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Sample records for viral gene flow

  1. Physical non-viral gene delivery methods for tissue engineering.

    Science.gov (United States)

    Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

    2013-03-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

  2. Physical non-viral gene delivery methods for tissue engineering

    Science.gov (United States)

    Mellott, Adam J.; Forrest, M. Laird; Detamore, Michael S.

    2016-01-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications. PMID:23099792

  3. Junk DNA enhances pEI-based non-viral gene delivery

    NARCIS (Netherlands)

    Gaal, E.V.B. van; Oosting, R.S.; Hennink, W.E.; Crommelin, D.J.A.; Mastrobattista, E.

    Gene therapy aims at delivering exogenous DNA into the nuclei of target cells to establish expression of a therapeutic protein. Non-viral gene delivery is examined as a safer alternative to viral approaches, but is presently characterized by a low efficiency. In the past years several non-viral

  4. Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stiehler, Maik; Duch, Mogens; Mygind, Tina

    2006-01-01

    INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors...... viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month......-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno...

  5. Massive activation of archaeal defense genes during viral infection.

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    Quax, Tessa E F; Voet, Marleen; Sismeiro, Odile; Dillies, Marie-Agnes; Jagla, Bernd; Coppée, Jean-Yves; Sezonov, Guennadi; Forterre, Patrick; van der Oost, John; Lavigne, Rob; Prangishvili, David

    2013-08-01

    Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo.

  6. Core Gene Expression and Association of Genotypes with Viral ...

    African Journals Online (AJOL)

    Purpose: To determine genotypic distribution, ribonucleic acid (RNA) RNA viral load and express core gene from Hepatitis C Virus (HCV) infected patients in Punjab, Pakistan. Methods: A total of 1690 HCV RNA positive patients were included in the study. HCV genotyping was tested by type-specific genotyping assay, viral ...

  7. DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

    Science.gov (United States)

    Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

    2003-03-01

    We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

  8. Improved osteogenic vector for non-viral gene therapy

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    ARA Hacobian

    2016-03-01

    Full Text Available Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2 gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp. Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.

  9. Development of Viral Vectors for Gene Therapy for Chronic Pain

    Directory of Open Access Journals (Sweden)

    Yu Huang

    2011-01-01

    Full Text Available Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.

  10. Coupled Transcriptome and Proteome Analysis of Human Lymphotropic Tumor Viruses: Insights on the Detection and Discovery of Viral Genes

    Energy Technology Data Exchange (ETDEWEB)

    Dresang, Lindsay R.; Teuton, Jeremy R.; Feng, Huichen; Jacobs, Jon M.; Camp, David G.; Purvine, Samuel O.; Gritsenko, Marina A.; Li, Zhihua; Smith, Richard D.; Sugden, Bill; Moore, Patrick S.; Chang, Yuan

    2011-12-20

    Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions. Results The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels. Conclusions This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.

  11. Comparative Annotation of Viral Genomes with Non-Conserved Gene Structure

    DEFF Research Database (Denmark)

    de Groot, Saskia; Mailund, Thomas; Hein, Jotun

    2007-01-01

    Motivation: Detecting genes in viral genomes is a complex task. Due to the biological necessity of them being constrained in length, RNA viruses in particular tend to code in overlapping reading frames. Since one amino acid is encoded by a triplet of nucleic acids, up to three genes may be coded...... allows for coding in unidirectional nested and overlapping reading frames, to annotate two homologous aligned viral genomes. Our method does not insist on conserved gene structure between the two sequences, thus making it applicable for the pairwise comparison of more distantly related sequences. Results...... and HIV2, as well as of two different Hepatitis Viruses, attaining results of ~87% sensitivity and ~98.5% specificity. We subsequently incorporate prior knowledge by "knowing" the gene structure of one sequence and annotating the other conditional on it. Boosting accuracy close to perfect we demonstrate...

  12. Gene flow and pathogen transmission among bobcats (Lynx rufus) in a fragmented urban landscape

    Science.gov (United States)

    Lee, Justin S.; Ruell, Emily W.; Boydston, Erin E.; Lyren, Lisa M.; Alonso, Robert S.; Troyer, Jennifer L.; Crooks, Kevin R.; VandeWoude, Sue

    2012-01-01

    Urbanization can result in the fragmentation of once contiguous natural landscapes into a patchy habitat interspersed within a growing urban matrix. Animals living in fragmented landscapes often have reduced movement among habitat patches because of avoidance of intervening human development, which potentially leads to both reduced gene flow and pathogen transmission between patches. Mammalian carnivores with large home ranges, such as bobcats (Lynx rufus), may be particularly sensitive to habitat fragmentation. We performed genetic analyses on bobcats and their directly transmitted viral pathogen, feline immunodeficiency virus (FIV), to investigate the effects of urbanization on bobcat movement. We predicted that urban development, including major freeways, would limit bobcat movement and result in genetically structured host and pathogen populations. We analysed molecular markers from 106 bobcats and 19 FIV isolates from seropositive animals in urban southern California. Our findings indicate that reduced gene flow between two primary habitat patches has resulted in genetically distinct bobcat subpopulations separated by urban development including a major highway. However, the distribution of genetic diversity among FIV isolates determined through phylogenetic analyses indicates that pathogen genotypes are less spatially structured--exhibiting a more even distribution between habitat fragments. We conclude that the types of movement and contact sufficient for disease transmission occur with enough frequency to preclude structuring among the viral population, but that the bobcat population is structured owing to low levels of effective bobcat migration resulting in gene flow. We illustrate the utility in using multiple molecular markers that differentially detect movement and gene flow between subpopulations when assessing connectivity.

  13. Bioreducible poly(amido amine)s for non-viral gene delivery

    NARCIS (Netherlands)

    Lin, C.

    2008-01-01

    This thesis describes the design and development of bioreducible poly(amido amine)s as non-viral vectors for gene delivery in vitro and in vivo. The structural influences of these polymers on their physico-chemical properties and gene delivery properties, transfection capability and cytotoxicity in

  14. The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

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    Rattay, Stephanie; Trilling, Mirko; Megger, Dominik A; Sitek, Barbara; Meyer, Helmut E; Hengel, Hartmut; Le-Trilling, Vu Thuy Khanh

    2015-08-01

    Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted

  15. Progress in developing cationic vectors for non-viral systemic gene therapy against cancer.

    Science.gov (United States)

    Morille, Marie; Passirani, Catherine; Vonarbourg, Arnaud; Clavreul, Anne; Benoit, Jean-Pierre

    2008-01-01

    Initially, gene therapy was viewed as an approach for treating hereditary diseases, but its potential role in the treatment of acquired diseases such as cancer is now widely recognized. The understanding of the molecular mechanisms involved in cancer and the development of nucleic acid delivery systems are two concepts that have led to this development. Systemic gene delivery systems are needed for therapeutic application to cells inaccessible by percutaneous injection and for multi-located tumor sites, i.e. metastases. Non-viral vectors based on the use of cationic lipids or polymers appear to have promising potential, given the problems of safety encountered with viral vectors. Using these non-viral vectors, the current challenge is to obtain a similarly effective transfection to viral ones. Based on the advantages and disadvantages of existing vectors and on the hurdles encountered with these carriers, the aim of this review is to describe the "perfect vector" for systemic gene therapy against cancer.

  16. Viral vectors for cystic fibrosis gene therapy: What does the future hold?

    Directory of Open Access Journals (Sweden)

    Uta Griesenbach

    2010-12-01

    Full Text Available Uta Griesenbach1, Makoto Inoue2, Mamoru Hasegawa2, Eric WFW Alton11Department of Gene Therapy, Imperial College London, UK; The UK Cystic Fibrosis Gene Therapy Consortium; 2DNAVEC Corporation, Tsukuba, JapanAbstract: Gene transfer to the airway epithelium has been more difficult than originally anticipated, largely because of significant extra- and intracellular barriers in the lung. In general, viral vectors are more adapted to overcoming these barriers than nonviral gene transfer agents and are, therefore, more efficient in transferring genes into recipient cells. Viral vectors derived from adenovirus, adeno-associated virus, and Sendai virus, which all have a natural tropism for the airway epithelium, have been evaluated for cystic fibrosis (CF gene therapy. Although these vectors transduce airway epithelial cells efficiently, gene expression is transient and repeated administration is inefficient. They are, therefore, unlikely to be suitable for CF gene therapy. More recently, lentiviruses (LV have been assessed for lung gene transfer. In contrast to retroviruses, they transduce nondividing cells and randomly integrate into the genome. However, LVs do not have a natural tropism for the lung, and a significant amount of effort has been put into pseudotyping these vectors with proteins suitable for airway gene transfer. Several studies have shown that LV-mediated transduction leads to persistent gene expression (for the lifetime of the animal in the airways and, importantly, repeated administration is feasible. Thus, appropriately pseudotyped LV vectors are promising candidates for CF gene therapy. Here, we will review preclinical and clinical research related to viral CF gene therapy.Keywords: cystic fibrosis, gene therapy, adenovirus, AAV, lentivirus, Sendai virus

  17. Deep sequencing of the viral phoH gene reveals temporal variation, depth-specific composition, and persistent dominance of the same viral phoH genes in the Sargasso Sea

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    Dawn B. Goldsmith

    2015-06-01

    Full Text Available Deep sequencing of the viral phoH gene, a host-derived auxiliary metabolic gene, was used to track viral diversity throughout the water column at the Bermuda Atlantic Time-series Study (BATS site in the summer (September and winter (March of three years. Viral phoH sequences reveal differences in the viral communities throughout a depth profile and between seasons in the same year. Variation was also detected between the same seasons in subsequent years, though these differences were not as great as the summer/winter distinctions. Over 3,600 phoH operational taxonomic units (OTUs; 97% sequence identity were identified. Despite high richness, most phoH sequences belong to a few large, common OTUs whereas the majority of the OTUs are small and rare. While many OTUs make sporadic appearances at just a few times or depths, a small number of OTUs dominate the community throughout the seasons, depths, and years.

  18. Discovery of Cationic Polymers for Non-viral Gene Delivery using Combinatorial Approaches

    Science.gov (United States)

    Barua, Sutapa; Ramos, James; Potta, Thrimoorthy; Taylor, David; Huang, Huang-Chiao; Montanez, Gabriela; Rege, Kaushal

    2015-01-01

    Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems. PMID:21843141

  19. Highly Pathogenic H5N1 Avian Influenza Viruses Exhibit Few Barriers to Gene Flow in Vietnam

    Science.gov (United States)

    Carrel, Margaret; Wan, Xiu-Feng; Nguyen, Tung; Emch, Michael

    2013-01-01

    Locating areas where genetic change is inhibited can illuminate underlying processes that drive evolution of pathogens. The persistence of highly pathogenic H5N1 avian influenza in Vietnam since 2003, and the continuous molecular evolution of Vietnamese avian influenza viruses, indicates that local environmental factors are supportive not only of incidence but also of viral adaptation. This article explores whether gene flow is constant across Vietnam, or whether there exist boundary areas where gene flow exhibits discontinuity. Using a dataset of 125 highly pathogenic H5N1 avian influenza viruses, principal components analysis and wombling analysis are used to indicate the location, magnitude, and statistical significance of genetic boundaries. Results show that a small number of geographically minor boundaries to gene flow in highly pathogenic H5N1 avian influenza viruses exist in Vietnam, but that overall there is little division in genetic exchange. This suggests that differences in genetic characteristics of viruses from one region to another are not the result of barriers to H5N1 viral exchange in Vietnam, and that H5N1 avian influenza is able to spread relatively unimpeded across the country. PMID:22350419

  20. Nano-sized calcium phosphate (CaP) carriers for non-viral gene deilvery

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Donghyun, E-mail: dhlee@cau.ac.kr [Department of Biomedical Engineering, Division of Integrative Engineering, Chung-Ang University, 221 Heukseok-Dong, Dongjak-Gu, Seoul 156-756 (Korea, Republic of); Upadhye, Kalpesh [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Kumta, Prashant N., E-mail: pkumta@pitt.edu [Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Department of Mechanical Engineering and Materials Sceince, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Center for Complex Engineered Multifunctional Materials, University of Pittsburgh, Pittsburgh, PA 15261 (United States)

    2012-02-25

    Highlights: Black-Right-Pointing-Pointer Nanostructured calcium phosphates (NanoCaPs): comprehensive review. Black-Right-Pointing-Pointer Non viral gene delivery mechanisms: detailed mechanisms are outlined. Black-Right-Pointing-Pointer Barriers to non-viral gene delivery: detailed barriers are discussed. - Abstract: Gene therapy has garnered much interest due to the potential for curing multiple inherited and/or increases in the acquired diseases. As a result, there has been intense activity from multiple research groups for developing effective delivery methods and carriers, which is a critical step in advancing gene delivery technologies. In order for the carriers to effectively deliver the genetic payloads, multiple extracellular and intracellular barriers need to be overcome. Although overcoming these challenges to improve the effectiveness is critical, the development of safe gene delivery agents is even more vital to assure its use in clinical applications. The development of safe and effective strategies has therefore been a major challenge impeding gene therapy progress. In this regard, calcium phosphate (CaP) based nano-particles has been considered as one of the candidate non-viral gene delivery vehicles, but has been plagued by inconsistent and low transfection efficiencies limiting its progress. There has been major research effort to improve the consistency and effectiveness of CaP based vectors. Currently, it is therefore thought that by controlling the various synthesis factors such as Ca/P ratio, mode of mixing, and type of calcium phosphate phase, such variability and inefficiency could be modulated. This review attempts to provide a comprehensive analysis of the current research activity in the development of CaP based ceramic and polymer-ceramic hybrid systems for non-viral gene delivery. Preliminary transfection results of hydroxyapatite (HA or NanoCaPs), amorphous calcium phosphate (ACP) and brushite phases are also compared to assess the

  1. Gene Expression Profiles Link Respiratory Viral Infection, Platelet Response to Aspirin, and Acute Myocardial Infarction

    Science.gov (United States)

    Cyr, Derek D.; Lucas, Joseph E.; Zaas, Aimee K.; Woods, Christopher W.; Newby, L. Kristin; Kraus, William E.; Ginsburg, Geoffrey S.

    2015-01-01

    Background Influenza infection is associated with myocardial infarction (MI), suggesting that respiratory viral infection may induce biologic pathways that contribute to MI. We tested the hypotheses that 1) a validated blood gene expression signature of respiratory viral infection (viral GES) was associated with MI and 2) respiratory viral exposure changes levels of a validated platelet gene expression signature (platelet GES) of platelet function in response to aspirin that is associated with MI. Methods A previously defined viral GES was projected into blood RNA data from 594 patients undergoing elective cardiac catheterization and used to classify patients as having evidence of viral infection or not and tested for association with acute MI using logistic regression. A previously defined platelet GES was projected into blood RNA data from 81 healthy subjects before and after exposure to four respiratory viruses: Respiratory Syncytial Virus (RSV) (n=20), Human Rhinovirus (HRV) (n=20), Influenza A virus subtype H1N1 (H1N1) (n=24), Influenza A Virus subtype H3N2 (H3N2) (n=17). We tested for the change in platelet GES with viral exposure using linear mixed-effects regression and by symptom status. Results In the catheterization cohort, 32 patients had evidence of viral infection based upon the viral GES, of which 25% (8/32) had MI versus 12.2% (69/567) among those without evidence of viral infection (OR 2.3; CI [1.03-5.5], p=0.04). In the infection cohorts, only H1N1 exposure increased platelet GES over time (time course p-value = 1e-04). Conclusions A viral GES of non-specific, respiratory viral infection was associated with acute MI; 18% of the top 49 genes in the viral GES are involved with hemostasis and/or platelet aggregation. Separately, H1N1 exposure, but not exposure to other respiratory viruses, increased a platelet GES previously shown to be associated with MI. Together, these results highlight specific genes and pathways that link viral infection

  2. Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

    International Nuclear Information System (INIS)

    Kokusho, Ryuhei; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu

    2016-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

  3. Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.ac.jp; Koh, Yoshikazu; Fujimoto, Masaru; Shimada, Toru; Katsuma, Susumu, E-mail: katsuma@ss.ab.a.u-tokyo.ac.jp

    2016-11-15

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulation of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.

  4. Imaging living central neurones using viral gene transfer.

    Science.gov (United States)

    Teschemacher, A G; Paton, J F R; Kasparov, S

    2005-01-02

    Studies of central neurones and other cellular components of the brain, such as glial and vascular cells, can be greatly advanced by the use of the modern optical techniques such as confocal live cell imaging. Fluorescent proteins have allowed imaging of particular cell types or intracellular elements to be visualised and distinguished from irrelevant background structures. To introduce the genetic information encoding for fluorescent proteins into relevant cellular targets, molecular tools are required. Viral vectors are one of the best ways of gene delivery into differentiated postnatal brain neurones and glia. Current progress in this field allows targeting of various cell types and therefore makes it possible to express a variety of fluorescent constructs in selected subpopulations of neurones, for example. In this review, we will discuss and compare the properties of the most popular viral gene delivery systems and the advantages of different brain cell preparations to illustrate how they can be used for high-resolution live cell confocal imaging in order to study new aspects of central nervous system (CNS) structure and function.

  5. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  6. Biological species in the viral world.

    Science.gov (United States)

    Bobay, Louis-Marie; Ochman, Howard

    2018-06-05

    Due to their dependence on cellular organisms for metabolism and replication, viruses are typically named and assigned to species according to their genome structure and the original host that they infect. But because viruses often infect multiple hosts and the numbers of distinct lineages within a host can be vast, their delineation into species is often dictated by arbitrary sequence thresholds, which are highly inconsistent across lineages. Here we apply an approach to determine the boundaries of viral species based on the detection of gene flow within populations, thereby defining viral species according to the biological species concept (BSC). Despite the potential for gene transfer between highly divergent genomes, viruses, like the cellular organisms they infect, assort into reproductively isolated groups and can be organized into biological species. This approach revealed that BSC-defined viral species are often congruent with the taxonomic partitioning based on shared gene contents and host tropism, and that bacteriophages can similarly be classified in biological species. These results open the possibility to use a single, universal definition of species that is applicable across cellular and acellular lifeforms.

  7. Graphene materials as 2D non-viral gene transfer vector platforms.

    Science.gov (United States)

    Vincent, M; de Lázaro, I; Kostarelos, K

    2017-03-01

    Advances in genomics and gene therapy could offer solutions to many diseases that remain incurable today, however, one of the critical reasons halting clinical progress is due to the difficulty in designing efficient and safe delivery vectors for the appropriate genetic cargo. Safety and large-scale production concerns counter-balance the high gene transfer efficiency achieved with viral vectors, while non-viral strategies have yet to become sufficiently efficient. The extraordinary physicochemical, optical and photothermal properties of graphene-based materials (GBMs) could offer two-dimensional components for the design of nucleic acid carrier systems. We discuss here such properties and their implications for the optimization of gene delivery. While the design of such vectors is still in its infancy, we provide here an exhaustive and up-to-date analysis of the studies that have explored GBMs as gene transfer vectors, focusing on the functionalization strategies followed to improve vector performance and on the biological effects attained.

  8. Viral Genome DataBase: storing and analyzing genes and proteins from complete viral genomes.

    Science.gov (United States)

    Hiscock, D; Upton, C

    2000-05-01

    The Viral Genome DataBase (VGDB) contains detailed information of the genes and predicted protein sequences from 15 completely sequenced genomes of large (&100 kb) viruses (2847 genes). The data that is stored includes DNA sequence, protein sequence, GenBank and user-entered notes, molecular weight (MW), isoelectric point (pI), amino acid content, A + T%, nucleotide frequency, dinucleotide frequency and codon use. The VGDB is a mySQL database with a user-friendly JAVA GUI. Results of queries can be easily sorted by any of the individual parameters. The software and additional figures and information are available at http://athena.bioc.uvic.ca/genomes/index.html .

  9. Dynamic Changes in Host Gene Expression following In Vitro Viral Mimic Stimulation in Crocodile Cells

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    Subir Sarker

    2017-11-01

    Full Text Available The initial control of viral infection in a host is dominated by a very well orchestrated early innate immune system; however, very little is known about the ability of a host to control viral infection outside of mammals. The reptiles offer an evolutionary bridge between the fish and mammals, with the crocodile having evolved from the archosauria clade that included the dinosaurs, and being the largest living reptile species. Using an RNA-seq approach, we have defined the dynamic changes of a passaged primary crocodile cell line to stimulation with both RNA and DNA viral mimics. Cells displayed a marked upregulation of many genes known to be involved in the mammalian response to viral infection, including viperin, Mx1, IRF7, IRF1, and RIG-I with approximately 10% of the genes being uncharacterized transcripts. Both pathway and genome analysis suggested that the crocodile may utilize the main known mammalian TLR and cytosolic antiviral RNA signaling pathways, with the pathways being responsible for sensing DNA viruses less clear. Viral mimic stimulation upregulated the type I interferon, IFN-Omega, with many known antiviral interferon-stimulated genes also being upregulated. This work demonstrates for the first time that reptiles show functional regulation of many known and unknown antiviral pathways and effector genes. An enhanced knowledge of these ancient antiviral pathways will not only add to our understanding of the host antiviral innate response in non-mammalian species, but is critical to fully comprehend the complexity of the mammalian innate immune response to viral infection.

  10. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    Science.gov (United States)

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  11. EBV tegument protein BNRF1 disrupts DAXX-ATRX to activate viral early gene transcription.

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    Kevin Tsai

    2011-11-01

    Full Text Available Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs, suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus.

  12. EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription

    Science.gov (United States)

    Tsai, Kevin; Thikmyanova, Nadezhda; Wojcechowskyj, Jason A.; Delecluse, Henri-Jacques; Lieberman, Paul M.

    2011-01-01

    Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus. PMID:22102817

  13. Influenza virus gene expression: viral RNA replication in vivo and in vitro

    International Nuclear Information System (INIS)

    Shapiro, G.I.

    1987-01-01

    To develop an overall scheme for the control of influenza virus gene expression, single-stranded M13 DNAs specific for the various genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results showed that virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs (vRNAs), viral mRNAs, and viral proteins were coupled. This phase lasted for 2.5 hours in BHK-21 cells, the time when the rate of synthesis of all the viral mRNAs was maximal. During the late phase, the synthesis of all the vRNAs remained at or near maximum, whereas the rate of synthesis of all the viral mRNAs declined dramatically. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed principally by previously synthesized viral mRNAs. Pulses with [ 3 H]uridine and nonaqueous fractionation of cells were used to show that influenza vRNA, like viral mRNAs, are synthesized in the nucleus and efficiently transported to the cytoplasm. In contrast, the full-length transcripts of the vRNAs, the templates for new vRNA synthesis, were synthesized only at early times, and remained sequestered in the nucleus to direct vRNA synthesis throughout infection

  14. Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells.

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    Ilker Kudret Sariyer

    Full Text Available PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV, which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs. We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF. SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen.Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins.Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral reactivation leading to

  15. A viral microRNA down-regulates multiple cell cycle genes through mRNA 5'UTRs.

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    Finn Grey

    2010-06-01

    Full Text Available Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3' untranslated region (UTR. Using RNA induced silencing complex immunoprecipitation (RISC-IP techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5'UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5'UTRs.

  16. Non-viral gene delivery strategies for gene therapy: a “ménage à trois” among nucleic acids, materials, and the biological environment

    International Nuclear Information System (INIS)

    Pezzoli, Daniele; Candiani, Gabriele

    2013-01-01

    Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription–translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

  17. Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.

    2002-01-01

    The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

  18. Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication

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    Jiun-Yan Huang

    2017-09-01

    Full Text Available Members of the microRNA miR-10 family are highly conserved and play many important roles in diverse biological mechanisms, including immune-related responses and cancer-related processes in certain types of cancer. In this study, we found the most highly upregulated shrimp microRNA from Penaeus vannamei during white spot syndrome virus (WSSV infection was miR-10a. After confirming the expression level of miR-10a by northern blot and quantitative RT-PCR, an in vivo experiment showed that the viral copy number was decreased in miR-10a-inhibited shrimp. We found that miR-10a targeted the 5′ untranslated region (UTR of at least three viral genes (vp26, vp28, and wssv102, and plasmids that were controlled by the 5′ UTR of these genes produced enhanced luciferase signals in transfected SF9 cells. These results suggest a previously unreported role for shrimp miR-10a and even a new type of host–virus interaction, whereby a co-opts the key cellular regulator miR-10a to globally enhance the translation of viral proteins.

  19. Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication.

    Science.gov (United States)

    Huang, Jiun-Yan; Kang, Shih-Ting; Chen, I-Tung; Chang, Li-Kwan; Lin, Shih-Shun; Kou, Guang-Hsiung; Chu, Chia-Ying; Lo, Chu-Fang

    2017-01-01

    Members of the microRNA miR-10 family are highly conserved and play many important roles in diverse biological mechanisms, including immune-related responses and cancer-related processes in certain types of cancer. In this study, we found the most highly upregulated shrimp microRNA from Penaeus vannamei during white spot syndrome virus (WSSV) infection was miR-10a. After confirming the expression level of miR-10a by northern blot and quantitative RT-PCR, an in vivo experiment showed that the viral copy number was decreased in miR-10a-inhibited shrimp. We found that miR-10a targeted the 5' untranslated region (UTR) of at least three viral genes ( vp26, vp28 , and wssv102 ), and plasmids that were controlled by the 5' UTR of these genes produced enhanced luciferase signals in transfected SF9 cells. These results suggest a previously unreported role for shrimp miR-10a and even a new type of host-virus interaction, whereby a co-opts the key cellular regulator miR-10a to globally enhance the translation of viral proteins.

  20. Construction of a recombinant viral vector containing part of the nucleocapsid protein gene of newcastle disease virus

    Energy Technology Data Exchange (ETDEWEB)

    Bader, D.E.

    1995-09-01

    This report describes the procedures used to clone a 673 base pair gene fragment of the major nucleocapsid protein gene of Newcastle disease virus into a viral vector molecule for the purpose of maintaining a stable, long-term, renewable source of this target sequence for gene probe studies. The gene fragment was prepared by reverse-transcription polymerase chain reaction of Newcastle disease virus RNA and was cloned into the viral DNA vector Ml3mp18 RF to produce a recombinant DNA molecule. The cloned fragment was shown to be present in the recombinant clones based on (i) clonal selection on indicator plates; (ii) restriction enzyme analysis; (iii) gene probe analysis and (iv) nested PCR amplification.

  1. Recombination and population mosaic of a multifunctional viral gene, adeno-associated virus cap.

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    Yasuhiro Takeuchi

    Full Text Available Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred.

  2. Non-viral gene delivery strategies for cancer therapy, tissue engineering and regenerative medicine

    Science.gov (United States)

    Bhise, Nupura S.

    Gene therapy involves the delivery of deoxyribonucleic acid (DNA) into cells to override or replace a malfunctioning gene for treating debilitating genetic diseases, including cancer and neurodegenerative diseases. In addition to its use as a therapeutic, it can also serve as a technology to enable regenerative medicine strategies. The central challenge of the gene therapy research arena is developing a safe and effective delivery agent. Since viral vectors have critical immunogenic and tumorogenic safety issues that limit their clinical use, recent efforts have focused on developing non-viral biomaterial based delivery vectors. Cationic polymers are an attractive class of gene delivery vectors due to their structural versatility, ease of synthesis, biodegradability, ability to self-complex into nanoparticles with negatively charged DNA, capacity to carry large cargo, cellular uptake and endosomal escape capacity. In this thesis, we hypothesized that developing a biomaterial library of poly(betaamino esters) (PBAE), a newer class of cationic polymers consisting of biodegradable ester groups, would allow investigating vector design parameters and formulating effective non-viral gene delivery strategies for cancer drug delivery, tissue engineering and stem cell engineering. Consequently, a high-throughput transfection assay was developed to screen the PBAE-based nanoparticles in hard to transfect fibroblast cell lines. To gain mechanistic insights into the nanoparticle formulation process, biophysical properties of the vectors were characterized in terms of molecular weight (MW), nanoparticle size, zeta potential and plasmid per particle count. We report a novel assay developed for quantifying the plasmid per nanoparticle count and studying its implications for co-delivery of multiple genes. The MW of the polymers ranged from 10 kDa to 100 kDa, nanoparticle size was about 150 run, zeta potential was about 30 mV in sodium acetate buffer (25 mM, pH 5) and 30 to 100

  3. Extensive gene remodeling in the viral world: new evidence for nongradual evolution in the mobilome network.

    Science.gov (United States)

    Jachiet, Pierre-Alain; Colson, Philippe; Lopez, Philippe; Bapteste, Eric

    2014-08-07

    Complex nongradual evolutionary processes such as gene remodeling are difficult to model, to visualize, and to investigate systematically. Despite these challenges, the creation of composite (or mosaic) genes by combination of genetic segments from unrelated gene families was established as an important adaptive phenomena in eukaryotic genomes. In contrast, almost no general studies have been conducted to quantify composite genes in viruses. Although viral genome mosaicism has been well-described, the extent of gene mosaicism and its rules of emergence remain largely unexplored. Applying methods from graph theory to inclusive similarity networks, and using data from more than 3,000 complete viral genomes, we provide the first demonstration that composite genes in viruses are 1) functionally biased, 2) involved in key aspects of the arm race between cells and viruses, and 3) can be classified into two distinct types of composite genes in all viral classes. Beyond the quantification of the widespread recombination of genes among different viruses of the same class, we also report a striking sharing of genetic information between viruses of different classes and with different nucleic acid types. This latter discovery provides novel evidence for the existence of a large and complex mobilome network, which appears partly bound by the sharing of genetic information and by the formation of composite genes between mobile entities with different genetic material. Considering that there are around 10E31 viruses on the planet, gene remodeling appears as a hugely significant way of generating and moving novel sequences between different kinds of organisms on Earth. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Influenza NA and PB1 Gene Segments Interact during the Formation of Viral Progeny: Localization of the Binding Region within the PB1 Gene

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    Brad Gilbertson

    2016-08-01

    Full Text Available The influenza A virus genome comprises eight negative-sense viral RNAs (vRNAs that form individual ribonucleoprotein (RNP complexes. In order to incorporate a complete set of each of these vRNAs, the virus uses a selective packaging mechanism that facilitates co-packaging of specific gene segments but whose molecular basis is still not fully understood. Recently, we used a competitive transfection model where plasmids encoding the A/Puerto Rico/8/34 (PR8 and A/Udorn/307/72 (Udorn PB1 gene segments were competed to show that the Udorn PB1 gene segment is preferentially co-packaged into progeny virions with the Udorn NA gene segment. Here we created chimeric PB1 genes combining both Udorn and PR8 PB1 sequences to further define the location within the Udorn PB1 gene that drives co-segregation of these genes and show that nucleotides 1776–2070 of the PB1 gene are crucial for preferential selection. In vitro assays examining specific interactions between Udorn NA vRNA and purified vRNAs transcribed from chimeric PB1 genes also supported the importance of this region in the PB1-NA interaction. Hence, this work identifies an association between viral genes that are co-selected during packaging. It also reveals a region potentially important in the RNP-RNP interactions within the supramolecular complex that is predicted to form prior to budding to allow one of each segment to be packaged in the viral progeny. Our study lays the foundation to understand the co-selection of specific genes, which may be critical to the emergence of new viruses with pandemic potential.

  5. Non-viral gene therapy that targets motor neurons in vivo

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    Mary-Louise eRogers

    2014-10-01

    Full Text Available A major challenge in neurological gene therapy is safe delivery of transgenes to sufficient cell numbers from the circulation or periphery. This is particularly difficult for diseases involving spinal cord motor neurons such as amyotrophic lateral sclerosis (ALS. We have examined the feasibility of non-viral gene delivery to spinal motor neurons from intraperitoneal injections of plasmids carried by ‘immunogene’ nanoparticles targeted for axonal retrograde transport using antibodies. PEGylated polyethylenimine (PEI-PEG12 as DNA carrier was conjugated to an antibody (MLR2 to the neurotrophin receptor p75 (p75NTR. We used a plasmid (pVIVO2 designed for in vivo gene delivery that produces minimal immune responses, has improved nuclear entry into post mitotic cells and also expresses green fluorescent protein (GFP. MLR2-PEI-PEG12 carried pVIVO2 and was specific for mouse motor neurons in mixed cultures containing astrocytes. While only 8% of motor neurons expressed GFP 72 h post transfection in vitro, when the immunogene was given intraperitonealy to neonatal C57BL/6J mice GFP specific motor neuron expression was observed in 25.4% of lumbar, 18.3% of thoracic and 17.0 % of cervical motor neurons, 72 h post transfection. PEI-PEG12 carrying pVIVO2 by itself did not transfect motor neurons in vivo, demonstrating the need for specificity via the p75NTR antibody MLR2. This is the first time that specific transfection of spinal motor neurons has been achieved from peripheral delivery of plasmid DNA as part of a non-viral gene delivery agent. These results stress the specificity and feasibility of immunogene delivery targeted for p75NTR expressing motor neurons, but suggests that further improvements are required to increase the transfection efficiency of motor neurons in vivo.

  6. Endogenous viral elements in animal genomes.

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    Aris Katzourakis

    2010-11-01

    Full Text Available Integration into the nuclear genome of germ line cells can lead to vertical inheritance of retroviral genes as host alleles. For other viruses, germ line integration has only rarely been documented. Nonetheless, we identified endogenous viral elements (EVEs derived from ten non-retroviral families by systematic in silico screening of animal genomes, including the first endogenous representatives of double-stranded RNA, reverse-transcribing DNA, and segmented RNA viruses, and the first endogenous DNA viruses in mammalian genomes. Phylogenetic and genomic analysis of EVEs across multiple host species revealed novel information about the origin and evolution of diverse virus groups. Furthermore, several of the elements identified here encode intact open reading frames or are expressed as mRNA. For one element in the primate lineage, we provide statistically robust evidence for exaptation. Our findings establish that genetic material derived from all known viral genome types and replication strategies can enter the animal germ line, greatly broadening the scope of paleovirological studies and indicating a more significant evolutionary role for gene flow from virus to animal genomes than has previously been recognized.

  7. Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy.

    Science.gov (United States)

    Peterson, Christopher W; Wang, Jianbin; Deleage, Claire; Reddy, Sowmya; Kaur, Jasbir; Polacino, Patricia; Reik, Andreas; Huang, Meei-Li; Jerome, Keith R; Hu, Shiu-Lok; Holmes, Michael C; Estes, Jacob D; Kiem, Hans-Peter

    2018-04-01

    Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

  8. Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy.

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    Christopher W Peterson

    2018-04-01

    Full Text Available Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP model of latent infection using simian/human immunodeficiency virus (SHIV and combination antiretroviral therapy (cART in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05, relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

  9. Adenovirus-encoding virus-associated RNAs suppress HDGF gene expression to support efficient viral replication.

    Directory of Open Access Journals (Sweden)

    Saki Kondo

    Full Text Available Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs, are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

  10. Regulated expression of the human cytomegalovirus pp65 gene: Octamer sequence in the promoter is required for activation by viral gene products

    International Nuclear Information System (INIS)

    Depto, A.S.; Stenberg, R.M.

    1989-01-01

    To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, the authors examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene

  11. HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

    Directory of Open Access Journals (Sweden)

    Mikaël Boullé

    2016-11-01

    Full Text Available Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

  12. Hepatic transcriptome analysis of hepatitis C virus infection in chimpanzees defines unique gene expression patterns associated with viral clearance.

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    Santosh Nanda

    Full Text Available Hepatitis C virus infection leads to a high rate of chronicity. Mechanisms of viral clearance and persistence are still poorly understood. In this study, hepatic gene expression analysis was performed to identify any molecular signature associated with the outcome of hepatitis C virus (HCV infection in chimpanzees. Acutely HCV-infected chimpanzees with self-limited infection or progression to chronicity were studied. Interferon stimulated genes were induced irrespective of the outcome of infection. Early induction of a set of genes associated with cell proliferation and immune activation was associated with subsequent viral clearance. Specifically, two of the genes: interleukin binding factor 3 (ILF3 and cytotoxic granule-associated RNA binding protein (TIA1, associated with robust T-cell response, were highly induced early in chimpanzees with self-limited infection. Up-regulation of genes associated with CD8+ T cell response was evident only during the clearance phase of the acute self-limited infection. The induction of these genes may represent an initial response of cellular injury and proliferation that successfully translates to a "danger signal" leading to induction of adaptive immunity to control viral infection. This primary difference in hepatic gene expression between self-limited and chronic infections supports the concept that successful activation of HCV-specific T-cell response is critical in clearance of acute HCV infection.

  13. A reverse genetics system for the Great Lakes strain of viral hemorrhagic septicemia virus: the NV gene is required for pathogenicity

    Science.gov (United States)

    Ammayappan, Arun; Kurath, Gael; Thompson, Tarin M.; Vakharia, Vikram N.

    2011-01-01

    Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus in the family of Rhabdoviridae, causes a highly contagious disease of fresh and saltwater fish worldwide. Recently, a novel genotype of VHSV, designated IVb, has invaded the Great Lakes in North America, causing large-scale epidemics in wild fish. An efficient reverse genetics system was developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The recombinant VHSV (rVHSV) was comparable to the parental wild-type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified recombinant VHSV was generated in which the NV gene was substituted with an enhanced green fluorescent protein gene (rVHSV-ΔNV-EGFP), and another recombinant was made by inserting the EGFP gene into the full-length viral clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV; however, the rVHSV-ΔNV-EGFP grew 2 logs lower. In yellow perch challenges, wtVHSV and rVHSV induced 82-100% cumulative per cent mortality (CPM), respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-ΔNV-EGFP caused only 15% CPM. No reversion of mutation was detected in the recovered viruses and the recombinant viruses stably maintained the foreign gene after several passages. These results indicate that the NV gene of VHSV is not essential for viral replication in vitro and in vivo, but it plays an important role in viral replication efficiency and pathogenicity. This system will facilitate studies of VHSV replication, virulence, and production of viral vectored vaccines.

  14. Evidence of differential HLA class I-mediated viral evolution in functional and accessory/regulatory genes of HIV-1.

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    Zabrina L Brumme

    2007-07-01

    Full Text Available Despite the formidable mutational capacity and sequence diversity of HIV-1, evidence suggests that viral evolution in response to specific selective pressures follows generally predictable mutational pathways. Population-based analyses of clinically derived HIV sequences may be used to identify immune escape mutations in viral genes; however, prior attempts to identify such mutations have been complicated by the inability to discriminate active immune selection from virus founder effects. Furthermore, the association between mutations arising under in vivo immune selection and disease progression for highly variable pathogens such as HIV-1 remains incompletely understood. We applied a viral lineage-corrected analytical method to investigate HLA class I-associated sequence imprinting in HIV protease, reverse transcriptase (RT, Vpr, and Nef in a large cohort of chronically infected, antiretrovirally naïve individuals. A total of 478 unique HLA-associated polymorphisms were observed and organized into a series of "escape maps," which identify known and putative cytotoxic T lymphocyte (CTL epitopes under selection pressure in vivo. Our data indicate that pathways to immune escape are predictable based on host HLA class I profile, and that epitope anchor residues are not the preferred sites of CTL escape. Results reveal differential contributions of immune imprinting to viral gene diversity, with Nef exhibiting far greater evidence for HLA class I-mediated selection compared to other genes. Moreover, these data reveal a significant, dose-dependent inverse correlation between HLA-associated polymorphisms and HIV disease stage as estimated by CD4(+ T cell count. Identification of specific sites and patterns of HLA-associated polymorphisms across HIV protease, RT, Vpr, and Nef illuminates regions of the genes encoding these products under active immune selection pressure in vivo. The high density of HLA-associated polymorphisms in Nef compared to other

  15. The immune gene repertoire of an important viral reservoir, the Australian black flying fox

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    Papenfuss Anthony T

    2012-06-01

    Full Text Available Abstract Background Bats are the natural reservoir host for a range of emerging and re-emerging viruses, including SARS-like coronaviruses, Ebola viruses, henipaviruses and Rabies viruses. However, the mechanisms responsible for the control of viral replication in bats are not understood and there is little information available on any aspect of antiviral immunity in bats. Massively parallel sequencing of the bat transcriptome provides the opportunity for rapid gene discovery. Although the genomes of one megabat and one microbat have now been sequenced to low coverage, no transcriptomic datasets have been reported from any bat species. In this study, we describe the immune transcriptome of the Australian flying fox, Pteropus alecto, providing an important resource for identification of genes involved in a range of activities including antiviral immunity. Results Towards understanding the adaptations that have allowed bats to coexist with viruses, we have de novo assembled transcriptome sequence from immune tissues and stimulated cells from P. alecto. We identified about 18,600 genes involved in a broad range of activities with the most highly expressed genes involved in cell growth and maintenance, enzyme activity, cellular components and metabolism and energy pathways. 3.5% of the bat transcribed genes corresponded to immune genes and a total of about 500 immune genes were identified, providing an overview of both innate and adaptive immunity. A small proportion of transcripts found no match with annotated sequences in any of the public databases and may represent bat-specific transcripts. Conclusions This study represents the first reported bat transcriptome dataset and provides a survey of expressed bat genes that complement existing bat genomic data. In addition, these data provide insight into genes relevant to the antiviral responses of bats, and form a basis for examining the roles of these molecules in immune response to viral infection.

  16. Bombyx mori nucleopolyhedrovirus ORF54, a viral desmoplakin gene, is associated with the infectivity of budded virions.

    Science.gov (United States)

    Zhang, Min-Juan; Tian, Cai-Hong; Fan, Xiao-Ying; Lou, Yi-Han; Cheng, Ruo-Lin; Zhang, Chuan-Xi

    2012-07-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

  17. An Optimized Protocol to Increase Virus-Induced Gene Silencing Efficiency and Minimize Viral Symptoms in Petunia

    OpenAIRE

    Broderick, Shaun R.; Jones, Michelle L.

    2013-01-01

    Virus-induced gene silencing (VIGS) is used to down-regulate endogenous plant genes. VIGS efficiency depends on viral proliferation and systemic movement throughout the plant. Although tobacco rattle virus (TRV)-based VIGS has been successfully used in petunia (Petunia × hybrida), the protocol has not been thoroughly optimized for efficient and uniform gene down-regulation in this species. Therefore, we evaluated six parameters that improved VIGS in petunia. Inoculation of mechanically wounde...

  18. Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

    Science.gov (United States)

    Hanning, Jennifer E; Saini, Harpreet K; Murray, Matthew J; Caffarel, Maria M; van Dongen, Stijn; Ward, Dawn; Barker, Emily M; Scarpini, Cinzia G; Groves, Ian J; Stanley, Margaret A; Enright, Anton J; Pett, Mark R; Coleman, Nicholas

    2013-11-01

    In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral

  19. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.

    Science.gov (United States)

    Diner, Benjamin A; Lum, Krystal K; Toettcher, Jared E; Cristea, Ileana M

    2016-11-15

    The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16

  20. Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells).

    Science.gov (United States)

    Howarth, Joanna L; Lee, Youn Bok; Uney, James B

    2010-02-01

    In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described.

  1. An integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus.

    Directory of Open Access Journals (Sweden)

    Shaoying Lee

    2011-10-01

    Full Text Available Genome-wide yeast two-hybrid (Y2H screens were conducted to elucidate the molecular functions of open reading frames (ORFs encoded by murine γ-herpesvirus 68 (MHV-68. A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs. Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP and low priority/not-scored (Y2H-LP/NS groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p<0.05. Enriched Gene Ontology (GO terms in the Y2H-HP group included regulation of apoptosis, protein kinase cascade, post-translational protein modification, transcription from RNA polymerase II promoter, and IκB kinase/NFκB cascade. Functional validation assays indicated that PCBP1, which interacted with MHV-68 ORF34, may be involved in regulating late virus gene expression in a manner consistent with the effects of its viral interacting partner. Our study integrated Y2H screening with multiple functional validation approaches to create

  2. Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

    Science.gov (United States)

    Lv, Lulu; Zhao, Huiqing

    2014-01-01

    The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes. PMID:24392152

  3. BRD4 is associated with raccoon polyomavirus genome and mediates viral gene transcription and maintenance of a stem cell state in neuroglial tumour cells.

    Science.gov (United States)

    Church, Molly E; Estrada, Marko; Leutenegger, Christian M; Dela Cruz, Florante N; Pesavento, Patricia A; Woolard, Kevin D

    2016-11-01

    Polyomavirus infection often results in persistence of the viral genome with little or no virion production. However, infection of certain cell types can result in high viral gene transcription and either cytolysis or neoplastic transformation. While infection by polyomavirus is common in humans and many animals, major questions regarding viral persistence of most polyomaviruses remain unanswered. Specifically, identification of target cells for viral infection and the mechanisms polyomaviruses employ to maintain viral genomes within cells are important not only in ascribing causality to polyomaviruses in disease, but in understanding specific mechanisms by which they cause disease. Here, we characterize the cell of origin in raccoon polyomavirus (RacPyV)-associated neuroglial brain tumours as a neural stem cell. Moreover, we identify an association between the viral genome and the host cell bromodomain protein, BRD4, which is involved in numerous cellular functions, including cell cycle progression, differentiation of stem cells, tethering of persistent DNA viruses, and regulation of viral and host-cell gene transcription. We demonstrate that inhibition of BRD4 by the small molecule inhibitors (+)-JQ1 and IBET-151 (GSK1210151A) results in reduced RacPyV genome within cells in vitro, as well as significant reduction of viral gene transcripts LT and VP1, highlighting its importance in both maintenance of the viral genome and in driving oncogenic transformation by RacPyV. This work implicates BRD4 as a central protein involved in RacPyV neuroglial tumour cell proliferation and in the maintenance of a stem cell state.

  4. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

    Directory of Open Access Journals (Sweden)

    Benjamin A. Diner

    2016-11-01

    Full Text Available The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS. Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1 and human cytomegalovirus (HCMV infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.

  5. Advances in Viral Vector-Based TRAIL Gene Therapy for Cancer

    International Nuclear Information System (INIS)

    Norian, Lyse A.; James, Britnie R.; Griffith, Thomas S.

    2011-01-01

    Numerous biologic approaches are being investigated as anti-cancer therapies in an attempt to induce tumor regression while circumventing the toxic side effects associated with standard chemo- or radiotherapies. Among these, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown particular promise in pre-clinical and early clinical trials, due to its preferential ability to induce apoptotic cell death in cancer cells and its minimal toxicity. One limitation of TRAIL use is the fact that many tumor types display an inherent resistance to TRAIL-induced apoptosis. To circumvent this problem, researchers have explored a number of strategies to optimize TRAIL delivery and to improve its efficacy via co-administration with other anti-cancer agents. In this review, we will focus on TRAIL-based gene therapy approaches for the treatment of malignancies. We will discuss the main viral vectors that are being used for TRAIL gene therapy and the strategies that are currently being attempted to improve the efficacy of TRAIL as an anti-cancer therapeutic

  6. MicroRNA-Mediated Gene Silencing in Plant Defense and Viral Counter-Defense

    Directory of Open Access Journals (Sweden)

    Sheng-Rui Liu

    2017-09-01

    Full Text Available MicroRNAs (miRNAs are non-coding RNAs of approximately 20–24 nucleotides in length that serve as central regulators of eukaryotic gene expression by targeting mRNAs for cleavage or translational repression. In plants, miRNAs are associated with numerous regulatory pathways in growth and development processes, and defensive responses in plant–pathogen interactions. Recently, significant progress has been made in understanding miRNA-mediated gene silencing and how viruses counter this defense mechanism. Here, we summarize the current knowledge and recent advances in understanding the roles of miRNAs involved in the plant defense against viruses and viral counter-defense. We also document the application of miRNAs in plant antiviral defense. This review discusses the current understanding of the mechanisms of miRNA-mediated gene silencing and provides insights on the never-ending arms race between plants and viruses.

  7. On the relation between gene flow theory and genetic gain

    Directory of Open Access Journals (Sweden)

    Woolliams John A

    2000-01-01

    Full Text Available Abstract In conventional gene flow theory the rate of genetic gain is calculated as the summed products of genetic selection differential and asymptotic proportion of genes deriving from sex-age groups. Recent studies have shown that asymptotic proportions of genes predicted from conventional gene flow theory may deviate considerably from true proportions. However, the rate of genetic gain predicted from conventional gene flow theory was accurate. The current note shows that the connection between asymptotic proportions of genes and rate of genetic gain that is embodied in conventional gene flow theory is invalid, even though genetic gain may be predicted correctly from it.

