradation of genomic RNA by the cell 5'–3' exoribonuclease XRN1 which stalls at highly ordered secondary RNA structures at the beginning of the 3'UTR. Generation of sfRNA results in inhibition of XRN1 activity leading to an increase in stability of many cellular mRNAs. Mutant WNV deficient in sfRNA generation was highly attenuated displaying a marked decrease in cytopathicity in cells and pathogenicity in mice. sfRNA has also been shown to inhibit the antiviral activity of IFN-α/β by yet unknown mechanism and of the RNAi pathway by likely serving as a decoy substrate for Dicer. Thus, sfRNA is involved in modulating multiple cellular pathways to facilitate viral pathogenicity; however the overlying mechanism linking all these multiple functions of sfRNA remains to be elucidated.

  5. Flock House virus subgenomic RNA3 is replicated and its replication correlates with transactivation of RNA2

    International Nuclear Information System (INIS)

    Eckerle, Lance D.; Albarino, Cesar G.; Ball, L. Andrew.

    2003-01-01

    The nodavirus Flock House virus has a bipartite genome composed of RNAs 1 and 2, which encode the catalytic component of the RNA-dependent RNA polymerase (RdRp) and the capsid protein precursor, respectively. In addition to catalyzing replication of the viral genome, the RdRp also transcribes from RNA1 a subgenomic RNA3, which is both required for and suppressed by RNA2 replication. Here, we show that in the absence of RNA1 replication, FHV RdRp replicated positive-sense RNA3 transcripts fully and copied negative-sense RNA3 transcripts into positive strands. The two nonstructural proteins encoded by RNA3 were dispensable for replication, but sequences in the 3'-terminal 58 nucleotides were required. RNA3 variants that failed to replicate also failed to transactivate RNA2. These results imply that RNA3 is naturally produced both by transcription from RNA1 and by subsequent RNA1-independent replication and that RNA3 replication may be necessary for transactivation of RNA2

  6. High throughput multiplex real time PCR assay for the simultaneous quantification of DNA and RNA viruses infecting cassava plants

    OpenAIRE

    Otti, Gerald; Bouvaine, Sophie; Kimata, Bernadetha; Mkamillo, Geoffrey; Kumar, Lava; Tomlins, Keith; Maruthi, M.N.

    2016-01-01

    Aims: To develop a multiplex TaqMan-based real-time PCR assay (qPCR) for the simultaneous detection and quantification of both RNA and DNA viruses affecting cassava (Manihot esculenta) in eastern Africa.\\ud \\ud Methods and Results: The diagnostic assay was developed for two RNA viruses; Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) and two predominant DNA viruses; African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV), which cause t...

  7. Analysis of double-stranded RNA from microbial communities identifies double-stranded RNA virus-like elements.

    Science.gov (United States)

    Decker, Carolyn J; Parker, Roy

    2014-05-08

    Double-stranded RNA (dsRNA) can function as genetic information and may have served as genomic material before the existence of DNA-based life. By developing a method to purify dsRNA, we have investigated the diversity of dsRNA in microbial populations. We detect large dsRNAs in multiple microbial populations. Analysis of an aquatic microbial population reveals that some dsRNA sequences match metagenomic DNA, suggesting that microbes contain pools of sense-antisense transcripts. In addition, ∼30% of the dsRNA sequences are not present in the corresponding DNA pool and are strongly biased toward encoding novel proteins. Of these "dsRNA unique" sequences, only a small percentage share similarity to known viruses, a large fraction assemble into RNA virus-like contigs, and the remaining fraction has an unexplained origin. These results have uncovered dsRNA virus-like elements and underscore that dsRNA potentially represents an additional reservoir of genetic information in microbial populations. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Screening for the Location of RNA using the Chloride Ion Distribution in Simulations of Virus Capsids.

    Science.gov (United States)

    Larsson, Daniel S D; van der Spoel, David

    2012-07-10

    The complete structure of the genomic material inside a virus capsid remains elusive, although a limited amount of symmetric nucleic acid can be resolved in the crystal structure of 17 icosahedral viruses. The negatively charged sugar-phosphate backbone of RNA and DNA as well as the large positive charge of the interior surface of the virus capsids suggest that electrostatic complementarity is an important factor in the packaging of the genomes in these viruses. To test how much packing information is encoded by the electrostatic and steric envelope of the capsid interior, we performed extensive all-atom molecular dynamics (MD) simulations of virus capsids with explicit water molecules and solvent ions. The model systems were two small plant viruses in which significant amounts of RNA has been observed by X-ray crystallography: satellite tobacco mosaic virus (STMV, 62% RNA visible) and satellite tobacco necrosis virus (STNV, 34% RNA visible). Simulations of half-capsids of these viruses with no RNA present revealed that the binding sites of RNA correlated well with regions populated by chloride ions, suggesting that it is possible to screen for the binding sites of nucleic acids by determining the equilibrium distribution of negative ions. By including the crystallographically resolved RNA in addition to ions, we predicted the localization of the unresolved RNA in the viruses. Both viruses showed a hot-spot for RNA binding at the 5-fold symmetry axis. The MD simulations were compared to predictions of the chloride density based on nonlinear Poisson-Boltzmann equation (PBE) calculations with mobile ions. Although the predictions are superficially similar, the PBE calculations overestimate the ion concentration close to the capsid surface and underestimate it far away, mainly because protein dynamics is not taken into account. Density maps from chloride screening can be used to aid in building atomic models of packaged virus genomes. Knowledge of the principles of

  9. Nucleotide composition of the Zika virus RNA genome and its codon usage

    NARCIS (Netherlands)

    van Hemert, Formijn; Berkhout, Ben

    2016-01-01

    RNA viruses have genomes with a distinct nucleotide composition and codon usage. We present the global characteristics of the RNA genome of Zika virus (ZIKV), an emerging pathogen within the Flavivirus genus. ZIKV was first isolated in 1947 in Uganda, caused a widespread epidemic in South and

  10. HIV-1 genomic RNA diversification following sexual and parenteral virus transmission

    NARCIS (Netherlands)

    Wolfs, T. F.; Zwart, G.; Bakker, M.; Goudsmit, J.

    1992-01-01

    Human immunodeficiency virus type 1 (HIV-1) genomic RNA variation was studied in seven presumed donor-recipient pairs directly following sexual (6/7) or parenteral (1/7) transmission. The first RNA-positive serum sample of each recipient and the serum sample of the virus transmitter, identified by

  11. Promotion of Hendra Virus Replication by MicroRNA 146a

    OpenAIRE

    Stewart, Cameron R.; Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.

    2013-01-01

    Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate imm...

  12. A new virus discovered by immunocapture of double-stranded RNA, a rapid method for virus enrichment in metagenomic studies.

    Science.gov (United States)

    Blouin, Arnaud G; Ross, Howard A; Hobson-Peters, Jody; O'Brien, Caitlin A; Warren, Ben; MacDiarmid, Robin

    2016-09-01

    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.

  13. Infection and RNA recombination of Brome mosaic virus in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Dzianott, Aleksandra; Bujarski, Jozef J.

    2004-01-01

    Ecotypes of Arabidopsis thaliana supported the replication and systemic spread of Brome mosaic virus (BMV) RNAs. Infection was induced either by manual inoculation with viral RNA or by BMV virions, demonstrating that virus disassembly did not prevent infection. When in vitro-transcribed BMV RNAs 1-3 were used, production of subgenomic RNA4 was observed, showing that BMV RNA replication and transcription had occurred. Furthermore, inoculations of the transgenic Arabidopsis line that expressed a suppressor of RNA interference (RNAi) pathway markedly increased the BMV RNA concentrations. Inoculations with designed BMV RNA3 recombination vectors generated both homologous and nonhomologous BMV RNA-RNA recombinants. Thus, all cellular factors essential for BMV RNA replication, transcription, and RNA recombination were shown to be present in Arabidopsis. The current scope of understanding of the model Arabidopsis plant system should facilitate the identification of these factors governing the BMV life cycle

  14. The utility of siRNA transcripts produced by RNA polymerase i in down regulating viral gene expression and replication of negative- and positive-strand RNA viruses

    International Nuclear Information System (INIS)

    McCown, Matthew; Diamond, Michael S.; Pekosz, Andrew

    2003-01-01

    Short interfering double-stranded RNAs (siRNAs) expressed under the control of an RNA polymerase I promoter system were used to target gene expression of influenza A and West Nile virus. Decreased RNA and protein expression was induced in a sequence-specific manner--reducing sequence complementarity from 21 to 17 nucleotides abrogated the siRNA effect. Reduced M 2 expression resulted in a decrease in total and infectious influenza A virus production. WNV protein expression, genomic RNA, and infectious virus production were all dramatically reduced by siRNAs targeting two distinct viral sequences. The data demonstrate the utility of plasmid-driven siRNAs in regulating the expression of single viral genes, global viral gene expression, as a potential antiviral treatment, and as a genetic tool for viruses whose genomes are difficult to manipulate

  15. Virus-specific mRNA capping enzyme encoded by hepatitis E virus.

    Science.gov (United States)

    Magden, J; Takeda, N; Li, T; Auvinen, P; Ahola, T; Miyamura, T; Merits, A; Kääriäinen, L

    2001-07-01

    Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.

  16. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis.

    Science.gov (United States)

    Ives, Annette; Ronet, Catherine; Prevel, Florence; Ruzzante, Giulia; Fuertes-Marraco, Silvia; Schutz, Frederic; Zangger, Haroun; Revaz-Breton, Melanie; Lye, Lon-Fye; Hickerson, Suzanne M; Beverley, Stephen M; Acha-Orbea, Hans; Launois, Pascal; Fasel, Nicolas; Masina, Slavica

    2011-02-11

    Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus-1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

  17. Simplified methods for the construction of RNA and DNA virus infectious clones.

    Science.gov (United States)

    Nagata, Tatsuya; Inoue-Nagata, Alice Kazuko

    2015-01-01

    Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.

  18. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

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    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  19. Examination for double-stranded RNA viruses in Trichomonas gallinae and identification of a novel sequence of a Trichomonas vaginalis virus.

    Science.gov (United States)

    Gerhold, Richard W; Allison, Andrew B; Sellers, Holly; Linnemann, Erich; Chang, T-H; Alderete, John F

    2009-09-01

    To determine if double-stranded RNA (dsRNA) viruses exist and are potential virulence factors in Trichomonas gallinae, virus purification via ultracentrifugation was attempted for 12 T. gallinae isolates recovered from wild birds. Following purification, virus-like particles were not observed by transmission electron microscopy, nor were dsRNA segments visualized in agarose gels after electrophoresis of extracted RNA from any of the 12 T. gallinae isolates. However, virus particles and dsRNA segments were detected from a previously determined virus-infected T. vaginalis isolate as a control using identical purification procedures. Subsequent reverse transcription-polymerase chain reaction analysis of the dsRNA of the virus in this isolate revealed a novel sequence of the RNA-dependent RNA polymerase gene of T. vaginalis viruses.

  20. Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing.

    Science.gov (United States)

    Xu, Chenxi; Sun, Xuepeng; Taylor, Angela; Jiao, Chen; Xu, Yimin; Cai, Xiaofeng; Wang, Xiaoli; Ge, Chenhui; Pan, Guanghui; Wang, Quanxi; Fei, Zhangjun; Wang, Quanhua

    2017-06-01

    Tomato is a major vegetable crop that has tremendous popularity. However, viral disease is still a major factor limiting tomato production. Here, we report the tomato virome identified through sequencing small RNAs of 170 field-grown samples collected in China. A total of 22 viruses were identified, including both well-documented and newly detected viruses. The tomato viral community is dominated by a few species, and they exhibit polymorphisms and recombination in the genomes with cold spots and hot spots. Most samples were coinfected by multiple viruses, and the majority of identified viruses are positive-sense single-stranded RNA viruses. Evolutionary analysis of one of the most dominant tomato viruses, Tomato yellow leaf curl virus (TYLCV), predicts its origin and the time back to its most recent common ancestor. The broadly sampled data have enabled us to identify several unreported viruses in tomato, including a completely new virus, which has a genome of ∼13.4 kb and groups with aphid-transmitted viruses in the genus Cytorhabdovirus Although both DNA and RNA viruses can trigger the biogenesis of virus-derived small interfering RNAs (vsiRNAs), we show that features such as length distribution, paired distance, and base selection bias of vsiRNA sequences reflect different plant Dicer-like proteins and Argonautes involved in vsiRNA biogenesis. Collectively, this study offers insights into host-virus interaction in tomato and provides valuable information to facilitate the management of viral diseases. IMPORTANCE Tomato is an important source of micronutrients in the human diet and is extensively consumed around the world. Virus is among the major constraints on tomato production. Categorizing virus species that are capable of infecting tomato and understanding their diversity and evolution are challenging due to difficulties in detecting such fast-evolving biological entities. Here, we report the landscape of the tomato virome in China, the leading country in

  1. Transfer of the 3' non-translated region of grapevine chrome mosaic virus RNA-1 by recombination to tomato black ring virus RNA-2 in pseudorecombinant isolates.

    Science.gov (United States)

    Le Gall, O; Candresse, T; Dunez, J

    1995-05-01

    In grapevine chrome mosaic and tomato black ring viruses (GCMV and TBRV), as in many other nepoviruses, the 3' non-translated regions (3'NTR) are identical between the two genomic RNAs. We have investigated the structure of the 3'NTR of two recombinant isolates which contain GCMV RNA-1 and TBRV RNA-2. In these isolates, the 3'NTR of RNA-1 was transferred to RNA-2, thus restoring the 3' identity. The transfer occurred within three passages, and probably contributes to the spread of randomly appearing mutations from one genomic RNA to the other. The site of recombination is near the 3' end of the open reading frame.

  2. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway.

    Directory of Open Access Journals (Sweden)

    Irma Sánchez-Vargas

    2009-02-01

    Full Text Available A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi, is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA, which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs. These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2 infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

  3. Intermolecular RNA Recombination Occurs at Different Frequencies in Alternate Forms of Brome Mosaic Virus RNA Replication Compartments

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    Hernan Garcia-Ruiz

    2018-03-01

    Full Text Available Positive-strand RNA viruses replicate their genomes in membrane-bound replication compartments. Brome mosaic virus (BMV replicates in vesicular invaginations of the endoplasmic reticulum membrane. BMV has served as a productive model system to study processes like virus-host interactions, RNA replication and recombination. Here we present multiple lines of evidence showing that the structure of the viral RNA replication compartments plays a fundamental role and that recruitment of parental RNAs to a common replication compartment is a limiting step in intermolecular RNA recombination. We show that a previously defined requirement for an RNA recruitment element on both parental RNAs is not to function as a preferred crossover site, but in order for individual RNAs to be recruited into the replication compartments. Moreover, modulating the form of the replication compartments from spherular vesicles (spherules to more expansive membrane layers increased intermolecular RNA recombination frequency by 200- to 1000-fold. We propose that intermolecular RNA recombination requires parental RNAs to be recruited into replication compartments as monomers, and that recruitment of multiple RNAs into a contiguous space is much more common for layers than for spherules. These results could explain differences in recombination frequencies between viruses that replicate in association with smaller spherules versus larger double-membrane vesicles and convoluted membranes.

  4. Broad RNA interference-mediated antiviral immunity and virus-specific inducible responses in Drosophila.

    Science.gov (United States)

    Kemp, Cordula; Mueller, Stefanie; Goto, Akira; Barbier, Vincent; Paro, Simona; Bonnay, François; Dostert, Catherine; Troxler, Laurent; Hetru, Charles; Meignin, Carine; Pfeffer, Sébastien; Hoffmann, Jules A; Imler, Jean-Luc

    2013-01-15

    The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity and has led to some important discoveries about the sensing and signaling of microbial infections. The response of Drosophila to virus infections remains poorly characterized and appears to involve two facets. On the one hand, RNA interference involves the recognition and processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible response controlled by the evolutionarily conserved JAK-STAT pathway contributes to the antiviral host defense. To clarify the contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by Drosophila C virus and cricket paralysis virus, two members of the Dicistroviridae family, which contrasts with the susceptibility of Dcr-2 mutant flies to many viruses, including the DNA virus invertebrate iridescent virus 6. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by Drosophila C virus or by two unrelated RNA viruses, Flock House virus and Sindbis virus. Overall, our data reveal that RNA interference is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK-STAT pathway appears to be virus specific.

  5. Error baseline rates of five sample preparation methods used to characterize RNA virus populations.

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    Jeffrey R Kugelman

    Full Text Available Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic "no amplification" method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a "targeted" amplification method, sequence-independent single-primer amplification (SISPA as a "random" amplification method, rolling circle reverse transcription sequencing (CirSeq as an advanced "no amplification" method, and Illumina TruSeq RNA Access as a "targeted" enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4-5 of all compared methods.

  6. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    International Nuclear Information System (INIS)

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F.; Verdaguer, Núria

    2012-01-01

    The RNA-dependent RNA polymerase of Thosea asigna virus has been purified and crystallized in two different crystal forms. Preliminary characterization of P2 1 2 1 2 and C222 1 crystals is reported. Co-crystallization experiments in the presence of lutetium produced a heavy-atom derivative suitable for structure determination. Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2 1 2 1 2 and diffracts up to 2.1 Å and the RdRp-Lu 3+ derivative co-crystals belong to the C222 1 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses

  7. Detection of Leishmania RNA virus in Leishmania parasites.

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    Haroun Zangger

    Full Text Available 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.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.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.

  8. RNA synthesis by cells or protoplasts isolated from broadbean leaves infected with broadbean mottle virus

    International Nuclear Information System (INIS)

    Romero, J.

    1973-01-01

    Cells or protoplasts isolated from leaves of Vicia faba infected with broadbean mottle virus incorporated specifically uridine- 14 C into genomic viral RNA, and into an RNA which corresponds to the rapidly-labelled unencapsulated RNA synthesized by leaf tissue of infected broadbean [fr

  9. Cloning and profiling of small RNAs from cucumber mosaic virus satellite RNA.

    Science.gov (United States)

    Fang, Yuan-Yuan; Smith, Neil A; Zhao, Jian-Hua; Lee, Joanne R M; Guo, Hui-Shan; Wang, Ming-Bo

    2015-01-01

    RNA silencing is not only a gene regulation mechanism that is conserved in a broad range of eukaryotes but also an adaptive immune response against foreign nucleic acids including viruses in plants. A major feature of RNA silencing is the production of small RNA (sRNA) of 21-24 nucleotides (nt) in length from double-stranded (ds) or hairpin-like (hp) RNA by Dicer-like (DCL) proteins. These sRNAs guide the binding and cleavage of cognate single-stranded (ss) RNA by an RNA silencing complex. Like all plant viruses and subviral agents, replication of viral satellite RNAs (satRNAs) is associated with the accumulation of 21-24 nt viral small interfering RNA (vsiRNA) derived from the whole region of a satRNA genome in both plus and minus-strand polarities. These satRNA-derived siRNAs (satsiRNAs) have recently been shown to play an important role in the trilateral interactions among host plants, helper viruses and satRNAs. Here, we describe the cloning and profile analysis of satsiRNAs from satRNAs of Cucumber mosaic virus (CMV). We also describe a method to minimize the strand bias that often occurs during vsiRNA cloning and sequencing.

  10. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

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    Adrian Valli

    Full Text Available RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  11. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Science.gov (United States)

    Valli, Adrian; Busnadiego, Idoia; Maliogka, Varvara; Ferrero, Diego; Castón, José R; Rodríguez, José Francisco; García, Juan Antonio

    2012-01-01

    RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  12. Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

    Science.gov (United States)

    Samuel, Glady Hazitha; Wiley, Michael R; Badawi, Atif; Adelman, Zach N; Myles, Kevin M

    2016-11-29

    Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

  13. The cis-acting replication signal at the 3' end of Flock House virus RNA2 is RNA3-dependent

    International Nuclear Information System (INIS)

    Albarino, Cesar G.; Eckerle, Lance D.; Ball, L. Andrew

    2003-01-01

    The nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3' 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3' termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3

  14. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

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    Ying Wen Huang

    Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

  15. Emaravirus-specific degenerate PCR primers allowed the identification of partial RNA-dependent RNA polymerase sequences of Maize red stripe virus and Pigeonpea sterility mosaic virus.

    Science.gov (United States)

    Elbeaino, Toufic; Whitfield, Anna; Sharma, Mamta; Digiaro, Michele

    2013-03-01

    Emaravirus is a recently established viral genus that includes two approved virus species: European mountain ash ringspot-associated virus (EMARaV) and Fig mosaic virus (FMV). Other described but unclassified viruses appear to share biological characteristics similar to emaraviruses, including segmented, negative-single stranded RNA genomes with enveloped virions approximately 80-200nm in diameter. Sequence analysis of emaravirus genomes revealed the presence of conserved amino acid sequences in the RNA-dependent RNA polymerase gene (RdRp) denoted as pre-motif A, motifs A and C. Degenerate oligonucleotide primers were developed to these conserved sequences and were shown to amplify in reverse transcription-polymerase chain reaction assay (RT-PCR) DNA fragments of 276bp and 360bp in size. These primers efficiently detected emaraviruses with known sequences available in the database (FMV and EMARaV); they also detected viruses with limited sequence information such as Pigeonpea sterility mosaic virus (PPSMV) and Maize red stripe virus (MRSV). The degenerate primers designed on pre-motif A and motif A sequences successfully amplified the four species used as positive controls (276bp), whereas those of motifs A and C failed to detect only MRSV. The amino acid sequences obtained from PPSMV and MRSV shared the highest identity with those of two other tentative species of the Emaravirus genus, Rose rosette virus (RRV) (69%) and Redbud yellow ringspot virus (RYRV) (60%), respectively. The phylogenetic tree constructed with 92 amino acid-long portions of polypeptide putatively encoded by RNA1 of definitive and tentative emaravirus species clustered PPSMV and MRSV in two separate clades close to RRV and Raspberry leaf blotch virus (RLBV), respectively. The newly developed degenerate primers have proved their efficacy in amplifying new emaravirus-specific sequences; accordingly, they could be useful in identifying new emaravirus-like species in nature. Copyright © 2012

  16. In silico discovery and modeling of non-coding RNA structure in viruses.

    Science.gov (United States)

    Moss, Walter N; Steitz, Joan A

    2015-12-01

    This review covers several computational methods for discovering structured non-coding RNAs in viruses and modeling their putative secondary structures. Here we will use examples from two target viruses to highlight these approaches: influenza A virus-a relatively small, segmented RNA virus; and Epstein-Barr virus-a relatively large DNA virus with a complex transcriptome. Each system has unique challenges to overcome and unique characteristics to exploit. From these particular cases, generically useful approaches can be derived for the study of additional viral targets. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Inhibition of RNA Helicases of ssRNA+ Virus Belonging to Flaviviridae, Coronaviridae and Picornaviridae Families

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    Irene Briguglio

    2011-01-01

    Full Text Available Many viral pathogens encode the motor proteins named RNA helicases which display various functions in genome replication. General strategies to design specific and selective drugs targeting helicase for the treatment of viral infections could act via one or more of the following mechanisms: inhibition of the NTPase activity, by interferences with ATP binding and therefore by limiting the energy required for the unwinding and translocation, or by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state; inhibition of nucleic acids binding to the helicase; inhibition of coupling of ATP hydrolysis to unwinding; inhibition of unwinding by sterically blocking helicase translocation. Recently, by in vitro screening studies, it has been reported that several benzotriazole, imidazole, imidazodiazepine, phenothiazine, quinoline, anthracycline, triphenylmethane, tropolone, pyrrole, acridone, small peptide, and Bananin derivatives are endowed with helicase inhibition of pathogen viruses belonging to Flaviviridae, Coronaviridae, and Picornaviridae families.

  18. Identification of Novel Positive-Strand RNA Viruses by Metagenomic Analysis of Archaea-Dominated Yellowstone Hot Springs

    Science.gov (United States)

    Bolduc, Benjamin; Shaughnessy, Daniel P.; Wolf, Yuri I.; Koonin, Eugene V.; Roberto, Francisco F.

    2012-01-01

    There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes. PMID:22379100

  19. Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes.

    Science.gov (United States)

    Carissimo, Guillaume; Eiglmeier, Karin; Reveillaud, Julie; Holm, Inge; Diallo, Mawlouth; Diallo, Diawo; Vantaux, Amélie; Kim, Saorin; Ménard, Didier; Siv, Sovannaroth; Belda, Eugeni; Bischoff, Emmanuel; Antoniewski, Christophe; Vernick, Kenneth D

    2016-01-01

    Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O'nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.

  20. In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus

    Science.gov (United States)

    Zhang, Xing; Ding, Ke; Yu, Xuekui; Chang, Winston; Sun, Jingchen; Hong Zhou, Z.

    2015-11-01

    Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.

  1. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

    Directory of Open Access Journals (Sweden)

    Olga V Viktorovskaya

    2016-08-01

    Full Text Available There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses replication.Seventy-nine novel RNA binding proteins for dengue virus (DENV were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated.The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps

  2. Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains

    International Nuclear Information System (INIS)

    Bedows, E.; Payne, F.E.; Kohne, D.E.; Tourtellotte, W.W.

    1982-01-01

    A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs. 70-100% of the measles virus-specific RNA present in these SSPE brain samples were recovered in this enriched fraction. (Auth.)

  3. New Kids on the Block: RNA-Based Influenza Virus Vaccines.

    Science.gov (United States)

    Scorza, Francesco Berlanda; Pardi, Norbert

    2018-04-01

    RNA-based immunization strategies have emerged as promising alternatives to conventional vaccine approaches. A substantial body of published work demonstrates that RNA vaccines can elicit potent, protective immune responses against various pathogens. Consonant with its huge impact on public health, influenza virus is one of the best studied targets of RNA vaccine research. Currently licensed influenza vaccines show variable levels of protection against seasonal influenza virus strains but are inadequate against drifted and pandemic viruses. In recent years, several types of RNA vaccines demonstrated efficacy against influenza virus infections in preclinical models. Additionally, comparative studies demonstrated the superiority of some RNA vaccines over the currently used inactivated influenza virus vaccines in animal models. Based on these promising preclinical results, clinical trials have been initiated and should provide valuable information about the translatability of the impressive preclinical data to humans. This review briefly describes RNA-based vaccination strategies, summarizes published preclinical and clinical data, highlights the roadblocks that need to be overcome for clinical applications, discusses the landscape of industrial development, and shares the authors' personal perspectives about the future of RNA-based influenza virus vaccines.

  4. Computational identification of hepatitis C virus associated microRNA-mRNA regulatory modules in human livers

    OpenAIRE

    Peng, Xinxia; Li, Yu; Walters, Kathie-Anne; Rosenzweig, Elizabeth R; Lederer, Sharon L; Aicher, Lauri D; Proll, Sean; Katze, Michael G

    2009-01-01

    Abstract Background Hepatitis C virus (HCV) is a major cause of chronic liver disease by infecting over 170 million people worldwide. Recent studies have shown that microRNAs (miRNAs), a class of small non-coding regulatory RNAs, are involved in the regulation of HCV infection, but their functions have not been systematically studied. We propose an integrative strategy for identifying the miRNA-mRNA regulatory modules that are associated with HCV infection. This strategy combines paired expre...

  5. RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions.

    Science.gov (United States)

    Silva, Amanda Perse da; Lopes, Juliana Freitas; Paula, Vanessa Salete de

    2014-01-01

    The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative. Copyright © 2014. Published by Elsevier Editora Ltda.

  6. The icosahedral RNA virus as a grotto: organizing the genome into stalagmites and stalactites.

    Science.gov (United States)

    Harvey, Stephen C; Zeng, Yingying; Heitsch, Christine E

    2013-03-01

    There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure--often one or more stem-loops--either promotes assembly or is required for assembly, but there are others where specific packaging signals are apparently not required. With regard to the assembly pathway, in those cases where stem-loops are involved, the first step is generally believed to be binding of the capsid proteins to these "fingers" of the RNA secondary structure. In the mature virus, the core of the RNA would then occupy the center of the viral particle, and the stem-loops would reach outward, towards the capsid, like stalagmites reaching up from the floor of a grotto towards the ceiling. Those viruses whose assembly does not depend on protein binding to stem-loops could have a different structure, with the core of the RNA lying just under the capsid, and the fingers reaching down into the interior of the virus, like stalactites. We review the literature on these alternative structures, focusing on RNA selectivity and the assembly mechanism, and we propose experiments aimed at determining, in a given virus, which of the two structures actually occurs.

  7. A human torque teno virus encodes a microRNA that inhibits interferon signaling.

    Directory of Open Access Journals (Sweden)

    Rodney P Kincaid

    Full Text Available Torque teno viruses (TTVs are a group of viruses with small, circular DNA genomes. Members of this family are thought to ubiquitously infect humans, although causal disease associations are currently lacking. At present, there is no understanding of how infection with this diverse group of viruses is so prevalent. Using a combined computational and synthetic approach, we predict and identify miRNA-coding regions in diverse human TTVs and provide evidence for TTV miRNA production in vivo. The TTV miRNAs are transcribed by RNA polymerase II, processed by Drosha and Dicer, and are active in RISC. A TTV mutant defective for miRNA production replicates as well as wild type virus genome; demonstrating that the TTV miRNA is dispensable for genome replication in a cell culture model. We demonstrate that a recombinant TTV genome is capable of expressing an exogenous miRNA, indicating the potential utility of TTV as a small RNA vector. Gene expression profiling of host cells identifies N-myc (and STAT interactor (NMI as a target of a TTV miRNA. NMI transcripts are directly regulated through a binding site in the 3'UTR. SiRNA knockdown of NMI contributes to a decreased response to interferon signaling. Consistent with this, we show that a TTV miRNA mediates a decreased response to IFN and increased cellular proliferation in the presence of IFN. Thus, we add Annelloviridae to the growing list of virus families that encode miRNAs, and suggest that miRNA-mediated immune evasion can contribute to the pervasiveness associated with some of these viruses.

  8. RNA structural constraints in the evolution of the influenza A virus genome NP segment

    NARCIS (Netherlands)

    A.P. Gultyaev (Alexander); A. Tsyganov-Bodounov (Anton); M.I. Spronken (Monique); S. Van Der Kooij (Sander); R.A.M. Fouchier (Ron); R.C.L. Olsthoorn (René)

    2014-01-01

    textabstractConserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length,

  9. Characterization of murine hepatitis virus (JHM) RNA from rats with experimental encephalomyelitis.

    Science.gov (United States)

    Jackson, D P; Percy, D H; Morris, V L

    1984-09-01

    When Wistar Furth rats are inoculated intracerebrally with the murine hepatitis virus JHM they often develop a demyelinating disease with resulting hind leg paralysis. Using an RNA transfer procedure and hybridization kinetic analysis, the virus-specific RNA in these rats was characterized. The pattern of JHM-specific RNA varied with individual infections of Wistar Furth rats. However, two species of JHM-specific RNA, the nucleocapsid and a 2.1-2.4 X 10(6)-Da RNA species were generally present. A general decrease in JHM-specific RNA in brains and spinal cord samples taken later than 20 days postinoculation was observed; however, JHM-specific RNA persisted in the spinal cord longer than in the brain of these rats.

  10. A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses

    Science.gov (United States)

    2012-01-01

    Background Viruses are known to be the most abundant organisms on earth, yet little is known about their collective origin and evolutionary history. With exceptionally high rates of genetic mutation and mosaicism, it is not currently possible to resolve deep evolutionary histories of the known major virus groups. Metagenomics offers a potential means of establishing a more comprehensive view of viral evolution as vast amounts of new sequence data becomes available for comparative analysis. Results Bioinformatic analysis of viral metagenomic sequences derived from a hot, acidic lake revealed a circular, putatively single-stranded DNA virus encoding a major capsid protein similar to those found only in single-stranded RNA viruses. The presence and circular configuration of the complete virus genome was confirmed by inverse PCR amplification from native DNA extracted from lake sediment. The virus genome appears to be the result of a RNA-DNA recombination event between two ostensibly unrelated virus groups. Environmental sequence databases were examined for homologous genes arranged in similar configurations and three similar putative virus genomes from marine environments were identified. This result indicates the existence of a widespread but previously undetected group of viruses. Conclusions This unique viral genome carries implications for theories of virus emergence and evolution, as no mechanism for interviral RNA-DNA recombination has yet been identified, and only scant evidence exists that genetic exchange occurs between such distinct virus lineages. Reviewers This article was reviewed by EK, MK (nominated by PF) and AM. For the full reviews, please go to the Reviewers' comments section. PMID:22515485

  11. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, Amr [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Present address: Genomics Facility, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619 (Egypt); Hutchens, Heather M. [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Howard Berg, R. [Integrated Microscopy Facility, Donald Danforth Plant Science Center, Saint Louis, MO 63132 (United States); Sue Loesch-Fries, L., E-mail: loeschfr@purdue.edu [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States)

    2012-11-25

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  12. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    International Nuclear Information System (INIS)

    Ibrahim, Amr; Hutchens, Heather M.; Howard Berg, R.; Sue Loesch-Fries, L.

    2012-01-01

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  13. Vector-Borne Transmission Imposes a Severe Bottleneck on an RNA Virus Population

    OpenAIRE

    Forrester, Naomi L.; Guerbois, Mathilde; Seymour, Robert L.; Spratt, Heidi; Weaver, Scott C.

    2012-01-01

    RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via...

  14. The ATP-Dependent RNA Helicase DDX3X Modulates Herpes Simplex Virus 1 Gene Expression.

    Science.gov (United States)

    Khadivjam, Bita; Stegen, Camille; Hogue-Racine, Marc-Aurèle; El Bilali, Nabil; Döhner, Katinka; Sodeik, Beate; Lippé, Roger

    2017-04-15

    The human protein DDX3X is a DEAD box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, and mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication, and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection, but, rather, the protein controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway. IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, an RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export, and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here, we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and, consequently, virion assembly by a process that is independent of its ability to promote the interferon pathway. Copyright © 2017 American Society for Microbiology.

  15. Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén).

    Science.gov (United States)

    Xu, Qiufang; Liu, Haoqiu; Yuan, Pingping; Zhang, Xiaoxia; Chen, Qingqing; Jiang, Xuanli; Zhou, Yijun

    2017-05-03

    The small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples. We established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 10 3 fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation. A simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.

  16. Patterns of evolution and host gene mimicry in influenza and other RNA viruses.

    Directory of Open Access Journals (Sweden)

    Benjamin D Greenbaum

    2008-06-01

    Full Text Available It is well known that the dinucleotide CpG is under-represented in the genomic DNA of many vertebrates. This is commonly thought to be due to the methylation of cytosine residues in this dinucleotide and the corresponding high rate of deamination of 5-methycytosine, which lowers the frequency of this dinucleotide in DNA. Surprisingly, many single-stranded RNA viruses that replicate in these vertebrate hosts also have a very low presence of CpG dinucleotides in their genomes. Viruses are obligate intracellular parasites and the evolution of a virus is inexorably linked to the nature and fate of its host. One therefore expects that virus and host genomes should have common features. In this work, we compare evolutionary patterns in the genomes of ssRNA viruses and their hosts. In particular, we have analyzed dinucleotide patterns and found that the same patterns are pervasively over- or under-represented in many RNA viruses and their hosts suggesting that many RNA viruses evolve by mimicking some of the features of their host's genes (DNA and likely also their corresponding mRNAs. When a virus crosses a species barrier into a different host, the pressure to replicate, survive and adapt, leaves a footprint in dinucleotide frequencies. For instance, since human genes seem to be under higher pressure to eliminate CpG dinucleotide motifs than avian genes, this pressure might be reflected in the genomes of human viruses (DNA and RNA viruses when compared to those of the same viruses replicating in avian hosts. To test this idea we have analyzed the evolution of the influenza virus since 1918. We find that the influenza A virus, which originated from an avian reservoir and has been replicating in humans over many generations, evolves in a direction strongly selected to reduce the frequency of CpG dinucleotides in its genome. Consistent with this observation, we find that the influenza B virus, which has spent much more time in the human population, has

  17. The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection.

    Science.gov (United States)

    Wisskirchen, Christian; Ludersdorfer, Thomas H; Müller, Dominik A; Moritz, Eva; Pavlovic, Jovan

    2011-09-01

    The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.

  18. Phomopsis longicolla RNA virus 1-Novel virus at the edge of myco- and plant viruses

    Czech Academy of Sciences Publication Activity Database

    Hrabáková, Lenka; Koloniuk, Igor; Petrzik, Karel

    2017-01-01

    Roč. 506, June (2017), s. 14-18 ISSN 0042-6822 R&D Projects: GA MŠk LH13136; GA MŠk(CZ) EE2.3.30.0032 Grant - others:GA MŠk(CZ) LM2010005 Institutional support: RVO:60077344 Keywords : double-stranded-rna * molecular characterization * genus ourmiavirus Subject RIV: EE - Microbiology, Virology OBOR OECD: Virology Impact factor: 3.353, year: 2016

  19. Degradation of nucleic acids with ozone. II. Degradation of yeast RNA, yeast phenylalanine tRNA and tobacco mosaic virus RNA.

    Science.gov (United States)

    Shinriki, N; Ishizaki, K; Ikehata, A; Yoshizaki, T; Nomura, A; Miura, K; Mizuno, Y

    1981-10-27

    The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.

  20. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps

    Directory of Open Access Journals (Sweden)

    Überla Klaus

    2007-06-01

    Full Text Available Abstract Background Proteins of human and animal viruses are frequently expressed from RNA polymerase II dependent expression cassettes to study protein function and to develop gene-based vaccines. Initial attempts to express the G protein of vesicular stomatitis virus (VSV and the F protein of respiratory syncytial virus (RSV by eukaryotic promoters revealed restrictions at several steps of gene expression. Results Insertion of an intron flanked by exonic sequences 5'-terminal to the open reading frames (ORF of VSV-G and RSV-F led to detectable cytoplasmic mRNA levels of both genes. While the exonic sequences were sufficient to stabilise the VSV-G mRNA, cytoplasmic mRNA levels of RSV-F were dependent on the presence of a functional intron. Cytoplasmic VSV-G mRNA levels led to readily detectable levels of VSV-G protein, whereas RSV-F protein expression remained undetectable. However, RSV-F expression was observed after mutating two of four consensus sites for polyadenylation present in the RSV-F ORF. Expression levels could be further enhanced by codon optimisation. Conclusion Insufficient cytoplasmic mRNA levels and premature polyadenylation prevent expression of RSV-F by RNA polymerase II dependent expression plasmids. Since RSV replicates in the cytoplasm, the presence of premature polyadenylation sites and elements leading to nuclear instability should not interfere with RSV-F expression during virus replication. The molecular mechanisms responsible for the destabilisation of the RSV-F and VSV-G mRNAs and the different requirements for their rescue by insertion of an intron remain to be defined.

  1. RNA viruses can hijack vertebrate microRNAs to suppress innate immunity

    Science.gov (United States)

    Trobaugh, Derek W.; Gardner, Christina L.; Sun, Chengqun; Haddow, Andrew D.; Wang, Eryu; Chapnik, Elik; Mildner, Alexander; Weaver, Scott C.; Ryman, Kate D.; Klimstra, William B.

    2014-02-01

    Currently, there is little evidence for a notable role of the vertebrate microRNA (miRNA) system in the pathogenesis of RNA viruses. This is primarily attributed to the ease with which these viruses mutate to disrupt recognition and growth suppression by host miRNAs. Here we report that the haematopoietic-cell-specific miRNA miR-142-3p potently restricts the replication of the mosquito-borne North American eastern equine encephalitis virus in myeloid-lineage cells by binding to sites in the 3' non-translated region of its RNA genome. However, by limiting myeloid cell tropism and consequent innate immunity induction, this restriction directly promotes neurologic disease manifestations characteristic of eastern equine encephalitis virus infection in humans. Furthermore, the region containing the miR-142-3p binding sites is essential for efficient virus infection of mosquito vectors. We propose that RNA viruses can adapt to use antiviral properties of vertebrate miRNAs to limit replication in particular cell types and that this restriction can lead to exacerbation of disease severity.

  2. Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus.

    Science.gov (United States)

    Le, Dung Tien; Chu, Ha Duc; Sasaya, Takahide

    2015-01-01

    Rice tungro spherical virus (RTSV), also known as Rice waika virus, does not cause visible symptoms in infected rice plants. However, the virus plays a critical role in spreading Rice tungro bacilliform virus (RTBV), which is the major cause of severe symptoms of rice tungro disease. Recent studies showed that RNA interference (RNAi) can be used to develop virus-resistance transgenic rice plants. In this report, we presented simple procedures and protocols needed for the creation of transgenic rice plants capable of producing small interfering RNA specific against RTSV sequences. Notably, our study showed that 60 out of 64 individual hygromycin-resistant lines (putative transgenic lines) obtained through transformation carried transgenes designed for producing hairpin double-stranded RNA. Northern blot analyses revealed the presence of small interfering RNA of 21- to 24-mer in 46 out of 56 confirmed transgenic lines. Taken together, our study indicated that transgenic rice plants carrying an inverted repeat of 500-bp fragments encoding various proteins of RTSV can produce small interfering RNA from the hairpin RNA transcribed from that transgene. In light of recent studies with other viruses, it is possible that some of these transgenic rice lines might be resistant to RTSV.

  3. Efficient cellular release of Rift Valley fever virus requires genomic RNA.

    Directory of Open Access Journals (Sweden)

    Mary E Piper

    2011-03-01

    Full Text Available The Rift Valley fever virus is responsible for periodic, explosive epizootics throughout sub-Saharan Africa. The development of therapeutics targeting this virus is difficult due to a limited understanding of the viral replicative cycle. Utilizing a virus-like particle system, we have established roles for each of the viral structural components in assembly, release, and virus infectivity. The envelope glycoprotein, Gn, was discovered to be necessary and sufficient for packaging of the genome, nucleocapsid protein and the RNA-dependent RNA polymerase into virus particles. Additionally, packaging of the genome was found to be necessary for the efficient release of particles, revealing a novel mechanism for the efficient generation of infectious virus. Our results identify possible conserved targets for development of anti-phlebovirus therapies.

  4. Computational identification of hepatitis C virus associated microRNA-mRNA regulatory modules in human livers.

    Science.gov (United States)

    Peng, Xinxia; Li, Yu; Walters, Kathie-Anne; Rosenzweig, Elizabeth R; Lederer, Sharon L; Aicher, Lauri D; Proll, Sean; Katze, Michael G

    2009-08-11

    Hepatitis C virus (HCV) is a major cause of chronic liver disease by infecting over 170 million people worldwide. Recent studies have shown that microRNAs (miRNAs), a class of small non-coding regulatory RNAs, are involved in the regulation of HCV infection, but their functions have not been systematically studied. We propose an integrative strategy for identifying the miRNA-mRNA regulatory modules that are associated with HCV infection. This strategy combines paired expression profiles of miRNAs and mRNAs and computational target predictions. A miRNA-mRNA regulatory module consists of a set of miRNAs and their targets, in which the miRNAs are predicted to coordinately regulate the level of the target mRNA. We simultaneously profiled the expression of cellular miRNAs and mRNAs across 30 HCV positive or negative human liver biopsy samples using microarray technology. We constructed a miRNA-mRNA regulatory network, and using a graph theoretical approach, identified 38 miRNA-mRNA regulatory modules in the network that were associated with HCV infection. We evaluated the direct miRNA regulation of the mRNA levels of targets in regulatory modules using previously published miRNA transfection data. We analyzed the functional roles of individual modules at the systems level by integrating a large-scale protein interaction network. We found that various biological processes, including some HCV infection related canonical pathways, were regulated at the miRNA level during HCV infection. Our regulatory modules provide a framework for future experimental analyses. This report demonstrates the utility of our approach to obtain new insights into post-transcriptional gene regulation at the miRNA level in complex human diseases.

  5. Computational identification of hepatitis C virus associated microRNA-mRNA regulatory modules in human livers

    Directory of Open Access Journals (Sweden)

    Aicher Lauri D

    2009-08-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is a major cause of chronic liver disease by infecting over 170 million people worldwide. Recent studies have shown that microRNAs (miRNAs, a class of small non-coding regulatory RNAs, are involved in the regulation of HCV infection, but their functions have not been systematically studied. We propose an integrative strategy for identifying the miRNA-mRNA regulatory modules that are associated with HCV infection. This strategy combines paired expression profiles of miRNAs and mRNAs and computational target predictions. A miRNA-mRNA regulatory module consists of a set of miRNAs and their targets, in which the miRNAs are predicted to coordinately regulate the level of the target mRNA. Results We simultaneously profiled the expression of cellular miRNAs and mRNAs across 30 HCV positive or negative human liver biopsy samples using microarray technology. We constructed a miRNA-mRNA regulatory network, and using a graph theoretical approach, identified 38 miRNA-mRNA regulatory modules in the network that were associated with HCV infection. We evaluated the direct miRNA regulation of the mRNA levels of targets in regulatory modules using previously published miRNA transfection data. We analyzed the functional roles of individual modules at the systems level by integrating a large-scale protein interaction network. We found that various biological processes, including some HCV infection related canonical pathways, were regulated at the miRNA level during HCV infection. Conclusion Our regulatory modules provide a framework for future experimental analyses. This report demonstrates the utility of our approach to obtain new insights into post-transcriptional gene regulation at the miRNA level in complex human diseases.

  6. Stability of RNA silencing-based traits after virus infection

    DEFF Research Database (Denmark)

    Jørgensen, Bodil; Albrechtsen, Merete

    2007-01-01

    with constructs based on virus coat protein (CP) genes or other viral genes has been successfully used to engineer PTGS-mediated virus resistance into a large number of crop plants and some transgenic lines have been commercially exploited. However the discovery that plant viruses encode suppressors of gene...

  7. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition

    NARCIS (Netherlands)

    Das, Atze T.; Brummelkamp, Thijn R.; Westerhout, Ellen M.; Vink, Monique; Madiredjo, Mandy; Bernards, René; Berkhout, Ben

    2004-01-01

    Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We

  8. Human Immunodeficiency Virus Type 1 Escapes from RNA Interference-Mediated Inhibition

    NARCIS (Netherlands)

    Das, A.T.; Brummelkamp, T.R.; Westerhout, E.M.; Vink, M.; Madiredjo, M.; Bernards, R.A.; Berkhout, Ben

    2004-01-01

    Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We

  9. Genome transcription/translation of segmented, negative-strand RNA viruses

    NARCIS (Netherlands)

    Geerts-Dimitriadou, C.

    2011-01-01

    The requirements for alignment of capped RNA leader sequences along the viral genome during influenza transcription initiation (“cap-snatching”) have long been an enigma. Previous work on Tomato spotted wilt virus (TSWV) transcription initiation has revealed that this virus displays a

  10. A Novel Astrovirus-Like RNA Virus Detected in Human Stool

    NARCIS (Netherlands)

    Oude Munnink, Bas B.; Cotten, Matthew; Canuti, Marta; Deijs, Martin; Jebbink, Maarten F.; van Hemert, Formijn J.; Phan, My V. T.; Bakker, Margreet; Jazaeri Farsani, Seyed Mohammad; Kellam, Paul; van der Hoek, Lia

    2016-01-01

    Several novel clades of astroviruses have recently been identified in human faecal samples. Here, we describe a novel astrovirus-like RNA virus detected in human stools, which we have tentatively named bastrovirus. The genome of this novel virus consists of 6,300 nucleotides organized in three open

  11. HumanViCe: Host ceRNA network in virus infected cells in human

    Directory of Open Access Journals (Sweden)

    Suman eGhosal

    2014-07-01

    Full Text Available Host-virus interaction via host cellular components has been an important field of research in recent times. RNA interference mediated by short interfering RNAs and microRNAs (miRNA, is a widespread anti-viral defence strategy. Importantly, viruses also encode their own miRNAs. In recent times miRNAs were identified as key players in host-virus interaction. Furthermore, viruses were shown to exploit the host miRNA networks to suite their own need. The complex cross-talk between host and viral miRNAs and their cellular and viral targets forms the environment for viral pathogenesis. Apart from protein-coding mRNAs, non-coding RNAs may also be targeted by host or viral miRNAs in virus infected cells, and viruses can exploit the host miRNA mediated gene regulatory network via the competing endogenous RNA effect. A recent report showed that viral U-rich non-coding RNAs called HSUR, expressed in primate virus herpesvirus saimiri (HVS infected T cells, were able to bind to three host miRNAs, causing significant alteration in cellular level for one of the miRNAs. We have predicted protein coding and non protein-coding targets for viral and human miRNAs in virus infected cells. We identified viral miRNA targets within host non-coding RNA loci from AGO interacting regions in three different virus infected cells. Gene ontology (GO and pathway enrichment analysis of the genes comprising the ceRNA networks in the virus infected cells revealed enrichment of key cellular signalling pathways related to cell fate decisions and gene transcription, like Notch and Wnt signalling pathways, as well as pathways related to viral entry, replication and virulence. We identified a vast number of non-coding transcripts playing as potential ceRNAs to the immune response associated genes; e.g. APOBEC family genes, in some virus infected cells. All these information are compiled in HumanViCe, a comprehensive database that provides the potential ceRNA networks in virus

  12. Identification of novel RNA viruses in alfalfa (Medicago sativa): an Alphapartitivirus, a Deltapartitivirus, and a Marafivirus.

    Science.gov (United States)

    Kim, Hyein; Park, Dongbin; Hahn, Yoonsoo

    2018-01-05

    Genomic RNA molecules of plant RNA viruses are often co-isolated with the host RNAs, and their sequences can be detected in plant transcriptome datasets. Here, an alfalfa (Medicago sativa) transcriptome dataset was analyzed and three new RNA viruses were identified, which were named Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), and Medicago sativa marafivirus 1 (MsMV1). The RNA-dependent RNA polymerases of MsAPV1, MsDPV1, and MsMV1 showed about 68%, 58%, and 46% amino acid sequence identity, respectively, with their closest virus species. Sequence similarity and phylogenetic analyses indicated that MsAPV1, MsDPV1, and MsMV1 were novel RNA virus species that belong to the genus Alphapartitivirus of the family Partitiviridae, the genus Deltapartitivirus of the family Partitiviridae, and the genus Marafivirus of the family Tymoviridae, respectively. The bioinformatics procedure applied in this study may facilitate the identification of novel RNA viruses from plant transcriptome data. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Convergent Evolution of Argonaute-2 Slicer Antagonism in Two Distinct Insect RNA Viruses

    OpenAIRE

    van Mierlo, Joël T.; Bronkhorst, Alfred W.; Overheul, Gijs J.; Sadanandan, Sajna A.; Ekström, Jens-Ola; Heestermans, Marco; Hultmark, Dan; Antoniewski, Christophe; van Rij, Ronald P.

    2012-01-01

    Author Summary Multi-cellular organisms require a potent immune response to ensure survival under the ongoing assault by microbial pathogens. Co-evolution of virus and host shapes the genome of both pathogen and host. Using Drosophila melanogaster as a model, we study virus-host interactions in infections by Nora virus, a non-lethal natural pathogen of fruit flies. Insects depend on the RNA interference (RNAi) pathway for antiviral defense. A hallmark of the antiviral RNAi response is the pro...

  14. Generation of Constructs for DNA-Directed RNA Interference of Venezuelan Equine Encephalitis Virus Genes

    Science.gov (United States)

    2006-12-01

    the viral genome of the attenuated vaccine strain of VEE [14], TC-83, based on their suitability for siRNA design. Using the Silencer Express system...of an effective vaccine or therapeutic for VEE, a highly infectious virus, underscores the need for research in this area. In addition, the potential... poliovirus [5], influenza virus [6], Ebola virus [7], Coxsackievirus B3 [8], among others. RNAi involves sequence-specific gene silencing, or gene knockdown

  15. Mechanisms of Innate Immune Evasion In Re-Emerging RNA Viruses

    OpenAIRE

    Ma, Daphne Y.; Suthar, Mehul S.

    2015-01-01

    Recent outbreaks of Ebola, West Nile, Chikungunya, Middle Eastern Respiratory and other emerging/re-emerging RNA viruses continue to highlight the need to further understand the virus-host interactions that govern disease severity and infection outcome. As part of the early host antiviral defense, the innate immune system mediates pathogen recognition and initiation of potent antiviral programs that serve to limit virus replication and spread and activate adaptive immune responses. Concordant...

  16. Emaravirus: A Novel Genus of Multipartite, Negative Strand RNA Plant Viruses

    Science.gov (United States)

    Mielke-Ehret, Nicole; Mühlbach, Hans-Peter

    2012-01-01

    Ringspot symptoms in European mountain ash (Sorbus aucuparia L.), fig mosaic, rose rosette, raspberry leaf blotch, pigeonpea sterility mosaic (Cajanus cajan) and High Plains disease of maize and wheat were found to be associated with viruses that share several characteristics. They all have single-stranded multipartite RNA genomes of negative orientation. In some cases, double membrane-bound virus-like particles of 80 to 200 nm in diameter were found in infected tissue. Furthermore, at least five of these viruses were shown to be vectored by eriophyid mites. Sequences of European mountain ash ringspot-associated virus (EMARaV), Fig mosaic virus (FMV), rose rosette virus (RRV), raspberry leaf blotch virus (RLBV), pigeonpea sterility mosaic virus and High Plains virus strongly support their potential phylogenetic relationship. Therefore, after characterization of EMARaV, the novel genus Emaravirus was established, and FMV was the second virus species assigned to this genus. The recently sequenced RRV and RLBV are supposed to be additional members of this new group of plant RNA viruses. PMID:23170170

  17. Emaravirus: A Novel Genus of Multipartite, Negative Strand RNA Plant Viruses

    Directory of Open Access Journals (Sweden)

    Hans-Peter Mühlbach

    2012-09-01

    Full Text Available Ringspot symptoms in European mountain ash (Sorbus aucuparia L., fig mosaic, rose rosette, raspberry leaf blotch, pigeonpea sterility mosaic (Cajanus cajan and High Plains disease of maize and wheat were found to be associated with viruses that share several characteristics. They all have single-stranded multipartite RNA genomes of negative orientation. In some cases, double membrane-bound virus-like particles of 80 to 200 nm in diameter were found in infected tissue. Furthermore, at least five of these viruses were shown to be vectored by eriophyid mites. Sequences of European mountain ash ringspot-associated virus (EMARaV, Fig mosaic virus (FMV, rose rosette virus (RRV, raspberry leaf blotch virus (RLBV, pigeonpea sterility mosaic virus and High Plains virus strongly support their potential phylogenetic relationship. Therefore, after characterization of EMARaV, the novel genus Emaravirus was established, and FMV was the second virus species assigned to this genus. The recently sequenced RRV and RLBV are supposed to be additional members of this new group of plant RNA viruses.

  18. Haiku: New paradigm for the reverse genetics of emerging RNA viruses.

    Science.gov (United States)

    Atieh, Thérèse; El Ayoubi, Miriam Diala; Aubry, Fabien; Priet, Stéphane; de Lamballerie, Xavier; Nougairède, Antoine

    2018-01-01

    Reverse genetics is key technology for producing wild-type and genetically modified viruses. The ISA (Infectious Subgenomic Amplicons) method is a recent versatile and user-friendly reverse genetics method to rescue RNA viruses. The main constraint of its canonic protocol was the requirement to produce (e.g., by DNA synthesis or fusion PCR) 5' and 3' modified genomic fragments encompassing the human cytomegalovirus promoter (pCMV) and the hepatitis delta virus ribozyme/simian virus 40 polyadenylation signal (HDR/SV40pA), respectively. Here, we propose the ultimately simplified "Haiku" designs in which terminal pCMV and HDR/SV40pA sequences are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses belonging to genera Flavivirus, Alphavirus and Enterovirus in mosquito or mammalian cells using only standard PCR amplification techniques and starting from a variety of original materials including viral RNAs extracted from cell supernatant media or animal samples. We also demonstrate that, in specific experimental conditions, the presence of the HDR/SV40pA is not necessary to rescue the targeted viruses. These ultimately simplified "Haiku" designs provide an even more simple, rapid, versatile and cost-effective tool to rescue RNA viruses since only generation of overlapping amplicons encompassing the entire viral genome is now required to generate infectious virus. This new approach may completely modify our capacity to obtain infectious RNA viruses.

  19. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection

    DEFF Research Database (Denmark)

    Lanford, Robert E; Hildebrandt-Eriksen, Elisabeth S; Petri, Andreas

    2010-01-01

    The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA...

  20. Stability of hepatitis C virus RNA during specimen handling and storage prior to NASBA amplification

    NARCIS (Netherlands)

    Damen, M.; Sillekens, P.; Sjerps, M.; Melsert, R.; Frantzen, I.; Reesink, H. W.; Lelie, P. N.; Cuypers, H. T.

    1998-01-01

    The influence of different anticoagulants and pre-amplification storage conditions on the stability of hepatitis C virus (HCV)-RNA, as detected by the quantitative HCV NASBA assay (NASBA-QT), was studied. The HCV-RNA load remained stable for at least 15 months when serum or plasma samples (EDTA and

  1. Patterns of hepatitis C virus RNA levels during acute infection: the InC3 study

    NARCIS (Netherlands)

    Hajarizadeh, Behzad; Grady, Bart; Page, Kimberly; Kim, Arthur Y.; McGovern, Barbara H.; Cox, Andrea L.; Rice, Thomas M.; Sacks-Davis, Rachel; Bruneau, Julie; Morris, Meghan; Amin, Janaki; Schinkel, Janke; Applegate, Tanya; Maher, Lisa; Hellard, Margaret; Lloyd, Andrew R.; Prins, Maria; Dore, Gregory J.; Grebely, Jason; Lauer, Georg; Shoukry, Naglaa H.; Hahn, Judy; Shiboski, Steve; Alavi, Maryam; Bouchard, Rachel; Evans, Jennifer; May, Linda; Aneja, Jasneet; Teutsch, Suzy; White, Bethany; Wells, Brittany; Zang, Geng; Matthews, Gail; Yeung, Barbara; Prince, Leslie Erin; Roy, Elise; Bates, Anna; Enriquez, Jarliene; Chow, Sammy; McCredie, Luke; Aitken, Campbell; Doyle, Joseph; Spelman, Tim

    2015-01-01

    Understanding the patterns of HCV RNA levels during acute hepatitis C virus (HCV) infection provides insights into immunopathogenesis and is important for vaccine design. This study evaluated patterns of HCV RNA levels and associated factors among individuals with acute infection. Data were from an

  2. RNA virus interference via CRISPR/Cas13a system in plants

    KAUST Repository

    Aman, Rashid

    2018-01-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

  3. Complementation and recombination between alfalfa mosaic virus RNA3 mutants in tobacco plants

    NARCIS (Netherlands)

    van der Kuyl, A. C.; Neeleman, L.; Bol, J. F.

    1991-01-01

    Deletions were made in an infectious cDNA clone of alfalfa mosaic virus (AIMV) RNA3 and the replication of RNA transcripts of these cDNAs was studied in tobacco plants transformed with AIMV replicase genes (P12 plants). Previously, we found that deletions in the P3 gene did not affect accumulation

  4. A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions

    NARCIS (Netherlands)

    Ooms, Marcel; Huthoff, Hendrik; Russell, Rodney; Liang, Chen; Berkhout, Ben

    2004-01-01

    The genome of retroviruses, including human immunodeficiency virus type I (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the Psi

  5. RNA recombination in Hepatitis delta virus: Identification of a novel naturally occurring recombinant

    Directory of Open Access Journals (Sweden)

    Chia-Chi Lin

    2017-12-01

    Full Text Available Background/Purpose: Hepatitis delta virus (HDV is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity. It replicates in the nucleus by host RNA polymerase via a rolling circle mechanism. Similar to many RNA viruses encoding their own RNA-dependent RNA polymerases, homologous recombination of HDV occurs in mixed-genotype infections and in cultured cells cotransfected with two HDV sequences, as demonstrated by molecular analyses. Methods: Among 237 published complete genomic sequences, 34 sequences were reported from the small and isolated Miyako Island, Japan, and belonged to the Asia-specific genotypes, HDV-2 and HDV-4 (the majority of them belonged to the known Miyako Island-specific subgroup, HDV-4M. We investigated the presence of naturally occurring HDV recombinant in Miyako Island using phylogenetic and recombination analyses. Results: We identified a two-switch HDV-4/4M intersubtype recombinant with an unbranched rod-like RNA genome. Conclusion: Our data suggest that RNA recombination plays an important role in the rapid evolution of HDV, allowing the production of new HDV strains with correct genomic structures. Keywords: hepatitis delta virus, RNA recombination

  6. High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs

    NARCIS (Netherlands)

    Chen, Y.K.; Lohuis, H.; Goldbach, R.W.; Prins, M.W.

    2004-01-01

    The application of RNA-mediated resistance against Cucumber mosaic virus (CMV) by using single transgene constructs generally results in only a small portion of resistant individuals. Inverted repeat constructs encoding self-complementary double-stranded RNA have been demonstrated a potential way to

  7. Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus bisporus

    International Nuclear Information System (INIS)

    Sriskantha, A.; Wach, P.; Schlagnhaufer, B.; Romaine, C.P.

    1986-01-01

    Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from health sporophores. Enzyme activity was dependent upon the presence of Mg 2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The 3 H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . Cs 2 SO 4 equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 /times/ 10 6 and 1.4 /times/ 10 6 . The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs

  8. Error baseline rates of five sample preparation methods used to characterize RNA virus populations

    Science.gov (United States)

    Kugelman, Jeffrey R.; Wiley, Michael R.; Nagle, Elyse R.; Reyes, Daniel; Pfeffer, Brad P.; Kuhn, Jens H.; Sanchez-Lockhart, Mariano; Palacios, Gustavo F.

    2017-01-01

    Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic “no amplification” method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a “targeted” amplification method, sequence-independent single-primer amplification (SISPA) as a “random” amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced “no amplification” method, and Illumina TruSeq RNA Access as a “targeted” enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4−5) of all compared methods. PMID:28182717

  9. The RNA-dependent-RNA polymerase, an emerging antiviral drug target for the Hendra virus.

    Science.gov (United States)

    Velkov, Tony; Carbone, Vincenzo; Akter, Jesmin; Sivanesan, Sivashangarie; Li, Jian; Beddoe, Travis; Marsh, Glenn A

    2014-01-01

    Australia is facing a major national medical challenge with the emergence of the Hendra virus (HeV) as a medically and economically important pathogen of humans and animals. Clinical symptoms of human HeV infection can include fever, hypotension, dizziness, encephalitis, respiratory haemorrhage and edema. The window of opportunity for successful patient treatment remains unknown, but is likely to be very narrow. Currently, very few effective therapeutic options are available for the case management of severe HeV infections or the rapid silencing of local outbreaks. This underscores the need for more activity in the drug discovery arena to develop much needed therapeutics that specifically targets this deadly disease. The structural analysis of HeV is very much in its infancy, which leaves many gaps in our understanding of the biology of HeV and makes structure-guided drug design difficult. Structural studies of the viral RNAdependent- RNA polymerase (RdRp), which is the heart of the viral replication machinery, will set the stage for rational drug design and fill a major gap in our understanding of the HeV replication machinery. This review examines the current knowledge based on the multi-domain architecture of the Hendra RdRp and highlights which essential domain functions represent tangible targets for drug development against this deadly disease.

  10. Identification and validation of a virus-inducible ta-siRNA-generating ...

    Indian Academy of Sciences (India)

    2016-02-01

    Feb 1, 2016 ... approach, we identified a new locus-producing ta-siRNA in tomato. We have also identified the putative miRNA regulating the production of ta-siRNA from this locus. The ta-siRNAs generated from TAS4 were up-regulated upon infection with a DNA virus. The potential targets of ta-siRNAs were predicted to ...

  11. Polymyositis and dermatomyositis: no persistence of enterovirus or encephalomyocarditis virus RNA in muscle.

    OpenAIRE

    Jongen, P J; Zoll, G J; Beaumont, M; Melchers, W J; van de Putte, L B; Galama, J M

    1993-01-01

    OBJECTIVES--A persistent infection of enteroviruses and cardioviruses has been implicated in polymyositis and dermatomyositis, but conventional hybridisation studies of the presence of enterovirus RNA and encephalomyocarditis (EMC) virus RNA in affected muscle have yielded conflicting results. To investigate further the possibility of viral persistence, the presence of viral RNA in muscle from patients with adult onset polymyositis and dermatomyositis was investigated using a polymerase chain...

  12. Detection and molecular characterization of double-stranded RNA viruses in Philippine Trichomonas vaginalis isolates.

    Science.gov (United States)

    Rivera, Windell L; Justo, Christine Aubrey C; Relucio-San Diego, Mary Ann Cielo V; Loyola, Lorenz M

    2017-10-01

    The flagellated protozoon Trichomonas vaginalis that parasitizes the urogenital tract of humans was reported to harbor double-stranded RNA (dsRNA) viruses. These viruses, identified as Trichomonas vaginalis virus (TVV), belong to the genus Trichomonasvirus of the family Totiviridae. Four species, formally recognized by the International Committee on Taxonomy of Viruses (ICTV), have been reported and distinguished by pairwise comparisons of the sequences of genes coding for major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the complimentary DNA of target virus genes coding for CP and RdRp. Sequence analyses confirmed the identity of the TVV isolates from T. vaginalis cultures. A total of 35 dsRNA viruses were identified from 18 (19%) T. vaginalis isolates. Multiple TVV species were observed in six of the 18 T. vaginalis cultures. Phylogenetic analyses show monophyly in TVV1 and TVV2 whereas TVV3 and TVV4 appear paraphyletic. The phylogeny of Philippine Trichomonasvirus reflects the global distribution of its host. This is the first study in the Philippines and one of the two reports worldwide to detect the four TVVs and their concurrent infection in a single T. vaginalis isolate. Copyright © 2015. Published by Elsevier B.V.

  13. A double-stranded RNA as the genome of a potential virus infecting Vicia faba.

    Science.gov (United States)

    Liu, Weixia; Chen, Jishuang

    2009-08-01

    Preparations of double-stranded (ds) RNAs extracted from naturally infected Vicia faba Linn. growing in Hangzhou, Zhejiang Province, Eastern China displayed 3 dominant bands (FaR1, FaR2, and FaR3). FaR2 and FaR3 were found to be identical to the genomic dsRNAs of a recently reported Vicia cryptic virus (VCV). The positive strand of FaR1 contained two large open reading frames (ORFs), ORF1 and ORF2. The putative proteins encoded by these ORFs were found to have certain similarities to the putative capsid protein [ABO36237] and RNA-dependent RNA polymerase [ABC96788], respectively, of Tomato yellow stunt virus. Thus, FaR1 may represent the genome of a new dsRNA virus, which we have named Vicia cryptic virus M.

  14. Nuclear trafficking of proteins from RNA viruses: potential target for antivirals?

    Science.gov (United States)

    Caly, Leon; Wagstaff, Kylie M; Jans, David A

    2012-09-01

    A key aspect of the infectious cycle of many viruses is the transport of specific viral proteins into the host cell nucleus to perturb the antiviral response. Examples include a number of RNA viruses that are significant human pathogens, such as human immunodeficiency virus (HIV)-1, influenza A, dengue, respiratory syncytial virus and rabies, as well agents that predominantly infect livestock, such as Rift valley fever virus and Venezuelan equine encephalitis virus. Inhibiting the nuclear trafficking of viral proteins as a therapeutic strategy offers an attractive possibility, with important recent progress having been made with respect to HIV-1 and dengue. The results validate nuclear protein import as an antiviral target, and suggest the identification and development of nuclear transport inhibitors as a viable therapeutic approach for a range of human and zoonotic pathogenic viruses. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Cell Type-Dependent RNA Recombination Frequency in the Japanese Encephalitis Virus

    Directory of Open Access Journals (Sweden)

    Wei-Wei Chiang

    2014-01-01

    Full Text Available Japanese encephalitis virus (JEV is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously.