  8. Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

    Science.gov (United States)

    Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-04-01

    Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

  9. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  10. A versatile family of degradable non-viral gene carriers based on hyperbranched poly(ester amine)s

    NARCIS (Netherlands)

    Zhong, Zhiyuan; Song, Y.; Engbersen, Johannes F.J.; Lok, Martin C.; Hennink, Wim E.; Feijen, Jan

    2005-01-01

    A variety of degradable hyperbranched poly(ester amine)s containing primary, secondary and tertiary amino groups, were synthesized and evaluated as non-viral gene carriers. The polymers were obtained in high yields through a Michael-type conjugate addition of diacrylate monomers with trifunctional

  11. Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

    Science.gov (United States)

    Prang, N; Wolf, H; Schwarzmann, F

    1999-12-01

    The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo. Copyright 1999 Wiley-Liss, Inc.

  12. Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus▿

    Science.gov (United States)

    Cosme, Ruth S. Cruz; Yamamura, Yasuhiro; Tang, Qiyi

    2009-01-01

    Human cytomegalovirus (HCMV), a member of the β subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns. PMID:19144709

  13. Viral Disease Networks?

    Science.gov (United States)

    Gulbahce, Natali; Yan, Han; Vidal, Marc; Barabasi, Albert-Laszlo

    2010-03-01

    Viral infections induce multiple perturbations that spread along the links of the biological networks of the host cells. Understanding the impact of these cascading perturbations requires an exhaustive knowledge of the cellular machinery as well as a systems biology approach that reveals how individual components of the cellular system function together. Here we describe an integrative method that provides a new approach to studying virus-human interactions and its correlations with diseases. Our method involves the combined utilization of protein - protein interactions, protein -- DNA interactions, metabolomics and gene - disease associations to build a ``viraldiseasome''. By solely using high-throughput data, we map well-known viral associated diseases and predict new candidate viral diseases. We use microarray data of virus-infected tissues and patient medical history data to further test the implications of the viral diseasome. We apply this method to Epstein-Barr virus and Human Papillomavirus and shed light into molecular development of viral diseases and disease pathways.

  14. A comprehensive and quantitative exploration of thousands of viral genomes

    Science.gov (United States)

    Mahmoudabadi, Gita

    2018-01-01

    The complete assembly of viral genomes from metagenomic datasets (short genomic sequences gathered from environmental samples) has proven to be challenging, so there are significant blind spots when we view viral genomes through the lens of metagenomics. One approach to overcoming this problem is to leverage the thousands of complete viral genomes that are publicly available. Here we describe our efforts to assemble a comprehensive resource that provides a quantitative snapshot of viral genomic trends – such as gene density, noncoding percentage, and abundances of functional gene categories – across thousands of viral genomes. We have also developed a coarse-grained method for visualizing viral genome organization for hundreds of genomes at once, and have explored the extent of the overlap between bacterial and bacteriophage gene pools. Existing viral classification systems were developed prior to the sequencing era, so we present our analysis in a way that allows us to assess the utility of the different classification systems for capturing genomic trends. PMID:29624169

  15. Multiple-integrations of HPV16 genome and altered transcription of viral oncogenes and cellular genes are associated with the development of cervical cancer.

    Directory of Open Access Journals (Sweden)

    Xulian Lu

    Full Text Available The constitutive expression of the high-risk HPV E6 and E7 viral oncogenes is the major cause of cervical cancer. To comprehensively explore the composition of HPV16 early transcripts and their genomic annotation, cervical squamous epithelial tissues from 40 HPV16-infected patients were collected for analysis of papillomavirus oncogene transcripts (APOT. We observed different transcription patterns of HPV16 oncogenes in progression of cervical lesions to cervical cancer and identified one novel transcript. Multiple-integration events in the tissues of cervical carcinoma (CxCa are significantly more often than those of low-grade squamous intraepithelial lesions (LSIL and high-grade squamous intraepithelial lesions (HSIL. Moreover, most cellular genes within or near these integration sites are cancer-associated genes. Taken together, this study suggests that the multiple-integrations of HPV genome during persistent viral infection, which thereby alters the expression patterns of viral oncogenes and integration-related cellular genes, play a crucial role in progression of cervical lesions to cervix cancer.

  16. Novel non-viral vectors for gene delivery: synthesis of a second-generation library of mono-functionalized poly-(guanidinium)amines and their introduction into cationic lipids.

    Science.gov (United States)

    Byk, G; Soto, J; Mattler, C; Frederic, M; Scherman, D

    1998-01-01

    The development of new gene delivery technologies is a prerequisite towards gene therapy clinical trials. Because gene delivery mediated by viral vectors remains of limited scope due to immunological and propagation risks, the development of new non-viral gene delivery systems is of crucial importance. We have synthesized a secondary library of mono-functionalized poly-(guanidinium)amines generated from a library of mono-functionalized polyamines applying the concept of "libraries from libraries." The method allows a quick and easy access to mono-functionalized geometrically varied poly-(guanidinium)amines. The new building blocks were introduced into cationic lipids to obtain novel poly-(guanidinium)amine lipids, which are potential DNA vectors for gene delivery. Copyright 1998 John Wiley & Sons, Inc.

  17. Zn(II)-dipicolylamine-based metallo-lipids as novel non-viral gene vectors.

    Science.gov (United States)

    Su, Rong-Chuan; Liu, Qiang; Yi, Wen-Jing; Zhao, Zhi-Gang

    2017-08-01

    In this study, a series of Zn(II)-dipicolylamine (Zn-DPA) based cationic lipids bearing different hydrophobic tails (long chains, α-tocopherol, cholesterol or diosgenin) were synthesized. Structure-activity relationship (SAR) of these lipids was studied in detail by investigating the effects of several structural aspects including the type of hydrophobic tails, the chain length and saturation degree. In addition, several assays were used to study their interactions with plasmid DNA, and results reveal that these lipids could condense DNA into nanosized particles with appropriate size and zeta-potentials. MTT-based cell viability assays showed that lipoplexes 5 had low cytotoxicity. The in vitro gene transfection studies showed the hydrophobic tails clearly affected the TE, and hexadecanol-containing lipid 5b gives the best TE, which was 2.2 times higher than bPEI 25k in the presence of 10% serum. The results not only demonstrate that these lipids might be promising non-viral gene vectors, but also afford us clues for further optimization of lipidic gene delivery materials.

  18. Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression.

    Science.gov (United States)

    Bastian, Thomas W; Rice, Stephen A

    2009-01-01

    Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.

  19. Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

    OpenAIRE

    Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

    2013-01-01

    Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize deliver...

  20. Recent Advances in Non-viral Vectors for Gene Delivery

    Science.gov (United States)

    Guo, Xia; Huang, Leaf

    2011-01-01

    CONSPECTUS Non-viral vectors, typically based on cationic lipids or polymers, are preferred due to safety concerns with viral vectors. So far, non-viral vectors can proficiently transfect cells in culture, but obtaining efficient nanomedicines is far from evident. To overcome the hurdles associated with non-viral vectors is significant for improving delivery efficiency and therapeutic effect of nucleic acid. The drawbacks include the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, targeting ability of the carriers to the cells of interest, and so on. PEGylation is the predominant method used to reduce the binding of plasma proteins with non-viral vectors and minimize the clearance by RES after intravenous administration. The nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors due to the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral or anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal model. Designing and synthesizing novel cationic lipids and polymers, and binding nucleic acid with peptides, targeting ligands, polymers, or environmentally sensitive moieties also attract many attentions for resolving the problems encountered by non-viral vectors. The application of inorganic nanoparticles in nucleic acid delivery is an emerging field, too. Recently, different classes of non-viral vectors appear to be converging and the features of different classes of non-viral vectors could be combined in one strategy. More hurdles associated with efficient nucleic acid delivery therefore might be expected to be overcome. In this account, we will focus on these novel non-viral vectors, which are classified into multifunctional hybrid nucleic acid vectors, novel

  1. Sex-biased gene flow among elk in the greater Yellowstone ecosystem

    Science.gov (United States)

    Hand, Brian K.; Chen, Shanyuan; Anderson, Neil; Beja-Pereira, Albano; Cross, Paul C.; Ebinger, Michael R.; Edwards, Hank; Garrott, Robert A.; Kardos, Marty D.; Kauffman, Matthew J.; Landguth, Erin L.; Middleton, Arthur; Scurlock, Brandon M.; White, P.J.; Zager, Pete; Schwartz, Michael K.; Luikart, Gordon

    2014-01-01

    We quantified patterns of population genetic structure to help understand gene flow among elk populations across the Greater Yellowstone Ecosystem. We sequenced 596 base pairs of the mitochondrial control region of 380 elk from eight populations. Analysis revealed high mitochondrial DNA variation within populations, averaging 13.0 haplotypes with high mean gene diversity (0.85). The genetic differentiation among populations for mitochondrial DNA was relatively high (FST  =  0.161; P  =  0.001) compared to genetic differentiation for nuclear microsatellite data (FST  =  0.002; P  =  0.332), which suggested relatively low female gene flow among populations. The estimated ratio of male to female gene flow (mm/mf  =  46) was among the highest we have seen reported for large mammals. Genetic distance (for mitochondrial DNA pairwise FST) was not significantly correlated with geographic (Euclidean) distance between populations (Mantel's r  =  0.274, P  =  0.168). Large mitochondrial DNA genetic distances (e.g., FST > 0.2) between some of the geographically closest populations (<65 km) suggested behavioral factors and/or landscape features might shape female gene flow patterns. Given the strong sex-biased gene flow, future research and conservation efforts should consider the sexes separately when modeling corridors of gene flow or predicting spread of maternally transmitted diseases. The growing availability of genetic data to compare male vs. female gene flow provides many exciting opportunities to explore the magnitude, causes, and implications of sex-biased gene flow likely to occur in many species.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

    2016-05-11

    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.

  4. Kinetics of viral shedding provide insights into the epidemiology of viral hemorrhagic septicemia in Pacific herring

    Science.gov (United States)

    Hershberger, Paul K.; Gregg, Jacob L.; Winton, James R.; Grady, Courtney; Collins, Rachael

    2010-01-01

    Losses from infectious diseases are an important component of natural mortality among marine fish species, but factors controlling the ecology of these diseases and their potential responses to anthropogenic changes are poorly understood. We used viral hemorrhagic septicemia virus (VHSV) and a laboratory stock of Pacific herring Clupea pallasii to investigate the kinetics of viral shedding and its effect on disease transmission and host mortality. Outbreaks of acute disease, accompanied by mortality and viral shedding, were initiated after waterborne exposure of herring to concentrations of VHSV as low as 101 plaque-forming units (pfu) ml–1. Shed virus in flow-through tanks was first detected 4 to 5 d post-exposure, peaked after 6 to 10 d, and was no longer detected after 16 d. Shedding rates, calculated from density, flow and waterborne virus titer reached 1.8 to 5.0 × 108 pfu fish–1 d–1. Onset of viral shedding was dose-dependent and preceded initial mortality by 2 d. At 21 d, cumulative mortality in treatment groups ranged from 81 to 100% and was dependent not on challenge dose, but on the kinetics and level of viral shedding by infected fish in the tank. Possible consequences of the viral shedding and disease kinetics are discussed in the context of epizootic initiation and perpetuation among populations of wild Pacific herring.

  5. Patterns of gene flow define species of thermophilic Archaea.

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    Hinsby Cadillo-Quiroz

    2012-02-01

    Full Text Available Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

  6. Patterns of gene flow define species of thermophilic Archaea.

    Science.gov (United States)

    Cadillo-Quiroz, Hinsby; Didelot, Xavier; Held, Nicole L; Herrera, Alfa; Darling, Aaron; Reno, Michael L; Krause, David J; Whitaker, Rachel J

    2012-02-01

    Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

  7. Genetics and Molecular Biology of Epstein-Barr Virus-Encoded BART MicroRNA: A Paradigm for Viral Modulation of Host Immune Response Genes and Genome Stability

    Directory of Open Access Journals (Sweden)

    David H. Dreyfus

    2017-01-01

    Full Text Available Epstein-Barr virus, a ubiquitous human herpesvirus, is associated through epidemiologic evidence with common autoimmune syndromes and cancers. However, specific genetic mechanisms of pathogenesis have been difficult to identify. In this review, the author summarizes evidence that recently discovered noncoding RNAs termed microRNA encoded by Epstein-Barr virus BARF (BamHI A right frame termed BART (BamHI A right transcripts are modulators of human immune response genes and genome stability in infected and bystander cells. BART expression is apparently regulated by complex feedback loops with the host immune response regulatory NF-κB transcription factors. EBV-encoded BZLF-1 (ZEBRA protein could also regulate BART since ZEBRA contains a terminal region similar to ankyrin proteins such as IκBα that regulate host NF-κB. BALF-2 (BamHI A left frame transcript, a viral homologue of the immunoglobulin and T cell receptor gene recombinase RAG-1 (recombination-activating gene-1, may also be coregulated with BART since BALF-2 regulatory sequences are located near the BART locus. Viral-encoded microRNA and viral mRNA transferred to bystander cells through vesicles, defective viral particles, or other mechanisms suggest a new paradigm in which bystander or hit-and-run mechanisms enable the virus to transiently or chronically alter human immune response genes as well as the stability of the human genome.

  8. Speciation and gene flow between snails of opposite chirality.

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    Angus Davison

    2005-09-01

    Full Text Available Left-right asymmetry in snails is intriguing because individuals of opposite chirality are either unable to mate or can only mate with difficulty, so could be reproductively isolated from each other. We have therefore investigated chiral evolution in the Japanese land snail genus Euhadra to understand whether changes in chirality have promoted speciation. In particular, we aimed to understand the effect of the maternal inheritance of chirality on reproductive isolation and gene flow. We found that the mitochondrial DNA phylogeny of Euhadra is consistent with a single, relatively ancient evolution of sinistral species and suggests either recent "single-gene speciation" or gene flow between chiral morphs that are unable to mate. To clarify the conditions under which new chiral morphs might evolve and whether single-gene speciation can occur, we developed a mathematical model that is relevant to any maternal-effect gene. The model shows that reproductive character displacement can promote the evolution of new chiral morphs, tending to counteract the positive frequency-dependent selection that would otherwise drive the more common chiral morph to fixation. This therefore suggests a general mechanism as to how chiral variation arises in snails. In populations that contain both chiral morphs, two different situations are then possible. In the first, gene flow is substantial between morphs even without interchiral mating, because of the maternal inheritance of chirality. In the second, reproductive isolation is possible but unstable, and will also lead to gene flow if intrachiral matings occasionally produce offspring with the opposite chirality. Together, the results imply that speciation by chiral reversal is only meaningful in the context of a complex biogeographical process, and so must usually involve other factors. In order to understand the roles of reproductive character displacement and gene flow in the chiral evolution of Euhadra, it will be

  9. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

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    Janet M. Doolittle-Hall

    2015-11-01

    Full Text Available Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV, hepatitis B virus (HBV or Merkel cell polyomavirus (MCPyV. These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

  10. Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

    Science.gov (United States)

    Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.

    2017-01-01

    A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

  11. Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population

    Directory of Open Access Journals (Sweden)

    Jessica eLabonté

    2015-04-01

    Full Text Available A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a three km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32 % of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

  12. The evolutionary history of bears is characterized by gene flow across species

    Science.gov (United States)

    Kumar, Vikas; Lammers, Fritjof; Bidon, Tobias; Pfenninger, Markus; Kolter, Lydia; Nilsson, Maria A.; Janke, Axel

    2017-01-01

    Bears are iconic mammals with a complex evolutionary history. Natural bear hybrids and studies of few nuclear genes indicate that gene flow among bears may be more common than expected and not limited to polar and brown bears. Here we present a genome analysis of the bear family with representatives of all living species. Phylogenomic analyses of 869 mega base pairs divided into 18,621 genome fragments yielded a well-resolved coalescent species tree despite signals for extensive gene flow across species. However, genome analyses using different statistical methods show that gene flow is not limited to closely related species pairs. Strong ancestral gene flow between the Asiatic black bear and the ancestor to polar, brown and American black bear explains uncertainties in reconstructing the bear phylogeny. Gene flow across the bear clade may be mediated by intermediate species such as the geographically wide-spread brown bears leading to large amounts of phylogenetic conflict. Genome-scale analyses lead to a more complete understanding of complex evolutionary processes. Evidence for extensive inter-specific gene flow, found also in other animal species, necessitates shifting the attention from speciation processes achieving genome-wide reproductive isolation to the selective processes that maintain species divergence in the face of gene flow. PMID:28422140

  13. The evolutionary history of bears is characterized by gene flow across species.

    Science.gov (United States)

    Kumar, Vikas; Lammers, Fritjof; Bidon, Tobias; Pfenninger, Markus; Kolter, Lydia; Nilsson, Maria A; Janke, Axel

    2017-04-19

    Bears are iconic mammals with a complex evolutionary history. Natural bear hybrids and studies of few nuclear genes indicate that gene flow among bears may be more common than expected and not limited to polar and brown bears. Here we present a genome analysis of the bear family with representatives of all living species. Phylogenomic analyses of 869 mega base pairs divided into 18,621 genome fragments yielded a well-resolved coalescent species tree despite signals for extensive gene flow across species. However, genome analyses using different statistical methods show that gene flow is not limited to closely related species pairs. Strong ancestral gene flow between the Asiatic black bear and the ancestor to polar, brown and American black bear explains uncertainties in reconstructing the bear phylogeny. Gene flow across the bear clade may be mediated by intermediate species such as the geographically wide-spread brown bears leading to large amounts of phylogenetic conflict. Genome-scale analyses lead to a more complete understanding of complex evolutionary processes. Evidence for extensive inter-specific gene flow, found also in other animal species, necessitates shifting the attention from speciation processes achieving genome-wide reproductive isolation to the selective processes that maintain species divergence in the face of gene flow.

  14. A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts

    Directory of Open Access Journals (Sweden)

    Hannah L. Fox

    2017-06-01

    Full Text Available Herpes simplex virus 1 (HSV-1 genes are transcribed by cellular RNA polymerase II (RNA Pol II. While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22 function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16 was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq. The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq, we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production.

  15. Niewirusowy transfer genów do komórek skóry – wybrane metody = Non-viral gene transfer into skin cells – selected methods

    Directory of Open Access Journals (Sweden)

    Ewelina Wędrowska

    2016-01-01

    3Department of Pathobiochemistry and Clinical Chemistry, Nicolaus Copernicus University Collegium Medicum, Bydgoszcz, Poland       Streszczenie   Wprowadzenie: Skóra jako największy i najłatwiej dostępny narząd stanowi atrakcyjny cel dla terapii genowej, która od wielu lat budzi ogromne nadzieje środowiska naukowego. Jednakże, próby terapii przeprowadzone z wykorzystaniem wektorów wirusowych wykazały szereg wad i ograniczeń m.in. obserwowano indukcję odpowiedzi immunologicznej, losową integrację transgenu z genomem gospodarza i/lub niską wydajność jego ekspresji. Dlatego, wciąż poszukuje się alternatywnych, skuteczniejszych i jednocześnie bezpieczniejszych metod transferu genów. Atrakcyjnej alternatywy upatruje się w metodach niewirusowych. Cel pracy: Przedstawienie wybranych metod niewirusowego transferu genów wykorzystywanych w terapii genowej chorób skóry. Skrócony opis stanu wiedzy: Terapia genowa chorób skóry obejmuje wykorzystanie wektorów plazmidowych jako nośnika genów terapeutycznych, a także metod ich dostarczania do komórek takich jak: elektroporacja, mikroiniekcja, sonikacja, wykorzystanie nośników lipidowych i polimerów kationowych. Podsumowanie: Niewirusowe metody transferu genów oferują pewne zalety włączając niską toksyczność, brak infekcyjności oraz łatwość i niskie koszty produkcji w porównaniu z technikami wirusowymi. Niewirusowe metody wydają się być obiecującym narzędziem terapii genowej chorób skóry w szczególności nowotworów tego narządu.   Słowa kluczowe: plazmid, transfer genów, skóra, wirus, terapia genowa.     Summary Introduction: Skin, the largest and most accessible organ of the human body is considered as an ideal gene therapy target. However, various types of viral vectors used in classical gene therapy have a number of disadvantages, such as possibility of immune response induction, random integration of inserts into the host genome or low expression efficiency

  16. In vitro efficacy of a gene-activated nerve guidance conduit incorporating non-viral PEI-pDNA nanoparticles carrying genes encoding for NGF, GDNF and c-Jun.

    Science.gov (United States)

    Lackington, William A; Raftery, Rosanne M; O'Brien, Fergal J

    2018-06-07

    Despite the success of tissue engineered nerve guidance conduits (NGCs) for the treatment of small peripheral nerve injuries, autografts remain the clinical gold standard for larger injuries. The delivery of neurotrophic factors from conduits might enhance repair for more effective treatment of larger injuries but the efficacy of such systems is dependent on a safe, effective platform for controlled and localised therapeutic delivery. Gene therapy might offer an innovative approach to control the timing, release and level of neurotrophic factor production by directing cells to transiently sustain therapeutic protein production in situ. In this study, a gene-activated NGC was developed by incorporating non-viral polyethyleneimine-plasmid DNA (PEI-pDNA) nanoparticles (N/P 7 ratio, 2μg dose) with the pDNA encoding for nerve growth factor (NGF), glial derived neurotrophic factor (GDNF) or the transcription factor c-Jun. The physicochemical properties of PEI-pDNA nanoparticles, morphology, size and charge, were shown to be suitable for gene delivery and demonstrated high Schwann cell transfection efficiency (60±13%) in vitro. While all three genes showed therapeutic potential in terms of enhancing neurotrophic cytokine production while promoting neurite outgrowth, delivery of the gene encoding for c-Jun showed the greatest capacity to enhance regenerative cellular processes in vitro. Ultimately, this gene-activated NGC construct was shown to be capable of transfecting both Schwann cells (S42 cells) and neuronal cells (PC12 and dorsal root ganglia) in vitro, demonstrating potential for future therapeutic applications in vivo. The basic requirements of biomaterial-based nerve guidance conduits have now been well established and include being able to bridge a nerve injury to support macroscopic guidance between nerve stumps, while being strong enough to withstand longitudinal tension and circumferential compression, in addition to being mechanically sound to facilitate

  17. Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies

    Directory of Open Access Journals (Sweden)

    Beatriz Santos-Carballal

    2018-04-01

    Full Text Available Non-viral gene delivery vectors have lagged far behind viral ones in the current pipeline of clinical trials of gene therapy nanomedicines. Even when non-viral nanovectors pose less safety risks than do viruses, their efficacy is much lower. Since the early studies to deliver pDNA, chitosan has been regarded as a highly attractive biopolymer to deliver nucleic acids intracellularly and induce a transgenic response resulting in either upregulation of protein expression (for pDNA, mRNA or its downregulation (for siRNA or microRNA. This is explained as the consequence of a multi-step process involving condensation of nucleic acids, protection against degradation, stabilization in physiological conditions, cellular internalization, release from the endolysosome (“proton sponge” effect, unpacking and enabling the trafficking of pDNA to the nucleus or the siRNA to the RNA interference silencing complex (RISC. Given the multiple steps and complexity involved in the gene transfection process, there is a dearth of understanding of the role of chitosan’s structural features (Mw and degree of acetylation, DA% on each step that dictates the net transfection efficiency and its kinetics. The use of fully characterized chitosan samples along with the utilization of complementary biophysical and biological techniques is key to bridging this gap of knowledge and identifying the optimal chitosans for delivering a specific gene. Other aspects such as cell type and administration route are also at play. At the same time, the role of chitosan structural features on the morphology, size and surface composition of synthetic virus-like particles has barely been addressed. The ongoing revolution brought about by the recent discovery of CRISPR-Cas9 technology will undoubtedly be a game changer in this field in the short term. In the field of rare diseases, gene therapy is perhaps where the greatest potential lies and we anticipate that chitosans will be key players

  18. Polyphyly and gene flow between non-sibling Heliconius species

    Directory of Open Access Journals (Sweden)

    Jiggins Chris D

    2006-04-01

    Full Text Available Abstract Background The view that gene flow between related animal species is rare and evolutionarily unimportant largely antedates sensitive molecular techniques. Here we use DNA sequencing to investigate a pair of morphologically and ecologically divergent, non-sibling butterfly species, Heliconius cydno and H. melpomene (Lepidoptera: Nymphalidae, whose distributions overlap in Central and Northwestern South America. Results In these taxa, we sequenced 30–45 haplotypes per locus of a mitochondrial region containing the genes for cytochrome oxidase subunits I and II (CoI/CoII, and intron-spanning fragments of three unlinked nuclear loci: triose-phosphate isomerase (Tpi, mannose-6-phosphate isomerase (Mpi and cubitus interruptus (Ci genes. A fifth gene, dopa decarboxylase (Ddc produced sequence data likely to be from different duplicate loci in some of the taxa, and so was excluded. Mitochondrial and Tpi genealogies are consistent with reciprocal monophyly, whereas sympatric populations of the species in Panama share identical or similar Mpi and Ci haplotypes, giving rise to genealogical polyphyly at the species level despite evidence for rapid sequence divergence at these genes between geographic races of H. melpomene. Conclusion Recent transfer of Mpi haplotypes between species is strongly supported, but there is no evidence for introgression at the other three loci. Our results demonstrate that the boundaries between animal species can remain selectively porous to gene flow long after speciation, and that introgression, even between non-sibling species, can be an important factor in animal evolution. Interspecific gene flow is demonstrated here for the first time in Heliconius and may provide a route for the transfer of switch-gene adaptations for Müllerian mimicry. The results also forcefully demonstrate how reliance on a single locus may give an erroneous picture of the overall genealogical history of speciation and gene flow.

  19. PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival

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    Amanda R. Panfil

    2015-12-01

    Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL. This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5 on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment.

  20. Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes.

    Science.gov (United States)

    Rangarajan, P N; Vatsala, P G; Ashok, M S; Srinivas, V K; Habibullah, C M; Padmanaban, G

    1997-04-29

    Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.

  1. A new pathway for developing in vitro nanostructured non-viral gene carriers

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Benjamin [Chemistry Department, Stony Brook University, Stony Brook, NY 11794-3400 (United States); Liang Dehai [Chemistry Department, Stony Brook University, Stony Brook, NY 11794-3400 (United States); Hadjiargyrou, Michael [Biomedical Engineering Department, Stony Brook University, Stony Brook, NY 11794-3400 (United States); Hsiao, Benjamin S [Chemistry Department, Stony Brook University, Stony Brook, NY 11794-3400 (United States)

    2006-09-13

    Extracellular and intracellular barriers typically prevent the efficient transfection of non-viral gene vectors. The formulation of a gene delivery carrier that can overcome the barriers would be a key for successful gene therapy. We have developed a novel pathway for the preparation of core-shelled DNA nanoparticles by invoking solvent-induced condensation of plasmid DNA ({beta}-galactosidase) in a poor solvent mixture and subsequent encapsulation of the condensed DNA globule in a tri-block copolymer (e.g. polylactide-poly(ethylene glycol)-polylactide, L{sub 8}E{sub 78}L{sub 8}). The polylactide shell can protect the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a non-woven nanofibrous DNA-containing scaffold. The bioactive plasmid DNA can then be released in an intact form and in sufficient quantity from the scaffold with a controlled release rate and to transfect cells in vitro. Further consideration of the stability of the DNA in extracellular and intracellular environments is proposed. In particular, the use of block copolymers with a positively charged block and a hydrophilic block, as well as tri-arm block copolymers with an additional hydrophobic, biodegradable block to stabilize the DNA chain of interest, is discussed.

  2. Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability.

    Science.gov (United States)

    Kraus, Armin; Täger, Joachim; Kohler, Konrad; Haerle, Max; Werdin, Frank; Schaller, Hans-Eberhard; Sinis, Nektarios

    2010-11-01

    To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC). The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined. Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-L-ornithine coated plates was compromised by either method. Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

  3. A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

    Science.gov (United States)

    Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

    2015-10-01

    During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Ecological Divergence and the Origins of Intrinsic Postmating Isolation with Gene Flow

    Directory of Open Access Journals (Sweden)

    Aneil F. Agrawal

    2011-01-01

    Full Text Available The evolution of intrinsic postmating isolation has received much attention, both historically and in recent studies of speciation genes. Intrinsic isolation often stems from between-locus genetic incompatibilities, where alleles that function well within species are incompatible with one another when brought together in the genome of a hybrid. It can be difficult for such incompatibilities to originate when populations diverge with gene flow, because deleterious genotypic combinations will be created and then purged by selection. However, it has been argued that if genes underlying incompatibilities are themselves subject to divergent selection, then they might overcome gene flow to diverge between populations, resulting in the origin of incompatibilities. Nonetheless, there has been little explicit mathematical exploration of such scenarios for the origin of intrinsic incompatibilities during ecological speciation with gene flow. Here we explore theoretical models for the origin of intrinsic isolation where genes subject to divergent natural selection also affect intrinsic isolation, either directly or via linkage disequilibrium with other loci. Such genes indeed overcome gene flow, diverge between populations, and thus result in the evolution of intrinsic isolation. We also examine barriers to neutral gene flow. Surprisingly, we find that intrinsic isolation sometimes weakens this barrier, by impeding differentiation via ecologically based divergent selection.

  5. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    Directory of Open Access Journals (Sweden)

    Jonathan M.O. Rawson

    2014-09-01

    Full Text Available Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved.

  6. A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons

    NARCIS (Netherlands)

    Fagoe, N D; Eggers, R; Verhaagen, J; Mason, M R J

    Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the

  7. Identification of an ovine atadenovirus gene whose product activates the viral E2 promoter: possible involvement of E2F-1

    International Nuclear Information System (INIS)

    Kuemin, Daniel; Hofmann, Christian; Uckert, Wolfgang; Both, Gerald W.; Loeser, Peter

    2004-01-01

    Activation of the adenoviral E2 promoter is an early step in adenovirus gene expression. For members of the mast- and aviadenoviruses, this requires induction of the cellular transcription factor E2F by virally encoded gene products such as E1A, E4orf6/7 and orf22/GAM-1. The newly recognized genus atadenovirus, of which the ovine isolate OAdV is the prototype, lacks any sequence homology to those genes. To find a possible link between E2 promoter activation and OAdV gene expression, we utilized a screening method to search for genes within the OAdV genome that were capable of stimulating the viral E2 promoter. One such gene, E43, was identified within the proposed E4 region toward the right-hand end of the OAdV genome. The E43 gene product was also found to be capable of stimulating E2F-1-dependent gene expression. A closer inspection of the E2 promoter revealed the presence of a non-palindromic E2F binding site within the OAdV E2 promoter. Mutation of this site markedly reduced both E2F-1- and E43-dependent promoter activation. Moreover, a direct protein-protein interaction of the E43 gene product with E2F, but not with the retinoblastoma protein pRb, suggested a possible cooperation between these two proteins in activating the E2 promoter. The importance of the E43 gene product for virus replication is also underlined by the finding that an OAdV recombinant with a functionally inactivated E43 gene showed severely inhibited virus growth

  8. Viral gene products and replication of the human immunodeficiency type 1 virus.

    Science.gov (United States)

    Morrow, C D; Park, J; Wakefield, J K

    1994-05-01

    The acquired immunodeficiency syndrome (AIDS) epidemic represents a modern-day plague that has not only resulted in a tragic loss of people from a wide spectrum of society but has reshaped our viewpoints regarding health care, the treatment of infectious diseases, and social issues regarding sexual behavior. There is little doubt now that the cause of the disease AIDS is a virus known as the human immunodeficiency virus (HIV). The HIV virus is a member of a large family of viruses termed retroviruses, which have as a hallmark the capacity to convert their RNA genome into a DNA form that then undergoes a process of integration into the host cell chromosome, followed by the expression of the viral genome and translation of viral proteins in the infected cell. This review describes the organization of the HIV-1 viral genome, the expression of viral proteins, as well as the functions of the accessory viral proteins in HIV replication. The replication of the viral genome is divided into two phases, the early phase and the late phase. The early phase consists of the interaction of the virus with the cell surface receptor (CD4 molecule in most cases), the uncoating and conversion of the viral RNA genome into a DNA form, and the integration into the host cell chromosome. The late phase consists of the expression of the viral proteins from the integrated viral genome, the translation of viral proteins, and the assembly and release of the virus. Points in the HIV-1 life cycle that are targets for therapeutic intervention are also discussed.

  9. Gene expression of herpes simplex virus. II. Uv radiological analysis of viral transcription units

    International Nuclear Information System (INIS)

    Millette, R. L.; Klaiber, R.

    1980-01-01

    The transcriptional organization of the genome of herpes simplex virus type 1 was analyzed by measuring the sensitivity of viral polypeptide synthesis to uv irradiation of the infecting virus. Herpes simplex virus type 1 was irradiated with various doses of uv light and used to infect xeroderma pigmentosum fibroblasts. Immediate early transcription units were analyzed by having cycloheximide present throughout the period of infection, removing the drug at 8 h postinfection, and pulse-labeling proteins with [355]methionine. Delayed early transcription units were analyzed in similar studies by having 9-beta-D-arabinofuranosyladenine present during the experiment to block replication of the input irradiated genome. The results indicate that none of the immediate early genes analyzed can be cotranscribed, whereas some of the delayed early genes might be cotranscribed. No evidence was found for the existence of large, multigene transcription units

  10. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

    Science.gov (United States)

    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

  11. A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

    Science.gov (United States)

    Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

    2017-06-13

    Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription

  12. Viral impacts on microbial carbon cycling in thawing permafrost soils

    Science.gov (United States)

    Trubl, G. G.; Roux, S.; Bolduc, B.; Jang, H. B.; Emerson, J. B.; Solonenko, N.; Li, F.; Solden, L. M.; Vik, D. R.; Wrighton, K. C.; Saleska, S. R.; Sullivan, M. B.; Rich, V. I.

    2017-12-01

    Permafrost contains 30-50% of global soil carbon (C) and is rapidly thawing. While the fate of this C is unknown, it will be shaped in part by microbes and their associated viruses, which modulate host activities via mortality and metabolic control. To date, viral research in soils has been outpaced by that in aquatic environments, due to the technical challenges of accessing viruses as well as the dramatic physicochemical heterogeneity in soils. Here, we describe advances in soil viromics from our research on permafrost-associated soils, and their implications for associated terrestrial C cycling. First, we optimized viral resuspension-DNA extraction methods for a range of soil types. Second, we applied cutting-edge viral-specific informatics methods to recover viral populations, define their gene content, connect them to potential hosts, and analyze their relationships to environmental parameters. A total of 781 viral populations were recovered from size-fractionated virus samples of three soils along a permafrost thaw gradient. Ecological analyses revealed endemism as recovered viral populations were largely unique to each habitat and unlike those in aquatic communities. Genome- and network-based classification assigned these viruses into 226 viral clusters (VCs; genus-level taxonomy), 55% of which were novel. This increases the number of VCs by a third and triples the number of soil viral populations in the RefSeq database (currently contains 256 VCs and 316 soil viral populations). Genomic analyses revealed 85% of the genes were functionally unknown, though 5% of the annotatable genes contained C-related auxiliary metabolic genes (AMGs; e.g. glycoside hydrolases). Using sequence-based features and microbial population genomes, we were able to in silico predict hosts for 30% of the viral populations. The identified hosts spanned 3 phyla and 6 genera but suggested these viruses have species-specific host ranges as >80% of hosts for a given virus were in the same

  13. Low-frequency ultrasound increases non-viral gene transfer to the mouse lung.

    Science.gov (United States)

    Xenariou, Stefania; Liang, Hai-Dong; Griesenbach, Uta; Zhu, Jie; Farley, Raymond; Somerton, Lucinda; Singh, Charanjit; Jeffery, Peter K; Scheule, Ronald K; Cheng, Seng H; Geddes, Duncan M; Blomley, Martin; Alton, Eric W F W

    2010-01-01

    The aim of the study was to assess if low-frequency ultrasound (US), in the range of 30-35 kHz, increases non-viral gene transfer to the mouse lung. US is greatly attenuated in the lung due to large energy losses at the air/tissue interfaces. The advantages of low-frequency US, compared with high-frequency US are: (i) increased cavitation (responsible for the formation of transient pores in the cell membrane) and (ii) reduced energy losses during lung penetration. Cationic lipid GL67/plasmid DNA (pDNA), polyethylenimine (PEI)/pDNA and naked pDNA were delivered via intranasal instillation and the animals were then exposed to US (sonoporation) at 0.07 or 0.1 MPa for 10 min. Under these conditions, US did not enhance GL67 or PEI-mediated transfection. It did, however, increase naked pDNA gene transfer by approximately 4 folds. Importantly, this was achieved in the absence of microbubbles, which are crucial for the commonly used high-frequency (1 MHz) sonoporation but may not be able to withstand nebulization in a clinically relevant setup. Lung hemorrhage was also assessed and shown to increase with US pressure in a dose-dependent manner. We have thus, established that low-frequency US can enhance lung gene transfer with naked pDNA and this enhancement is more effective than the previously reported 1 MHz US.

  14. Microbial and viral-like rhodopsins present in coastal marine sediments from four polar and subpolar regions

    Energy Technology Data Exchange (ETDEWEB)

    López, José L.; Golemba, Marcelo; Hernández, Edgardo; Lozada, Mariana; Dionisi, Hebe; Jansson, Janet K.; Carroll, Jolynn; Lundgren, Leif; Sjöling, Sara; Mac Cormack, Walter P.; Sobecky, Patricia

    2016-11-03

    Rhodopsins are broadly distributed. In this work, we analyzed 23 metagenomes corresponding to marine sediment samples from four regions that share cold climate conditions (Norway; Sweden; Argentina and Antarctica). In order to investigate the genes evolution of viral rhodopsins, an initial set of 6224 bacterial rhodopsin sequences according to COG5524 were retrieved from the 23 metagenomes. After selection by the presence of transmembrane domains and alignment, 123 viral (51) and non-viral (72) sequences (>50 amino acids) were finally included in further analysis. Viral rhodopsin genes were homologs of Phaeocystis globosa virus and Organic lake Phycodnavirus. Non-viral microbial rhodopsin genes were ascribed to Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Proteobacteria, Deinococcus-Thermus and Cryptophyta and Fungi. A rescreening using Blastp, using as queries the viral sequences previously described, retrieved 30 sequences (>100 amino acids). Phylogeographic analysis revealed a geographical clustering of the sequences affiliated to the viral group. This clustering was not observed for the microbial non-viral sequences. The phylogenetic reconstruction allowed us to propose the existence of a putative ancestor of viral rhodopsin genes related to Actinobacteria and Chloroflexi. This is the first report about the existence of a phylogeographic association of the viral rhodopsin sequences from marine sediments.

  15. A conceptual framework that links pollinator foraging behavior to gene flow

    Science.gov (United States)

    In insect-pollinated crops such as alfalfa, a better understanding of how pollinator foraging behavior affects gene flow could lead to the development of management strategies to reduce gene flow and facilitate the coexistence of distinct seed-production markets. Here, we introduce a conceptual fram...

  16. Viral Inhibition of Bacterial Phagocytosis by Human Macrophages: Redundant Role of CD36.

    Directory of Open Access Journals (Sweden)

    Grace E Cooper

    Full Text Available Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of Streptococcus pneumoniae, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of S. pneumoniae by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFNβ gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031 and CD36 gene expression (p = 0.031 by MDM cultured for 24 h in 50IU/ml IFNβ. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFNβ production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency.

  17. GeneXpert HIV-1 quant assay, a new tool for scale up of viral load monitoring in the success of ART programme in India.

    Science.gov (United States)

    Kulkarni, Smita; Jadhav, Sushama; Khopkar, Priyanka; Sane, Suvarna; Londhe, Rajkumar; Chimanpure, Vaishali; Dhilpe, Veronica; Ghate, Manisha; Yelagate, Rajendra; Panchal, Narayan; Rahane, Girish; Kadam, Dilip; Gaikwad, Nitin; Rewari, Bharat; Gangakhedkar, Raman

    2017-07-21

    Recent WHO guidelines identify virologic monitoring for diagnosing and confirming ART failure. In view of this, validation and scale up of point of care viral load technologies is essential in resource limited settings. A systematic validation of the GeneXpert® HIV-1 Quant assay (a point-of-care technology) in view of scaling up HIV-1 viral load in India to monitor the success of national ART programme was carried out. Two hundred nineteen plasma specimens falling in nine viral load ranges (5 L copies/ml) were tested by the Abbott m2000rt Real Time and GeneXpert HIV-1 Quant assays. Additionally, 20 seronegative; 16 stored specimens and 10 spiked controls were also tested. Statistical analysis was done using Stata/IC and sensitivity, specificity, PPV, NPV and %misclassification rates were calculated as per DHSs/AISs, WHO, NACO cut-offs for virological failure. The GeneXpert assay compared well with the Abbott assay with a higher sensitivity (97%), specificity (97-100%) and concordance (91.32%). The correlation between two assays (r = 0.886) was statistically significant (p performance and rapidity will aid in timely diagnosis of ART failures, integrated HIV-TB management and will facilitate the UNAIDS 90-90-90 target.

  18. Host-derived viral transporter protein for nitrogen uptake in infected marine phytoplankton

    Science.gov (United States)

    Chambouvet, Aurélie; Milner, David S.; Attah, Victoria; Terrado, Ramón; Lovejoy, Connie; Moreau, Hervé; Derelle, Évelyne; Richards, Thomas A.

    2017-01-01

    Phytoplankton community structure is shaped by both bottom–up factors, such as nutrient availability, and top–down processes, such as predation. Here we show that marine viruses can blur these distinctions, being able to amend how host cells acquire nutrients from their environment while also predating and lysing their algal hosts. Viral genomes often encode genes derived from their host. These genes may allow the virus to manipulate host metabolism to improve viral fitness. We identify in the genome of a phytoplankton virus, which infects the small green alga Ostreococcus tauri, a host-derived ammonium transporter. This gene is transcribed during infection and when expressed in yeast mutants the viral protein is located to the plasma membrane and rescues growth when cultured with ammonium as the sole nitrogen source. We also show that viral infection alters the nature of nitrogen compound uptake of host cells, by both increasing substrate affinity and allowing the host to access diverse nitrogen sources. This is important because the availability of nitrogen often limits phytoplankton growth. Collectively, these data show that a virus can acquire genes encoding nutrient transporters from a host genome and that expression of the viral gene can alter the nutrient uptake behavior of host cells. These results have implications for understanding how viruses manipulate the physiology and ecology of phytoplankton, influence marine nutrient cycles, and act as vectors for horizontal gene transfer. PMID:28827361

  19. Intracellular delivery of potential therapeutic genes: prospects in cancer gene therapy.

    Science.gov (United States)

    Bakhtiar, Athirah; Sayyad, Mustak; Rosli, Rozita; Maruyama, Atsushi; Chowdhury, Ezharul H

    2014-01-01

    Conventional therapies for malignant cancer such as chemotherapy and radiotherapy are associated with poor survival rates owing to the development of cellular resistance to cancer drugs and the lack of targetability, resulting in unwanted adverse effects on healthy cells and necessitating the lowering of therapeutic dose with consequential lower efficacy of the treatment. Gene therapy employing different types of viral and non-viral carriers to transport gene(s) of interest and facilitating production of the desirable therapeutic protein(s) has tremendous prospects in cancer treatments due to the high-level of specificity in therapeutic action of the expressed protein(s) with diminished off-target effects, although cancer cell-specific delivery of transgene(s) still poses some challenges to be addressed. Depending on the potential therapeutic target genes, cancer gene therapy could be categorized into tumor suppressor gene replacement therapy, immune gene therapy and enzyme- or prodrug-based therapy. This review would shed light on the current progress of delivery of potentially therapeutic genes into various cancer cells in vitro and animal models utilizing a variety of viral and non-viral vectors.

  20. Complete suppression of viral gene expression is associated with the onset and progression of lymphoid malignancy: observations in Bovine Leukemia Virus-infected sheep

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    Burny Arsène

    2007-07-01

    Full Text Available Abstract Background During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentally-infected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts. Results In the first animal, we observed the emergence of a genetically modified provirus simultaneously with leukemia onset. We found a Tax-mutated (TaxK303 replication-deficient provirus in the malignant B-cell clone while functional provirus (TaxE303 had been consistently monitored over the 17-month aleukemic period. In the second case, both non-transformed and transformed BLV-infected cells were present at the same time, but at distinct sites. While there was potentially-active provirus in the non-leukemic blood B-cell population, as demonstrated by ex-vivo culture and injection into naïve sheep, virus expression was completely suppressed in the malignant B-cells isolated from the lymphoid tumors despite the absence of genetic alterations in the proviral genome. These observations suggest that silencing of viral genes, including the oncoprotein Tax, is associated with tumor onset. Conclusion Our findings suggest that silencing is critical for tumor progression and identify two distinct mechanisms-genetic and epigenetic-involved in the complete suppression of virus and Tax expression. We demonstrate that, in contrast to systems that require sustained oncogene expression, the major viral transforming protein Tax can be turned-off without reversing the transformed phenotype. We propose that suppression

  1. Long-distance gene flow and adaptation of forest trees to rapid climate change

    Science.gov (United States)

    Kremer, Antoine; Ronce, Ophélie; Robledo-Arnuncio, Juan J; Guillaume, Frédéric; Bohrer, Gil; Nathan, Ran; Bridle, Jon R; Gomulkiewicz, Richard; Klein, Etienne K; Ritland, Kermit; Kuparinen, Anna; Gerber, Sophie; Schueler, Silvio

    2012-01-01

    Forest trees are the dominant species in many parts of the world and predicting how they might respond to climate change is a vital global concern. Trees are capable of long-distance gene flow, which can promote adaptive evolution in novel environments by increasing genetic variation for fitness. It is unclear, however, if this can compensate for maladaptive effects of gene flow and for the long-generation times of trees. We critically review data on the extent of long-distance gene flow and summarise theory that allows us to predict evolutionary responses of trees to climate change. Estimates of long-distance gene flow based both on direct observations and on genetic methods provide evidence that genes can move over spatial scales larger than habitat shifts predicted under climate change within one generation. Both theoretical and empirical data suggest that the positive effects of gene flow on adaptation may dominate in many instances. The balance of positive to negative consequences of gene flow may, however, differ for leading edge, core and rear sections of forest distributions. We propose future experimental and theoretical research that would better integrate dispersal biology with evolutionary quantitative genetics and improve predictions of tree responses to climate change. PMID:22372546

  2. Non-viral Nucleic Acid Delivery Strategies to the Central Nervous System

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    James-Kevin Tan

    2016-11-01

    Full Text Available With an increased prevalence and understanding of central nervous system injuries and neurological disorders, nucleic acid therapies are gaining promise as a way to regenerate lost neurons or halt disease progression. While more viral vectors have been used clinically as tools for gene delivery, non-viral vectors are gaining interest due to lower safety concerns and the ability to deliver all types of nucleic acids. Nevertheless, there are still a number of barriers to nucleic acid delivery. In this focused review, we explore the in vivo challenges hindering non-viral nucleic acid delivery to the central nervous system and the strategies and vehicles used to overcome them. Advantages and disadvantages of different routes of administration including: systemic injection, cerebrospinal fluid injection, intraparenchymal injection, and peripheral administration are discussed. Non-viral vehicles and treatment strategies that have overcome delivery barriers and demonstrated in vivo gene transfer to the central nervous system are presented. These approaches can be used as guidelines in developing synthetic gene delivery vectors for central nervous system applications and will ultimately bring non-viral vectors closer to clinical application.

  3. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

    Science.gov (United States)

    Herzog, R W; Yang, E Y; Couto, L B; Hagstrom, J N; Elwell, D; Fields, P A; Burton, M; Bellinger, D A; Read, M S; Brinkhous, K M; Podsakoff, G M; Nichols, T C; Kurtzman, G J; High, K A

    1999-01-01

    Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

  4. Study of gene flow from GM cotton (Gossypium hirsutum) varieties in El Espinal (Tolima, Colombia)

    International Nuclear Information System (INIS)

    Rache Cardenal, Leidy Yanira; Mora Oberlaender, Julian; Chaparro Giraldo, Alejandro

    2013-01-01

    In 2009, 4088 hectares of genetically modified (GM) cotton were planted in Tolima (Colombia), however there is some uncertainty about containment measures needed to prevent the flow of pollen and seed from regulated GM fields into adjacent fields. In this study, the gene flow from GM cotton varieties to conventional or feral cotton plants via seed and pollen was evaluated. ImmunostripTM, PCR and ELISA assays were used to detect gene flow. Fifty six refuges, 27 fields with conventional cotton and four feral individuals of the enterprise Remolinos Inc. located in El Espinal (Tolima) were analyzed in the first half of 2010. The results indicated seed mediated gene flow in 45 refuges (80.4 %) and 26 fields with conventional cotton (96 %), besides pollen mediated gene flow in one field with conventional cotton and nine refuges. All fields cultivated with conventional cotton showed gene flow from GM cotton. Two refuges and two feral individuals did not reveal gene flow from GM cotton.

  5. Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

    Science.gov (United States)

    Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

    2013-01-01

    Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

  6. Non-viral transfection methods optimized for gene delivery to a lung cancer cell line.

    Science.gov (United States)

    Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

    2013-04-01

    Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods.