  16. Viral quantitative capillary electrophoresis for counting and quality control of RNA viruses.

    Science.gov (United States)

    Azizi, Afnan; Mironov, Gleb G; Muharemagic, Darija; Wehbe, Mohamed; Bell, John C; Berezovski, Maxim V

    2012-11-06

    The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze-thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10(8) to 10(13) ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation.

  17. Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

    Science.gov (United States)

    Grubaugh, Nathan D.; McMenamy, Scott S.; Turell, Michael J.; Lee, John S.

    2013-01-01

    Background Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae). Methodology/Principal Findings The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. Conclusions/Significance We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health

  18. Multi-gene detection and identification of mosquito-borne RNA viruses using an oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Nathan D Grubaugh

    Full Text Available BACKGROUND: Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae, Alphavirus (Togaviridae, Orthobunyavirus (Bunyaviridae, and Phlebovirus (Bunyaviridae. METHODOLOGY/PRINCIPAL FINDINGS: The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish

  19. Mechanisms of innate immune evasion in re-emerging RNA viruses.

    Science.gov (United States)

    Ma, Daphne Y; Suthar, Mehul S

    2015-06-01

    Recent outbreaks of Ebola, West Nile, Chikungunya, Middle Eastern Respiratory and other emerging/re-emerging RNA viruses continue to highlight the need to further understand the virus-host interactions that govern disease severity and infection outcome. As part of the early host antiviral defense, the innate immune system mediates pathogen recognition and initiation of potent antiviral programs that serve to limit virus replication, limit virus spread and activate adaptive immune responses. Concordantly, viral pathogens have evolved several strategies to counteract pathogen recognition and cell-intrinsic antiviral responses. In this review, we highlight the major mechanisms of innate immune evasion by emerging and re-emerging RNA viruses, focusing on pathogens that pose significant risk to public health. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. RNA virus interference via CRISPR/Cas13a system in plants

    KAUST Repository

    Aman, Rashid

    2017-11-04

    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

  1. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    Directory of Open Access Journals (Sweden)

    Markus Hoffmann

    Full Text Available Bats (Chiroptera host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat or Yangochiroptera (genera Carollia and Tadarida for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV, a porcine coronavirus, or to infection mediated by the Spike (S protein of SARS-coronavirus (SARS-CoV incorporated into pseudotypes based on vesicular stomatitis virus (VSV. The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3 were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.

  2. The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

    OpenAIRE

    Certo, Jeanine L.; Kabdulov, Timur O.; Paulson, Michelle L.; Anderson, Jeffrey A.; Hu, Wei-Shau

    1999-01-01

    Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generati...

  3. Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R.

    Science.gov (United States)

    Pfaller, Christian K; Radeke, Monte J; Cattaneo, Roberto; Samuel, Charles E

    2014-01-01

    Measles virus (MV) lacking expression of C protein (C(KO)) is a potent activator of the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), whereas the isogenic parental virus expressing C protein is not. Here, we demonstrate that significant amounts of dsRNA accumulate during C(KO) mutant infection but not following parental virus infection. dsRNA accumulated during late stages of infection and localized with virus replication sites containing N and P proteins. PKR autophosphorylation and stress granule formation correlated with the timing of dsRNA appearance. Phospho-PKR localized to dsRNA-containing structures as revealed by immunofluorescence. Production of dsRNA was sensitive to cycloheximide but resistant to actinomycin D, suggesting that dsRNA is a viral product. Quantitative PCR (qPCR) analyses revealed reduced viral RNA synthesis and a steepened transcription gradient in C(KO) virus-infected cells compared to those in parental virus-infected cells. The observed alterations were further reflected in lower viral protein expression levels and reduced C(KO) virus infectious yield. RNA deep sequencing confirmed the viral RNA expression profile differences seen by qPCR between C(KO) mutant and parental viruses. After one subsequent passage of the C(KO) virus, defective interfering RNA (DI-RNA) with a duplex structure was obtained that was not seen with the parental virus. We conclude that in the absence of C protein, the amount of PKR activator RNA, including DI-RNA, is increased, thereby triggering innate immune responses leading to impaired MV growth.

  4. Characterization of the Zika virus induced small RNA response in Aedes aegypti cells.

    Directory of Open Access Journals (Sweden)

    Margus Varjak

    2017-10-01

    Full Text Available RNA interference (RNAi controls arbovirus infections in mosquitoes. Two different RNAi pathways are involved in antiviral responses: the PIWI-interacting RNA (piRNA and exogenous short interfering RNA (exo-siRNA pathways, which are characterized by the production of virus-derived small RNAs of 25-29 and 21 nucleotides, respectively. The exo-siRNA pathway is considered to be the key mosquito antiviral response mechanism. In Aedes aegypti-derived cells, Zika virus (ZIKV-specific siRNAs were produced and loaded into the exo-siRNA pathway effector protein Argonaute 2 (Ago2; although the knockdown of Ago2 did not enhance virus replication. Enhanced ZIKV replication was observed in a Dcr2-knockout cell line suggesting that the exo-siRNA pathway is implicated in the antiviral response. Although ZIKV-specific piRNA-sized small RNAs were detected, these lacked the characteristic piRNA ping-pong signature motif and were bound to Ago3 but not Piwi5 or Piwi6. Silencing of PIWI proteins indicated that the knockdown of Ago3, Piwi5 or Piwi6 did not enhance ZIKV replication and only Piwi4 displayed antiviral activity. We also report that the expression of ZIKV capsid (C protein amplified the replication of a reporter alphavirus; although, unlike yellow fever virus C protein, it does not inhibit the exo-siRNA pathway. Our findings elucidate ZIKV-mosquito RNAi interactions that are important for understanding its spread.

  5. Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

    Science.gov (United States)

    Brettmann, Erin A; Shaik, Jahangheer S; Zangger, Haroun; Lye, Lon-Fye; Kuhlmann, F Matthew; Akopyants, Natalia S; Oschwald, Dayna M; Owens, Katherine L; Hickerson, Suzanne M; Ronet, Catherine; Fasel, Nicolas; Beverley, Stephen M

    2016-10-25

    Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

  6. Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV).

    Science.gov (United States)

    Aktar, Suriya J; Vivet-Boudou, Valérie; Ali, Lizna M; Jabeen, Ayesha; Kalloush, Rawan M; Richer, Delphine; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

    2014-11-14

    two MMTV RNAs, leading to gRNA dimerization and its subsequent encapsidation into the assembling virus particles. The results presented here enhance our understanding of the MMTV gRNA dimerization and packaging processes and the role of structural motifs with respect to RNA-RNA and possibly RNA-protein interactions that might be taking place during MMTV life cycle.

  7. Short communication: Stability and integrity of classical swine fever virus RNA stored at room temperature

    Directory of Open Access Journals (Sweden)

    Damarys Relova

    2017-12-01

    Full Text Available Worldwide cooperation between laboratories working with classical swine fever virus (CSFV requires exchange of virus isolates. For this purpose, shipment of CSFV RNA is a safe alternative to the exchange of infectious material. New techniques using desiccation have been developed to store RNA at room temperature and are reported as effective means of preserving RNA integrity. In this study, we evaluated the stability and integrity of dried CSFV RNA stored at room temperature. First, we determined the stability of CSFV RNA covering CSFV genome regions used typically for the detection of viral RNA in diagnostic samples by reverse transcription-polymerase chain reaction (RT-PCR. To this end, different concentrations of in vitro-transcribed RNAs of the 5’-untranslated region and of the NS5B gene were stored as dried RNA at 4, 20, and 37oC for two months. Aliquots were analyzed every week by CSFV-specific quantitative real-time RT-PCR. Neither the RNA concentration nor the storage temperature did affect CSFV RNA yields at any of the time evaluated until the end of the experiment. Furthermore, it was possible to recover infectious CSFV after transfection of SK-6 cells with dried viral RNA stored at room temperature for one week. The full-length E2 of CSFV was amplified from all the recovered viruses, and nucleotide sequence analysis revealed 100% identity with the corresponding sequence obtained from RNA of the original material. These results show that CSFV RNA stored as dried RNA at room temperature is stable, maintaining its integrity for downstream analyses and applications.

  8. Oligonucleotide Antiviral Therapeutics: Antisense and RNA Interference for Highly Pathogenic RNA Viruses

    Science.gov (United States)

    2008-01-01

    intravenously (Kumar et al., 2007). The uthors utilized the ability of rabies virus glycoprotein G (RVG) o bind acetylcholine receptor on neuronal cells. A...derived from the glycoprotein f rabies virus (RVG) enables brain-specific delivery of conju- ated siRNAs when given IV. Treatment with RVG-conjugated...Johnson and Mueller, 002). H5N1 is a highly virulent avian influenza virus that can lso infect humans. Currently, the virus does not spread easily

  9. The Battle of RNA Synthesis: Virus versus Host

    NARCIS (Netherlands)

    Harwig, Alex; Landick, Robert; Berkhout, Ben

    2017-01-01

    Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular

  10. Quantitative detection for plant virus's RNA-loading by dot-blot

    International Nuclear Information System (INIS)

    Chai Lihong; Xu Bujin; Chen Jishuang

    2003-01-01

    A new method, RNA dot blot combined with direct determination of the radioactivity by BIO-Imaging Analyzer (dRH-dBIA) was used for detecting RNA of plant virus in infected plant tissue. This method was used for the influence of RNA-loading level of tobacco mosaic virus (TMV) in tobacco leave tissues after treatment of a plant hormone relatives (n-Propyl dihydro-jasmonate, PDJ) in the concentration range of 0.001-10 ppm. The results indicate that after PDJ application onto tobacco leaves for 3 days all PDJ treatments cause increase of TMV RNA-loading level except 0.001 ppm treatment, and the higher the concentration, the more obvious increase was observed. This phenomenon was confirmed with semi-leaf lesion spot on Nicotiana glutinosa as a local lesion host. The dRH-dBIA method is applicable in quantitative determination of RNA without obvious artificial influence

  11. Structural Basis for dsRNA Recognition by NS1 Protein of Influenza A Virus

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, A.; Wong, S; Yuan, Y

    2009-01-01

    Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel alpha-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

  12. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J. (Pharmasset); (Emerald)

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  13. Expression of IMP1 Enhances Production of Murine Leukemia Virus Vector by Facilitating Viral Genomic RNA Packaging

    OpenAIRE

    Mai, Yun; Gao, Guangxia

    2010-01-01

    Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1) enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulat...

  14. Widespread horizontal gene transfer from double-stranded RNA viruses to eukaryotic nuclear genomes.

    Science.gov (United States)

    Liu, Huiquan; Fu, Yanping; Jiang, Daohong; Li, Guoqing; Xie, Jiatao; Cheng, Jiasen; Peng, Youliang; Ghabrial, Said A; Yi, Xianhong

    2010-11-01

    Horizontal gene transfer commonly occurs from cells to viruses but rarely occurs from viruses to their host cells, with the exception of retroviruses and some DNA viruses. However, extensive sequence similarity searches in public genome databases for various organisms showed that the capsid protein and RNA-dependent RNA polymerase genes from totiviruses and partitiviruses have widespread homologs in the nuclear genomes of eukaryotic organisms, including plants, arthropods, fungi, nematodes, and protozoa. PCR amplification and sequencing as well as comparative evidence of junction coverage between virus and host sequences support the conclusion that these viral homologs are real and occur in eukaryotic genomes. Sequence comparison and phylogenetic analysis suggest that these genes were likely transferred horizontally from viruses to eukaryotic genomes. Furthermore, we present evidence showing that some of the transferred genes are conserved and expressed in eukaryotic organisms and suggesting that these viral genes are also functional in the recipient genomes. Our findings imply that horizontal transfer of double-stranded RNA viral genes is widespread among eukaryotes and may give rise to functionally important new genes, thus entailing that RNA viruses may play significant roles in the evolution of eukaryotes.

  15. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity

    Directory of Open Access Journals (Sweden)

    Gang Lu

    2016-05-01

    Full Text Available An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells.

  16. Cell tropism predicts long-term nucleotide substitution rates of mammalian RNA viruses.

    Directory of Open Access Journals (Sweden)

    Allison L Hicks

    2014-01-01

    Full Text Available The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P<0.0001 as the most significant predictors. Further, one-tailed t-tests showed that viruses primarily infecting epithelial cells evolve significantly faster than neurotropic viruses (P<0.0001 and P<0.001 for the structural genes and non-structural genes, respectively. These results provide strong evidence that the fastest evolving mammalian RNA viruses infect cells with the highest turnover rates: the highly proliferative epithelial cells. Estimated viral generation times suggest that epithelial-infecting viruses replicate more quickly than viruses with different cell tropisms. Our results indicate that cell tropism is a key factor in viral evolvability.

  17. Replication and encapsidation of the viroid-like satellite RNA of lucerne transient streak virus are supported in divergent hosts by cocksfoot mottle virus and turnip rosette virus.

    Science.gov (United States)

    Sehgal, O P; Sinha, R C; Gellatly, D L; Ivanov, I; AbouHaidar, M G

    1993-04-01

    Cocksfoot mottle sobemovirus supports replication and encapsidation of the viroid-like satellite RNA (sat-RNA) of lucerne transient streak virus (LTSV) in two monocotyledonous species, Triticum aestivum and Dactylis glomerata. Additionally, LTSV sat-RNA replicates effectively in the presence of turnip rosette sobemovirus in Brassica rapa, Raphanus raphanistrum and Sinapsis arvensis, but not in Thlaspi arvense or Nicotiana bigelovii, indicating that host species markedly influence this interaction. Previous reports of the association between LTSV sat-RNA and helper sobemoviruses were limited to dicotyledonous hosts. Our results demonstrate that the biological interaction between these two entities spans divergent dicotyledonous and monocotyledonous species.

  18. Effects of ultraviolet radiation on structural components of enveloped RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Mauro R.; Couceiro, Jose N.S.S.; Brito, Sieberth N.; Cabral, Maulori C. [Universidade Federal, Rio de janeiro, RJ (Brazil). Inst. de Microbiologia

    1995-01-01

    The interaction of ultraviolet radiation and virus particles of Western Equine Encephalomyelitis Virus (WEE) and Newcastle Disease Virus (NDV) which have respectively RNA of positive (RNA+) and negative (RNA-) polarity as genomes, was studied using purified particles. The purified virus preparations were irradiated at a range of 1,000 to 6,000 joules per m{sup 2} with posterior analysis of their propagation in primary cells cultures of chicken embryos. It could be observed that a radiation dose of 4,500 joules per m{sup 2} could induce 10{sup 9} TCID50 per ml as minimal loss of titer for WEE virus and NDV. The hemagglutination assay was used as a tool for evaluate the alterations caused by UV radiation on the molecular arrangement of virus proteins. Alterations of the virus hemagglutinating activity were only observed when radiation levels higher than 6,000 joules per m{sup 2} were used. The results from hemolysis assays showed the importance of the loss of the envelope integrity and the damages to nucleoprotein structures during the inactivation process, when we use radiation doses higher than 6,000 joules per m{sup 2}. This model of study can increase our comprehension of the radiation effects on the cell physiology and biological components of the cell membranes. (author). 34 refs., 3 figs., 1 tab.

  19. Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses.

    Science.gov (United States)

    Yanagisawa, Hironobu; Tomita, Reiko; Katsu, Koji; Uehara, Takuya; Atsumi, Go; Tateda, Chika; Kobayashi, Kappei; Sekine, Ken-Taro

    2016-03-07

    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.

  20. Functional specialization of the small interfering RNA pathway in response to virus infection.

    Directory of Open Access Journals (Sweden)

    Joao Trindade Marques

    Full Text Available In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA is processed into small interfering RNAs (siRNAs by Dicer-2 (Dcr-2 in association with a dsRNA-binding protein (dsRBP cofactor called Loquacious (Loqs-PD. siRNAs are then loaded onto Argonaute-2 (Ago2 by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

  1. Analysis of the RNA species isolated from defective particles of vesicular stomatitis virus.

    Science.gov (United States)

    Adler, R; Banerjee, A K

    1976-10-01

    Serial high multiplicity passage of a cloned stock of vesicular stomatitis virus was found to generate defective interfering particles containing three size classes of RNA, with sedimentaiton coefficients of 31 S, 23 S and 19 S. The 31 S and 23 S RNA species were found to be complementary to both the 12 to 18 S and 31 S size classes of VSV mRNAs. The 19 S class of RNA was found to be partially base-paired. All three RNA species were found to contain ppAp at their 5' termini.

  2. The icosahedral RNA virus as a grotto: organizing the genome into stalagmites and stalactites

    OpenAIRE

    Harvey, Stephen C.; Zeng, Yingying; Heitsch, Christine E.

    2013-01-01

    There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure—often one or more stem-loops—either promotes assembly or is required for assembly, but there are others where specific packagin...

  3. Preliminary studies about multiplication of cucumber mosaic virus RNA-4

    International Nuclear Information System (INIS)

    Douine, Laurence; Marchoux, G.; Clement, Marcelle

    1977-01-01

    Component 4 of CMV-RNA is not required for infectivity, nevertheless it multiplies even when it is not in the inoculum. This component is really a viral RNA for it is synthetised in presence of actinomycin D. It accumulates during the infection and perhaps it acts in regulation of CMV synthesis [fr

  4. Ecological Drivers of Virus Evolution: Astrovirus as a Case Study.

    Science.gov (United States)

    Mendenhall, Ian H; Smith, Gavin J D; Vijaykrishna, Dhanasekaran

    2015-07-01

    Although RNA viruses exhibit a high frequency of host jumps, major differences exist among the different virus families. Astroviruses infect a wide range of hosts, affecting both public health systems and economic production chains. Here we delineate the ecological and adaptive processes that drive the cross-species transmission of astroviruses. We observe that distinct transmission zones determine the prevailing astrovirus host and virus diversity, which in turn suggests that no single host group (e.g., bats) can be the natural reservoir, as illustrated through our phylogenetic analysis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Use of S1 nuclease in deep sequencing for detection of double-stranded RNA viruses.

    Science.gov (United States)

    Shimada, Saya; Nagai, Makoto; Moriyama, Hiromitsu; Fukuhara, Toshiyuki; Koyama, Satoshi; Omatsu, Tsutomu; Furuya, Tetsuya; Shirai, Junsuke; Mizutani, Tetsuya

    2015-09-01

    Metagenomic approach using next-generation DNA sequencing has facilitated the detection of many pathogenic viruses from fecal samples. However, in many cases, majority of the detected sequences originate from the host genome and bacterial flora in the gut. Here, to improve efficiency of the detection of double-stranded (ds) RNA viruses from samples, we evaluated the applicability of S1 nuclease on deep sequencing. Treating total RNA with S1 nuclease resulted in 1.5-28.4- and 10.1-208.9-fold increases in sequence reads of group A rotavirus in fecal and viral culture samples, respectively. Moreover, increasing coverage of mapping to reference sequences allowed for sufficient genotyping using analytical software. These results suggest that library construction using S1 nuclease is useful for deep sequencing in the detection of dsRNA viruses.

  6. Efficient double-stranded RNA production methods for utilization in plant virus control.

    Science.gov (United States)

    Voloudakis, Andreas E; Holeva, Maria C; Sarin, L Peter; Bamford, Dennis H; Vargas, Marisol; Poranen, Minna M; Tenllado, Francisco

    2015-01-01

    Double-stranded RNA (dsRNA) is an inducer molecule of the RNA silencing (RNA interference, RNAi) pathway that is present in all higher eukaryotes and controls gene expression at the posttranscriptional level. This mechanism allows the cell to recognize aberrant genetic material in a highly sequence specific manner. This ultimately leads to degradation of the homologous target sequence, rendering the plant cell resistant to subcellular pathogens. Consequently, dsRNA-mediated resistance has been exploited in transgenic plants to convey resistance against viruses. In addition, it has been shown that enzymatically synthesized specific dsRNA molecules can be applied directly onto plant tissue to induce resistance against the cognate virus. This strongly implies that dsRNA molecules are applicable as efficacious agents in crop protection, which will fuel the demand for cost-effective dsRNA production methods. In this chapter, the different methods for dsRNA production-both in vitro and in vivo-are described in detail.

  7. Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation.

    Science.gov (United States)

    Meyerson, Nicholas R; Zhou, Ligang; Guo, Yusong R; Zhao, Chen; Tao, Yizhi J; Krug, Robert M; Sawyer, Sara L

    2017-11-08

    TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Adenosine Deaminases Acting on RNA (ADARs) are both Antiviral and Proviral Dependent upon the Virus

    Science.gov (United States)

    Samuel, Charles E.

    2010-01-01

    A-to-I RNA editing, the deamination of adenosine (A) to inosine (I) that occurs in regions of RNA with double-stranded character, is catalyzed by a family of Adenosine Deaminases Acting on RNA (ADARs). In mammals there are three ADAR genes. Two encode proteins that possess demonstrated deaminase activity: ADAR1, which is interferon-inducible, and ADAR2 which is constitutively expressed. ADAR3, by contrast, has not yet been shown to bean active enzyme. The specificity of the ADAR1 and ADAR2 deaminases ranges from highly site-selective to non-selective, dependent on the duplex structure of the substrate RNA. A-to-I editing is a form of nucleotide substitution editing, because I is decoded as guanosine (G) instead of A by ribosomes during translation and by polymerases during RNA-dependent RNA replication. Additionally, A-to-I editing can alter RNA structure stability as I:U mismatches are less stable than A:U base pairs. Both viral and cellular RNAs are edited by ADARs. A-to-I editing is of broad physiologic significance. Among the outcomes of A-to-I editing are biochemical changes that affect how viruses interact with their hosts, changes that can lead to either enhanced or reduced virus growth and persistence dependent upon the specific virus. PMID:21211811

  9. RNA immunization can protect mice against foot-and-mouth disease virus.

    Science.gov (United States)

    Pulido, Miguel Rodríguez; Sobrino, Francisco; Borrego, Belén; Sáiz, Margarita

    2010-03-01

    In previous work we have reported the immunization of swine using in vitro-transcribed foot-and-mouth disease virus (FMDV) RNA. With the aim of testing whether RNA-induced immunization can mediate protection against viral infection, a group of Swiss adult mice was inoculated with FMDV infectious transcripts. In most inoculated animals viral RNA was detected in serum at 48-72h postinoculation. A group of the RNA-inoculated mice (11 out of 19) developed significant titers of neutralizing antibodies against FMDV. Among those animals that were successfully challenged with infectious virus (15 out of 19), three out of the eight animals immunized upon RNA inoculation were protected, as infectious virus could not be isolated from sera but specific anti-FMDV antibodies could be readily detected. These results suggest the potential of the inoculation of genetically engineered FMDV RNA for virulence and protection assays in the murine model and allow to explore the suitability of RNA-based FMDV vaccination in natural host animals.

  10. Dengue subgenomic flaviviral RNA disrupts immunity in mosquito salivary glands to increase virus transmission.

    Science.gov (United States)

    Pompon, Julien; Manuel, Menchie; Ng, Geok Kee; Wong, Benjamin; Shan, Chao; Manokaran, Gayathri; Soto-Acosta, Ruben; Bradrick, Shelton S; Ooi, Eng Eong; Missé, Dorothée; Shi, Pei-Yong; Garcia-Blanco, Mariano A

    2017-07-01

    Globally re-emerging dengue viruses are transmitted from human-to-human by Aedes mosquitoes. While viral determinants of human pathogenicity have been defined, there is a lack of knowledge of how dengue viruses influence mosquito transmission. Identification of viral determinants of transmission can help identify isolates with high epidemiological potential. Additionally, mechanistic understanding of transmission will lead to better understanding of how dengue viruses harness evolution to cycle between the two hosts. Here, we identified viral determinants of transmission and characterized mechanisms that enhance production of infectious saliva by inhibiting immunity specifically in salivary glands. Combining oral infection of Aedes aegypti mosquitoes and reverse genetics, we identified two 3' UTR substitutions in epidemic isolates that increased subgenomic flaviviral RNA (sfRNA) quantity, infectious particles in salivary glands and infection rate of saliva, which represents a measure of transmission. We also demonstrated that various 3'UTR modifications similarly affect sfRNA quantity in both whole mosquitoes and human cells, suggesting a shared determinism of sfRNA quantity. Furthermore, higher relative quantity of sfRNA in salivary glands compared to midgut and carcass pointed to sfRNA function in salivary glands. We showed that the Toll innate immune response was preferentially inhibited in salivary glands by viruses with the 3'UTR substitutions associated to high epidemiological fitness and high sfRNA quantity, pointing to a mechanism for higher saliva infection rate. By determining that sfRNA is an immune suppressor in a tissue relevant to mosquito transmission, we propose that 3'UTR/sfRNA sequence evolution shapes dengue epidemiology not only by influencing human pathogenicity but also by increasing mosquito transmission, thereby revealing a viral determinant of epidemiological fitness that is shared between the two hosts.

  11. Relation of type-C RNA virus infectivity and leukemogenesis in rats and mice

    International Nuclear Information System (INIS)

    Nagao, Kenji; Ito, Takaaki; Yokoro, Kenjiro

    1976-01-01

    Observation was made as to movement of type-C RNA virus infectivity in the process of leukemogensis induced by Gross virus, N-nitrosoethylurea (NEU), or, x-ray. Total dose of 680 R in 4 times was given to the whole body or parts of the body at intervals of 5 days. Thymic leukemia occurred in 100% or rats which were inoculated with type-C RNA virus at the period of newborn 64 days after, on the average. Infectious titer of virus rose only in thymus toward leukemogenesis. Thymic leukemia was induced 100% in mice by NEU 122 days after, but its incidence was 9% of mice of which thymus was extracted. Leukemia virus was not detected in non-extracted thymus of mice, and pattern of virus infectivity in other organs did not show any difference with that of mice of which thymus was extracted. Virus showed high infectious titer in uterus of mice of both groups. Leukemia occurred 87% in the whole body irradiated mice, 15% in partially irradiated mice, and 39% in mice of which thymus was extracted and the whole body was irradiated. Virus did not show any homeostatic infectious titer in three kinds of leukemia, but it showed high infectious titer in uterus. (Kanao, N.)

  12. MicroRNA and the innate immune response toinfluenza A virus infection in pigs

    DEFF Research Database (Denmark)

    Brogaard, Louise

    response to influenza A virus infection requires the joint expression profiling of protein-coding gene and microRNA expression. Paper 1 is a review which emphasizes the importance of the pig in the study of influenza Avirus infections. Pigs are themselves natural hosts for influenza A virus, and our close......’ in the infected cell as well as neighboring cells. As with numerous other cellular processes, the innate host response is modulated by microRNAs, a class of short non-coding RNAs important for the regulation of translation of protein-coding gene transcripts. Comprehensive assessment of the transcriptional host...... influenza A virus infection. Paper 2 presents results demonstrating the temporal dynamics of microRNA expression in circulating leukocytes from pigs after influenza A virus challenge, and emphasizes the need for control of the timeparameter in suitable animal models for the evaluation of the biomarker...

  13. Stimulation of Vesicular Stomatitis Virus in vitro RNA Synthesis by Microtubule-Associated Proteins

    Science.gov (United States)

    Hill, Virginia M.; Harmon, Shirley A.; Summers, Donald F.

    1986-08-01

    Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.

  14. Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Chuang, C.-K.; Chen, W.-J.

    2009-01-01

    Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.

  15. Mimicry of molecular pretenders: the terminal structures of satellites associated with plant RNA viruses.

    Science.gov (United States)

    Huang, Ying-Wen; Hu, Chung-Chi; Lin, Na-Sheng; Hsu, Yau-Heiu

    2010-01-01

    Satellite RNAs (satRNAs) and satellite viruses depend on the replicase complexes provided by their cognate helper viruses and host plants for replication, pretending that they are part of the viral genomes. Although satRNAs and satellite viruses do not share significant nucleotide sequence similarity with the helper viruses, the essential cis-acting elements recognized by the replicase complexes must reside on their genomes, acting as the mimicry for the molecular pretenders. By understanding how this molecular mimicry deceives the helper viruses into supporting the satellites, a significant amount of knowledge of the basic requirements and mechanisms for replication of viruses and satellites has been obtained. Here we review the recent advances in understanding the effects of the cis elements at the termini of satRNAs and satellite viruses on their accumulation. Several well-characterized satellite/helper virus systems, representing the non-coding short satRNAs, mRNA-type long satRNAs, circular satRNAs and satellite viruses, are compared and contrasted. It is concluded that different satellites may adopt different strategies to exploit the replication/transcription/translation machineries of their helper viruses, and different mimicries may be implemented by the same molecular pretender for different biological functions.

  16. Aedes aegypti uses RNA interference in defense against Sindbis virus infection

    Directory of Open Access Journals (Sweden)

    Wilusz Jeffrey

    2008-03-01

    Full Text Available Abstract Background RNA interference (RNAi is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus. Results SINV (TR339-eGFP (+ strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate Ago2, Dcr2, or TSN mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut Ago2, Dcr2, and TSN transcript levels during infection revealed that only TSN transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner. Conclusion We show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication. Furthermore, Ae. aegypti RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in Ae. aegypti.

  17. The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.; Saphire, Erica Ollmann (Scripps)

    2016-10-18

    Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.

  18. Ultrastructure of the replication sites of positive-strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

    2015-05-15

    Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.

  19. Varieties of charge distributions in coat proteins of ssRNA+  viruses

    Science.gov (United States)

    Lošdorfer Božič, Anže; Podgornik, Rudolf

    2018-01-01

    A major part of the interactions involved in the assembly and stability of icosahedral, positive-sense single-stranded RNA (ssRNA+) viruses is electrostatic in nature, as can be inferred from the strong pH- and salt-dependence of their assembly phase diagrams. Electrostatic interactions do not act only between the capsid coat proteins (CPs), but just as often provide a significant contribution to the interactions of the CPs with the genomic RNA, mediated to a large extent by positively charged, flexible N-terminal tails of the CPs. In this work, we provide two clear and complementary definitions of an N-terminal tail of a protein, and use them to extract the tail sequences of a large number of CPs of ssRNA+  viruses. We examine the pH-dependent interplay of charge on both tails and CPs alike, and show that—in contrast to the charge on the CPs—the net positive charge on the N-tails persists even to very basic pH values. In addition, we note a limit to the length of the wild-type genomes of those viruses which utilize positively charged tails, when compared to viruses without charged tails and similar capsid size. At the same time, we observe no clear connection between the charge on the N-tails and the genome lengths of the viruses included in our study.

  20. Looking for inhibitors of the dengue virus NS5 RNA-dependent RNA-polymerase using a molecular docking approach

    Directory of Open Access Journals (Sweden)

    Galiano V

    2016-10-01

    Full Text Available Vicente Galiano,1 Pablo Garcia-Valtanen,2 Vicente Micol,3,4 José Antonio Encinar3 1Physics and Computer Architecture Department, Miguel Hernández University (UMH, Elche, Spain; 2Experimental Therapeutics Laboratory, Hanson and Sansom Institute for Health Research, School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia; 3Molecular and Cell Biology Institute, Miguel Hernández University (UMH, Elche, Spain; 4CIBER: CB12/03/30038, Physiopathology of the Obesity and Nutrition, CIBERobn, Instituto de Salud Carlos III, Palma de Mallorca, Spain Abstract: The dengue virus (DENV nonstructural protein 5 (NS5 contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <-10.5 kcal/mol were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo. Keywords: virtual screening, molecular

  1. FKBP8 interact with classical swine fever virus NS5A protein and promote virus RNA replication.

    Science.gov (United States)

    Li, Helin; Zhang, Chengcheng; Cui, Hongjie; Guo, Kangkang; Wang, Fang; Zhao, Tianyue; Liang, Wulong; Lv, Qizhuang; Zhang, Yanming

    2016-02-01

    The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling and host cellular responses via to its ability to interact with various cellular proteins. FKBP8 is also reported to promote virus replication. Here, we show that NS5A specifically interacts with FKBP8 through coimmunoprecipitation and GST-pulldown studies. Additionally, confocal microscopy study showed that NS5A and FKBP8 colocalized in the cytoplasm. Overexpression of FKBP8 via the eukaryotic expression plasmid pDsRED N1 significantly promoted viral RNA synthesis. The cells knockdown of FKBP8 by lentivirus-mediated shRNA markedly decreased the virus replication when infected with CSFV. These data suggest that FKBP8 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of FKBP8 protein functions may be beneficial for developing new strategies to treat CSFV infection.

  2. Comparative analysis of hepatitis B virus polymerase sequences required for viral RNA binding, RNA packaging, and protein priming.

    Science.gov (United States)

    Jones, Scott A; Clark, Daniel N; Cao, Feng; Tavis, John E; Hu, Jianming

    2014-02-01

    Hepatitis B virus replicates a DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific association with a viral RNA signal, termed ε (Hε), located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template. HP is made up of four domains, including the terminal protein (TP), the spacer, the reverse transcriptase (RT), and the RNase H domains. A recently developed, Hε-dependent, in vitro protein priming assay was used in this study to demonstrate that almost the entire TP and RT domains and most of the RNase H domain were required for protein priming. Specific residues within TP, RT, and the spacer were identified as being critical for HP-Hε binding and/or protein priming. Comparison of HP sequence requirements for Hε binding, pgRNA packaging, and protein priming allowed the classification of the HP mutants into five groups, each with distinct effects on these complex and related processes. Detailed characterization of HP requirements for these related and essential functions of HP will further elucidate the mechanisms of its multiple functions and aid in the targeting of these functions for antiviral therapy.