  7. Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system

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    Peixin Zhu

    2009-12-01

    Full Text Available The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function.

  8. Interspecific and interploidal gene flow in Central European Arabidopsis (Brassicaceae

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    Jørgensen Marte H

    2011-11-01

    Full Text Available Abstract Background Effects of polyploidisation on gene flow between natural populations are little known. Central European diploid and tetraploid populations of Arabidopsis arenosa and A. lyrata are here used to study interspecific and interploidal gene flow, using a combination of nuclear and plastid markers. Results Ploidal levels were confirmed by flow cytometry. Network analyses clearly separated diploids according to species. Tetraploids and diploids were highly intermingled within species, and some tetraploids intermingled with the other species, as well. Isolation with migration analyses suggested interspecific introgression from tetraploid A. arenosa to tetraploid A. lyrata and vice versa, and some interploidal gene flow, which was unidirectional from diploid to tetraploid in A. arenosa and bidirectional in A. lyrata. Conclusions Interspecific genetic isolation at diploid level combined with introgression at tetraploid level indicates that polyploidy may buffer against negative consequences of interspecific hybridisation. The role of introgression in polyploid systems may, however, differ between plant species, and even within the small genus Arabidopsis, we find very different evolutionary fates when it comes to introgression.

  9. Reverse transcriptase directs viral evolution in a deep ocean methane seep

    Science.gov (United States)

    Paul, B. G.; Bagby, S. C.

    2013-12-01

    Deep ocean methane seeps are sites of intense microbial activity, with complex communities fueled by aerobic and anaerobic methanotrophy. Methane consumption in these communities has a substantial impact on the global carbon cycle, yet little is known about their evolutionary history or their likely evolutionary trajectories in a warming ocean. As in other marine systems, viral predation and virally mediated horizontal gene transfer are expected to be major drivers of evolutionary change in these communities; however, the host cells' resistance to cultivation has impeded direct study of the viral population. We conducted a metagenomic study of viruses in the anoxic sediments of a deep methane seep in the Santa Monica Basin in the Southern California Bight. We retrieved 1660 partial viral genomes, tentatively assigning 1232 to bacterial hosts and 428 to archaea. One abundant viral genome, likely hosted by Clostridia species present in the sediment, was found to encode a diversity-generating retroelement (DGR), a module for reverse transcriptase-mediated directed mutagenesis of a distal tail fiber protein. While DGRs have previously been described in the viruses of human pathogens, where diversification of viral tail fibers permits infection of a range of host cell types, to our knowledge this is the first description of such an element in a marine virus. By providing a mechanism for massively broadening potential host range, the presence of DGRs in these systems may have a major impact on the prevalence of virally mediated horizontal gene transfer, and even on the phylogenetic distances across which genes are moved.

  10. Impact of Bee Species and Plant Density on Alfalfa Pollination and Potential for Gene Flow

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    Johanne Brunet

    2010-01-01

    Full Text Available In outcrossing crops like alfalfa, various bee species can contribute to pollination and gene flow in seed production fields. With the increasing use of transgenic crops, it becomes important to determine the role of these distinct pollinators on alfalfa pollination and gene flow. The current study examines the relative contribution of honeybees, three bumble bee species, and three solitary bee species to pollination and gene flow in alfalfa. Two wild solitary bee species and one wild bumble bee species were best at tripping flowers, while the two managed pollinators commonly used in alfalfa seed production, honeybees and leaf cutting bees, had the lowest tripping rate. Honeybees had the greatest potential for gene flow and risk of transgene escape relative to the other pollinators. For honeybees, gene flow and risk of transgene escape were not affected by plant density although for the three bumble bee species gene flow and risk of transgene escape were the greatest in high-density fields.

  11. VIRmiRNA: a comprehensive resource for experimentally validated viral miRNAs and their targets.

    Science.gov (United States)

    Qureshi, Abid; Thakur, Nishant; Monga, Isha; Thakur, Anamika; Kumar, Manoj

    2014-01-01

    Viral microRNAs (miRNAs) regulate gene expression of viral and/or host genes to benefit the virus. Hence, miRNAs play a key role in host-virus interactions and pathogenesis of viral diseases. Lately, miRNAs have also shown potential as important targets for the development of novel antiviral therapeutics. Although several miRNA and their target repositories are available for human and other organisms in literature, but a dedicated resource on viral miRNAs and their targets are lacking. Therefore, we have developed a comprehensive viral miRNA resource harboring information of 9133 entries in three subdatabases. This includes 1308 experimentally validated miRNA sequences with their isomiRs encoded by 44 viruses in viral miRNA ' VIRMIRNA: ' and 7283 of their target genes in ' VIRMIRTAR': . Additionally, there is information of 542 antiviral miRNAs encoded by the host against 24 viruses in antiviral miRNA ' AVIRMIR': . The web interface was developed using Linux-Apache-MySQL-PHP (LAMP) software bundle. User-friendly browse, search, advanced search and useful analysis tools are also provided on the web interface. VIRmiRNA is the first specialized resource of experimentally proven virus-encoded miRNAs and their associated targets. This database would enhance the understanding of viral/host gene regulation and may also prove beneficial in the development of antiviral therapeutics. Database URL: http://crdd.osdd.net/servers/virmirna. © The Author(s) 2014. Published by Oxford University Press.

  12. Biodegradable nanoparticles for gene therapy technology

    International Nuclear Information System (INIS)

    Hosseinkhani, Hossein; He, Wen-Jie; Chiang, Chiao-Hsi; Hong, Po-Da; Yu, Dah-Shyong; Domb, Abraham J.; Ou, Keng-Liang

    2013-01-01

    Rapid propagations in materials technology together with biology have initiated great hopes in the possibility of treating many diseases by gene therapy technology. Viral and non-viral gene carriers are currently applied for gene delivery. Non-viral technology is safe and effective for the delivery of genetic materials to cells and tissues. Non-viral systems are based on plasmid expression containing a gene encoding a therapeutic protein and synthetic biodegradable nanoparticles as a safe carrier of gene. Biodegradable nanoparticles have shown great interest in drug and gene delivery systems as they are easy to be synthesized and have no side effect in cells and tissues. This review provides a critical view of applications of biodegradable nanoparticles on gene therapy technology to enhance the localization of in vitro and in vivo and improve the function of administered genes

  13. Tombusvirus-yeast interactions identify conserved cell-intrinsic viral restriction factors

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    Zsuzsanna eSasvari

    2014-08-01

    Full Text Available To combat viral infections, plants possess innate and adaptive immune pathways, such as RNA silencing, R gene and recessive gene-mediated resistance mechanisms. However, it is likely that additional cell-intrinsic restriction factors (CIRF are also involved in limiting plant virus replication. This review discusses novel CIRFs with antiviral functions, many of them RNA-binding proteins or affecting the RNA binding activities of viral replication proteins. The CIRFs against tombusviruses have been identified in yeast (Saccharomyces cerevisiae, which is developed as an advanced model organism. Grouping of the identified CIRFs based on their known cellular functions and subcellular localization in yeast reveals that TBSV replication is limited by a wide variety of host gene functions. Yeast proteins with the highest connectivity in the network map include the well-characterized Xrn1p 5’-3’ exoribonuclease, Act1p actin protein and Cse4p centromere protein. The protein network map also reveals an important interplay between the pro-viral Hsp70 cellular chaperone and the antiviral co-chaperones, and possibly key roles for the ribosomal or ribosome-associated factors. We discuss the antiviral functions of selected CIRFs, such as the RNA binding nucleolin, ribonucleases, WW-domain proteins, single- and multi-domain cyclophilins, TPR-domain co-chaperones and cellular ion pumps. These restriction factors frequently target the RNA-binding region in the viral replication proteins, thus interfering with the recruitment of the viral RNA for replication and the assembly of the membrane-bound viral replicase. Although many of the characterized CIRFs act directly against TBSV, we propose that the TPR-domain co-chaperones function as guardians of the cellular Hsp70 chaperone system, which is subverted efficiently by TBSV for viral replicase assembly in the absence of the TPR-domain co-chaperones.

  14. Divergence and gene flow in the globally distributed blue-winged ducks

    Science.gov (United States)

    Nelson, Joel; Wilson, Robert E.; McCracken, Kevin G.; Cumming, Graeme; Joseph, Leo; Guay, Patrick-Jean; Peters, Jeffrey

    2017-01-01

    The ability to disperse over long distances can result in a high propensity for colonizing new geographic regions, including uninhabited continents, and lead to lineage diversification via allopatric speciation. However, high vagility can also result in gene flow between otherwise allopatric populations, and in some cases, parapatric or divergence-with-gene-flow models might be more applicable to widely distributed lineages. Here, we use five nuclear introns and the mitochondrial control region along with Bayesian models of isolation with migration to examine divergence, gene flow, and phylogenetic relationships within a cosmopolitan lineage comprising six species, the blue-winged ducks (genus Anas), which inhabit all continents except Antarctica. We found two primary sub-lineages, the globally-distributed shoveler group and the New World blue-winged/cinnamon teal group. The blue-winged/cinnamon sub-lineage is composed of sister taxa from North America and South America, and taxa with parapatric distributions are characterized by low to moderate levels of gene flow. In contrast, our data support strict allopatry for most comparisons within the shovelers. However, we found evidence of gene flow from the migratory, Holarctic northern shoveler (A. clypeata) and the more sedentary, African Cape shoveler (A. smithii) into the Australasian shoveler (A. rhynchotis), although we could not reject strict allopatry. Given the diverse mechanisms of speciation within this complex, the shovelers and blue-winged/cinnamon teals can serve as an effective model system for examining how the genome diverges under different evolutionary processes and how genetic variation is partitioned among highly dispersive taxa.

  15. Viral evasion of intracellular DNA and RNA sensing

    Science.gov (United States)

    Chan, Ying Kai; Gack, Michaela U.

    2016-01-01

    The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP–AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals. PMID:27174148

  16. Substance P Receptor Antagonism: A Potential Novel Treatment Option for Viral-Myocarditis

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    Prema Robinson

    2015-01-01

    Full Text Available Viral-myocarditis is an important cause of heart failure for which no specific treatment is available. We previously showed the neuropeptide substance P (SP is associated with the pathogenesis of murine myocarditis caused by encephalomyocarditis virus (EMCV. The current studies determined if pharmacological inhibition of SP-signaling via its high affinity receptor, NK1R and downstream G-protein, Ras homolog gene family, member-A (RhoA, will be beneficial in viral-myocarditis. Aprepitant (1.2 mg/kg, a SP-receptor antagonist, or fasudil (10 mg/kg, a RhoA inhibitor, or saline control was administered daily to mice orally for 3 days, prior to, or 5 days following, intraperitoneal infection with and without 50 PFU of EMCV, following which disease assessment studies, including echocardiogram and cardiac Doppler were performed in day 14 after infection. Pretreatment and posttreatment with aprepitant significantly reduced mortality, heart and cardiomyocyte size, and cardiac viral RNA levels (P<0.05 all, ANOVA. Only aprepitant pretreatment improved heart functions; it significantly decreased end systolic diameter, improved fractional shortening, and increased peak aortic flow velocity (P<0.05 all, ANOVA. Pre- or posttreatment with fasudil did not significantly impact disease manifestations. These findings indicate that SP contributes to cardiac-remodeling and dysfunction following ECMV infection via its high affinity receptor, but not through the Rho-A pathway. These studies suggest that SP-receptor antagonism may be a novel therapeutic-option for patients with viral-myocarditis.

  17. Vaccines prepared from translation products of cloned viral genes

    International Nuclear Information System (INIS)

    Patzer, J.; Obijeski, J.F.

    1985-01-01

    With the advent of recombinant DNA (rDNA) techniques and their application to viruses for vaccine research, there has been an explosion of information about the molecular structure and replication of many viruses. rDNA technology in conjunction with several other emerging technologies, e.g. monoclonal antibodies, solid phase synthesis of peptides and prediction of protein conformation on the basis of amino acid sequence, has provided a powerful battery of techniques that in many cases has allowed the identification of specific sites on the virion surface that elicit neutralizing antibodies. Knowledge of these sites allows one to design a subunit vaccine that utilizes one of the virion proteins or regions of a particular protein in the absence of any other viral proteins or the viral nucleic acid. The advantages of this approach are: that there are no potentially infectious agents contained in the vaccine if the inactivation procedure is incomplete, there is less chance of complications from the vaccine due to nonessential viral components in the vaccine, a purified protein or polypeptide is usually more stable than virus particles during storage, and many times larger quanitities of an antigen can be produced by rDNA techniques than by classical vaccine methods

  18. The Pacific Ocean virome (POV: a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology.

    Directory of Open Access Journals (Sweden)

    Bonnie L Hurwitz

    Full Text Available Bacteria and their viruses (phage are fundamental drivers of many ecosystem processes including global biogeochemistry and horizontal gene transfer. While databases and resources for studying function in uncultured bacterial communities are relatively advanced, many fewer exist for their viral counterparts. The issue is largely technical in that the majority (often 90% of viral sequences are functionally 'unknown' making viruses a virtually untapped resource of functional and physiological information. Here, we provide a community resource that organizes this unknown sequence space into 27 K high confidence protein clusters using 32 viral metagenomes from four biogeographic regions in the Pacific Ocean that vary by season, depth, and proximity to land, and include some of the first deep pelagic ocean viral metagenomes. These protein clusters more than double currently available viral protein clusters, including those from environmental datasets. Further, a protein cluster guided analysis of functional diversity revealed that richness decreased (i from deep to surface waters, (ii from winter to summer, (iii and with distance from shore in surface waters only. These data provide a framework from which to draw on for future metadata-enabled functional inquiries of the vast viral unknown.

  19. The Pacific Ocean virome (POV): a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology.

    Science.gov (United States)

    Hurwitz, Bonnie L; Sullivan, Matthew B

    2013-01-01

    Bacteria and their viruses (phage) are fundamental drivers of many ecosystem processes including global biogeochemistry and horizontal gene transfer. While databases and resources for studying function in uncultured bacterial communities are relatively advanced, many fewer exist for their viral counterparts. The issue is largely technical in that the majority (often 90%) of viral sequences are functionally 'unknown' making viruses a virtually untapped resource of functional and physiological information. Here, we provide a community resource that organizes this unknown sequence space into 27 K high confidence protein clusters using 32 viral metagenomes from four biogeographic regions in the Pacific Ocean that vary by season, depth, and proximity to land, and include some of the first deep pelagic ocean viral metagenomes. These protein clusters more than double currently available viral protein clusters, including those from environmental datasets. Further, a protein cluster guided analysis of functional diversity revealed that richness decreased (i) from deep to surface waters, (ii) from winter to summer, (iii) and with distance from shore in surface waters only. These data provide a framework from which to draw on for future metadata-enabled functional inquiries of the vast viral unknown.

  20. Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut.

    Directory of Open Access Journals (Sweden)

    Vincent Raquin

    2017-12-01

    Full Text Available Dengue virus (DENV causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP, which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

  1. Association of coral algal symbionts with a diverse viral community responsive to heat shock

    KAUST Repository

    Brüwer, Jan D.

    2017-08-17

    Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin.Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host\\'s antiviral response.Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

  2. Novel chaperonins are prevalent in the virioplankton and demonstrate links to viral biology and ecology.

    Science.gov (United States)

    Marine, Rachel L; Nasko, Daniel J; Wray, Jeffrey; Polson, Shawn W; Wommack, K Eric

    2017-11-01

    Chaperonins are protein-folding machinery found in all cellular life. Chaperonin genes have been documented within a few viruses, yet, surprisingly, analysis of metagenome sequence data indicated that chaperonin-carrying viruses are common and geographically widespread in marine ecosystems. Also unexpected was the discovery of viral chaperonin sequences related to thermosome proteins of archaea, indicating the presence of virioplankton populations infecting marine archaeal hosts. Virioplankton large subunit chaperonin sequences (GroELs) were divergent from bacterial sequences, indicating that viruses have carried this gene over long evolutionary time. Analysis of viral metagenome contigs indicated that: the order of large and small subunit genes was linked to the phylogeny of GroEL; both lytic and temperate phages may carry group I chaperonin genes; and viruses carrying a GroEL gene likely have large double-stranded DNA (dsDNA) genomes (>70 kb). Given these connections, it is likely that chaperonins are critical to the biology and ecology of virioplankton populations that carry these genes. Moreover, these discoveries raise the intriguing possibility that viral chaperonins may more broadly alter the structure and function of viral and cellular proteins in infected host cells.

  3. Gene flow among wild and domesticated almond species: insights from chloroplast and nuclear markers

    Science.gov (United States)

    Delplancke, Malou; Alvarez, Nadir; Espíndola, Anahí; Joly, Hélène; Benoit, Laure; Brouck, Elise; Arrigo, Nils

    2012-01-01

    Hybridization has played a central role in the evolutionary history of domesticated plants. Notably, several breeding programs relying on gene introgression from the wild compartment have been performed in fruit tree species within the genus Prunus but few studies investigated spontaneous gene flow among wild and domesticated Prunus species. Consequently, a comprehensive understanding of genetic relationships and levels of gene flow between domesticated and wild Prunus species is needed. Combining nuclear and chloroplastic microsatellites, we investigated the gene flow and hybridization among two key almond tree species, the cultivated Prunus dulcis and one of the most widespread wild relative Prunus orientalis in the Fertile Crescent. We detected high genetic diversity levels in both species along with substantial and symmetric gene flow between the domesticated P. dulcis and the wild P. orientalis. These results were discussed in light of the cultivated species diversity, by outlining the frequent spontaneous genetic contributions of wild species to the domesticated compartment. In addition, crop-to-wild gene flow suggests that ad hoc transgene containment strategies would be required if genetically modified cultivars were introduced in the northwestern Mediterranean. PMID:25568053

  4. Recombinant viruses as vaccines against viral diseases

    Directory of Open Access Journals (Sweden)

    A.P.D. Souza

    2005-04-01

    Full Text Available Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

  5. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity

    Science.gov (United States)

    Schoggins, John W.; MacDuff, Donna A.; Imanaka, Naoko; Gainey, Maria D.; Shrestha, Bimmi; Eitson, Jennifer L.; Mar, Katrina B.; Richardson, R. Blake; Ratushny, Alexander V.; Litvak, Vladimir; Dabelic, Rea; Manicassamy, Balaji; Aitchison, John D.; Aderem, Alan; Elliott, Richard M.; García-Sastre, Adolfo; Racaniello, Vincent; Snijder, Eric J.; Yokoyama, Wayne M.; Diamond, Michael S.; Virgin, Herbert W.; Rice, Charles M.

    2014-01-01

    The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

  6. Ancestry dynamics in a South American population: The impact of gene flow and preferential mating.

    Science.gov (United States)

    Hedrick, Philip W

    2017-07-01

    European ancestry in many populations in Latin America at autosomal loci is often higher than that from X-linked loci indicating more European male ancestry and more Amerindian female ancestry. Generally, this has been attributed to more European male gene flow but could also result from an advantage to European mating or reproductive success. Population genetic models were developed to investigate the dynamics of gene flow and mating or reproductive success. Using estimates of autosomal and X-chromosome European ancestry, the amount of male gene flow or mating or reproductive advantage for Europeans, or those with European ancestry, was estimated. In a population from Antioquia, Colombia with an estimated 79% European autosomal ancestry and an estimated 69% European X-chromosome ancestry, about 15% male gene flow from Europe or about 20% mating or reproductive advantage of Europeans over Amerindians resulted in these levels of European ancestry in the contemporary population. Combinations of gene flow and mating advantage were nearly additive in their impact. Gene flow, mating advantage, or a combination of both factors, are consistent with observed levels of European ancestry in a Latin American population. This approach provides a general methodology to determine the levels of gene flow and mating differences that can explain the observed contemporary differences in ancestry from autosomes and X-chromosomes. © 2017 Wiley Periodicals, Inc.

  7. Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation.

    Science.gov (United States)

    Crow, Marni S; Cristea, Ileana M

    2017-04-01

    The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral

  8. Species-specific deletion of the viral attachment glycoprotein of avian metapneumovirus.

    Science.gov (United States)

    Kong, Byung-Whi; Foster, Linda K; Foster, Douglas N

    2008-03-01

    The avian metapneumovirus (AMPV) genome encodes the fusion (F), small hydrophobic (SH), and attachment glycoprotein (G) as envelope glycoproteins. The F and G proteins mainly function to allow viral entry into host cells during the early steps of the virus life cycle. The highly variable AMPV G protein is a major determinant for distinguishing virus subtypes. Sequence analysis was used to determine if any differences between avian or mammalian cell propagated subtype C AMPV could be detected for the 1.8kb G gene. As a result, the complete 1.8kb G gene was found to be present when AMPV was propagated in our immortal turkey turbinate (TT-1) cell line regardless of passage number. Surprisingly, AMPV propagated for 15 or more passages in mammalian Vero cells revealed an essentially deleted G gene in the viral genome, resulting in no G gene mRNA expression. Although the Vero cell propagated AMPV genome contained a small 122 nucleotide fragment of the G gene, no other mRNA variants were detected from either mammalian or avian propagated AMPV. The G gene truncation might be caused by cellular molecular mechanisms that are species-specific. The lack of viral gene deletions suggests that avian cell propagated AMPV will provide a better alternative host for live recombinant vaccine development based on a reverse genetics system.

  9. Gene flow in genetically modified wheat.

    Directory of Open Access Journals (Sweden)

    Silvan Rieben

    Full Text Available Understanding gene flow in genetically modified (GM crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting "phytometers" of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5-2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7-0.03% over the test distances of 0.5-2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer

  10. Gene flow in genetically modified wheat.

    Science.gov (United States)

    Rieben, Silvan; Kalinina, Olena; Schmid, Bernhard; Zeller, Simon L

    2011-01-01

    Understanding gene flow in genetically modified (GM) crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting "phytometers" of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5-2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7-0.03% over the test distances of 0.5-2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer approach and that gene-flow

  11. Coordinate viral induction of tumor necrosis factor α and interferon β in human B cells and monocytes

    International Nuclear Information System (INIS)

    Goldfeld, A.E.; Maniatis, T.

    1989-01-01

    Human tumor necrosis factor α (TNF-α) gene expression can be induced primarily in cells of the monocyte/macrophage lineage by a variety of inducers, including lipopolysaccharide, phorbol esters such as phorbol 12-myristate 13-acetate, and virus or synthetic double-stranded RNA [poly(I)·poly(C)]. In this paper the authors show that the TNF-α gene also responds to virus and phorbol 12-myristate 13-acetate in B lymphocytes and that virus is the most potent inducer of TNF-α mRNA in both monocyte and B-cell lines. In addition, they show that viral infection coinduces the expression of TNF-α and interferon β mRNA and that viral induction of both genes is blocked by the kinase inhibitor 2-aminopurine. Inhibition of protein synthesis with cycloheximide had no effect on mRNA expression of the genes in one of three cell lines tested (U937) but blocked the viral induction of both genes in another (Namalwa). Thus, the regulatory factors required for mRNA induction of both genes are present prior to the addition of virus in U937 but not in Namalwa cells. However, in a third cell line (JY), cycloheximide blocked viral induction of the interferon β gene but not the TNF-α gene. Taken together, these observations suggest that viral induction of TNF-α and interferon β gene expression may involve overlapping pathways with both common and distinct regulatory factors

  12. STUDY OF GENE FLOW FROM GM COTTON (Gossypium hirsutum VARIETIES IN “EL ESPINAL” (TOLIMA, COLOMBIA.

    Directory of Open Access Journals (Sweden)

    Alejandro Chaparro Giraldo

    2013-09-01

    Full Text Available In 2009, 4088 hectares of genetically modified (GM cotton were planted in Tolima (Colombia, however there is some uncertainty about containment measures needed to prevent the flow of pollen and seed from regulated GM fields into adjacent fields. In this study, the gene flow from GM cotton varieties to conventional or feral cotton plants via seed and pollen was evaluated. ImmunostripTM, PCR and ELISA assays were used to detect gene flow. Fifty six refuges, 27 fields with conventional cotton and four feral individuals of the enterprise “Remolinos Inc.” located in El Espinal (Tolima were analyzed in the first half of 2010. The results indicated seeds mediated gene flow in 45 refuges (80,4 % and 26 fields with conventional cotton (96 %, besides a pollen mediated gene flow in one field with conventional cotton and nine refuges. All fields cultivated with conventional cotton showed gene flow from GM cotton. Two refuges and two feral individuals did not reveal gene flow from GM cotton.

  13. Viral infection of the marine alga Emiliania huxleyi triggers lipidome remodeling and induces the production of highly saturated triacylglycerol.

    Science.gov (United States)

    Malitsky, Sergey; Ziv, Carmit; Rosenwasser, Shilo; Zheng, Shuning; Schatz, Daniella; Porat, Ziv; Ben-Dor, Shifra; Aharoni, Asaph; Vardi, Assaf

    2016-04-01

    Viruses that infect marine photosynthetic microorganisms are major ecological and evolutionary drivers of microbial food webs, estimated to turn over more than a quarter of the total photosynthetically fixed carbon. Viral infection of the bloom-forming microalga Emiliania huxleyi induces the rapid remodeling of host primary metabolism, targeted towards fatty acid metabolism. We applied a liquid chromatography-mass spectrometry (LC-MS)-based lipidomics approach combined with imaging flow cytometry and gene expression profiling to explore the impact of viral-induced metabolic reprogramming on lipid composition. Lytic viral infection led to remodeling of the cellular lipidome, by predominantly inducing the biosynthesis of highly saturated triacylglycerols (TAGs), coupled with a significant accumulation of neutral lipids within lipid droplets. Furthermore, TAGs were found to be a major component (77%) of the lipidome of isolated virions. Interestingly, viral-induced TAGs were significantly more saturated than TAGs produced under nitrogen starvation. This study highlights TAGs as major products of the viral-induced metabolic reprogramming during the host-virus interaction and indicates a selective mode of membrane recruitment during viral assembly, possibly by budding of the virus from specialized subcellular compartments. These findings provide novel insights into the role of viruses infecting microalgae in regulating metabolism and energy transfer in the marine environment and suggest their possible biotechnological application in biofuel production. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  14. Gene flow analysis method, the D-statistic, is robust in a wide parameter space.

    Science.gov (United States)

    Zheng, Yichen; Janke, Axel

    2018-01-08

    We evaluated the sensitivity of the D-statistic, a parsimony-like method widely used to detect gene flow between closely related species. This method has been applied to a variety of taxa with a wide range of divergence times. However, its parameter space and thus its applicability to a wide taxonomic range has not been systematically studied. Divergence time, population size, time of gene flow, distance of outgroup and number of loci were examined in a sensitivity analysis. The sensitivity study shows that the primary determinant of the D-statistic is the relative population size, i.e. the population size scaled by the number of generations since divergence. This is consistent with the fact that the main confounding factor in gene flow detection is incomplete lineage sorting by diluting the signal. The sensitivity of the D-statistic is also affected by the direction of gene flow, size and number of loci. In addition, we examined the ability of the f-statistics, [Formula: see text] and [Formula: see text], to estimate the fraction of a genome affected by gene flow; while these statistics are difficult to implement to practical questions in biology due to lack of knowledge of when the gene flow happened, they can be used to compare datasets with identical or similar demographic background. The D-statistic, as a method to detect gene flow, is robust against a wide range of genetic distances (divergence times) but it is sensitive to population size. The D-statistic should only be applied with critical reservation to taxa where population sizes are large relative to branch lengths in generations.

  15. Impact of viral E2-gene status on outcome after radiotherapy for patients with human papillomavirus 16-positive cancer of the uterine cervix

    International Nuclear Information System (INIS)

    Lindel, Katja; Villiers, Ethel-Michele de; Burri, Philipp; Studer, Ueli; Altermatt, Hans J.; Greiner, Richard H.; Gruber, Guenther

    2006-01-01

    Purpose: Integration of high-risk papillomavirus DNA has been considered an important step in oncogenic progression to cervical carcinoma. Disruption of the human papillomavirus (HPV) genome within the E2 gene is frequently a consequence. This study investigated the influence of episomal viral DNA on outcome in patients with advanced cervical cancer treated with primary radiotherapy. Methods and Materials: Paraffin-embedded biopsies of 82 women with locally advanced cervical cancer could be analyzed for HPV infection by multiplex polymerase chain reaction (PCR) by use of SPF1/2 primers. E2-gene intactness of HPV-16-positive samples was analyzed in 3 separate amplification reactions by use of the E2A, E2B, E2C primers. Statistical analyses (Kaplan-Meier method; log-rank test) were performed for overall survival (OS), disease-free survival (DFS), local progression-free survival (LPFS), and distant metastases-free survival (DMFS). Results: Sixty-one (75%) of 82 carcinomas were HPV positive, 44 of them for HPV-16 (72%). Seventeen of the 44 HPV-16-positive tumors (39%) had an intact E2 gene. Patients with a HPV-16-positive tumor and an intact E2 gene showed a trend for a better DFS (58% vs. 38%, p = 0.06) compared with those with a disrupted E2 gene. A nonsignificant difference occurred regarding OS (87% vs. 66%, p = 0.16) and DMFS (57% vs. 48%, p = 0.15). Conclusion: E2-gene status may be a promising new target, but more studies are required to elucidate the effect of the viral E2 gene on outcome after radiotherapy in HPV-positive tumors

  16. Pleistocene land bridges act as semipermeable agents of avian gene flow in Wallacea.

    Science.gov (United States)

    Garg, Kritika M; Chattopadhyay, Balaji; Wilton, Peter R; Malia Prawiradilaga, Dewi; Rheindt, Frank E

    2018-08-01

    Cyclical periods of global cooling have been important drivers of biotic differentiation throughout the Quaternary. Ice age-induced sea level fluctuations can lead to changing patterns of land connections, both facilitating and disrupting gene flow. In this study, we test if species with differing life histories are differentially affected by Quaternary land connections. We used genome-wide SNPs in combination with mitochondrial gene sequences to analyse levels of divergence and gene flow between two songbird complexes across two Wallacean islands that have been repeatedly connected during glaciations. Although the two bird complexes are similar in ecological attributes, the forest and edge-inhabiting golden whistler Pachycephala pectoralis is comparatively flexible in its diet and niche requirements as compared to the henna-tailed jungle-flycatcher Cyornis colonus, which is largely restricted to the forest interior. Using population-genomic and coalescent approaches, we estimated levels of gene flow, population differentiation and divergence time between the two island populations. We observed higher levels of differentiation, an approximately two to four times deeper divergence time and near-zero levels of gene flow between the two island populations of the more forest-dependent henna-tailed jungle-flycatcher as compared to the more generalist golden whistler. Our results suggest that Quaternary land bridges act as semipermeable agents of gene flow in Wallacea, allowing only certain taxa to connect between islands while others remain isolated. Quaternary land bridges do not accommodate all terrestrial species equally, differing in suitability according to life history and species biology. More generalist species are likely to use Quaternary land connections as a conduit for gene flow between islands whereas island populations of more specialist species may continue to be reproductively isolated even during periods of Quaternary land bridges. Copyright © 2018

  17. A Test for Gene Flow among Sympatric and Allopatric Hawaiian Picture-Winged Drosophila.

    Science.gov (United States)

    Kang, Lin; Garner, Harold R; Price, Donald K; Michalak, Pawel

    2017-06-01

    The Hawaiian Drosophila are one of the most species-rich endemic groups in Hawaii and a spectacular example of adaptive radiation. Drosophila silvestris and D. heteroneura are two closely related picture-winged Drosophila species that occur sympatrically on Hawaii Island and are known to hybridize in nature, yet exhibit highly divergent behavioral and morphological traits driven largely through sexual selection. Their closest-related allopatric species, D. planitibia from Maui, exhibits hybrid male sterility and reduced behavioral reproductive isolation when crossed experimentally with D. silvestris or D. heteroneura. A modified four-taxon test for gene flow was applied to recently obtained genomes of the three Hawaiian Drosophila species. The analysis indicates recent gene flow in sympatry, but also, although less extensive, between allopatric species. This study underscores the prevalence of gene flow, even in taxonomic groups considered classic examples of allopatric speciation on islands. The potential confounding effects of gene flow in phylogenetic and population genetics inference are discussed, as well as the implications for conservation.

  18. Cryptic species? Patterns of maternal and paternal gene flow in eight neotropical bats.

    Directory of Open Access Journals (Sweden)

    Elizabeth L Clare

    Full Text Available Levels of sequence divergence at mitochondrial loci are frequently used in phylogeographic analysis and species delimitation though single marker systems cannot assess bi-parental gene flow. In this investigation I compare the phylogeographic patterns revealed through the maternally inherited mitochondrial COI region and the paternally inherited 7(th intron region of the Dby gene on the Y-chromosome in eight common Neotropical bat species. These species are diverse and include members of two families from the feeding guilds of sanguivores, nectarivores, frugivores, carnivores and insectivores. In each case, the currently recognized taxon is comprised of distinct, substantially divergent intraspecific mitochondrial lineages suggesting cryptic species complexes. In Chrotopterus auritus, and Saccopteryx bilineata I observed congruent patterns of divergence in both genetic regions suggesting a cessation of gene flow between intraspecific groups. This evidence supports the existence of cryptic species complexes which meet the criteria of the genetic species concept. In Glossophaga soricina two intraspecific groups with largely sympatric South American ranges show evidence for incomplete lineage sorting or frequent hybridization while a third group with a Central American distribution appears to diverge congruently at both loci suggesting speciation. Within Desmodus rotundus and Trachops cirrhosus the paternally inherited region was monomorphic and thus does not support or refute the potential for cryptic speciation. In Uroderma bilobatum, Micronycteris megalotis and Platyrrhinus helleri the gene regions show conflicting patterns of divergence and I cannot exclude ongoing gene flow between intraspecific groups. This analysis provides a comprehensive comparison across taxa and employs both maternally and paternally inherited gene regions to validate patterns of gene flow. I present evidence for previously unrecognized species meeting the criteria of

  19. Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

    Directory of Open Access Journals (Sweden)

    Yang Lin

    2013-02-01

    Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

  20. Analysis of viral protein-2 encoding gene of avian encephalomyelitis virus from field specimens in Central Java region, Indonesia

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2016-01-01

    Full Text Available Aim: Avian encephalomyelitis (AE is a viral disease which can infect various types of poultry, especially chicken. In Indonesia, the incidence of AE infection in chicken has been reported since 2009, the AE incidence tends to increase from year to year. The objective of this study was to analyze viral protein 2 (VP-2 encoding gene of AE virus (AEV from various species of birds in field specimen by reverse transcription polymerase chain reaction (RT-PCR amplification using specific nucleotides primer for confirmation of AE diagnosis. Materials and Methods: A total of 13 AEV samples are isolated from various species of poultry which are serologically diagnosed infected by AEV from some areas in central Java, Indonesia. Research stage consists of virus samples collection from field specimens, extraction of AEV RNA, amplification of VP-2 protein encoding gene by RT-PCR, separation of RT-PCR product by agarose gel electrophoresis, DNA sequencing and data analysis. Results: Amplification products of the VP-2 encoding gene of AEV by RT-PCR methods of various types of poultry from field specimens showed a positive results on sample code 499/4/12 which generated DNA fragment in the size of 619 bp. Sensitivity test of RT-PCR amplification showed that the minimum concentration of RNA template is 127.75 ng/μl. The multiple alignments of DNA sequencing product indicated that positive sample with code 499/4/12 has 92% nucleotide homology compared with AEV with accession number AV1775/07 and 85% nucleotide homology with accession number ZCHP2/0912695 from Genbank database. Analysis of VP-2 gene sequence showed that it found 46 nucleotides difference between isolate 499/4/12 compared with accession number AV1775/07 and 93 nucleotides different with accession number ZCHP2/0912695. Conclusions: Analyses of the VP-2 encoding gene of AEV with RT-PCR method from 13 samples from field specimen generated the DNA fragment in the size of 619 bp from one sample with

  1. Analysis of metagenomic data reveals common features of halophilic viral communities across continents.

    Science.gov (United States)

    Roux, Simon; Enault, Francois; Ravet, Viviane; Colombet, Jonathan; Bettarel, Yvan; Auguet, Jean-Christophe; Bouvier, Thierry; Lucas-Staat, Soizick; Vellet, Agnès; Prangishvili, David; Forterre, Patrick; Debroas, Didier; Sime-Ngando, Telesphore

    2016-03-01

    Microbial communities from hypersaline ponds, dominated by halophilic archaea, are considered specific of such extreme conditions. The associated viral communities have accordingly been shown to display specific features, such as similar morphologies among different sites. However, little is known about the genetic diversity of these halophilic viral communities across the Earth. Here, we studied viral communities in hypersaline ponds sampled on the coast of Senegal (8-36% of salinity) using metagenomics approach, and compared them with hypersaline viromes from Australia and Spain. The specificity of hyperhalophilic viruses could first be demonstrated at a community scale, salinity being a strong discriminating factor between communities. For the major viral group detected in all samples (Caudovirales), only a limited number of halophilic Caudovirales clades were highlighted. These clades gather viruses from different continents and display consistent genetic composition, indicating that they represent related lineages with a worldwide distribution. Non-tailed hyperhalophilic viruses display a greater rate of gene transfer and recombination, with uncharacterized genes conserved across different kind of viruses and plasmids. Thus, hypersaline viral communities around the world appear to form a genetically consistent community that are likely to harbour new genes coding for enzymes specifically adapted to these environments. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Molecular imaging of oncolytic viral therapy

    Directory of Open Access Journals (Sweden)

    Dana Haddad

    2014-01-01

    Full Text Available Oncolytic viruses have made their mark on the cancer world as a potential therapeutic option, with the possible advantages of reduced side effects and strengthened treatment efficacy due to higher tumor selectivity. Results have been so promising, that oncolytic viral treatments have now been approved for clinical trials in several countries. However, clinical studies may benefit from the ability to noninvasively and serially identify sites of viral targeting via molecular imaging in order to provide safety, efficacy, and toxicity information. Furthermore, molecular imaging of oncolytic viral therapy may provide a more sensitive and specific diagnostic technique to detect tumor origin and, more importantly, presence of metastases. Several strategies have been investigated for molecular imaging of viral replication broadly categorized into optical and deep tissue imaging, utilizing several reporter genes encoding for fluorescence proteins, conditional enzymes, and membrane protein and transporters. Various imaging methods facilitate molecular imaging, including computer tomography, magnetic resonance imaging, positron emission tomography, single photon emission CT, gamma-scintigraphy, and photoacoustic imaging. In addition, several molecular probes are used for medical imaging, which act as targeting moieties or signaling agents. This review will explore the preclinical and clinical use of in vivo molecular imaging of replication-competent oncolytic viral therapy.

  3. Introgression in the Drosophila subobscura--D. Madeirensis sister species: evidence of gene flow in nuclear genes despite mitochondrial differentiation.

    Science.gov (United States)

    Herrig, Danielle K; Modrick, Alec J; Brud, Evgeny; Llopart, Ana

    2014-03-01

    Species hybridization, and thus the potential for gene flow, was once viewed as reproductive mistake. However, recent analysis based on large datasets and newly developed models suggest that gene exchange is not as rare as originally suspected. To investigate the history and speciation of the closely related species Drosophila subobscura, D. madeirensis, and D. guanche, we obtained polymorphism and divergence data for 26 regions throughout the genome, including the Y chromosome and mitochondrial DNA. We found that the D. subobscura X/autosome ratio of silent nucleotide diversity is significantly smaller than the 0.75 expected under neutrality. This pattern, if held genomewide, may reflect a faster accumulation of beneficial mutations on the X chromosome than on autosomes. We also detected evidence of gene flow in autosomal regions, while sex chromosomes remain distinct. This is consistent with the large X effect on hybrid male sterility seen in this system and the presence of two X chromosome inversions fixed between species. Overall, our data conform to chromosomal speciation models in which rearrangements are proposed to serve as gene flow barriers. Contrary to other observations in Drosophila, the mitochondrial genome appears resilient to gene flow in the presence of nuclear exchange. © 2013 The Authors. Evolution published by Wiley Periodicals, Inc. on behalf of The Society for the Study of Evolution.

  4. Oxygen tension level and human viral infections

    Energy Technology Data Exchange (ETDEWEB)

    Morinet, Frédéric, E-mail: frederic.morinet@sls.aphp.fr [Centre des Innovations Thérapeutiques en Oncologie et Hématologie (CITOH), CHU Saint-Louis, Paris (France); Université Denis Diderot, Sorbonne Paris Cité Paris, Paris (France); Casetti, Luana [Institut Cochin INSERM U1016, Paris (France); François, Jean-Hugues; Capron, Claude [Institut Cochin INSERM U1016, Paris (France); Laboratoire d' Hématologie, Hôpital Ambroise Paré, Boulogne (France); Université de Versailles Saint-Quentin en Yvelynes, Versailles (France); Pillet, Sylvie [Laboratoire de Bactériologie-Virologie-Hygiène, CHU de Saint-Etienne, Saint-Etienne (France); Université de Lyon et Université de Saint-Etienne, Jean Monnet, GIMAP EA3064, F-42023 Saint-Etienne, Lyon (France)

    2013-09-15

    The role of oxygen tension level is a well-known phenomenon that has been studied in oncology and radiotherapy since about 60 years. Oxygen tension may inhibit or stimulate propagation of viruses in vitro as well as in vivo. In turn modulating oxygen metabolism may constitute a novel approach to treat viral infections as an adjuvant therapy. The major transcription factor which regulates oxygen tension level is hypoxia-inducible factor-1 alpha (HIF-1α). Down-regulating the expression of HIF-1α is a possible method in the treatment of chronic viral infection such as human immunodeficiency virus infection, chronic hepatitis B and C viral infections and Kaposi sarcoma in addition to classic chemotherapy. The aim of this review is to supply an updating concerning the influence of oxygen tension level in human viral infections and to evoke possible new therapeutic strategies regarding this environmental condition. - Highlights: • Oxygen tension level regulates viral replication in vitro and possibly in vivo. • Hypoxia-inducible factor 1 (HIF-1α) is the principal factor involved in Oxygen tension level. • HIF-1α upregulates gene expression for example of HIV, JC and Kaposi sarcoma viruses. • In addition to classical chemotherapy inhibition of HIF-1α may constitute a new track to treat human viral infections.

  5. Pollen-mediated gene flow in flax (Linum usitatissimum L.): can genetically engineered and organic flax coexist?

    Science.gov (United States)

    Jhala, A J; Bhatt, H; Topinka, K; Hall, L M

    2011-04-01

    Coexistence allows growers and consumers the choice of producing or purchasing conventional or organic crops with known standards for adventitious presence of genetically engineered (GE) seed. Flax (Linum usitatissimum L.) is multipurpose oilseed crop in which product diversity and utility could be enhanced for industrial, nutraceutical and pharmaceutical markets through genetic engineering. If GE flax were released commercially, pollen-mediated gene flow will determine in part whether GE flax could coexist without compromising other markets. As a part of pre-commercialization risk assessment, we quantified pollen-mediated gene flow between two cultivars of flax. Field experiments were conducted at four locations during 2006 and 2007 in western Canada using a concentric donor (20 × 20 m) receptor (120 × 120 m) design. Gene flow was detected through the xenia effect of dominant alleles of high α-linolenic acid (ALA; 18:3(cisΔ9,12,15)) to the low ALA trait. Seeds were harvested from the pollen recipient plots up to a distance of 50 m in eight directions from the pollen donor. High ALA seeds were identified using a thiobarbituric acid test and served as a marker for gene flow. Binomial distribution and power analysis were used to predict the minimum number of seeds statistically required to detect the frequency of gene flow at specific α (confidence interval) and power (1-β) values. As a result of the low frequency of gene flow, approximately 4 million seeds were screened to derive accurate quantification. Frequency of gene flow was highest near the source: averaging 0.0185 at 0.1 m but declined rapidly with distance, 0.0013 and 0.00003 at 3 and 35 m, respectively. Gene flow was reduced to 50% (O₅₀) and 90% (O₉₀) between 0.85 to 2.64 m, and 5.68 to 17.56 m, respectively. No gene flow was detected at any site or year > 35 m distance from the pollen source, suggesting that frequency of gene flow was ≤ 0.00003 (P = 0.95). Although it is not possible to

  6. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

    DEFF Research Database (Denmark)

    Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

    2011-01-01

    The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

  7. Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

    Science.gov (United States)

    Raisin, Sophie; Belamie, Emmanuel; Morille, Marie

    2016-10-01

    Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

    Science.gov (United States)

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-09-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. © 2014 Wiley Periodicals, Inc.

  9. Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stiehler, Maik; Duch, Mogens R.; Mygind, Tina

    2006-01-01

    -old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno...

  10. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

    Directory of Open Access Journals (Sweden)

    Boyce Mark

    2012-08-01

    Full Text Available Abstract Background Bluetongue virus (BTV is a double-stranded RNA (dsRNA virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Results Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1 as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3′ poly(A sequence identifying the 3′ end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. Conclusions NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed

  11. Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis.

    Science.gov (United States)

    Boyce, Mark; Celma, Cristina C P; Roy, Polly

    2012-08-29

    Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3' poly(A) sequence identifying the 3' end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein

  12. Intercontinental gene flow among western arctic populations of Lesser Snow Geese

    Science.gov (United States)

    Shorey, Rainy I.; Scribner, Kim T.; Kanefsky, Jeannette; Samuel, Michael D.; Libants, Scot V.

    2011-01-01

    Quantifying the spatial genetic structure of highly vagile species of birds is important in predicting their degree of population demographic and genetic independence during changing environmental conditions, and in assessing their abundance and distribution. In the western Arctic, Lesser Snow Geese (Chen caerulescens caerulescens) provide an example useful for evaluating spatial population genetic structure and the relative contribution of male and female philopatry to breeding and wintering locales. We analyzed biparentally inherited microsatellite loci and maternally inherited mtDNA sequences from geese breeding at Wrangel Island (Russia) and Banks Island (Canada) to estimate gene flow among populations whose geographic overlap during breeding and winter differ. Significant differences in the frequencies of mtDNA haplotypes contrast with the homogeneity of allele frequencies for microsatellite loci. Coalescence simulations revealed high variability and asymmetry between males and females in rates and direction of gene flow between populations. Our results highlight the importance of wintering areas to demographic independence and spatial genetic structure of these populations. Male-mediated gene flow among the populations on northern Wrangel Island, southern Wrangel Island, and Banks Island has been substantial. A high rate of female-mediated gene flow from southern Wrangel Island to Banks Island suggests that population exchange can be achieved when populations winter in a common area. Conversely, when birds from different breeding populations do not share a common wintering area, the probability of population exchange is likely to be dramatically reduced.

  13. Massive activation of archaeal defense genes during viral infection

    NARCIS (Netherlands)

    Quax, T.E.F.; Voet, M.; Sismeiro, O.; Dillies, M.A.; Jagla, B.; Coppée, J.Y.; Sezonov, G.; Forterre, P.; Oost, van der J.; Lavigne, R.; Prangishvili, D.

    2013-01-01

    Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in

  14. TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery.

    Science.gov (United States)

    Wang, Bing; Yi, Wen-Jing; Zhang, Ji; Zhang, Qin-Fang; Xun, Miao-Miao; Yu, Xiao-Qi

    2014-04-01

    Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Gene flow from transgenic common beans expressing the bar gene.

    Science.gov (United States)

    Faria, Josias C; Carneiro, Geraldo E S; Aragão, Francisco J L

    2010-01-01

    Gene flow is a common phenomenon even in self-pollinated plant species. With the advent of genetically modified plants this subject has become of the utmost importance due to the need for controlling the spread of transgenes. This study was conducted to determine the occurrence and intensity of outcrossing in transgenic common beans. In order to evaluate the outcross rates, four experiments were conducted in Santo Antonio de Goiás (GO, Brazil) and one in Londrina (PR, Brazil), using transgenic cultivars resistant to the herbicide glufosinate ammonium and their conventional counterparts as recipients of the transgene. Experiments with cv. Olathe Pinto and the transgenic line Olathe M1/4 were conducted in a completely randomized design with ten replications for three years in one location, whereas the experiments with cv. Pérola and the transgenic line Pérola M1/4 were conducted at two locations for one year, with the transgenic cultivar surrounded on all sides by the conventional counterpart. The outcross occurred at a negligible rate of 0.00741% in cv. Pérola, while none was observed (0.0%) in cv. Olathe Pinto. The frequency of gene flow was cultivar dependent and most of the observed outcross was within 2.5 m from the edge of the pollen source. Index terms: Phaseolus vulgaris, outcross, glufosinate ammonium.

  16. Delivery of viral vectors to tumor cells: extracellular transport, systemic distribution, and strategies for improvement.