  3. Adenosine triphosphate analogs can efficiently inhibit the Zika virus RNA-dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Hercík, Kamil; Kozák, Jaroslav; Šála, Michal; Dejmek, Milan; Hřebabecký, Hubert; Zborníková, Eva; Smola, Miroslav; Růžek, Daniel; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 137, Jan (2017), s. 131-133 ISSN 0166-3542 R&D Projects: GA ČR GA15-09310S Institutional support: RVO:61388963 ; RVO:60077344 Keywords : hepatitis C virus * borne encephalitis virus * crystal structure Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 4.271, year: 2016

  4. Identification of small RNA in polyhedra of Bombyx mori nuclear polyhedrosis virus

    Directory of Open Access Journals (Sweden)

    T. V. Shirina

    2014-04-01

    Full Text Available It has been shown by bioinformatic methods­ that regions of the Bombyx mori viral nuclear poly­hedrosis genome encoded two small RNA – snc RNA-1 and snc RNA-2, which could perform a structural function in polyhedra crystals formation. The aim of this work was identification of the nucleo­tide sequence of small non-coding RNAs, predicted by bioinformatic methods in B. mori polyhedra. The following methods have been used: polymera­se chain reaction, agarose gel electrophoresis, the cloning­ of PCR products, sequencing. There were first determined nucleotide sequences of snc RNA-1 and snc RNA-2 of polyhedrin mRNA complementary regions which are included in B. mori polyhedra. These RNAs have 100% identity with bioinformatic predicted sequences. These results confirmed our bioinformatic approach to the search for small RNAs encoded in B. mori nuclear polyhedrosis virus genome.

  5. Clinical performance of the VERIS HCV assay for hepatitis C virus RNA quantification.

    Science.gov (United States)

    Izquierdo, Laure; Prégermain, Corinne; Hottelet, Corinne; Decombe, Gwenaëlle; Roque-Afonso, Anne-Marie

    2017-08-01

    Diagnosis of hepatitis C virus (HCV) infection and treatment monitoring rely on detection/quantification of HCV RNA and real-time polymerase chain reaction (PCR) techniques are expected to equivalently quantify the different HCV genotypes. The clinical performance of the VERIS HCV assay for HCV RNA quantification was compared to that of the Abbott RealTime HCV assay. Qualitative concordance and quantitative comparison were evaluated on a first panel of 286 clinical samples containing HCV genotypes 1-6. Forty additional genotype 4 samples were tested to explore genotype 4 HCV RNA underquantification. Qualitative discrepancies were observed for low viral loads (HCV RNA quantification by the VERIS HCV assay and the Abbott RealTime HCV assay was well correlated for all HCV genotypes, except genotype 4 where 5' UTR RNA folding may impact quantification. Nevertheless, this underestimation of HCV RNA levels had no impact on clinical use. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Identification and validation of a virus-inducible ta-siRNA-generating ...

    Indian Academy of Sciences (India)

    We have also identified the putative miRNA regulating the production of ta-siRNA from this locus. The ta-siRNAs generated from TAS4 were up-regulated upon infection with a DNA virus. The potential targets of ta-siRNAs were predicted to be variety of proteins including MYB transcription factors and cell cycle regulators for ...

  7. Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

    Science.gov (United States)

    Friebe, Peter; Lohmann, Volker; Krieger, Nicole; Bartenschlager, Ralf

    2001-01-01

    Sequences in the 5′ and 3′ termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5′ end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5′ NTR required for RNA replication. We show that deletions introduced into the 5′ terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5′ NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5′ NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5′ NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus. PMID:11711595

  8. Protection Against Lethal Marburg Virus Infection Mediated by Lipid Encapsulated Small Interfering RNA

    OpenAIRE

    Ursic-Bedoya, Raul; Mire, Chad E.; Robbins, Marjorie; Geisbert, Joan B.; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W.

    2013-01-01

    Background.  Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. Methods.  The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro t...

  9. Differential expression of miRNA-423-5p in serum from cattle challenged with bovine viral diarrhea virus

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV) is an RNA virus that causes respiratory disease in cattle. MicroRNAs have been proposed as indicators of exposure to respiratory pathogens. However, microRNA profiles in cattle exposed to BVDV are currently nonexistent and few studies have been reported; therefore,...

  10. Statoviruses, A novel taxon of RNA viruses present in the gastrointestinal tracts of diverse mammals.

    Science.gov (United States)

    Janowski, Andrew B; Krishnamurthy, Siddharth R; Lim, Efrem S; Zhao, Guoyan; Brenchley, Jason M; Barouch, Dan H; Thakwalakwa, Chrissie; Manary, Mark J; Holtz, Lori R; Wang, David

    2017-04-01

    Next-generation sequencing has expanded our understanding of the viral populations that constitute the mammalian virome. We describe a novel taxon of viruses named Statoviruses, for Stool associated Tombus-like viruses, present in multiple metagenomic datasets. These viruses define a novel clade that is phylogenetically related to the RNA virus families Tombusviridae and Flaviviridae. Five distinct statovirus types were identified in human, macaque, mouse, and cow gastrointestinal tract samples. The prototype genome, statovirus A, was frequently identified in macaque stool samples from multiple geographically distinct cohorts. Another genome, statovirus C1, was discovered in a stool sample from a human child with fever, cough, and rash. Further experimental data will clarify whether these viruses are infectious to mammals or if they originate from another source present in the mammalian gastrointestinal tract. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Expanding our Understanding of the Seaweed Holobiont: RNA Viruses of the Red Alga Delisea pulchra.

    Science.gov (United States)

    Lachnit, Tim; Thomas, Torsten; Steinberg, Peter

    2015-01-01

    Marine seaweeds are holobionts comprised of the macroalgal hosts and their associated microbiota. While the composition of the bacterial component of seaweed microbiomes is increasingly studied, almost nothing is known about the presence, diversity and composition of viruses in macroalgae in situ. In this study, we characterize for the first time the viruses associated with a red macroalga, Delisea pulchra. Using transmission electron microscopy we identified diverse morphotypes of virus-like particles in D. pulchra ranging from icosahedral to bacilliform to coiled pleomorphic as well as bacteriophages. Virome sequencing revealed the presence of a diverse group of dsRNA viruses affiliated to the genus Totivirus, known to infect plant pathogenic fungi. We further identified a ssRNA virus belonging to the order Picornavirales with a close phylogenetic relationship to a pathogenic virus infecting marine diatoms. The results of this study shed light on a so far neglected part of the seaweed holobiont, and suggest that some of the identified viruses may be possible pathogens for a host that is already known to be significantly impacted by bacterial infections.

  12. Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

    DEFF Research Database (Denmark)

    Nayak, A.; Goodfellow, I. G.; Woolaway, K. E.

    2006-01-01

    The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3D(pol)), the precursor 3CD, and an RNA template containing the cre....../bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV X protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg...... precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues...

  13. High sensitive method detection of plant RNA viruses by electrochemiluminescence reverse transcription PCR

    Science.gov (United States)

    Tang, Ya-bing; Xing, Da; Zhu, De-bin; Zhou, Xiao-ming

    2007-05-01

    It is well known that plant and animal viruses had widely spread the whole of world, and made a big loss in farming and husbandry. It is necessary that a highly efficient and accurate virus's detection method was developed. This research combines reverse transcription polymerase chain reaction (RT-PCR) technique with electrochemiluminescence method, to detect plant RNA viruses for the first time. Biotin-probe hybridizes with PCR product to specific select the target for detection, thus can avoid pseudo-positive result. TBR-probe hybridizes with PCR product to emit light for ECL detection. Specific nucleic acid sequences (20bp) were added to 5' terminal all of the primers, which can improve the chance of hybridization between TBR-probe and PCR product. At the same time, one of the PCR product chain can hybridize two Ru-probes, the ECL signal is intensified. The method was used to detect Odntoglossum ringspot virus ORSV, Sugarcane mosaic virus ScMV, Sorghum mosaic virus SrMV, and Maize dwarf mosaic virus MDMV, the experiment results show that this method could reliably identity virus infected plant samples. In a word, this method has higher sensitivity and lower cost than others. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.

  14. Protection from RNA and DNA viruses by IL-32.

    NARCIS (Netherlands)

    Zepp, J.A.; Nold-Petry, C.A.; Dinarello, C.A.; Nold, M.F.

    2011-01-01

    Several studies have documented a proinflammatory role for IL-32, which induces IL-1alpha, IL-1beta, IL-6, TNF, and chemokines via NF-kappaB, p38MAPK, and AP-1. However, IL-32 also participates in the responses to infection with viruses such as HIV-1 and influenza. In this study, we explored these

  15. High diversity of RNA viruses in rodents, Ethiopia

    Czech Academy of Sciences Publication Activity Database

    Meheretu, Yonas; Čížková, Dagmar; Těšíková, Jana; Welegerima, K.; Tomas, Z.; Kidane, D.; Girmay, K.; Schmidt-Chanasit, J.; Bryja, Josef; Günther, S.; Bryjová, Anna; Leirs, H.; Goüy de Bellocq, Joëlle

    2012-01-01

    Roč. 18, č. 12 (2012), s. 2047-2050 ISSN 1080-6040 R&D Projects: GA ČR GCP502/11/J070 Institutional support: RVO:68081766 Keywords : white-footed mouse * Mobala virus Subject RIV: EE - Microbiology, Virology Impact factor: 5.993, year: 2012

  16. Improving miRNA Delivery by Optimizing miRNA Expression Cassettes in Diverse Virus Vectors

    NARCIS (Netherlands)

    Herrera-Carrillo, Elena; Liu, Ying Poi; Berkhout, Ben

    2017-01-01

    The RNA interference pathway is an evolutionary conserved post-transcriptional gene regulation mechanism that is exclusively triggered by double-stranded RNA inducers. RNAi-based methods and technologies have facilitated the discovery of many basic science findings and spurred the development of

  17. Cloning and sequencing of full-length cDNAs of RNA1 and RNA2 of a Tomato black ring virus isolate from Poland.

    Science.gov (United States)

    Jończyk, M; Le Gall, O; Pałucha, A; Borodynko, N; Pospieszny, H

    2004-04-01

    Full-length cDNA clones corresponding to the RNA1 and RNA2 of the Polish isolate MJ of Tomato black ring virus (TBRV, genus Nepovirus) were obtained using a direct recombination strategy in yeast, and their complete nucleotide sequences were established. RNA1 is 7358 nucleotides and RNA2 is 4633 nucleotides in length, excluding the poly(A) tails. Both RNAs contain a single open reading frame encoding polyproteins of 254 kDa and 149 kDa for RNA1 and RNA2 respectively. Putative cleavage sites were identified, and the relationships between TBRV and related nepoviruses were studied by sequence comparison.

  18. A single-stranded RNA copy of the Giardia lamblia virus double-stranded RNA genome is present in the infected Giardia lamblia.

    OpenAIRE

    Furfine, E S; White, T C; Wang, A L; Wang, C C

    1989-01-01

    An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the sin...

  19. The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3'-translational enhancer

    International Nuclear Information System (INIS)

    Matsuda, Daiki; Dreher, Theo W.

    2004-01-01

    Many positive stand RNA viral genomes lack the poly(A) tail that is characteristic of cellular mRNAs and that promotes translation in cis. The 3' untranslated regions (UTRs) of such genomes are expected to provide similar translation-enhancing properties as a poly(A) tail, yet the great variety of 3' sequences suggests that this is accomplished in a range of ways. We have identified a translational enhancer present in the 3' UTR of Turnip yellow mosaic virus (TYMV) RNA using luciferase reporter RNAs with generic 5' sequences transfected into plant cells. The 3' terminal 109 nucleotides comprising the tRNA-like structure (TLS) and an upstream pseudoknot (UPSK) act in synergy with a 5'-cap to enhance translation, with a minor contribution in stabilizing the RNA. Maximum enhancement requires that the RNA be capable of aminoacylation, but either the native valine or engineered methionine is acceptable. Mutations that decrease the affinity for translation elongation factor eEF1A (but also diminish aminoacylation efficiency) strongly decrease translational enhancement, suggesting that eEF1A is mechanistically involved. The UPSK seems to act as an important, though nonspecific, spacer element ensuring proper presentation of a functional TLS. Our studies have uncovered a novel type of translational enhancer and a new role for a plant viral TLS

  20. Cellular microRNA networks regulate host dependency of hepatitis C virus infection

    OpenAIRE

    Li, Qisheng; Lowey, Brianna; Sodroski, Catherine; Krishnamurthy, Siddharth; Alao, Hawwa; Cha, Helen; Chiu, Stephan; El-Diwany, Ramy; Ghany, Marc G.; Liang, T. Jake

    2017-01-01

    Cellular microRNAs (miRNAs) have been shown to regulate hepatitis C virus (HCV) replication, yet a systematic interrogation of the repertoire of miRNAs impacting HCV life cycle is lacking. Here we apply integrative functional genomics strategies to elucidate global HCV–miRNA interactions. Through genome-wide miRNA mimic and hairpin inhibitor phenotypic screens, and miRNA–mRNA transcriptomics analyses, we identify three proviral and nine antiviral miRNAs that interact with HCV. These miRNAs ar...

  1. Complete Genome Sequence of a Double-Stranded RNA Virus from Avocado

    Science.gov (United States)

    Villanueva, Francisco; Sabanadzovic, Sead; Valverde, Rodrigo A.

    2012-01-01

    A number of avocado (Persea americana) cultivars are known to contain high-molecular-weight double-stranded RNA (dsRNA) molecules for which a viral nature has been suggested, although sequence data are not available. Here we report the cloning and complete sequencing of a 13.5-kbp dsRNA virus isolated from avocado and show that it corresponds to the genome of a new species of the genus Endornavirus (family Endornaviridae), tentatively named Persea americana endornavirus (PaEV). PMID:22205720

  2. Identification of Novel Influenza A Virus Proteins Translated from PA mRNA

    Science.gov (United States)

    Muramoto, Yukiko; Noda, Takeshi; Kawakami, Eiryo; Akkina, Ramesh

    2013-01-01

    Many replication events are involved in the influenza A virus life cycle, and they are accomplished by different virus proteins with specific functions. However, because the size of the influenza virus genome is limited, the virus uses different mechanisms to express multiple viral proteins from a single gene segment. The M2 and NS2 proteins are produced by splicing, and several novel influenza A virus proteins, such as PB1-F2, PB1-N40, and PA-X, have recently been identified. Here, we identified novel PA-related proteins in influenza A virus-infected cells. These newly identified proteins are translated from the 11th and 13th in-frame AUG codons in the PA mRNA and are, therefore, N-terminally truncated forms of PA, which we named PA-N155 and PA-N182, respectively. The 11th and 13th AUG codons are highly conserved among influenza A viruses, and the PA-N155 and PA-N182 proteins were detected in cells infected with various influenza A viruses isolated from different host species, suggesting the expression of these N-truncated PAs is universal in nature among influenza A viruses. These N-truncated PAs did not show polymerase activity when expressed together with PB1 and PB2; however, mutant viruses lacking the N-truncated PAs replicated more slowly in cell culture and had lower pathogenicity in mice than did wild-type virus. These results suggest that these novel PA-related proteins likely possess important functions in the replication cycle of influenza A virus. PMID:23236060

  3. Role of Electrostatics in the assembly pathway of a single-stranded RNA virus

    NARCIS (Netherlands)

    Garmann, R.F.; Comas-Garcia, M.; Koay, M.S.T.; Cornelissen, Jeroen Johannes Lambertus Maria; Knobler, C.M.; Gelbart, W.M.

    2014-01-01

    We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318–3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of

  4. Undetectable hepatitis C virus RNA during syphilis infection in two HIV/HCV-co-infected patients

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Knudsen, Andreas; Krarup, Henrik Bygum

    2014-01-01

    BACKGROUND: Treponema pallidum, the causative agent of syphilis, elicits a vigorous immune response in the infected host. This study sought to describe the impact of syphilis infection on hepatitis C virus (HCV) RNA levels in patients with HIV and chronic HCV infection. METHODS: Patients...

  5. MicroRNA-mediated interactions between host and hepatitis C virus.

    Science.gov (United States)

    Li, Hu; Jiang, Jian-Dong; Peng, Zong-Gen

    2016-01-28

    MicroRNAs (miRNAs) are small noncoding RNAs. More than 2500 mature miRNAs are detected in plants, animals and several types of viruses. Hepatitis C virus (HCV), which is a positive-sense, single-stranded RNA virus, does not encode viral miRNA. However, HCV infection alters the expression of host miRNAs, either in cell culture or in patients with liver disease progression, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma. In turn, host miRNAs regulate HCV life cycle through directly binding to HCV RNAs or indirectly targeting cellular mRNAs. Increasing evidence demonstrates that miRNAs are one of the centered factors in the interaction network between virus and host. The competitive viral and host RNA hypothesis proposes a latent cross-regulation pattern between host mRNAs and HCV RNAs. High loads of HCV RNA sequester and de-repress host miRNAs from their normal host targets and thus disturb host gene expression, indicating a means of adaptation for HCV to establish a persistent infection. Some special miRNAs are closely correlated with liver-specific disease progression and the changed levels of miRNAs are even higher sensitivity and specificity than those of traditional proteins. Therefore, some of them can serve as novel diagnostic/prognostic biomarkers in HCV-infected patients with liver diseases. They are also attractive therapeutic targets for development of new anti-HCV agents.

  6. West Nile Virus RNA in Tissues from Donor Associated with Transmission to Organ Transplant Recipients

    Centers for Disease Control (CDC) Podcasts

    2013-11-19

    William Hale reads an abridged version of the Emerging Infectious Diseases’ dispatch, West Nile Virus RNA in Tissues from Donor Associated with Transmission to Organ Transplant Recipients.  Created: 11/19/2013 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 11/21/2013.

  7. De novo reconstruction of plant RNA and DNA virus genomes from viral siRNAs

    Science.gov (United States)

    In antiviral defense, plants produce massive quantities of 21-24 nucleotide siRNAs. Here we demonstrate that the complete genomes of DNA and RNA viruses and viroids can be reconstructed by deep sequencing and de novo assembly of viral/viroid siRNAs from experimentally- and naturally-infected plants....

  8. First Experimental Assessment of Protein Intrinsic Disorder Involvement in an RNA Virus Natural Adaptive Process.

    Science.gov (United States)

    Charon, Justine; Barra, Amandine; Walter, Jocelyne; Millot, Pauline; Hébrard, Eugénie; Moury, Benoît; Michon, Thierry

    2018-01-01

    Intrinsic disorder (ID) in proteins is defined as a lack of stable structure in physiological conditions. Intrinsically disordered regions (IDRs) are highly abundant in some RNA virus proteomes. Low topological constraints exerted on IDRs are expected to buffer the effect of numerous deleterious mutations and could be related to the remarkable adaptive potential of RNA viruses to overcome resistance of their host. To experimentally test this hypothesis in a natural pathosystem, a set of four variants of Potato virus Y (PVY; Potyvirus genus) containing various ID degrees in the Viral genome-linked (VPg) protein, a key determinant of potyvirus adaptation, was designed. To estimate the ID contribution to the VPg-based PVY adaptation, the adaptive ability of the four PVY variants was monitored in the pepper host (Capsicum annuum) carrying a recessive resistance gene. Intriguingly, the two mutants with the highest ID content displayed a significantly higher ability to restore infection in the resistant host, whereas the less intrinsically disordered mutant was unable to restore infection. The role of ID on virus adaptation may be due either to a larger exploration of evolutionary pathways or the minimization of fitness penalty caused by resistance-breaking mutations. This pioneering study strongly suggests the positive impact of ID in an RNA virus adaptive capacity. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Protection Against Lethal Marburg Virus Infection Mediated by Lipid Encapsulated Small Interfering RNA

    Science.gov (United States)

    Ursic-Bedoya, Raul; Mire, Chad E.; Robbins, Marjorie; Geisbert, Joan B.; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W.

    2014-01-01

    Background. Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. Methods. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Results. Treatment resulted in 60%–100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. Conclusions. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection. PMID:23990568

  10. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    Science.gov (United States)

    Huang, Yong; Xing, Na; Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  11. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus and PCV2 (DNA virus from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29% and TGEV (11.7% preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility.

  12. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    Science.gov (United States)

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  13. RNA Viruses that Cause Hemorrhagic, Encephalitic, and Febrile Disease

    Science.gov (United States)

    1990-01-01

    commercial priority. Further, fund- - The views of the authors do not purport to reflect ing for studying many of these viruses is the position of the...tion of headache, meningismus, vertigo , mated 200,000 cases occurred with 598 re- confusion, hallucinations, and recrudes- ported deaths. Most cases...latter. Patients surviving this disease require several weeks for conva- Clinical Features lescence, and paroxysmal and orthostatic hypotension

  14. Plant viruses of the Amalgaviridae family evolved via recombination between viruses with double-stranded and negative-strand RNA genomes.

    Science.gov (United States)

    Krupovic, Mart; Dolja, Valerian V; Koonin, Eugene V

    2015-03-29

    Plant viruses of the recently recognized family Amalgaviridae have monopartite double-stranded (ds) RNA genomes and encode two proteins: an RNA-dependent RNA polymerase (RdRp) and a putative capsid protein (CP). Whereas the RdRp of amalgaviruses has been found to be most closely related to the RdRps of dsRNA viruses of the family Partitiviridae, the provenance of their CP remained obscure. Here we show that the CP of amalgaviruses is homologous to the nucleocapsid proteins of negative-strand RNA viruses of the genera Phlebovirus (Bunyaviridae) and Tenuivirus. The chimeric genomes of amalgaviruses are a testament to the effectively limitless gene exchange between viruses that shaped the evolution of the virosphere.

  15. Characterisation of several heterogeneous species of defective RNAs derived from RNA 3 of cucumber mosaic virus.

    Science.gov (United States)

    López, C; Aramburu, J; Galipienso, L; Nuez, F

    2007-01-01

    Preparations of double-stranded RNAs (dsRNAs) extracted from Nicotiana tabacum cv Xanthi plants infected with a subgroup IB isolate of Cucumber mosaic virus (CMV) were found to contain a heterogeneous population of defective RNAs (D-RNAs) derived from RNA 3. Characterised D-RNAs ranged in size from 1.5 to 1.9 kb and were derived either by a single in-frame deletion within the 3a or 3b genes or by means of double in-frame deletions within both genes. Also, northern blot hybridisation showed two other types of RNA derived from RNA 3: (a) RNA species of ca. 0.7 kb containing the 3'-terminus but lacking the 5'-terminus, which could be 3'-coterminal subgenomic of D-RNAs derived from the 3b gene and (b) RNA species of unknown origin of ca. 0.8 kb containing the 5'-terminus but lacking the 3'-terminus.

  16. HIV and hepatitis C virus RNA in seronegative organ and tissue donors.

    Science.gov (United States)

    Challine, Dominique; Pellegrin, Bertrand; Bouvier-Alias, Magali; Rigot, Pierrette; Laperche, Liliane; Pawlotsky, Jean-Michel

    The objective of our study was to determine whether nucleic acid testing could detect HIV RNA or hepatitis C virus (HCV) RNA in a large series of seronegative organ and tissue donors, and whether this technique should be routinely used to improve viral safety of grafts. We studied 2236 organ donors, 636 tissue donors, and 177 cornea donors. We identified five HCV RNA-positive donors in 2119 HCV-seronegative organ donors, and one HCV RNA-positive donor in 631 HCV-seronegative tissue donors. No HIV-seronegative, HIV RNA-positive donor was identified. Our data suggest that routine nucleic acid testing of organ and tissue donors might increase viral safety in transplantation.

  17. RNA-Free and Ribonucleoprotein-Associated Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal Domain of Host RNA Polymerase II

    Science.gov (United States)

    Martínez-Alonso, Mónica; Hengrung, Narin

    2016-01-01

    ABSTRACT Influenza viruses subvert the transcriptional machinery of their hosts to synthesize their own viral mRNA. Ongoing transcription by cellular RNA polymerase II (Pol II) is required for viral mRNA synthesis. By a process known as cap snatching, the virus steals short 5′ capped RNA fragments from host capped RNAs and uses them to prime viral transcription. An interaction between the influenza A virus RNA polymerase and the C-terminal domain (CTD) of the large subunit of Pol II has been established, but the molecular details of this interaction remain unknown. We show here that the influenza virus ribonucleoprotein (vRNP) complex binds to the CTD of transcriptionally engaged Pol II. Furthermore, we provide evidence that the viral polymerase binds directly to the serine-5-phosphorylated form of the Pol II CTD, both in the presence and in the absence of viral RNA, and show that this interaction is conserved in evolutionarily distant influenza viruses. We propose a model in which direct binding of the viral RNA polymerase in the context of vRNPs to Pol II early in infection facilitates cap snatching, while we suggest that binding of free viral polymerase to Pol II late in infection may trigger Pol II degradation. IMPORTANCE Influenza viruses cause yearly epidemics and occasional pandemics that pose a threat to human health, as well as represent a large economic burden to health care systems globally. Existing vaccines are not always effective, as they may not exactly match the circulating viruses. Furthermore, there are a limited number of antivirals available, and development of resistance to these is a concern. New measures to combat influenza are needed, but before they can be developed, it is necessary to better understand the molecular interactions between influenza viruses and their host cells. By providing further insights into the molecular details of how influenza viruses hijack the host transcriptional machinery, we aim to uncover novel targets for

  18. Frequent Zika Virus Sexual Transmission and Prolonged Viral RNA Shedding in an Immunodeficient Mouse Model

    Directory of Open Access Journals (Sweden)

    Nisha K. Duggal

    2017-02-01

    Full Text Available Circulation of Zika virus (ZIKV was first identified in the Western hemisphere in late 2014. Primarily transmitted through mosquito bite, ZIKV can also be transmitted through sex and from mother to fetus, and maternal ZIKV infection has been associated with fetal malformations. We assessed immunodeficient AG129 mice for their capacity to shed ZIKV in semen and to infect female mice via sexual transmission. Infectious virus was detected in semen between 7 and 21 days post-inoculation, and ZIKV RNA was detected in semen through 58 days post-inoculation. During mating, 73% of infected males transmitted ZIKV to uninfected females, and 50% of females became infected, with evidence of fetal infection in resulting pregnancies. Semen from vasectomized mice contained significantly lower levels of infectious virus, though sexual transmission still occurred. This model provides a platform for studying the kinetics of ZIKV sexual transmission and prolonged RNA shedding also observed in human semen.

  19. Varroa destructor Macula-like virus, Lake Sinai virus and other new RNA viruses in wild bumblebee hosts (Bombus pascuorum, Bombus lapidarius and Bombus pratorum).

    Science.gov (United States)

    Parmentier, Laurian; Smagghe, Guy; de Graaf, Dirk C; Meeus, Ivan

    2016-02-01

    Pollinators such as bumblebees (Bombus spp.) are in decline worldwide which poses a threat not only for ecosystem biodiversity but also to human crop production services. One main cause of pollinator decline may be the infection and transmission of diseases including RNA viruses. Recently, new viruses have been discovered in honeybees, but information on the presence of these in wild bumblebees is largely not available. In this study, we investigated the prevalence of new RNA viruses in Bombus species, and can report for the first time Varroa destructor Macula-like virus (VdMLV) and Lake Sinai virus (LSV) infection in multiple wild bumblebee hosts of Bombus pascuorum, Bombus lapidarius and Bombus pratorum. We sampled in 4 locations in Flanders, Belgium. Besides, we confirmed Slow bee paralysis virus (SBPV) in wild bumblebees, but no positive samples were obtained for Big Sioux river virus (BSRV). Secondly, we screened for the influence of apiaries on the prevalence of these viruses. Our results indicated a location effect for the prevalence of VdMLV in Bombus species, with a higher prevalence in the proximity of honeybee apiaries mainly observed in one location. For LSV, the prevalence was not different in the proximity or at a 1.5 km-distance of apiaries, but we reported a different isolate with similarities to LSV-2 and "LSV-clade A" as described by Ravoet et al. (2015), which was detected both in Apis mellifera and Bombus species. In general, our results indicate the existence of a disease pool of new viruses that seems to be associated to a broad range of Apoidae hosts, including multiple Bombus species. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA.

    Science.gov (United States)

    Ono, Ekaterina Alexandrovna Durymanova; Taniwaki, Sueli Akemi; Brandão, Paulo

    The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1log decrease in virus titter and 5.16-fold reduction in P mRNA, 24h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  1. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    International Nuclear Information System (INIS)

    Tzeng, W.-P.; Frey, Teryl K.

    2005-01-01

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA

  2. Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication.

    Science.gov (United States)

    Esser-Nobis, Katharina; Harak, Christian; Schult, Philipp; Kusov, Yuri; Lohmann, Volker

    2015-08-01

    Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive-strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell-culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver-derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7-Lunet cells supported HAV- and HCV-RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA-122 and phosphatidylinositol 4-kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine-triphosphate-binding cassette transporters and FK506-binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. We established a cell-culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. © 2015 by the American Association for the

  3. Physicochemical properties of double-stranded RNA used to discover a reo-like virus from blue crab Callinectes sapidus.

    Science.gov (United States)

    Bowers, Holly A; Messick, Gretchen A; Hanif, Ammar; Jagus, Rosemary; Carrion, Lee; Zmora, Oded; Schott, Eric J

    2010-12-07

    Mortality among blue crab Callinectes sapidus in soft shell production facilities is typically 25% or greater. The harvest, handling, and husbandry practices of soft shell crab production have the potential to spread or exacerbate infectious crab diseases. To investigate the possible role of viruses in soft shell crab mortalities, we took advantage of the physicochemical properties of double-stranded RNA (dsRNA) to isolate a putative virus genome. Further characterization confirmed the presence of a reo-like virus that possesses 12 dsRNA genome segments. The virus was present in >50% of dead or dying soft shell crabs, but fewer than 5% of healthy hard crabs. Injection of the virus caused mortality and resulted in the appearance of viral RNA and virus inclusions in hemocytes. The genome of the virus was partially sequenced and the information used to develop a reverse transcription polymerase chain reaction (RT-PCR) assay that is able to detect the virus genome in as little as 7.5 pg of total RNA. The molecular tools developed during this study will allow us to quantify prevalence of the blue crab reo-like virus in captive (soft shell facilities, aquaculture operations) and wild populations and facilitate understanding of the role this virus has in blue crab life history.

  4. Expanding our understanding of the seaweed holobiont: RNA viruses of the red alga Delisea pulchra.

    Directory of Open Access Journals (Sweden)

    Tim eLachnit

    2016-01-01

    Full Text Available Marine seaweeds are holobionts comprised of the macroalgal hosts and their associated microbiota. While the composition of the bacterial component of seaweed microbiomes is increasingly studied, almost nothing is known about the presence, diversity and composition of viruses in macroalgae in situ. In this study, we characterise for the first time the viruses associated with a red macroalga, Delisea pulchra. Using transmission electron microscopy we identified diverse morphotypes of virus-like particles in D. pulchra ranging from icosahedral to bacilliform to coiled pleomorphic as well as bacteriophages. Virome sequencing revealed the presence of a diverse group of dsRNA viruses affiliated to the genus Totivirus, known to infect plant pathogenic fungi. We further identified a ssRNA virus belonging to the order Picornavirales with a close phylogenetic relationship to a pathogenic virus infecting marine diatoms. The results of this study shed light on a so far neglected part of the seaweed holobiont, and suggest that some of these may be possible pathogens for a host that is already known to be significantly impacted by bacterial infections.

  5. Generalized selection to overcome innate immunity selects for host breadth in an RNA virus.

    Science.gov (United States)

    Wasik, Brian R; Muñoz-Rojas, Andrés R; Okamoto, Kenichi W; Miller-Jensen, Kathryn; Turner, Paul E

    2016-02-01

    Virus-host coevolution has selected for generalized host defense against viruses, exemplified by interferon production/signaling and other innate immune function in eukaryotes such as humans. Although cell-surface binding primarily limits virus infection success, generalized adaptation to counteract innate immunity across disparate hosts may contribute to RNA virus emergence potential. We examined this idea using vesicular stomatitis virus (VSV) populations previously evolved on strictly immune-deficient (HeLa) cells, strictly immune competent (MDCK) cells, or on alternating deficient/competent cells. By measuring viral fitness in unselected human cancer cells of differing innate immunity, we confirmed that HeLa-adapted populations were specialized for innate immune-deficient hosts, whereas MDCK-adapted populations were relatively more generalized for fitness on hosts of differing innate immune capacity and of different species origin. We also confirmed that HeLa-evolved populations maintained fitness in immune-deficient nonhuman primate cells. These results suggest that innate immunity is more prominent than host species in determining viral fitness at the host-cell level. Finally, our prediction was inexact that selection on alternating deficient/competent hosts should produce innate viral generalists. Rather, fitness differences among alternating host-evolved VSV populations indicated variable capacities to evade innate immunity. Our results suggest that the evolutionary history of innate immune selection can affect whether RNA viruses evolve greater host-breadth. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

  6. Characterization of Botrytis cinerea negative-stranded RNA virus 1, a new mycovirus related to plant viruses, and a reconstruction of host pattern evolution in negative-sense ssRNA viruses.

    Science.gov (United States)

    Donaire, Livia; Pagán, Israel; Ayllón, María A

    2016-12-01

    The molecular characterization of a novel negative single-stranded RNA virus infecting the plant pathogenic fungus Botrytis cinerea is reported here. Comparison of the sequence of Botrytis cinerea negative-stranded RNA virus 1 (BcNSRV-1) showed a strong identity with RNA dependent RNA polymerases (RdRps) of plant pathogenic emaraviruses and tospoviruses. We have also found all the molecular signatures present in the RdRp of the genus Emaravirus and in other genera of family Bunyaviridae: the conserved TPD triplet and RY dinucleotide, the three basic residues in premotif A and the conserved motifs A, B, C, D, and E. Our results showed that BcNSRV-1 is phylogenetically close to members of the genus Emaravirus and of the family Bunyaviridae, and an ancestral state reconstruction using the conserved RdRp motifs of type members of each family of (-)ssRNA viruses indicated that BcNSRV-1 could possibly derive from an invertebrate and vertebrate-infecting virus. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA

    Science.gov (United States)

    Ursic-Bedoya, Raul; Geisbert, Joan B.; Lee, Amy C. H.; Agans, Krystle N.; Robbins, Marjorie; Deer, Daniel J.; Fenton, Karla A.; MacLachlan, Ian; Geisbert, Thomas W.