    Science.gov (United States)

    Wang, Yong; Yuan, Fan

    2006-01-01

    It is a challenge to deliver therapeutic genes to tumor cells using viral vectors because (i) the size of these vectors are close to or larger than the space between fibers in extracellular matrix and (ii) viral proteins are potentially toxic in normal tissues. In general, gene delivery is hindered by various physiological barriers to virus transport from the site of injection to the nucleus of tumor cells and is limited by normal tissue tolerance of toxicity determined by local concentrations of transgene products and viral proteins. To illustrate the obstacles encountered in the delivery and yet limit the scope of discussion, this review focuses only on extracellular transport in solid tumors and distribution of viral vectors in normal organs after they are injected intravenously or intratumorally. This review also discusses current strategies for improving intratumoral transport and specificity of viral vectors.

  17. An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene.

    Science.gov (United States)

    Cui, Hongguang; Wang, Aiming

    2017-03-01

    RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  18. Multi-resistance strategy for viral diseases and short hairpin RNA verification method in pigs

    Directory of Open Access Journals (Sweden)

    Jong-nam Oh

    2018-04-01

    Full Text Available Objective Foot and mouth disease (FMD and porcine reproductive and respiratory syndrome (PRRS are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV and PRRS virus (PRRSV, the present study introduced two genetic modification techniques to porcine cells. Methods First, cluster of differentiation 163 (CD163, the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7 gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

  19. High rates of gene flow by pollen and seed in oak populations across Europe

    NARCIS (Netherlands)

    Gerber, S.; Chadoeuf, J.; Gugerli, F.; Lascoux, M.; Buiteveld, J.; Cottrell, J.; Dounavi, A.; Fineschi, S.; Forrest, L.; Fogelqvist, J.; Goicoechea, P.G.; Jensen, J.S.; Salvini, D.; Vendramin, G.G.; Kremer, A.

    2014-01-01

    Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult

  20. Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

    Science.gov (United States)

    Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

    2013-10-17

    Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

  1. Adaptive divergence with gene flow in incipient speciation of Miscanthus floridulus / sinensis complex (Poaceae)

    KAUST Repository

    Huang, Chao-Li; Ho, Chuan-Wen; Chiang, Yu-Chung; Shigemoto, Yasumasa; Hsu, Tsai-Wen; Hwang, Chi-Chuan; Ge, Xue-Jun; Chen, Charles; Wu, Tai-Han; Chou, Chang-Hung; Huang, Hao-Jen; Gojobori, Takashi; Osada, Naoki; Chiang, Tzen-Yuh

    2014-01-01

    Young incipient species provide ideal materials for untangling the process of ecological speciation in the presence of gene flow. The Miscanthus floridulus/sinensis complex exhibits diverse phenotypic and ecological differences despite recent divergence (approximately 1.59million years ago). To elucidate the process of genetic differentiation during early stages of ecological speciation, we analyzed genomic divergence in the Miscanthus complex using 72 randomly selected genes from a newly assembled transcriptome. In this study, rampant gene flow was detected between species, estimated as M=3.36x10(-9) to 1.20x10(-6), resulting in contradicting phylogenies across loci. Nevertheless, beast analyses revealed the species identity and the effects of extrinsic cohesive forces that counteracted the non-stop introgression. As expected, early in speciation with gene flow, only 3-13 loci were highly diverged; two to five outliers (approximately 2.78-6.94% of the genome) were characterized by strong linkage disequilibrium, and asymmetrically distributed among ecotypes, indicating footprints of diversifying selection. In conclusion, ecological speciation of incipient species of Miscanthus probably followed the parapatric model, whereas allopatric speciation cannot be completely ruled out, especially between the geographically isolated northern and southern M.sinensis, for which no significant gene flow across oceanic barriers was detected. Divergence between local ecotypes in early-stage speciation began at a few genomic regions under the influence of natural selection and divergence hitchhiking that overcame gene flow.

  2. Adaptive divergence with gene flow in incipient speciation of Miscanthus floridulus / sinensis complex (Poaceae)

    KAUST Repository

    Huang, Chao-Li

    2014-11-11

    Young incipient species provide ideal materials for untangling the process of ecological speciation in the presence of gene flow. The Miscanthus floridulus/sinensis complex exhibits diverse phenotypic and ecological differences despite recent divergence (approximately 1.59million years ago). To elucidate the process of genetic differentiation during early stages of ecological speciation, we analyzed genomic divergence in the Miscanthus complex using 72 randomly selected genes from a newly assembled transcriptome. In this study, rampant gene flow was detected between species, estimated as M=3.36x10(-9) to 1.20x10(-6), resulting in contradicting phylogenies across loci. Nevertheless, beast analyses revealed the species identity and the effects of extrinsic cohesive forces that counteracted the non-stop introgression. As expected, early in speciation with gene flow, only 3-13 loci were highly diverged; two to five outliers (approximately 2.78-6.94% of the genome) were characterized by strong linkage disequilibrium, and asymmetrically distributed among ecotypes, indicating footprints of diversifying selection. In conclusion, ecological speciation of incipient species of Miscanthus probably followed the parapatric model, whereas allopatric speciation cannot be completely ruled out, especially between the geographically isolated northern and southern M.sinensis, for which no significant gene flow across oceanic barriers was detected. Divergence between local ecotypes in early-stage speciation began at a few genomic regions under the influence of natural selection and divergence hitchhiking that overcame gene flow.

  3. Targeting of breast metastases using a viral gene vector with tumour-selective transcription.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    BACKGROUND: Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression. MATERIALS AND METHODS: We investigated use of the C-X-C chemokine receptor type 4 (CXCR4) promoter to restrict AAV expression to tumour cells, in subcutaneous MCF-7 xenograft mouse models of breast cancer and in patient samples, using bioluminescent imaging and flow cytometric analysis. RESULTS: Higher transgene expression levels were observed in subcutaneous MCF-7 tumours relative to normal tissue (muscle) using the CXCR4 promoter, unlike a ubiquitously expressing Cytomegalovirus promoter construct, with preferential AAVCXCR4 expression in epithelial tumour and CXCR4-positive cells. Transgene expression following intravenously administered AAVCXCR4 in a model of liver metastasis was detected specifically in livers of tumour bearing mice. Ex vivo analysis using patient samples also demonstrated higher AAVCXCR4 expression in tumour compared with normal liver tissue. CONCLUSION: This study demonstrates for the first time, the potential for systemic administration of AAV2 vector for tumour-selective gene therapy.

  4. Illegal gene flow from transgenic creeping bentgrass: the saga continues.

    Science.gov (United States)

    Snow, Allison A

    2012-10-01

    Ecologists have paid close attention to environmental effects that fitness-enhancing transgenes might have following crop-to-wild gene flow (e.g. Snow et al. 2003). For some crops, gene flow also can lead to legal problems,especially when government agencies have not approved transgenic events for unrestricted environmental release.Creeping bentgrass (Agrostis stolonifera), a common turf grass used in golf courses, is the focus of both areas of concern. In 2002, prior to expected deregulation (still pending), The Scotts Company planted creeping bentgrass with transgenic resistance to the herbicide glyphosate,also known as RoundUp, on 162 ha in a designated control area in central Oregon (Fig. 1).Despite efforts to restrict gene flow, wind-dispersed pollen carried transgenes to florets of local A. stolonifera and A. gigantea as far as 14 km away, and to sentinel plants placed as far as 21 km away (Watrud et al. 2004).Then, in August 2003, a strong wind event moved transgenic seeds from wind rows of cut bentgrass into nearby areas. The company’s efforts to kill all transgenic survivors in the area failed: feral glyphosate-resistant populations of A. stolonifera were found by Reichman et al.(2006), and 62% of 585 bentgrass plants had the telltale CP4 EPSPS transgene in 2006 (Zapiola et al. 2008; Fig. 2).Now, in this issue, the story gets even more interesting as Zapiola & Mallory-Smith (2012) describe a transgenic,intergeneric hybrid produced on a feral, transgenic creeping bentgrass plant that received pollen from Polypogon monspeliensis (rabbitfoot grass). Their finding raises a host of new questions about the prevalence and fitness of intergeneric hybrids, as well as how to evaluate the full extent of gene flow from transgenic crops.

  5. Spatial and temporal assessment of pollen- and seed-mediated gene flow from genetically engineered plum Prunus domestica.

    Directory of Open Access Journals (Sweden)

    Ralph Scorza

    Full Text Available Pollen flow from a 0.46 ha plot of genetically engineered (GE Prunus domestica located in West Virginia, USA was evaluated from 2000-2010. Sentinel plum trees were planted at distances ranging from 132 to 854 m from the center of the GE orchard. Plots of mixed plum varieties and seedlings were located at 384, 484 and 998 m from the GE plot. Bee hives (Apis mellifera were dispersed between the GE plum plot and the pollen flow monitoring sites. Pollen-mediated gene flow from out of the GE plum plot to non-GE plums under the study conditions was low, only occurring at all in 4 of 11 years and then in only 0.31% of the 12,116 seeds analyzed. When it occurred, gene flow, calculated as the number of GUS positive embryos/total embryos sampled, ranged from 0.215% at 132 m from the center of the GE plum plot (28 m from the nearest GE plum tree to 0.033-0.017% at longer distances (384-998 m. Based on the percentage of GUS positive seeds per individual sampled tree the range was 0.4% to 12%. Within the GE field plot, gene flow ranged from 4.9 to 39%. Gene flow was related to distance and environmental conditions. A single year sample from a sentinel plot 132 m from the center of the GE plot accounted for 65% of the total 11-year gene flow. Spatial modeling indicated that gene flow dramatically decreased at distances over 400 m from the GE plot. Air temperature and rainfall were, respectively, positively and negatively correlated with gene flow, reflecting the effects of weather conditions on insect pollinator activity. Seed-mediated gene flow was not detected. These results support the feasibility of coexistence of GE and non-GE plum orchards.

  6. Interspecific gene flow and maintenance of species integrity in oaks

    Directory of Open Access Journals (Sweden)

    Oliver Gailing

    2014-07-01

    Full Text Available Oak species show a wide variation in morphological and physiological characters, and species boundaries between closely related species are often not clear-cut. Still, despite frequent interspecific gene flow, oaks maintain distinct morphological and physiological adaptations. In sympatric stands, spatial distribution of species with different ecological requirements is not random but constrained by soil and other microenvironmental factors. Pre-zygotic isolation (e.g. cross incompatibilities, asynchrony in flowering, pollen competition and post-zygotic isolation (divergent selection contribute to the maintenance of species integrity in sympatric oak stands. The antagonistic effects of interspecific gene flow and divergent selection are reflected in the low genetic differentiation between hybridizing oak species at most genomic regions interspersed by regions with signatures of divergent selection (outlier regions. In the near future, the availability of high-density genetic linkage maps anchored to scaffolds of a sequenced Q. robur genome will allow to characterize the underlying genes in these outlier regions and their putative role in reproductive isolation between species. Reciprocal transplant experiments of seedlings between parental environments can be used to characterize selection on outlier genes. High transferability of gene-based markers will enable comparative outlier screens in different oak species.

  7. Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

    International Nuclear Information System (INIS)

    Rivera, Angel A.; Wang Minghui; Suzuki, Kaori; Uil, Taco G.; Krasnykh, Victor; Curiel, David T.; Nettelbeck, Dirk M.

    2004-01-01

    The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdΔE1AIL), the early gene E2B (AdΔE2BIL), or the late fiber gene (AdΔfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdΔE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdΔE1AIL) to six (AdΔfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdΔE2BIL, AdΔfiberIL, and AdTL. AdΔfiberIL and the parental virus, Ad5-Δ24, resulted in similar cytotoxicity, but AdΔE2BIL and AdΔE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdΔE2BIL and AdΔfiberIL, but substantial for AdΔE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression

  8. Viral infection of human lung macrophages increases PDL1 expression via IFNβ.

    Directory of Open Access Journals (Sweden)

    Karl J Staples

    Full Text Available Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.

  9. Expression kinetics of key genes in the early innate immune response to Great Lakes viral hemorrhagic septicemia virus IVb infection in yellow perch (Perca flavescens)

    Science.gov (United States)

    Olson, Wendy; Emmenegger, Eveline; Glenn, Jolene; Simchick, Crystal; Winton, Jim; Goetz, Frederick

    2013-01-01

    The recently discovered strain of viral hemorrhagic septicemia virus, VHSV-IVb, represents an example of the introduction of an extremely pathogenic rhabdovirus capable of infecting a wide variety of new fish species in a new host-environment. The goal of the present study was to delineate the expression kinetics of key genes in the innate immune response relative to the very early stages of VHSV-IVb infection using the yellow perch (Perca flavescens) as a model. Administration of VHSV-IVb by IP-injection into juvenile yellow perch resulted in 84% cumulative mortality, indicating their high susceptibility to this disease. In fish sampled in the very early stages of infection, a significant up-regulation of Mx gene expression in the liver, as well as IL-1β and SAA activation in the head kidney, spleen, and liver was directly correlated to viral load. The potential down-regulation of Mx in the hematopoietic tissues, head kidney and spleen, may represent a strategy utilized by the virus to increase replication.

  10. Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

    Science.gov (United States)

    Lee, Jong-Hwan; Seo, Hyuk Seong; Kwon, Jung-Hyuk; Kim, Hee-Tae; Kwon, Koo Chul; Sim, Sang Jun; Cha, Young Joo; Lee, Jeewon

    2015-07-15

    Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

    International Nuclear Information System (INIS)

    Kolakofsky, Daniel; Le Mercier, Philippe; Iseni, Frederic; Garcin, Dominique

    2004-01-01

    mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in that the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template

  12. Time and flow-dependent changes in the p27(kip1) gene network drive maladaptive vascular remodeling.

    Science.gov (United States)

    DeSart, Kenneth M; Butler, Khayree; O'Malley, Kerri A; Jiang, Zhihua; Berceli, Scott A

    2015-11-01

    Although clinical studies have identified that a single nucleotide polymorphism in the p27(kip1) gene is associated with success or failure after vein bypass grafting, the underlying mechanisms for this difference are not well defined. Using a high-throughput approach in a flow-dependent vein graft model, we explored the differences in p27(kip1)-related genes that drive the enhanced hyperplastic response under low-flow conditions. Bilateral rabbit carotid artery interposition grafts with jugular vein were placed with a unilateral distal outflow branch ligation to create differential flow states. Grafts were harvested at 2 hours and at 1, 3, 7, 14, and 28 days after implantation, measured for neointimal area, and assayed for cell proliferation. Whole-vessel messenger RNA was isolated and analyzed using an Affymetrix (Santa Clara, Calif) gene array platform. Ingenuity Pathway Analysis (Ingenuity, Redwood City, Calif) was used to identify the gene networks surrounding p27(kip1). This gene set was then analyzed for temporal expression changes after graft placement and for differential expression in the alternate flow conditions. Outflow branch ligation resulted in an eightfold difference in mean flow rates throughout the 28-day perfusion period (P Flow reduction led to a robust hyperplastic response, resulting in a significant increase in intimal area by 7 days (0.13 ± 0.04 mm(2) vs 0.014 ± 0.006 mm(2); P flow grafts demonstrated a burst of actively dividing intimal cells (36.4 ± 9.4 cells/mm(2) vs 11.5 ± 1.9 cells/mm(2); P = .04). Sixty-five unique genes within the microarray were identified as components of the p27(kip1) network. At a false discovery rate of 0.05, 26 genes demonstrated significant temporal changes, and two dominant patterns of expression were identified. Class comparison analysis identified differential expression of 11 genes at 2 hours and seven genes and 14 days between the high-flow and low-flow grafts (P flow and shear stress result in

  13. Modified montmorillonite as vector for gene delivery.

    Science.gov (United States)

    Lin, Feng-Huei; Chen, Chia-Hao; Cheng, Winston T K; Kuo, Tzang-Fu

    2006-06-01

    Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system. Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si-O tetrahedrons on the bottom of the layer and Al-O(OH)2 octahedrons on the top. The inter-layer space is about 12 A. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA-HDTMA-MMT is as follows: 1 mg of 1.5CEC HDTMA-MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA-MMT has a great potential as a vector for gene delivery in the future.

  14. Applying gene flow science to environmental policy needs: a boundary work perspective.

    Science.gov (United States)

    Ridley, Caroline E; Alexander, Laurie C

    2016-08-01

    One application of gene flow science is the policy arena. In this article, we describe two examples in which the topic of gene flow has entered into the U.S. national environmental policymaking process: regulation of genetically engineered crops and clarification of the jurisdictional scope of the Clean Water Act. We summarize both current scientific understanding and the legal context within which gene flow science has relevance. We also discuss the process by which scientific knowledge has been synthesized and communicated to decision-makers in these two contexts utilizing the concept of 'boundary work'. Boundary organizations, the work they engage in to bridge the worlds of science, policy, and practice, and the boundary objects they produce to translate scientific knowledge existed in both examples. However, the specific activities and attributes of the objects produced varied based on the needs of the decision-makers. We close with suggestions for how scientists can contribute to or engage in boundary work with policymakers.

  15. Speciation with gene flow in equids despite extensive chromosomal plasticity.

    Science.gov (United States)

    Jónsson, Hákon; Schubert, Mikkel; Seguin-Orlando, Andaine; Ginolhac, Aurélien; Petersen, Lillian; Fumagalli, Matteo; Albrechtsen, Anders; Petersen, Bent; Korneliussen, Thorfinn S; Vilstrup, Julia T; Lear, Teri; Myka, Jennifer Leigh; Lundquist, Judith; Miller, Donald C; Alfarhan, Ahmed H; Alquraishi, Saleh A; Al-Rasheid, Khaled A S; Stagegaard, Julia; Strauss, Günter; Bertelsen, Mads Frost; Sicheritz-Ponten, Thomas; Antczak, Douglas F; Bailey, Ernest; Nielsen, Rasmus; Willerslev, Eske; Orlando, Ludovic

    2014-12-30

    Horses, asses, and zebras belong to a single genus, Equus, which emerged 4.0-4.5 Mya. Although the equine fossil record represents a textbook example of evolution, the succession of events that gave rise to the diversity of species existing today remains unclear. Here we present six genomes from each living species of asses and zebras. This completes the set of genomes available for all extant species in the genus, which was hitherto represented only by the horse and the domestic donkey. In addition, we used a museum specimen to characterize the genome of the quagga zebra, which was driven to extinction in the early 1900s. We scan the genomes for lineage-specific adaptations and identify 48 genes that have evolved under positive selection and are involved in olfaction, immune response, development, locomotion, and behavior. Our extensive genome dataset reveals a highly dynamic demographic history with synchronous expansions and collapses on different continents during the last 400 ky after major climatic events. We show that the earliest speciation occurred with gene flow in Northern America, and that the ancestor of present-day asses and zebras dispersed into the Old World 2.1-3.4 Mya. Strikingly, we also find evidence for gene flow involving three contemporary equine species despite chromosomal numbers varying from 16 pairs to 31 pairs. These findings challenge the claim that the accumulation of chromosomal rearrangements drive complete reproductive isolation, and promote equids as a fundamental model for understanding the interplay between chromosomal structure, gene flow, and, ultimately, speciation.

  16. High gene flow in epiphytic ferns despite habitat loss and fragmentation.

    Science.gov (United States)

    Winkler, Manuela; Koch, Marcus; Hietz, Peter

    2011-01-01

    Tropical montane forests suffer from increasing fragmentation and replacement by other types of land-use such as coffee plantations. These processes are known to affect gene flow and genetic structure of plant populations. Epiphytes are particularly vulnerable because they depend on their supporting trees for their entire life-cycle. We compared population genetic structure and genetic diversity derived from AFLP markers of two epiphytic fern species differing in their ability to colonize secondary habitats. One species, Pleopeltis crassinervata , is a successful colonizer of shade trees and isolated trees whereas the other species, Polypodium rhodopleuron , is restricted to forests with anthropogenic separation leading to significant isolation between populations. By far most genetic variation was distributed within rather than among populations in both species, and a genetic admixture analysis did not reveal any clustering. Gene flow exceeded by far the benchmark of one migrant per generation to prevent genetic divergence between populations in both species. Though populations are threatened by habitat loss, long-distance dispersal is likely to support gene flow even between distant populations, which efficiently delays genetic isolation. Consequently, populations may rather be threatened by ecological consequences of habitat loss and fragmentation.

  17. Speciation with gene flow in whiptail lizards from a Neotropical xeric biome.

    Science.gov (United States)

    Oliveira, Eliana F; Gehara, Marcelo; São-Pedro, Vinícius A; Chen, Xin; Myers, Edward A; Burbrink, Frank T; Mesquita, Daniel O; Garda, Adrian A; Colli, Guarino R; Rodrigues, Miguel T; Arias, Federico J; Zaher, Hussam; Santos, Rodrigo M L; Costa, Gabriel C

    2015-12-01

    Two main hypotheses have been proposed to explain the diversification of the Caatinga biota. The riverine barrier hypothesis (RBH) claims that the São Francisco River (SFR) is a major biogeographic barrier to gene flow. The Pleistocene climatic fluctuation hypothesis (PCH) states that gene flow, geographic genetic structure and demographic signatures on endemic Caatinga taxa were influenced by Quaternary climate fluctuation cycles. Herein, we analyse genetic diversity and structure, phylogeographic history, and diversification of a widespread Caatinga lizard (Cnemidophorus ocellifer) based on large geographical sampling for multiple loci to test the predictions derived from the RBH and PCH. We inferred two well-delimited lineages (Northeast and Southwest) that have diverged along the Cerrado-Caatinga border during the Mid-Late Miocene (6-14 Ma) despite the presence of gene flow. We reject both major hypotheses proposed to explain diversification in the Caatinga. Surprisingly, our results revealed a striking complex diversification pattern where the Northeast lineage originated as a founder effect from a few individuals located along the edge of the Southwest lineage that eventually expanded throughout the Caatinga. The Southwest lineage is more diverse, older and associated with the Cerrado-Caatinga boundaries. Finally, we suggest that C. ocellifer from the Caatinga is composed of two distinct species. Our data support speciation in the presence of gene flow and highlight the role of environmental gradients in the diversification process. © 2015 John Wiley & Sons Ltd.

  18. Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

    Directory of Open Access Journals (Sweden)

    El Andaloussi Samir

    2011-05-01

    Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

  19. Mucosal immunization with recombinant adenoviral vectors expressing murine gammaherpesvirus-68 genes M2 and M3 can reduce latent viral load

    DEFF Research Database (Denmark)

    Hoegh-Petersen, Mette; Thomsen, Allan R; Christensen, Jan P

    2009-01-01

    -68 (MHV-68) is a member of the Gammaherpesvirinae subfamily and represents a useful murine model for this category of infections, in which new vaccination strategies may initially be evaluated. Two attenuated variants of MHV-68 have successfully been used as vaccines, but the oncogenic potential...... of the gammaherpesvirinae speaks against using a similar approach in humans. DNA immunization with plasmids encoding the MHV-68 genes M2 or M3 caused a reduction in either acute or early latent viral load, respectively, but neither immunization had an effect at times later than 14 days post-infection. Adenovirus......-based vaccines are substantially more immunogenic than DNA vaccines and can be applied to induce mucosal immunity. Here we show that a significant reduction of the late viral load in the spleens, at 60 days post-infection, was achieved when immunizing mice both intranasally and subcutaneously with adenoviral...

  20. Gene flow of common ash (Fraxinus excelsior L. in a fragmented landscape.

    Directory of Open Access Journals (Sweden)

    Devrim Semizer-Cuming

    Full Text Available Gene flow dynamics of common ash (Fraxinus excelsior L. is affected by several human activities in Central Europe, including habitat fragmentation, agroforestry expansion, controlled and uncontrolled transfer of reproductive material, and a recently introduced emerging infectious disease, ash dieback, caused by Hymenoscyphus fraxineus. Habitat fragmentation may alter genetic connectivity and effective population size, leading to loss of genetic diversity and increased inbreeding in ash populations. Gene flow from cultivated trees in landscapes close to their native counterparts may also influence the adaptability of future generations. The devastating effects of ash dieback have already been observed in both natural and managed populations in continental Europe. However, potential long-term effects of genetic bottlenecks depend on gene flow across fragmented landscapes. For this reason, we studied the genetic connectivity of ash trees in an isolated forest patch of a fragmented landscape in Rösenbeck, Germany. We applied two approaches to parentage analysis to estimate gene flow patterns at the study site. We specifically investigated the presence of background pollination at the landscape level and the degree of genetic isolation between native and cultivated trees. Local meteorological data was utilized to understand the effect of wind on the pollen and seed dispersal patterns. Gender information of the adult trees was considered for calculating the dispersal distances. We found that the majority of the studied seeds (55-64% and seedlings (75-98% in the forest patch were fathered and mothered by the trees within the same patch. However, we determined a considerable amount of pollen flow (26-45% from outside of the study site, representing background pollination at the landscape level. Limited pollen flow was observed from neighbouring cultivated trees (2%. Both pollen and seeds were dispersed in all directions in accordance with the local

  1. Gene flow of common ash (Fraxinus excelsior L.) in a fragmented landscape.

    Science.gov (United States)

    Semizer-Cuming, Devrim; Kjær, Erik Dahl; Finkeldey, Reiner

    2017-01-01

    Gene flow dynamics of common ash (Fraxinus excelsior L.) is affected by several human activities in Central Europe, including habitat fragmentation, agroforestry expansion, controlled and uncontrolled transfer of reproductive material, and a recently introduced emerging infectious disease, ash dieback, caused by Hymenoscyphus fraxineus. Habitat fragmentation may alter genetic connectivity and effective population size, leading to loss of genetic diversity and increased inbreeding in ash populations. Gene flow from cultivated trees in landscapes close to their native counterparts may also influence the adaptability of future generations. The devastating effects of ash dieback have already been observed in both natural and managed populations in continental Europe. However, potential long-term effects of genetic bottlenecks depend on gene flow across fragmented landscapes. For this reason, we studied the genetic connectivity of ash trees in an isolated forest patch of a fragmented landscape in Rösenbeck, Germany. We applied two approaches to parentage analysis to estimate gene flow patterns at the study site. We specifically investigated the presence of background pollination at the landscape level and the degree of genetic isolation between native and cultivated trees. Local meteorological data was utilized to understand the effect of wind on the pollen and seed dispersal patterns. Gender information of the adult trees was considered for calculating the dispersal distances. We found that the majority of the studied seeds (55-64%) and seedlings (75-98%) in the forest patch were fathered and mothered by the trees within the same patch. However, we determined a considerable amount of pollen flow (26-45%) from outside of the study site, representing background pollination at the landscape level. Limited pollen flow was observed from neighbouring cultivated trees (2%). Both pollen and seeds were dispersed in all directions in accordance with the local wind directions

  2. Bears in a forest of gene trees: phylogenetic inference is complicated by incomplete lineage sorting and gene flow.

    Science.gov (United States)

    Kutschera, Verena E; Bidon, Tobias; Hailer, Frank; Rodi, Julia L; Fain, Steven R; Janke, Axel

    2014-08-01

    Ursine bears are a mammalian subfamily that comprises six morphologically and ecologically distinct extant species. Previous phylogenetic analyses of concatenated nuclear genes could not resolve all relationships among bears, and appeared to conflict with the mitochondrial phylogeny. Evolutionary processes such as incomplete lineage sorting and introgression can cause gene tree discordance and complicate phylogenetic inferences, but are not accounted for in phylogenetic analyses of concatenated data. We generated a high-resolution data set of autosomal introns from several individuals per species and of Y-chromosomal markers. Incorporating intraspecific variability in coalescence-based phylogenetic and gene flow estimation approaches, we traced the genealogical history of individual alleles. Considerable heterogeneity among nuclear loci and discordance between nuclear and mitochondrial phylogenies were found. A species tree with divergence time estimates indicated that ursine bears diversified within less than 2 My. Consistent with a complex branching order within a clade of Asian bear species, we identified unidirectional gene flow from Asian black into sloth bears. Moreover, gene flow detected from brown into American black bears can explain the conflicting placement of the American black bear in mitochondrial and nuclear phylogenies. These results highlight that both incomplete lineage sorting and introgression are prominent evolutionary forces even on time scales up to several million years. Complex evolutionary patterns are not adequately captured by strictly bifurcating models, and can only be fully understood when analyzing multiple independently inherited loci in a coalescence framework. Phylogenetic incongruence among gene trees hence needs to be recognized as a biologically meaningful signal. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  3. Anti-viral RNA silencing: do we look like plants ?

    Directory of Open Access Journals (Sweden)

    Lecellier Charles-Henri

    2006-01-01

    Full Text Available Abstract The anti-viral function of RNA silencing was first discovered in plants as a natural manifestation of the artificial 'co-suppression', which refers to the extinction of endogenous gene induced by homologous transgene. Because silencing components are conserved among most, if not all, eukaryotes, the question rapidly arose as to determine whether this process fulfils anti-viral functions in animals, such as insects and mammals. It appears that, whereas the anti-viral process seems to be similarly conserved from plants to insects, even in worms, RNA silencing does influence the replication of mammalian viruses but in a particular mode: micro(miRNAs, endogenous small RNAs naturally implicated in translational control, rather than virus-derived small interfering (siRNAs like in other organisms, are involved. In fact, these recent studies even suggest that RNA silencing may be beneficial for viral replication. Accordingly, several large DNA mammalian viruses have been shown to encode their own miRNAs. Here, we summarize the seminal studies that have implicated RNA silencing in viral infection and compare the different eukaryotic responses.

  4. Genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV)

    DEFF Research Database (Denmark)

    Snow, M.; Bain, N.; Black, J.

    2004-01-01

    The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders this the m......The nucleotide sequences of a specific region of the nucleoprotein gene were compared in order to investigate the genetic population structure of marine viral haemorrhagic septicaemia virus (VHSV). Analysis of the sequence from 128 isolates of diverse geographic and host origin renders...... this the most comprehensive molecular epidemiological study of marine VHSV conducted to date. Phylogenetic analysis of nucleoprotein gene sequences confirmed the existence of the 4 major genotypes previously identified based on N- and subsequent G-gene based analyses. The range of Genotype I included subgroups...... of isolates associated with rainbow trout aquaculture (Genotype la) and those from the Baltic marine environment (Genotype Ib) to emphasise the relatively close genetic relationship between these isolates. The existence of an additional genotype circulating within the Baltic Sea (Genotype II) was also...

  5. Mucosal immunization with recombinant adenoviral vectors expressing murine gammaherpesvirus-68 genes M2 and M3 can reduce latent viral load.

    Science.gov (United States)

    Hoegh-Petersen, Mette; Thomsen, Allan R; Christensen, Jan P; Holst, Peter J

    2009-11-12

    Gammaherpesviruses establish life-long latent infections in their hosts. If the host becomes immunosuppressed, these viruses may reactivate and cause severe disease, and even in immunocompetent individuals the gammaherpesviruses are presumed to have an oncogenic potential. Murine gammaherpesvirus-68 (MHV-68) is a member of the Gammaherpesvirinae subfamily and represents a useful murine model for this category of infections, in which new vaccination strategies may initially be evaluated. Two attenuated variants of MHV-68 have successfully been used as vaccines, but the oncogenic potential of the gammaherpesvirinae speaks against using a similar approach in humans. DNA immunization with plasmids encoding the MHV-68 genes M2 or M3 caused a reduction in either acute or early latent viral load, respectively, but neither immunization had an effect at times later than 14 days post-infection. Adenovirus-based vaccines are substantially more immunogenic than DNA vaccines and can be applied to induce mucosal immunity. Here we show that a significant reduction of the late viral load in the spleens, at 60 days post-infection, was achieved when immunizing mice both intranasally and subcutaneously with adenoviral vectors encoding both M2 and M3. Additionally we show that M3 immunization prevented the usual development of virus-induced splenomegaly at 2-3 weeks post-infection. This is the first time that immunization with a non-replicating vaccine has lead to a significantly reduced viral load at time points beyond 14 days post-infection, and thus demonstrates that a non-replicating vaccine may successfully be employed to reduce the viral burden during chronic gammaherpesvirus infection.

  6. Viral MicroRNAs Repress the Cholesterol Pathway, and 25-Hydroxycholesterol Inhibits Infection.

    Science.gov (United States)

    Serquiña, Anna K P; Kambach, Diane M; Sarker, Ontara; Ziegelbauer, Joseph M

    2017-07-11

    From various screens, we found that Kaposi's sarcoma-associated herpesvirus (KSHV) viral microRNAs (miRNAs) target several enzymes in the mevalonate/cholesterol pathway. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR [a rate-limiting step in the mevalonate pathway]), and farnesyl-diphosphate farnesyltransferase 1 (FDFT1 [a committed step in the cholesterol branch]) are repressed by multiple KSHV miRNAs. Transfection of viral miRNA mimics in primary endothelial cells (human umbilical vein endothelial cells [HUVECs]) is sufficient to reduce intracellular cholesterol levels; however, small interfering RNAs (siRNAs) targeting only HMGCS1 did not reduce cholesterol levels. This suggests that multiple targets are needed to perturb this tightly regulated pathway. We also report here that cholesterol levels were decreased in de novo -infected HUVECs after 7 days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild-type infections. We hypothesized that KSHV is downregulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the cholesterol 25-hydroxylase (CH25H) gene, which is responsible for generating 25HC, had increased expression in de novo -infected HUVECs but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of 25HC. IMPORTANCE A subset of viruses express unique microRNAs (miRNAs), which act like cellular miRNAs to generally repress host gene

  7. Extracellular Hsp90 serves as a co-factor for MAPK activation and latent viral gene expression during de novo infection by KSHV

    International Nuclear Information System (INIS)

    Qin Zhiqiang; DeFee, Michael; Isaacs, Jennifer S.; Parsons, Chris

    2010-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an important cause of morbidity and mortality in immunocompromised patients. KSHV interaction with the cell membrane triggers activation of specific intracellular signal transduction pathways to facilitate virus entry, nuclear trafficking, and ultimately viral oncogene expression. Extracellular heat shock protein 90 localizes to the cell surface (csHsp90) and facilitates signal transduction in cancer cell lines, but whether csHsp90 assists in the coordination of KSHV gene expression through these or other mechanisms is unknown. Using a recently characterized non-permeable inhibitor specifically targeting csHsp90 and Hsp90-specific antibodies, we show that csHsp90 inhibition suppresses KSHV gene expression during de novo infection, and that this effect is mediated largely through the inhibition of mitogen-activated protein kinase (MAPK) activation by KSHV. Moreover, we show that targeting csHsp90 reduces constitutive MAPK expression and the release of infectious viral particles by patient-derived, KSHV-infected primary effusion lymphoma cells. These data suggest that csHsp90 serves as an important co-factor for KSHV-initiated MAPK activation and provide proof-of-concept for the potential benefit of targeting csHsp90 for the treatment or prevention of KSHV-associated illnesses.

  8. Genetic diversity and gene flow revealed by microsatellite DNA ...

    African Journals Online (AJOL)

    Dacryodes edulis is a multipurpose tree integrated in the cropping system of Central African region still dominated by subsistence agriculture. Some populations grown are wild which can provide information on the domestication process, and could also represent a potential source of gene flow. Leaves samples for DNA ...

  9. Gene flow among established Puerto Rican populations of the exotic tree species, Albizia lebbeck.

    Science.gov (United States)

    Dunphy, B K; Hamrick, J L

    2005-04-01

    We estimate gene flow and patterns of genetic diversity in Albizia lebbeck, an invasive leguminous tree in the dry forest of southwestern Puerto Rico. Genetic diversity estimates calculated for 10 populations of 24 trees each indicated that these populations may have been formed from multiple introductions. The presence of unique genotypes in the northernmost populations suggests that novel genotypes are still immigrating into the area. This combination of individuals from disparate locations led to high estimates of genetic diversity (He = 0.266, P = 0.67). Indirect estimates of gene flow indicate that only 0.69 migrants per generation move between populations, suggesting that genetic diversity within populations should decrease due to genetic drift. Since migration-drift equilibrium was not found, however, this estimate needs to be viewed with caution. The regular production of pods in this outcrossing species (tm = 0.979) indicates that sufficient outcross pollen is received to insure successful reproduction. Direct estimates of gene flow indicate that between 44 and 100% of pollen received by trees in four small stands of trees (n < 11) was foreign. The role of gene flow in facilitating the spread of this invasive plant species is discussed.

  10. 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

  11. Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta.

    Science.gov (United States)

    Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I; Mäkinen, Kristiina

    2011-09-01

    Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

  12. Potyviral VPg Enhances Viral RNA Translation and Inhibits Reporter mRNA Translation In Planta▿

    Science.gov (United States)

    Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I.; Mäkinen, Kristiina

    2011-01-01

    Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5′ untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation. PMID:21697470

  13. Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

    Science.gov (United States)

    Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

    2009-03-09

    The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate

  14. Beekeeping practices and geographic distance, not land use, drive gene flow across tropical bees.

    Science.gov (United States)

    Jaffé, Rodolfo; Pope, Nathaniel; Acosta, André L; Alves, Denise A; Arias, Maria C; De la Rúa, Pilar; Francisco, Flávio O; Giannini, Tereza C; González-Chaves, Adrian; Imperatriz-Fonseca, Vera L; Tavares, Mara G; Jha, Shalene; Carvalheiro, Luísa G

    2016-11-01

    Across the globe, wild bees are threatened by ongoing natural habitat loss, risking the maintenance of plant biodiversity and agricultural production. Despite the ecological and economic importance of wild bees and the fact that several species are now managed for pollination services worldwide, little is known about how land use and beekeeping practices jointly influence gene flow. Using stingless bees as a model system, containing wild and managed species that are presumed to be particularly susceptible to habitat degradation, here we examine the main drivers of tropical bee gene flow. We employ a novel landscape genetic approach to analyse data from 135 populations of 17 stingless bee species distributed across diverse tropical biomes within the Americas. Our work has important methodological implications, as we illustrate how a maximum-likelihood approach can be applied in a meta-analysis framework to account for multiple factors, and weight estimates by sample size. In contrast to previously held beliefs, gene flow was not related to body size or deforestation, and isolation by geographic distance (IBD) was significantly affected by management, with managed species exhibiting a weaker IBD than wild ones. Our study thus reveals the critical importance of beekeeping practices in shaping the patterns of genetic differentiation across bee species. Additionally, our results show that many stingless bee species maintain high gene flow across heterogeneous landscapes. We suggest that future efforts to preserve wild tropical bees should focus on regulating beekeeping practices to maintain natural gene flow and enhancing pollinator-friendly habitats, prioritizing species showing a limited dispersal ability. © 2016 John Wiley & Sons Ltd.

  15. Spread of a new parasitic B chromosome variant is facilitated by high gene flow.

    Directory of Open Access Journals (Sweden)

    María Inmaculada Manrique-Poyato

    Full Text Available The B24 chromosome variant emerged several decades ago in a Spanish population of the grasshopper Eyprepocnemis plorans and is currently reaching adjacent populations. Here we report, for the first time, how a parasitic B chromosome (a strictly vertically transmitted parasite expands its geographical range aided by high gene flow in the host species. For six years we analyzed B frequency in several populations to the east and west of the original population and found extensive spatial variation, but only a slight temporal trend. The highest B24 frequency was found in its original population (Torrox and it decreased closer to both the eastern and the western populations. The analysis of Inter Simple Sequence Repeat (ISSR markers showed the existence of a low but significant degree of population subdivision, as well as significant isolation by distance (IBD. Pairwise Nem estimates suggested the existence of high gene flow between the four populations located in the Torrox area, with higher values towards the east. No significant barriers to gene flow were found among these four populations, and we conclude that high gene flow is facilitating B24 diffusion both eastward and westward, with minor role for B24 drive due to the arrival of drive suppressor genes which are also frequent in the donor population.

  16. Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

    OpenAIRE

    Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

    2003-01-01

    For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...

  17. AN UNUSUAL PATTERN OF GENE FLOW BETWEEN THE TWO SOCIAL FORMS OF THE FIRE ANT SOLENOPSIS INVICTA.

    Science.gov (United States)

    Ross, Kenneth G; Shoemaker, D DeWayne

    1993-10-01

    Uncertainty over the role of shifts in social behavior in the process of speciation in social insects has stimulated interest in determining the extent of gene flow between conspecific populations differing in colony social organization. Allele and genotype frequencies at 12 neutral polymorphic protein markers, as well as the numbers of alleles at the sex-determining locus (loci), are shown here to be consistent with significant ongoing gene flow between two geographically adjacent populations of Solenopsis invicta that differ in colony queen number. Data from a thirteenth protein marker that is under strong differential selection in the two social forms confirm that such gene flow occurs. Data from this selected locus, combined with knowledge of the reproductive biology of the two social forms, further suggest that interform gene flow is largely unidirectional and mediated through males only. This unusual pattern of gene flow results from the influence of the unique social enviroments of the two forms on the behavior of workers and on the reproductive physiology of sexuals. © 1993 The Society for the Study of Evolution.

  18. Compartmentalization of the gut viral reservoir in HIV-1 infected patients

    Directory of Open Access Journals (Sweden)

    Grant Tannika

    2007-12-01

    Full Text Available Abstract Background Recently there has been an increasing interest and appreciation for the gut as both a viral reservoir as well as an important host-pathogen interface in human immunodefiency virus type 1 (HIV-1 infection. The gut associated lymphoid tissue (GALT is the largest lymphoid organ infected by HIV-1. In this study we examined if different HIV-1 quasispecies are found in different parts of the gut of HIV-1 infected individuals. Results Gut biopsies (esophagus, stomach, duodenum and colorectum were obtained from eight HIV-1 infected preHAART (highly active antiretroviral therapy patients. HIV-1 Nef and Reverse transcriptase (RT encoding sequences were obtained through nested PCR amplification from DNA isolated from the gut biopsy tissues. The PCR fragments were cloned and sequenced. The resulting sequences were subjected to various phylogenetic analyses. Expression of the nef gene and viral RNA in the different gut tissues was determined using real-time RT-PCR. Phylogenetic analysis of the Nef protein-encoding region revealed compartmentalization of viral replication in the gut within patients. Viral diversity in both the Nef and RT encoding region varied in different parts of the gut. Moreover, increased nef gene expression (p Conclusion Our results indicated that different HIV-1 quasispecies populate different parts of the gut, and that viral replication in the gut is compartmentalized. These observations underscore the importance of the gut as a host-pathogen interface in HIV-1 infection.

  19. Raw Sewage Harbors Diverse Viral Populations

    Science.gov (United States)

    Cantalupo, Paul G.; Calgua, Byron; Zhao, Guoyan; Hundesa, Ayalkibet; Wier, Adam D.; Katz, Josh P.; Grabe, Michael; Hendrix, Roger W.; Girones, Rosina; Wang, David; Pipas, James M.

    2011-01-01

    ABSTRACT At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity. Importance At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that

  20. Endocrine aspects of cancer gene therapy.

    Science.gov (United States)

    Barzon, Luisa; Boscaro, Marco; Palù, Giorgio

    2004-02-01

    The field of cancer gene therapy is in continuous expansion, and technology is quickly moving ahead as far as gene targeting and regulation of gene expression are concerned. This review focuses on the endocrine aspects of gene therapy, including the possibility to exploit hormone and hormone receptor functions for regulating therapeutic gene expression, the use of endocrine-specific genes as new therapeutic tools, the effects of viral vector delivery and transgene expression on the endocrine system, and the endocrine response to viral vector delivery. Present ethical concerns of gene therapy and the risk of germ cell transduction are also discussed, along with potential lines of innovation to improve cell and gene targeting.

  1. p53 Activation following Rift Valley fever virus infection contributes to cell death and viral production.

    Directory of Open Access Journals (Sweden)

    Dana Austin

    Full Text Available Rift Valley fever virus (RVFV is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production.

  2. Viral Hepatitis

    Science.gov (United States)

    ... Home A-Z Health Topics Viral hepatitis Viral hepatitis > A-Z Health Topics Viral hepatitis (PDF, 90 ... liver. Source: National Cancer Institute Learn more about hepatitis Watch a video. Learn who is at risk ...

  3. Inactivation of the host lipin gene accelerates RNA virus replication through viral exploitation of the expanded endoplasmic reticulum membrane.

    Directory of Open Access Journals (Sweden)

    Chingkai Chuang

    2014-02-01

    Full Text Available RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs that drive viral replication. The host lipins (phosphatidate phosphatases are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase, which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.

  4. Consequences of gene flow between oilseed rape (Brassica napus) and its relatives.

    Science.gov (United States)

    Liu, Yongbo; Wei, Wei; Ma, Keping; Li, Junsheng; Liang, Yuyong; Darmency, Henri

    2013-10-01

    Numerous studies have focused on the probability of occurrence of gene flow between transgenic crops and their wild relatives and the likelihood of transgene escape, which should be assessed before the commercial release of transgenic crops. This review paper focuses on this issue for oilseed rape, Brassica napus L., a species that produces huge numbers of pollen grains and seeds. We analyze separately the distinct steps of gene flow: (1) pollen and seeds as vectors of gene flow; (2) spontaneous hybridization; (3) hybrid behavior, fitness cost due to hybridization and mechanisms of introgression; (4) and fitness benefit due to transgenes (e.g. herbicide resistance and Bt toxin). Some physical, biological and molecular means of transgene containment are also described. Although hybrids and first generation progeny are difficult to identify in fields and non-crop habitats, the literature shows that transgenes could readily introgress into Brassica rapa, Brassica juncea and Brassica oleracea, while introgression is expected to be rare with Brassica nigra, Hirschfeldia incana and Raphanus raphanistrum. The hybrids grow well but produce less seed than their wild parent. The difference declines with increasing generations. However, there is large uncertainty about the evolution of chromosome numbers and recombination, and many parameters of life history traits of hybrids and progeny are not determined with satisfactory confidence to build generic models capable to really cover the wide diversity of situations. We show that more studies are needed to strengthen and organize biological knowledge, which is a necessary prerequisite for model simulations to assess the practical and evolutionary outputs of introgression, and to provide guidelines for gene flow management. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Landscape genetics as a tool for conservation planning: predicting the effects of landscape change on gene flow.

    Science.gov (United States)

    van Strien, Maarten J; Keller, Daniela; Holderegger, Rolf; Ghazoul, Jaboury; Kienast, Felix; Bolliger, Janine

    2014-03-01

    For conservation managers, it is important to know whether landscape changes lead to increasing or decreasing gene flow. Although the discipline of landscape genetics assesses the influence of landscape elements on gene flow, no studies have yet used landscape-genetic models to predict gene flow resulting from landscape change. A species that has already been severely affected by landscape change is the large marsh grasshopper (Stethophyma grossum), which inhabits moist areas in fragmented agricultural landscapes in Switzerland. From transects drawn between all population pairs within maximum dispersal distance (landscape composition as well as some measures of habitat configuration. Additionally, a complete sampling of all populations in our study area allowed incorporating measures of population topology. These measures together with the landscape metrics formed the predictor variables in linear models with gene flow as response variable (F(ST) and mean pairwise assignment probability). With a modified leave-one-out cross-validation approach, we selected the model with the highest predictive accuracy. With this model, we predicted gene flow under several landscape-change scenarios, which simulated construction, rezoning or restoration projects, and the establishment of a new population. For some landscape-change scenarios, significant increase or decrease in gene flow was predicted, while for others little change was forecast. Furthermore, we found that the measures of population topology strongly increase model fit in landscape genetic analysis. This study demonstrates the use of predictive landscape-genetic models in conservation and landscape planning.

  6. Activation of erythropoietin receptors by Friend viral gp55 and by erythropoietin and down-modulation by the murine Fv-2r resistance gene

    International Nuclear Information System (INIS)

    Hoatlin, M.E.; Kozak, S.L.; Kabat, D.; Lilly, F.; Chakraborti, A.; Kozak, C.A.

    1990-01-01

    The leukemogenic membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus appears to bind to erythropoietin receptors (EpoR) to stimulate erythroblastosis. To directly compare the effects of gp55 with erythropoietin (Epo), the authors produced retrovirions that encode either gp55, Epo, or EpoR. After infection with EpoR virus, interleukin 3-dependent DA-3 cells bound 125 I-labeled Epo and grew without interleukin 3 in the presence of Epo. These latter cells, but not parental DA-3 cells, became factor-independent after superinfection either with Epo virus or with Friend spleen focus-forming virus. In addition, Epo virus caused a disease in mice that mimicked Friend erythroleukemia. Although Fv-2 r homozygotes are susceptible to all other retroviral diseases, they are resistant to both Epo viral and Friend viral erythroleukemia. These results indicate that both gp55 and Epo stimulate EpoR and that the Fv-2 gene encodes a protein that controls response to these ligands. However, the Fv-2 protein is not EpoR because the corresponding genes map to opposite ends of mouse chromosome 9. These results have important implications for understanding signal transduction by EpoR and the role of host genetic variation in controlling susceptibility to an oncogenic protein

  7. Recent long-distance transgene flow into wild populations conforms to historical patterns of gene flow in cotton (Gossypium hirsutum) at its centre of origin.

    Science.gov (United States)

    Wegier, A; Piñeyro-Nelson, A; Alarcón, J; Gálvez-Mariscal, A; Alvarez-Buylla, E R; Piñero, D

    2011-10-01

    Over 95% of the currently cultivated cotton was domesticated from Gossypium hirsutum, which originated and diversified in Mexico. Demographic and genetic studies of this species at its centre of origin and diversification are lacking, although they are critical for cotton conservation and breeding. We investigated the actual and potential distribution of wild cotton populations, as well as the contribution of historical and recent gene flow in shaping cotton genetic diversity and structure. We evaluated historical gene flow using chloroplast microsatellites and recent gene flow through the assessment of transgene presence in wild cotton populations, exploiting the fact that genetically modified cotton has been planted in the North of Mexico since 1996. Assessment of geographic structure through Bayesian spatial analysis, BAPS and Genetic Algorithm for Rule-set Production (GARP), suggests that G. hirsutum seems to conform to a metapopulation scheme, with eight distinct metapopulations. Despite evidence for long-distance gene flow, genetic variation among the metapopulations of G. hirsutum is high (He = 0.894 ± 0.01). We identified 46 different haplotypes, 78% of which are unique to a particular metapopulation, in contrast to a single haplotype detected in cotton cultivars. Recent gene flow was also detected (m = 66/270 = 0.24), with four out of eight metapopulations having transgenes. We discuss the implications of the data presented here with respect to the conservation and future breeding of cotton populations and genetic diversity at its centre of crop origin. © 2011 Blackwell Publishing Ltd.

  8. MicroRNA Roles in the NF-κB Signaling Pathway during Viral Infections

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    Zeqian Gao

    2014-01-01

    Full Text Available NF-κB signaling network is a crucial component of innate immunity. miRNAs are a subtype of small noncoding RNAs, involved in regulation of gene expression at the posttranscriptional level. Increasing evidence has emerged that miRNAs play an important role in regulation of NF-κB signaling pathway during viral infections. Both host and viral miRNAs are attributed to modulation of NF-κB activity, thus affecting viral infection and clearance. Understandings of the mechanisms of these miRNAs will open a direction for development of novel antivirus drugs.

  9. Gene flow from North Africa contributes to differential human genetic diversity in southern Europe

    Science.gov (United States)

    Botigué, Laura R.; Henn, Brenna M.; Gravel, Simon; Maples, Brian K.; Gignoux, Christopher R.; Corona, Erik; Atzmon, Gil; Burns, Edward; Ostrer, Harry; Flores, Carlos; Bertranpetit, Jaume; Comas, David; Bustamante, Carlos D.

    2013-01-01

    Human genetic diversity in southern Europe is higher than in other regions of the continent. This difference has been attributed to postglacial expansions, the demic diffusion of agriculture from the Near East, and gene flow from Africa. Using SNP data from 2,099 individuals in 43 populations, we show that estimates of recent shared ancestry between Europe and Africa are substantially increased when gene flow from North Africans, rather than Sub-Saharan Africans, is considered. The gradient of North African ancestry accounts for previous observations of low levels of sharing with Sub-Saharan Africa and is independent of recent gene flow from the Near East. The source of genetic diversity in southern Europe has important biomedical implications; we find that most disease risk alleles from genome-wide association studies follow expected patterns of divergence between Europe and North Africa, with the principal exception of multiple sclerosis. PMID:23733930

  10. Parallel epigenomic and transcriptomic responses to viral infection in honey bees (Apis mellifera).

    Science.gov (United States)

    Galbraith, David A; Yang, Xingyu; Niño, Elina Lastro; Yi, Soojin; Grozinger, Christina

    2015-03-01

    Populations of honey bees are declining throughout the world, with US beekeepers losing 30% of their colonies each winter. Though multiple factors are driving these colony losses, it is increasingly clear that viruses play a major role. However, information about the molecular mechanisms mediating antiviral immunity in honey bees is surprisingly limited. Here, we examined the transcriptional and epigenetic (DNA methylation) responses to viral infection in honey bee workers. One-day old worker honey bees were fed solutions containing Israeli Acute Paralysis Virus (IAPV), a virus which causes muscle paralysis and death and has previously been associated with colony loss. Uninfected control and infected, symptomatic bees were collected within 20-24 hours after infection. Worker fat bodies, the primary tissue involved in metabolism, detoxification and immune responses, were collected for analysis. We performed transcriptome- and bisulfite-sequencing of the worker fat bodies to identify genome-wide gene expression and DNA methylation patterns associated with viral infection. There were 753 differentially expressed genes (FDR<0.05) in infected versus control bees, including several genes involved in epigenetic and antiviral pathways. DNA methylation status of 156 genes (FDR<0.1) changed significantly as a result of the infection, including those involved in antiviral responses in humans. There was no significant overlap between the significantly differentially expressed and significantly differentially methylated genes, and indeed, the genomic characteristics of these sets of genes were quite distinct. Our results indicate that honey bees have two distinct molecular pathways, mediated by transcription and methylation, that modulate protein levels and/or function in response to viral infections.

  11. Parallel epigenomic and transcriptomic responses to viral infection in honey bees (Apis mellifera.

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    David A Galbraith

    2015-03-01

    Full Text Available Populations of honey bees are declining throughout the world, with US beekeepers losing 30% of their colonies each winter. Though multiple factors are driving these colony losses, it is increasingly clear that viruses play a major role. However, information about the molecular mechanisms mediating antiviral immunity in honey bees is surprisingly limited. Here, we examined the transcriptional and epigenetic (DNA methylation responses to viral infection in honey bee workers. One-day old worker honey bees were fed solutions containing Israeli Acute Paralysis Virus (IAPV, a virus which causes muscle paralysis and death and has previously been associated with colony loss. Uninfected control and infected, symptomatic bees were collected within 20-24 hours after infection. Worker fat bodies, the primary tissue involved in metabolism, detoxification and immune responses, were collected for analysis. We performed transcriptome- and bisulfite-sequencing of the worker fat bodies to identify genome-wide gene expression and DNA methylation patterns associated with viral infection. There were 753 differentially expressed genes (FDR<0.05 in infected versus control bees, including several genes involved in epigenetic and antiviral pathways. DNA methylation status of 156 genes (FDR<0.1 changed significantly as a result of the infection, including those involved in antiviral responses in humans. There was no significant overlap between the significantly differentially expressed and significantly differentially methylated genes, and indeed, the genomic characteristics of these sets of genes were quite distinct. Our results indicate that honey bees have two distinct molecular pathways, mediated by transcription and methylation, that modulate protein levels and/or function in response to viral infections.

  12. Acute Viral Respiratory Infection Rapidly Induces a CD8+ T Cell Exhaustion-like Phenotype.

    Science.gov (United States)

    Erickson, John J; Lu, Pengcheng; Wen, Sherry; Hastings, Andrew K; Gilchuk, Pavlo; Joyce, Sebastian; Shyr, Yu; Williams, John V

    2015-11-01

    Acute viral infections typically generate functional effector CD8(+) T cells (TCD8) that aid in pathogen clearance. However, during acute viral lower respiratory infection, lung TCD8 are functionally impaired and do not optimally control viral replication. T cells also become unresponsive to Ag during chronic infections and cancer via signaling by inhibitory receptors such as programmed cell death-1 (PD-1). PD-1 also contributes to TCD8 impairment during viral lower respiratory infection, but how it regulates TCD8 impairment and the connection between this state and T cell exhaustion during chronic infections are unknown. In this study, we show that PD-1 operates in a cell-intrinsic manner to impair lung TCD8. In light of this, we compared global gene expression profiles of impaired epitope-specific lung TCD8 to functional spleen TCD8 in the same human metapneumovirus-infected mice. These two populations differentially regulate hundreds of genes, including the upregulation of numerous inhibitory receptors by lung TCD8. We then compared the gene expression of TCD8 during human metapneumovirus infection to those in acute or chronic lymphocytic choriomeningitis virus infection. We find that the immunophenotype of lung TCD8 more closely resembles T cell exhaustion late into chronic infection than do functional effector T cells arising early in acute infection. Finally, we demonstrate that trafficking to the infected lung alone is insufficient for TCD8 impairment or inhibitory receptor upregulation, but that viral Ag-induced TCR signaling is also required. Our results indicate that viral Ag in infected lungs rapidly induces an exhaustion-like state in lung TCD8 characterized by progressive functional impairment and upregulation of numerous inhibitory receptors. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. Lineage divergence and historical gene flow in the Chinese horseshoe bat (Rhinolophus sinicus.

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    Xiuguang Mao

    Full Text Available Closely related taxa living in sympatry provide good opportunities to investigate the origin of barriers to gene flow as well as the extent of reproductive isolation. The only two recognized subspecies of the Chinese rufous horseshoe bat Rhinolophus sinicus are characterized by unusual relative distributions in which R. s. septentrionalis is restricted to a small area within the much wider range of its sister taxon R. s. sinicus. To determine the history of lineage divergence and gene flow between these taxa, we applied phylogenetic, demographic and coalescent analyses to multi-locus datasets. MtDNA gene genealogies and microsatellite-based clustering together revealed three divergent lineages of sinicus, corresponding to Central China, East China and the offshore Hainan Island. However, the central lineage of sinicus showed a closer relationship with septentrionalis than with other lineages of R. s. sinicus, in contrary to morphological data. Paraphyly of sinicus could result from either past asymmetric mtDNA introgression between these two taxa, or could suggest septentrionalis evolved in situ from its more widespread sister subspecies. To test between these hypotheses, we applied coalescent-based phylogenetic reconstruction and Approximate Bayesian Computation (ABC. We found that septentrionalis is likely to be the ancestral taxon and therefore a recent origin of this subspecies can be ruled out. On the other hand, we found a clear signature of asymmetric mtDNA gene flow from septentrionalis into central populations of sinicus yet no nuclear gene flow, thus strongly pointing to historical mtDNA introgression. We suggest that the observed deeply divergent lineages within R. sinicus probably evolved in isolation in separate Pleistocene refugia, although their close phylogeographic correspondence with distinct eco-environmental zones suggests that divergent selection might also have promoted broad patterns of population genetic structure.

  14. P53-mediated rapid induction of apoptosis conveys resistance to viral infection in Drosophila melanogaster.

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    Bo Liu

    2013-02-01

    Full Text Available Arthropod-borne pathogens account for millions of deaths each year. Understanding the genetic mechanisms controlling vector susceptibility to pathogens has profound implications for developing novel strategies for controlling insect-transmitted infectious diseases. The fact that many viruses carry genes that have anti-apoptotic activity has long led to the hypothesis that induction of apoptosis could be a fundamental innate immune response. However, the cellular mechanisms mediating the induction of apoptosis following viral infection remained enigmatic, which has prevented experimental verification of the functional significance of apoptosis in limiting viral infection in insects. In addition, studies with cultured insect cells have shown that there is sometimes a lack of apoptosis, or the pro-apoptotic response happens relatively late, thus casting doubt on the functional significance of apoptosis as an innate immunity. Using in vivo mosquito models and the native route of infection, we found that there is a rapid induction of reaper-like pro-apoptotic genes within a few hours following exposure to DNA or RNA viruses. Recapitulating a similar response in Drosophila, we found that this rapid induction of apoptosis requires the function of P53 and is mediated by a stress-responsive regulatory region upstream of reaper. More importantly, we showed that the rapid induction of apoptosis is responsible for preventing the expression of viral genes and blocking the infection. Genetic changes influencing this rapid induction of reaper-like pro-apoptotic genes led to significant differences in susceptibility to viral infection.

  15. Gene therapy: An overview

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    Sudip Indu

    2013-01-01

    Full Text Available Gene therapy "the use of genes as medicine" involves the transfer of a therapeutic or working copy of a gene into specific cells of an individual in order to repair a faulty gene copy. The technique may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. The objective of gene therapy is to introduce new genetic material into target cells while causing no damage to the surrounding healthy cells and tissues, hence the treatment related morbidity is decreased. The delivery system includes a vector that delivers a therapeutic gene into the patient′s target cell. Functional proteins are created from the therapeutic gene causing the cell to return to a normal stage. The vectors used in gene therapy can be viral and non-viral. Gene therapy, an emerging field of biomedicine, is still at infancy and much research remains to be done before this approach to the treatment of condition will realize its full potential.

  16. Evidence for gene flow between two sympatric mealybug species (Insecta; Coccoidea; Pseudococcidae.

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    Hofit Kol-Maimon

    Full Text Available Occurrence of inter-species hybrids in natural populations might be evidence of gene flow between species. In the present study we found evidence of gene flow between two sympatric, genetically related scale insect species--the citrus mealybug Planococcus citri (Risso and the vine mealybug Planococcus ficus (Signoret. These species can be distinguished by morphological, behavioral, and molecular traits. We employed the sex pheromones of the two respective species to study their different patterns of male attraction. We also used nuclear ITS2 (internal transcribed spacer 2 and mitochondrial COI (Cytochrome c oxidase sub unit 1 DNA sequences to characterize populations of the two species, in order to demonstrate the outcome of a possible gene flow between feral populations of the two species. Our results showed attraction to P. ficus pheromones of all tested populations of P. citri males but not vice versa. Furthermore, ITS2 sequences revealed the presence of 'hybrid females' among P. citri populations but not among those of P. ficus. 'hybrid females' from P. citri populations identified as P. citri females according to COI sequences. We offer two hypotheses for these results. 1 The occurrence of phenotypic and genotypic traits of P. ficus in P. citri populations may be attributed to both ancient and contemporary gene flow between their populations; and 2 we cannot rule out that an ancient sympatric speciation by which P. ficus emerged from P. citri might have led to the present situation of shared traits between these species. In light of these findings we also discuss the origin of the studied species and the importance of the pherotype phenomenon as a tool with which to study genetic relationships between congener scale insects.

  17. Genetic clusters and sex-biased gene flow in a unicolonial Formica ant

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    Chapuisat Michel

    2009-03-01

    Full Text Available Abstract Background Animal societies are diverse, ranging from small family-based groups to extraordinarily large social networks in which many unrelated individuals interact. At the extreme of this continuum, some ant species form unicolonial populations in which workers and queens can move among multiple interconnected nests without eliciting aggression. Although unicoloniality has been mostly studied in invasive ants, it also occurs in some native non-invasive species. Unicoloniality is commonly associated with very high queen number, which may result in levels of relatedness among nestmates being so low as to raise the question of the maintenance of altruism by kin selection in such systems. However, the actual relatedness among cooperating individuals critically depends on effective dispersal and the ensuing pattern of genetic structuring. In order to better understand the evolution of unicoloniality in native non-invasive ants, we investigated the fine-scale population genetic structure and gene flow in three unicolonial populations of the wood ant F. paralugubris. Results The analysis of geo-referenced microsatellite genotypes and mitochondrial haplotypes revealed the presence of cryptic clusters of genetically-differentiated nests in the three populations of F. paralugubris. Because of this spatial genetic heterogeneity, members of the same clusters were moderately but significantly related. The comparison of nuclear (microsatellite and mitochondrial differentiation indicated that effective gene flow was male-biased in all populations. Conclusion The three unicolonial populations exhibited male-biased and mostly local gene flow. The high number of queens per nest, exchanges among neighbouring nests and restricted long-distance gene flow resulted in large clusters of genetically similar nests. The positive relatedness among clustermates suggests that kin selection may still contribute to the maintenance of altruism in unicolonial

  18. The population genomics of begomoviruses: global scale population structure and gene flow

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    Prasanna HC

    2010-09-01

    Full Text Available Abstract Background The rapidly growing availability of diverse full genome sequences from across the world is increasing the feasibility of studying the large-scale population processes that underly observable pattern of virus diversity. In particular, characterizing the genetic structure of virus populations could potentially reveal much about how factors such as geographical distributions, host ranges and gene flow between populations combine to produce the discontinuous patterns of genetic diversity that we perceive as distinct virus species. Among the richest and most diverse full genome datasets that are available is that for the dicotyledonous plant infecting genus, Begomovirus, in the Family Geminiviridae. The begomoviruses all share the same whitefly vector, are highly recombinogenic and are distributed throughout tropical and subtropical regions where they seriously threaten the food security of the world's poorest people. Results We focus here on using a model-based population genetic approach to identify the genetically distinct sub-populations within the global begomovirus meta-population. We demonstrate the existence of at least seven major sub-populations that can further be sub-divided into as many as thirty four significantly differentiated and genetically cohesive minor sub-populations. Using the population structure framework revealed in the present study, we further explored the extent of gene flow and recombination between genetic populations. Conclusions Although geographical barriers are apparently the most significant underlying cause of the seven major population sub-divisions, within the framework of these sub-divisions, we explore patterns of gene flow to reveal that both host range differences and genetic barriers to recombination have probably been major contributors to the minor population sub-divisions that we have identified. We believe that the global Begomovirus population structure revealed here could

  19. Integration, viral charge and E2 mRNA levels in the progresion of intraepitelial cervical lesions

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    Esperanza Trujillo

    2018-01-01

    Full Text Available It is important to distinguish among squamous intraepithelial lesions (SIL those associated with increased risk cervical cancer. Our aim was to evaluate if the expression level of gen E2 in women with SIL and evidence of viral integration is associated to the grade of lesion. Cervical scrapes HPV16 positive from 19 women with normal histology, 45 women with low-grade SIL (LSIL and 45 women with high-grade SIL (HSIL were analyzed. Real-time PCR was used to quantify the mRNA of E2 and E2 and E6 genes to calculate viral load (E6 and the ratio E2/E6 to assess viral integration. Similar frequencies of E2 expression were found in LSIL and HSIL15/45 (33 %, the frequency in women without SIL was lower 3/19 (15.8 %, and all cases with E2 gene expression had mixed episomal and integrated viral DNA. The viral load increased significantly with the grade of SIL (p= 0.049, while E2/E6 ratio decreased (p=0.049. The ROC analysis showed low capacity of the three viral parameters analyzed to distinguish between low and high grade SIL. In conclusion although SIL with mixed and integrated viral DNA with E2 expression could be at lower risk of progression, and viral load and integration were associated with higher severity of the lesion, its clinical value as biomarkers of HSIL is limited.

  20. Viral Cre-LoxP tools aid genome engineering in mammalian cells.

    Science.gov (United States)

    Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao

    2017-01-01

    Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

  1. Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

    Science.gov (United States)

    Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

    2013-11-01

    The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

  2. Immune Modulation of NYVAC-Based HIV Vaccines by Combined Deletion of Viral Genes that Act on Several Signalling Pathways

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    Carmen Elena Gómez

    2017-12-01

    Full Text Available An HIV-1 vaccine continues to be a major target to halt the AIDS pandemic. The limited efficacy of the RV144 phase III clinical trial with the canarypox virus-based vector ALVAC and a gp120 protein component led to the conclusion that improved immune responses to HIV antigens are needed for a more effective vaccine. In non-human primates, the New York vaccinia virus (NYVAC poxvirus vector has a broader immunogenicity profile than ALVAC and has been tested in clinical trials. We therefore analysed the HIV immune advantage of NYVAC after removing viral genes that act on several signalling pathways (Toll-like receptors—TLR—interferon, cytokines/chemokines, as well as genes of unknown immune function. We generated a series of NYVAC deletion mutants and studied immune behaviour (T and B cell to HIV antigens and to the NYVAC vector in mice. Our results showed that combined deletion of selected vaccinia virus (VACV genes is a valuable strategy for improving the immunogenicity of NYVAC-based vaccine candidates. These immune responses were differentially modulated, positive or negative, depending on the combination of gene deletions. The deletions also led to enhanced antigen- or vector-specific cellular and humoral responses. These findings will facilitate the development of optimal NYVAC-based vaccines for HIV and other diseases.

  3. Human leukocyte antigen and cytokine receptor gene polymorphisms associated with heterogeneous immune responses to mumps viral vaccine.

    Science.gov (United States)

    Ovsyannikova, Inna G; Jacobson, Robert M; Dhiman, Neelam; Vierkant, Robert A; Pankratz, V Shane; Poland, Gregory A

    2008-05-01

    Mumps outbreaks continue to occur throughout the world, including in highly vaccinated populations. Vaccination against mumps has been successful; however, humoral and cellular immune responses to mumps vaccines vary significantly from person to person. We set out to assess whether HLA and cytokine gene polymorphisms are associated with variations in the immune response to mumps viral vaccine. To identify genetic factors that might contribute to variations in mumps vaccine-induced immune responses, we performed HLA genotyping in a group of 346 healthy schoolchildren (12-18 years of age) who previously received 2 doses of live mumps vaccine. Single-nucleotide polymorphisms (minor allele frequency of >5%) in cytokine and cytokine receptor genes were genotyped for a subset of 118 children. Median values for mumps-specific antibody titers and lymphoproliferative stimulation indices were 729 IU/mL and 4.8, respectively. Girls demonstrated significantly higher mumps antibody titers than boys, indicating gender-linked genetic differences in humoral immune response. Significant associations were found between the HLA-DQB1*0303 alleles and lower mumps-specific antibody titers. An interesting finding was the association of several HLA class II alleles with mumps-specific lymphoproliferation. Alleles of the DRB1 (*0101, *0301, *0801, *1001, *1201, and *1302), DQA1 (*0101, *0105, *0401, and *0501), and DQB1 (*0201, *0402, and *0501) loci were associated with significant variations in lymphoproliferative immune responses to mumps vaccine. Additional associations were observed with single-nucleotide polymorphisms in the interleukin-10RA, interleukin-12RB1, and interleukin-12RB2 cytokine receptor genes. Minor alleles for 4 single-nucleotide polymorphisms within interleukin-10RA and interleukin-12RB genes were associated with variations in humoral and cellular immune responses to mumps vaccination. These data suggest the important role of HLA and immunoregulatory cytokine receptor

  4. A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus.

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    Cui Zhao

    Full Text Available A DNA-binding protein (DBP [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05, indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01. The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01. The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.

  5. IPNV with high and low virulence: host immune responses and viral mutations during infection

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    Skjesol Astrid

    2011-08-01

    Full Text Available Abstract Background Infectious pancreatic necrosis virus (IPNV is an aquatic member of the Birnaviridae family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR, which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described. Methods In this study we compared two field isolates of IPNV (NFH-Ar and NFH-El. The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney. Results Significant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i. was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin was observed at 13 d p.i. (NFH-Ar and 29 d p.i. (both isolates

  6. Interleukin 28B gene variation at rs12979860 determines early viral kinetics during treatment in patients carrying genotypes 2 or 3 of hepatitis C virus

    DEFF Research Database (Denmark)

    Lindh, Magnus; Lagging, Martin; Färkkilä, Martti

    2011-01-01

    Single-nucleotide polymorphisms upstream of the interleukin 28B (interferon λ3) gene (IL28B) strongly influence treatment efficacy in patients carrying hepatitis C virus (HCV) of genotype 1. In patients receiving 12 or 24 weeks of interferon-ribavirin therapy for infection with genotype 2 or 3 (n...... = 341), we found that rs12979860 strikingly determined the first phase of viral elimination (P

  7. Study of male–mediated gene flow across a hybrid zone in the common shrew (Sorex araneus using Y chromosome

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    Andrei V. Polyakov

    2017-06-01

    Full Text Available Despite many studies, the impact of chromosome rearrangements on gene flow between chromosome races of the common shrew (Sorex araneus Linnaeus, 1758 remains unclear. Interracial hybrids form meiotic chromosome complexes that are associated with reduced fertility. Nevertheless comprehensive investigations of autosomal and mitochondrial markers revealed weak or no barrier to gene flow between chromosomally divergent populations. In a narrow zone of contact between the Novosibirsk and Tomsk races hybrids are produced with extraordinarily complex configurations at meiosis I. Microsatellite markers have not revealed any barrier to gene flow, but the phenotypic differentiation between races is greater than may be expected if gene flow was unrestricted. To explore this contradiction we analyzed the distribution of the Y chromosome SNP markers within this hybrid zone. The Y chromosome variants in combination with race specific autosome complements allow backcrosses to be distinguished and their proportion among individuals within the hybrid zone to be evaluated. The balanced ratio of the Y variants observed among the pure race individuals as well as backcrosses reveals no male mediated barrier to gene flow. The impact of reproductive unfitness of backcrosses on gene flow is discussed as a possible mechanism of the preservation of race-specific morphology within the hybrid zone.

  8. Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

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    Dixon Melissa

    2005-07-01

    Full Text Available Abstract Background Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. Results A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. Conclusion These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

  9. Forest corridors maintain historical gene flow in a tiger metapopulation in the highlands of central India.

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    Sharma, Sandeep; Dutta, Trishna; Maldonado, Jesús E; Wood, Thomas C; Panwar, Hemendra Singh; Seidensticker, John

    2013-09-22

    Understanding the patterns of gene flow of an endangered species metapopulation occupying a fragmented habitat is crucial for landscape-level conservation planning and devising effective conservation strategies. Tigers (Panthera tigris) are globally endangered and their populations are highly fragmented and exist in a few isolated metapopulations across their range. We used multi-locus genotypic data from 273 individual tigers (Panthera tigris tigris) from four tiger populations of the Satpura-Maikal landscape of central India to determine whether the corridors in this landscape are functional. This 45 000 km(2) landscape contains 17% of India's tiger population and 12% of its tiger habitat. We applied Bayesian and coalescent-based analyses to estimate contemporary and historical gene flow among these populations and to infer their evolutionary history. We found that the tiger metapopulation in central India has high rates of historical and contemporary gene flow. The tests for population history reveal that tigers populated central India about 10 000 years ago. Their population subdivision began about 1000 years ago and accelerated about 200 years ago owing to habitat fragmentation, leading to four spatially separated populations. These four populations have been in migration-drift equilibrium maintained by high gene flow. We found the highest rates of contemporary gene flow in populations that are connected by forest corridors. This information is highly relevant to conservation practitioners and policy makers, because deforestation, road widening and mining are imminent threats to these corridors.

  10. The full-length E1-circumflexE4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

    International Nuclear Information System (INIS)

    Wilson, Regina; Ryan, Gordon B.; Knight, Gillian L.; Laimins, Laimonis A.; Roberts, Sally

    2007-01-01

    Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1-circumflexE4 protein. To determine the role of E1-circumflexE4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1-circumflexE4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1-circumflexE4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1-circumflexE4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1-circumflexE4 expression, and to HPV16 where only C-terminal truncations in E1-circumflexE4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1-circumflexE4 proteins

  11. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

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    Yarong Liu

    2014-01-01

    Full Text Available Adeno-associated virus type 2 (AAV2 is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs. We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.

  12. Genealogy-based methods for inference of historical recombination and gene flow and their application in Saccharomyces cerevisiae.

    Science.gov (United States)

    Jenkins, Paul A; Song, Yun S; Brem, Rachel B

    2012-01-01

    Genetic exchange between isolated populations, or introgression between species, serves as a key source of novel genetic material on which natural selection can act. While detecting historical gene flow from DNA sequence data is of much interest, many existing methods can be limited by requirements for deep population genomic sampling. In this paper, we develop a scalable genealogy-based method to detect candidate signatures of gene flow into a given population when the source of the alleles is unknown. Our method does not require sequenced samples from the source population, provided that the alleles have not reached fixation in the sampled recipient population. The method utilizes recent advances in algorithms for the efficient reconstruction of ancestral recombination graphs, which encode genealogical histories of DNA sequence data at each site, and is capable of detecting the signatures of gene flow whose footprints are of length up to single genes. Further, we employ a theoretical framework based on coalescent theory to test for statistical significance of certain recombination patterns consistent with gene flow from divergent sources. Implementing these methods for application to whole-genome sequences of environmental yeast isolates, we illustrate the power of our approach to highlight loci with unusual recombination histories. By developing innovative theory and methods to analyze signatures of gene flow from population sequence data, our work establishes a foundation for the continued study of introgression and its evolutionary relevance.

  13. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    Science.gov (United States)

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  14. Early viral replication and induced or constitutive immunity in rainbow trout families with differential resistance to Infectious hematopoietic necrosis virus (IHNV)

    Science.gov (United States)

    Purcell, M.K.; LaPatra, S.E.; Woodson, J.C.; Kurath, G.; Winton, J.R.

    2010-01-01

    The main objective of this study was to assess correlates of innate resistance in rainbow trout full-sibling families that differ in susceptibility to Infectious hematopoietic necrosis virus (IHNV). As part of a commercial breeding program, full-sibling families were challenged with IHNV by waterborne exposure at the 1 g size to determine susceptibility to IHNV. Progeny from select families (N = 7 families) that varied in susceptibility (ranging from 32 to 90% cumulative percent mortality (CPM)) were challenged again at the 10 g size by intra-peritoneal injection and overall mortality, early viral replication and immune responses were evaluated. Mortality challenges included 20–40 fish per family while viral replication and immune response studies included 6 fish per family at each time point (24, 48 and 72 h post-infection (hpi)). CPM at the 1 g size was significantly correlated with CPM at the 10 g size, indicating that inherent resistance was a stable trait irrespective of size. In the larger fish, viral load was measured by quantitative reverse-transcriptase PCR in the anterior kidney and was a significant predictor of family disease outcome at 48 hpi. Type I interferon (IFN) transcript levels were significantly correlated with an individual's viral load at 48 and 72 hpi, while type II IFN gene expression was significantly correlated with an individual's viral load at 24 and 48 hpi. Mean family type I but not type II IFN gene expression was weakly associated with susceptibility at 72 hpi. There was no association between mean family susceptibility and the constitutive expression of a range of innate immune genes (e.g. type I and II IFN pathway genes, cytokine and viral recognition receptor genes). The majority of survivors from the challenge had detectable serum neutralizing antibody titers but no trend was observed among families. This result suggests that even the most resistant families experienced sufficient levels of viral replication to trigger specific

  15. Stem cell gene therapy for HIV: strategies to inhibit viral entry and replication.

    Science.gov (United States)

    DiGiusto, David L

    2015-03-01

    Since the demonstration of a cure of an HIV+ patient with an allogeneic stem cell transplant using naturally HIV-resistant cells, significant interest in creating similar autologous products has fueled the development of a variety of "cell engineering" approaches to stem cell therapy for HIV. Among the more well-studied strategies is the inhibition of viral entry through disruption of expression of viral co-receptors or through competitive inhibitors of viral fusion with the cell membrane. Preclinical evaluation of these approaches often starts in vitro but ultimately is tested in animal models prior to clinical implementation. In this review, we trace the development of several key approaches (meganucleases, short hairpin RNA (shRNA), and fusion inhibitors) to modification of hematopoietic stem cells designed to impart resistance to HIV to their T-cell and monocytic progeny. The basic evolution of technologies through in vitro and in vivo testing is discussed as well as the pros and cons of each approach and how the addition of postentry inhibitors may enhance the overall antiviral efficacy of these approaches.

  16. Integrating gene flow, crop biology, and farm management in on-farm conservation of avocado (Persea americana, Lauraceae).

    Science.gov (United States)

    Birnbaum, Kenneth; Desalle, Rob; Peters, Charles M; Benfey, Philip N

    2003-11-01

    Maintaining crop diversity on farms where cultivars can evolve is a conservation goal, but few tools are available to assess the long-term maintenance of genetic diversity on farms. One important issue for on-farm conservation is gene flow from crops with a narrow genetic base into related populations that are genetically diverse. In a case study of avocado (Persea americana var. americana) in one of its centers of diversity (San Jerónimo, Costa Rica), we used 10 DNA microsatellite markers in a parentage analysis to estimate gene flow from commercialized varieties into a traditional crop population. Five commercialized genotypes comprised nearly 40% of orchard trees, but they contributed only about 14.5% of the gametes to the youngest cohort of trees. Although commercialized varieties and the diverse population were often planted on the same farm, planting patterns appeared to keep the two types of trees separated on small scales, possibly explaining the limited gene flow. In a simulation that combined gene flow estimates, crop biology, and graft tree management, loss of allelic diversity was less than 10% over 150 yr, and selection was effective in retaining desirable alleles in the diverse subpopulation. Simulations also showed that, in addition to gene flow, managing the genetic makeup and life history traits of the invasive commercialized varieties could have a significant impact on genetic diversity in the target population. The results support the feasibility of on-farm crop conservation, but simulations also showed that higher levels of gene flow could lead to severe losses of genetic diversity even if farmers continue to plant diverse varieties.

  17. High rates of gene flow by pollen and seed in oak populations across Europe.

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    Sophie Gerber

    Full Text Available Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423, mapped, and acorns were collected ([17,147], 51 from several mother trees ([3], [47], 23. Seedlings ([65,387], 178 were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5-8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21-88%. Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20-66%. Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.

  18. Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

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    Busch Melanie

    2009-12-01

    Full Text Available Abstract Background Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC on complete Freund's adjuvant-induced inflammatory pain in rats. Results POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin. Conclusion Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.

  19. POLYMORPHIC VARIANTS OF THE GENE OF INTERFERON LAMBDA 3 AND FEATURES OF IMMUNE RESPONSE IN CHILDREN WITH CHRONIC VIRAL HEPATITIS C

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    T. B. Sentsova

    2017-01-01

    Full Text Available To study the immune manifestations of the interferon-lambda 3 genepolymorphism in chronic viral hepatitis C, 110 Russian children (54 girls and 56 boys with chronic HCV infection aged from 3 to 17 years were examined. All children were on combined therapy (pegylated interferon + ribavirin. It was found that among the studied polymorphic variants of the IFN-λ 3 gene in children with chronic HCV infection, T allele of the marker rs12979860 is associated with infection and chronization of HCV. The T/T rs12979860 genotype of the IFN-λ3 gene is unfavorable for the course of chronic HCV infection due to low levels of activated T-lymphocytes, intactness of the proinflammatory cytokines TNF-α, IL-6, IL-1α, and interferon-γ inducible protein IP-10. The revealed relation of the polymorphic variants of C/C + C/T locus rs12979860 of INF-λ3 gene with the expression of activated T-lymphocytes discloses the protective nature of these genotypes to the development of chronic HCV infection in children. 

  20. Morphological differentiation despite gene flow in an endangered grasshopper.

    Science.gov (United States)

    Dowle, Eddy J; Morgan-Richards, Mary; Trewick, Steven A

    2014-10-16

    Gene flow is traditionally considered a limitation to speciation because selection is required to counter the homogenising effect of allele exchange. Here we report on two sympatric short-horned grasshoppers species in the South Island of New Zealand; one (Sigaus australis) widespread and the other (Sigaus childi) a narrow endemic. Of the 79 putatively neutral markers (mtDNA, microsatellite loci, ITS sequences and RAD-seq SNPs) all but one marker we examined showed extensive allele sharing, and similar or identical allele frequencies in the two species where they co-occur. We found no genetic evidence of deviation from random mating in the region of sympatry. However, analysis of morphological and geometric traits revealed no evidence of morphological introgression. Based on phenotype the two species are clearly distinct, but their genotypes thus far reveal no divergence. The best explanation for this is that some loci associated with the distinguishing morphological characters are under strong selection, but exchange of neutral loci is occurring freely between the two species. Although it is easier to define species as requiring a barrier between them, a dynamic model that accommodates gene flow is a biologically more reasonable explanation for these grasshoppers.

  1. Viral RNAi suppressor reversibly binds siRNA to outcompete Dicer and RISC via multiple turnover.

    Science.gov (United States)

    Rawlings, Renata A; Krishnan, Vishalakshi; Walter, Nils G

    2011-04-29

    RNA interference is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response to viruses and retrotransposons. During viral infection, the RNase-III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs) 21-24 nucleotides in length and helps load them into the RNA-induced silencing complex (RISC) to guide the cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressors (RSS) that tightly, and presumably quantitatively, bind siRNAs to thwart RNA-interference-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus, as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding [(1.69 ± 0.07) × 10(8) M(-)(1) s(-1)] and marked dissociation (k(off)=0.062 ± 0.002 s(-1)). We also observe that p19 efficiently competes with recombinant Dicer and inhibits the formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Viral RNAi suppressor reversibly binds siRNA to outcompete Dicer and RISC via multiple-turnover

    Science.gov (United States)

    Rawlings, Renata A.; Krishnan, Vishalakshi; Walter, Nils G.

    2011-01-01

    RNA interference (RNAi) is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response against viruses and retrotransposons. During viral infection, the RNase III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs), 21–24 nucleotides in length, and helps load them into the RNA-induced silencing complex (RISC) to guide cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressor (RSS) proteins that tightly, and presumably quantitatively, bind siRNAs to thwart RNAi-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus (CIRV), as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding ((1.69 ± 0.07)×108 M−1s−1) and marked dissociation (koff = 0.062 ± 0.002 s−1). We also observe that p19 efficiently competes with recombinant Dicer and inhibits formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple-turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy. PMID:21354178

  3. Genetic structure and gene flow among Komodo dragon populations inferred by microsatellite loci analysis.

    Science.gov (United States)

    Ciofi, C; Bruford, M W

    1999-12-01

    A general concern for the conservation of endangered species is the maintenance of genetic variation within populations, particularly when they become isolated and reduced in size. Estimates of gene flow and effective population size are therefore important for any conservation initiative directed to the long-term persistence of a species in its natural habitat. In the present study, 10 microsatellite loci were used to assess the level of genetic variability among populations of the Komodo dragon Varanus komodoensis. Effective population size was calculated and gene flow estimates were compared with palaeogeographic data in order to assess the degree of vulnerability of four island populations. Rinca and Flores, currently separated by an isthmus of about 200 m, retained a high level of genetic diversity and showed a high degree of genetic similarity, with gene flow values close to one migrant per generation. The island of Komodo showed by far the highest levels of genetic divergence, and its allelic distinctiveness was considered of great importance in the maintenance of genetic variability within the species. A lack of distinct alleles and low levels of gene flow and genetic variability were found for the small population of Gili Motang island, which was identified as vulnerable to stochastic threats. Our results are potentially important for both the short- and long-term management of the Komodo dragon, and are critical in view of future re-introduction or augmentation in areas where the species is now extinct or depleted.

  4. Asian wild rice is a hybrid swarm with extensive gene flow and feralization from domesticated rice.

    Science.gov (United States)

    Wang, Hongru; Vieira, Filipe G; Crawford, Jacob E; Chu, Chengcai; Nielsen, Rasmus

    2017-06-01

    The domestication history of rice remains controversial, with multiple studies reaching different conclusions regarding its origin(s). These studies have generally assumed that populations of living wild rice, O. rufipogon , are descendants of the ancestral population that gave rise to domesticated rice, but relatively little attention has been paid to the origins and history of wild rice itself. Here, we investigate the genetic ancestry of wild rice by analyzing a diverse panel of rice genomes consisting of 203 domesticated and 435 wild rice accessions. We show that most modern wild rice is heavily admixed with domesticated rice through both pollen- and seed-mediated gene flow. In fact, much presumed wild rice may simply represent different stages of feralized domesticated rice. In line with this hypothesis, many presumed wild rice varieties show remnants of the effects of selective sweeps in previously identified domestication genes, as well as evidence of recent selection in flowering genes possibly associated with the feralization process. Furthermore, there is a distinct geographical pattern of gene flow from aus , indica , and japonica varieties into colocated wild rice. We also show that admixture from aus and indica is more recent than gene flow from japonica , possibly consistent with an earlier spread of japonica varieties. We argue that wild rice populations should be considered a hybrid swarm, connected to domesticated rice by continuous and extensive gene flow. © 2017 Wang et al.; Published by Cold Spring Harbor Laboratory Press.

  5. Sex− and species−biased gene flow in a spotted eagle hybrid zone

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    Väli Ülo

    2011-04-01

    Full Text Available Abstract Background Recent theoretical and empirical work points toward a significant role for sex-chromosome linked genes in the evolution of traits that induce reproductive isolation and for traits that evolve under influence of sexual selection. Empirical studies including recently diverged (Pleistocene, short-lived avian species pairs with short generation times have found that introgression occurs on the autosomes but not on the Z-chromosome. Here we study genetic differentiation and gene flow in the long-lived greater spotted eagle (Aquila clanga and lesser spotted eagle (A. pomarina, two species with comparatively long generation times. Results Our data suggest that there is a directional bias in migration rates between hybridizing spotted eagles in eastern Europe. We find that a model including post divergence gene flow fits our data best for both autosomal and Z-chromosome linked loci but, for the Z-chromosome, the rate is reduced in the direction from A. pomarina to A. clanga. Conclusions The fact that some introgression still occurs on the Z-chromosome between these species suggests that the differentiation process is in a more premature phase in our study system than in previously studied avian species pairs and that could be explained by a shorter divergence time and/or a longer average generation time in the spotted eagles. The results are in agreement with field observations and provide further insight into the role of sex-linked loci for the build-up of barriers to gene flow among diverging populations and species.

  6. Myxovirus resistance 1 gene polymorphisms and outcomes of viral hepatitis B and C infections in Moroccan patients.

    Science.gov (United States)

    Rebbani, Khadija; Ababou, Mostafa; Nadifi, Sellama; Kandil, Mostafa; Marchio, Agnès; Pineau, Pascal; Ezzikouri, Sayeh; Benjelloun, Soumaya

    2017-04-01

    Host genetic factors may influence the establishment of chronicity or spontaneous clearance in viral hepatitis B and C infections. More light was shed on the role played by interferon-stimulated genes in the innate immunity. Myxovirus resistance 1 (MX1) is one of those key genes that have reported to inhibit several viruses. The present study aims to explore the possible association of -88G/T and -123C/A promoter variants of MX1 with susceptibility to chronic hepatitis B and C and/or with spontaneous clearance in a Moroccan population. The -88G/T and -123C/A SNPs were genotyped by PCR-RFLP in 538 individuals stratified into HBV chronically infected patients (n = 120), HCV-chronically infected patients (n = 115), HBV spontaneously resolved subjects (n = 114), HCV spontaneously resolved group (n = 52), and healthy controls (n = 137). A significant association of -123C allele with HBV spontaneous clearance has been found (P = 0.002, OR = 2.34; 95%CI [1.36-4]). In addition, a significant correlation between the MX1-GC haplotype and HBV spontaneous clearance (P C/A polymorphisms with regard to HCV infection was observed in this study. Here, we show that for North African patients with chronic hepatitis, MX1 gene variation at position -123 may influence the outcome of HBV infection but not HCV infection. J. Med. Virol. 89:647-652, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Intrinsic incompatibilities evolving as a by-product of divergent ecological selection: Considering them in empirical studies on divergence with gene flow.

    Science.gov (United States)

    Kulmuni, J; Westram, A M

    2017-06-01

    The possibility of intrinsic barriers to gene flow is often neglected in empirical research on local adaptation and speciation with gene flow, for example when interpreting patterns observed in genome scans. However, we draw attention to the fact that, even with gene flow, divergent ecological selection may generate intrinsic barriers involving both ecologically selected and other interacting loci. Mechanistically, the link between the two types of barriers may be generated by genes that have multiple functions (i.e., pleiotropy), and/or by gene interaction networks. Because most genes function in complex networks, and their evolution is not independent of other genes, changes evolving in response to ecological selection can generate intrinsic barriers as a by-product. A crucial question is to what extent such by-product barriers contribute to divergence and speciation-that is whether they stably reduce gene flow. We discuss under which conditions by-product barriers may increase isolation. However, we also highlight that, depending on the conditions (e.g., the amount of gene flow and the strength of selection acting on the intrinsic vs. the ecological barrier component), the intrinsic incompatibility may actually destabilize barriers to gene flow. In practice, intrinsic barriers generated as a by-product of divergent ecological selection may generate peaks in genome scans that cannot easily be interpreted. We argue that empirical studies on divergence with gene flow should consider the possibility of both ecological and intrinsic barriers. Future progress will likely come from work combining population genomic studies, experiments quantifying fitness and molecular studies on protein function and interactions. © 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

  8. Distribution of T Cells in Rainbow Trout (Oncorhynchus mykiss) Skin and Responsiveness to Viral Infection

    Science.gov (United States)

    Leal, Esther; Granja, Aitor G.; Zarza, Carlos; Tafalla, Carolina

    2016-01-01

    Although the skin constitutes the first line of defense against waterborne pathogens, there is a great lack of information regarding the skin associated lymphoid tissue (SALT) and whether immune components of the skin are homogeneously distributed through the surface of the fish is still unknown. In the current work, we have analyzed the transcription of several immune genes throughout different rainbow trout (Oncorhynchus mykiss) skin areas. We found that immunoglobulin and chemokine gene transcription levels were higher in a skin area close to the gills. Furthermore, this skin area as well as other anterior sections also transcribed significantly higher levels of many different immune genes related to T cell immunity such as T cell receptor α (TCRα), TCRγ, CD3, CD4, CD8, perforin, GATA3, Tbet, FoxP3, interferon γ (IFNγ), CD40L and Eomes in comparison to posterior skin sections. In agreement with these results, immunohistochemical analysis revealed that anterior skin areas had a higher concentration of CD3+ T cells and flow cytometry analysis confirmed that the percentage of CD8+ T lymphocytes was also higher in anterior skin sections. These results demonstrate for the first time that T cells are not homogeneously distributed throughout the teleost skin. Additionally, we studied the transcriptional regulation of these and additional T cell markers in response to a bath infection with viral hemorrhagic septicemia virus (VHSV). We found that VHSV regulated the transcription of several of these T cell markers in both the skin and the spleen; with some differences between anterior and posterior skin sections. Altogether, our results point to skin T cells as major players of teleost skin immunity in response to waterborne viral infections. PMID:26808410

  9. Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral hemorrhagic septicemia virus, a fish rhabdovirus

    Science.gov (United States)

    Benmansour, A.; Bascuro, B.; Monnier, A.F.; Vende, P.; Winton, J.R.; de Kinkelin, P.

    1997-01-01

    To evaluate the genetic diversity of viral haemorrhagic septicaemia virus (VHSV), the sequence of the glycoprotein genes (G) of 11 North American and European isolates were determined. Comparison with the G protein of representative members of the family Rhabdoviridae suggested that VHSV was a different virus species from infectious haemorrhagic necrosis virus (IHNV) and Hirame rhabdovirus (HIRRV). At a higher taxonomic level, VHSV, IHNV and HIRRV formed a group which was genetically closest to the genus Lyssavirus. Compared with each other, the G genes of VHSV displayed a dissimilar overall genetic diversity which correlated with differences in geographical origin. The multiple sequence alignment of the complete G protein, showed that the divergent positions were not uniformly distributed along the sequence. A central region (amino acid position 245-300) accumulated substitutions and appeared to be highly variable. The genetic heterogeneity within a single isolate was high, with an apparent internal mutation frequency of 1.2 x 10(-3) per nucleotide site, attesting the quasispecies nature of the viral population. The phylogeny separated VHSV strains according to the major geographical area of isolation: genotype I for continental Europe, genotype II for the British Isles, and genotype III for North America. Isolates from continental Europe exhibited the highest genetic variability, with sub-groups correlated partially with the serological classification. Neither neutralizing polyclonal sera, nor monoclonal antibodies, were able to discriminate between the genotypes. The overall structure of the phylogenetic tree suggests that VHSV genetic diversity and evolution fit within the model of random change and positive selection operating on quasispecies.

  10. [Investigation of RNA viral genome amplification by multiple displacement amplification technique].

    Science.gov (United States)

    Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

    2013-06-01

    In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

  11. Viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis.

    Directory of Open Access Journals (Sweden)

    Ashish V Chintakuntlawar

    2010-04-01

    Full Text Available Human adenovirus (HAdV infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9 signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/- mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD. These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.

  12. MicroRNAs: role and therapeutic targets in viral hepatitis

    NARCIS (Netherlands)

    van der Ree, Meike H.; de Bruijne, Joep; Kootstra, Neeltje A.; Jansen, Peter Lm; Reesink, Hendrik W.

    2014-01-01

    MicroRNAs regulate gene expression by binding to the 3'-untranslated region (UTR) of target messenger RNAs (mRNAs). The importance of microRNAs has been shown for several liver diseases, for example, viral hepatitis. MicroRNA-122 is highly abundant in the liver and is involved in the regulation of

  13. CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b

    Science.gov (United States)

    He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin

    2017-01-01

    Abstract CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. PMID:27980065

  14. A viral microRNA functions as an ortholog of cellular miR-155

    Science.gov (United States)

    Gottwein, Eva; Mukherjee, Neelanjan; Sachse, Christoph; Frenzel, Corina; Majoros, William H.; Chi, Jen-Tsan A.; Braich, Ravi; Manoharan, Muthiah; Soutschek, Jürgen; Ohler, Uwe; Cullen, Bryan R.