    2015-01-01

    Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)–targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti–MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease. PMID:25143366

  8. Characteristics of enzyme hydrolyzing natural covalent bond between RNA and protein VPg of encephalomyocarditis virus

    International Nuclear Information System (INIS)

    Drygin, Yu.F.; Siyanova, E.Yu.

    1986-01-01

    The isolation and a preliminary characterization of the enzyme specifically hydrolyzing the phosphodiester bond between protein VPg and the RNA of encephalomyocarditis virus was the goal of the present investigation. The enzyme was isolated from a salt extract of Krebs II mouse ascites carcinoma cells by ion-exchange and affinity chromatography. It was found that the enzyme actually specifically cleaves the covalent bond between the RNA and protein, however, the isolation procedure does not free the enzyme from impurities which partially inhibit it. The enzyme cleaves the RNA-protein VPg complex of polio virus at a high rate, it is completely inactivated at 55 0 C, and is partially inhibited by EDTA

  9. Detection of eastern equine encephalomyelitis virus RNA in North American snakes.

    Science.gov (United States)

    Bingham, Andrea M; Graham, Sean P; Burkett-Cadena, Nathan D; White, Gregory S; Hassan, Hassan K; Unnasch, Thomas R

    2012-12-01

    The role of non-avian vertebrates in the ecology of eastern equine encephalomyelitis virus (EEEV) is unresolved, but mounting evidence supports a potential role for snakes in the EEEV transmission cycle, especially as over-wintering hosts. To determine rates of exposure and infection, we examined serum samples from wild snakes at a focus of EEEV in Alabama for viral RNA using quantitative reverse transcription polymerase chain reaction. Two species of vipers, the copperhead (Agkistrodon contortrix) and the cottonmouth (Agkistrodon piscivorus), were found to be positive for EEEV RNA using this assay. Prevalence of EEEV RNA was more frequent in seropositive snakes than seronegative snakes. Positivity for the quantitative reverse transcription polymerase chain reaction in cottonmouths peaked in April and September. Body size and sex ratios were not significantly different between infected and uninfected snakes. These results support the hypothesis that snakes are involved in the ecology of EEEV in North America, possibly as over-wintering hosts for the virus.

  10. Genotypes of Pestivirus RNA detected n anti influenza virus vaccines for human use

    Directory of Open Access Journals (Sweden)

    M. Giangaspero

    2004-02-01

    Full Text Available Nine polyvalent human influenza virus vaccines were tested by reverse transcriptase-polymerase chain reaction (RT-PCR for the presence of pestivirus RNA. Samples were selected from manufacturers in Europe and the USA. Three samples of the nine vaccines tested (33.3% gave positive results for pestivirus RNA. The 5´-untranslated genomic region sequence of the contaminant pestivirus RNA was analysed based on primary nucleotide sequence homology and on secondary sequence structures characteristic to genotypes. Two sequences belonged to Pestivirus type-1 (bovine viral diarrhoea virus [BVDV] species, genotypes BVDV-1b and BVDV-1e. These findings confirm previous reports, suggesting an improvement in preventive measures against contamination of biological products for human use.

  11. The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

    Directory of Open Access Journals (Sweden)

    Dutartre Hélène

    2005-10-01

    Full Text Available Abstract Background The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C. Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. Results We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Conclusion Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA

  12. Brome mosaic virus Infection of Rice Results in Decreased Accumulation of RNA1.

    Science.gov (United States)

    Kitayama, Masahiko; Hoover, Haley; Middleton, Stefani; Kao, C Cheng

    2015-05-01

    Brome mosaic virus (BMV) (the Russian strain) infects monocot plants and has been studied extensively in barley and wheat. Here, we report BMV can systemically infect rice (Oryza sativa var. japonica), including cultivars in which the genomes have been determined. The BMV capsid protein can be found throughout the inoculated plants. However, infection in rice exhibits delayed symptom expression or no symptoms when compared with wheat (Triticum aestivum). The sequences of BMV RNAs isolated from rice did not reveal any nucleotide changes in RNA1 or RNA2, while RNA3 had only one synonymous nucleotide change from the inoculum sequence. Preparations of purified BMV virions contained RNA1 at a significantly reduced level relative to the other two RNAs. Analysis of BMV RNA replication in rice revealed that minus-strand RNA1 was replicated at a reduced rate when compared with RNA2. Thus, rice appears to either inhibit RNA1 replication or lacks a sufficient amount of a factor needed to support efficient RNA1 replication.

  13. Molecular characterization of a bipartite double-stranded RNA virus and its satellite-like RNA co-infecting the phytopathogenic fungus Sclerotinia sclerotiorum

    Directory of Open Access Journals (Sweden)

    Lijiang eLiu

    2015-05-01

    Full Text Available A variety of mycoviruses have been found in Sclerotinia sclerotiorum. In this study, we report a novel mycovirus Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1 that was originally isolated from the hypovirulent strain SCH941 of S. sclerotiorum. SsBRV1 has rigid spherical virions that are ~38 nm in diameter, and three dsRNA segments (dsRNA1, 2 and 3 with lengths of 6.4, 6.0 and 1.7 kbp, respectively were packaged in the virions. dsRNA1 encodes a cap-pol fusion protein, and dsRNA2 encodes a polyprotein with unknown functions but contributes to the formation of virus particles. The dsRNA3 is dispensable and may be a satellite-like RNA (SatlRNA of SsBRV1. Although phylogenetic analysis of the RdRp domain demonstrated that SsBRV1 is related to Botrytis porri RNA virus 1 (BpRV1 and Ustilago maydis dsRNA virus-H1 (UmV-H1, the structure proteins of SsBRV1 do not have any significant sequence similarities with other known viral proteins with the exception of those of BpRV1. SsBRV1 carrying dsRNA3 seems to have no obvious effects on the colony morphology, but can significantly reduce the growth rate and virulence of S. sclerotiorum. Notably, a growth hormone receptor binding domain (GHBP, Pfam12772 is detected in ORF2-encoded protein of SsBRV1, which have not been reported in any other viruses. These findings provide new insights into the virus taxonomy, virus evolution and the interactions between SsBRV1 and the fungal hosts.

  14. Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

    Science.gov (United States)

    Kaushik, Nidhi; Subramani, Chandru; Anang, Saumya; Muthumohan, Rajagopalan; Shalimar; Nayak, Baibaswata; Ranjith-Kumar, C T; Surjit, Milan

    2017-11-01

    Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used

  15. Virus-induced gene silencing of RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana

    Science.gov (United States)

    In eukaryotic cells, RNA polymerase III is highly conserved, contains 17 subunits and transcribes housekeeping genes such as ribosomal 50S rRNA, tRNA and other small RNAs. Functional roles of the RPC5 are poorly characterized in the literature. In this work, we report that virus-induced gene silenci...

  16. Small angle scattering study of the structure and organization of RNA and protein in Brome Mosaic Virus (BMV)

    Science.gov (United States)

    Das, Narayan C.; Warren, Garfield T.; Cheng, Si; Kao, C. Cheng; Ni, Peng; Dragnea, Bogdan; Sokol, Paul E.

    2012-02-01

    Brome mosaic virus (BMV) is a small icosahedral of the alpha virus-like superfamily of RNA with a segmented positive-strand RNA genome and a mean diameter ˜ 268å that offers high levels of RNA synthesis and virus production in plants. BMV also tightly regulates the packaging of its four RNAs (RNA1 through RNA4) into three separate particles; RNA1 and RNA2 are encapsidated separately while one copy each of RNA3 and RNA4 are normally packaged together. Small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) were applied to study the size, shape and protein-RNA organization of BMV. D2O/H2O mixture was used to enhance contrast in SANS measurement. The radial distribution of BMV from the Fourier transform of scattering spectrum gives a clear indication of RNA packing, and distribution and their structure in the BMV. The result reveals that the virus is about 266 å in diameter and is composed of RNA inside the virion coated with a protein shell.

  17. Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1

    NARCIS (Netherlands)

    SenGupta, D. N.; Berkhout, B.; Gatignol, A.; Zhou, A. M.; Silverman, R. H.

    1990-01-01

    Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids.

  18. The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes.

    Directory of Open Access Journals (Sweden)

    Kun Zhang

    2017-04-01

    Full Text Available RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+ RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes.

  19. The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes

    Science.gov (United States)

    Yang, Meng; Liu, Songyu; Li, Zhenggang; Wang, Xianbing; Han, Chenggui; Yu, Jialin

    2017-01-01

    RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes. PMID:28388677

  20. Role of electrostatics in the assembly pathway of a single-stranded RNA virus.

    Science.gov (United States)

    Garmann, Rees F; Comas-Garcia, Mauricio; Koay, Melissa S T; Cornelissen, Jeroen J L M; Knobler, Charles M; Gelbart, William M

    2014-09-01

    We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of

  1. Strategies of highly pathogenic RNA viruses to block dsRNA detection by RIG-I-like receptors: hide, mask, hit.

    Science.gov (United States)

    Zinzula, Luca; Tramontano, Enzo

    2013-12-01

    Double-stranded RNA (dsRNA) is synthesized during the course of infection by RNA viruses as a byproduct of replication and transcription and acts as a potent trigger of the host innate antiviral response. In the cytoplasm of the infected cell, recognition of the presence of viral dsRNA as a signature of "non-self" nucleic acid is carried out by RIG-I-like receptors (RLRs), a set of dedicated helicases whose activation leads to the production of type I interferon α/β (IFN-α/β). To overcome the innate antiviral response, RNA viruses encode suppressors of IFN-α/β induction, which block RLRs recognition of dsRNA by means of different mechanisms that can be categorized into: (i) dsRNA binding and/or shielding ("hide"), (ii) dsRNA termini processing ("mask") and (iii) direct interaction with components of the RLRs pathway ("hit"). In light of recent functional, biochemical and structural findings, we review the inhibition mechanisms of RLRs recognition of dsRNA displayed by a number of highly pathogenic RNA viruses with different disease phenotypes such as haemorrhagic fever (Ebola, Marburg, Lassa fever, Lujo, Machupo, Junin, Guanarito, Crimean-Congo, Rift Valley fever, dengue), severe respiratory disease (influenza, SARS, Hendra, Hantaan, Sin Nombre, Andes) and encephalitis (Nipah, West Nile). Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Characterization of soluble RNA-dependent RNA polymerase from dengue virus serotype 2: The polyhistidine tag compromises the polymerase activity.

    Science.gov (United States)

    Kamkaew, Maliwan; Chimnaronk, Sarin

    2015-08-01

    The viral RNA polymerase is an attractive target for inhibition in the treatment of viral infections. In the case of dengue virus (DENV), a member of the genus Flavivirus, the RNA-dependent RNA polymerase (RdRp) activity resides in the C-terminal two-thirds of non-structural protein (NS) 5 responsible for the de novo synthesis of the viral RNA genome. Among four distinct, but closely related dengue serotypes, serotype 2 (DENV-2) produces more severe diseases than other serotypes. It has been reported that bacterial production of the recombinant DENV-2 RdRp was difficult due to its low expression and solubility levels. To facilitate functional and structural analyses, we here demonstrate complete protocols for overexpression and purification of soluble DENV-2 RdRp, increasing protein yields by a remarkable 10 times compared to earlier reports. Three different forms of DENV-2 RdRp as either N- or C-terminally His-tagged fusions, or without tag, were purified to homogeneity. We show here that the presence of both the N- and C-terminal His-tag had a deleterious effect on polymerase activity and, in contrast to earlier studies, our non-tagged RdRp did not require manganese ions to activate RNA polymerization. We also determined an apparent Kd value of 53nM for binding to the 5'-UTR RNA by surface plasmon resonance (SPR). Our work provide a more suitable material for basic research of viral RdRp and for drug development. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Unbiased RNA Shotgun Metagenomics in Social and Solitary Wild Bees Detects Associations with Eukaryote Parasites and New Viruses.

    Science.gov (United States)

    Schoonvaere, Karel; De Smet, Lina; Smagghe, Guy; Vierstraete, Andy; Braeckman, Bart P; de Graaf, Dirk C

    2016-01-01

    The diversity of eukaryote organisms and viruses associated with wild bees remains poorly characterized in contrast to the well-documented pathosphere of the western honey bee, Apis mellifera. Using a deliberate RNA shotgun metagenomic sequencing strategy in combination with a dedicated bioinformatics workflow, we identified the (micro-)organisms and viruses associated with two bumble bee hosts, Bombus terrestris and Bombus pascuorum, and two solitary bee hosts, Osmia cornuta and Andrena vaga. Ion Torrent semiconductor sequencing generated approximately 3.8 million high quality reads. The most significant eukaryote associations were two protozoan, Apicystis bombi and Crithidia bombi, and one nematode parasite Sphaerularia bombi in bumble bees. The trypanosome protozoan C. bombi was also found in the solitary bee O. cornuta. Next to the identification of three honey bee viruses Black queen cell virus, Sacbrood virus and Varroa destructor virus-1 and four plant viruses, we describe two novel RNA viruses Scaldis River bee virus (SRBV) and Ganda bee virus (GABV) based on their partial genomic sequences. The novel viruses belong to the class of negative-sense RNA viruses, SRBV is related to the order Mononegavirales whereas GABV is related to the family Bunyaviridae. The potential biological role of both viruses in bees is discussed in the context of recent advances in the field of arthropod viruses. Further, fragmentary sequence evidence for other undescribed viruses is presented, among which a nudivirus in O. cornuta and an unclassified virus related to Chronic bee paralysis virus in B. terrestris. Our findings extend the current knowledge of wild bee parasites in general and addsto the growing evidence of unexplored arthropod viruses in valuable insects.

  4. Unbiased RNA Shotgun Metagenomics in Social and Solitary Wild Bees Detects Associations with Eukaryote Parasites and New Viruses.

    Directory of Open Access Journals (Sweden)

    Karel Schoonvaere

    Full Text Available The diversity of eukaryote organisms and viruses associated with wild bees remains poorly characterized in contrast to the well-documented pathosphere of the western honey bee, Apis mellifera. Using a deliberate RNA shotgun metagenomic sequencing strategy in combination with a dedicated bioinformatics workflow, we identified the (micro-organisms and viruses associated with two bumble bee hosts, Bombus terrestris and Bombus pascuorum, and two solitary bee hosts, Osmia cornuta and Andrena vaga. Ion Torrent semiconductor sequencing generated approximately 3.8 million high quality reads. The most significant eukaryote associations were two protozoan, Apicystis bombi and Crithidia bombi, and one nematode parasite Sphaerularia bombi in bumble bees. The trypanosome protozoan C. bombi was also found in the solitary bee O. cornuta. Next to the identification of three honey bee viruses Black queen cell virus, Sacbrood virus and Varroa destructor virus-1 and four plant viruses, we describe two novel RNA viruses Scaldis River bee virus (SRBV and Ganda bee virus (GABV based on their partial genomic sequences. The novel viruses belong to the class of negative-sense RNA viruses, SRBV is related to the order Mononegavirales whereas GABV is related to the family Bunyaviridae. The potential biological role of both viruses in bees is discussed in the context of recent advances in the field of arthropod viruses. Further, fragmentary sequence evidence for other undescribed viruses is presented, among which a nudivirus in O. cornuta and an unclassified virus related to Chronic bee paralysis virus in B. terrestris. Our findings extend the current knowledge of wild bee parasites in general and addsto the growing evidence of unexplored arthropod viruses in valuable insects.

  5. Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein

    International Nuclear Information System (INIS)

    Boulo, Sebastien; Akarsu, Hatice; Lotteau, Vincent; Mueller, Christoph W.; Ruigrok, Rob W.H.; Baudin, Florence

    2011-01-01

    Influenza virus has a segmented genome composed of eight negative stranded RNA segments. Each segment is covered with NP forming ribonucleoproteins (vRNPs) and carries a copy of the heterotrimeric polymerase complex. As a rare phenomenon among the RNA viruses, the viral replication occurs in the nucleus and therefore implies interactions between host and viral factors, such as between importin alpha and nucleoprotein. In the present study we report that through binding with the human nuclear receptor importin α5 (Impα5), the viral NP is no longer oligomeric but maintained as a monomer inside the complex. In this regard, Impα5 acts as a chaperone until NP is delivered in the nucleus for viral RNA encapsidation. Moreover, we show that the association of NP with the host transporter does not impair the binding of NP to RNA. The complex human Impα5-NP binds RNA with the same affinity as wt NP alone, whereas engineered monomeric NP through point mutations binds RNA with a strongly reduced affinity.

  6. Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

    Science.gov (United States)

    Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

  7. A structured RNA motif is involved in correct placement of the tRNA(3)(Lys) primer onto the human immunodeficiency virus genome

    NARCIS (Netherlands)

    Beerens, N.; Klaver, B.; Berkhout, B.

    2000-01-01

    Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA(3)(Lys) molecule that binds with its 3'-terminal 18 nucleotides to the fully complementary primer-binding site (PBS) on the viral RNA genome. Besides this complementarity, annealing of the primer may be

  8. Hepatitis C virus (HCV) RNA profiles among chronic HIV/HCV-coinfected individuals in ESPRIT; spontaneous HCV RNA clearance observed in nine individuals

    DEFF Research Database (Denmark)

    Grint, D; Tedaldi, Ellen; Peters, L

    2017-01-01

    OBJECTIVES: Studies have shown that hepatitis C virus (HCV) RNA levels remain stable over time in HIV/HCV-coinfected individuals taking combination antiretroviral therapy (cART), while spontaneous clearance of HCV RNA during the persistent infection phase has been documented only rarely among tho...

  9. Control of African swine fever virus replication by small interfering RNA targeting the A151R and VP72 genes.

    Science.gov (United States)

    Keita, Djénéba; Heath, Livio; Albina, Emmanuel

    2010-01-01

    African swine fever virus (ASFV) is the unique member of the Asfarviridae family and Asfivirus genus. It is an enveloped double-stranded DNA arbovirus that replicates in the cell cytoplasm, similar to poxviruses. There is no vaccine and no treatment available to control this virus. We describe the use of small interfering RNA (siRNA) targeting the A151R and VP72 (B646L) genes to control the ASFV replication in vitro. Results suggest that siRNA targeting the A151R and VP72 genes can reduce both the virus replication and its levels of messenger RNA transcripts. The reduction was up to 4 log(10) copies on the virus titre and up to 3 log(10) copies on virus RNA transcripts levels. The combination of multiple siRNA did not improve the antiviral effect significantly, compared with use of individual siRNAs. The function of the A151R gene product in the virus replication cycle is yet unclear, but is essential. We also demonstrate that it is possible to inhibit, using small interfering RNA, a virus that replicates exclusively in the cell cytoplasm in specific viral factories.

  10. De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs.

    Science.gov (United States)

    Seguin, Jonathan; Rajeswaran, Rajendran; Malpica-López, Nachelli; Martin, Robert R; Kasschau, Kristin; Dolja, Valerian V; Otten, Patricia; Farinelli, Laurent; Pooggin, Mikhail M

    2014-01-01

    Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.

  11. A raspberry bushy dwarf virus isolate from Ecuadorean Rubus glaucus contains an additional RNA that is a rearrangement of RNA-2.

    Science.gov (United States)

    Quito-Avila, D F; Ibarra, M A; Alvarez, R; Peralta, E L; Martin, R R

    2014-09-01

    Sequencing of the complete genome of a raspberry bushy dwarf virus isolate from Rubus glaucus in Ecuador revealed that its RNA-1 and RNA-2 were 5449 and 2231 nucleotides (nt) long, respectively, and phylogenetically closest to isolates from Sweden and Slovenia. In dsRNA analysis of infected plants, an additional band of 3 kbp was observed. Sequencing of this band revealed that it was 3279 nt long. BLAST searches revealed that this band contained a modified version of RNA-2, which consisted of RNA-2 (2231 nt) plus an additional 1048-nt fragment that was concatenated in a reverse-complement fashion to its 5' terminus.

  12. Role of human GRP75 in miRNA mediated regulation of dengue virus replication.

    Science.gov (United States)

    Kakumani, Pavan Kumar; Medigeshi, Guruprasad R; Kaur, Inderjeet; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2016-07-15

    In recent times, RNAi has emerged as an important defence system that regulates replication of pathogens in host cells. Many RNAi related host factors especially the host miRNAs play important roles in all intrinsic cellular functions, including viral infection. We have been working on identification of mammalian host factors involved in Dengue virus infection. In the present study, we identified Glucose Regulated Protein 75kDa (GRP75), as a host factor that is associated with dicer complex, in particular with HADHA (trifunctional enzyme subunit alpha, mitochondrial), an auxiliary component of dicer complex. Knockdown of GRP75 by respective siRNAs in Huh-7 cells resulted in the accumulation of dengue viral genomic RNA suggesting a role of GRP75 in regulating dengue virus replication in human cell lines. To elucidate the mode of action of GRP75, we over expressed the protein in Huh-7 cells and analysed the host miRNAs processing. The results revealed that, GRP75 is involved in processing of host miRNA, hsa-mir-126, that down regulates dengue virus replication. These findings suggest a regulatory role of human miRNA pathway especially GRP75 protein and hsa-mir-126 in dengue virus replication. These results thus provide insights into the role of miRNAs and RNAi machinery in dengue life cycle. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Comparison of various methods of detection of different forms of dengue virus type 2 RNA in cultured cells

    International Nuclear Information System (INIS)

    Liu, H.S.; Lin, Y.L.; Chen, C.C.

    1997-01-01

    In this report, the sensitivity of various methods of detection of dengue virus type 2 (DEN-2) sense, antisense, replicative intermediate (RI) and replicative form (RF) RNAs in infected mosquito Aedes pseudoscutellaris AP-61 and mammalian baby hamster kidney BHK-21 cells is compared. LiCl precipitation was used for separation of viral RF RNA from RI RNA. Our results show that reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis and slot blot hybridisation of LiCl-fractionated RNA were the most sensitive methods of detection of viral RNA and determination of its single-stranded form. Northern blot analysis was the least sensitive method of detection of any form of viral RNA. U sing slot blot hybridisation of LiCl-precipitated RNA, viral RI RNA containing de novo synthesised negative strand viral RNA was first detected 30 min after virus inoculation in both cell lines. This is the earliest time of detection of DEN viral RNA synthesis in host cells so far reported. However, RF RNA could not be detected until 24 hrs post infection (p.i.) in AP-61 and 2 days p.i. in BHK-21 cells, respectively. The sequential order of individual forms of viral RNA detected in the infected cells was RI, RF and genomic RNAs. Viral RNA was detected in AP-61 cells always earlier than in BHK-21 cells. Moreover, the level of viral RNA in AP-61 cells was higher than that in BHK-21 cells, suggesting that the virus replicated more actively in AP-61 cells. In conclusion, the LiCl separation of viral RNA followed by slot blot hybridisation was found to be the most sensitive and reliable method of detection of DEN virus RI, RF and genomic RNAs in the infected cells. Moreover, this method can be applied to determine the replication status of any single-stranded RNA virus in the host. (authors)

  14. Ammonia disinfection of hatchery waste for elimination of single-stranded RNA viruses.

    Science.gov (United States)

    Emmoth, Eva; Ottoson, Jakob; Albihn, Ann; Belák, Sándor; Vinnerås, Björn

    2011-06-01

    Hatchery waste, an animal by-product of the poultry industry, needs sanitation treatment before further use as fertilizer or as a substrate in biogas or composting plants, owing to the potential presence of opportunistic pathogens, including zoonotic viruses. Effective sanitation is also important in viral epizootic outbreaks and as a routine, ensuring high hygiene standards on farms. This study examined the use of ammonia at different concentrations and temperatures to disinfect hatchery waste. Inactivation kinetics of high-pathogenic avian influenza virus H7N1 and low-pathogenic avian influenza virus H5N3, as representatives of notifiable avian viral diseases, were determined in spiked hatchery waste. Bovine parainfluenza virus type 3, feline coronavirus, and feline calicivirus were used as models for other important avian pathogens, such as Newcastle disease virus, infectious bronchitis virus, and avian hepatitis E virus. Bacteriophage MS2 was also monitored as a stable indicator. Coronavirus was the most sensitive virus, with decimal reduction (D) values of 1.2 and 0.63 h after addition of 0.5% (wt/wt) ammonia at 14 and 25°C, respectively. Under similar conditions, high-pathogenic avian influenza H7N1 was the most resistant, with D values of 3.0 and 1.4 h. MS2 was more resistant than the viruses to all treatments and proved to be a suitable indicator of viral inactivation. The results indicate that ammonia treatment of hatchery waste is efficient in inactivating enveloped and naked single-stranded RNA viruses. Based on the D values and confidence intervals obtained, guidelines for treatment were proposed, and one was successfully validated at full scale at a hatchery, with MS2 added to hatchery waste.

  15. Three-dimensional structure of a protozoal double-stranded RNA virus that infects the enteric pathogen Giardia lamblia.

    Science.gov (United States)

    Janssen, Mandy E W; Takagi, Yuko; Parent, Kristin N; Cardone, Giovanni; Nibert, Max L; Baker, Timothy S

    2015-01-15

    Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans. Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia

  16. Zika Virus RNA Replication and Persistence in Brain and Placental Tissue

    Science.gov (United States)

    Rabeneck, Demi B.; Martines, Roosecelis B.; Reagan-Steiner, Sarah; Ermias, Yokabed; Estetter, Lindsey B.C.; Suzuki, Tadaki; Ritter, Jana; Keating, M. Kelly; Hale, Gillian; Gary, Joy; Muehlenbachs, Atis; Lambert, Amy; Lanciotti, Robert; Oduyebo, Titilope; Meaney-Delman, Dana; Bolaños, Fernando; Saad, Edgar Alberto Parra; Shieh, Wun-Ju; Zaki, Sherif R.

    2017-01-01

    Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections. PMID:27959260

  17. Stable hepatitis C virus RNA detection by RT-PCR during four days storage

    Directory of Open Access Journals (Sweden)

    Horsmans Yves

    2002-10-01

    Full Text Available Abstract Background Suboptimal specimen processing and storage conditions of samples which contain hepatitis C virus (HCV RNA may result in a decline of HCV RNA concentration or false-negative results in the detection of HCV RNA in serum. We evaluated the stability of HCV RNA in serum and clotted blood samples stored at room temperature or at 4°C for 4 days with the aim of optimizing the standard procedures of processing and storage of samples. Methods Blood from five HCV RNA positive patients was collected in tubes with and without separator gel, centrifuged 1 or 6 hours after collection. Samples were then left 6, 24, 48, 72 or 96 h at room temperature (21.5 – 25.4°C or at 4°C before determining their HCV RNA level using the COBAS AMPLICOR HCV MONITOR Test, vs 2.0 (Roche Diagnostic Systems. Results The logarithm of the HCV RNA level measurements remained within a 0.3 value of the means for 4 days at both temperatures (room temperature or 4°C. Conclusions We conclude that blood samples may be collected and aliquoted within 6 h of collection and can be stored at 4°C for 72 hours as proposed by the manufacturer without significant differences in measured HCV RNA level. Our results indicate that lapses in this scheme may still yield reliable results.

  18. Genetic variability and evolution of the satellite RNA of cucumber mosaic virus during natural epidemics.

    Science.gov (United States)

    Aranda, M A; Fraile, A; Garcia-Arenal, F

    1993-01-01

    The genetic structure of populations of cucumber mosaic virus (CMV) satellite RNA (satRNA) and its evolution were analyzed during the course of a CMV epidemic in tomatoes in eastern Spain. A total of 62 variants of CMV-satRNA from epidemic episodes in 1989, 1990, and 1991 were characterized by RNase protection assay (RPA); RPA patterns defined 60 haplotypes in the CMV-satRNA population. RPA of nine CMV-satRNAs of known sequences showed that numbers of nucleotide substitutions per site (dij) between different satRNAs can be estimated from RPA data. Thus, dij were estimated for any possible pair of field CMV-satRNA types, and nucleotide diversities within and between yearly subpopulations were calculated. Also, phylogenetic relationships among CMV-satRNAs were derived from RPA data (by parsimony) or from dij (by neighbor joining). From these analyses, a model for the evolution of CMV-satRNAs in field epidemics can be built. High genetic variability of CMV-satRNA results in very heterogeneous populations, even compared with those of other RNA genomes. The high diversity of the population is maintained through time by the continuous generation of variants by mutation, counterbalanced by negative selection; this results in a certain replacement of haplotypes from year to year. The sequential accumulation of mutations in CMV-satRNA leads to fast genetic divergence to reach what appears to be an upper permitted threshold. Images PMID:7690414

  19. The effect of RNA stiffness on the self-assembly of virus particles

    Science.gov (United States)

    Li, Siyu; Erdemci-Tandogan, Gonca; van der Schoot, Paul; Zandi, Roya

    2018-01-01

    Under many in vitro conditions, some small viruses spontaneously encapsidate a single stranded (ss) RNA into a protein shell called the capsid. While viral RNAs are found to be compact and highly branched because of long distance base-pairing between nucleotides, recent experiments reveal that in a head-to-head competition between an ssRNA with no secondary or higher order structure and a viral RNA, the capsid proteins preferentially encapsulate the linear polymer! In this paper, we study the impact of genome stiffness on the encapsidation free energy of the complex of RNA and capsid proteins. We show that an increase in effective chain stiffness because of base-pairing could be the reason why under certain conditions linear chains have an advantage over branched chains when it comes to encapsidation efficiency. While branching makes the genome more compact, RNA base-pairing increases the effective Kuhn length of the RNA molecule, which could result in an increase of the free energy of RNA confinement, that is, the work required to encapsidate RNA, and thus less efficient packaging.

  20. Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

    International Nuclear Information System (INIS)

    Brock, K.V.

    1987-01-01

    Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32 P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus

  1. Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA.

    Science.gov (United States)

    Khan, R; Giedroc, D P

    1992-04-05

    The nucleocapsid protein (NC) of all animal retroviruses, encoded by the gag gene, is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. NC is also thought to play one or more accessory roles in reverse transcription. Mature NC (p7NC) from human immunodeficiency virus type 1 (HIV-1) is a 71-amino acid, basic protein which contains two Cys3His Zn(II) retroviral-type zinc finger domains. Herein, we describe the subcloning and expression of HIV-1 NC, denoted NC71, from an inducible phage T7 RNA polymerase promoter in Escherichia coli. Purified NC71 can be reversibly reconstituted with 2 g.at Zn(II) determined by atomic absorption. Ultraviolet circulation dichroism spectroscopy has been used to characterize the complexes between highly purified NC71 and the RNA homopolynucleotide poly(A) and E. coli tRNA(mixed). On poly(A), Zn2 NC71 is characterized by an apparent site size n = 15 +/- 3 nucleotides and high affinity (Kapp = 3 x 10(7) M-1) and moderately cooperative (omega approximately 170 +/- 25) binding. A mixture of E. coli tRNA species (tRNA(mixed) was used to probe the conformational changes induced in tRNA upon binding of HIV-1 NC71. Two structural forms of tRNA(mixed), which differ in their degree of tertiary structure, were assayed for their susceptibility to denaturation by NC71. Five molar monomer equivalents of NC71 are required to denature the "inactive" tRNA in the absence of Mg2+. A Zn(II)-free, oxidized form of NC71 was also shown to unwind inactive tRNA with the same efficiency and stoichiometry. The detailed spectral changes which occur on NC-induced denaturation closely mimic temperature-induced denaturation of inactive tRNA(mixed). The prototype helix-destabilizing protein, T4 gene 32 protein, is unable to unwind this form of tRNA under the same conditions. The stoichiometry of unwinding of inactive tRNA by NC71 is consistent with the site size determined with poly(A). An "active" form of tRNA

  2. Viral RNA intermediates as targets for detection and discovery of novel and emerging mosquito-borne viruses.

    Science.gov (United States)

    O'Brien, Caitlin A; Hobson-Peters, Jody; Yam, Alice Wei Yee; Colmant, Agathe M G; McLean, Breeanna J; Prow, Natalie A; Watterson, Daniel; Hall-Mendelin, Sonja; Warrilow, David; Ng, Mah-Lee; Khromykh, Alexander A; Hall, Roy A

    2015-03-01

    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

  3. The use of RNA-dependent RNA polymerase for the taxonomic assignment of Picorna-like viruses (order Picornavirales infecting Apis mellifera L. populations

    Directory of Open Access Journals (Sweden)

    Schroeder Declan C

    2008-01-01

    Full Text Available Abstract Background Single-stranded RNA viruses, infectious to the European honeybee, Apis mellifera L. are known to reside at low levels in colonies, with typically no apparent signs of infection observed in the honeybees. Reverse transcription-PCR (RT-PCR of regions of the RNA-dependent RNA polymerase (RdRp is often used to diagnose their presence in apiaries and also to classify the type of virus detected. Results Analysis of RdRp conserved domains was undertaken on members of the newly defined order, the Picornavirales; focusing in particular on the amino acid residues and motifs known to be conserved. Consensus sequences were compiled using partial and complete honeybee virus sequences published to date. Certain members within the iflaviruses, deformed wing virus (DWV, Kakugo virus (KV and Varroa destructor virus (VDV; and the dicistroviruses, acute bee paralysis virus (ABPV, Israeli paralysis virus (IAPV and Kashmir bee virus (KBV, shared greater than 98% and 92% homology across the RdRp conserved domains, respectively. Conclusion RdRp was validated as a suitable taxonomic marker for the assignment of members of the order Picornavirales, with the potential for use independent of other genetic or phenotypic markers. Despite the current use of the RdRp as a genetic marker for the detection of specific honeybee viruses, we provide overwhelming evidence that care should be taken with the primer set design. We demonstrated that DWV, VDV and KV, or ABPV, IAPV and KBV, respectively are all recent descendents or variants of each other, meaning caution should be applied when assigning presence or absence to any of these viruses when using current RdRp primer sets. Moreover, it is more likely that some primer sets (regardless of what gene is used are too specific and thus are underestimating the diversity of honeybee viruses.

  4. Ectromelia virus accumulates less double-stranded RNA compared to vaccinia virus in BS-C-1 cells.

    Science.gov (United States)

    Frey, Tiffany R; Lehmann, Michael H; Ryan, Colton M; Pizzorno, Marie C; Sutter, Gerd; Hersperger, Adam R

    2017-09-01

    Most orthopoxviruses, including vaccinia virus (VACV), contain genes in the E3L and K3L families. The protein products of these genes have been shown to combat PKR, a host defense pathway. Interestingly, ectromelia virus (ECTV) contains an E3L ortholog but does not possess an intact K3L gene. Here, we gained insight into how ECTV can still efficiently evade PKR despite lacking K3L. Relative to VACV, we found that ECTV-infected BS-C-1 cells accumulated considerably less double-stranded (ds) RNA, which was due to lower mRNA levels and less transcriptional read-through of some genes by ECTV. The abundance of dsRNA in VACV-infected cells, detected using a monoclonal antibody, was able to activate the RNase L pathway at late time points post-infection. Historically, the study of transcription by orthopoxviruses has largely focused on VACV as a model. Our data suggest that there could be more to learn by studying other members of this genus. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Inhibition of porcine transmissible gastroenteritis virus (TGEV) replication in mini-pigs by shRNA.

    Science.gov (United States)

    Zhou, Junfang; Huang, Fen; Hua, Xiuguo; Cui, Li; Zhang, Wen; Shen, Yan; Yan, Yijia; Chen, Piren; Ding, Dezhong; Mou, Jing; Chen, Qi; Lan, Daoliang; Yang, Zhibiao

    2010-04-01

    Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis (TGE), characterized by high mortality and severely retarded growth in piglets that dramatically affects the porcine industry. Previously, we have identified two shRNA-expressing plasmids pEGFP-U6/P1 and pEGFP-U6/P2 that target RNA-dependent RNA polymerase (RdRP) gene of TGEV with more than 95% of virus inhibition in vitro. In this study, inhibition of the TGEV replication by pEGFP-U6/P1 and pEGFP-U6/P2 was tested in mini-pigs. SPF mini-pigs at 25 days old were injected with the shRNA-expressing plasmids and then infected with TGEV. The results from the analyses of clinical signs, histopathology, indirect immunofluorescence (IIF) and RT-PCR show that the two shRNA-expressing plasmids could significantly decrease the quantity of TGEV in different organs and protect mini-pigs from TGEV infection. These findings illustrate the prospect for TGEV-specific shRNAs to be new anti-TGEV agents. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  6. Binding of RNA by the Nucleoproteins of Influenza Viruses A and B.

    Science.gov (United States)

    Labaronne, Alice; Swale, Christopher; Monod, Alexandre; Schoehn, Guy; Crépin, Thibaut; Ruigrok, Rob W H

    2016-09-13

    This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)₆-FAM(3') RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.

  7. Binding of RNA by the Nucleoproteins of Influenza Viruses A and B

    Directory of Open Access Journals (Sweden)

    Alice Labaronne

    2016-09-01

    Full Text Available This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt, starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM-labelled RNAs. By using a (UC6-FAM3′ RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.

  8. Superiority of West Nile Virus RNA Detection in Whole Blood for Diagnosis of Acute Infection.

    Science.gov (United States)

    Lustig, Yaniv; Mannasse, Batya; Koren, Ravit; Katz-Likvornik, Shiri; Hindiyeh, Musa; Mandelboim, Michal; Dovrat, Sara; Sofer, Danit; Mendelson, Ella

    2016-09-01

    The current diagnosis of West Nile virus (WNV) infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia and absence of detectable virus in cerebrospinal fluid (CSF). Recent studies have shown that WNV RNA can be detected in urine for a longer period and at higher concentrations than in plasma. In this study, we examined the presence of WNV RNA in serum, plasma, whole-blood, CSF, and urine samples obtained from patients diagnosed with acute WNV infection during an outbreak which occurred in Israel in 2015. Our results demonstrate that 33 of 38 WNV patients had detectable WNV RNA in whole blood at the time of diagnosis, a higher rate than in any of the other sample types tested. Overall, whole blood was superior to all other samples, with 86.8% sensitivity, 100% specificity, 100% positive predictive value, and 83.9% negative predictive value. Interestingly, WNV viral load in urine was higher than in whole blood, CSF, serum, and plasma despite the lower sensitivity than that of whole blood. This study establishes the utility of whole blood in the routine diagnosis of acute WNV infection and suggests that it may provide the highest sensitivity for WNV RNA detection in suspected cases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Targeting microRNA-122 to Treat Hepatitis C Virus Infection.

    Science.gov (United States)

    Jopling, Catherine L

    2010-07-01

    An important host factor for hepatitis C virus (HCV) is microRNA-122 (miR-122). miR-122 is a liver-specific member of a family of small, non-coding RNA molecules known as microRNAs that play major roles in the regulation of gene expression by direct interaction with RNA targets. miR-122 binds directly to two sites in the 5' untranslated region (UTR) of HCV RNA and positively regulates the viral life cycle. The mechanism by which this regulation occurs is still not fully understood. There has been a great deal of interest in potential therapeutics based on small RNAs, and targeting miR-122 to combat HCV is one of the furthest advanced. Chemical inhibitors of miR-122 can be introduced into mammals intravenously and result in potent and specific knockdown of the microRNA, with no detectable adverse effects on liver physiology. This strategy was recently applied to chimpanzees chronically infected with HCV and resulted in a sustained reduction in viral load in the animals. Inhibition of miR-122 therefore presents a very attractive novel approach to treating HCV, a virus for which improved therapeutics are urgently needed.

  10. Targeting microRNA-122 to Treat Hepatitis C Virus Infection

    Directory of Open Access Journals (Sweden)

    Catherine L. Jopling

    2010-07-01

    Full Text Available An important host factor for hepatitis C virus (HCV is microRNA-122 (miR-122. miR-122 is a liver-specific member of a family of small, non-coding RNA molecules known as microRNAs that play major roles in the regulation of gene expression by direct interaction with RNA targets. miR-122 binds directly to two sites in the 5′ untranslated region (UTR of HCV RNA and positively regulates the viral life cycle. The mechanism by which this regulation occurs is still not fully understood. There has been a great deal of interest in potential therapeutics based on small RNAs, and targeting miR-122 to combat HCV is one of the furthest advanced. Chemical inhibitors of miR-122 can be introduced into mammals intravenously and result in potent and specific knockdown of the microRNA, with no detectable adverse effects on liver physiology. This strategy was recently applied to chimpanzees chronically infected with HCV and resulted in a sustained reduction in viral load in the animals. Inhibition of miR-122 therefore presents a very attractive novel approach to treating HCV, a virus for which improved therapeutics are urgently needed.

  11. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Deepika Bhullar

    Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

  12. Computational fitness landscape for all gene-order permutations of an RNA virus.

    Directory of Open Access Journals (Sweden)

    Kwang-il Lim

    2009-02-01

    Full Text Available How does the growth of a virus depend on the linear arrangement of genes in its genome? Answering this question may enhance our basic understanding of virus evolution and advance applications of viruses as live attenuated vaccines, gene-therapy vectors, or anti-tumor therapeutics. We used a mathematical model for vesicular stomatitis virus (VSV, a prototype RNA virus that encodes five genes (N-P-M-G-L, to simulate the intracellular growth of all 120 possible gene-order variants. Simulated yields of virus infection varied by 6,000-fold and were found to be most sensitive to gene-order permutations that increased levels of the L gene transcript or reduced levels of the N gene transcript, the lowest and highest expressed genes of the wild-type virus, respectively. Effects of gene order on virus growth also depended upon the host-cell environment, reflecting different resources for protein synthesis and different cell susceptibilities to infection. Moreover, by computationally deleting intergenic attenuations, which define a key mechanism of transcriptional regulation in VSV, the variation in growth associated with the 120 gene-order variants was drastically narrowed from 6,000- to 20-fold, and many variants produced higher progeny yields than wild-type. These results suggest that regulation by intergenic attenuation preceded or co-evolved with the fixation of the wild type gene order in the evolution of VSV. In summary, our models have begun to reveal how gene functions, gene regulation, and genomic organization of viruses interact with their host environments to define processes of viral growth and evolution.

  13. The brome mosaic virus 3' untranslated sequence regulates RNA replication, recombination, and virion assembly.

    Science.gov (United States)

    Rao, A L N; Cheng Kao, C

    2015-08-03

    The 3' untranslated region in each of the three genomic RNAs of Brome mosaic virus (BMV) is highly homologous and contains a sequence that folds into a tRNA-like structure (TLS). Experiments performed over the past four decades revealed that the BMV 3' TLS regulates many important steps in BMV infection. This review summarizes in vitro and in vivo studies of the roles of the BMV 3' TLS functioning as a minus-strand promoter, in RNA recombination, and to nucleate virion assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Defective RNA sensing by RIG-I in severe influenza virus infection

    DEFF Research Database (Denmark)

    Jørgensen, Sofie Eg; Christiansen, Mette; Ryø, Laura Barrett

    2018-01-01

    Influenza virus infection causes worldwide seasonal epidemics. Although influenza usually is a mild disease, a minority of patients experience very severe fulminating disease courses. Previous studies have demonstrated a role for type I interferon (IFN) in antiviral responses during influenza...... and p.P885S, located in the CARD and RNA binding domains, respectively, of DDX58 encoding the RNA sensor RIG-I. These variants significantly impair the signaling activity of RIG-I. Similarly, patient cells demonstrate decreased antiviral responses to RIG-I ligands as well as increased pro...

  15. Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.

    Science.gov (United States)

    Zhang, Jianhua; Nie, Xianzhou; Boquel, Sébastien; Al-Daoud, Fadi; Pelletier, Yvan

    2015-12-01

    The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was sulfite RNA isolation methods, respectively. Copyright © 2015. Published by Elsevier B.V.

  16. Intracellular localization of rice stripe virus RNA-dependent RNA polymerase and its interaction with nucleocapsid protein.

    Science.gov (United States)

    Zhao, Shuling; Hao, Jiahui; Xue, Yanan; Liang, Changyong

    2015-12-01

    The RNA-dependent RNA polymerase (RdRp) of rice stripe virus (RSV) is critical for both the transcription and replication of the viral genome. Despite its importance, little is known about how it functions in cells. In the present study, RSV RdRp was split into three pieces, since expression of the full protein could not be achieved. Then, the intracellular localization of these three RdRp fragments and their interactions with nucleocapsid protein (NP) were investigated, which is another viral protein required for viral RNA synthesis. The data showed that all three RdRp fragments displayed punctuate staining patterns in the cytoplasm, and the C-terminal fragment co-localized with NP in the perinuclear region. Both bimolecular fluorescence complementation and co-immunoprecipitation experiments demonstrated that of the three RdRp fragments, only the C-terminal fragment could interact with NP. Further analysis using a series of truncated NPs identified the N-terminal 50-amino-acid region within NP as the determinant for its interaction with the C-terminus of RdRp.

  17. Identification and molecular characterization of a new nonsegmented double-stranded RNA virus isolated from Culex mosquitoes in Japan.

    Science.gov (United States)

    Isawa, Haruhiko; Kuwata, Ryusei; Hoshino, Keita; Tsuda, Yoshio; Sakai, Kouji; Watanabe, Shumpei; Nishimura, Miho; Satho, Tomomitsu; Kataoka, Michiyo; Nagata, Noriyo; Hasegawa, Hideki; Bando, Hisanori; Yano, Kazuhiko; Sasaki, Toshinori; Kobayashi, Mutsuo; Mizutani, Tetsuya; Sawabe, Kyoko

    2011-01-01

    Two infectious agents were isolated from Culex species mosquitoes in Japan and were identified as distinct strains of a new RNA virus by a method for sequence-independent amplification of viral nucleic acids. The virus designated Omono River virus (OMRV) replicated in mosquito cells in which it produced a severe cytopathic effect. Icosahedral virus particles of approximately 40 nm in diameter were detected in the cytoplasm of infected cells. The OMRV genome was observed to consist of a nonsegmented, 7.6-kb double-stranded RNA (dsRNA) and contain two overlapping open reading frames (ORFs), namely ORF1 and ORF2. ORF1 was found to encode a putative dsRNA-binding protein, a major capsid protein, and other putative proteins, which might be generated by co- and/or post-translational processing of the ORF1 polyprotein precursor, and ORF2 was found to encode a putative RNA-dependent RNA polymerase (RdRp), which could be translated as a fusion with the ORF1 product by a -1 ribosomal frameshift. Phylogenetic analysis based on RdRp revealed that OMRV is closely related to penaeid shrimp infectious myonecrosis virus and Drosophila totivirus, which are tentatively assigned to the family Totiviridae. These results indicated that OMRV is a new member of the family of nonsegmented dsRNA viruses infecting arthropod hosts, but not fungal or protozoan hosts. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Detection of hepatitis A virus by hybridization with single-stranded RNA probes

    International Nuclear Information System (INIS)

    Xi, J.; Estes, M.K.; Metcalf, T.G.

    1987-01-01

    An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32 P-labeled ssRNA probes were at least eightfold more sensitive than the 32 P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32 P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted an semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay

  19. Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

    Science.gov (United States)

    Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

    2015-11-01

    To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

  20. Hepatitis delta antigen requires a flexible quasi-double-stranded RNA structure to bind and condense hepatitis delta virus RNA in a ribonucleoprotein complex.

    Science.gov (United States)

    Griffin, Brittany L; Chasovskikh, Sergey; Dritschilo, Anatoly; Casey, John L

    2014-07-01

    The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2'OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg. RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed

  1. The role of microRNAs in hepatitis C virus RNA replication.

    Science.gov (United States)

    Conrad, K Dominik; Niepmann, Michael

    2014-05-01

    Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5' end of the 5' untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA's 5' end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5'-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.

  2. The untranslated regions of classic swine fever virus RNA trigger apoptosis.

    Directory of Open Access Journals (Sweden)

    Wei-Li Hsu

    Full Text Available Classical swine fever virus (CSFV causes a broad range of disease in pigs, from acute symptoms including high fever and hemorrhages, to chronic disease or unapparent infection, depending on the virus strain. CSFV belongs to the genus Pestivirus of the family Flaviviridae. It carries a single-stranded positive-sense RNA genome. An internal ribosomal entry site (IRES in the 5' untranslated region (UTR drives the translation of a single open reading frame encoding a 3898 amino acid long polypeptide chain. The open reading frame is followed by a 3' UTR comprising four highly structured stem-loops. In the present study, a synthetic RNA composed of the 5' and 3' UTRs of the CSFV genome devoid of any viral coding sequence and separated by a luciferase gene cassette (designated 5'UTR-Luc-3'UTR triggered apoptotic cell death as early as 4 h post-transfection. The apoptosis was measured by DNA laddering analysis, TUNEL assay, annexin-V binding determined by flow cytometry, and by analysis of caspase activation. Contrasting with this, only trace DNA laddering was observed in cells transfected with the individual 5' or 3' UTR RNA; even when the 5' UTR and 3' UTR were co-transfected as separate RNA molecules, DNA laddering did not reach the level induced by the chimeric 5'UTR-Luc-3'UTR RNA. Interestingly, RNA composed of the 5'UTR and of stem-loop I of the 3'UTR triggered much stronger apoptosis than the 5' or 3'UTR alone. These results indicate that the 5' and 3' UTRs act together in cis induce apoptosis. We furthered obtained evidence that the UTR-mediated apoptosis required double-stranded RNA and involved translation shutoff possibly through activation of PKR.

  3. Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses.

    Science.gov (United States)

    Spear, Allyn; Sisterson, Mark S; Yokomi, Raymond; Stenger, Drake C

    2010-09-01

    Novel double-stranded RNAs (approximately 8 kbp) were isolated from threecornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA polymerase (RdRp). Expression of the 3'-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3. Published by Elsevier Inc.

  4. Longitudinal follow-up of Zika virus RNA in semen of a traveller returning from Barbados to the Netherlands with Zika virus disease, March 2016

    NARCIS (Netherlands)

    C.B.E.M. Reusken (Chantal); S.D. Pas (Suzan); C.H. Geurts van Kessel (Corine); R. Mögling (Ramona); J.J.A. van Kampen (Jeroen); T. Langerak (Thomas); M.P.G. Koopmans D.V.M. (Marion); A.A. Eijck (Annemiek); E.C.M. van Gorp (Eric)

    2016-01-01

    textabstractWe report the longitudinal follow-up of Zika virus (ZIKV) RNA in semen of a traveller who developed ZIKV disease after return to the Netherlands from Barbados, March 2016. Persistence of ZIKV RNA in blood, urine, saliva and semen was followed until the loads reached undetectable levels.

  5. Longitudinal follow-up of Zika virus RNA in semen of a traveller returning from Barbados to the Netherlands with Zika virus disease, march 2016

    NARCIS (Netherlands)

    C.B.E.M. Reusken (Chantal); S.D. Pas (Suzan); C.H. Geurts van Kessel (Corine); R. Mögling (Ramona); J.J.A. van Kampen (Jeroen); T. Langerak (Thomas); M.P.G. Koopmans D.V.M. (Marion); A.A. Eijck (Annemiek); E.C.M. van Gorp (Eric)

    2016-01-01

    textabstractWe report the longitudinal follow-up of Zika virus (ZIKV) RNA in semen of a traveller who developed ZIKV disease after return to the Netherlands from Barbados, March 2016. Persistence of ZIKV RNA in blood, urine, saliva and semen was followed until the loads reached undetectable levels.

  6. Quantitative multi-target RNA profiling in Epstein-Barr virus infected tumor cells.

    Science.gov (United States)

    Greijer, A E; Ramayanti, O; Verkuijlen, S A W M; Novalić, Z; Juwana, H; Middeldorp, J M

    2017-03-01

    Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Discovery of a dsRNA virus infecting the marine photosynthetic protist Micromonas pusilla

    International Nuclear Information System (INIS)

    Brussaard, C.P.D.; Noordeloos, A.A.M.; Sandaa, R.-A.; Heldal, M.; Bratbak, G.

    2004-01-01

    We report the isolation of the first double-stranded (ds) RNA virus in the family Reoviridae that infects a protist (microalga Micromonas pusilla, Prasinophyceae). The dsRNA genome was composed of 11 segments ranging between 0.8 and 5.8 kb, with a total size of approximately 25.5 kb. The virus (MpRNAV-01B) could not be assigned to the genus level because host type, genome size, and number of segments smaller than 2 kb did not correspond to either of the two existing 11-segmented dsRNA genera Rotavirus and Aquareovirus. MpRNAV-01B has a particle size of 65-80 nm, a narrow host range, a latent period of 36 h, and contains five major proteins (120, 95, 67, 53, and 32 kDa). MpRNAV-01B was stable to freeze-thawing, resistant to chloroform, ether, nonionic detergents, chelating and reducing agents. The virus was inactivated at temperatures above 35 deg. C and by ionic detergent, ethanol, acetone, and acidic conditions (pH 2-5)

  8. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    Science.gov (United States)

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2).

  9. Positive-Strand RNA Viruses Infecting the Red Imported Fire Ant, Solenopsis invicta

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    Steven M. Valles

    2012-01-01

    Full Text Available The imported fire ants, Solenopsis invicta and S. richteri were introduced into the USA between 1918 and 1945. Since that time, they have expanded their USA range to include some 138 million hectares. Their introduction has had significant economic consequences with costs associated with damage and control efforts estimated at 6 billion dollars annually in the USA. The general consensus of entomologists and myrmecologists is that permanent, sustainable control of these ants in the USA will likely depend on self-sustaining biological control agents. A metagenomics approach successfully resulted in discovery of three viruses infecting S. invicta. Solenopsis invicta virus 1 (SINV-1, SINV-2, and SINV-3 are all positive, single-stranded RNA viruses and represent the first viral discoveries in any ant species. Molecular characterization, host relationships, and potential development and use of SINV-1, SINV-2, and SINV-3 as biopesticides are discussed.

  10. Biochemical characterization of enzyme fidelity of influenza A virus RNA polymerase complex.

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    Shilpa Aggarwal

    2010-04-01

    Full Text Available It is widely accepted that the highly error prone replication process of influenza A virus (IAV, together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol, is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized.Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT of human immunodeficiency virus (HIV-1 and murine leukemia virus (MuLV, which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg(++ with Mn(++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn(++. Finally, when the IAV nucleoprotein (NP was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity.Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of

  11. Active RNA replication of hepatitis C virus downregulates CD81 expression.

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    Po-Yuan Ke

    Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  12. Vector-borne transmission imposes a severe bottleneck on an RNA virus population.

    Science.gov (United States)

    Forrester, Naomi L; Guerbois, Mathilde; Seymour, Robert L; Spratt, Heidi; Weaver, Scott C

    2012-09-01

    RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller's ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study.

  13. Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation.

    Science.gov (United States)

    Zhu, Hui; Duan, Cheng-Guo; Hou, Wei-Na; Du, Quan-Sheng; Lv, Dian-Qiu; Fang, Rong-Xiang; Guo, Hui-Shan

    2011-12-01

    RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.

  14. VP1, the RNA-dependent RNA polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4AII

    NARCIS (Netherlands)

    Tacken, M.G.J.; Thomas, A.A.M.; Peeters, B.P.H.; Rottier, P.J.M.; Boot, H.J.

    2004-01-01

    Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight

  15. A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells.

    Science.gov (United States)

    Zhang, Qianyi; Xu, Lu; Zhang, Yujie; Wang, Tuanjie; Zou, Xingqi; Zhu, Yuanyuan; Zhao, Yan; Li, Cui; Chen, Kai; Sun, Yongfang; Sun, Junxiang; Zhao, Qizu; Wang, Qin

    2017-04-18

    Classical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization method, quantitatively localization and visualization of the virus RNA molecular in cultured cell or tissue section becomes very important tool to address these pivotal pathogenic questions. In this study, we established ViewRNA ISH method to reveal the dynamic distribution of CSFV RNA in PK15 cells. We designed several specific probes of CSFV RNA and reference gene β-actin for host PK15 cells to monitor the relative location of CSFV RNA and house-keeping gene in the infected cells. After determining the titer of reference strain CSFV (HeBHH1/95) with the 50% tissue culture infective dose (TCID50), we optimized the protease K concentration and formalin fixation time to analyze the hybridization efficiency, fluorescence intensity and repeatability. In order to measure the sensitivity of this assay, we compared it with the fluorescent antibody test (FAT) and immunohistochemical(IHC) method. Specificity of the ViewRNA ISH was tested by detecting several sub genotypes of CSFV (sub genotype 1.1, 2.1, 2.2 and 2.3) which are present in China and other normal pig infectious virus (bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirusII(PCV-2). The lowest detection threshold of the ViewRNA ISH method was 10 -8 , while the sensitivity of FAT and IHC were 10 -5 and 10 -4 , respectively. The ViewRNA ISH was specific for CSFV RNA including 1.1, 2.1, 2.2 and 2.3 subtypes, meanwhile, there was no cross-reaction with negative control and other viruses including BVDV, PPV, PRV and PCV-2. Our results showed that after infection at 0.5 hpi (hours post inoculation, hpi), the CSFV RNA can be detected in nucleus and cytoplasm; during 3-9 hpi, RNA

  16. Hepatitis C virus double-stranded RNA is the predominant form in human liver and in interferon-treated cells.

    Science.gov (United States)

    Klepper, Arielle; Eng, Francis J; Doyle, Erin H; El-Shamy, Ahmed; Rahman, Adeeb H; Fiel, M Isabel; Avino, Gonzalo Carrasco; Lee, Moonju; Ye, Fei; Roayaie, Sasan; Bansal, Meena B; MacDonald, Margaret R; Schiano, Thomas D; Branch, Andrea D

    2017-08-01

    Hepatitis C virus (HCV) is unique among RNA viruses in its ability to establish chronic infection in the majority of exposed adults. HCV persists in the liver despite interferon (IFN)-stimulated gene (ISG) induction; robust induction actually predicts treatment failure and viral persistence. It is unclear which forms of HCV RNA are associated with ISG induction and IFN resistance during natural infections. To thoroughly delineate HCV RNA populations, we developed conditions that fully separate the strands of long double-stranded RNA (dsRNA) and allow the released RNAs to be quantified in reverse transcription/polymerase chain reaction assays. These methods revealed that dsRNA, a pathogen-associated molecular pattern (PAMP), comprised 52% (standard deviation, 28%) of the HCV RNA in the livers of patients with chronic infection. HCV dsRNA was proportionally higher in patients with the unfavorable IL28B TT (rs12979860) genotype. Higher ratios of HCV double-stranded to single-stranded RNA (ssRNA) correlated positively with ISG induction. In Huh-7.5 cells, IFN treatment increased the total amount of HCV dsRNA through a process that required de novo viral RNA synthesis and shifted the ratio of viral dsRNA/ssRNA in favor of dsRNA. This shift was blocked by ribavirin (RBV), an antiviral drug that reduces relapse in HCV patients. Northern blotting established that HCV dsRNA contained genome-length minus strands. HCV dsRNA is the predominant form in the HCV-infected liver and has features of both a PAMP and a genomic reservoir. Interferon treatment increased rather than decreased HCV dsRNA. This unexpected finding suggests that HCV produces dsRNA in response to IFN, potentially to antagonize antiviral defenses. (Hepatology 2017;66:357-370). © 2016 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

  17. Respiratory Syncytial Virus (RSV RNA loads in peripheral blood correlates with disease severity in mice

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    Torres Juan

    2010-09-01

    Full Text Available Abstract Background Respiratory Syncytial Virus (RSV infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity. Methods Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood. Results RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes. Conclusions RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.

  18. siRNA rescues nonhuman primates from advanced Marburg and Ravn virus disease

    Science.gov (United States)

    Thi, Emily P.; Mire, Chad E.; Lee, Amy C.H.; Geisbert, Joan B.; Ursic-Bedoya, Raul; Agans, Krystle N.; Robbins, Marjorie; Deer, Daniel J.; Cross, Robert W.; Kondratowicz, Andrew S.; Fenton, Karla A.; MacLachlan, Ian; Geisbert, Thomas W.

    2017-01-01

    Ebolaviruses and marburgviruses belong to the family Filoviridae and cause high lethality in infected patients. There are currently no licensed filovirus vaccines or antiviral therapies. The development of broad-spectrum therapies against members of the Marburgvirus genus, including Marburg virus (MARV) and Ravn virus (RAVV), is difficult because of substantial sequence variability. RNAi therapeutics offer a potential solution, as identification of conserved target nucleotide sequences may confer activity across marburgvirus variants. Here, we assessed the therapeutic efficacy of lipid nanoparticle (LNP) delivery of a single nucleoprotein–targeting (NP-targeting) siRNA in nonhuman primates at advanced stages of MARV or RAVV disease to mimic cases in which patients begin treatment for fulminant disease. Sixteen rhesus monkeys were lethally infected with MARV or RAVV and treated with NP siRNA-LNP, with MARV-infected animals beginning treatment four or five days after infection and RAVV-infected animals starting treatment three or six days after infection. While all untreated animals succumbed to disease, NP siRNA-LNP treatment conferred 100% survival of RAVV-infected macaques, even when treatment began just 1 day prior to the death of the control animals. In MARV-infected animals, day-4 treatment initiation resulted in 100% survival, and day-5 treatment resulted in 50% survival. These results identify a single siRNA therapeutic that provides broad-spectrum protection against both MARV and RAVV. PMID:29106386

  19. Cryphonectria nitschkei virus 1 structure shows that the capsid protein of chrysoviruses is a duplicated helix-rich fold conserved in fungal double-stranded RNA viruses.

    Science.gov (United States)

    Gómez-Blanco, Josué; Luque, Daniel; González, José M; Carrascosa, José L; Alfonso, Carlos; Trus, Benes; Havens, Wendy M; Ghabrial, Said A; Castón, José R

    2012-08-01

    Cryoelectron microscopy reconstruction of Cryphonectria nitschkei virus 1, a double-stranded RNA (dsRNA) virus, shows that the capsid protein (60 copies/particle) is formed by a repeated helical core, indicative of gene duplication. This unusual organization is common to chrysoviruses. The arrangement of many of these putative α-helices is conserved in the totivirus L-A capsid protein, suggesting a shared motif. Our results indicate that a 120-subunit T=1 capsid is a conserved architecture that optimizes dsRNA replication and organization.

  20. Cellular microRNA networks regulate host dependency of hepatitis C virus infection.

    Science.gov (United States)

    Li, Qisheng; Lowey, Brianna; Sodroski, Catherine; Krishnamurthy, Siddharth; Alao, Hawwa; Cha, Helen; Chiu, Stephan; El-Diwany, Ramy; Ghany, Marc G; Liang, T Jake

    2017-11-27

    Cellular microRNAs (miRNAs) have been shown to regulate hepatitis C virus (HCV) replication, yet a systematic interrogation of the repertoire of miRNAs impacting HCV life cycle is lacking. Here we apply integrative functional genomics strategies to elucidate global HCV-miRNA interactions. Through genome-wide miRNA mimic and hairpin inhibitor phenotypic screens, and miRNA-mRNA transcriptomics analyses, we identify three proviral and nine antiviral miRNAs that interact with HCV. These miRNAs are functionally linked to particular steps of HCV life cycle and related viral host dependencies. Further mechanistic studies demonstrate that miR-25, let-7, and miR-130 families repress essential HCV co-factors, thus restricting viral infection at multiple stages. HCV subverts the antiviral actions of these miRNAs by dampening their expression in cell culture models and HCV-infected human livers. This comprehensive HCV-miRNA interaction map provides fundamental insights into HCV-mediated pathogenesis and unveils molecular pathways linking RNA biology to viral infections.

  1. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.

    Science.gov (United States)

    Pardi, Norbert; Hogan, Michael J; Pelc, Rebecca S; Muramatsu, Hiromi; Andersen, Hanne; DeMaso, Christina R; Dowd, Kimberly A; Sutherland, Laura L; Scearce, Richard M; Parks, Robert; Wagner, Wendeline; Granados, Alex; Greenhouse, Jack; Walker, Michelle; Willis, Elinor; Yu, Jae-Sung; McGee, Charles E; Sempowski, Gregory D; Mui, Barbara L; Tam, Ying K; Huang, Yan-Jang; Vanlandingham, Dana; Holmes, Veronica M; Balachandran, Harikrishnan; Sahu, Sujata; Lifton, Michelle; Higgs, Stephen; Hensley, Scott E; Madden, Thomas D; Hope, Michael J; Karikó, Katalin; Santra, Sampa; Graham, Barney S; Lewis, Mark G; Pierson, Theodore C; Haynes, Barton F; Weissman, Drew

    2017-03-09

    Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

  2. A +RNA virus diptych : Chikungunya virus-host interactions and arteriviral programmed ribosomal frameshifting

    NARCIS (Netherlands)

    Treffers, Emma Elisabeth (Emmely)

    2015-01-01

    In part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus (CHIKV). Several proteins were identified that may be specifically downregulated during CHIKV infection to

  3. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    International Nuclear Information System (INIS)

    Li Guiqiu; Gu Hongxi; Li Di; Xu Weizhen

    2007-01-01

    The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies

  4. Targeting microRNA-122: walking on cutting edge of hepatitis C virus infection therapy.

    Science.gov (United States)

    Motavaf, M; Safari, S; Alavian, S M

    2014-01-01

    Hepatitis C virus (HCV) infection, with an estimated 170 million carriers worldwide, remains a major cause of chronic liver disease. Current anti-HCV treatments have significant side effects and have met with only partial success. Therefore, a more effective therapeutic modality for HCV infection is needed. The stability and propagation of HCV is dependent on the interaction between its genome and a highly abundant liver microRNA (miRNA), known as microRNA-122 (miR-122). As a conserved host factor that would not be expected to evolve resistance mutations, miR-122 makes an attractive antiviral target. In this review we will discuss how targeting miR-122, using antisense oligonucleotides (ASOs), can be a new anti-HCV treatment.

  5. Interference of hepatitis C virus RNA replication by short interfering RNAs

    Science.gov (United States)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  6. Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

    Science.gov (United States)

    Mirzakhanyan, Yeva; Gershon, Paul D

    2017-09-01

    The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size. Copyright © 2017 American Society for Microbiology.

  7. Data mining cDNAs reveals three new single stranded RNA viruses in Nasonia (Hymenopetera:Pteromalidae)

    Science.gov (United States)

    Hymenopteran viruses may provide insights into colony collapse disorder in honey bees and other insect species. Three novel small RNA viruses were discovered during the genomics effort for the beneficial parasitoid of flies in the genus Nasonia (Hymenoptera). Genomics provides a great deal of inform...

  8. Promoter melting by a stage-specific vaccinia virus transcription factor is independent of the presence of RNA polymerase

    NARCIS (Netherlands)

    Vos, J C; Sasker, M; Stunnenberg, H.G.

    1991-01-01

    Fractionation of an extract prepared from HeLa cells infected with vaccinia virus resulted in the separation of factors involved in vaccinia virus intermediate transcription. Two activities, VITF-A and VITF-B, in addition to the viral RNA polymerase are necessary and sufficient to direct

  9. Transformation of Cowpea Vigna unguiculata with a Full-Length DNA Copy of Cowpea Mosaic Virus M-RNA

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1987-01-01

    A full-length DNA copy of the M-RNA of cowpea mosaic virus (CPMV), supplied with either the 35S promoter from cauliflower mosaic virus (CaMV) or the nopaline synthase promoter from Agrobacterium tumefaciens, was introduced into the T-DNA region of a Ti-plasmid-derived gene vector and transferred to

  10. Hepatitis B Virus-Encoded MicroRNA Controls Viral Replication.

    Science.gov (United States)

    Yang, Xi; Li, Hongfeng; Sun, Huahui; Fan, Hongxia; Hu, Yaqi; Liu, Min; Li, Xin; Tang, Hua

    2017-05-15

    MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding, functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and Northern blotting. HBV-miR-3 is located at nucleotides (nt) 373 to 393 of the HBV genome and was generated from 3.5-kb, 2.4-kb, and 2.1-kb HBV in a classic miRNA biogenesis (Drosha-Dicer-dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV + hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released into the circulation by exosomes and HBV virions, and HBV-miR-3 expression had a positive correlation with HBV titers in the sera of patients in the acute phase of HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg, and replication of HBV. HBV-miR-3 targets the unique site of the HBV 3.5-kb transcript to specifically reduce HBc protein expression, levels of pregenomic RNA (pgRNA), and HBV replication intermediate (HBV-RI) generation but does not affect the HBV DNA polymerase level, thus suppressing HBV virion production (replication). This may explain the low levels of HBV virion generation with abundant subviral particles lacking core during HBV replication, which may contribute to the development of persistent infection in patients. Taken together, our findings shed light on novel mechanisms by which HBV-encoded miRNA controls the process of self-replication by regulating HBV transcript during infection. IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV) that can become a long-term, chronic infection and lead to cirrhosis or liver cancer. HBV is a small DNA virus that belongs to the hepadnavirus family, with virions and subviral forms of particles that lack a core. MicroRNA (miRNA), a small (∼22-nt) noncoding RNA

  11. A shift in plant proteome profile for a Bromodomain containing RNA binding Protein (BRP1) in plants infected with Cucumber mosaic virus and its satellite RNA.

    Science.gov (United States)

    Chaturvedi, Sonali; Rao, A L N

    2016-01-10

    Host proteins are the integral part of a successful infection caused by a given RNA virus pathogenic to plants. Therefore, identification of crucial host proteins playing an important role in establishing the infection process is likely to help in devising approaches to curbing disease spread. Cucumber mosaic virus (Q-CMV) and its satellite RNA (QsatRNA) are important pathogens of many economically important crop plants worldwide. In a previous study, we demonstrated the biological significance of a Bromodomain containing RNA-binding Protein (BRP1) in the infection cycle of QsatRNA, making BRP1 an important host protein to study. To further shed a light on the mechanistic role of BRP1 in the replication of Q-CMV and QsatRNA, we analyzed the Nicotiana benthamiana host protein interactomes either for BRP1 alone or in the presence of Q-CMV or QsatRNA. Co-immunoprecipitation, followed by LC-MS/MS analysis of BRP1-FLAG on challenging with Q-CMV or QsatRNA has led us to observe a shift in the host protein interactome of BRP1. We discuss the significance of these results in relation to Q-CMV and its QsatRNA infection cycle. Host proteins play an important role in replication and infection of eukaryotic cells by a wide-range of RNA viruses pathogenic to humans, animals and plants. Since a given eukaryotic cell typically contains ~30,000 different proteins, recent advances made in proteomics and bioinformatics approaches allowed the identification of host proteins critical for viral replication and pathogenesis. Although Cucumber mosaic virus (CMV) and its satRNA are well characterized at molecular level, information concerning the network of host factors involved in their replication and pathogenesis is still on its infancy. We have recently observed that a Bromodomain containing host protein (BRP1) is obligatory to transport satRNA to the nucleus. Consequently, it is imperative to apply proteomics and bioinformatics approaches in deciphering how host interactome network

  12. Competitive selection in vivo by a cell for one variant over another: implications for RNA virus quasispecies in vivo.

    OpenAIRE

    Dockter, J; Evans, C F; Tishon, A; Oldstone, M B

    1996-01-01

    Infidelity of genome applications of RNA viruses leads to the generation of viral quasispecies both in vitro and in vivo. However, the biological significance of such generated variants in vivo is largely unknown and controversial. To study this issue, we continued our evaluation of the tropism of a lymphocytic choriomeningitis virus (LCMV) variant termed clone 13 with its parental virus clonal pool ARM 53b (wild-type parent) for neuronal cells in vivo. Earlier in vivo and in vitro studies no...

  13. The role of virulence for in vivo superinfection fitness of a vertebrate RNA virus

    Science.gov (United States)

    Kell, Alison M.; Wargo, Andrew R.; Kurath, Gael

    2013-01-01

    We have developed a novel, in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural, vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a co-evolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 hours to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal virulence and unequal virulence genotype pairs, the frequency of superinfection was the same regardless of which genotype was used in the primary exposure. However, the ability to replicate in the context of superinfection did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection, while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV, and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural, vertebrate host.

  14. Adaptation to fluctuating temperatures in an RNA virus is driven by the most stringent selective pressure.

    Directory of Open Access Journals (Sweden)

    María Arribas

    Full Text Available The frequency of change in the selective pressures is one of the main factors driving evolution. It is generally accepted that constant environments select specialist organisms whereas changing environments favour generalists. The particular outcome achieved in either case also depends on the relative strength of the selective pressures and on the fitness costs of mutations across environments. RNA viruses are characterized by their high genetic diversity, which provides fast adaptation to environmental changes and helps them evade most antiviral treatments. Therefore, the study of the adaptive possibilities of RNA viruses is highly relevant for both basic and applied research. In this study we have evolved an RNA virus, the bacteriophage Qβ, under three different temperatures that either were kept constant or alternated periodically. The populations obtained were analyzed at the phenotypic and the genotypic level to characterize the evolutionary process followed by the virus in each case and the amount of convergent genetic changes attained. Finally, we also investigated the influence of the pre-existent genetic diversity on adaptation to high temperature. The main conclusions that arise from our results are: i under periodically changing temperature conditions, evolution of bacteriophage Qβ is driven by the most stringent selective pressure, ii there is a high degree of evolutionary convergence between replicated populations and also among populations evolved at different temperatures, iii there are mutations specific of a particular condition, and iv adaptation to high temperatures in populations differing in their pre-existent genetic diversity takes place through the selection of a common set of mutations.

  15. A sting in the spit: widespread cross-infection of multiple RNA viruses across wild and managed bees.

    Science.gov (United States)

    McMahon, Dino P; Fürst, Matthias A; Caspar, Jesicca; Theodorou, Panagiotis; Brown, Mark J F; Paxton, Robert J

    2015-05-01

    Declining populations of bee pollinators are a cause of concern, with major repercussions for biodiversity loss and food security. RNA viruses associated with honeybees represent a potential threat to other insect pollinators, but the extent of this threat is poorly understood. This study aims to attain a detailed understanding of the current and ongoing risk of emerging infectious disease (EID) transmission between managed and wild pollinator species across a wide range of RNA viruses. Within a structured large-scale national survey across 26 independent sites, we quantify the prevalence and pathogen loads of multiple RNA viruses in co-occurring managed honeybee (Apis mellifera) and wild bumblebee (Bombus spp.) populations. We then construct models that compare virus prevalence between wild and managed pollinators. Multiple RNA viruses associated with honeybees are widespread in sympatric wild bumblebee populations. Virus prevalence in honeybees is a significant predictor of virus prevalence in bumblebees, but we remain cautious in speculating over the principle direction of pathogen transmission. We demonstrate species-specific differences in prevalence, indicating significant variation in disease susceptibility or tolerance. Pathogen loads within individual bumblebees may be high and in the case of at least one RNA virus, prevalence is higher in wild bumblebees than in managed honeybee populations. Our findings indicate widespread transmission of RNA viruses between managed and wild bee pollinators, pointing to an interconnected network of potential disease pressures within and among pollinator species. In the context of the biodiversity crisis, our study emphasizes the importance of targeting a wide range of pathogens and defining host associations when considering potential drivers of population decline. © 2015 The Authors. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.

  16. Supervised learning classification models for prediction of plant virus encoded RNA silencing suppressors.

    Directory of Open Access Journals (Sweden)

    Zeenia Jagga

    Full Text Available Viral encoded RNA silencing suppressor proteins interfere with the host RNA silencing machinery, facilitating viral infection by evading host immunity. In plant hosts, the viral proteins have several basic science implications and biotechnology applications. However in silico identification of these proteins is limited by their high sequence diversity. In this study we developed supervised learning based classification models for plant viral RNA silencing suppressor proteins in plant viruses. We developed four classifiers based on supervised learning algorithms: J48, Random Forest, LibSVM and Naïve Bayes algorithms, with enriched model learning by correlation based feature selection. Structural and physicochemical features calculated for experimentally verified primary protein sequences were used to train the classifiers. The training features include amino acid composition; auto correlation coefficients; composition, transition, and distribution of various physicochemical properties; and pseudo amino acid composition. Performance analysis of predictive models based on 10 fold cross-validation and independent data testing revealed that the Random Forest based model was the best and achieved 86.11% overall accuracy and 86.22% balanced accuracy with a remarkably high area under the Receivers Operating Characteristic curve of 0.95 to predict viral RNA silencing suppressor proteins. The prediction models for plant viral RNA silencing suppressors can potentially aid identification of novel viral RNA silencing suppressors, which will provide valuable insights into the mechanism of RNA silencing and could be further explored as potential targets for designing novel antiviral therapeutics. Also, the key subset of identified optimal features may help in determining compositional patterns in the viral proteins which are important determinants for RNA silencing suppressor activities. The best prediction model developed in the study is available as a

  17. Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

    Science.gov (United States)

    Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

    2017-12-01

    Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

  18. High rate of hepatitis C virus (HCV) recurrence in HIV-infected individuals with spontaneous HCV RNA clearance

    NARCIS (Netherlands)

    Peters, L.; Mocroft, A.; Soriano, V.; Rockstroh, J. K.; Kirkby, N.; Reiss, P.; Katlama, C.; Zakharova, N.; Flisiak, R.; Lundgren, J. D.

    2014-01-01

    Following resolution of hepatitis C virus (HCV) infection, recurrence has been shown to occur in some persons with repeated exposure to HCV. We aimed to investigate the rate and factors associated with HCV RNA recurrence among HIV-1-infected patients with prior spontaneous HCV RNA clearance in the

  19. Factors associated with hepatitis C virus RNA levels in early chronic infection: the InC3 study

    NARCIS (Netherlands)

    Hajarizadeh, B.; Grady, B.; Page, K.; Kim, A. Y.; McGovern, B. H.; Cox, A. L.; Rice, T. M.; Sacks-Davis, R.; Bruneau, J.; Morris, M.; Amin, J.; Schinkel, J.; Applegate, T.; Maher, L.; Hellard, M.; Lloyd, A. R.; Prins, M. [= Maria; Geskus, R. B.; Dore, G. J.; Grebely, J.; Page, Kimberly; Bruneau, Julie; Cox, Andrea L.; Dore, Gregory J.; Grebely, Jason; Hellard, Margaret; Lauer, Georg; Kim, Arthur Y.; Lloyd, Andrew R.; Maher, Lisa; McGovern, Barbara H.; Shoukry, Naglaa H.; Morris, Meghan; Hahn, Judy; Shiboski, Steve; Rice, Thomas M.; Alavi, Maryam; Bouchard, Rachel; Evans, Jennifer; Grady, Bart; Aneja, Jasneet; Sacks-Davis, Rachel; Teutsch, Suzy; White, Bethany; Wells, Brittany; Zang, Geng; Matthews, Gail; Yeung, Barbara; Prince, Leslie Erin; Roy, Elise; Bates, Anna; Enriquez, Jarliene; Chow, Sammy; McCredie, Luke; Aitken, Campbell; Doyle, Joseph; Spelman, Tim

    2015-01-01

    Improved understanding of natural history of hepatitis C virus (HCV) RNA levels in chronic infection provides enhanced insights into immunopathogenesis of HCV and has implications for the clinical management of chronic HCV infection. This study assessed factors associated with HCV RNA levels during

  20. Deletion analysis of cis- and trans-acting elements involved in replication of alfalfa mosaic virus RNA 3 in vivo

    NARCIS (Netherlands)

    van der Kuyl, A. C.; Neeleman, L.; Bol, J. F.

    1991-01-01

    DNA copies of alfalfa mosaic virus (AIMV) RNA 3 were transcribed in vitro into RNA molecules with deletions in coding and noncoding sequences. The replication of these transcripts was studied in protoplasts from transgenic tobacco plants expressing DNA copies of AIMV RNAs 1 and 2. Deletions in the

  1. West Nile virus noncoding subgenomic RNA contributes to viral evasion of the type I interferon-mediated antiviral response.

    Science.gov (United States)

    Schuessler, Andrea; Funk, Anneke; Lazear, Helen M; Cooper, Daphne A; Torres, Shessy; Daffis, Stephane; Jha, Babal Kant; Kumagai, Yutaro; Takeuchi, Osamu; Hertzog, Paul; Silverman, Robert; Akira, Shizuo; Barton, David J; Diamond, Michael S; Khromykh, Alexander A

    2012-05-01

    We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.

  2. Structure and Dynamics of the tRNA-like Structure Domain of Brome Mosaic Virus

    Science.gov (United States)

    Vieweger, Mario; Nesbitt, David

    2014-03-01

    Conformational switching is widely accepted as regulatory mechanism in gene expression in bacterial systems. More recently, similar regulation mechanisms are emerging for viral systems. One of the most abundant and best studied systems is the tRNA-like structure domain that is found in a number of plant viruses across eight genera. In this work, the folding dynamics of the tRNA-like structure domain of Brome Mosaic Virus are investigated using single-molecule Fluorescence Resonance Energy Transfer techniques. In particular, Burst fluorescence is applied to observe metal-ion induced folding in freely diffusing RNA constructs resembling the 3'-terminal 169nt of BMV RNA3. Histograms of EFRET probabilities reveal a complex equilibrium of three distinct populations. A step-wise kinetic model for TLS folding is developed in accord with the evolution of conformational populations and structural information in the literature. In this mechanism, formation of functional TLS domains from unfolded RNAs requires two consecutive steps; 1) hybridization of a long-range stem interaction followed by 2) formation of a 3' pseudoknot. This three-state equilibrium is well described by step-wise dissociation constants K1(328(30) μM) and K2(1092(183) μM) for [Mg2+] and K1(74(6) mM) and K2(243(52) mM) for [Na+]-induced folding. The kinetic model is validated by oligo competition with the STEM interaction. Implications of this conformational folding mechanism are discussed in regards to regulation of virus replication.

  3. Attenuation of Dengue Virus Infection by Adeno-Associated Virus-Mediated siRNA Delivery

    Science.gov (United States)

    2004-08-09

    and effective prophylaxis or treatment for dengue virus (DEN) infection, a category A mosquito - borne human pathogen, is a critical global priority...through Aedes aegypti mosquito bites, and resident skin DCs are regarded as the targets of DEN infection [12]. DCs are thought to be 10-fold more per...do not induce production of neutralizing antibodies that could reduce transgene function. They possess a broad-range of tissue tropism and the

  4. Urbanization and the dynamics of RNA viruses in Mallards (Anas platyrhynchos).

    Science.gov (United States)

    Wille, Michelle; Lindqvist, Kristine; Muradrasoli, Shaman; Olsen, Björn; Järhult, Josef D

    2017-07-01

    Urbanization is intensifying worldwide, and affects the epidemiology of infectious diseases. However, the effect of urbanization on natural host-pathogen systems remains poorly understood. Urban ducks occupy an interesting niche in that they directly interact with both humans and wild migratory birds, and either directly or indirectly with food production birds. Here we have collected samples from Mallards (Anas platyrhynchos) residing in a pond in central Uppsala, Sweden, from January 2013 to January 2014. This artificial pond is kept ice-free during the winter months, and is a popular location where the ducks are fed, resulting in a resident population of ducks year-round. Nine hundred and seventy seven (977) fecal samples were screened for RNA viruses including: influenza A virus (IAV), avian paramyxovirus 1, avian coronavirus (CoV), and avian astrovirus (AstroV). This intra-annual dataset illustrates that these RNA viruses exhibit similar annual patterns to IAV, suggesting similar ecological factors are at play. Furthermore, in comparison to wild ducks, autumnal prevalence of IAV and CoV are lower in this urban population. We also demonstrate that AstroV might be a larger burden to urban ducks than IAV, and should be better assessed to demonstrate the degree to which wild birds contribute to the epidemiology of these viruses. The presence of economically relevant viruses in urban Mallards highlights the importance of elucidating the ecology of wildlife pathogens in urban environments, which will become increasingly important for managing disease risks to wildlife, food production animals, and humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Biotechnological approaches for plant viruses resistance: from general to the modern RNA silencing pathway

    Directory of Open Access Journals (Sweden)

    Silas Pessini Rodrigues

    2009-08-01

    Full Text Available Virus diseases are significant threats to modern agriculture and their control remains a challenge to the management of cultivation. The main virus resistance strategies are based on either natural resistance or engineered virus-resistant plants. Recent progress in understanding the molecular mechanisms underlying the roles of resistance genes has promoted the development of new anti-virus strategies. Engineered plants, in particular plants expressing RNA-silencing nucleotides, are becoming increasingly important and are likely to provide more effective strategies in future. A general discussion on the biotechnology of plant responses to virus infection is followed by recent advances in engineered plant resistance.As viroses são problemas importantes para a agricultura moderna e o seu controle representa um desafio para o manejo de áreas cultivadas. As principais estratégias de resistência a vírus se baseiam em mecanismos naturais ou em engenharia genética. Recentemente, a maior compreensão dos mecanismos moleculares envolvidos na função de genes de resistência da planta facilitou o desenvolvimento de novas estratégias antivirais. Plantas modificadas geneticamente, em particular aquelas expressando a via de silenciamento de RNA, são alvo de interesse crescente e representam a possibilidade de estratégias futuras mais efetivas. Neste trabalho são discutidos diferentes aspectos relacionados à resistência a viroses em plantas. Adicionalmente, a perspectiva de aplicação biotecnológica das diferentes vias de resistência é apresentada.

  6. Indeterminate RIBA results were associated with the absence of hepatitis C virus RNA (HCV-RNA in blood donors

    Directory of Open Access Journals (Sweden)

    Felicidade Mota Pereira

    2014-01-01

    Full Text Available Introduction: Hepatitis C virus (HCV infection is diagnosed by the presence of antibodies and is supplemented by confirmatory testing methods, such as recombinant immunoblot assay (RIBA and HCV-RNA detection. This study aimed to evaluate the efficacy of RIBA testing to diagnose HCV infection in blood donors positive for anti-HCV antibodies. Methods: A total of 102 subjects positive for anti-HCV determined by enzyme-linked immunosorbent assay (ELISA at the Hematology and Hemotherapy Foundation of Bahia (HEMOBA were later assessed with new samples using the Abbott Architect anti-HCV test (Abbott Diagnostics, Wiesbaden, Germany, the RIBA III test (Chiron RIBA HCV 3.0 SIA, Chiron Corp., Emeryville, CA, USA, the polymerase chain reaction (PCR; COBAS® AMPLICOR HCV Roche Diagnostics Corp., Indianapolis, IN, USA and line probe assay (LiPA - Siemens, Tarrytown, NY, USA genotyping for HCV diagnosis. Results: Of these new samples, 38.2% (39/102 were positive, 57.8% (59/102 were negative and 3.9% (4/102 were indeterminate for anti-HCV; HCV-RNA was detected in 22.5% (23/102 of the samples. RIBA results were positive in 58.1% (25/43, negative in 9.3% (4/43 and indeterminate in 32.6% (14/43 of the samples. The prevailing genotypes were 1 (78.3%, 18/23, 3 (17.4%, 4/23 and 2 (4.3%, 1/23. All 14 samples with indeterminate RIBA results had undetectable viral loads (detection limit ≤50 IU/mL. Of these samples, 71.4% (10/14 were reevaluated six months later. Eighty percent (8/10 of these samples remained indeterminate by RIBA, and 20% (2/10 were negative. Conclusions: In this study, individuals with indeterminate RIBA results had no detectable HCV-RNA.

  7. Sequence analysis of RNA 2 and RNA 3 of lilac leaf chlorosis virus: a putative new member of the genus Ilarvirus.

    Science.gov (United States)

    James, D; Varga, A; Leippi, L; Godkin, S; Masters, C

    2010-06-01

    RNA 2 and RNA 3 of lilac leaf chlorosis virus (LLCV) were sequenced and shown to be 2,762 nucleotides (nt) and 2,117 nts in length, respectively. RNA 2 encodes a putative 807-amino-acid (aa) RNA-dependent RNA polymerase associated protein with an estimated M (r) of 92.75 kDa. RNA 3 is bicistronic, with ORF1 encoding a putative movement protein (277 aa, M (r) 31.45 kDa) and ORF2 encoding the putative coat protein (221 aa, M (r) 24.37 kDa). The genome organization is similar to that typical for members of the genus Ilarvirus. Phylogenetic analyses indicate a close evolutionary relationship between LLCV, ApMV, and PNRSV.

  8. MicroRNA 130a Regulates both Hepatitis C Virus and Hepatitis B Virus Replication through a Central Metabolic Pathway.

    Science.gov (United States)

    Duan, Xiaoqiong; Li, Shilin; Holmes, Jacinta A; Tu, Zeng; Li, Yujia; Cai, Dachuan; Liu, Xiao; Li, Wenting; Yang, Chunhui; Jiao, Baihai; Schaefer, Esperance A; Fusco, Dahlene N; Salloum, Shadi; Chen, Limin; Lin, Wenyu; Chung, Raymond T

    2018-04-01

    Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication. IMPORTANCE We identified

  9. Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses.

    Science.gov (United States)

    Lennefors, Britt-Louise; van Roggen, Petra M; Yndgaard, Flemming; Savenkov, Eugene I; Valkonen, Jari P T

    2008-04-01

    Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is one of the most devastating sugar beet diseases. Sugar beet plants engineered to express a 0.4 kb inverted repeat construct based on the BNYVV replicase gene accumulated the transgene mRNA to similar levels in leaves and roots, whereas accumulation of the transgene-homologous siRNA was more pronounced in roots. The roots expressed high levels of resistance to BNYVV transmitted by the vector, Polymyxa betae. Resistance to BNYVV was not decreased following co-infection of the plants with Beet soil borne virus and Beet virus Q that share the same vector with BNYVV. Similarly, co-infection with the aphid-transmitted Beet mild yellowing virus, Beet yellows virus (BYV), or with all of the aforementioned viruses did not affect the resistance to BNYVV, while they accumulated in roots. These viruses are common in most of the sugar beet growing areas in Europe and world wide. However, there was a competitive interaction between BYV and BMYV in sugar beet leaves, as infection with BYV decreased the titres of BMYV. Other interactions between the viruses studied were not observed. The results suggest that the engineered resistance to BNYVV expressed in the sugar beets of this study is efficient in roots and not readily compromised following infection of the plants with heterologous viruses.

  10. Analysis of the in vitro secondary structure of cucumber mosaic virus satellite RNA.

    Science.gov (United States)

    Bernal, J J; García-Arenal, F

    1997-01-01

    The solution conformation of two variants of cucumber mosaic virus satellite RNA (CMV satRNA) was analyzed using several enzymatic and chemical probes. Ribonuclease T1 and nuclease S1 were used to map unpaired nucleotides, and nuclease V1 was used to detect double-stranded, or stacked, bases. Chemical probing with dimethylsulphate and diethylpyrocarbonate also identified unpaired and unstacked nucleotides, respectively. Modified or cleaved positions were identified by direct gel electrophoresis of radioactively labeled RNA, or by analysis of DNA sequence patterns generated by primer-extension with reverse transcriptase. Additional information was obtained by a gel-fractionation method under nondenaturing conditions for the identification of base paired fragments. On these data, a model for the in vitro secondary structure of CMV satRNA is proposed. Results support the existence of a complex structure with 51% of nucleotides involved in base pairs (40 G:C, 28 G:U, and 18 A:U pairs). Several structural elements, numbered I-VI, were defined, and interactions between separate domains are suggested. Comparisons of experimental data and a formerly reported secondary structure model for CMV satRNA support the validity of the structure we propose. PMID:9292503

  11. Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.

    Science.gov (United States)

    Matranga, Christian B; Andersen, Kristian G; Winnicki, Sarah; Busby, Michele; Gladden, Adrianne D; Tewhey, Ryan; Stremlau, Matthew; Berlin, Aaron; Gire, Stephen K; England, Eleina; Moses, Lina M; Mikkelsen, Tarjei S; Odia, Ikponmwonsa; Ehiane, Philomena E; Folarin, Onikepe; Goba, Augustine; Kahn, S Humarr; Grant, Donald S; Honko, Anna; Hensley, Lisa; Happi, Christian; Garry, Robert F; Malboeuf, Christine M; Birren, Bruce W; Gnirke, Andreas; Levin, Joshua Z; Sabeti, Pardis C

    2014-01-01

    We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

  12. Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity.

    Science.gov (United States)

    Turkington, Hannah L; Juozapaitis, Mindaugas; Kerry, Philip S; Aydillo, Teresa; Ayllon, Juan; García-Sastre, Adolfo; Schwemmle, Martin; Hale, Benjamin G

    2015-10-01

    We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. RNA interference mediated inhibition of dengue virus multiplication and entry in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Abdelfatah Alhoot

    Full Text Available BACKGROUND: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. METHODOLOGY/PRINCIPAL FINDINGS: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78 and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4% and extracellular viral RNA load (71.4% compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7% in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells. CONCLUSIONS/SIGNIFICANCE: Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS

  14. Two Negative-Strand RNA Viruses Identified in Watermelon Represent a Novel Clade in the Order Bunyavirales

    Directory of Open Access Journals (Sweden)

    Min Xin

    2017-08-01

    Full Text Available Two novel negative-sense, single-stranded (ss RNA viruses were identified in watermelon plants and named watermelon crinkle leaf-associated virus 1 and 2 (WCLaV-1 and -2, respectively. The multipartite genomes consist of three RNA molecules of ~6.8, 1.4, and 1.3 kb. The genomes and the deduced proteins of RNA1 and RNA3 show features resembling those of members in the genus Phlebovirus and Tenuivirus; however, the predicted proteins encoded by RNA2 are related to the movement protein (MP in the genus Ophiovirus and Emaravirus. Furthermore, these two viruses define a novel clade in the family Phenuiviridae, order Bunyavirales, which is phylogenetically related to the viruses in the above four genera. Moreover, after mechanical inoculation with WCLaV-1 seedlings of the natural host watermelon plants develop crinkling similar to those observed in the field. These findings enhance our understanding of the evolution and the classification of ssRNA viruses.

  15. The nucleocapsid domain is responsible for the ability of spleen necrosis virus (SNV) Gag polyprotein to package both SNV and murine leukemia virus RNA.

    Science.gov (United States)

    Certo, J L; Kabdulov, T O; Paulson, M L; Anderson, J A; Hu, W S

    1999-11-01

    Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.

  16. Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Sudiro, T M; Zivny, J; Ishiko, H; Green, S; Vaughn, D W; Kalayanarooj, S; Nisalak, A; Norman, J E; Ennis, F A; Rothman, A L

    2001-01-01

    There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

  17. The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

    Science.gov (United States)

    Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

    2017-11-24

    To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus , family Closteroviridae ) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana . Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

  18. Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation.

    Science.gov (United States)

    McCaskill, Jana L; Marsh, Glenn A; Monaghan, Paul; Wang, Lin-Fa; Doran, Timothy; McMillan, Nigel A J

    2013-01-01

    Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi) based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs) targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.

  19. Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation.

    Directory of Open Access Journals (Sweden)

    Jana L McCaskill

    Full Text Available Hendra virus (HeV is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.

  20. Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture

    Science.gov (United States)

    Lohmann, Volker; Hoffmann, Sandra; Herian, Ulrike; Penin, Francois; Bartenschlager, Ralf

    2003-01-01

    Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself. PMID:12584326

  1. Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

    Science.gov (United States)

    Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

    2013-01-01

    RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001

  2. Biopolymers under large external forces and mean-field RNA virus evolutionary dynamics

    Science.gov (United States)

    Ahsan, Syed Amir

    The modeling of the mechanical response of single-molecules of DNA and RNA under large external forces through statistical mechanical methods is central to this thesis with a small portion devoted to modeling the evolutionary dynamics of positive-sense single-stranded RNA viruses. In order to develop and test models of biopolymer mechanics and illuminate the mechanisms underlying biological processes where biopolymers undergo changes in energy on the order of the thermal energy, , entails measuring forces and lengths on the scale of piconewtons (pN) and nanometers (nm), respectively. A capacity achieved in the past two decades at the single-molecule level through the development of micromanipulation techniques such as magnetic and optical tweezers, atomic force microscopy, coupled with advances in micro- and nanofabrication. The statistical mechanical models of biopolymers developed in this dissertation are dependent upon and the outcome of these advancements and resulting experiments. The dissertation begins in chapter 1 with an introduction to the structure and thermodynamics of DNA and RNA, highlighting the importance and effectiveness of simple, two-state models in their description as a prelude to the emergence of two-state models in the research manuscripts. In chapter 2 the standard models of the elasticity of polymers and of a polymer gel are reviewed, characterizing the continuum and mean-field models, including the scaling behavior of DNA in confined spaces. The research manuscript presented in the last section of chapter 2 (section 2.5), subsequent to a review of a Flory gel and in contrast to it, is a model of the elasticity of RNA as a gel, with viral RNA illustrating an instance of such a network, and shown to exhibit anomalous elastic behavior, a negative Poisson ratio, and capable of facilitating viral RNA encapsidation with further context provided in section 5.1. In chapter 3 the experimental methods and behavior of DNA and RNA under mechanical

  3. Complete nucleotide sequence of a South African isolate of Grapevine fanleaf virus and its associated satellite RNA.

    Science.gov (United States)

    Lamprecht, Renate L; Spaltman, Monique; Stephan, Dirk; Wetzel, Thierry; Burger, Johan T

    2013-07-17

    The complete sequences of RNA1, RNA2 and satellite RNA have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SACH44). The two RNAs of GFLV-SACH44 are 7,341 nucleotides (nt) and 3,816 nt in length, respectively, and its satellite RNA (satRNA) is 1,104 nt in length, all excluding the poly(A) tail. Multiple sequence alignment of these sequences showed that GFLV-SACH44 RNA1 and RNA2 were the closest to the South African isolate, GFLV-SAPCS3 (98.2% and 98.6% nt identity, respectively), followed by the French isolate, GFLV-F13 (87.3% and 90.1% nt identity, respectively). Interestingly, the GFLV-SACH44 satRNA is more similar to three Arabis mosaic virus satRNAs (85%-87.4% nt identity) than to the satRNA of GFLV-F13 (81.8% nt identity) and was most distantly related to the satRNA of GFLV-R2 (71.0% nt identity). Full-length infectious clones of GFLV-SACH44 satRNA were constructed. The infectivity of the clones was tested with three nepovirus isolates, GFLV-NW, Arabis mosaic virus (ArMV)-NW and GFLV-SAPCS3. The clones were mechanically inoculated in Chenopodium quinoa and were infectious when co-inoculated with the two GFLV helper viruses, but not when co-inoculated with ArMV-NW.

  4. High Variety of Known and New RNA and DNA Viruses of Diverse Origins in Untreated Sewage

    Science.gov (United States)

    Ng, Terry Fei Fan; Marine, Rachel; Wang, Chunlin; Simmonds, Peter; Kapusinszky, Beatrix; Bodhidatta, Ladaporn; Oderinde, Bamidele Soji; Wommack, K. Eric

    2012-01-01

    Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for high-throughput viral discovery. A metagenomics analysis of purified viral particles in untreated sewage from the United States (San Francisco, CA), Nigeria (Maiduguri), Thailand (Bangkok), and Nepal (Kathmandu) revealed sequences related to 29 eukaryotic viral families infecting vertebrates, invertebrates, and plants (BLASTx E score, 90% protein identities) in numerous viral families infecting humans (Adenoviridae, Astroviridae, Caliciviridae, Hepeviridae, Parvoviridae, Picornaviridae, Picobirnaviridae, and Reoviridae), plants (Alphaflexiviridae, Betaflexiviridae, Partitiviridae, Sobemovirus, Secoviridae, Tombusviridae, Tymoviridae, Virgaviridae), and insects (Dicistroviridae, Nodaviridae, and Parvoviridae). The full and partial genomes of a novel kobuvirus, salivirus, and sapovirus are described. A novel astrovirus (casa astrovirus) basal to those infecting mammals and birds, potentially representing a third astrovirus genus, was partially characterized. Potential new genera and families of viruses distantly related to members of the single-stranded RNA picorna-like virus superfamily were genetically characterized and named Picalivirus, Secalivirus, Hepelivirus, Nedicistrovirus, Cadicistrovirus, and Niflavirus. Phylogenetic analysis placed these highly divergent genomes near the root of the picorna-like virus superfamily, with possible vertebrate, plant, or arthropod hosts inferred from nucleotide composition analysis. Circular DNA genomes distantly related to the plant-infecting Geminiviridae family were named Baminivirus, Nimivirus, and Niminivirus. These results highlight the utility of analyzing sewage to monitor shedding of viral pathogens and the high viral diversity found in this common pollutant and provide genetic information to facilitate future studies of these newly characterized viruses. PMID:22933275

  5. Molecular Mechanisms of Innate Immune Inhibition by Non-Segmented Negative-Sense RNA Viruses

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, Srirupa; Basler, Christopher F.; Amarasinghe, Gaya K.; Leung, Daisy W.

    2016-08-01

    The host innate immune system serves as the first line of defense against viral infections. Germline-encoded pattern recognition receptors detect molecular patterns associated with pathogens and activate innate immune responses. Of particular relevance to viral infections are those pattern recognition receptors that activate type I interferon responses, which establish an antiviral state. The order Mononegavirales is composed of viruses that possess single-stranded, non-segmented negative-sense (NNS) RNA genomes and are important human pathogens that consistently antagonize signaling related to type I interferon responses. NNS viruses have limited encoding capacity compared to many DNA viruses, and as a likely consequence, most open reading frames encode multifunctional viral proteins that interact with host factors in order to evade host cell defenses while promoting viral replication. In this review, we will discuss the molecular mechanisms of innate immune evasion by select NNS viruses. A greater understanding of these interactions will be critical in facilitating the development of effective therapeutics and viral countermeasures.

  6. Diversity of DNA and RNA Viruses in Indoor Air As Assessed via Metagenomic Sequencing.

    Science.gov (United States)

    Rosario, Karyna; Fierer, Noah; Miller, Shelly; Luongo, Julia; Breitbart, Mya

    2018-02-06

    Diverse bacterial and fungal communities inhabit human-occupied buildings and circulate in indoor air; however, viral diversity in these man-made environments remains largely unknown. Here we investigated DNA and RNA viruses circulating in the air of 12 university dormitory rooms by analyzing dust accumulated over a one-year period on heating, ventilation, and air conditioning (HVAC) filters. A metagenomic sequencing approach was used to determine the identity and diversity of viral particles extracted from the HVAC filters. We detected a broad diversity of viruses associated with a range of hosts, including animals, arthropods, bacteria, fungi, humans, plants, and protists, suggesting that disparate organisms can contribute to indoor airborne viral communities. Viral community composition and the distribution of human-infecting papillomaviruses and polyomaviruses were distinct in the different dormitory rooms, indicating that airborne viral communities are variable in human-occupied spaces and appear to reflect differential rates of viral shedding from room occupants. This work significantly expands the known airborne viral diversity found indoors, enabling the design of sensitive and quantitative assays to further investigate specific viruses of interest and providing new insight into the likely sources of viruses found in indoor air.

  7. Molecular Mechanisms of Innate Immune Inhibition by Non-Segmented Negative-Sense RNA Viruses.

    Science.gov (United States)

    Chatterjee, Srirupa; Basler, Christopher F; Amarasinghe, Gaya K; Leung, Daisy W

    2016-08-28

    The host innate immune system serves as the first line of defense against viral infections. Germline-encoded pattern recognition receptors detect molecular patterns associated with pathogens and activate innate immune responses. Of particular relevance to viral infections are those pattern recognition receptors that activate type I interferon responses, which establish an antiviral state. The order Mononegavirales is composed of viruses that possess single-stranded, non-segmented negative-sense (NNS) RNA genomes and are important human pathogens that consistently antagonize signaling related to type I interferon responses. NNS viruses have limited encoding capacity compared to many DNA viruses, and as a likely consequence, most open reading frames encode multifunctional viral proteins that interact with host factors in order to evade host cell defenses while promoting viral replication. In this review, we will discuss the molecular mechanisms of innate immune evasion by select NNS viruses. A greater understanding of these interactions will be critical in facilitating the development of effective therapeutics and viral countermeasures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. RNA viruses in hymenopteran pollinators: evidence of inter-Taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species.

    Directory of Open Access Journals (Sweden)

    Rajwinder Singh

    2010-12-01

    Full Text Available Although overall pollinator populations have declined over the last couple of decades, the honey bee (Apis mellifera malady, colony collapse disorder (CCD, has caused major concern in the agricultural community. Among honey bee pathogens, RNA viruses are emerging as a serious threat and are suspected as major contributors to CCD. Recent detection of these viral species in bumble bees suggests a possible wider environmental spread of these viruses with potential broader impact. It is therefore vital to study the ecology and epidemiology of these viruses in the hymenopteran pollinator community as a whole. We studied the viral distribution in honey bees, in their pollen loads, and in other non-Apis hymenopteran pollinators collected from flowering plants in Pennsylvania, New York, and Illinois in the United States. Viruses in the samples were detected using reverse transcriptase-PCR and confirmed by sequencing. For the first time, we report the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies. These viruses were detected in eleven other non-Apis hymenopteran species, ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of our ecosystem. Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD harbored honey bees with Israeli Acute Paralysis virus (IAPV, nearby

  9. RNA viruses in hymenopteran pollinators: evidence of inter-Taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species.

    Science.gov (United States)

    Singh, Rajwinder; Levitt, Abby L; Rajotte, Edwin G; Holmes, Edward C; Ostiguy, Nancy; Vanengelsdorp, Dennis; Lipkin, W Ian; Depamphilis, Claude W; Toth, Amy L; Cox-Foster, Diana L

    2010-12-22

    Although overall pollinator populations have declined over the last couple of decades, the honey bee (Apis mellifera) malady, colony collapse disorder (CCD), has caused major concern in the agricultural community. Among honey bee pathogens, RNA viruses are emerging as a serious threat and are suspected as major contributors to CCD. Recent detection of these viral species in bumble bees suggests a possible wider environmental spread of these viruses with potential broader impact. It is therefore vital to study the ecology and epidemiology of these viruses in the hymenopteran pollinator community as a whole. We studied the viral distribution in honey bees, in their pollen loads, and in other non-Apis hymenopteran pollinators collected from flowering plants in Pennsylvania, New York, and Illinois in the United States. Viruses in the samples were detected using reverse transcriptase-PCR and confirmed by sequencing. For the first time, we report the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus) in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies. These viruses were detected in eleven other non-Apis hymenopteran species, ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of our ecosystem. Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD harbored honey bees with Israeli Acute Paralysis virus (IAPV), nearby non

  10. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

    Directory of Open Access Journals (Sweden)

    Philippa M Beard

    Full Text Available Vaccinia virus (VACV is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

  11. Attenuation of the influenza virus by microRNA response element in vivo and protective efficacy against 2009 pandemic H1N1 virus in mice.

    Science.gov (United States)

    Feng, Chunlai; Tan, Mingming; Sun, Wenkui; Shi, Yi; Xing, Zheng

    2015-09-01

    The 2009 influenza pandemics underscored the need for effective vaccines to block the spread of influenza virus infection. Most live attenuated vaccines utilize cold-adapted, temperature-sensitive virus. An alternative to live attenuated virus is presented here, based on microRNA-induced gene silencing. In this study, miR-let-7b target sequences were inserted into the H1N1 genome to engineer a recombinant virus - miRT-H1N1. Female BALB/c mice were vaccinated intranasally with the miRT-H1N1 and challenged with a lethal dose of homologous virus. This miRT-H1N1 virus was attenuated in mice, while it exhibited wild-type characteristics in chicken embryos. Mice vaccinated intranasally with the miRT-H1N1 responded with robust immunity that protected the vaccinated mice from a lethal challenge with the wild-type 2009 pandemic H1N1 virus. These results indicate that the influenza virus containing microRNA response elements (MREs) is attenuated in vivo and can be used to design a live attenuated vaccine. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Luthra, Priya; Jordan, David S.; Leung, Daisy W.; Amarasinghe, Gaya K.; Basler, Christopher F.; Lyles, D. S.

    2015-03-04

    Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

  13. Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

    Directory of Open Access Journals (Sweden)

    Isabel Weinheimer

    2015-03-01

    Full Text Available Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant and Caenorhabditis elegans (nematode and found that it cleaves double-stranded small interfering RNA (ds-siRNA molecules that are pivotal in the host RNA interference (RNAi pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3 produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are

  14. A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

    Directory of Open Access Journals (Sweden)

    Rui eZhang

    2014-07-01

    Full Text Available Here we report a biological and molecular characterization of a novel positive-sense RNA virus isolated from a field isolate (NW10 of a filamentous phytopathogenic fungus, the white root rot fungus that is designated as Rosellinia necatrix fusarivirus 1 (RnFV1. A recently developed technology using zinc ions allowed us to transfer RnFV1 to two mycelially incompatible Rosellinia necatrix strains. A biological comparison of the virus-free and -recipient isogenic fungal strains suggested that RnFV1 infects latently and thus has no potential as a virocontrol agent. The virus has an undivided positive-sense RNA genome of 6286 nucleotides excluding a poly (A tail. The genome possesses two non-overlapping open reading frames (ORFs: a large ORF1 that encodes polypeptides with RNA replication functions and a smaller ORF2 that encodes polypeptides of unknown function. A lack of coat protein genes was suggested by the failure of virus particles from infected mycelia. No evidence was obtained by Northern analysis or classical 5'-RACE for the presence of subgenomic RNA for the downstream ORF. Sequence similarities were found in amino-acid sequence between RnFV1 putative proteins and counterparts of a previously reported mycovirus, Fusarium graminearum virus 1 (FgV1. Interestingly, several related sequences were detected by BLAST searches of independent transcriptome assembly databases one of which probably represents an entire virus genome. Phylogenetic analysis based on the conserved RNA-dependent RNA polymerase showed that RnFV1, FgV1, and these similar sequences are grouped in a cluster distinct from distantly related hypoviruses. It is proposed that a new taxonomic family termed Fusariviridae be created to include RnFV1and FgV1.

  15. Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

    Science.gov (United States)

    Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

    2017-08-01

    Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

  16. Post-transcriptional inhibition of hepatitis C virus replication through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-03-01

    Full Text Available Abstract Background Hepatitis C Virus (HCV infection is a major health problem throughout world that causes acute and chronic infection which resulted in liver fibrosis, hepatocellular carcinoma and death. The only therapy currently available for HCV infection is the combination of pegylated interferon alpha (PEG-IFN α and ribavirin. This therapy can effectively clear the virus infection in only 50% of infected individuals. Hence, there is a dire need to develop antiviral agents against HCV. Results This study was design to examine the ability of exogenous small interfering RNAs (siRNAs to block the replication of HCV in human liver cells. In the present study six 21-bp siRNAs were designed against different regions of HCV non-structural genes (NS2, NS3 serine protease/helicase, NS4Band NS5B RNA dependent RNA polymerase. siRNAs were labeled as NS2si241, NS3si-229, NS3si-858, NS4Bsi-166, NS5Bsi-241 and NS5Bsi-1064. We found that siRNAs against HCV NS2- NS5B efficiently inhibit HCV replication in Huh-7 cells. Our results demonstrated that siRNAs directed against HCV NS3 (NS3si-229 and NS3si-858 showed 58% and 88% reduction in viral titer respectively. Moreover, NS4Bsi-166 and NS5Bsi-1064 exhibited a dramatic reduction in HCV viral RNA and resulted in greater than 90% inhibition at a 20 μM concentration, while NS2si-241 showed 27% reduction in viral titer. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggest that siRNAs targeting against HCV non-structural genes (NS2-NS5B efficiently inhibit HCV replication and combination of these siRNAs of different targets and interferon will be better option to treat HCV infection throughout the world.

  17. Comparison of Hepatitis C Virus RNA and antibody detection in dried blood spots and plasma specimens.

    Science.gov (United States)

    Dokubo, E Kainne; Evans, Jennifer; Winkelman, Valerie; Cyrus, Sherri; Tobler, Leslie H; Asher, Alice; Briceno, Alya; Page, Kimberly

    2014-04-01

    Current diagnostic tests for Hepatitis C Virus (HCV) involve phlebotomy and serologic testing for HCV antibodies (anti-HCV) and RNA, which are not always feasible. Dried blood spots (DBS) present a minimally invasive sampling method and are suitable for sample collection, storage and testing. To assess the utility of DBS in HCV detection, we evaluated the sensitivity and specificity of DBS for anti-HCV and HCV RNA detection compared to plasma specimens. This cross-sectional validation study was conducted in the context of an existing prospective study of HCV in young injection drug users. Blood samples were collected by venipuncture into serum separator tubes (SST) and via finger stick onto Whatman 903(®) protein-saver cards. Plasma samples and eluates from the DBS were tested for anti-HCV using either a third generation enzyme-linked or chemiluminescent immunoassay (IA), and HCV RNA using discriminatory HCV transcription-mediated amplification assay (dHCV TMA). DBS results were compared to their corresponding plasma sample results. 148 participants were tested for anti-HCV and 132 participants were tested for HCV RNA. For anti-HCV, the sensitivity of DBS was 70%, specificity was 100%, positive predictive value (PPV) was 100%, negative predictive value (NPV) was 76% and Kappa was 0.69. For HCV RNA, the sensitivity of DBS was 90%, specificity was 100%, PPV was 100%, NPV was 94% and Kappa was 0.92. DBS are sensitive and very specific in detecting anti-HCV and HCV RNA, demonstrate good correlation with plasma results, and have potential to facilitate diagnosis of HCV infection. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. The cricket paralysis virus suppressor inhibits microRNA silencing mediated by the Drosophila Argonaute-2 protein.

    Directory of Open Access Journals (Sweden)

    Corinne Besnard-Guérin

    Full Text Available Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs. Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2. Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.

  19. Characterization of a novel single-stranded RNA virus, closely related to fusariviruses, infecting the plant pathogenic fungus Alternaria brassicicola.

    Science.gov (United States)

    Zhong, Jie; Shang, Hong Hong; Zhu, Chuan Xia; Zhu, Jun Zi; Zhu, Hong Jian; Hu, Yan; Gao, Bi Da

    2016-06-02

    The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as "Alternaria brassicicola fusarivirus 1" (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host. Copyright © 2016. Published by Elsevier B.V.

  20. Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

    Energy Technology Data Exchange (ETDEWEB)

    Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.; Reynard, Olivier; Volchkova, Valentina A.; Reid, St. Patrick; Ramanan, Parameshwaran; Cárdenas, Washington B.; Amarasinghe, Gaya K.; Volchkov, Viktor E.; Basler, Christopher F. (CNRS-INSERM); (Mount Sinai Hospital); (LB-Ecuador); (Iowa State)

    2010-10-11

    Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.

  1. Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

    Science.gov (United States)

    Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

    2014-01-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

  2. Topical treatment of herpes simplex virus infection with enzymatically created siRNA swarm.

    Science.gov (United States)

    Paavilainen, Henrik; Lehtinen, Jenni; Romanovskaya, Alesia; Nygårdas, Michaela; Bamford, Dennis H; Poranen, Minna M; Hukkanen, Veijo

    2017-01-01

    Herpes simplex virus (HSV) is a common human pathogen. Despite current antivirals, it causes a significant medical burden. Drug resistant strains exist and they are especially prevalent in immunocompromised patients and in HSV eye infections. New treatment modalities are needed. BALB/c mice were corneally infected with HSV and subsequently treated with a swarm of enzymatically created, Dicer-substrate small interfering RNA (siRNA) molecules that targeted the HSV gene UL29. Two infection models were used, one in which the infection was predominantly peripheral and another in which it spread to the central nervous system. Mouse survival, as well as viral spread, load, latency and peripheral shedding, was studied. The anti-HSV-UL29 siRNA swarm alleviated HSV infection symptoms, inhibited viral shedding and replication and had a favourable effect on mouse survival. Treatment with anti-HSV-UL29 siRNA swarm reduced symptoms and viral spread in HSV infection of mice and also inhibited local viral replication in mouse corneas.

  3. Correlation between particle multiplicity and location on virion RNA of the assembly initiation site for viruses of the tobacco mosaic virus group.

    Science.gov (United States)

    Fukuda, M; Meshi, T; Okada, Y; Otsuki, Y; Takebe, I

    1981-07-01

    The initiation site for reconstitution on genome RNA was determined by electron microscopic serology for a watermelon strain of cucumber green mottle mosaic virus (CGMMV-W), which is chemically and serologically related to tobacco mosaic virus (TMV). The initiation site was located at the same position as that of the cowpea strain, a virus that produces short rods of encapsidated subgenomic messenger RNA for the coat protein (a two-component TMV), being about 320 nucleotides away from the 3' terminus, and hence within the coat protein cistron. Although CGMMV-W was until now believed to be a single-component TMV, the location of the initiation site indicated the presence of short rods containing coat protein messenger RNA in CGMMV-W-infected tissue, as in the case for the cowpea strain. We found such short rods in CGMMV-W-infected tissue. The results confirmed our previous hypothesis that the site of the initiation region for reconstitution determines the rod multiplicity of TMV. The finding of the second two-component TMV, CGMMV, indicates that the cowpea strain of TMV is not unique in being a two-component virus and that the location of the assembly initiation site on the genome RNA can be a criterion for grouping of viruses.

  4. Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single-stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva

    Energy Technology Data Exchange (ETDEWEB)

    Valles, Steven M., E-mail: steven.valles@ars.usda.gov [Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608 (United States); Oi, David H.; Becnel, James J. [Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608 (United States); Wetterer, James K. [Wilkes Honors College, Florida Atlantic University, 5353 Parkside Drive, Jupiter, FL 33458 (United States); LaPolla, John S. [Department of Biological Sciences, Towson University, 8000 York Road, Towson, MD 21252 (United States); Firth, Andrew E. [Department of Pathology, University of Cambridge, Cambridge CB2 1QP (United Kingdom)

    2016-09-15

    We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1 nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species. - Highlights: • A new positive-strand RNA virus was discovered in the ant, Nylanderia fulva. • The Nylanderia fulva virus 1 genome was comprised of 10,881 nucleotides. • NfV-1 was detected in larval, pupal, queen and worker ants, but not eggs. • Replication of NfV-1 appeared to be limited to the larval stage.

  5. Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boiling vs. conventional RNA extraction.

    Science.gov (United States)

    Fereidouni, Sasan R; Starick, Elke; Ziller, Mario; Harder, Timm C; Unger, Hermann; Hamilton, Keith; Globig, Anja

    2015-09-01

    RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commercial lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTqPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing materials, including diluted virus positive allantoic fluid or cell culture supernatant, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Cowpea mosaic virus RNA-1 acts as an amplicon whose effects can be counteracted by a RNA-2-encoded suppressor of silencing

    International Nuclear Information System (INIS)

    Liu Li; Grainger, Jef; Canizares, M. Carmen; Angell, Susan M.; Lomonossoff, George P.

    2004-01-01

    Lines of Nicotiana benthamiana transgenic for full-length copies of both Cowpea mosaic virus (CPMV) genomic RNAs, either singly or together, have been produced. Plants transgenic for both RNAs developed symptoms characteristic of a CPMV infection. When plants transgenic for RNA-1 were agro-inoculated with RNA-2, no infection developed and the plants were also resistant to challenge with CPMV. By contrast, plants transgenic for RNA-2 became infected when agro-inoculated with RNA-1 and were fully susceptible to CPMV infection. The resistance of RNA-1 transgenic plants was shown to be related to the ability of RNA-1 to self-replicate and act as an amplicon. The ability of transgenically expressed RNA-2 to counteract the amplicon effect suggested that it encodes a suppressor of posttranscriptional gene silencing (PTGS). By examining the ability of portions of RNA-2 to reverse PTGS in N. benthamiana, we have identified the small (S) coat protein as the CPMV RNA-2-encoded suppressor of PTGS

  7. Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X.

    Science.gov (United States)

    Choi, Hoseong; Jo, Yeonhwa; Lian, Sen; Jo, Kyoung-Min; Chu, Hyosub; Yoon, Ju-Yeon; Choi, Seung-Kook; Kim, Kook-Hyung; Cho, Won Kyong

    2015-06-01

    The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

  8. Structural requirements for the binding of tRNA Lys3 to reverse transcriptase of the human immunodeficiency virus type 1

    NARCIS (Netherlands)

    Oude Essink, B. B.; Das, A. T.; Berkhout, B.

    1995-01-01

    Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA

  9. A new Defective Helper RNA to produce recombinant Sindbis virus that infects neurons but does not propagate

    Directory of Open Access Journals (Sweden)

    Justus M Kebschull

    2016-05-01

    Full Text Available Recombinant Sindbis viruses are important tools in neuroscience because they combine rapid and high transgene expression with a capacity to carry large transgenes. Currently, two packaging systems based on the DH(26S5’SIN and the DH-BB(tRNA;TE12 Defective Helper (DH RNAs are available for generating recombinant Sindbis virus that is neurotropic (able to infect neurons and potentially other cells. Both systems produce a fraction of viral particles that can propagate beyond the primary infected neuron. When injected into mouse brain, viruses produced using these DH RNAs produce transgene expression at the injection site, but also elsewhere in the brain. Such ectopic labeling caused recombinant Sindbis viruses to be classified as anterograde viruses with limited retrograde spread, and can complicate the interpretation of neuroanatomical and other experiments.Here we describe a new DH RNA, DH-BB(5’SIN;TE12ORF, that can be used to produce virus that is both neurotropic and propagation-incompetent. We show in mice that DH-BB(5’SIN;TE12ORF-packaged virus eliminates infection of cells outside the injection site. We also provide evidence that ectopically labeled cells observed in previous experiments with recombinant Sindbis virus resulted from secondary infection by propagation-competent virus, rather than from inefficient retrograde spread. Virus produced with our new packaging system retains all the advantages of previous recombinant Sindbis viruses, but minimizes the risks of confounding results with unwanted ectopic labeling. It should therefore be considered in future studies in which a neurotropic, recombinant Sindbis virus is needed.

  10. Simultaneous identification of DNA and RNA viruses present in pig faeces using process-controlled deep sequencing.

    Directory of Open Access Journals (Sweden)

    Jana Sachsenröder

    Full Text Available BACKGROUND: Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2 with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. RESULTS: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9% and mammalian viruses (23.9%; 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV, represents a novel pig virus. CONCLUSION: The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures

  11. Cloning and physical mapping of DNA complementary to potato leafroll virus RNA

    International Nuclear Information System (INIS)

    Smith, O.P.

    1987-01-01

    Potato leafroll virus (PLRV) was aphid-transmitted from potato (Solanum tuberosum cultivar Russett Burbank) to ground cherry (Physalis floridana), where it was maintained by serial aphid transmission. Serological and plant differential tests indicated that the isolate was not contaminated with beet western yellows virus. Purified PLRV RNA was poly(A)-tailed in vitro and used as a template for reverse transcriptase, primed with oligo(dT). Alkaline gel electrophoresis of 32 P-labeled first-strand complementary DNA (cDNA) indicated a major size range of 0.1 to 3.5 kilobases (kb). A small percentage of transcripts corresponded to full length PLRV RNA. Following RNase H and DNA polymerase I-mediated second strand synthesis, double-stranded cDNA was cloned into the Pst I site of the plasmid pUC9 using oligo (dC)-oligo(dG) tailing methodology. Escherichia coli JM109 transformants were screened with first-strand 32 P-cDNA in colony hybridization experiments to confirm that recombinants contained PLRV-specific sequences

  12. Vascular movement of beet necrotic yellow vein virus in Beta macrocarpa is probably dependent on an RNA 3 sequence domain rather than a gene product.

    Science.gov (United States)

    Lauber, E; Guilley, H; Tamada, T; Richards, K E; Jonard, G

    1998-02-01

    RNAs 1 and 2 of beet necrotic yellow vein virus (BNYVV) carry the functions enabling viral RNA replication, cell-to-cell movement, virus assembly and vascular movement of the virus in the systemic host Spinacea oleracea. In Beta macrocarpa, on the other hand, BNYVV RNA 3 is required for vascular movement. Replication-competent RNA 3 transcripts carrying various point mutations and deletions were coinnoculated with RNAs 1 and 2 to young leaves of B. macrocarpa and the ability of the virus to multiply on the inoculated leaves and to invade the plant systemically was examined. None of the RNA 3 mutants tested interfered with virus multiplication in the inoculated leaves. Point mutations designed to specifically block or truncate translation of the ORFs of the two known RNA 3 gene products, P25 and N, did not interfere with vascular movement. Vascular movement was not inhibited by deletions eliminating the short 5'-proximal ORF on RNA 3 (ORF A) or by point mutations blocking putative translation of the short 5'-proximal ORF (ORF S) on RNA 3sub, a subgenomic RNA derived from RNA 3. On the other hand, deletions in a 'core region' encompassing nucleotides 1033-1257 of RNA 3 completely blocked vascular movement of the virus while removal of sequences flanking the core region lowered its efficiency. The observations suggest that some feature of the RNA 3 sequence rather than an RNA-3 coded protein is important for vascular movement of BNYVV in B. macrocarpa.

  13. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    Energy Technology Data Exchange (ETDEWEB)

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-02-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, /sup 32/P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

  14. Mechanism of Human Influenza Virus RNA Persistence and Virion Survival in Feces: Mucus Protects Virions From Acid and Digestive Juices.

    Science.gov (United States)

    Hirose, Ryohei; Nakaya, Takaaki; Naito, Yuji; Daidoji, Tomo; Watanabe, Yohei; Yasuda, Hiroaki; Konishi, Hideyuki; Itoh, Yoshito

    2017-07-01

    Although viral RNA or infectious virions have been detected in the feces of individuals infected with human influenza A and B viruses (IAV/IBV), the mechanism of viral survival in the gastrointestinal tract remains unclear. We developed a model that attempts to recapitulate the conditions encountered by a swallowed virus. While IAV/IBV are vulnerable to simulated digestive juices (gastric acid and bile/pancreatic juice), highly viscous mucus protects viral RNA and virions, allowing the virus to retain its infectivity. Our results suggest that virions and RNA present in swallowed mucus are not inactivated or degraded by the gastrointestinal environment, allowing their detection in feces. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  15. Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein

    International Nuclear Information System (INIS)

    Rozenblatt, S.; Eizenberg, O.; Englund, G.; Bellini, W.J.

    1985-01-01

    Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32 P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1500, 1850, 1850 and 2500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV

  16. Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).

    Science.gov (United States)

    Jothikumar, N; Sobsey, M D; Cromeans, T L

    2010-10-01

    RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food. Published by Elsevier B.V.

  17. In Silico Reconstruction of Viral Genomes from Small RNAs Improves Virus-Derived Small Interfering RNA Profiling ▿ † ‡

    Science.gov (United States)

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-01-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly. PMID:21880776

  18. The cytopathic 18f strain of Hepatitis A virus induces RNA degradation in FrhK4 cells.

    Science.gov (United States)

    Kulka, M; Chen, A; Ngo, D; Bhattacharya, S S; Cebula, T A; Goswami, B B

    2003-07-01

    The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

  19. Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis.

    Science.gov (United States)

    Ito, Marcos Massayuki; Catanhêde, Lilian Motta; Katsuragawa, Tony Hiroshi; Silva Junior, Cipriano Ferreira da; Camargo, Luis Marcelo Aranha; Mattos, Ricardo de Godoi; Vilallobos-Salcedo, Juan Miguel

    2015-01-01

    Mucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. A cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. Out of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. Correlation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area. Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  20. The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

    Science.gov (United States)

    Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

    2011-01-01

    The ability of human cells to defend against viruses originating from distant species has long been ignored. Owing to the pressure of natural evolution and human exploration, some of these viruses may be able to invade human beings. If their ‘fresh’ host had no defences, the viruses could cause a serious pandemic, as seen with HIV, SARS (severe acute respiratory syndrome) and avian influenza virus that originated from chimpanzees, the common palm civet and birds, respectively. It is unknown whether the human immune system could tolerate invasion with a plant virus. To model such an alien virus invasion, we chose TMV (tobacco mosaic virus) and used human epithelial carcinoma cells (HeLa cells) as its ‘fresh’ host. We established a reliable system for transfecting TMV-RNA into HeLa cells and found that TMV-RNA triggered autophagy in HeLa cells as shown by the appearance of autophagic vacuoles, the conversion of LC3-I (light chain protein 3-I) to LC3-II, the up-regulated expression of Beclin1 and the accumulation of TMV protein on autophagosomal membranes. We observed suspected TMV virions in HeLa cells by TEM (transmission electron microscopy). Furthermore, we found that TMV-RNA was translated into CP (coat protein) in the ER (endoplasmic reticulum) and that TMV-positive RNA translocated from the cytoplasm to the nucleolus. Finally, we detected greatly increased expression of GRP78 (78 kDa glucose-regulated protein), a typical marker of ERS (ER stress) and found that the formation of autophagosomes was closely related to the expanded ER membrane. Taken together, our data indicate that HeLa cells used ERS and ERS-related autophagy to defend against TMV-RNA. PMID:21729006

  1. Enhanced efficiency in single-host white organic light-emitting diode by triplet exciton conversion

    International Nuclear Information System (INIS)

    Wu, Qingyang; Zhang, Shiming; Yue, Shouzhen; Zhang, Zhensong; Xie, Guohua; Zhao, Yi; Liu, Shiyong

    2013-01-01

    The authors observe that the external quantum efficiency (EQE) of the Iridium (III) bis(4-phenylthieno [3,2-c]pyridinato-N,C 2′ )acetylacetonate (PO-01) based yellow organic light-emitting diode (OLED) is significantly increased by uniformly co-doping Iridium (III)bis[(4,6-difluorophenyl)-pyridinato-N,C 2− ] (FIrpic) and PO-01 into the same wide band-gap host of N,N ′ -dicarbazolyl-3, 5-benzene (mCP). Detailed investigation indicates that the efficiency enhancement is ascribed to effective triplet exciton gathering by FIrpic, followed by energy transfer to PO-01. Compared to the control device, which has maximum EQE of 10.5%, an improved maximum EQE of 13.2% is obtained in the optimization white device based on FIrpic and PO-01 emission according to this principle. This work makes it easier for a single host white OLED to simultaneously harvest high efficiency in both blue and yellow units. Comprehensive experimental results show that this phenomenon can also be found and utilized in other popular hosts to realize more efficient white devices. -- Highlights: • This work makes easier for a single host white OLED to harvest high efficiency in both blue and yellow units. • Efficiency enhancement is ascribed to effective triplet exciton gathering by FIrpic, followed by energy transfer to PO-01. • This phenomenon can also be found and utilized in other popular hosts to realize more efficient white devices

  2. Inhibition of full length Hepatitis C Virus particles of 1a genotype through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV, a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection. Results This study was design to assess or explore the silencing effect of small interference RNAs (siRNAs against full length HCV particles of genotype 1a. In the present study six 21-bp siRNAs were designed against different regions of HCV structural genes (Core, E1 and E2. Selected siRNAs were labeled as Csi 301, Csi 29, E1si 52, E1si 192, E2si 86 and E2si 493. Our results demonstrated that siRNAs directed against HCV core gene showed 70% reduction in viral titer in HCV infected liver cells. Moreover, siRNAs against E1 and E2 envelop genes showed a dramatic reduction in HCV viral RNA, E2si 86 exhibited 93% inhibition, while E1si 192, E2si 493 and E1si 52 showed 87%, 80%, and 66% inhibition respectively. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggested that siRNAs targeted against HCV structural genes efficiently silence full length HCV particles and provide an effective therapeutic option against HCV infection.

  3. Improvement of resistance to maize dwarf mosaic virus mediated by transgenic RNA interference.

    Science.gov (United States)

    Zhang, Zhi-Yong; Yang, Lin; Zhou, Shu-Feng; Wang, Han-Guang; Li, Wan-Chen; Fu, Feng-Ling

    2011-05-20

    To overcome the low efficiency of agronomic protection from maize dwarf mosaic disease, susceptible maize inbred line was transformed by Agrobacterium tumefaciens harboring hpRNA expression vectors containing inverted-repeat sequences of different lengths targeting coat protein gene (CP) of maize dwarf mosaic virus (MDMV). After PCR screening and Southern blotting, the flanking sequences of the integration sites were amplified by thermal asymmetric interlaced PCR (TAIL-PCR) and used for analysis of T-DNA integration patterns. The T₂ plant lines were evaluated for their MDMV resistance in field inoculation trials under two environments. Of the nineteen T₂ plant lines positive in Southern blotting, six were evaluated as resistant to MDMV, and four of them had resistance non-significantly different from the highly resistant control "H9-21", while the resistance of the other eleven was proved to be significantly improved when compared to their non-transformed parent line. These improvements in MDMV resistance were verified by the relative amount of virus CP gene expression measured by quantitative real time PCR. Comparing the results of Southern blotting and TAIL-PCR analysis, different integration patterns of one or two copies of the inverted-repeat sequences were identified from non-repetitive and repetitive sequences of the maize genome. The MDMV resistance mediated by RNA interference is relative to the length of the inverted-repeat sequence, the copy number of T-DNA integration and the repeatability of integration sites. A longer hpRNA expression construct shows more efficiency than a shorter one. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. The intrinsically disordered N-terminal arm of the brome mosaic virus coat protein specifically recognizes the RNA motif that directs the initiation of viral RNA replication.

    Science.gov (United States)

    Jacobs, Alexander; Hoover, Haley; Smith, Edward; Clemmer, David E; Kim, Chul-Hyun; Kao, C Cheng

    2018-01-09

    In the brome mosaic virus (BMV) virion, the coat protein (CP) selectively contacts the RNA motifs that regulate translation and RNA replication (Hoover et al., 2016. J. Virol. 90, 7748). We hypothesize that the unstructured N-terminal arm (NTA) of the BMV CP can specifically recognize RNA motifs. Using ion mobility spectrometry-mass spectrometry, we demonstrate that peptides containing the NTA of the CP were found to preferentially bind to an RNA hairpin motif that directs the initiation of BMV RNA synthesis. RNA binding causes the peptide to change from heterogeneous structures to a single family of structures. Fluorescence anisotropy, fluorescence quenching and size exclusion chromatography experiments all confirm that the NTA can specific recognize the RNA motif. The peptide introduced into plants along with BMV virion increased accumulation of the BMV CP and accelerated the rate of minus-strand RNA synthesis. The intrinsically disordered BMV NTA could thus specifically recognize BMV RNAs to affect viral infection. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Identification and molecular characterization of a naturally occurring RNA virus mutant defective in the initiation of host recovery

    International Nuclear Information System (INIS)

    Xin Hongwu; Ding Shouwei

    2003-01-01

    The host recovery response is characterized by the disappearance of disease symptoms and activation of the RNA silencing virus