    2008-01-01

    All metazoan eukaryotes express microRNAs (miRNAs), ∼22 nt regulatory RNAs that can repress the expression of mRNAs bearing complementary sequences1. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis2. While specific viral miRNAs have been shown to autoregulate viral mRNAs3,4 or downregulate cellular mRNAs5,6, the function of the majority of viral miRNAs remains unknown. Here, we report that the miR-K12−11 miRNA encoded by Kaposi's Sarcoma Associated Herpesvirus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA “seed” region7. Using a range of assays, we demonstrate that expression of physiological levels of miR-K12−11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12−11 functions as an ortholog of cellular miR-155 and has likely evolved to exploit a pre-existing gene regulatory pathway in B-cells. Moreover, the known etiological role of miR-155 in B-cell transformation8-10 suggests that miR-K12−11 may contribute to the induction of KSHV-positive B-cell tumors in infected patients. PMID:18075594

  15. Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy.

    Directory of Open Access Journals (Sweden)

    Jitka Holcakova

    Full Text Available Cyclin-dependent kinases (CDKs are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional

  16. Viral vector-based tools advance knowledge of basal ganglia anatomy and physiology.

    Science.gov (United States)

    Sizemore, Rachel J; Seeger-Armbruster, Sonja; Hughes, Stephanie M; Parr-Brownlie, Louise C

    2016-04-01

    Viral vectors were originally developed to deliver genes into host cells for therapeutic potential. However, viral vector use in neuroscience research has increased because they enhance interpretation of the anatomy and physiology of brain circuits compared with conventional tract tracing or electrical stimulation techniques. Viral vectors enable neuronal or glial subpopulations to be labeled or stimulated, which can be spatially restricted to a single target nucleus or pathway. Here we review the use of viral vectors to examine the structure and function of motor and limbic basal ganglia (BG) networks in normal and pathological states. We outline the use of viral vectors, particularly lentivirus and adeno-associated virus, in circuit tracing, optogenetic stimulation, and designer drug stimulation experiments. Key studies that have used viral vectors to trace and image pathways and connectivity at gross or ultrastructural levels are reviewed. We explain how optogenetic stimulation and designer drugs used to modulate a distinct pathway and neuronal subpopulation have enhanced our mechanistic understanding of BG function in health and pathophysiology in disease. Finally, we outline how viral vector technology may be applied to neurological and psychiatric conditions to offer new treatments with enhanced outcomes for patients. Copyright © 2016 the American Physiological Society.

  17. Effects of Interferon-α/β on HBV Replication Determined by Viral Load

    Science.gov (United States)

    Tian, Yongjun; Chen, Wen-ling; Ou, Jing-hsiung James

    2011-01-01

    Interferons α and β (IFN-α/β) are type I interferons produced by the host to control microbial infections. However, the use of IFN-α to treat hepatitis B virus (HBV) patients generated sustained response to only a minority of patients. By using HBV transgenic mice as a model and by using hydrodynamic injection to introduce HBV DNA into the mouse liver, we studied the effect of IFN-α/β on HBV in vivo. Interestingly, our results indicated that IFN-α/β could have opposite effects on HBV: they suppressed HBV replication when viral load was high and enhanced HBV replication when viral load was low. IFN-α/β apparently suppressed HBV replication via transcriptional and post-transcriptional regulations. In contrast, IFN-α/β enhanced viral replication by inducing the transcription factor HNF3γ and activating STAT3, which together stimulated HBV gene expression and replication. Further studies revealed an important role of IFN-α/β in stimulating viral growth and prolonging viremia when viral load is low. This use of an innate immune response to enhance its replication and persistence may represent a novel strategy that HBV uses to enhance its growth and spread in the early stage of viral infection when the viral level is low. PMID:21829354

  18. Interferon Response and Viral Evasion by Members of the Family Rhabdoviridae

    Directory of Open Access Journals (Sweden)

    Matthias J. Schnell

    2009-11-01

    Full Text Available Like many animal viruses, those of the Rhabdoviridae family, are able to antagonize the type I interferon response and cause disease in mammalian hosts. Though these negative-stranded RNA viruses are very simple and code for as few as five proteins, they have been seen to completely abrogate the type I interferon response early in infection. In this review, we will discuss the viral organization and type I interferon evasion of rhabdoviruses, focusing on vesicular stomatitis virus (VSV and rabies virus (RABV. Despite their structural similarities, VSV and RABV have completely different mechanisms by which they avert the host immune response. VSV relies on the matrix protein to interfere with host gene transcription and nuclear export of anti-viral mRNAs. Alternatively, RABV uses its phosphoprotein to interfere with IRF-3 phosphorylation and STAT1 signaling. Understanding the virus-cell interactions and viral proteins necessary to evade the immune response is important in developing effective vaccines and therapeutics for this viral family.

  19. A Viral RNA Structural Element Alters Host Recognition of Nonself RNA

    Energy Technology Data Exchange (ETDEWEB)

    Hyde, J. L.; Gardner, C. L.; Kimura, T.; White, J. P.; Liu, G.; Trobaugh, D. W.; Huang, C.; Tonelli, M.; Paessler, S.; Takeda, K.; Klimstra, W. B.; Amarasinghe, G. K.; Diamond, M. S.

    2014-01-30

    Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response. As an example, 2'-O methylation of the 5' cap of viral RNA subverts mammalian antiviral responses by evading restriction of Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing Ifit1 even though their genomic RNA has a 5' cap lacking 2'-O methylation. We show that pathogenic alphaviruses use secondary structural motifs within the 5' untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5'-UTR affecting RNA structural elements enabled restriction by or antagonism of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid immune restriction.

  20. A flood-based information flow analysis and network minimization method for gene regulatory networks.

    Science.gov (United States)

    Pavlogiannis, Andreas; Mozhayskiy, Vadim; Tagkopoulos, Ilias

    2013-04-24

    Biological networks tend to have high interconnectivity, complex topologies and multiple types of interactions. This renders difficult the identification of sub-networks that are involved in condition- specific responses. In addition, we generally lack scalable methods that can reveal the information flow in gene regulatory and biochemical pathways. Doing so will help us to identify key participants and paths under specific environmental and cellular context. This paper introduces the theory of network flooding, which aims to address the problem of network minimization and regulatory information flow in gene regulatory networks. Given a regulatory biological network, a set of source (input) nodes and optionally a set of sink (output) nodes, our task is to find (a) the minimal sub-network that encodes the regulatory program involving all input and output nodes and (b) the information flow from the source to the sink nodes of the network. Here, we describe a novel, scalable, network traversal algorithm and we assess its potential to achieve significant network size reduction in both synthetic and E. coli networks. Scalability and sensitivity analysis show that the proposed method scales well with the size of the network, and is robust to noise and missing data. The method of network flooding proves to be a useful, practical approach towards information flow analysis in gene regulatory networks. Further extension of the proposed theory has the potential to lead in a unifying framework for the simultaneous network minimization and information flow analysis across various "omics" levels.

  1. Reading the viral signature by Toll-like receptors and other pattern recognition receptors.

    Science.gov (United States)

    Mogensen, Trine H; Paludan, Søren R

    2005-03-01

    Successful host defense against viral infections relies on early production of type I interferon (IFN) and subsequent activation of a cellular cytotoxic response. The acute IFN and inflammatory response against virus infections is mediated by cellular pattern-recognition receptors (PRRs) that recognize specific molecular structures on viral particles or products of viral replication. Toll-like receptors (TLRs) constitute a class of membrane-bound PRRs capable of detecting microbial infections. While TLR2 and TLR4, which were first identified to recognize Gram-positive and Gram-negative bacteria, respectively, sense specific viral proteins on the cell surface, TLRs 3, 7, 8, and 9 serve as receptors for viral nucleic acids in endosomic compartments. In addition to TLRs, cells express cytoplasmic PRRs such as the RNA helicase retinoic acid inducible gene I and the kinase double-stranded RNA-activated protein kinase R, both of which sense dsRNA, a characteristic signature of viral replication, and initiate a protective cellular response. Here we review the recent progress in our understanding of PRRs and viral infections and discuss the molecular and cellular responses evoked by virus-activated PRRs. Finally, we look into what is currently known about the role of PRRs in viral infections in vivo.

  2. A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

    Directory of Open Access Journals (Sweden)

    Melanie D. White

    2011-07-01

    Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

  3. Gene therapy in cystic fibrosis.

    Science.gov (United States)

    Flotte, T R; Laube, B L

    2001-09-01

    Theoretically, cystic fibrosis transmembrane conductance regulator (CFTR) gene replacement during the neonatal period can decrease morbidity and mortality from cystic fibrosis (CF). In vivo gene transfers have been accomplished in CF patients. Choice of vector, mode of delivery to airways, translocation of genetic information, and sufficient expression level of the normalized CFTR gene are issues that currently are being addressed in the field. The advantages and limitations of viral vectors are a function of the parent virus. Viral vectors used in this setting include adenovirus (Ad) and adeno-associated virus (AAV). Initial studies with Ad vectors resulted in a vector that was efficient for gene transfer with dose-limiting inflammatory effects due to the large amount of viral protein delivered. The next generation of Ad vectors, with more viral coding sequence deletions, has a longer duration of activity and elicits a lesser degree of cell-mediated immunity in mice. A more recent generation of Ad vectors has no viral genes remaining. Despite these changes, the problem of humoral immunity remains with Ad vectors. A variety of strategies such as vector systems requiring single, or widely spaced, administrations, pharmacologic immunosuppression at administration, creation of a stealth vector, modification of immunogenic epitopes, or tolerance induction are being considered to circumvent humoral immunity. AAV vectors have been studied in animal and human models. They do not appear to induce inflammatory changes over a wide range of doses. The level of CFTR messenger RNA expression is difficult to ascertain with AAV vectors since the small size of the vector relative to the CFTR gene leaves no space for vector-specific sequences on which to base assays to distinguish endogenous from vector-expressed messenger RNA. In general, AAV vectors appear to be safe and have superior duration profiles. Cationic liposomes are lipid-DNA complexes. These vectors generally have been

  4. Consequences of population topology for studying gene flow using link-based landscape genetic methods.

    Science.gov (United States)

    van Strien, Maarten J

    2017-07-01

    Many landscape genetic studies aim to determine the effect of landscape on gene flow between populations. These studies frequently employ link-based methods that relate pairwise measures of historical gene flow to measures of the landscape and the geographical distance between populations. However, apart from landscape and distance, there is a third important factor that can influence historical gene flow, that is, population topology (i.e., the arrangement of populations throughout a landscape). As the population topology is determined in part by the landscape configuration, I argue that it should play a more prominent role in landscape genetics. Making use of existing literature and theoretical examples, I discuss how population topology can influence results in landscape genetic studies and how it can be taken into account to improve the accuracy of these results. In support of my arguments, I have performed a literature review of landscape genetic studies published during the first half of 2015 as well as several computer simulations of gene flow between populations. First, I argue why one should carefully consider which population pairs should be included in link-based analyses. Second, I discuss several ways in which the population topology can be incorporated in response and explanatory variables. Third, I outline why it is important to sample populations in such a way that a good representation of the population topology is obtained. Fourth, I discuss how statistical testing for link-based approaches could be influenced by the population topology. I conclude the article with six recommendations geared toward better incorporating population topology in link-based landscape genetic studies.

  5. Identification of the two rotavirus genes determining neutralization specificities

    International Nuclear Information System (INIS)

    Offit, P.A.; Blavat, G.

    1986-01-01

    Bovine rotavirus NCDV and simian rotavirus SA-11 represent two distinct rotavirus serotypes. A genetic approach was used to determine which viral gene segments segregated with serotype-specific viral neutralization. There were 16 reassortant rotarviruses derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant rotavirus double-stranded RNA segments was determined by gene segment mobility in polyacrylamide gels and by hybridization with radioactively labeled parental viral transcripts. The authors found that two rotavirus gene segments found previously to code for outer capsid proteins vp3 and vp7 cosegreated with virus neutralization specificities

  6. Identification of the two rotavirus genes determining neutralization specificities

    Energy Technology Data Exchange (ETDEWEB)

    Offit, P.A.; Blavat, G.

    1986-01-01

    Bovine rotavirus NCDV and simian rotavirus SA-11 represent two distinct rotavirus serotypes. A genetic approach was used to determine which viral gene segments segregated with serotype-specific viral neutralization. There were 16 reassortant rotarviruses derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant rotavirus double-stranded RNA segments was determined by gene segment mobility in polyacrylamide gels and by hybridization with radioactively labeled parental viral transcripts. The authors found that two rotavirus gene segments found previously to code for outer capsid proteins vp3 and vp7 cosegreated with virus neutralization specificities.

  7. MAINTENANCE OF ECOLOGICALLY SIGNIFICANT GENETIC VARIATION IN THE TIGER SWALLOWTAIL BUTTERFLY THROUGH DIFFERENTIAL SELECTION AND GENE FLOW.

    Science.gov (United States)

    Bossart, J L; Scriber, J M

    1995-12-01

    Differential selection in a heterogeneous environment is thought to promote the maintenance of ecologically significant genetic variation. Variation is maintained when selection is counterbalanced by the homogenizing effects of gene flow and random mating. In this study, we examine the relative importance of differential selection and gene flow in maintaining genetic variation in Papilio glaucus. Differential selection on traits contributing to successful use of host plants (oviposition preference and larval performance) was assessed by comparing the responses of southern Ohio, north central Georgia, and southern Florida populations of P. glaucus to three hosts: Liriodendron tulipifera, Magnolia virginiana, and Prunus serotina. Gene flow among populations was estimated using allozyme frequencies from nine polymorphic loci. Significant genetic differentiation was observed among populations for both oviposition preference and larval performance. This differentiation was interpreted to be the result of selection acting on Florida P. glaucus for enhanced use of Magnolia, the prevalent host in Florida. In contrast, no evidence of population differentiation was revealed by allozyme frequencies. F ST -values were very small and Nm, an estimate of the relative strengths of gene flow and genetic drift, was large, indicating that genetic exchange among P. glaucus populations is relatively unrestricted. The contrasting patterns of spatial differentiation for host-use traits and lack of differentiation for electrophoretically detectable variation implies that differential selection among populations will be counterbalanced by gene flow, thereby maintaining genetic variation for host-use traits. © 1995 The Society for the Study of Evolution.

  8. Local evolution of pyrethroid resistance offsets gene flow among Aedes aegypti collections in Yucatan State, Mexico.

    Science.gov (United States)

    Saavedra-Rodriguez, Karla; Beaty, Meaghan; Lozano-Fuentes, Saul; Denham, Steven; Garcia-Rejon, Julian; Reyes-Solis, Guadalupe; Machain-Williams, Carlos; Loroño-Pino, Maria Alba; Flores-Suarez, Adriana; Ponce-Garcia, Gustavo; Beaty, Barry; Eisen, Lars; Black, William C

    2015-01-01

    The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1-4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow. © The American Society of Tropical Medicine and Hygiene.

  9. CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice

    Directory of Open Access Journals (Sweden)

    Oscar Meca-Cortés

    2017-09-01

    Full Text Available The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs, using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene, under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells. Keywords: CRISPR/Cas9, cell therapy, mesenchymal stem cells, bystander suicide therapy, glioblastoma, non-invasive bioluminescence imaging, CRISPR/Cas9, CRISPR/Cas9 knockin

  10. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    International Nuclear Information System (INIS)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-01-01

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  11. Does gene flow constrain adaptive divergence or vice versa? A test using ecomorphology and sexual isolation in Timema cristinae walking-sticks.

    Science.gov (United States)

    Nosil, P; Crespi, B J

    2004-01-01

    Population differentiation often reflects a balance between divergent natural selection and the opportunity for homogenizing gene flow to erode the effects of selection. However, during ecological speciation, trait divergence results in reproductive isolation and becomes a cause, rather than a consequence, of reductions in gene flow. To assess both the causes and the reproductive consequences of morphological differentiation, we examined morphological divergence and sexual isolation among 17 populations of Timema cristinae walking-sticks. Individuals from populations adapted to using Adenostoma as a host plant tended to exhibit smaller overall body size, wide heads, and short legs relative to individuals using Ceonothus as a host. However, there was also significant variation in morphology among populations within host-plant species. Mean trait values for each single population could be reliably predicted based upon host-plant used and the potential for homogenizing gene flow, inferred from the size of the neighboring population using the alternate host and mitochondrial DNA estimates of gene flow. Morphology did not influence the probability of copulation in between-population mating trials. Thus, morphological divergence is facilitated by reductions in gene flow, but does not cause reductions in gene flow via the evolution of sexual isolation. Combined with rearing data indicating that size and shape have a partial genetic basis, evidence for parallel origins of the host-associated forms, and inferences from functional morphology, these results indicate that morphological divergence in T. cristinae reflects a balance between the effects of host-specific natural selection and gene flow. Our findings illustrate how data on mating preferences can help determine the causal associations between trait divergence and levels of gene flow.

  12. ORF18 is a transfactor that is essential for late gene transcription of a gammaherpesvirus.

    Science.gov (United States)

    Arumugaswami, Vaithilingaraja; Wu, Ting-Ting; Martinez-Guzman, DeeAnn; Jia, Qingmei; Deng, Hongyu; Reyes, Nichole; Sun, Ren

    2006-10-01

    Lytic replication of the tumor-associated human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus has important implications in pathogenesis and tumorigenesis. Herpesvirus lytic genes have been temporally classified as exhibiting immediate-early (IE), early, and late expression kinetics. Though the regulation of IE and early gene expression has been studied extensively, very little is known regarding the regulation of late gene expression. Late genes, which primarily encode virion structural proteins, require viral DNA replication for their expression. We have identified a murine gammaherpesvirus 68 (MHV-68) early lytic gene, ORF18, essential for viral replication. ORF18 is conserved in both beta- and gammaherpesviruses. By generating an MHV-68 ORF18-null virus, we characterized the stage of the virus lytic cascade that requires the function of ORF18. Gene expression profiling and quantitation of viral DNA synthesis of the ORF18-null virus revealed that the expression of early genes and viral DNA replication were not affected; however, the transcription of late genes was abolished. Hence, we have identified a gammaherpesvirus-encoded factor essential for the expression of late genes independently of viral DNA synthesis.

  13. Viral (hepatitis C virus, hepatitis B virus, HIV) persistence and immune homeostasis

    Science.gov (United States)

    Zhou, Yun; Zhang, Ying; Moorman, Jonathan P; Yao, Zhi Q; Jia, Zhan S

    2014-01-01

    Immune homeostasis is a host characteristic that maintains biological balance within a host. Humans have evolved many host defence mechanisms that ensure the survival of individuals upon encountering a pathogenic infection, with recovery or persistence from a viral infection being determined by both viral factors and host immunity. Chronic viral infections, such as hepatitis B virus, hepatitis C virus and HIV, often result in chronic fluctuating viraemia in the face of host cellular and humoral immune responses, which are dysregulated by multi-faceted mechanisms that are incompletely understood. This review attempts to illuminate the mechanisms involved in this process, focusing on immune homeostasis in the setting of persistent viral infection from the aspects of host defence mechanism, including interferon-stimulated genes, apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3), autophagy and interactions of various immune cells, cytokines and regulatory molecules. PMID:24965611

  14. Options to Expand HIV Viral Load Testing in South Africa: Evaluation of the GeneXpert® HIV-1 Viral Load Assay.

    Directory of Open Access Journals (Sweden)

    Natasha Gous

    Full Text Available Expansion of HIV viral load (VL testing services are required to meet increased targets for monitoring patients on antiretroviral treatment. South Africa currently tests >4million VLs per annum in 16 highly centralised, automated high-throughput laboratories. The Xpert HIV-1 VL assay (Cepheid was evaluated against in-country predicates, the Roche Cobas Taqmanv2 and Abbott HIV-1RT, to investigate options for expanding VL testing using GeneXpert's random access, polyvalent capabilities and already established footprint in South Africa with the Xpert MTB/RIF assay (207 sites. Additionally, the performance of Xpert HIV-1VL on alternative, off-label specimen types, Dried Blood Spots (DBS and whole blood, was investigated.Precision, accuracy (agreement and clinical misclassification (1000cp/ml of Xpert HIV-1VL plasma was compared to Taqmanv2 (n = 155 and Abbott HIV-1 RT (n = 145. Misclassification of Xpert HIV-1VL was further tested on DBS (n = 145 and whole blood (n = 147.Xpert HIV-1VL demonstrated 100% concordance with predicate platforms on a standardised frozen, plasma panel (n = 42 and low overall percentage similarity CV of 1.5% and 0.9% compared to Taqmanv2 and Abbott HIV-1 RT, respectively. On paired plasma clinical specimens, Xpert HIV-1VL had low bias (SD 0.32-0.37logcp/ml and 3% misclassification at the 1000cp/ml threshold compared to Taqmanv2 (fresh and Abbott HIV-1 RT (frozen, respectively. Xpert HIV-1VL on whole blood and DBS increased misclassification (upward by up to 14% with increased invalid rate. All specimen testing was easy to perform and compatible with concurrent Xpert MTB/RIF Tuberculosis testing on the same instrument.The Xpert HIV-1VL on plasma can be used interchangeably with existing predicate platforms in South Africa. Whole blood and DBS testing requires further investigation, but polyvalency of the GeneXpert offers a solution to extending VL testing services.

  15. Innate immune restriction and antagonism of viral RNA lacking 2'-O methylation

    Energy Technology Data Exchange (ETDEWEB)

    Hyde, Jennifer L. [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Diamond, Michael S., E-mail: diamond@borcim.wustl.edu [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Molecular Microbiology, Washington University School of Medicine, St Louis., MO 63110 (United States); Pathology & Immunology, Washington University School of Medicine, St Louis., MO 63110 (United States); The Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St Louis., MO 63110 (United States)

    2015-05-15

    N-7 and 2′-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m{sup 7}GpppN; cap 1, m{sup 7}GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2′-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on their RNA through cap-snatching or virally-encoded 2′-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5′-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.

  16. VirSorter: mining viral signal from microbial genomic data

    Directory of Open Access Journals (Sweden)

    Simon Roux

    2015-05-01

    Full Text Available Viruses of microbes impact all ecosystems where microbes drive key energy and substrate transformations including the oceans, humans and industrial fermenters. However, despite this recognized importance, our understanding of viral diversity and impacts remains limited by too few model systems and reference genomes. One way to fill these gaps in our knowledge of viral diversity is through the detection of viral signal in microbial genomic data. While multiple approaches have been developed and applied for the detection of prophages (viral genomes integrated in a microbial genome, new types of microbial genomic data are emerging that are more fragmented and larger scale, such as Single-cell Amplified Genomes (SAGs of uncultivated organisms or genomic fragments assembled from metagenomic sequencing. Here, we present VirSorter, a tool designed to detect viral signal in these different types of microbial sequence data in both a reference-dependent and reference-independent manner, leveraging probabilistic models and extensive virome data to maximize detection of novel viruses. Performance testing shows that VirSorter’s prophage prediction capability compares to that of available prophage predictors for complete genomes, but is superior in predicting viral sequences outside of a host genome (i.e., from extrachromosomal prophages, lytic infections, or partially assembled prophages. Furthermore, VirSorter outperforms existing tools for fragmented genomic and metagenomic datasets, and can identify viral signal in assembled sequence (contigs as short as 3kb, while providing near-perfect identification (>95% Recall and 100% Precision on contigs of at least 10kb. Because VirSorter scales to large datasets, it can also be used in “reverse” to more confidently identify viral sequence in viral metagenomes by sorting away cellular DNA whether derived from gene transfer agents, generalized transduction or contamination. Finally, VirSorter is made

  17. VirSorter: mining viral signal from microbial genomic data

    Science.gov (United States)

    Roux, Simon; Enault, Francois; Hurwitz, Bonnie L.

    2015-01-01

    Viruses of microbes impact all ecosystems where microbes drive key energy and substrate transformations including the oceans, humans and industrial fermenters. However, despite this recognized importance, our understanding of viral diversity and impacts remains limited by too few model systems and reference genomes. One way to fill these gaps in our knowledge of viral diversity is through the detection of viral signal in microbial genomic data. While multiple approaches have been developed and applied for the detection of prophages (viral genomes integrated in a microbial genome), new types of microbial genomic data are emerging that are more fragmented and larger scale, such as Single-cell Amplified Genomes (SAGs) of uncultivated organisms or genomic fragments assembled from metagenomic sequencing. Here, we present VirSorter, a tool designed to detect viral signal in these different types of microbial sequence data in both a reference-dependent and reference-independent manner, leveraging probabilistic models and extensive virome data to maximize detection of novel viruses. Performance testing shows that VirSorter’s prophage prediction capability compares to that of available prophage predictors for complete genomes, but is superior in predicting viral sequences outside of a host genome (i.e., from extrachromosomal prophages, lytic infections, or partially assembled prophages). Furthermore, VirSorter outperforms existing tools for fragmented genomic and metagenomic datasets, and can identify viral signal in assembled sequence (contigs) as short as 3kb, while providing near-perfect identification (>95% Recall and 100% Precision) on contigs of at least 10kb. Because VirSorter scales to large datasets, it can also be used in “reverse” to more confidently identify viral sequence in viral metagenomes by sorting away cellular DNA whether derived from gene transfer agents, generalized transduction or contamination. Finally, VirSorter is made available through the i

  18. Molecular Tracing of Viral Pathogen in Aquaculture (MOLTRAQ): a new EMIDA project

    DEFF Research Database (Denmark)

    Jensen, B. Bang; Aldrin, M.; Avarre, M. C.

    2012-01-01

    a generic approach to viral disease control by using information on epidemiological and phylogenetic attributes from several important aquatic animal viruses. The project will i) generate and use spatio-temporal epidemiological data, phylogeographic data and gene expression data for important host......-viral pathogen systems to identify important factors affecting the spread of diseases in aquaculture, and ii) integrate these in scenario simulation models to assess effects of various control strategies for selected host-pathogen systems. The project consists of six workpackages: WP 1: Project co...

  19. Pharyngitis - viral

    Science.gov (United States)

    ... throat is due to a viral infection. The antibiotics will not help. Using them to treat viral infections helps bacteria become resistant to antibiotics. With some sore throats (such as those caused ...

  20. Improved detection of Bovine Viral Diarrhea Virus in Bovine lymphoid cell lines using PrimeFlow RNA assay

    Science.gov (United States)

    Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. BVDV can be identified by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely di...

  1. The role of gene flow in shaping genetic structures of the subtropical conifer species Araucaria angustifolia.

    Science.gov (United States)

    Stefenon, V M; Gailing, O; Finkeldey, R

    2008-05-01

    The morphological features of pollen and seed of Araucaria angustifolia have led to the proposal of limited gene dispersal for this species. We used nuclear microsatellite and AFLP markers to assess patterns of genetic variation in six natural populations at the intra- and inter-population level, and related our findings to gene dispersal in this species. Estimates of both fine-scale spatial genetic structure (SGS) and migration rate suggest relatively short-distance gene dispersal. However, gene dispersal differed among populations, and effects of more efficient dispersal within population were observed in at least one stand. In addition, even though some seed dispersal may be aggregated in this principally barochorous species, reasonable secondary seed dispersal, presumably facilitated by animals, and overlap of seed shadows within populations is suggested. Overall, no correlation was observed between levels of SGS and inbreeding, density or age structure, except that a higher level of SGS was revealed for the population with a higher number of juvenile individuals. A low estimate for the number of migrants per generation between two neighbouring populations implies limited gene flow. We expect that stepping-stone pollen flow may have contributed to low genetic differentiation among populations observed in a previous survey. Thus, strategies for maintenance of gene flow among remnant populations should be considered in order to avoid degrading effects of population fragmentation on the evolution of A. angustifolia.

  2. High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.

    Directory of Open Access Journals (Sweden)

    Sandra J Kuhlman

    2008-04-01

    Full Text Available We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs that express GFP, dsRedExpress, or channelrhodopsin (ChR2 upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 expression allowed light activation of neuronal spiking. The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv fast-spiking GABAergic interneuron, was monitored over the course of a week. We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex. With the generation of an increasing number of Cre knockin mice and because viral transfection can be delivered to defined brain regions at defined developmental stages, this strategy represents a general method to systematically visualize the structure and manipulate the function of different cell types in the mouse brain.

  3. The immunomodulatory gene products of myxoma virus

    Indian Academy of Sciences (India)

    Unknown

    273. Keywords. Gene products; myxoma virus; Oryctolagus cuniculus; poxvirus; skin lesions ...... these data is that these viral proteins do not promote class .... Cudmore S, Reckmann I and Way M 1997 Viral manipulations of the actin ...

  4. CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b.

    Science.gov (United States)

    He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin; Peng, Xu

    2017-02-28

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Transcriptomic study of 39 ostreid herpesvirus 1 genes during an experimental infection.

    Science.gov (United States)

    Segarra, Amélie; Faury, Nicole; Pépin, Jean-François; Renault, Tristan

    2014-06-01

    Massive mortality outbreaks have been reported in France since 2008 among Pacific oysters, Crassostrea gigas, with the detection of a particular OsHV-1 variant called μVar. Virus infection can be induced in healthy spat in experimental conditions allowing to better understand the disease process, including viral gene expression. Although gene expression of other herpesviruses has been widely studied, we provide the first study following viral gene expression of OsHV-1 over time. In this context, an in vivo transcriptomic study targeting 39 OsHV-1 genes was carried out during an experimental infection of Pacific oyster spat. For the first time, several OsHV-1 mRNAs were detected by real-time PCR at 0 h, 2 h, 4 h, 18 h, 26 h and 42 h post-injection. Several transcripts were detected at 2h post-infection and at 18 h post-infection for all selected ORFs. Quantification of virus gene expression at different times of infection was also carried out using an oyster housekeeping gene, Elongation factor. Developing an OsHV-1-specific reverse transcriptase real time PCR targeting 39 viral gene appears a new tool in terms of diagnosis and can be used to complement viral DNA detection in order to monitor viral replication. Copyright © 2014. Published by Elsevier Inc.

  6. Barriers to gene flow in the marine environment: insights from two common intertidal limpet species of the Atlantic and Mediterranean.

    Directory of Open Access Journals (Sweden)

    Alexandra Sá-Pinto

    Full Text Available Knowledge of the scale of dispersal and the mechanisms governing gene flow in marine environments remains fragmentary despite being essential for understanding evolution of marine biota and to design management plans. We use the limpets Patella ulyssiponensis and Patella rustica as models for identifying factors affecting gene flow in marine organisms across the North-East Atlantic and the Mediterranean Sea. A set of allozyme loci and a fragment of the mitochondrial gene cytochrome C oxidase subunit I were screened for genetic variation through starch gel electrophoresis and DNA sequencing, respectively. An approach combining clustering algorithms with clinal analyses was used to test for the existence of barriers to gene flow and estimate their geographic location and abruptness. Sharp breaks in the genetic composition of individuals were observed in the transitions between the Atlantic and the Mediterranean and across southern Italian shores. An additional break within the Atlantic cluster separates samples from the Alboran Sea and Atlantic African shores from those of the Iberian Atlantic shores. The geographic congruence of the genetic breaks detected in these two limpet species strongly supports the existence of transpecific barriers to gene flow in the Mediterranean Sea and Northeastern Atlantic. This leads to testable hypotheses regarding factors restricting gene flow across the study area.

  7. Viral Meningitis

    Science.gov (United States)

    ... better from treatment such as an antiviral medicine. Antibiotics do not help viral infections, so they are not useful in the treatment of viral meningitis. However, antibiotics do fight bacteria, so they are very important ...

  8. Direct inhibition of RNAse T2 expression by the HTLV-1 viral protein Tax.

    Science.gov (United States)

    Polakowski, Nicholas; Han, Hongjin; Lemasson, Isabelle

    2011-08-01

    Adult T-cell leukemia (ATL) is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1) infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP) assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development.

  9. Direct Inhibition of RNAse T2 Expression by the HTLV-1 Viral Protein Tax

    Directory of Open Access Journals (Sweden)

    Isabelle Lemasson

    2011-08-01

    Full Text Available Adult T-cell leukemia (ATL is one of the primary diseases caused by Human T-cell Leukemia Virus type 1 (HTLV-1 infection. The virally-encoded Tax protein is believed to initiate early events in the development of this disease, as it is able to promote immortalization of T-cells and transformation of other cell types. These processes may be aided by the ability of the viral protein to directly deregulate expression of specific cellular genes through interactions with numerous transcriptional regulators. To identify gene promoters where Tax is localized, we isolated Tax-DNA complexes from an HTLV-1-infected T-cell line through a chromatin immunoprecipitation (ChIP assay and used the DNA to probe a CpG island microarray. A site within the RNASET2 gene was found to be occupied by Tax. Real-time PCR analysis confirmed this result, and transient expression of Tax in uninfected cells led to the recruitment of the viral protein to the promoter. This event correlated with a decrease in the level of RNase T2 mRNA and protein, suggesting that Tax represses expression of this gene. Loss of RNase T2 expression occurs in certain hematological malignancies and other forms of cancer, and RNase T2 was recently reported to function as a tumor suppressor. Consequently, a reduction in the level of RNase T2 by Tax may play a role in ATL development.

  10. Viral-bacterial associations in acute apical abscesses.

    Science.gov (United States)

    Ferreira, Dennis C; Rôças, Isabela N; Paiva, Simone S M; Carmo, Flávia L; Cavalcante, Fernanda S; Rosado, Alexandre S; Santos, Kátia R N; Siqueira, José F

    2011-08-01

    Viral-bacterial and bacterial synergism have been suggested to contribute to the pathogenesis of several human diseases. This study sought to investigate the possible associations between 9 candidate endodontic bacterial pathogens and 9 human viruses in samples from acute apical abscesses. DNA extracts from purulent exudate aspirates of 33 cases of acute apical abscess were surveyed for the presence of 9 selected bacterial species using a 16S ribosomal RNA gene-based nested polymerase chain reaction (PCR) approach. Single or nested PCR assays were used for detection of the human papillomavirus (HPV) and herpesviruses types 1 to 8. Two-thirds of the abscess samples were positive for at least one of the target viruses. Specifically, the most frequently detected viruses were HHV-8 (54.5%); HPV (9%); and varicella zoster virus (VZV), Epstein-Barr virus (EBV), and HHV-6 (6%). Bacterial DNA was present in all cases and the most prevalent bacterial species were Treponema denticola (70%), Tannerella forsythia (67%), Porphyromonas endodontalis (67%), Dialister invisus (61%), and Dialister pneumosintes (57.5%). HHV-8 was positively associated with 7 of the target bacterial species and HPV with 4, but all these associations were weak. Several bacterial pairs showed a moderate positive association. Viral coinfection was found in 6 abscess cases, but no significant viral association could be determined. Findings demonstrated that bacterial and viral DNA occurred concomitantly in two-thirds of the samples from endodontic abscesses. Although this may suggest a role for viruses in the etiology of apical abscesses, the possibility also exists that the presence of viruses in abscess samples is merely a consequence of the bacterially induced disease process. Further studies are necessary to clarify the role of these viral-bacterial interactions, if any, in the pathogenesis of acute apical abscesses. Copyright © 2011 Mosby, Inc. All rights reserved.

  11. Therapeutic genes for anti-HIV/AIDS gene therapy.

    Science.gov (United States)

    Bovolenta, Chiara; Porcellini, Simona; Alberici, Luca

    2013-01-01

    The multiple therapeutic approaches developed so far to cope HIV-1 infection, such as anti-retroviral drugs, germicides and several attempts of therapeutic vaccination have provided significant amelioration in terms of life-quality and survival rate of AIDS patients. Nevertheless, no approach has demonstrated efficacy in eradicating this lethal, if untreated, infection. The curative power of gene therapy has been proven for the treatment of monogenic immunodeficiensies, where permanent gene modification of host cells is sufficient to correct the defect for life-time. No doubt, a similar concept is not applicable for gene therapy of infectious immunodeficiensies as AIDS, where there is not a single gene to be corrected; rather engineered cells must gain immunotherapeutic or antiviral features to grant either short- or long-term efficacy mostly by acquisition of antiviral genes or payloads. Anti-HIV/AIDS gene therapy is one of the most promising strategy, although challenging, to eradicate HIV-1 infection. In fact, genetic modification of hematopoietic stem cells with one or multiple therapeutic genes is expected to originate blood cell progenies resistant to viral infection and thereby able to prevail on infected unprotected cells. Ultimately, protected cells will re-establish a functional immune system able to control HIV-1 replication. More than hundred gene therapy clinical trials against AIDS employing different viral vectors and transgenes have been approved or are currently ongoing worldwide. This review will overview anti-HIV-1 infection gene therapy field evaluating strength and weakness of the transgenes and payloads used in the past and of those potentially exploitable in the future.

  12. The interaction between endogenous 30S ribosomal subunit protein S11 and Cucumber mosaic virus LS2b protein affects viral replication, infection and gene silencing suppressor activity.

    Directory of Open Access Journals (Sweden)

    Ruilin Wang

    Full Text Available Cucumber mosaic virus (CMV is a model virus for plant-virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11 was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC assays observed by confocal laser microscopy and Glutathione S-transferase (GST pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.

  13. Virally encoded 7TM receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Waldhoer, M; Lüttichau, H R

    2001-01-01

    expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets.......A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion...

  14. pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

    Science.gov (United States)

    Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

    2018-05-01

    The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Genomic evidence of geographically widespread effect of gene flow from polar bears into brown bears.

    Science.gov (United States)

    Cahill, James A; Stirling, Ian; Kistler, Logan; Salamzade, Rauf; Ersmark, Erik; Fulton, Tara L; Stiller, Mathias; Green, Richard E; Shapiro, Beth

    2015-03-01

    Polar bears are an arctic, marine adapted species that is closely related to brown bears. Genome analyses have shown that polar bears are distinct and genetically homogeneous in comparison to brown bears. However, these analyses have also revealed a remarkable episode of polar bear gene flow into the population of brown bears that colonized the Admiralty, Baranof and Chichagof islands (ABC islands) of Alaska. Here, we present an analysis of data from a large panel of polar bear and brown bear genomes that includes brown bears from the ABC islands, the Alaskan mainland and Europe. Our results provide clear evidence that gene flow between the two species had a geographically wide impact, with polar bear DNA found within the genomes of brown bears living both on the ABC islands and in the Alaskan mainland. Intriguingly, while brown bear genomes contain up to 8.8% polar bear ancestry, polar bear genomes appear to be devoid of brown bear ancestry, suggesting the presence of a barrier to gene flow in that direction. © 2014 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

  16. Staphylococcus aureus α-toxin modulates skin host response to viral infection.

    Science.gov (United States)

    Bin, Lianghua; Kim, Byung Eui; Brauweiler, Anne; Goleva, Elena; Streib, Joanne; Ji, Yinduo; Schlievert, Patrick M; Leung, Donald Y M

    2012-09-01

    Patients with atopic dermatitis (AD) with a history of eczema herpeticum have increased staphylococcal colonization and infections. However, whether Staphylococcus aureus alters the outcome of skin viral infection has not been determined. We investigated whether S aureus toxins modulated host response to herpes simplex virus (HSV) 1 and vaccinia virus (VV) infections in normal human keratinocytes (NHKs) and in murine infection models. NHKs were treated with S aureus toxins before incubation of viruses. BALB/c mice were inoculated with S aureus 2 days before VV scarification. Viral loads of HSV-1 and VV were evaluated by using real-time PCR, a viral plaque-forming assay, and immunofluorescence staining. Small interfering RNA duplexes were used to knockdown the gene expression of the cellular receptor of α-toxin, a disintegrin and metalloprotease 10 (ADAM10). ADAM10 protein and α-toxin heptamers were detected by using Western blot assays. We demonstrate that sublytic staphylococcal α-toxin increases viral loads of HSV-1 and VV in NHKs. Furthermore, we demonstrate in vivo that the VV load is significantly greater (P skin inoculated with an α-toxin-producing S aureus strain compared with murine skin inoculated with the isogenic α-toxin-deleted strain. The viral enhancing effect of α-toxin is mediated by ADAM10 and is associated with its pore-forming property. Moreover, we demonstrate that α-toxin promotes viral entry in NHKs. The current study introduces the novel concept that staphylococcal α-toxin promotes viral skin infection and provides a mechanism by which S aureus infection might predispose the host toward disseminated viral infections. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  17. Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging.

    Science.gov (United States)

    Pocock, Ginger M; Zimdars, Laraine L; Yuan, Ming; Eliceiri, Kevin W; Ahlquist, Paul; Sherer, Nathan M

    2017-02-01

    Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include "burst" RNA nuclear export dynamics regulated by HIV-1's Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element-specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation. © 2017 Pocock et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Evaluation of the Universal Viral Transport system for long-term storage of virus specimens for microbial forensics.

    Science.gov (United States)

    Hosokawa-Muto, Junji; Fujinami, Yoshihito; Mizuno, Natsuko

    2015-08-01

    Forensic microbial specimens, including bacteria and viruses, are collected at biocrime and bioterrorism scenes. Although it is preferable that the pathogens in these samples are alive and kept in a steady state, the samples may be stored for prolonged periods before analysis. Therefore, it is important to understand the effects of storage conditions on the pathogens contained within such samples. To evaluate the capacity to preserve viable virus and the viral genome, influenza virus was added to the transport medium of the Universal Viral Transport system and stored for over 3 months at various temperatures, after which virus titrations and quantitative analysis of the influenza hemagglutinin gene were performed. Although viable viruses became undetectable 29 days after the medium was stored at room temperature, viruses in the medium stored at 4°C were viable even after 99 days. A quantitative PCR analysis indicated that the hemagglutinin gene was maintained for 99 days at both 4°C and room temperature. Therefore, long-term storage at 4°C has little effect on viable virus and viral genes, so the Universal Viral Transport system can be useful for microbial forensics. This study provides important information for the handling of forensic virus specimens. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  19. Contrasting Patterns of Gene Flow for Amazonian Snakes That Actively Forage and Those That Wait in Ambush.

    Science.gov (United States)

    de Fraga, Rafael; Lima, Albertina P; Magnusson, William E; Ferrão, Miquéias; Stow, Adam J

    2017-07-01

    Knowledge of genetic structure, geographic distance and environmental heterogeneity can be used to identify environmental features and natural history traits that influence dispersal and gene flow. Foraging mode is a trait that might predict dispersal capacity in snakes, because actively foragers typically have greater movement rates than ambush predators. Here, we test the hypothesis that 2 actively foraging snakes have higher levels of gene flow than 2 ambush predators. We evaluated these 4 co-distributed species of snakes in the Brazilian Amazon. Snakes were sampled along an 880 km transect from the central to the southwest of the Amazon basin, which covered a mosaic of vegetation types and seasonal differences in climate. We analyzed thousands of single nucleotide polymorphisms to compare patterns of neutral gene flow based on isolation by geographic distance (IBD) and environmental resistance (IBR). We show that IBD and IBR were only evident in ambush predators, implying lower levels of dispersal than the active foragers. Therefore, gene flow was high enough in the active foragers analyzed here to prevent any build-up of spatial genotypic structure with respect to geographic distance and environmental heterogeneity. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. A novel homologous model for gene therapy of dwarfism by non-viral transfer of the mouse growth hormone gene into immunocompetent dwarf mice.

    Science.gov (United States)

    Cecchi, Claudia R; Higuti, Eliza; Oliveira, Nelio A J; Lima, Eliana R; Jakobsen, Maria; Dagnaes-Hansen, Frederick; Gissel, Hanne; Aagaard, Lars; Jensen, Thomas G; Jorge, Alexander A L; Bartolini, Paolo; Peroni, Cibele N

    2014-02-01

    The possibilities for non-viral GH gene therapy are studied in immunocompetent dwarf mice (lit/lit). As expression vector we used a plasmid previously employed in immunodeficient dwarf mice (pUBI-hGH-gDNA) by replacing the human GH gene with the genomic sequence of mouse-GH DNA (pUBI-mGH-gDNA). HEK-293 human cells transfected with pUBI-mGH-gDNA produced 3.0 µg mGH/10(6) cells/day compared to 3.7 µg hGH/10(6) cells/day for pUBIhGH- gDNA transfected cells. The weight of lit/lit mice treated with the same two plasmids (50 µg DNA/mouse) by electrotransfer into the quadriceps muscle was followed for 3 months. The weight increase up to 15 days for mGH, hGH and saline treated mice were 0.130, 0.112 and 0.027 g/mouse/day. Most sera from hGH-treated mice contained anti-hGH antibodies already on day 15, with the highest titers on day 45, while no significant anti-mGH antibodies were observed in mGH-treated mice. At the end of 3 months, the weight increase for mGH-treated mice was 34.3%, while the nose-to-tail and femur lengths increased 9.5% and 24.3%. Mouse-GH and hGH circulating levels were 4-5 ng/mL 15 days after treatment, versus control levels of ~0.7 ng GH/mL (Pdeficiency.

  1. Circumpolar Genetic Structure and Recent Gene Flow of Polar Bears: A Reanalysis.

    Science.gov (United States)

    Malenfant, René M; Davis, Corey S; Cullingham, Catherine I; Coltman, David W

    2016-01-01

    Recently, an extensive study of 2,748 polar bears (Ursus maritimus) from across their circumpolar range was published in PLOS ONE, which used microsatellites and mitochondrial haplotypes to apparently show altered population structure and a dramatic change in directional gene flow towards the Canadian Archipelago-an area believed to be a future refugium for polar bears as their southernmost habitats decline under climate change. Although this study represents a major international collaborative effort and promised to be a baseline for future genetics work, methodological shortcomings and errors of interpretation undermine some of the study's main conclusions. Here, we present a reanalysis of this data in which we address some of these issues, including: (1) highly unbalanced sample sizes and large amounts of systematically missing data; (2) incorrect calculation of FST and of significance levels; (3) misleading estimates of recent gene flow resulting from non-convergence of the program BayesAss. In contrast to the original findings, in our reanalysis we find six genetic clusters of polar bears worldwide: the Hudson Bay Complex, the Western and Eastern Canadian Arctic Archipelago, the Western and Eastern Polar Basin, and-importantly-we reconfirm the presence of a unique and possibly endangered cluster of bears in Norwegian Bay near Canada's expected last sea-ice refugium. Although polar bears' abundance, distribution, and population structure will certainly be negatively affected by ongoing-and increasingly rapid-loss of Arctic sea ice, these genetic data provide no evidence of strong directional gene flow in response to recent climate change.

  2. Circumpolar Genetic Structure and Recent Gene Flow of Polar Bears: A Reanalysis.

    Directory of Open Access Journals (Sweden)

    René M Malenfant

    Full Text Available Recently, an extensive study of 2,748 polar bears (Ursus maritimus from across their circumpolar range was published in PLOS ONE, which used microsatellites and mitochondrial haplotypes to apparently show altered population structure and a dramatic change in directional gene flow towards the Canadian Archipelago-an area believed to be a future refugium for polar bears as their southernmost habitats decline under climate change. Although this study represents a major international collaborative effort and promised to be a baseline for future genetics work, methodological shortcomings and errors of interpretation undermine some of the study's main conclusions. Here, we present a reanalysis of this data in which we address some of these issues, including: (1 highly unbalanced sample sizes and large amounts of systematically missing data; (2 incorrect calculation of FST and of significance levels; (3 misleading estimates of recent gene flow resulting from non-convergence of the program BayesAss. In contrast to the original findings, in our reanalysis we find six genetic clusters of polar bears worldwide: the Hudson Bay Complex, the Western and Eastern Canadian Arctic Archipelago, the Western and Eastern Polar Basin, and-importantly-we reconfirm the presence of a unique and possibly endangered cluster of bears in Norwegian Bay near Canada's expected last sea-ice refugium. Although polar bears' abundance, distribution, and population structure will certainly be negatively affected by ongoing-and increasingly rapid-loss of Arctic sea ice, these genetic data provide no evidence of strong directional gene flow in response to recent climate change.

  3. MicroRNA-Related Polymorphisms in Infectious Diseases—Tiny Changes With a Huge Impact on Viral Infections and Potential Clinical Applications

    Directory of Open Access Journals (Sweden)

    Joel Henrique Ellwanger

    2018-06-01

    Full Text Available MicroRNAs (miRNAs are single-stranded sequences of non-coding RNA with approximately 22 nucleotides that act posttranscriptionally on gene expression. miRNAs are important gene regulators in physiological contexts, but they also impact the pathogenesis of various diseases. The role of miRNAs in viral infections has been explored by different authors in both population-based as well as in functional studies. However, the effect of miRNA polymorphisms on the susceptibility to viral infections and on the clinical course of these diseases is still an emerging topic. Thus, this review will compile and organize the findings described in studies that evaluated the effects of genetic variations on miRNA genes and on their binding sites, in the context of human viral diseases. In addition to discussing the basic aspects of miRNAs biology, we will cover the studies that investigated miRNA polymorphisms in infections caused by hepatitis B virus, hepatitis C virus, human immunodeficiency virus, Epstein–Barr virus, and human papillomavirus. Finally, emerging topics concerning the importance of miRNA genetic variants will be presented, focusing on the context of viral infectious diseases.

  4. Viral Diversity Threshold for Adaptive Immunity in Prokaryotes

    Science.gov (United States)

    Weinberger, Ariel D.; Wolf, Yuri I.; Lobkovsky, Alexander E.; Gilmore, Michael S.; Koonin, Eugene V.

    2012-01-01

    ABSTRACT Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological

  5. Passive larval transport explains recent gene flow in a Mediterranean gorgonian

    Science.gov (United States)

    Padrón, Mariana; Costantini, Federica; Baksay, Sandra; Bramanti, Lorenzo; Guizien, Katell

    2018-06-01

    Understanding the patterns of connectivity is required by the Strategic Plan for Biodiversity 2011-2020 and will be used to guide the extension of marine protection measures. Despite the increasing accuracy of ocean circulation modelling, the capacity to model the population connectivity of sessile benthic species with dispersal larval stages can be limited due to the potential effect of filters acting before or after dispersal, which modulates offspring release or settlement, respectively. We applied an interdisciplinary approach that combined demographic surveys, genetic methods (assignment tests and coalescent-based analyses) and larval transport simulations to test the relative importance of demographics and ocean currents in shaping the recent patterns of gene flow among populations of a Mediterranean gorgonian ( Eunicella singularis) in a fragmented rocky habitat (Gulf of Lion, NW Mediterranean Sea). We show that larval transport is a dominant driver of recent gene flow among the populations, and significant correlations were found between recent gene flow and larval transport during an average single dispersal event when the pelagic larval durations (PLDs) ranged from 7 to 14 d. Our results suggest that PLDs that efficiently connect populations distributed over a fragmented habitat are filtered by the habitat layout within the species competency period. Moreover, a PLD ranging from 7 to 14 d is sufficient to connect the fragmented rocky substrate of the Gulf of Lion. The rocky areas located in the centre of the Gulf of Lion, which are currently not protected, were identified as essential hubs for the distribution of migrants in the region. We encourage the use of a range of PLDs instead of a single value when estimating larval transport with biophysical models to identify potential connectivity patterns among a network of Marine Protected Areas or even solely a seascape.

  6. The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.

    Directory of Open Access Journals (Sweden)

    Jasdave S Chahal

    Full Text Available Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

  7. Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor.

    Science.gov (United States)

    Dong, Shanshan; Liu, Yan; Yu, Cigang; Zhang, Zhenhua; Chen, Ming; Wang, Changyong

    2016-01-01

    Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.

  8. Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor.

    Directory of Open Access Journals (Sweden)

    Shanshan Dong

    Full Text Available Pollen-mediated gene flow (PMGF is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.

  9. Prospects for Foamy Viral Vector Anti-HIV Gene Therapy

    Directory of Open Access Journals (Sweden)

    Arun K. Nalla

    2016-03-01

    Full Text Available Stem cell gene therapy approaches for Human Immunodeficiency Virus (HIV infection have been explored in clinical trials and several anti-HIV genes delivered by retroviral vectors were shown to block HIV replication. However, gammaretroviral and lentiviral based retroviral vectors have limitations for delivery of anti-HIV genes into hematopoietic stem cells (HSC. Foamy virus vectors have several advantages including efficient delivery of transgenes into HSC in large animal models, and a potentially safer integration profile. This review focuses on novel anti-HIV transgenes and the potential of foamy virus vectors for HSC gene therapy of HIV.

  10. Phage and Nucleocytoplasmic Large Viral Sequences Dominate Coral Viromes from the Arabian Gulf.

    Science.gov (United States)

    Mahmoud, Huda; Jose, Liny

    2017-01-01

    Corals that naturally thrive under extreme conditions are gaining increasing attention due to their importance as living models to understand the impact of global warming on world corals. Here, we present the first metagenomic study of viral communities in corals thriving in a thermally variable water body in which the temperature fluctuates between 11 and 39°C in different seasons. The viral assemblages of two of the most abundant massive ( Porites harrisoni ) and branching ( Acropora downingi ) corals in offshore and inshore reef systems in the northern Arabian Gulf were investigated. Samples were collected from five reef systems during summer, autumn and winter of 2011/2012. The two coral viromes contain 12 viral families, including 10 dsDNA viral families [Siphoviridae, Podoviridae, Myoviridae, Phycodnaviridae, Baculoviridae, Herpesviridae, Adenoviridae, Alloherpesviridae, Mimiviridae and one unclassified family], one-ssDNA viral family (Microviridae) and one RNA viral family (Retroviridae). Overall, sequences significantly similar to Podoviridae were the most abundant in the P. harrisoni and A. downingi viromes. Various morphological types of virus-like particles (VLPs) were confirmed in the healthy coral tissue by transmission electron microscopy, including large tailless VLPs and electron-dense core VLPs. Tailed bacteriophages were isolated from coral tissue using a plaque assay. Higher functional gene diversity was recorded in A. downingi than in P. harrisoni , and comparative metagenomics revealed that the Gulf viral assemblages are functionally distinct from Pacific Ocean coral viral communities.

  11. Phage and Nucleocytoplasmic Large Viral Sequences Dominate Coral Viromes from the Arabian Gulf

    Directory of Open Access Journals (Sweden)

    Huda Mahmoud

    2017-10-01

    Full Text Available Corals that naturally thrive under extreme conditions are gaining increasing attention due to their importance as living models to understand the impact of global warming on world corals. Here, we present the first metagenomic study of viral communities in corals thriving in a thermally variable water body in which the temperature fluctuates between 11 and 39°C in different seasons. The viral assemblages of two of the most abundant massive (Porites harrisoni and branching (Acropora downingi corals in offshore and inshore reef systems in the northern Arabian Gulf were investigated. Samples were collected from five reef systems during summer, autumn and winter of 2011/2012. The two coral viromes contain 12 viral families, including 10 dsDNA viral families [Siphoviridae, Podoviridae, Myoviridae, Phycodnaviridae, Baculoviridae, Herpesviridae, Adenoviridae, Alloherpesviridae, Mimiviridae and one unclassified family], one-ssDNA viral family (Microviridae and one RNA viral family (Retroviridae. Overall, sequences significantly similar to Podoviridae were the most abundant in the P. harrisoni and A. downingi viromes. Various morphological types of virus-like particles (VLPs were confirmed in the healthy coral tissue by transmission electron microscopy, including large tailless VLPs and electron-dense core VLPs. Tailed bacteriophages were isolated from coral tissue using a plaque assay. Higher functional gene diversity was recorded in A. downingi than in P. harrisoni, and comparative metagenomics revealed that the Gulf viral assemblages are functionally distinct from Pacific Ocean coral viral communities.

  12. Nuclear sensing of viral DNA, epigenetic regulation of herpes simplex virus infection, and innate immunity

    International Nuclear Information System (INIS)

    Knipe, David M.

    2015-01-01

    Herpes simplex virus (HSV) undergoes a lytic infection in epithelial cells and a latent infection in neuronal cells, and epigenetic mechanisms play a major role in the differential gene expression under the two conditions. HSV viron DNA is not associated with histones but is rapidly loaded with heterochromatin upon entry into the cell. Viral proteins promote reversal of the epigenetic silencing in epithelial cells while the viral latency-associated transcript promotes additional heterochromatin in neuronal cells. The cellular sensors that initiate the chromatinization of foreign DNA have not been fully defined. IFI16 and cGAS are both essential for innate sensing of HSV DNA, and new evidence shows how they work together to initiate innate signaling. IFI16 also plays a role in the heterochromatinization of HSV DNA, and this review will examine how IFI16 integrates epigenetic regulation and innate sensing of foreign viral DNA to show how these two responses are related. - Highlights: • HSV lytic and latent gene expression is regulated differentially by epigenetic processes. • The sensors of foreign DNA have not been defined fully. • IFI16 and cGAS cooperate to sense viral DNA in HSV-infected cells. • IFI16 plays a role in both innate sensing of HSV DNA and in restricting its expression

  13. Nuclear sensing of viral DNA, epigenetic regulation of herpes simplex virus infection, and innate immunity

    Energy Technology Data Exchange (ETDEWEB)

    Knipe, David M., E-mail: david_knipe@hms.harvard.edu

    2015-05-15

    Herpes simplex virus (HSV) undergoes a lytic infection in epithelial cells and a latent infection in neuronal cells, and epigenetic mechanisms play a major role in the differential gene expression under the two conditions. HSV viron DNA is not associated with histones but is rapidly loaded with heterochromatin upon entry into the cell. Viral proteins promote reversal of the epigenetic silencing in epithelial cells while the viral latency-associated transcript promotes additional heterochromatin in neuronal cells. The cellular sensors that initiate the chromatinization of foreign DNA have not been fully defined. IFI16 and cGAS are both essential for innate sensing of HSV DNA, and new evidence shows how they work together to initiate innate signaling. IFI16 also plays a role in the heterochromatinization of HSV DNA, and this review will examine how IFI16 integrates epigenetic regulation and innate sensing of foreign viral DNA to show how these two responses are related. - Highlights: • HSV lytic and latent gene expression is regulated differentially by epigenetic processes. • The sensors of foreign DNA have not been defined fully. • IFI16 and cGAS cooperate to sense viral DNA in HSV-infected cells. • IFI16 plays a role in both innate sensing of HSV DNA and in restricting its expression.

  14. A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

    Directory of Open Access Journals (Sweden)

    V.L. Quadros

    2006-07-01

    Full Text Available Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV and an antigenically identical but cytopathic virus (cpBVDV can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98% to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

  15. Extensive gene flow over Europe and possible speciation

    Energy Technology Data Exchange (ETDEWEB)

    VINCENOT, Dr. LUCIE [Centre d’Ecologie Fonctionnelle et Evolutive Montpellier, France; NARA, Dr. KAZUHIDE [Department of Natural Environmental Studies, The University of Tokyo, Japan; STHULTZ, CHRISTOPHER [Centre d’Ecologie Fonctionnelle et Evolutive Montpellier, France; Labbe, Jessy L [ORNL; DUBOIS, MARIE-PIERRE [Centre d’Ecologie Fonctionnelle et Evolutive Montpellier, France; TEDERSOO, LEHO [University of Tartu, Estonia; Martin, Francis [INRA, Nancy, France; SELOSSE, Dr. MARC-ANDRE [Centre d’Ecologie Fonctionnelle et Evolutive Montpellier, France

    2012-01-01

    Biogeographical patterns and large-scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (104 km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average FST = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (FST = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.

  16. Inferred vs realized patterns of gene flow: an analysis of population structure in the Andros Island Rock Iguana.

    Science.gov (United States)

    Colosimo, Giuliano; Knapp, Charles R; Wallace, Lisa E; Welch, Mark E

    2014-01-01

    Ecological data, the primary source of information on patterns and rates of migration, can be integrated with genetic data to more accurately describe the realized connectivity between geographically isolated demes. In this paper we implement this approach and discuss its implications for managing populations of the endangered Andros Island Rock Iguana, Cyclura cychlura cychlura. This iguana is endemic to Andros, a highly fragmented landmass of large islands and smaller cays. Field observations suggest that geographically isolated demes were panmictic due to high, inferred rates of gene flow. We expand on these observations using 16 polymorphic microsatellites to investigate the genetic structure and rates of gene flow from 188 Andros Iguanas collected across 23 island sites. Bayesian clustering of specimens assigned individuals to three distinct genotypic clusters. An analysis of molecular variance (AMOVA) indicates that allele frequency differences are responsible for a significant portion of the genetic variance across the three defined clusters (Fst =  0.117, p<0.01). These clusters are associated with larger islands and satellite cays isolated by broad water channels with strong currents. These findings imply that broad water channels present greater obstacles to gene flow than was inferred from field observation alone. Additionally, rates of gene flow were indirectly estimated using BAYESASS 3.0. The proportion of individuals originating from within each identified cluster varied from 94.5 to 98.7%, providing further support for local isolation. Our assessment reveals a major disparity between inferred and realized gene flow. We discuss our results in a conservation perspective for species inhabiting highly fragmented landscapes.

  17. Inferred vs Realized Patterns of Gene Flow: An Analysis of Population Structure in the Andros Island Rock Iguana

    Science.gov (United States)

    Colosimo, Giuliano; Knapp, Charles R.; Wallace, Lisa E.; Welch, Mark E.

    2014-01-01

    Ecological data, the primary source of information on patterns and rates of migration, can be integrated with genetic data to more accurately describe the realized connectivity between geographically isolated demes. In this paper we implement this approach and discuss its implications for managing populations of the endangered Andros Island Rock Iguana, Cyclura cychlura cychlura. This iguana is endemic to Andros, a highly fragmented landmass of large islands and smaller cays. Field observations suggest that geographically isolated demes were panmictic due to high, inferred rates of gene flow. We expand on these observations using 16 polymorphic microsatellites to investigate the genetic structure and rates of gene flow from 188 Andros Iguanas collected across 23 island sites. Bayesian clustering of specimens assigned individuals to three distinct genotypic clusters. An analysis of molecular variance (AMOVA) indicates that allele frequency differences are responsible for a significant portion of the genetic variance across the three defined clusters (Fst =  0.117, p0.01). These clusters are associated with larger islands and satellite cays isolated by broad water channels with strong currents. These findings imply that broad water channels present greater obstacles to gene flow than was inferred from field observation alone. Additionally, rates of gene flow were indirectly estimated using BAYESASS 3.0. The proportion of individuals originating from within each identified cluster varied from 94.5 to 98.7%, providing further support for local isolation. Our assessment reveals a major disparity between inferred and realized gene flow. We discuss our results in a conservation perspective for species inhabiting highly fragmented landscapes. PMID:25229344

  18. Inferred vs realized patterns of gene flow: an analysis of population structure in the Andros Island Rock Iguana.

    Directory of Open Access Journals (Sweden)

    Giuliano Colosimo

    Full Text Available Ecological data, the primary source of information on patterns and rates of migration, can be integrated with genetic data to more accurately describe the realized connectivity between geographically isolated demes. In this paper we implement this approach and discuss its implications for managing populations of the endangered Andros Island Rock Iguana, Cyclura cychlura cychlura. This iguana is endemic to Andros, a highly fragmented landmass of large islands and smaller cays. Field observations suggest that geographically isolated demes were panmictic due to high, inferred rates of gene flow. We expand on these observations using 16 polymorphic microsatellites to investigate the genetic structure and rates of gene flow from 188 Andros Iguanas collected across 23 island sites. Bayesian clustering of specimens assigned individuals to three distinct genotypic clusters. An analysis of molecular variance (AMOVA indicates that allele frequency differences are responsible for a significant portion of the genetic variance across the three defined clusters (Fst =  0.117, p<<0.01. These clusters are associated with larger islands and satellite cays isolated by broad water channels with strong currents. These findings imply that broad water channels present greater obstacles to gene flow than was inferred from field observation alone. Additionally, rates of gene flow were indirectly estimated using BAYESASS 3.0. The proportion of individuals originating from within each identified cluster varied from 94.5 to 98.7%, providing further support for local isolation. Our assessment reveals a major disparity between inferred and realized gene flow. We discuss our results in a conservation perspective for species inhabiting highly fragmented landscapes.

  19. The ins and outs of eukaryotic viruses: Knowledge base and ontology of a viral infection.

    Directory of Open Access Journals (Sweden)

    Chantal Hulo

    Full Text Available Viruses are genetically diverse, infect a wide range of tissues and host cells and follow unique processes for replicating themselves. All these processes were investigated and indexed in ViralZone knowledge base. To facilitate standardizing data, a simple ontology of viral life-cycle terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address unique viral replication cycle processes, and existing terminology was modified and adapted. The virus life-cycle is classically described by schematic pictures. Using this ontology, it can be represented by a combination of successive terms: "entry", "latency", "transcription", "replication" and "exit". Each of these parts is broken down into discrete steps. For example Zika virus "entry" is broken down in successive steps: "Attachment", "Apoptotic mimicry", "Viral endocytosis/ macropinocytosis", "Fusion with host endosomal membrane", "Viral factory". To demonstrate the utility of a standard ontology for virus biology, this work was completed by annotating virus data in the ViralZone, UniProtKB and Gene Ontology databases.

  20. Chitosan nanoparticles as non-viral gene delivery systems: determination of loading efficiency.

    Science.gov (United States)

    Carrillo, Carolina; Suñé, Josep Maria; Pérez-Lozano, Pilar; García-Montoya, Encarna; Sarrate, Rocío; Fàbregas, Anna; Miñarro, Montserrat; Ticó, Josep Ramon

    2014-07-01

    Chitosan has been studied for use in particle delivery systems for therapeutic purposes, since one of its most important applications is as a non-viral vector in gene therapy. Due to its positive charge, it is capable of forming DNA complexes (polyplexes) obtained through several methods and with the property of protecting nucleic acids. Two methods for obtaining the nanoparticles of chitosan-nucleic acids are reported in this study: simple complexation (of depolymerized chitosan or of different chitosan salts with plasmid) and ionic gelation (by adsorption of plasmid in the nanoparticles or by encapsulation of plasmid into nanoparticles). The determination of the loading efficiency of chitosan nanoparticles with the plasmid is carried out by electrophoretic mobility of the samples on agarose gel. Furthermore, the nanoparticles have been characterized according to their morphology, size and surface charge using AFM, TEM, laser diffraction and dynamic light scattering techniques. The polyplexes obtained have been found to be spherical and nanometric in size (between 100-230nm) with a zeta potential between 37 and 48mV. Positive results have been obtained by agarose gel electrophoresis for all studied cases: a concentration of between 20 and 30μg/mL of chitosan salts is required while for the remaining chitosan samples studied, 100% loading efficiency does not occur until a concentration equal to 100μg/mL (regardless of previous depolymerisation and the method performed). Chitosan-plasmid nanocapsules have been obtained at the polymer concentrations worked with (between 0.025 and 0.2%). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Comprehensive Screening for Naturally Occurring Hepatitis C Virus Resistance to Direct-Acting Antivirals in the NS3, NS5A, and NS5B Genes in Worldwide Isolates of Viral Genotypes 1 to 6.

    Science.gov (United States)

    Patiño-Galindo, Juan Ángel; Salvatierra, Karina; González-Candelas, Fernando; López-Labrador, F Xavier

    2016-04-01

    There is no comprehensive study available on the natural hepatitis C virus (HCV) polymorphism in sites associated with resistance including all viral genotypes which may present variable susceptibilities to particular direct-acting antivirals (DAAs). This study aimed to analyze the frequencies, genetic barriers, and evolutionary histories of naturally occurring resistance-associated variants (RAVs) in the six main HCV genotypes. A comprehensive analysis of up to 103 RAVs was performed in 2,901, 2,216, and 1,344 HCV isolates for the NS3, NS5A, and NS5B genes, respectively. We report significant intergenotypic differences in the frequencies of natural RAVs for these three HCV genes. In addition, we found a low genetic barrier for the generation of new RAVs, irrespective of the viral genotype. Furthermore, in 1,126 HCV genomes, including sequences spanning the three genes, haplotype analysis revealed a remarkably high frequency of viruses carrying more than one natural RAV to DAAs (53% of HCV-1a, 28.5% of HCV-1b, 67.1% of HCV-6, and 100% of genotype 2, 3, 4, and 5 haplotypes). With the exception of HCV-1a, the most prevalent haplotypes showed RAVs in at least two different viral genes. Finally, evolutionary analyses revealed that, while most natural RAVs appeared recently, others have been efficiently transmitted over time and cluster in well-supported clades. In summary, and despite the observed high efficacy of DAA-based regimens, we show that naturally occurring RAVs are common in all HCV genotypes and that there is an overall low genetic barrier for the selection of resistance mutations. There is a need for natural DAA resistance profiling specific for each HCV genotype. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Hepatitis viral aguda

    Directory of Open Access Journals (Sweden)

    Héctor Rubén Hernández Garcés

    1998-10-01

    Full Text Available Se realizó una revisión bibliográfica de las hepatitis virales agudas sobre aspectos vinculados a su etiología. Se tuvieron en cuenta además algunos datos epidemiológicos, las formas clínicas más importantes, los exámenes complementarios con especial énfasis en los marcadores virales y el diagnóstico positivoA bibliographical review of acute viral hepatitis was made taking into account those aspects connected with its etiology. Some epidemiological markers, the most important clinical forms, and the complementary examinations with special emphasis on the viral markers and the positive diagnosis were also considered

  3. Quantitative molecular viral loads in 7 horses with naturally occurring equine herpesvirus-1 infection.

    Science.gov (United States)

    Estell, K E; Dawson, D R; Magdesian, K G; Swain, E; Laing, S T; Siso, S; Mapes, S; Pusterla, N

    2015-11-01

    Data associating quantitative viral load with severity, clinical signs and survival in equine herpesvirus-1 myeloencephalopathy (EHM) have not been reported. To report the clinical signs, treatment, and temporal progression of viral loads in 7 horses with naturally occurring EHM and to examine the association of these factors with survival. Retrospective case series. The population included 7 horses with EHM presented to the University of California, Davis William R. Pritchard Veterinary Medical Teaching Hospital from May to September 2011. Horses were graded using a neurological grading scale. Daily quantitative PCR was performed on nasal secretions and whole blood. Treatment, survival, outcome and histopathology were reported. At presentation, one horse was neurological grade 5/5, 3 were grade 4/5 and 3 were grade 3/5. All were treated with anti-inflammatory drugs, valacyclovir and management in a sling if necessary. All were infected with equine herpesvirus-1 of DNA polymerase D752 genotype. Peak viral load in nasal secretions and blood of 5 survivors ranged from 6.9 × 10(3) to 2.81 × 10(5) (median 5.11 × 10(4) ) and from 143 to 4340 gB gene copies/million eukaryotic cells (median 3146), respectively. The 2 nonsurvivors presented with grade 3/5 neurological signs and progressed to encephalopathy. Peak viral load was higher in nonsurvivors, with levels in nasal secretions of 1.9 × 10(9) and 2.2 × 10(9) and in blood of 2.05 × 10(4) and 1.02 × 10(5) gB gene copies/million eukaryotic cells. Case fatality was 2/7. Nonsurvivors had viral loads 1000-fold higher in nasal secretions and 10-fold higher in blood than survivors. There was no relationship between severity of clinical signs at presentation and survival. Thus, encephalopathy and high viral load were negatively associated with survival in this population. Further research should be performed to determine whether high viral loads are associated with encephalopathy and poor prognosis. The Summary is

  4. Virale commercials: de consument als marketeer. Onderzoek naar de redenen waarom consumenten virale commercials doorsturen: hun motieven, de inhoudskenmerken van viral commercials en de mediumcontext waarin virale commercials verschijnen

    NARCIS (Netherlands)

    Ketelaar, P.E.; Lucassen, P.; Kregting, G.H.J.

    2010-01-01

    Research into the reasons why consumers pass along viral commercials: their motives, the content characteristics of viral commercials and the medium context in which viral commercials appear. Based on the uses and gratifications perspective this study has determined which motives of consumers,

  5. Avian influenza H5N1 viral and bird migration networks in Asia

    Science.gov (United States)

    Tian, Huaivu; Zhou, Sen; Dong, Lu; Van Boeckel, Thomas P.; Cui, Yujun; Newman, Scott H.; Takekawa, John Y.; Prosser, Diann J.; Xiao, Xiangming; Wu, Yarong; Cazelles, Bernard; Huang, Shanqian; Yang, Ruifu; Grenfell, Bryan T.; Xu, Bing

    2015-01-01

    The spatial spread of the highly pathogenic avian influenza virus H5N1 and its long-term persistence in Asia have resulted in avian influenza panzootics and enormous economic losses in the poultry sector. However, an understanding of the regional long-distance transmission and seasonal patterns of the virus is still lacking. In this study, we present a phylogeographic approach to reconstruct the viral migration network. We show that within each wild fowl migratory flyway, the timing of H5N1 outbreaks and viral migrations are closely associated, but little viral transmission was observed between the flyways. The bird migration network is shown to better reflect the observed viral gene sequence data than other networks and contributes to seasonal H5N1 epidemics in local regions and its large-scale transmission along flyways. These findings have potentially far-reaching consequences, improving our understanding of how bird migration drives the periodic reemergence of H5N1 in Asia.

  6. Adaptive genetic markers discriminate migratory runs of Chinook salmon (Oncorhynchus tshawytscha) amid continued gene flow.

    Science.gov (United States)

    O'Malley, Kathleen G; Jacobson, Dave P; Kurth, Ryon; Dill, Allen J; Banks, Michael A

    2013-12-01

    Neutral genetic markers are routinely used to define distinct units within species that warrant discrete management. Human-induced changes to gene flow however may reduce the power of such an approach. We tested the efficiency of adaptive versus neutral genetic markers in differentiating temporally divergent migratory runs of Chinook salmon (Oncorhynchus tshawytscha) amid high gene flow owing to artificial propagation and habitat alteration. We compared seven putative migration timing genes to ten microsatellite loci in delineating three migratory groups of Chinook in the Feather River, CA: offspring of fall-run hatchery broodstock that returned as adults to freshwater in fall (fall run), spring-run offspring that returned in spring (spring run), and fall-run offspring that returned in spring (FRS). We found evidence for significant differentiation between the fall and federally listed threatened spring groups based on divergence at three circadian clock genes (OtsClock1b, OmyFbxw11, and Omy1009UW), but not neutral markers. We thus demonstrate the importance of genetic marker choice in resolving complex life history types. These findings directly impact conservation management strategies and add to previous evidence from Pacific and Atlantic salmon indicating that circadian clock genes influence migration timing.

  7. Viral Hybrid Vectors for Somatic Integration - Are They the Better Solution?

    Directory of Open Access Journals (Sweden)

    Anja Ehrhardt

    2009-12-01

    Full Text Available The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system.

  8. Pollen-mediated gene flow and seed exchange in small-scale Zambian maize farming, implications for biosafety assessment.

    Science.gov (United States)

    Bøhn, Thomas; Aheto, Denis W; Mwangala, Felix S; Fischer, Klara; Bones, Inger Louise; Simoloka, Christopher; Mbeule, Ireen; Schmidt, Gunther; Breckling, Broder

    2016-10-03

    Gene flow in agricultural crops is important for risk assessment of genetically modified (GM) crops, particularly in countries with a large informal agricultural sector of subsistence cultivation. We present a pollen flow model for maize (Zea mays), a major staple crop in Africa. We use spatial properties of fields (size, position) in three small-scale maize farming communities in Zambia and estimate rates of cross-fertilisation between fields sown with different maize varieties (e.g. conventional and transgene). As an additional factor contributing to gene flow, we present data on seed saving and sharing among farmers that live in the same communities. Our results show that: i) maize fields were small and located in immediate vicinity of neighboring fields; ii) a majority of farmers saved and shared seed; iii) modeled rates of pollen-mediated gene flow showed extensive mixing of germplasm between fields and farms and iv) as a result, segregation of GM and non-GM varieties is not likely to be an option in these systems. We conclude that the overall genetic composition of maize, in this and similar agricultural contexts, will be strongly influenced both by self-organised ecological factors (pollen flow), and by socially mediated intervention (seed recycling and sharing).

  9. Combining genomic sequencing methods to explore viral diversity and reveal potential virus-host interactions

    Directory of Open Access Journals (Sweden)

    Cheryl-Emiliane Tien Chow

    2015-04-01

    Full Text Available Viral diversity and virus-host interactions in oxygen-starved regions of the ocean, also known as oxygen minimum zones (OMZs, remain relatively unexplored. Microbial community metabolism in OMZs alters nutrient and energy flow through marine food webs, resulting in biological nitrogen loss and greenhouse gas production. Thus, viruses infecting OMZ microbes have the potential to modulate community metabolism with resulting feedback on ecosystem function. Here, we describe viral communities inhabiting oxic surface (10m and oxygen-starved basin (200m waters of Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia using viral metagenomics and complete viral fosmid sequencing on samples collected between April 2007 and April 2010. Of 6459 open reading frames (ORFs predicted across all 34 viral fosmids, 77.6% (n=5010 had no homology to reference viral genomes. These fosmids recruited a higher proportion of viral metagenomic sequences from Saanich Inlet than from nearby northeastern subarctic Pacific Ocean (Line P waters, indicating differences in the viral communities between coastal and open ocean locations. While functional annotations of fosmid ORFs were limited, recruitment to NCBI’s non-redundant ‘nr’ database and publicly available single-cell genomes identified putative viruses infecting marine thaumarchaeal and SUP05 proteobacteria to provide potential host linkages with relevance to coupled biogeochemical cycling processes in OMZ waters. Taken together, these results highlight the power of coupled analyses of multiple sequence data types, such as viral metagenomic and fosmid sequence data with prokaryotic single cell genomes, to chart viral diversity, elucidate genomic and ecological contexts for previously unclassifiable viral sequences, and identify novel host interactions in natural and engineered ecosystems.

  10. Sexual selection and magic traits in speciation with gene flow

    Directory of Open Access Journals (Sweden)

    Maria R. SERVEDIO, Michael KOPP

    2012-06-01

    Full Text Available The extent to which sexual selection is involved in speciation with gene flow remains an open question and the subject of much research. Here, we propose that some insight can be gained from considering the concept of magic traits (i.e., traits involved in both reproductive isolation and ecological divergence. Both magic traits and other, “non-magic”, traits can contribute to speciation via a number of specific mechanisms. We argue that many of these mechanisms are likely to differ widely in the extent to which they involve sexual selection. Furthermore, in some cases where sexual selection is present, it may be prone to inhibit rather than drive speciation. Finally, there are a priori reasons to believe that certain categories of traits are much more effective than others in driving speciation. The combination of these points suggests a classification of traits that may shed light on the broader role of sexual selection in speciation with gene flow. In particular, we suggest that sexual selection can act as a driver of speciation in some scenarios, but may play a negligible role in potentially common categories of magic traits, and may be likely to inhibit speciation in common categories of non-magic traits [Current Zoology 58 (3: 507–513, 2012].

  11. Viral infections as controlling factors for the deep biosphere? (Invited)

    Science.gov (United States)

    Engelen, B.; Engelhardt, T.; Sahlberg, M.; Cypionka, H.

    2009-12-01

    The marine deep biosphere represents the largest biotope on Earth. Throughout the last years, we have obtained interesting insights into its microbial community composition. However, one component that was completely overlooked so far is the viral inventory of deep-subsurface sediments. While viral infections were identified to have a major impact on the benthic microflora of deep-sea surface sediments (Danavaro et al. 2008), no studies were performed on deep-biosphere samples, so far. As grazers probably play only a minor role in anoxic and highly compressed deep sediments, viruses might be the main “predators” for indigenous microorganisms. Furthermore, the release of cell components, called “the viral shunt”, could have a major impact on the deep biosphere in providing labile organic compounds to non-infected microorganisms in these generally nutrient depleted sediments. However, direct counting of viruses in sediments is highly challenging due to the small size of viruses and the high background of small particles. Even molecular surveys using “universal” PCR primers that target phage-specific genes fail due to the vast phage diversity. One solution for this problem is the lysogenic viral life cycle as many bacteriophages integrate their DNA into the host genome. It is estimated that up to 70% of cultivated bacteria contain prophages within their genome. Therefore, culture collections (Batzke et al. 2007) represent an archive of the viral composition within the respective habitat. These prophages can be induced to become free phage particles in stimulation experiments in which the host cells are set under certain stress situations such as a treatment with UV exposure or DNA-damaging antibiotics. The study of the viral component within the deep biosphere offers to answer the following questions: To which extent are deep-biosphere populations controlled by viral infections? What is the inter- and intra-specific diversity and the host-specific viral

  12. The Himalayas: barrier and conduit for gene flow.

    Science.gov (United States)

    Gayden, Tenzin; Perez, Annabel; Persad, Patrice J; Bukhari, Areej; Chennakrishnaiah, Shilpa; Simms, Tanya; Maloney, Trisha; Rodriguez, Kristina; Herrera, Rene J

    2013-06-01

    The Himalayan mountain range is strategically located at the crossroads of the major cultural centers in Asia, the Middle East and Europe. Although previous Y-chromosome studies indicate that the Himalayas served as a natural barrier for gene flow from the south to the Tibetan plateau, this region is believed to have played an important role as a corridor for human migrations between East and West Eurasia along the ancient Silk Road. To evaluate the effects of the Himalayan mountain range in shaping the maternal lineages of populations residing on either side of the cordillera, we analyzed mitochondrial DNA variation in 344 samples from three Nepalese collections (Newar, Kathmandu and Tamang) and a general population of Tibet. Our results revealed a predominantly East Asian-specific component in Tibet and Tamang, whereas Newar and Kathmandu are both characterized by a combination of East and South Central Asian lineages. Interestingly, Newar and Kathmandu harbor several deep-rooted Indian lineages, including M2, R5, and U2, whose coalescent times from this study (U2, >40 kya) and previous reports (M2 and R5, >50 kya) suggest that Nepal was inhabited during the initial peopling of South Central Asia. Comparisons with our previous Y-chromosome data indicate sex-biased migrations in Tamang and a founder effect and/or genetic drift in Tamang and Newar. Altogether, our results confirm that while the Himalayas acted as a geographic barrier for human movement from the Indian subcontinent to the Tibetan highland, it also served as a conduit for gene flow between Central and East Asia. Copyright © 2013 Wiley Periodicals, Inc.

  13. Current status of gene therapy for motor neuron disease

    Institute of Scientific and Technical Information of China (English)

    Xingkai An; Rong Peng; Shanshan Zhao

    2006-01-01

    OBJECTIVE: Although the etiology and pathogenesis of motor neuron disease is still unknown, there are many hypotheses on motor neuron mitochondrion, cytoskeleton structure and functional injuries. Thus, gene therapy of motor neuron disease has become a hot topic to apply in viral vector, gene delivery and basic gene techniques.DATA SOURCES: The related articles published between January 2000 and October 2006 were searched in Medline database and ISl database by computer using the keywords "motor neuron disease, gene therapy", and the language is limited to English. Meanwhile, the related references of review were also searched by handiwork. STUDY SELECTION: Original articles and referred articles in review were chosen after first hearing, then the full text which had new ideas were found, and when refer to the similar study in the recent years were considered first.DATA EXTRACTION: Among the 92 related articles, 40 ones were accepted, and 52 were excluded because of repetitive study or reviews.DATA SYNTHESIS: The viral vectors of gene therapy for motor neuron disease include adenoviral, adeno-associated viral vectors, herpes simplex virus type 1 vectors and lentiviral vectors. The delivery of them can be achieved by direct injection into the brain, or by remote delivery after injection vectors into muscle or peripheral nerves, or by ex vivo gene transfer. The viral vectors of gene therapy for motor neuron disease have been successfully developed, but the gene delivery of them is hampered by some difficulties. The RNA interference and neuroprotection are the main technologies for gene-based therapy in motor neuron disease. CONCLUSION : The RNA interference for motor neuron disease has succeeded in animal models, and the neuroprotection also does. But, there are still a lot of questions for gene therapy in the clinical treatment of motor neuron disease.

  14. MicroRNAs in the host response to viral infections of veterinary importance

    Directory of Open Access Journals (Sweden)

    Mohamed Samir Ahmed

    2016-10-01

    Full Text Available The discovery of small regulatory non-coding RNAs has been an exciting advance in the field of genomics. MicroRNAs (miRNAs are endogenous RNA molecules, approximately 22 nucleotides in length that regulate gene expression, mostly at the post-transcriptional level. MiRNA profiling technologies have made it possible to identify and quantify novel miRNAs and to study their regulation and potential roles in disease pathogenesis. Although miRNAs have been extensively investigated in viral infections of humans, their implications in viral diseases affecting animals of veterinary importance are much less understood. The number of annotated miRNAs in different animal species is growing continuously, and novel roles in regulating host-pathogen interactions are being discovered, for instance miRNA-mediated augmentation of viral transcription and replication. In this review, we present an overview of synthesis and function of miRNAs and an update on the current state of research on host-encoded miRNAs in the genesis of viral infectious diseases in their natural animal host as well as in selected in vivo and in vitro laboratory models.

  15. Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

    Science.gov (United States)

    Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

    2017-06-15

    Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    International Nuclear Information System (INIS)

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory

    2007-01-01

    Frog virus 3 (FV3) is a large DNA virus that encodes ∼ 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIα). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIα triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins

  17. Targeting Herpetic Keratitis by Gene Therapy

    Directory of Open Access Journals (Sweden)

    Hossein Mostafa Elbadawy

    2012-01-01

    Full Text Available Ocular gene therapy is rapidly becoming a reality. By November 2012, approximately 28 clinical trials were approved to assess novel gene therapy agents. Viral infections such as herpetic keratitis caused by herpes simplex virus 1 (HSV-1 can cause serious complications that may lead to blindness. Recurrence of the disease is likely and cornea transplantation, therefore, might not be the ideal therapeutic solution. This paper will focus on the current situation of ocular gene therapy research against herpetic keratitis, including the use of viral and nonviral vectors, routes of delivery of therapeutic genes, new techniques, and key research strategies. Whereas the correction of inherited diseases was the initial goal of the field of gene therapy, here we discuss transgene expression, gene replacement, silencing, or clipping. Gene therapy of herpetic keratitis previously reported in the literature is screened emphasizing candidate gene therapy targets. Commonly adopted strategies are discussed to assess the relative advantages of the protective therapy using antiviral drugs and the common gene therapy against long-term HSV-1 ocular infections signs, inflammation and neovascularization. Successful gene therapy can provide innovative physiological and pharmaceutical solutions against herpetic keratitis.

  18. Gene delivery to the lungs: pulmonary gene therapy for cystic fibrosis.

    Science.gov (United States)

    Villate-Beitia, Ilia; Zarate, Jon; Puras, Gustavo; Pedraz, José Luis

    2017-07-01

    Cystic fibrosis (CF) is a monogenic autosomal recessive disorder where the defective gene, the cystic fibrosis transmembrane conductance regulator (CFTR), is well identified. Moreover, the respiratory tract can be targeted through noninvasive aerosolized formulations for inhalation. Therefore, gene therapy is considered a plausible strategy to address this disease. Conventional gene therapy strategies rely on the addition of a correct copy of the CFTR gene into affected cells in order to restore the channel activity. In recent years, genome correction strategies have emerged, such as zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases. These gene editing tools aim to repair the mutated gene at its original genomic locus with high specificity. Besides, the success of gene therapy critically depends on the nucleic acids carriers. To date, several clinical studies have been carried out to add corrected copies of the CFTR gene into target cells using viral and non-viral vectors, some of them with encouraging results. Regarding genome editing systems, preliminary in vitro studies have been performed in order to repair the CFTR gene. In this review, after briefly introducing the basis of CF, we discuss the up-to-date gene therapy strategies to address the disease. The review focuses on the main factors to take into consideration when developing gene delivery strategies, such as the design of vectors and plasmid DNA, in vitro/in vivo tests, translation to human use, administration methods, manufacturing conditions and regulatory issues.

  19. Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

    Science.gov (United States)

    Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

    2018-03-01

    Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

  20. Amplified fragment length polymorphism fingerprints support limited gene flow among social spider populations

    NARCIS (Netherlands)

    Smith, Deborah; van Rijn, Sander; Henschel, Joh; Bilde, Trine; Lubin, Yael

    We used DNA fingerprints to determine whether the population structure and colony composition of the cooperative social spider Stegodyphus dumicola are compatible with requirements of interdemic ('group') selection: differential proliferation of demes or groups and limited gene flow among groups. To

  1. Mobil Viral Pazarlama

    OpenAIRE

    Barutçu, Süleyman

    2011-01-01

    OBJECTIVE: Mobile Viral Marketing, with using mobile phones, is one of the most importantinnovations after Word of Mouth Marketing performed by face to face amongpeople and Viral Marketing performed in the İnternet. The main objective of thisstudy is to call marketing communicators’ and academicians’ attentions whowant to increase the recognition of companies’ products, services and brands tobecome a current issue in the marketplace using Mobile Viral Marketingapplications by reason of techno...

  2. A method for quantifying mechanical properties of tissue following viral infection.

    Directory of Open Access Journals (Sweden)

    Vy Lam

    Full Text Available Viral infection and replication involves the reorganization of the actin network within the host cell. Actin plays a central role in the mechanical properties of cells. We have demonstrated a method to quantify changes in mechanical properties of fabricated model three-dimensional (3D connective tissue following viral infection. Using this method, we have characterized the impact of infection by the human herpesvirus, cytomegalovirus (HCMV. HCMV is a member of the herpesvirus family and infects a variety of cell types including fibroblasts. In the body, fibroblasts are necessary for maintaining connective tissue and function by creating mechanical force. Using this 3D connective tissue model, we observed that infection disrupted the cell's ability to generate force and reduced the cumulative contractile force of the tissue. The addition of HCMV viral particles in the absence of both viral gene expression and DNA replication was sufficient to disrupt tissue function. We observed that alterations of the mechanical properties are, in part, due to a disruption of the underlying complex actin microfilament network established by the embedded fibroblasts. Finally, we were able to prevent HCMV-mediated disruption of tissue function by the addition of human immune globulin against HCMV. This study demonstrates a method to quantify the impact of viral infection on mechanical properties which are not evident using conventional cell culture systems.

  3. Protection against Acute Lethal Viral Infections with the Native Steroid Dehydroepiandrosterone (DHEA)

    Science.gov (United States)

    1988-01-01

    of gout , hyperlipemia, and in post-coronary patients Protection Against Viral Inftiom With DHEA 311 [Regelson, 19881. In animal models [Yen, 19771 and...3333. Johnson DA, Schultz LD, Bedigian HG (1982): Immunodeficiency and reticulum cell sarcoma in mice segregating for HRS/J and SJL/J genes . Leukemia

  4. Chromosomal rearrangements and gene flow over time in an inter-specific hybrid zone of the Sorex araneus group.

    Science.gov (United States)

    Yannic, G; Basset, P; Hausser, J

    2009-06-01

    Most hybrid zones have existed for hundreds or thousands of years but have generally been observed for only a short time period. Studies extending over periods long enough to track evolutionary changes in the zones or assess the ultimate outcome of hybridization are scarce. Here, we describe the evolution over time of the level of genetic isolation between two karyotypically different species of shrews (Sorex araneus and Sorex antinorii) at a hybrid zone located in the Swiss Alps. We first evaluated hybrid zone movement by contrasting patterns of gene flow and changes in cline parameters (centre and width) using 24 microsatellite loci, between two periods separated by 10 years apart. Additionally, we tested the role of chromosomal rearrangements on gene flow by analysing microsatellite loci located on both rearranged and common chromosomes to both species. We did not detect any movement of the hybrid zone during the period analysed, suggesting that the zone is a typical tension zone. However, the gene flow was significantly lower among the rearranged than the common chromosomes for the second period, whereas the difference was only marginally significant for the first period. This further supports the role of chromosomal rearrangements on gene flow between these taxa.

  5. Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays.

    Science.gov (United States)

    Kim, John E; Beckthold, Brenda; Chen, Zhaoxia; Mihowich, Jennifer; Malloch, Laurie; Gill, Michael John

    2007-11-01

    The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.

  6. The Therapeutic Potential of CRISPR/Cas9 Systems in Oncogene-Addicted Cancer Types: Virally Driven Cancers as a Model System

    Directory of Open Access Journals (Sweden)

    Luqman Jubair

    2017-09-01

    Full Text Available The field of gene editing is undergoing unprecedented growth. The first ex vivo human clinical trial in China started in 2016, more than 1000 US patents have been filed, and there is exponential growth in publications. The ability to edit genes with high fidelity is promising for the development of new treatments for a range of diseases, particularly inherited conditions, infectious diseases, and cancers. For cancer, a major issue is the identification of driver mutations and oncogenes to target for therapeutic effect, and this requires the development of robust models with which to prove their efficacy. The challenge is that there is rarely a single critical gene. However, virally driven cancers, in which cells are addicted to the expression of a single viral oncogene in some cases, may serve as model systems for CRISPR/Cas therapies, as they did for RNAi. These models and systems offer an excellent opportunity to test both preclinical models and clinical conditions to examine the effectiveness of gene editing, and here we review the options and offer a way forward. Keywords: CRISPR/Cas9, virally-driven cancers, cervical cancer, oncogene-addiction

  7. Synthetic sustained gene delivery systems.

    Science.gov (United States)

    Agarwal, Ankit; Mallapragada, Surya K

    2008-01-01

    Gene therapy today is hampered by the need of a safe and efficient gene delivery system that can provide a sustained therapeutic effect without cytotoxicity or unwanted immune responses. Bolus gene delivery in solution results in the loss of delivered factors via lymphatic system and may cause undesired effects by the escape of bioactive molecules to distant sites. Controlled gene delivery systems, acting as localized depot of genes, provide an extended sustained release of genes, giving prolonged maintenance of the therapeutic level of encoded proteins. They also limit the DNA degradation in the nuclease rich extra-cellular environment. While attempts have been made to adapt existing controlled drug delivery technologies, more novel approaches are being investigated for controlled gene delivery. DNA encapsulated in nano/micro spheres of polymers have been administered systemically/orally to be taken up by the targeted tissues and provide sustained release once internalized. Alternatively, DNA entrapped in hydrogels or scaffolds have been injected/implanted in tissues/cavities as platforms for gene delivery. The present review examines these different modalities for sustained delivery of viral and non-viral gene-delivery vectors. Design parameters and release mechanisms of different systems made with synthetic or natural polymers are presented along with their prospective applications and opportunities for continuous development.

  8. Understanding Image Virality

    Science.gov (United States)

    2015-06-07

    Example non-viral images. Figure 1: Top: Images with high viral scores in our dataset depict internet “celebrity” memes ex. “Grumpy Cat”; Bottom: Images...of images that is most similar to ours is the concurrently introduced viral meme generator of Wang et al., that combines NLP and Computer Vision (low...doing any of our tasks. The test included questions about widely spread Reddit memes and jargon so that anyone familiar with Reddit can easily get a high

  9. DNA cleavage enzymes for treatment of persistent viral infections: Recent advances and the pathway forward

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Nicholas D., E-mail: nweber@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Aubert, Martine, E-mail: maubert@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Dang, Chung H., E-mail: cdang@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Stone, Daniel, E-mail: dstone2@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Jerome, Keith R., E-mail: kjerome@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Department of Microbiology, University of Washington, Seattle, WA 98195 (United States)

    2014-04-15

    Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.

  10. The oncogenic potential of BK-polyomavirus is linked to viral integration into the human genome.

    Science.gov (United States)

    Kenan, Daniel J; Mieczkowski, Piotr A; Burger-Calderon, Raquel; Singh, Harsharan K; Nickeleit, Volker

    2015-11-01

    It has been suggested that BK-polyomavirus is linked to oncogenesis via high expression levels of large T-antigen in some urothelial neoplasms arising following kidney transplantation. However, a causal association between BK-polyomavirus, large T-antigen expression and oncogenesis has never been demonstrated in humans. Here we describe an investigation using high-throughput sequencing of tumour DNA obtained from an urothelial carcinoma arising in a renal allograft. We show that a novel BK-polyomavirus strain, named CH-1, is integrated into exon 26 of the myosin-binding protein C1 gene (MYBPC1) on chromosome 12 in tumour cells but not in normal renal cells. Integration of the BK-polyomavirus results in a number of discrete alterations in viral gene expression, including: (a) disruption of VP1 protein expression and robust expression of large T-antigen; (b) preclusion of viral replication; and (c) deletions in the non-coding control region (NCCR), with presumed alterations in promoter feedback loops. Viral integration disrupts one MYBPC1 gene copy and likely alters its expression. Circular episomal BK-polyomavirus gene sequences are not found, and the renal allograft shows no productive polyomavirus infection or polyomavirus nephropathy. These findings support the hypothesis that integration of polyomaviruses is essential to tumourigenesis. It is likely that dysregulation of large T-antigen, with persistent over-expression in non-lytic cells, promotes cell growth, genetic instability and neoplastic transformation. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

  11. The gene flow and mode of reproduction of Dothistroma septosporum in the Czech Republic

    Czech Academy of Sciences Publication Activity Database

    Tomšovský, M.; Tomešová, V.; Palovčíková, D.; Kostovčík, Martin; Rohrer, M.; Hanáček, P.; Jankovský, L.

    2014-01-01

    Roč. 62, č. 1 (2014), s. 59-68 ISSN 0032-0862 Institutional support: RVO:61388971 Keywords : Dothistroma * dothistroma needle blight * gene flow Subject RIV: EE - Microbiology, Virology Impact factor: 2.121, year: 2014

  12. Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression.

    Directory of Open Access Journals (Sweden)

    Francesca Bonvicini

    Full Text Available CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.

  13. Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

    Science.gov (United States)

    Bonvicini, Francesca; Manaresi, Elisabetta; Di Furio, Francesca; De Falco, Luisa; Gallinella, Giorgio

    2012-01-01

    CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression. The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections. The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19. PMID:22413013

  14. Functional Role of Infective Viral Particles on Metal Reduction

    Energy Technology Data Exchange (ETDEWEB)

    Coates, John D.

    2014-04-01

    A proposed strategy for the remediation of uranium (U) contaminated sites was based on the immobilization of U by reducing the oxidized soluble U, U(VI), to form a reduced insoluble end product, U(IV). Previous studies identified Geobacter sp., including G. sulfurreducens and G. metallireducens, as predominant U(VI)-reducing bacteria under acetate-oxidizing and U(VI)-reducing conditions. Examination of the finished genome sequence annotation of the canonical metal reducing species Geobacter sulfurreducens strain PCA and G. metallireduceans strain GS-15 as well as the draft genome sequence of G. uraniumreducens strain Rf4 identified phage related proteins. In addition, the completed genome for Anaeromyxobacter dehalogenans and the draft genome sequence of Desulfovibrio desulfuricans strain G20, two more model metal-reducing bacteria, also revealed phage related sequences. The presence of these gene sequences indicated that Geobacter spp., Anaeromyxobacter spp., and Desulfovibrio spp. are susceptible to viral infection. Furthermore, viral populations in soils and sedimentary environments in the order of 6.4×10{sup 6}–2.7×10{sup 10} VLP’s cm{sup -3} have been observed. In some cases, viral populations exceed bacterial populations in these environments suggesting that a relationship may exist between viruses and bacteria. Our preliminary screens of samples collected from the ESR FRC indicated that viral like particles were observed in significant numbers. The objective of this study was to investigate the potential functional role viruses play in metal reduction specifically Fe(III) and U(VI) reduction, the environmental parameters affecting viral infection of metal reducing bacteria, and the subsequent effects on U transport.

  15. Urban landscape genetics: canopy cover predicts gene flow between white-footed mouse (Peromyscus leucopus) populations in New York City.

    Science.gov (United States)

    Munshi-South, Jason

    2012-03-01

    In this study, I examine the influence of urban canopy cover on gene flow between 15 white-footed mouse (Peromyscus leucopus) populations in New York City parklands. Parks in the urban core are often highly fragmented, leading to rapid genetic differentiation of relatively nonvagile species. However, a diverse array of 'green' spaces may provide dispersal corridors through 'grey' urban infrastructure. I identify urban landscape features that promote genetic connectivity in an urban environment and compare the success of two different landscape connectivity approaches at explaining gene flow. Gene flow was associated with 'effective distances' between populations that were calculated based on per cent tree canopy cover using two different approaches: (i) isolation by effective distance (IED) that calculates the single best pathway to minimize passage through high-resistance (i.e. low canopy cover) areas, and (ii) isolation by resistance (IBR), an implementation of circuit theory that identifies all low-resistance paths through the landscape. IBR, but not IED, models were significantly associated with three measures of gene flow (Nm from F(ST) , BayesAss+ and Migrate-n) after factoring out the influence of isolation by distance using partial Mantel tests. Predicted corridors for gene flow between city parks were largely narrow, linear parklands or vegetated spaces that are not managed for wildlife, such as cemeteries and roadway medians. These results have implications for understanding the impacts of urbanization trends on native wildlife, as well as for urban reforestation efforts that aim to improve urban ecosystem processes. © 2012 Blackwell Publishing Ltd.

  16. Natural Killer Cells in Viral HepatitisSummary

    Directory of Open Access Journals (Sweden)

    Barbara Rehermann

    2015-11-01

    Full Text Available Natural killer (NK cells are traditionally regarded as first-line effectors of the innate immune response, but they also have a distinct role in chronic infection. Here, we review the role of NK cells against hepatitis C virus (HCV and hepatitis B virus (HBV, two agents that cause acute and chronic hepatitis in humans. Interest in NK cells was initially sparked by genetic studies that demonstrated an association between NK cell–related genes and the outcome of HCV infection. Viral hepatitis also provides a model to study the NK cell response to both endogenous and exogenous type I interferon (IFN. Levels of IFN-stimulated genes increase in both acute and chronic HCV infection and pegylated IFNα has been the mainstay of HCV and HBV treatment for decades. In chronic viral hepatitis, NK cells display decreased production of antiviral cytokines. This phenotype is found in both HCV and HBV infection but is induced by different mechanisms. Potent antivirals now provide the opportunity to study the reversibility of the suppressed cytokine production of NK cells in comparison with the antigen-induced defect in IFNγ and tumor necrosis factor-α production of virus-specific T cells. This has implications for immune reconstitution in other conditions of chronic inflammation and immune exhaustion, such as human immunodeficiency virus infection and cancer. Keywords: HBV, HCV, Infection, Interferon, T Cell

  17. gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes.

    Science.gov (United States)

    Nakagawa, So; Takahashi, Mahoko Ueda

    2016-01-01

    In mammals, approximately 10% of genome sequences correspond to endogenous viral elements (EVEs), which are derived from ancient viral infections of germ cells. Although most EVEs have been inactivated, some open reading frames (ORFs) of EVEs obtained functions in the hosts. However, EVE ORFs usually remain unannotated in the genomes, and no databases are available for EVE ORFs. To investigate the function and evolution of EVEs in mammalian genomes, we developed EVE ORF databases for 20 genomes of 19 mammalian species. A total of 736,771 non-overlapping EVE ORFs were identified and archived in a database named gEVE (http://geve.med.u-tokai.ac.jp). The gEVE database provides nucleotide and amino acid sequences, genomic loci and functional annotations of EVE ORFs for all 20 genomes. In analyzing RNA-seq data with the gEVE database, we successfully identified the expressed EVE genes, suggesting that the gEVE database facilitates studies of the genomic analyses of various mammalian species.Database URL: http://geve.med.u-tokai.ac.jp. © The Author(s) 2016. Published by Oxford University Press.

  18. Host Innate Immunity against Hepatitis E Virus and Viral Evasion Mechanisms.

    Science.gov (United States)

    Kang, Sangmin; Myoung, Jinjong

    2017-10-28

    Hepatitis E virus (HEV) infections cause epidemic or sporadic acute hepatitis, which are mostly self-limiting. However, viral infection in immunocompromised patients and pregnant women may result in serious consequences, such as chronic hepatitis and liver damage, mortality of the latter of which reaches up to 20-30%. Type I interferon (IFN)-induced antiviral immunity is known to be the first-line defense against virus infection. Upon HEV infection in the cell, the virus genome is recognized by pathogen recognition receptors, leading to rapid activation of intracellular signaling cascades. Expression of type I IFN triggers induction of a barrage of IFN-stimulated genes, helping the cells cope with viral infection. Interestingly, some of the HEV-encoded genes seem to be involved in disrupting signaling cascades for antiviral immune responses, and thus crippling cytokine/chemokine production. Antagonistic mechanisms of type I IFN responses by HEV have only recently begun to emerge, and in this review, we summarize known HEV evasion strategies and compare them with those of other hepatitis viruses.

  19. Herpesviral ICP0 Protein Promotes Two Waves of Heterochromatin Removal on an Early Viral Promoter during Lytic Infection

    Directory of Open Access Journals (Sweden)

    Jennifer S. Lee

    2016-01-01

    Full Text Available Herpesviruses must contend with host cell epigenetic silencing responses acting on their genomes upon entry into the host cell nucleus. In this study, we confirmed that unchromatinized herpes simplex virus 1 (HSV-1 genomes enter primary human foreskin fibroblasts and are rapidly subjected to assembly of nucleosomes and association with repressive heterochromatin modifications such as histone 3 (H3 lysine 9-trimethylation (H3K9me3 and lysine 27-trimethylation (H3K27me3 during the first 1 to 2 h postinfection. Kinetic analysis of the modulation of nucleosomes and heterochromatin modifications over the course of lytic infection demonstrates a progressive removal that coincided with initiation of viral gene expression. We obtained evidence for three phases of heterochromatin removal from an early gene promoter: an initial removal of histones and heterochromatin not dependent on ICP0, a second ICP0-dependent round of removal of H3K9me3 that is independent of viral DNA synthesis, and a third phase of H3K27me3 removal that is dependent on ICP0 and viral DNA synthesis. The presence of ICP0 in transfected cells is also sufficient to promote removal of histones and H3K9me3 modifications of cotransfected genes. Overall, these results show that ICP0 promotes histone removal, a reduction of H3K9me3 modifications, and a later indirect reduction of H3K27me3 modifications following viral early gene expression and DNA synthesis. Therefore, HSV ICP0 promotes the reversal of host epigenetic silencing mechanisms by several mechanisms.

  20. Genomic evidence for divergence with gene flow in host races of the larch budmoth

    Czech Academy of Sciences Publication Activity Database

    Emelianov, I.; Marec, František; Mallet, J.

    2003-01-01

    Roč. 271, - (2003), s. 97-105 ISSN 0962-8452 Institutional research plan: CEZ:AV0Z5007907 Keywords : speciation * gene flow * selection Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.544, year: 2003

  1. A Next-Generation Sequencing Approach Uncovers Viral Transcripts Incorporated in Poxvirus Virions

    Directory of Open Access Journals (Sweden)

    Marica Grossegesse

    2017-10-01

    Full Text Available Transcripts are known to be incorporated in particles of DNA viruses belonging to the families of Herpesviridae and Mimiviridae, but the presence of transcripts in other DNA viruses, such as poxviruses, has not been analyzed yet. Therefore, we first established a next-generation-sequencing (NGS-based protocol, enabling the unbiased identification of transcripts in virus particles. Subsequently, we applied our protocol to analyze RNA in an emerging zoonotic member of the Poxviridae family, namely Cowpox virus. Our results revealed the incorporation of 19 viral transcripts, while host identifications were restricted to ribosomal and mitochondrial RNA. Most viral transcripts had an unknown and immunomodulatory function, suggesting that transcript incorporation may be beneficial for poxvirus immune evasion. Notably, the most abundant transcript originated from the D5L/I1R gene that encodes a viral inhibitor of the host cytoplasmic DNA sensing machinery.

  2. Epigenetic control of viral life-cycle by a DNA-methylation dependent transcription factor.

    Directory of Open Access Journals (Sweden)

    Kirsty Flower

    Full Text Available Epstein-Barr virus (EBV encoded transcription factor Zta (BZLF1, ZEBRA, EB1 is the prototype of a class of transcription factor (including C/EBPalpha that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.

  3. siRNAs Targeting Viral Protein 5: The Major Capsid Protein of ...

    African Journals Online (AJOL)

    Purpose: To investigate whether siRNA targeting viral protein 5 (VP5) can become a new treatment for herpes simplex virus type 1 (HSV-1). Methods: Flow cytometry was performed to determine the ratio of siRNA and lipo2000 to reach the highest transfection efficiency. Western blot and q-PCR were performed to determine ...

  4. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    International Nuclear Information System (INIS)

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-01

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  5. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  6. Genotyping of the fish rhabdovirus, viral haemorrhagic septicaemia virus, by restriction fragment length polymorphisms

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Winton, J.; Lorenzen, Niels

    2005-01-01

    The aim of this study was to develop a standardized molecular assay that used limited resources and equipment for routine genotyping of isolates of the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Computer generated restriction maps, based on 62 unique full-length (1524 nt....... Experimental evaluation of the method consisted of three steps: (i) RT-PCR amplification of the G-gene of VHSV isolates using purified viral RNA as template, (ii) digestion of the PCR products with a panel of restriction endonucleases and (iii) interpretation of the resulting RFLP profiles. The RFLP analysis...

  7. Viral vectors for production of recombinant proteins in plants.

    Science.gov (United States)

    Lico, Chiara; Chen, Qiang; Santi, Luca

    2008-08-01

    Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano-particles for various applications. The majority of recombinant proteins are produced by traditional biological "factories," that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two decades, plants have been under intensive investigation to provide an alternative system for cost-effective, highly scalable, and safe production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral-based expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral-based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. (c) 2008 Wiley-Liss, Inc.

  8. Targeted gene insertion for molecular medicine.

    Science.gov (United States)

    Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

    2008-11-01

    Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

  9. The antibiotic resistome: gene flow in environments, animals and human beings.

    Science.gov (United States)

    Hu, Yongfei; Gao, George F; Zhu, Baoli

    2017-06-01

    The antibiotic resistance is natural in bacteria and predates the human use of antibiotics. Numerous antibiotic resistance genes (ARGs) have been discovered to confer resistance to a wide range of antibiotics. The ARGs in natural environments are highly integrated and tightly regulated in specific bacterial metabolic networks. However, the antibiotic selection pressure conferred by the use of antibiotics in both human medicine and agriculture practice leads to a significant increase of antibiotic resistance and a steady accumulation of ARGs in bacteria. In this review, we summarized, with an emphasis on an ecological point of view, the important research progress regarding the collective ARGs (antibiotic resistome) in bacterial communities of natural environments, human and animals, i.e., in the one health settings.We propose that the resistance gene flow in nature is "from the natural environments" and "to the natural environments"; human and animals, as intermediate recipients and disseminators, contribute greatly to such a resistance gene "circulation."

  10. Wide Awake and Ready to Move: 20 Years of Non-Viral Therapeutic Genome Engineering with the Sleeping Beauty Transposon System.

    Science.gov (United States)

    Hodge, Russ; Narayanavari, Suneel A; Izsvák, Zsuzsanna; Ivics, Zoltán

    2017-10-01

    Gene therapies will only become a widespread tool in the clinical treatment of human diseases with the advent of gene transfer vectors that integrate genetic information stably, safely, effectively, and economically. Two decades after the discovery of the Sleeping Beauty (SB) transposon, it has been transformed into a vector system that is fulfilling these requirements. SB may well overcome some of the limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are being used in the majority of ongoing clinical trials. The SB system has achieved a high level of stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, representing crucial steps that may permit its clinical use in the near future. This article reviews the most important aspects of SB as a tool for gene therapy, including aspects of its vectorization and genomic integration. As an illustration, the clinical development of the SB system toward gene therapy of age-related macular degeneration and cancer immunotherapy is highlighted.

  11. Bile acids for viral hepatitis

    DEFF Research Database (Denmark)

    Chen, Weikeng; Liu, J; Gluud, C

    2003-01-01

    The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness.......The viral hepatitides are common causes of liver diseases globally. Trials have assessed bile acids for patients with viral hepatitis, but no consensus was reached regarding their usefulness....

  12. Viral hemorrhagic septicemia

    Science.gov (United States)

    Batts, William N.; Winton, James R.

    2012-01-01

    Viral hemorrhagic septicemia (VHS) is one of the most important viral diseases of finfish worldwide. In the past, VHS was thought to affect mainly rainbow trout Oncorhynchus mykiss reared at freshwater facilities in Western Europe where it was known by various names including Egtved disease and infectious kidney swelling and liver degeneration (Wolf 1988). Today, VHS is known as an important source of mortality for cultured and wild fish in freshwater and marine environments in several regions of the northern hemisphere (Dixon 1999; Gagné et al. 2007; Kim and Faisal 2011; Lumsden et al. 2007; Marty et al. 1998, 2003; Meyers and Winton 1995; Skall et al. 2005b; Smail 1999; Takano et al. 2001). Viral hemorrhagic septicemia is caused by the fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus of the family Rhabdoviridae

  13. Association of TNF-alpha (-308 A/G) and IFN-gamma (+874 A/T) gene polymorphisms in response to spontaneous and treatment induced viral clearance in HCV infected multitransfused thalassemic patients.

    Science.gov (United States)

    Biswas, Aritra; Gupta, Nabyendu; Gupta, Debanjali; Datta, Abira; Firdaus, Rushna; Chowdhury, Prosanto; Bhattacharyya, Maitreyee; Sadhukhan, Provash C

    2018-06-01

    Multitransfused thalassemic individuals are at high risk of developing transfusion transmitted Hepatitis C virus (HCV) infection. The aim of the study was to correlate the effects of host cytokine single nucleotide polymorphisms of TNF-α (-308 A/G) and IFN-γ (+874 A/T) in spontaneous or IFN induced treatment response in the HCV infected thalassemic individuals. A total of 427 HCV sero-reactive thalassemic individuals were processed for HCV viral genomic diversity and host gene polymorphisms analysis of TNF-α (-308 A/G) and IFN-γ (+874 A/T). Out of 427 HCV sero-reactive individuals, 69.09% were found to be HCV RNA positive with genotype 3 as the predominant infecting strain (94.29%). Study highlighted that, A allele was significantly associated with (p < .05) spontaneous clearance of HCV infection and G allele was correlated with viral persistence at TNF-α (-308) gene polymorphism. Whereas in case of IFN-γ (+874) SNPs, A allele was significantly responsible (p < .05) for spontaneous clearance than T allele. Our study also indicated that in relapsed cases, IFN-γ (+874) T allele is more responsible than A allele. Though no significant correlation was found at both TNF-α (-308) and IFN-γ (+874) gene polymorphism among SVR and relapsed thalassemic patients. A allele at both TNF-α (-308) and IFN-γ (+874) were strongly associated with spontaneous clearance among this population. But in case of SVR and relapsed cases no significant association was found. This cytokine gene polymorphisms pattern will help clinicians to take an informed decision about therapeutic management of HCV infected thalassemic individuals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

    Science.gov (United States)

    Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M

    2017-02-01

    HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide

  15. ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

    Science.gov (United States)

    Pellet, J; Tafforeau, L; Lucas-Hourani, M; Navratil, V; Meyniel, L; Achaz, G; Guironnet-Paquet, A; Aublin-Gex, A; Caignard, G; Cassonnet, P; Chaboud, A; Chantier, T; Deloire, A; Demeret, C; Le Breton, M; Neveu, G; Jacotot, L; Vaglio, P; Delmotte, S; Gautier, C; Combet, C; Deleage, G; Favre, M; Tangy, F; Jacob, Y; Andre, P; Lotteau, V; Rabourdin-Combe, C; Vidalain, P O

    2010-01-01

    Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

  16. Differential Expression of Tomato Spotted Wilt Virus-Derived Viral Small RNAs in Infected Commercial and Experimental Host Plants

    Science.gov (United States)

    Mitter, Neena; Koundal, Vikas; Williams, Sarah; Pappu, Hanu

    2013-01-01

    Background Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. Principal Findings Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. Significance Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral

  17. Differential expression of tomato spotted wilt virus-derived viral small RNAs in infected commercial and experimental host plants.

    Directory of Open Access Journals (Sweden)

    Neena Mitter

    Full Text Available BACKGROUND: Viral small RNAs (vsiRNAs in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV, a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide. PRINCIPAL FINDINGS: Tomato spotted wilt virus (TSWV-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1 higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV. SIGNIFICANCE: Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsi

  18. UGGT1 enhances enterovirus 71 pathogenicity by promoting viral RNA synthesis and viral replication.

    Directory of Open Access Journals (Sweden)

    Peng-Nien Huang

    2017-05-01

    Full Text Available Positive-strand RNA virus infections can induce the stress-related unfolded protein response (UPR in host cells. This study found that enterovirus A71 (EVA71 utilizes host UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1, a key endoplasmic reticulum protein (ER involved in UPR, to enhance viral replication and virulence. EVA71 forms replication complexes (RCs on cellular membranes that contain a mix of host and viral proteins to facilitate viral replication, but the components and processes involved in the assembly and function of RCs are not fully understood. Using EVA71 as a model, this study found that host UGGT1 and viral 3D polymerase co-precipitate along with other factors on membranous replication complexes to enhance viral replication. Increased UGGT1 levels elevated viral growth rates, while viral pathogenicity was observed to be lower in heterozygous knockout mice (Uggt1 +/- mice. These findings provide important insight on the role of UPR and host UGGT1 in regulating RNA virus replication and pathogenicity.

  19. Role of the viral and cellular encoded thymidine kinase in the repair of UV-irradiated herpes simplex virus

    International Nuclear Information System (INIS)

    Rainbow, A.J.; McMaster Univ., Hamilton, ON

    1989-01-01

    A strain of herpes simplex type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk + ) gene and a thymidine kinase deficient (tk - ) mutant strain (HSC-1:PTK3B) were used as probes to examine the repair of UV-damaged viral DNA in one tk - (143) and two tk + (R970-5 and AC4) human cell lines. UV survival for each HSC-1 strain was similar for infection of both tk - and tk + cells suggesting that the repair of viral DNA was not dependent on the expression of a functional cellular tk gene. In contrast, UV survival of HSV-1:PTK3B was substantially reduced compared to HSV-1:KOS when infecting all 3 human cell lines, as well as Vero monkey kidney cells and LPM1A mouse cells. Tjese results suggest that the repair of UV-irradiated HSV-1 in lytically infected mammalian cells depends, in part at least, on the expression of the viral encoded tk. (author). 20 refs.; 1 fig

  20. Non-viral gene therapy for bone tissue engineering.

    Science.gov (United States)

    Wegman, Fiona; Oner, F Cumhur; Dhert, Wouter J A; Alblas, Jacqueline

    2013-01-01

    The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.

  1. Role of RNase MRP in viral RNA degradation and RNA recombination.

    Science.gov (United States)

    Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

    2011-01-01

    RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

  2. Empirical phylogenies and species abundance distributions are consistent with pre-equilibrium dynamics of neutral community models with gene flow

    KAUST Repository

    Bonnet-Lebrun, Anne-Sophie

    2017-03-17

    Community characteristics reflect past ecological and evolutionary dynamics. Here, we investigate whether it is possible to obtain realistically shaped modelled communities - i.e., with phylogenetic trees and species abundance distributions shaped similarly to typical empirical bird and mammal communities - from neutral community models. To test the effect of gene flow, we contrasted two spatially explicit individual-based neutral models: one with protracted speciation, delayed by gene flow, and one with point mutation speciation, unaffected by gene flow. The former produced more realistic communities (shape of phylogenetic tree and species-abundance distribution), consistent with gene flow being a key process in macro-evolutionary dynamics. Earlier models struggled to capture the empirically observed branching tempo in phylogenetic trees, as measured by the gamma statistic. We show that the low gamma values typical of empirical trees can be obtained in models with protracted speciation, in pre-equilibrium communities developing from an initially abundant and widespread species. This was even more so in communities sampled incompletely, particularly if the unknown species are the youngest. Overall, our results demonstrate that the characteristics of empirical communities that we have studied can, to a large extent, be explained through a purely neutral model under pre-equilibrium conditions. This article is protected by copyright. All rights reserved.

  3. Empirical phylogenies and species abundance distributions are consistent with pre-equilibrium dynamics of neutral community models with gene flow

    KAUST Repository

    Bonnet-Lebrun, Anne-Sophie; Manica, Andrea; Eriksson, Anders; Rodrigues, Ana S.L.

    2017-01-01

    Community characteristics reflect past ecological and evolutionary dynamics. Here, we investigate whether it is possible to obtain realistically shaped modelled communities - i.e., with phylogenetic trees and species abundance distributions shaped similarly to typical empirical bird and mammal communities - from neutral community models. To test the effect of gene flow, we contrasted two spatially explicit individual-based neutral models: one with protracted speciation, delayed by gene flow, and one with point mutation speciation, unaffected by gene flow. The former produced more realistic communities (shape of phylogenetic tree and species-abundance distribution), consistent with gene flow being a key process in macro-evolutionary dynamics. Earlier models struggled to capture the empirically observed branching tempo in phylogenetic trees, as measured by the gamma statistic. We show that the low gamma values typical of empirical trees can be obtained in models with protracted speciation, in pre-equilibrium communities developing from an initially abundant and widespread species. This was even more so in communities sampled incompletely, particularly if the unknown species are the youngest. Overall, our results demonstrate that the characteristics of empirical communities that we have studied can, to a large extent, be explained through a purely neutral model under pre-equilibrium conditions. This article is protected by copyright. All rights reserved.

  4. Archaeal Viruses Contribute to the Novel Viral Assemblage Inhabiting Oceanic, Basalt-Hosted Deep Subsurface Crustal Fluids

    Science.gov (United States)

    Nigro, O. D.; Rappe, M. S.; Jungbluth, S.; Lin, H. T.; Steward, G.

    2015-12-01

    Fluids contained in the basalt-hosted deep subsurface of the world's oceans represent one of the most inaccessible and understudied biospheres on earth. Recent improvements in sampling infrastructure have allowed us to collect large volumes of crustal fluids (~104 L) from Circulation Obviation Retrofit Kits (CORKs) placed in boreholes located on the eastern flank of the Juan de Fuca Ridge (JdFR). We detected viruses within these fluids by TEM and epifluorescence microscopy in samples collected from 2010 to 2014. Viral abundance, determined by epifluorescence counts, indicated that concentrations of viruses in subsurface basement fluids (~105 ml-1) are lower than the overlying seawater, but are higher in abundance than microbial cells in the same samples. Analysis of TEM images revealed distinct viral morphologies (rod and spindle-shaped) that resemble the morphologies of viral families infecting archaea. There are very few, if any, direct observations of these viral morphologies in marine samples, although they have been observed in enrichment cultures and their signature genes detected in metagenomic studies from hydrothermal vents and marine sediments. Analysis of metagenomes from the JdFR crustal fluids revealed sequences with homology to archaeal viruses from the rudiviridae, bicaudaviridae and fuselloviridae. Prokaryotic communities in fluids percolating through the basaltic basement rock of the JdFR flank are distinct from those inhabiting the overlying sediments and seawater. Similarly, our data support the idea that the viral assemblage in these fluids is distinct from viral assemblages in other marine and terrestrial aquatic environments. Our data also suggest that viruses contribute to the mortality of deep subsurface prokaryotes through cell lysis, and viruses may alter the genetic potential of their hosts through the processes of lysogenic conversion and horizontal gene transfer.

  5. Non-Invasive Gene Therapy of Experimental Parkinson's Disease

    National Research Council Canada - National Science Library

    Pardridge, William M

    2005-01-01

    The present research has developed a non-viral gene targeting technology, whereby the effects of a neurotoxin on the brain can be reversed shortly after the intravenous injection of a therapeutic gene...

  6. Synthesis, and Characterization, and Evaluation of Cellular Effects of the FOL-PEG-g-PEI-GAL Nanoparticles as a Potential Non-Viral Vector for Gene Delivery

    International Nuclear Information System (INIS)

    Ghiamkazemi, S.; Amanzadeh, A.; Dinarvand, R.; Rafiee-Tehrani, M.; Amini, M.; Ghiamkazemi, S.; Dinarvand, R.; Rafiee-Tehrani, M.; Ghiamkazemi, S.; Rafiee-Tehrani, M.; Amanzadeh, A.; Rafiee-Tehrani, M.

    2010-01-01

    In this manuscript, we synthesized the potential non viral vector for gene delivery with proper transfection efficiency and low cytotoxicity. Polyethylenimine (PEI) is a well-known cationic polymer which has high positive surface charge for condensing plasmid DNA. However; it is highly cytotoxic in many cell lines because of the high surface charge, non-biodegradability and non-biocompatibility. To enhance PEI biodegradability, the graft copolymer PEG-g-PEI was synthesized. To target cancer liver cells, two targeting ligands folic acid and galactose (lactobionic acid) which are over expressed on human hepatocyte carcinoma were attached to graft copolymer and FOL-PEG-g-PEI-GAL copolymer was synthesized. Composition of this grafted copolymer was characterized using 1H-NMR and FTIR spectra. The molecular weight and zeta potential of this copolymer was compared to PEI. The particle size and zeta potential of FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratio were measured using dynamic light scattering (DLS). Cytotoxicity of the copolymer was also studied in cultured HepG2 human hepatoblastoma cell line. The FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratios exhibited no cytotoxicity in HepG2 cell line compared to PEI 25K as a control. The novel copolymer showed enhanced biodegradability in physiological conditions in compared with PEI and targeted cultured HepG2 cells. More importantly, significant transfection efficiency was exhibited in cancer liver cells. Together, our results showed that FOL-PEG-g-PEI-GAL nanoparticles could be considered as a useful non-viral vector for targeted gene delivery.

  7. Viral Marketing

    OpenAIRE

    Sorina Raula Gîrboveanu; Silvia Puiu

    2008-01-01

    With consumers showing increasing resistance to traditional forms of advertising such as TV or newspaper ads, marketers have turned to alternate strategies, including viral marketing. Viral marketing exploits existing social networks by encouraging customers to share product information with their friends.In our study we are able to directly observe the effectiveness of person to person word of mouth advertising for hundreds of thousands of products for the first time

  8. Identification of landscape features influencing gene flow: How useful are habitat selection models?

    Science.gov (United States)

    Gretchen H. Roffler; Michael K. Schwartz; Kristine Pilgrim; Sandra L. Talbot; George K. Sage; Layne G. Adams; Gordon Luikart

    2016-01-01

    Understanding how dispersal patterns are influenced by landscape heterogeneity is critical for modeling species connectivity. Resource selection function (RSF) models are increasingly used in landscape genetics approaches. However, because the ecological factors that drive habitat selection may be different from those influencing dispersal and gene flow, it is...

  9. Geographical patterns of adaptation within a species' range : Interactions between drift and gene flow

    NARCIS (Netherlands)

    Alleaume-Benharira, M; Pen, IR; Ronce, O

    We use individual-based stochastic simulations and analytical deterministic predictions to investigate the interaction between drift, natural selection and gene flow on the patterns of local adaptation across a fragmented species' range under clinally varying selection. Migration between populations

  10. Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors

    Directory of Open Access Journals (Sweden)

    Cavirani Sandro

    2010-09-01

    Full Text Available Abstract Background Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated. Results Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to neo gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck. Conclusion This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.

  11. Intersectional gene flow between insular endemics of Ilex (Aquifoliaceae) on the Bonin Islands and the Ryukyu Islands.

    Science.gov (United States)

    Setoguchi, H; Watanabe, I

    2000-06-01

    Hybridization and introgression play important roles in plant evolution, and their occurrence on the oceanic islands provides good examples of plant speciation and diversification. Restriction fragment length polymorphisms (RFLPs) and trnL (UAA) 3'exon-trnF (GAA) intergenic spacer (IGS) sequences of chloroplast DNA (cpDNA), and the sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA were examined to investigate the occurrence of gene transfer in Ilex species on the Bonin Islands and the Ryukyu Islands in Japan. A gene phylogeny for the plastid genome is in agreement with the morphologically based taxonomy, whereas the nuclear genome phylogeny clusters putatively unrelated endemics both on the Bonin and the Ryukyu Islands. Intersectional hybridization and nuclear gene flow were independently observed in insular endemics of Ilex on both sets of islands without evidence of plastid introgression. Gene flow observed in these island systems can be explained by ecological features of insular endemics, i.e., limits of distribution range or sympatric distribution in a small land area.

  12. Use of profile hidden Markov models in viral discovery: current insights

    Directory of Open Access Journals (Sweden)

    Reyes A

    2017-07-01

    efficacy of the models. One of the interesting applications of viral profile HMMs is the detection and sequence reconstruction of specific viral genomes from metagenomic data. In fact, several DNA assembly programs that use profile HMMs as seeds have been developed to identify and build gene-sized assemblies or viral genome sequences of unrestrained length, using conventional and progressive assembly approaches, respectively. In this review, we address these aspects and cover some up-to-date information on viral genomics that should be considered in the choice of molecular markers for viral discovery. Finally, we propose a roadmap for rational development of viral profile HMMs and discuss the main challenges associated with this task. Keywords: profile hidden Markov models, viral discovery, DNA assembly, metagenomic analysis, molecular markers, de novo diagnosis 

  13. Gene therapy, early promises, subsequent problems, and recent breakthroughs.

    Science.gov (United States)

    Razi Soofiyani, Saeideh; Baradaran, Behzad; Lotfipour, Farzaneh; Kazemi, Tohid; Mohammadnejad, Leila

    2013-01-01

    Gene therapy is one of the most attractive fields in medicine. The concept of gene delivery to tissues for clinical applications has been discussed around half a century, but scientist's ability to manipulate genetic material via recombinant DNA technology made this purpose to reality. Various approaches, such as viral and non-viral vectors and physical methods, have been developed to make gene delivery safer and more efficient. While gene therapy initially conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, to date gene therapy is considered for many non-life-threatening conditions including those adversely influence on a patient's quality of life. Gene therapy has made significant progress, including tangible success, although much slower than was initially predicted. Although, gene therapies still at a fairly primitive stage, it is firmly science based. There is justifiable hope that with enhanced pathobiological understanding and biotechnological improvements, gene therapy will be a standard part of clinical practice within 20 years.

  14. Admixture and gene flow from Russia in the recovering Northern European brown bear (Ursus arctos).

    Science.gov (United States)

    Kopatz, Alexander; Eiken, Hans Geir; Aspi, Jouni; Kojola, Ilpo; Tobiassen, Camilla; Tirronen, Konstantin F; Danilov, Pjotr I; Hagen, Snorre B

    2014-01-01

    Large carnivores were persecuted to near extinction during the last centuries, but have now recovered in some countries. It has been proposed earlier that the recovery of the Northern European brown bear is supported by migration from Russia. We tested this hypothesis by obtaining for the first time continuous sampling of the whole Finnish bear population, which is located centrally between the Russian and Scandinavian bear populations. The Finnish population is assumed to experience high gene flow from Russian Karelia. If so, no or a low degree of genetic differentiation between Finnish and Russian bears could be expected. We have genotyped bears extensively from all over Finland using 12 validated microsatellite markers and compared their genetic composition to bears from Russian Karelia, Sweden, and Norway. Our fine masked investigation identified two overlapping genetic clusters structured by isolation-by-distance in Finland (pairwise FST = 0.025). One cluster included Russian bears, and migration analyses showed a high number of migrants from Russia into Finland, providing evidence of eastern gene flow as an important driver during recovery. In comparison, both clusters excluded bears from Sweden and Norway, and we found no migrants from Finland in either country, indicating that eastern gene flow was probably not important for the population recovery in Scandinavia. Our analyses on different spatial scales suggest a continuous bear population in Finland and Russian Karelia, separated from Scandinavia.

  15. High gene flow in epiphytic ferns despite habitat loss and fragmentation

    OpenAIRE

    Winkler, Manuela; Koch, Marcus; Hietz, Peter

    2011-01-01

    Tropical montane forests suffer from increasing fragmentation and replacement by other types of land-use such as coffee plantations. These processes are known to affect gene flow and genetic structure of plant populations. Epiphytes are particularly vulnerable because they depend on their supporting trees for their entire life-cycle. We compared population genetic structure and genetic diversity derived from AFLP markers of two epiphytic fern species differing in their ability to colonize sec...

  16. Value of Sharing: Viral Advertisement

    OpenAIRE

    Duygu Aydın; Aşina Gülerarslan; Süleyman Karaçor; Tarık Doğan

    2013-01-01

    Sharing motivations of viral advertisements by consumers and the impacts of these advertisements on the perceptions for brand will be questioned in this study. Three fundamental questions are answered in the study. These are advertisement watching and sharing motivations of individuals, criteria of liking viral advertisement and the impact of individual attitudes for viral advertisement on brand perception respectively. This study will be carried out via a viral advertise...

  17. Viral Marketing and Academic Institution

    OpenAIRE

    Koktová, Silvie

    2010-01-01

    This bachelor thesis examines modern and constantly developing kind of internet marketing -- the so called viral marketing. It deals with its origin, principle, process, advantages and disadvantages, types of viral marketing and presumptions of creating successful viral campaign. The aim of the theoretical part is especially the understanding of viral marketing as one of the effective instruments of contemporary marketing. In this theoretical part the thesis also elaborates a marketing school...

  18. The transcriptional regulator gene E2 of the Human Papillomavirus (HPV) 16 influences the radiosensitivity of cervical keratinocytes

    International Nuclear Information System (INIS)

    Lindel, Katja; Rieken, Stefan; Daffinger, Sigrid; Weber, Klaus J; Villiers, Ethel-Michele de; Debus, Jürgen

    2012-01-01

    Clinical studies have demonstrated that HPV induced tumors constitute a specific subclass of cancer with a better response to radiation treatment. The purpose of this study was to investigate meaning of viral E2-gene for radiosensitivity. W12 cells contain episomal HPV 16 genomes, whereas S12 cells, which derive from the W12 line, contain HPV DNA as integrated copies. Clonogenic survival was analyzed using 96-well in vitro test. Using flow cytometry cell cycle analyses were performed. Expression of pRb and p53 were analyzed using intracellular staining. W12 cells (intact E2 gene) showed a lower survival fraction than S12 cells. W12 cells developed a G2/M block 24 h after irradiation with 2 Gy whereas S12 showed no G2/M bloc. After irradiation S12 cells developed polyploidy and pRb-positive cells decreased. W12 cells showed no change of pRb-positive cells. Depending on E2 gene status differences in cell cycle regulation might cause radioresistance. The E2/E7/pRb pathway seems to influence HPV-induced radiosensitivity. Our experiments demonstrated an effect of HPV on radiosensitivity of cervical keratinocytes via viral transcription regulator E2 pathway

  19. Contribution of viral recombinants to the study of the immune response against the Epstein-Barr virus.

    Science.gov (United States)

    Delecluse, Henri-Jacques; Feederle, Regina; Behrends, Uta; Mautner, Josef

    2008-12-01

    Over the past two decades, Epstein-Barr virus (EBV) mutants have become valuable tools for the analysis of viral functions. Several experimental strategies are currently used to generate recombinant mutant genomes that carry alterations in one or several viral genes. The probably most versatile approach utilizes bacterial artificial chromosomes (BAC) carrying parts or the whole EBV genome, which permits extensive genetic manipulations in Escherichia coli cells. The 'mini-EBVs', for example, which contain roughly half of the wild type viral information, efficiently transform primary B cells and have been used as gene vectors for foreign antigens. After expression in lymphoblastoid cell lines (LCLs), these antigens are efficiently presented on MHC molecules and recognized by antigen-specific T cells. These vectors, however, cannot undergo lytic replication and require a helper cell line for efficient replication and DNA packaging. Further experimental systems include the complete viral genome cloned onto a BAC. These mutants can typically be complemented by expression plasmids, some of which are expressed on EBV-derived vectors and can be propagated without requirement of a helper cell line. Over the last years, these viral recombinants have been utilized increasingly to analyse different aspects of the immune response against EBV. Immunological applications are manifold and steadily growing and include crude screening of T cell clones for their specificity towards latent versus lytic antigens, or more detailed analyses in which the exact specificity of T cells is determined using EBV mutants that lack a single viral antigen. Other applications include detailed analysis of protein domains important for immune recognition, e.g. Gly-Ala repeats in the EBV nuclear antigen 1 (EBNA1) protein, expansion of T cell clones directed against virion structures using virus-like particles and phenotypic analysis of virus mutants defective in infection. Future developments might

  20. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

    Science.gov (United States)

    Bi, Yanwei; Sun, Le; Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

    2014-05-01

    A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

  1. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

    Directory of Open Access Journals (Sweden)

    Yanwei Bi

    2014-05-01

    Full Text Available A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR-associated (Cas RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ and homology-directed repair (HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

  2. Conservation of gene cassettes among diverse viruses of the human gut.

    Directory of Open Access Journals (Sweden)

    Samuel Minot

    Full Text Available Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.

  3. 77 FR 22333 - Prospective Grant of Exclusive License: Development of Oncolytic Viral Cancer Therapies

    Science.gov (United States)

    2012-04-13

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: Development of Oncolytic Viral Cancer Therapies AGENCY: National Institutes of Health... administration of the recombinant virus to a human or animal subject, the foreign gene is expressed in vivo to...

  4. [Emergent viral infections

    NARCIS (Netherlands)

    Galama, J.M.D.

    2001-01-01

    The emergence and re-emergence of viral infections is an ongoing process. Large-scale vaccination programmes led to the eradication or control of some viral infections in the last century, but new viruses are always emerging. Increased travel is leading to a rise in the importation of exotic

  5. Gene therapy as a potential tool for treating neuroblastoma-a focused review.

    Science.gov (United States)

    Kumar, M D; Dravid, A; Kumar, A; Sen, D

    2016-05-01

    Neuroblastoma, a solid tumor caused by rapid division of undifferentiated neuroblasts, is the most common childhood malignancy affecting children aged genes is restored to normalcy. Gene therapy is a powerful tool with the potential to inhibit the deleterious effects of oncogenes by inserting corrected/normal genes into the genome. Both viral and non-viral vector-based gene therapies have been developed and adopted to deliver the target genes into neuroblastoma cells. These attempts have given hope to bringing in a new regime of treatment against neuroblastoma. A few gene-therapy-based treatment strategies have been tested in limited clinical trials yielding some positive results. This mini review is an attempt to provide an overview of the available options of gene therapy to treat neuroblastoma.

  6. Genetic structure and gene flows within horses: a genealogical study at the french population scale.

    Science.gov (United States)

    Pirault, Pauline; Danvy, Sophy; Verrier, Etienne; Leroy, Grégoire

    2013-01-01

    Since horse breeds constitute populations submitted to variable and multiple outcrossing events, we analyzed the genetic structure and gene flows considering horses raised in France. We used genealogical data, with a reference population of 547,620 horses born in France between 2002 and 2011, grouped according to 55 breed origins. On average, individuals had 6.3 equivalent generations known. Considering different population levels, fixation index decreased from an overall species FIT of 1.37%, to an average [Formula: see text] of -0.07% when considering the 55 origins, showing that most horse breeds constitute populations without genetic structure. We illustrate the complexity of gene flows existing among horse breeds, a few populations being closed to foreign influence, most, however, being submitted to various levels of introgression. In particular, Thoroughbred and Arab breeds are largely used as introgression sources, since those two populations explain together 26% of founder origins within the overall horse population. When compared with molecular data, breeds with a small level of coancestry also showed low genetic distance; the gene pool of the breeds was probably impacted by their reproducer exchanges.

  7. Genetic structure, mating system, and long-distance gene flow in heart of palm (Euterpe edulis Mart.).

    Science.gov (United States)

    Gaiotto, F A; Grattapaglia, D; Vencovsky, R

    2003-01-01

    We report a detailed analysis of the population genetic structure, mating system, and gene flow of heart of palm (Euterpe edulis Mart.-Arecaceae) in central Brazil. This palm is considered a keystone species because it supplies fruits for birds and rodents all year and is intensively harvested for culinary purposes. Two populations of this palm tree were examined, using 18 microsatellite loci. The species displays a predominantly outcrossed mating system (tm = 0.94), with a probability of full sibship greater than 70% within open-pollinated families. The following estimates of interpopulation genetic variation were calculated and found significant: FIT = 0.17, FIS = 0.12, FST = 0.06, and RST = 0.07. This low but significant level of interpopulation genetic variation indicates high levels of gene flow. Two adult trees were identified as likely seed parents (P > 99.9%) of juveniles located at a distance of 22 km. Gene flow over such distances has not been reported before for tropical tree species. The establishment and management of in situ genetic reserves or ex situ conservation and breeding populations for E. edulis should contemplate the collection of several hundreds open-pollinated maternal families from relatively few distant populations to maximize the genetic sampling of a larger number of pollen parents.

  8. Image of HSV1-TK gene expression with {sup 123}IVDU

    Energy Technology Data Exchange (ETDEWEB)

    Kim, S. Y.; Woo, K. S.; Chung, W. S. [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2005-07-01

    The liver is an important target organ for gene transfer due to its capacity for synthesizing serum protein and its involvement in numerous genetic diseases. So livertargeted gene transfer is significant tool for expanding the treatment options and gene function studies. Gene transfer methods commonly use recombinant viral vector. However, viral vectors also have various disadvantages for example immune recognition after adenoviral vector delivery and potential viralassociated toxicity including helper virus replication and insertional mutagenesis. In contrast, nonviral vectors such as naked plasmid DNA(pDNA) and cationic liposomal systems exhibit low immunogenicity and repeated administration is possible(Ledley et al.,1992; Nabel et al.,1993). These are attractive vectors for in vivo gene transfer because of their suitable characteristics such as biodegradability, minimal toxicity, nonimmunogenicity, and simplicity of use. But non-viral gene delivery, has problems associated with limited efficiency at gene expression. hydrodynamic-based produce has very high level efficiency of gene extraction in liver or soild tumor. In mice, hydrodynamic-based produce was reported that a high level of transgene expression could be obtained in the liver by intravenous injection of large volume( 8{approx}10% of body weight) and high-speed ( Kobayashi N et al., 2004 ). HSV1-TK is one of the most widely use effect gene systems sued for imaging gene expression, in association with its use as a suicide gene, or as a reporter gene In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have developed as prodrug for tumor proliferation imaging or as anti-viral drugs. Several 5-substituted uracil nucleoside derivatives have been identified to have high sensitivity and selective accumulation in HSV1-TK expression cell. This producer has been used hydrodynamic-based produce, we investigated to image of herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene with (E

  9. [A method for genetic transformation of maize for resistance to viral diseases].

    Science.gov (United States)

    Valdez, Marta; Madriz, Kenneth; Ramírez, Pilar

    2004-09-01

    A system for the genetic transformation of maize was developed for two Costa Rican varieties: CR-7 and Diamantes 8843, that can allow the subsequent transfer of viral-derived genes in order to confer resistance to the disease caused by maize rayado fino virus (MRFV). The method is based on particle bombardment of organogenic calli derived from shoot tips. On the other hand, the molecular construction pRFcp-bar, containing the coat protein gene of MRFV and the marker gene bar, was elaborated. For the visual selection of the transformed material was used also the plasmid pDM803 that contains the reporter gene uidA (GUS). The results indicate that devices evaluated: the PIG ("Particle Inflow Gun") and the Bio-Rad are both enough efficient to transfer foreign genes to the genome of the maize.

  10. Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system.

    Science.gov (United States)

    Ikegami, Tetsuro; Peters, C J; Makino, Shinji

    2005-05-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

  11. Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

    NARCIS (Netherlands)

    Yang, X.; Walboomers, X.F.; Dolder, J. van den; Yang, F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2008-01-01

    Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and

  12. Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories.

    Directory of Open Access Journals (Sweden)

    Jovan Nikolic

    2016-10-01

    Full Text Available Stress granules (SGs are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1, T-cell intracellular antigen 1 (TIA-1 and poly(A-binding protein (PABP in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs. Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.

  13. Halting angiogenesis by non-viral somatic gene therapy alleviates psoriasis and murine psoriasiform skin lesions

    DEFF Research Database (Denmark)

    Zibert, John Robert; Wallbrecht, Katrin; Schön, Margarete

    2011-01-01

    Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice......-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein-coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue....... High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated...

  14. Limited contemporary gene flow and high self-replenishment drives peripheral isolation in an endemic coral reef fish.

    Science.gov (United States)

    van der Meer, Martin H; Horne, John B; Gardner, Michael G; Hobbs, Jean-Paul A; Pratchett, Morgan; van Herwerden, Lynne

    2013-06-01

    Extensive ongoing degradation of coral reef habitats worldwide has lead to declines in abundance of coral reef fishes and local extinction of some species. Those most vulnerable are ecological specialists and endemic species. Determining connectivity between locations is vital to understanding recovery and long-term persistence of these species following local extinction. This study explored population connectivity in the ecologically-specialized endemic three-striped butterflyfish (Chaetodon tricinctus) using mt and msatDNA (nuclear microsatellites) to distinguish evolutionary versus contemporary gene flow, estimate self-replenishment and measure genetic diversity among locations at the remote Australian offshore coral reefs of Middleton Reef (MR), Elizabeth Reef (ER), Lord Howe Island (LHI), and Norfolk Island (NI). Mt and msatDNA suggested genetic differentiation of the most peripheral location (NI) from the remaining three locations (MR, ER, LHI). Despite high levels of mtDNA gene flow, there is limited msatDNA gene flow with evidence of high levels of self-replenishment (≥76%) at all four locations. Taken together, this suggests prolonged population recovery times following population declines. The peripheral population (NI) is most vulnerable to local extinction due to its relative isolation, extreme levels of self-replenishment (95%), and low contemporary abundance.

  15. Interferon Lambda Genetics and Biology in Regulation of Viral Control

    Directory of Open Access Journals (Sweden)

    Emily A. Hemann

    2017-12-01

    Full Text Available Type III interferons, also known as interferon lambdas (IFNλs, are the most recent addition to the IFN family following their discovery in 2003. Initially, IFNλ was demonstrated to induce expression of interferon-stimulated genes and exert antiviral properties in a similar manner to type I IFNs. However, while IFNλ has been described to have largely overlapping expression and function with type I IFNs, it has become increasingly clear that type III IFNs also have distinct functions from type I IFNs. In contrast to type I IFNs, whose receptor is ubiquitously expressed, type III IFNs signal and function largely at barrier epithelial surfaces, such as the respiratory and gastrointestinal tracts, as well as the blood–brain barrier. In further support of unique functions for type III IFNs, single nucleotide polymorphisms in IFNL genes in humans are strongly associated with outcomes to viral infection. These biological linkages have also been more directly supported by studies in mice highlighting roles of IFNλ in promoting antiviral immune responses. In this review, we discuss the current understanding of type III IFNs, and how their functions are similar to, and different from, type I IFN in various immune cell subtypes and viral infections.

  16. Prospecting for viral natural enemies of the fire ant Solenopsis invicta in Argentina.

    Science.gov (United States)

    Valles, Steven M; Porter, Sanford D; Calcaterra, Luis A

    2018-01-01

    Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations.