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Sample records for choriomeningitis virus-induced immunopathology

  1. Lymphocytic Choriomeningitis Virus Differentially Affects the Virus-Induced Type I Interferon Response and Mitochondrial Apoptosis Mediated by RIG-I/MAVS

    PubMed Central

    Pythoud, Christelle; Rothenberger, Sylvia; Martínez-Sobrido, Luis; de la Torre, Juan Carlos

    2015-01-01

    ABSTRACT Arenaviruses are important emerging human pathogens maintained by noncytolytic persistent infection in their rodent reservoir hosts. Despite high levels of viral replication, persistently infected carrier hosts show only mildly elevated levels of type I interferon (IFN-I). Accordingly, the arenavirus nucleoprotein (NP) has been identified as a potent IFN-I antagonist capable of blocking activation of interferon regulatory factor 3 (IRF3) via the retinoic acid inducible gene (RIG)-I/mitochondrial antiviral signaling (MAVS) pathway. Another important mechanism of host innate antiviral defense is represented by virus-induced mitochondrial apoptosis via RIG-I/MAVS and IRF3. In the present study, we investigated the ability of the prototypic Old World arenavirus lymphocytic choriomeningitis virus (LCMV) to interfere with RIG-I/MAVS-dependent apoptosis. We found that LCMV does not induce apoptosis at any time during infection. While LCMV efficiently blocked induction of IFN-I via RIG-I/MAVS in response to superinfection with cytopathic RNA viruses, virus-induced mitochondrial apoptosis remained fully active in LCMV-infected cells. Notably, in LCMV-infected cells, RIG-I was dispensable for virus-induced apoptosis via MAVS. Our study reveals that LCMV infection efficiently suppresses induction of IFN-I but does not interfere with the cell's ability to undergo virus-induced mitochondrial apoptosis as a strategy of innate antiviral defense. The RIG-I independence of mitochondrial apoptosis in LCMV-infected cells provides the first evidence that arenaviruses can reshape apoptotic signaling according to their needs. IMPORTANCE Arenaviruses are important emerging human pathogens that are maintained in their rodent hosts by persistent infection. Persistent virus is able to subvert the cellular interferon response, a powerful branch of the innate antiviral defense. Here, we investigated the ability of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) to

  2. Prevention of Influenza Virus-Induced Immunopathology by TGF-β Produced during Allergic Asthma

    PubMed Central

    Furuya, Yoichi; Furuya, Andrea K. M.; Roberts, Sean; Sanfilippo, Alan M.; Salmon, Sharon L.; Metzger, Dennis W.

    2015-01-01

    Asthma is believed to be a risk factor for influenza infection, however little experimental evidence exists to directly demonstrate the impact of asthma on susceptibility to influenza infection. Using a mouse model, we now report that asthmatic mice are actually significantly more resistant to a lethal influenza virus challenge. Notably, the observed increased resistance was not attributable to enhanced viral clearance, but instead, was due to reduced lung inflammation. Asthmatic mice exhibited a significantly reduced cytokine storm, as well as reduced total protein levels and cytotoxicity in the airways, indicators of decreased tissue injury. Further, asthmatic mice had significantly increased levels of TGF-β1 and the heightened resistance of asthmatic mice was abrogated in the absence of TGF-β receptor II. We conclude that a transient increase in TGF-β expression following acute asthma can induce protection against influenza-induced immunopathology. PMID:26407325

  3. COPD immunopathology.

    PubMed

    Caramori, Gaetano; Casolari, Paolo; Barczyk, Adam; Durham, Andrew L; Di Stefano, Antonino; Adcock, Ian

    2016-07-01

    The immunopathology of chronic obstructive pulmonary disease (COPD) is based on the innate and adaptive inflammatory immune responses to the chronic inhalation of cigarette smoking. In the last quarter of the century, the analysis of specimens obtained from the lower airways of COPD patients compared with those from a control group of age-matched smokers with normal lung function has provided novel insights on the potential pathogenetic role of the different cells of the innate and acquired immune responses and their pro/anti-inflammatory mediators and intracellular signalling pathways, contributing to a better knowledge of the immunopathology of COPD both during its stable phase and during its exacerbations. This also has provided a scientific rationale for new drugs discovery and targeting to the lower airways. This review summarises and discusses the immunopathology of COPD patients, of different severity, compared with control smokers with normal lung function. PMID:27178410

  4. Reactive oxygen species delay control of lymphocytic choriomeningitis virus

    PubMed Central

    Lang, P A; Xu, H C; Grusdat, M; McIlwain, D R; Pandyra, A A; Harris, I S; Shaabani, N; Honke, N; Kumar Maney, S; Lang, E; Pozdeev, V I; Recher, M; Odermatt, B; Brenner, D; Häussinger, D; Ohashi, P S; Hengartner, H; Zinkernagel, R M; Mak, T W; Lang, K S

    2013-01-01

    Cluster of differentiation (CD)8+ T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8+ T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1−/−) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage. PMID:23328631

  5. Immunopathology of multiple sclerosis.

    PubMed

    Dendrou, Calliope A; Fugger, Lars; Friese, Manuel A

    2015-09-15

    Two decades of clinical experience with immunomodulatory treatments for multiple sclerosis point to distinct immunological pathways that drive disease relapses and progression. In light of this, we discuss our current understanding of multiple sclerosis immunopathology, evaluate long-standing hypotheses regarding the role of the immune system in the disease and delineate key questions that are still unanswered. Recent and anticipated advances in the field of immunology, and the increasing recognition of inflammation as an important component of neurodegeneration, are shaping our conceptualization of disease pathophysiology, and we explore the potential implications for improved healthcare provision to patients in the future. PMID:26250739

  6. Vaccine-Elicited CD4 T Cells Induce Immunopathology Following Chronic LCMV Infection

    PubMed Central

    Penaloza-MacMaster, Pablo; Barber, Daniel L.; Wherry, E. John; Provine, Nicholas M.; Teigler, Jeffrey E.; Parenteau, Lily; Blackmore, Stephen; Borducchi, Erica N.; Larocca, Rafael A.; Yates, Kathleen B.; Shen, Hao; Haining, W. Nicholas; Sommerstein, Rami; Pinschewer, Daniel D.; Ahmed, Rafi; Barouch, Dan. H.

    2015-01-01

    CD4 T cells promote innate and adaptive immune responses, but how vaccine-elicited CD4 T cells contribute to immune protection remains unclear. Here we evaluated whether induction of virus-specific CD4 T cells by vaccination would protect mice against infection with chronic lymphocytic choriomeningitis virus (LCMV). Immunization with vaccines that selectively induced CD4 T cell responses resulted in catastrophic inflammation and mortality following challenge with a persistent strain of LCMV. Immunopathology required antigen-specific CD4 T cells and was associated with a cytokine storm, generalized inflammation, and multi-organ system failure. Virus-specific CD8 T cells or antibodies abrogated the pathology. These data demonstrate that vaccine-elicited CD4 T cells in the absence of effective antiviral immune responses can trigger lethal immunopathology. PMID:25593185

  7. The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles

    PubMed Central

    Ziegler, Christopher M.; Eisenhauer, Philip; Bruce, Emily A.; Weir, Marion E.; King, Benjamin R.; Klaus, Joseph P.; Krementsov, Dimitry N.; Shirley, David J.; Ballif, Bryan A.; Botten, Jason

    2016-01-01

    Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation. PMID:27010636

  8. Immunopathology of Schistosoma mansoni infection.

    PubMed Central

    Boros, D L

    1989-01-01

    Schistosomiasis mansoni is a chronic helminthic disease that affects about 100 million people in the tropics. The worms have a life span of 5 to 10 years, and they live in the mesenteric veins of the host. Lightly infected individuals are asymptomatic or manifest mild intestinal symptoms. Heavily infected individuals often develop severe morbidity with hepatosplenomegaly, sometimes with a fatal outcome. Morbidity is attributed to the strong humoral and T-cell-mediated host immune responses developed to a variety of parasite antigens and expressed as tissue inflammations. The immunopathology includes dermatitis, immune complex-mediated kidney disease, and, chiefly, T-cell-mediated granuloma formation and fibrosis around disseminated parasite eggs. This review describes the mechanisms of induction and expression of immunopathology in infected persons and experimental animals. Immunoregulatory mechanisms that modulate the enhanced immune responses and may ameliorate excessive morbidity are discussed. PMID:2504481

  9. Dendritic cells in lung immunopathology.

    PubMed

    Cook, Peter C; MacDonald, Andrew S

    2016-07-01

    Dendritic cells (DCs) lie at the heart of the innate immune system, specialised at recognising danger signals in many forms including foreign material, infection or tissue damage and initiating powerful adaptive immune and inflammatory responses. In barrier sites such as the lung, the instrumental role that DCs play at the interface between the environment and the host places them in a pivotal position in determining the severity of inflammatory disease. The past few years has seen a significant increase in our fundamental understanding of the subsets of DCs involved in pulmonary immunity, as well as the mechanisms by which they are activated and which they may use to coordinate downstream inflammation and pathology. In this review, we will summarise current understanding of the multi-faceted role that DCs play in the induction, maintenance and regulation of lung immunopathology, with an emphasis on allergic pulmonary disease. PMID:27256370

  10. 21 CFR 866.3360 - Lymphocytic choriomeningitis virus serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Lymphocytic choriomeningitis virus serological reagents. 866.3360 Section 866.3360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  11. 21 CFR 866.3360 - Lymphocytic choriomeningitis virus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lymphocytic choriomeningitis virus serological reagents. 866.3360 Section 866.3360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  12. 21 CFR 866.3360 - Lymphocytic choriomeningitis virus serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lymphocytic choriomeningitis virus serological reagents. 866.3360 Section 866.3360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  13. 21 CFR 866.3360 - Lymphocytic choriomeningitis virus serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lymphocytic choriomeningitis virus serological reagents. 866.3360 Section 866.3360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  14. 21 CFR 866.3360 - Lymphocytic choriomeningitis virus serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lymphocytic choriomeningitis virus serological reagents. 866.3360 Section 866.3360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological...

  15. Narcolepsy with cataplexy and comorbid immunopathological diseases.

    PubMed

    Martínez-Orozco, Francisco J; Vicario, José L; Villalibre-Valderrey, Isabel; De Andrés, Clara; Fernández-Arquero, Miguel; Peraita-Adrados, Rosa

    2014-08-01

    Evidence suggests that autoimmune diseases tend to co-occur so that patients with an autoimmune disorder are at higher risk of a second autoimmune disease. The association between allergic and autoimmune diseases is also of considerable interest. There are no reports on the association between sporadic or familial narcolepsy with cataplexy and other non-neurological immune-mediated diseases. This study reported on the comorbid immunopathological diseases associated with narcolepsy. One-hundred and fifty six narcoleptic patients with a mean age at diagnosis of 39.1 ± 17.8 years (range, 6-70 years) were assessed using the clinical history, physical and neurological examinations, sleep questionnaires, neuroimaging and human leucocyte antigen typing. Diagnosis was confirmed by polysomnography followed by a multiple sleep latency test or by measuring hypocretin-1 levels. Patients with immunopathological diseases were matched for gender and age at the onset of narcoleptic symptoms with narcoleptic patients without immunopathological diseases. Twenty-six patients (16.6%; 50% women; one familial, 25 sporadic) had one or more immunopathological diseases associated: autoimmune diseases, such as idiopathic thrombocytopenic purpura, multiple sclerosis, systemic lupus erythematosus, psoriasis, Crohn's disease, ulcerative colitis, autoimmune thyroid disease, Peyronie's disease and idiopathic recurrent facial palsy; other immunopathological diseases, like atopic dermatitis, allergic asthma and allergic rhinitis. Although not significant, the age at diagnosis of narcolepsy was 9.3 years earlier in patients with narcolepsy + immunopathological diseases. The results demonstrate that the prevalence of comorbid immunopathological diseases is high in narcolepsy, and cataplexy is significantly more severe in patients with narcolepsy + immunopathological diseases. PMID:24645699

  16. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon.

    PubMed

    Le Bon, Agnes; Etchart, Nathalie; Rossmann, Cornelia; Ashton, Miranda; Hou, Sam; Gewert, Dirk; Borrow, Persephone; Tough, David F

    2003-10-01

    CD8+ T cell responses can be generated against antigens that are not expressed directly within antigen-presenting cells (APCs), through a process known as cross-priming. To initiate cross-priming, APCs must both capture extracellular antigen and receive specific activation signals. We have investigated the nature of APC activation signals associated with virus infection that stimulate cross-priming. We show that infection with lymphocytic choriomeningitis virus induces cross-priming by a mechanism dependent on type I interferon (IFN-alpha/beta). Activation of cross-priming by IFN-alpha/beta was independent of CD4+ T cell help or interaction of CD40 and CD40 ligand, and involved direct stimulation of dendritic cells. These data identify expression of IFN-alpha/beta as a mechanism for the induction of cross-priming during virus infections. PMID:14502286

  17. On the origin of immunopathology.

    PubMed

    Vaz, Nelson M; Carvalho, Claudia R

    2015-06-21

    Stranded between medicine and experimental biology, immunology is buried in its own problems and remains distant from important areas of current biology, such as evolutionary theory, developmental biology and cognitive sciences. Immunology has treated the living system merely as the place or dimension in which immune activity takes place, inserted on a misleading axis (progressive responsiveness versus no response; memory versus tolerance) which neglects the analysis of a robustly stable dynamics which is always present and is neither tolerance nor immunity-a problem currently approached as one of "regulatory" activity. However, a regulatory response also demands regulation, leading to an endless recursion and the adoption of a stimulus-response framework inevitably drives us away from the physiological processes in which lymphocytes are involved. Herein, we propose that immunological physiology, like everything else in the body is dynamic and conservative. Immunopathology, including inherited immunodeficiencies, severe forms of infectious diseases, allergy and autoimmune diseases, are interferences with this stability which frequently include oligoclonal expansions of T lymphocytes. We suggest that this decrease in clonal diversity results from a loss of the stabilizing connectivity among lymphocytes and are not simply markers of immunopathology, but are rather expressions of basic pathogenic mechanisms. The so-called autoimmune diseases are examples of this disequilibrium. In the last decade the characterization of an enormous and diversified commensal microbiota has posed a new and pressing problem: how to explain the harmonic conviviality with trillions of foreign macromolecules. In addition, robustly stable relations towards macromolecular diet can be established by simple ingestion, a state presently labeled as "oral tolerance", a problem that has been buffered for decades as anti-inflammatory protection of the gut. A major change in terminology is necessary

  18. Congenital Viral Infections of the Brain: Lessons Learned from Lymphocytic Choriomeningitis Virus in the Neonatal Rat

    PubMed Central

    Bonthius, Daniel J; Perlman, Stanley

    2007-01-01

    -onset neuronal loss after the virus has been cleared, the neonatal rat infected with LCMV may be an excellent model system to study neurodegenerative or psychiatric diseases whose etiologies are hypothesized to be virus-induced, such as autism, schizophrenia, and temporal lobe epilepsy. PMID:18052527

  19. Virus-induced gene complementation in tomato

    PubMed Central

    Kong, Jinhua; Chen, Weiwei; Shen, Jiajia; Qin, Cheng; Lai, Tongfei; Zhang, Pengcheng; Wang, Ying; Wu, Chaoqun; Yang, Xin; Hong, Yiguo

    2013-01-01

    Virus-induced gene complementation (VIGC), a plant virus technology based on Potato virus X for transient overexpression of endogenous genes complemented tomato mutants, resulting in non-ripening fruits to ripen. This efficient “gain-of-function” approach involves no stable transformation, and reveals a fruit-specific transcriptional network that may exist among key transcription factors in modulating tomato ripening. Thus, VIGC represents a novel and feasible strategy for gene functional analysis in plants. PMID:24305652

  20. Inhibition of diacylglycerol kinase α restores restimulation-induced cell death and reduces immunopathology in XLP-1.

    PubMed

    Ruffo, Elisa; Malacarne, Valeria; Larsen, Sasha E; Das, Rupali; Patrussi, Laura; Wülfing, Christoph; Biskup, Christoph; Kapnick, Senta M; Verbist, Katherine; Tedrick, Paige; Schwartzberg, Pamela L; Baldari, Cosima T; Rubio, Ignacio; Nichols, Kim E; Snow, Andrew L; Baldanzi, Gianluca; Graziani, Andrea

    2016-01-13

    X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8(+) T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase α (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ (protein kinase Cθ). We show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKα prevents the excessive CD8(+) T cell expansion and interferon-γ production that occur in SAP-deficient mice after lymphocytic choriomeningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients. PMID:26764158

  1. Immunopathology of chronic lentivirus-induced arthritis.

    PubMed Central

    Wilkerson, M. J.; Davis, W. C.; Baszler, T. V.; Cheevers, W. P.

    1995-01-01

    This study evaluated histopathology and mononuclear cell phenotypes in synovial lesions of chronic arthritis induced by experimental infection of Saanen goats with caprine arthritis-encephalitis lentivirus. Histological examination of carpal joint synovium of three infected goats with clinical arthritis revealed progressive lesions consisting of membrane villus hypertrophy with extensive angiogenesis and mononuclear cell infiltration and degenerative changes of membrane villus necrosis associated with loss of vasculature and infiltrates. Changes in synovial tissue of five age-matched infected goats without clinical arthritis were limited to moderate synovial membrane hyperplasia also noted in an age-matched uninfected goat. Immunohistochemistry identified CD45R+ CD5- B lymphocytes as the principal component of most perivascular infiltrates in arthritic synovium. Other mononuclear cells included perivascular CD4+ and CD8+ T lymphocytes and macrophages with a prominent accumulation of CD8+ T lymphocytes at the lining surface of inflamed villi. T lymphocytes and macrophages as well as synovial lining cells were activated with respect to MHC class II but not for interleukin-2 receptors. Inflamed villi also contained lymphoid aggregates comprised of B cell germinal centers and activated T-cell mantles. B cells expressing immunoglobulin occurred around follicles and throughout inflamed villi. These findings indicate that memory immune responses that favor expansion and maturation of B cells and immunoglobulin production contribute to the immunopathology of chronic arthritis. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 PMID:7778682

  2. Trace-Forward Investigation of Mice in Response to Lymphocytic Choriomeningitis Virus Outbreak

    PubMed Central

    Knust, Barbara; Petersen, Bret; Gabel, Julie; Manning, Craig; Drenzek, Cherie; Ströher, Ute; Rollin, Pierre E.; Thoroughman, Douglas; Nichol, Stuart T.

    2014-01-01

    During follow-up of a 2012 US outbreak of lymphocytic choriomeningitis virus (LCMV), we conducted a trace-forward investigation. LCMV-infected feeder mice originating from a US rodent breeding facility had been distributed to >500 locations in 21 states. All mice from the facility were euthanized, and no additional persons tested positive for LCMV infection. PMID:24447898

  3. Expanding the range of immunopathology in neuromyelitis optica spectrum disorder.

    PubMed

    Massey, Jennifer; Buckland, Michael E; Barnett, Yael; Sutton, Ian

    2016-01-01

    Neuromyelitis optica (NMO) spectrum disorder typically associates with a pathognomonic antibody response directed against aquaporin 4 and a unique immunopathological signature characterised by loss of aquaporin 4 expression. We describe a tumefactive presentation of NMO in which a biopsy specimen demonstrated active demyelination and reactive astrocytes with preserved surface aquaporin 4 immunoreactivity and dystrophic processes. While the astrocytic pathology is more typical of classical multiple sclerosis, a positive NMO-IgG (1:80) and subsequent clinical presentation with an area postrema syndrome is consistent with a diagnosis of NMO spectrum disorder. This report expands the immunopathological features that have been reported in NMO spectrum disorder. PMID:27469383

  4. Isolation and characterization of a new strain of lymphocytic choriomeningitis virus from rodents in southwestern France.

    PubMed

    Yama, Ines N; Cazaux, Benoite; Britton-Davidian, Janice; Moureau, Grégory; Thirion, Laurence; de Lamballerie, Xavier; Dobigny, Gauthier; Charrel, Rémi N

    2012-10-01

    A total of 821 tissue samples from rodents trapped during field campaigns organized in Europe and Africa were screened for the presence of arenaviruses by molecular methods and cell culture inoculation when feasible. Two Mus musculus domesticus trapped in the southwestern part of France were infected with a potentially new strain of lymphocytic choriomeningitis virus (LCMV), here referred to as LCMV strain HP65-2009, which was isolated and genetically characterized by whole genome sequencing. Genetic and phylogenetic analyses comparing LCMV HP65-2009 with 26 other LCMV strains showed that it represents a novel highly-divergent strain within the group of Mus musculus-associated LCMV. PMID:22651393

  5. Newly Described Clinical and Immunopathological Feature of Dermatitis Herpetiformis

    PubMed Central

    Bonciolini, Veronica; Bonciani, Diletta; Verdelli, Alice; D'Errico, Antonietta; Antiga, Emiliano; Fabbri, Paolo; Caproni, Marzia

    2012-01-01

    Dermatitis herpetiformis (DH) is an inflammatory cutaneous disease with typical histopathological and immunopathological findings clinically characterized by intensely pruritic polymorphic lesions with a chronic-relapsing course. In addition to classic clinical manifestations of DH, atypical variants are more and more frequently reported and histological and immunological are added to them, whereas the impact on quality of life of patients with DH is increasingly important to a certain diagnosis. The aim of this paper is to describe all the possible clinical, histological, and immunological variants of DH in order to facilitate the diagnosis of a rare disease and, therefore, little known. PMID:22701503

  6. Virus-Induced Gene Silencing in Hexaploid Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional genomics analysis in hexaploid wheat is greatly impeded by the genetic redundancy of polyploidy and the difficulties in generating large numbers of transgenic plants required in insertional mutagenesis strategies. Virus-induced gene silencing (VIGS), however, is a strategy for creating g...

  7. Virus-Induced Gene Silencing in Ornametal Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-Induced Gene Silencing (VIGS) provides an attractive tool for high throughput analysis of the functional effects of gene knock-down. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defe...

  8. Virus-induced gene silencing (VIGS) in barley seedling leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterization. This method exploits a dsRNA-mediated antiviral defense mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid ...

  9. Measles Virus Induces Functional TRAIL Production by Human Dendritic Cells

    PubMed Central

    Vidalain, Pierre-Olivier; Azocar, Olga; Lamouille, Barbara; Astier, Anne; Rabourdin-Combe, Chantal; Servet-Delprat, Christine

    2000-01-01

    Measles virus infection induces a profound immunosuppression that can lead to serious secondary infections. Here we demonstrate that measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression in human monocyte-derived dendritic cells. Moreover, measles virus-infected dendritic cells are shown to be cytotoxic via the TRAIL pathway. PMID:10590149

  10. Virus-Induced gene silencing in ornamental plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-Induced Gene Silencing (VIGS) provides an attractive tool for high throughput analysis of the functional effects of gene knock-down. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defe...

  11. Lymphocytic Choriomeningitis Virus in Employees and Mice at Multipremises Feeder-Rodent Operation, United States, 2012

    PubMed Central

    Ströher, Ute; Edison, Laura; Albariño, César G.; Lovejoy, Jodi; Armeanu, Emilian; House, Jennifer; Cory, Denise; Horton, Clayton; Fowler, Kathy L.; Austin, Jessica; Poe, John; Humbaugh, Kraig E.; Guerrero, Lisa; Campbell, Shelley; Gibbons, Aridth; Reed, Zachary; Cannon, Deborah; Manning, Craig; Petersen, Brett; Metcalf, Douglas; Marsh, Bret; Nichol, Stuart T.; Rollin, Pierre E.

    2014-01-01

    We investigated the extent of lymphocytic choriomeningitis virus (LCMV) infection in employees and rodents at 3 commercial breeding facilities. Of 97 employees tested, 31 (32%) had IgM and/or IgG to LCMV, and aseptic meningitis was diagnosed in 4 employees. Of 1,820 rodents tested in 1 facility, 382 (21%) mice (Mus musculus) had detectable IgG, and 13 (0.7%) were positive by reverse transcription PCR; LCMV was isolated from 8. Rats (Rattus norvegicus) were not found to be infected. S-segment RNA sequence was similar to strains previously isolated in North America. Contact by wild mice with colony mice was the likely source for LCMV, and shipments of infected mice among facilities spread the infection. The breeding colonies were depopulated to prevent further human infections. Future outbreaks can be prevented with monitoring and management, and employees should be made aware of LCMV risks and prevention. PMID:24447605

  12. SHP-1-dependent macrophage differentiation exacerbates virus-induced myositis

    PubMed Central

    Watson, Neva B.; Schneider, Karin M.; Massa, Paul T.

    2015-01-01

    Virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. Moreover, muscle-tropic viruses often spread to the central nervous system causing dramatically increased morbidity. Therefore, there is an urgent need to explore genetic factors involved in this class of human disease. This report investigates critical innate immune pathways affecting murine virus-induced myositis. Of particular importance, the key immune regulator SHP-1, which normally suppresses macrophage-mediated inflammation, is a major factor in promoting clinical disease in muscle. We show that Theiler’s murine encephalomyelitis virus infection of skeletal myofibers induces inflammation and subsequent dystrophic calcification with loss of ambulation in wild type mice. Surprisingly, although similar extensive myofiber infection and inflammation is observed in SHP-1-deficient (SHP-1−/−) mice, these mice neither accumulate dead calcified myofibers nor lose ambulation. Macrophages were the predominant effector cells infiltrating WT and SHP-1−/− muscle, and an increased infiltration of immature monocytes/macrophages correlated with absence of clinical disease in SHP-1−/− mice, while mature M1-like macrophages corresponded with increased myofiber degeneration in WT mice. Furthermore, blocking SHP-1 activation in WT macrophages blocked virus-induced myofiber degeneration, and pharmacologic ablation of macrophages inhibited muscle calcification in TMEV-infected WT animals. These data suggest that following TMEV infection of muscle, SHP-1 promotes M1 differentiation of infiltrating macrophages, and these inflammatory macrophages are likely involved in damaging muscle fibers. These findings reveal a pathological role for SHP-1 in promoting inflammatory macrophage differentiation and myofiber damage in virus-infected skeletal muscle, thus identifying SHP-1 and M1 macrophages as essential mediators of virus-induced myopathy. PMID:25681345

  13. The immunopathology of canine vector-borne diseases

    PubMed Central

    2011-01-01

    The canine vector-borne infectious diseases (CVBDs) are an emerging problem in veterinary medicine and the zoonotic potential of many of these agents is a significant consideration for human health. The successful diagnosis, treatment and prevention of these infections is dependent upon firm understanding of the underlying immunopathology of the diseases in which there are unique tripartite interactions between the microorganism, the vector and the host immune system. Although significant advances have been made in the areas of molecular speciation and the epidemiology of these infections and their vectors, basic knowledge of the pathology and immunology of the diseases has lagged behind. This review summarizes recent studies of the pathology and host immune response in the major CVBDs (leishmaniosis, babesiosis, ehrlichiosis, hepatozoonosis, anaplasmosis, bartonellosis and borreliosis). The ultimate application of such immunological investigation is the development of effective vaccines. The current commercially available vaccines for canine leishmaniosis, babesiosis and borreliosis are reviewed. PMID:21489234

  14. Experimental Models of Microvascular Immunopathology: The Example of Cerebral Malaria

    PubMed Central

    El-Assaad, Fatima; Combes, Valery; Grau, Georges ER

    2015-01-01

    Human cerebral malaria is a severe and often lethal complication of Plasmodium falciparum infection. Complex host and parasite interactions should the precise mechanisms involved in the onset of this neuropathology. Adhesion of parasitised red blood cells and host cells to endothelial cells lead to profound endothelial alterations that trigger immunopathological changes, varying degrees of brain oedema and can compromise cerebral blood flow, cause cranial nerve dysfunction and hypoxia. Study of the cerebral pathology in human patients is limited to clinical and genetic field studies in endemic areas, thus cerebral malaria (CM) research relies heavily on experimental models. The availability of malaria models allows study from the inoculation of Plasmodium to the onset of disease and permit invasive experiments. Here, we discuss some aspects of our current understanding of CM, the experimental models available and some important recent findings extrapolated from these models. PMID:26430675

  15. Pulmonary immunity and immunopathology: lessons from respiratory syncytial virus

    PubMed Central

    Olson, Matthew R; Varga, Steven M

    2009-01-01

    Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and is an important source of morbidity and mortality in the elderly and immunocompromised. This review will discuss the humoral and cellular adaptive immune responses to RSV infection and how these responses are shaped in the immature immune system of the infant and the aged environment of the elderly. Furthermore, we will provide an overview of our current understanding of the role the various arms of the adaptive immune response play in mediating the delicate balance between the successful elimination of the virus from the host and the induction of immunopathology. Efficacious immunization against RSV remains a high priority within the field and we will highlight recent advances made in vaccine design. PMID:18844597

  16. Immunobiology of cytotoxic T-cell escape mutants of lymphocytic choriomeningitis virus.

    PubMed Central

    Moskophidis, D; Zinkernagel, R M

    1995-01-01

    Infection with virus variants exhibiting changes in the peptide sequences defining immunodominant determinants that abolish recognition by antiviral cytotoxic T cells (CTL) presents a considerable challenge to the antiviral T-cell immune system and may enable some viruses to persist in hosts. The potential importance of such variants with respect to mechanisms of viral persistence and disease pathogenesis was assessed by infecting adult mice with variants of lymphocytic choriomeningitis virus (LCMV) strain WE. These variants were selected in vivo or in vitro for resistance to lysis by CD8+ H-2b-restricted antiviral CTL. The majority of anti-LCMV CTL in infected H-2b mice recognize epitopes defined by residues 32 to 42 and 275 to 289 (epitopes 32-42 and 275-289) of the LCMV glycoprotein or 397 to 407 of the viral nucleoprotein. The 8.7 variant exhibits a change in the epitope 32-42 (Val-35-->Leu), and variant CL1.2 exhibits a change in the epitope 275-289 (Asn-280-->Asp) of the wild-type LCMV-WE. The double-mutated 8.7-B23 variant had the variation of 8.7 and an additional change located in the epitope 275-289 (Asn-280-->Ser). The 8.7 variant peptide with unchanged anchor positions bound efficiently to H-2Db and H-2Kb molecules but induced only a very weak CTL response. CTL epitope 275-289 of CL1.2 and 8.7-B23 altered at predicted anchor residues were unable to bind Db molecules and were also not recognized by antiviral CTL. Infection of C57BL/6 mice (H-2b) with the variants exhibiting mutations of one of the CTL epitopes, i.e., 8.7 or CL1.2, induced CTL responses specific for the unmutated epitopes comparable with those induced by infection with WE, and these responses were sufficient to eliminate virus from the host. In contrast, infection with the double-mutated variant 8.7-B23 induced CTL activity that was reduced by a factor of about 50-fold compared with wild-type LCMV. Consequently, high doses (10(7) PFU intravenously) of this virus were eliminated slowly and

  17. The Role of Myeloid Cell Activation and Arginine Metabolism in the Pathogenesis of Virus-Induced Diseases

    PubMed Central

    Burrack, Kristina S.; Morrison, Thomas E.

    2014-01-01

    When an antiviral immune response is generated, a balance must be reached between two opposing pathways: the production of proinflammatory and cytotoxic effectors that drive a robust antiviral immune response to control the infection and regulators that function to limit or blunt an excessive immune response to minimize immune-mediated pathology and repair tissue damage. Myeloid cells, including monocytes and macrophages, play an important role in this balance, particularly through the activities of the arginine-hydrolyzing enzymes nitric oxide synthase 2 (Nos2; iNOS) and arginase 1 (Arg1). Nitric oxide (NO) production by iNOS is an important proinflammatory mediator, whereas Arg1-expressing macrophages contribute to the resolution of inflammation and wound repair. In the context of viral infections, expression of these enzymes can result in a variety of outcomes for the host. NO has direct antiviral properties against some viruses, whereas during other virus infections NO can mediate immunopathology and/or inhibit the antiviral immune response to promote chronic infection. Arg1 activity not only has important wound healing functions but can also inhibit the antiviral immune response during some viral infections. Thus, depending on the specific virus and the tissue(s) involved, the activity of both of these arginine-hydrolyzing enzymes can either exacerbate or limit the severity of virus-induced disease. In this review, we will discuss a variety of viral infections, including HIV, SARS-CoV, LCMV, HCV, RSV, and others, where myeloid cells influence the control and clearance of the virus from the host, as well as the severity and resolution of tissue damage, via the activities of iNOS and/or Arg1. Clearly, monocyte/macrophage activation and arginine metabolism will continue to be important areas of investigation in the context of viral infections. PMID:25250029

  18. Epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus

    PubMed Central

    Saraya, Takeshi; Kurai, Daisuke; Ishii, Haruyuki; Ito, Anri; Sasaki, Yoshiko; Niwa, Shoichi; Kiyota, Naoko; Tsukagoshi, Hiroyuki; Kozawa, Kunihisa; Goto, Hajime; Takizawa, Hajime

    2014-01-01

    Viral respiratory infections may be associated with the virus-induced asthma in adults as well as children. Particularly, human rhinovirus is strongly suggested a major candidate for the associations of the virus-induced asthma. Thus, in this review, we reviewed and focused on the epidemiology, pathophysiology, and treatment of virus-induced asthma with special reference on human rhinovirus. Furthermore, we added our preliminary data regarding the clinical and virological findings in the present review. PMID:24904541

  19. Lymphocytic choriomeningitis virus uses a novel endocytic pathway for infectious entry via late endosomes

    SciTech Connect

    Quirin, Katharina; Eschli, Bruno; Scheu, Isabella; Poort, Linda; Kartenbeck, Juergen; Helenius, Ari

    2008-08-15

    The endocytic entry of lymphocytic choriomeningitis virus (LCMV) into host cells was compared to the entry of viruses known to exploit clathrin or caveolae/raft-dependent pathways. Pharmacological inhibitors, expression of pathway-specific dominant-negative constructs, and siRNA silencing of clathrin together with electron and light microscopy provided evidence that although a minority population followed a classical clathrin-mediated mechanism of entry, the majority of these enveloped RNA viruses used a novel endocytic route to late endosomes. The pathway was clathrin, dynamin-2, actin, Arf6, flotillin-1, caveolae, and lipid raft independent but required membrane cholesterol. Unaffected by perturbation of Rab5 or Rab7 and apparently without passing through Rab5/EEA1-positive early endosomes, the viruses reached late endosomes and underwent acid-induced penetration. This membrane trafficking route between the plasma membrane and late endosomes may function in the turnover of a select group of surface glycoproteins such as the dystroglycan complex, which serves as the receptor of LCMV.

  20. Peptide motif analysis predicts lymphocytic choriomeningitis virus as trigger for multiple sclerosis.

    PubMed

    Hogeboom, Charissa

    2015-10-01

    The etiology of multiple sclerosis (MS) involves both genetic and environmental factors. Genetically, the strongest link is with HLA DRB1*1501, but the environmental trigger, probably a virus, remains uncertain. This investigation scans a panel of proteins from encephalitogenic viruses for peptides homologous to the primary autoantigen from myelin basic protein (MBP), then evaluates candidate peptides against a motif required for T cell cross-reactivity and compares viral prevalence patterns to epidemiological characteristics of MS. The only peptide meeting criteria for cross-reactivity with MBP was one from lymphocytic choriomeningitis virus (LCMV), a zoonotic agent. In contrast to current candidates such as Epstein-Barr virus, the distribution of LCMV is consistent with epidemiological features of MS, including concentration in the temperate zone, higher prevalence farther from the equator, and increased prevalence in proximity to regions of peak MS incidence, while lack of person-to-person transmission is consistent with low MS concordance across monozygotic twins. Further, LCMV blocks induction of type I interferon (IFN). Hypothetically this would dysregulate immune processes in favor of proinflammatory pathways as well as upregulating HLA class II and providing more binding sites for autoantigen. The combination of molecular mimicry with virally-induced immune dysregulation has the potential to explain aspects of autoimmunity not addressed by either mechanism alone. PMID:26319106

  1. Lymphocytic choriomeningitis virus (LCMV) infection of macaques: a model for Lassa fever.

    PubMed

    Zapata, Juan C; Pauza, C David; Djavani, Mahmoud M; Rodas, Juan D; Moshkoff, Dmitry; Bryant, Joseph; Ateh, Eugene; Garcia, Cybele; Lukashevich, Igor S; Salvato, Maria S

    2011-11-01

    Arenaviruses such as Lassa fever virus (LASV) and lymphocytic choriomeningitis virus (LCMV) are benign in their natural reservoir hosts, and can occasionally cause severe viral hemorrhagic fever (VHF) in non-human primates and in human beings. LCMV is considerably more benign for human beings than Lassa virus, however certain strains, like the LCMV-WE strain, can cause severe disease when the virus is delivered as a high-dose inoculum. Here we describe a rhesus macaque model for Lassa fever that employs a virulent strain of LCMV. Since LASV must be studied within Biosafety Level-4 (BSL-4) facilities, the LCMV-infected macaque model has the advantage that it can be used at BSL-3. LCMV-induced disease is rarely as severe as other VHF, but it is similar in cases where vascular leakage leads to lethal systemic failure. The LCMV-infected macaque has been valuable for describing the course of disease with differing viral strains, doses and routes of infection. By monitoring system-wide changes in physiology and gene expression in a controlled experimental setting, it is possible to identify events that are pathognomonic for developing VHF and potential treatment targets. PMID:21820469

  2. Lymphocytic choriomeningitis virus (LCMV) infection of macaques: a model for Lassa fever

    PubMed Central

    Zapata, Juan C.; Pauza, C. David; Djavani, Mahmoud M.; Rodas, Juan D.; Moshkoff, Dmitry; Bryant, Joseph; Ateh, Eugene; Garcia, Cybele; Lukashevich, Igor S.; Salvato, Maria S.

    2011-01-01

    Arenaviruses such as Lassa fever virus (LASV) and lymphocytic choriomeningitis virus (LCMV) are benign in their natural reservoir hosts, and can occasionally cause severe viral hemorrhagic fever (VHF) in non-human primates and in human beings. LCMV is considerably more benign for human beings than Lassa virus, however certain strains, like the LCMV-WE strain, can cause severe disease when the virus is delivered as a high-dose inoculum. Here we describe a rhesus macaque model for Lassa fever that employs a virulent strain of LCMV. Since LASV must be studied within Biosafety Level-4 (BSL-4) facilities, the LCMV-infected macaque model has the advantage that it can be used at BSL-3. LCMV-induced disease is rarely as severe as other VHF, but it is similar in cases where vascular leakage leads to lethal systemic failure. The LCMV-infected macaque has been valuable for describing the course of disease with differing viral strains, doses and routes of infection. By monitoring system-wide changes in physiology and gene expression in a controlled experimental setting, it is possible to identify events that are pathognomonic for developing VHF and potential treatment targets. PMID:21820469

  3. The immuno-pathological conversions of canine demodicosis.

    PubMed

    Singh, Shanker K; Dimri, Umesh

    2014-06-16

    Canine demodicosis is a common but exigent noncontagious parasitic dermatosis caused by overpopulation of the host-specific follicular mites of various Demodex species. Receptivity of dogs to demodicosis and progression of the clinical disease are influenced by numerous factors including; genetic defect, alteration of skin's structure and biochemistry, immunological disorders, hormonal status, breed, age, nutritional status, oxidative stress, length of hair coat, stage of oestrus cycle, parturition, endoparasitism and debilitating diseases. Of these, the immune status is thought to be the most significant. Thus, in the present review we intended to edify the immuno-pathological conversions of canine demodicosis. Generalized demodicosis requires a cutaneous environment that is ecologically and immunologically favorable for extreme colonization of demodectic mites. Demodex canis mites can down regulate the CD4+ T cells; possibly by an increased rate of apoptosis or immunological exhaustion of CD4+ T cells. An increased apoptosis of peripheral leukocytes confers progression of the clinical manifestations. Mites induced elevation of TGF-β and inhibition of TNF-α mRNA expression might be a key factor for revealing the difference in the mechanism of onset between localized and generalized demodicosis. Moreover, an elevated serum level of IL-10 could be accountable for the recurrence as well as occurrence of demodicosis in dogs. Over production of reactive oxygen species can corroborate immunological discrepancies in dogs with demodicosis. PMID:24680602

  4. Immunopathological Features of Canine Myocarditis Associated with Leishmania infantum Infection

    PubMed Central

    Piegari, Giuseppe; Otrocka-Domagala, Iwona; Ciccarelli, Davide; Iovane, Valentina; Oliva, Gaetano; Russo, Valeria; Rinaldi, Laura; Papparella, Serenella; Paciello, Orlando

    2016-01-01

    Myocarditis associated with infectious diseases may occur in dogs, including those caused by the protozoa Neospora caninum, Trypanosoma cruzi, Babesia canis, and Hepatozoon canis. However, although cardiac disease due to Leishmania infection has also been documented, the immunopathological features of myocarditis have not been reported so far. The aim of this study was to examine the types of cellular infiltrates and expression of MHC classes I and II in myocardial samples obtained at necropsy from 15 dogs with an established intravitam diagnosis of visceral leishmaniasis. Pathological features of myocardium were characterized by hyaline degeneration of cardiomyocytes, necrosis, and infiltration of mononuclear inflammatory cells consisting of lymphocytes and macrophages, sometimes with perivascular pattern; fibrosis was also present in various degrees. Immunophenotyping of inflammatory cells was performed by immunohistochemistry on cryostat sections obtained from the heart of the infected dogs. The predominant leukocyte population was CD8+ with a fewer number of CD4+ cells. Many cardiomyocytes expressed MHC classes I and II on the sarcolemma. Leishmania amastigote forms were not detected within macrophages or any other cell of the examined samples. Our study provided evidence that myocarditis in canine visceral leishmaniasis might be related to immunological alterations associated with Leishmania infection. PMID:27413751

  5. Wolbachia and its implications for the immunopathology of filariasis.

    PubMed

    Genchi, Claudio; Kramer, Laura H; Sassera, Davide; Bandi, Claudio

    2012-03-01

    Filarial infections are characterized by immunopathological phenomena, that are responsible for the onset of often dramatic pathological outcomes, such as blindness (Onchocerca volvulus) and elephantiasis (W. bancrofti). In addition, the long-term survival (as long as 10 years) of these parasites in otherwise immunocompetent hosts indicates that these nematodes are capable of manipulating the host immune response. The ground-breaking discovery of the bacterial endosymbiont Wolbachia, which resides in most filarial nematodes causing disease, has led to increasing interest in the role it may play in immuno-modulation, pro-inflammatory pathology and other aspects of filarial infection. Indeed, Wolbachia has been shown to be responsible for exacerbating inflammation (as in river blindness), while at the same time blocking efficient elimination of parasites through the host immune response (Onchocerca ochengi). While studies aimed at identifying Wolbachia as a potential target for anti-filarial therapy are at the forefront of current research, understanding its role in the immunology of filarial infection is a fascinating field that has yet to uncover many secrets. PMID:22214329

  6. Chemokine gene expression in the brains of mice with lymphocytic choriomeningitis.

    PubMed Central

    Asensio, V C; Campbell, I L

    1997-01-01

    Chemokines are pivotal in the trafficking of leukocytes. In the present study, we examined the expression of multiple chemokine genes during the course of lymphocytic choriomeningitis (LCM) in mice. In noninfected mice, no detectable chemokine gene expression was found in the brain; however, by day 3 postinfection, the induction of a number of chemokine mRNAs was observed as follows (in order from the greatest to the least): cytokine responsive gene-2 or interferon-inducible 10-kDa protein (Crg-2/IP-10), RANTES, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1beta), and MCP-3. At day 6 postinfection, the expression of these chemokine mRNAs was increased, and low expression of lymphotactin, C10, MIP-2, and MIP-1alpha mRNAs was detectable. Transcript for T-cell activation-3 was not detectable in the brain at any time following LCM virus (LCMV) infection. With some exceptions, a pattern of chemokine gene expression similar to that in the brain was observed in the peripheral organs of LCMV-infected mice. Mice that lacked expression of gamma interferon developed LCM and had a qualitatively similar but quantitatively reduced cerebral chemokine gene expression profile. In contrast, little or no chemokine gene expression was detectable in the brains of LCMV-infected athymic mice which did not develop LCM. Expression of Crg-2/IP-10 RNA was localized to predominantly resident cells of the central nervous system (CNS) and overlapped with sites of viral infection and immune cell infiltration. These findings demonstrate the expression of a number of chemokine genes in the brains of mice infected with LCMV. The pattern of chemokine gene expression in LCM may profoundly influence the characteristic phenotype and response of leukocytes in the brain and contribute to the immunopathogenesis of this fatal CNS infection. PMID:9311871

  7. Quantity and Quality of Inhaled Dose Predicts Immunopathology in Tuberculosis

    PubMed Central

    Fennelly, Kevin P.; Jones-López, Edward C.

    2015-01-01

    Experimental animal models of tuberculosis (TB) have convincingly demonstrated that inhaled dose predicts immunopathology and survival. In contrast, the importance of inhaled dose has generally not been appreciated in TB epidemiology, clinical science, or the practice of TB control. Infectiousness of TB patients has traditionally been assessed using microscopy for acid-fast bacilli in the sputum, which should be considered only a risk factor. We have recently demonstrated that cough aerosol cultures from index cases with pulmonary TB are the best predictors of new infection among household contacts. We suggest that cough aerosols of M. tuberculosis are the best surrogates of inhaled dose, and we hypothesize that the quantity of cough aerosols is associated with TB infection versus disease. Although several factors affect the quality of infectious aerosols, we propose that the particle size distribution of cough aerosols is an important predictor of primary upper airway disease and cervical lymphadenitis and of immune responses in exposed hosts. We hypothesize that large droplet aerosols (>5 μ) containing M. tuberculosis deposit in the upper airway and can induce immune responses without establishing infection. We suggest that this may partially explain the large proportion of humans who never develop TB disease in spite of having immunological evidence of M. tuberculosis infection (e.g., positive tuberculin skin test or interferon gamma release assay). If these hypotheses are proven true, they would alter the current paradigm of latent TB infection and reactivation, further demonstrating the need for better biomarkers or methods of assessing TB infection and the risk of developing disease. PMID:26175730

  8. Virus -induced plankton dynamic and sea spray oragnics

    NASA Astrophysics Data System (ADS)

    Facchini, Maria Cristina; O'Dowd, Colin; Danovaro, Roberto

    2015-04-01

    The processes that link phytoplankton biomass and productivity to the organic matter enrichment in sea spray aerosol are far from being understood and modelling predictions remain highly uncertain at the moment. While some studies have asserted that the enrichment of OM in sea spray aerosol is independent on marine productivity, others, on the contrary, have shown significant correlation with phytoplankton biomass and productivity (Chl-a retrieved by satellites). Here we show that viral infection of prokaryotes and phytoplankton, by inducing the release of large quantities of surfaceactive organic matter (cell debris, exudates and other colloidal gel-forming material), in part due to cell lysis and plankton defence reactions, and in part from rapid virus multiplication, triggers the organic matter (OM) enrichment in the sea-spray particles during blooms. We show that virus-induced bloom dynamics may explain the contrasting results present in literature on the link between primary productivity and OM sea spray enrichment.

  9. Immunopathology and Cytokine Responses in Commercial Broiler Chickens with Gangrenous Dermatitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gangrene dermatitis (GD) is an emerging disease of increasing economic importance in poultry that results from infection by Clostridium septicum and C. perfringens (CP) type A. Lack of a reproducible disease model has been a major obstacle in understanding the immunopathology of GD. To gain better u...

  10. TPL-2 Regulates Macrophage Lipid Metabolism and M2 Differentiation to Control TH2-Mediated Immunopathology

    PubMed Central

    Entwistle, Lewis J.; Khoury, Hania; Papoutsopoulou, Stamatia; Mahmood, Radma; Mansour, Nuha R.; Ching-Cheng Huang, Stanley; Pearce, Edward J.; Pedro S. de Carvalho, Luiz; Ley, Steven C.

    2016-01-01

    Persistent TH2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of TH2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (Map3k8; Cot) regulated TH2-mediated intestinal, hepatic and pulmonary immunopathology following Schistosoma mansoni infection or S. mansoni egg injection. Elevated inflammation, TH2 cell responses and exacerbated fibrosis in Map3k8–/–mice was observed in mice with myeloid cell-specific (LysM) deletion of Map3k8, but not CD4 cell-specific deletion of Map3k8, indicating that TPL-2 regulated myeloid cell function to limit TH2-mediated immunopathology. Transcriptional and metabolic assays of Map3k8–/–M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated TH2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing TH2 cell expansion and downstream immunopathology and fibrosis. PMID:27487182

  11. Timed action of IL-27 protects from immunopathology while preserving defense in influenza.

    PubMed

    Liu, Francesca Diane M; Kenngott, Elisabeth E; Schröter, Micha F; Kühl, Anja; Jennrich, Silke; Watzlawick, Ralf; Hoffmann, Ute; Wolff, Thorsten; Norley, Stephen; Scheffold, Alexander; Stumhofer, Jason S; Saris, Christiaan J M; Schwab, Jan M; Hunter, Christopher A; Debes, Gudrun F; Hamann, Alf

    2014-05-01

    Infection with influenza virus can result in massive pulmonary infiltration and potentially fatal immunopathology. Understanding the endogenous mechanisms that control immunopathology could provide a key to novel adjunct therapies for this disease. Here we show that the cytokine IL-27 plays a crucial role in protection from exaggerated inflammation during influenza virus infection. Using Il-27ra-/- mice, IL-27 was found to limit immunopathology, neutrophil accumulation, and dampened TH1 or TH17 responses via IL-10-dependent and -independent pathways. Accordingly, the absence of IL-27 signals resulted in a more severe disease course and in diminished survival without impacting viral loads. Consistent with the delayed expression of endogenous Il-27p28 during influenza, systemic treatment with recombinant IL-27 starting at the peak of virus load resulted in a major amelioration of lung pathology, strongly reduced leukocyte infiltration and improved survival without affecting viral clearance. In contrast, early application of IL-27 impaired virus clearance and worsened disease. These findings demonstrate the importance of IL-27 for the physiological control of immunopathology and the potential value of well-timed IL-27 application to treat life-threatening inflammation during lung infection. PMID:24809349

  12. TPL-2 Regulates Macrophage Lipid Metabolism and M2 Differentiation to Control TH2-Mediated Immunopathology.

    PubMed

    Kannan, Yashaswini; Perez-Lloret, Jimena; Li, Yanda; Entwistle, Lewis J; Khoury, Hania; Papoutsopoulou, Stamatia; Mahmood, Radma; Mansour, Nuha R; Ching-Cheng Huang, Stanley; Pearce, Edward J; Pedro S de Carvalho, Luiz; Ley, Steven C; Wilson, Mark S

    2016-08-01

    Persistent TH2 cytokine responses following chronic helminth infections can often lead to the development of tissue pathology and fibrotic scarring. Despite a good understanding of the cellular mechanisms involved in fibrogenesis, there are very few therapeutic options available, highlighting a significant medical need and gap in our understanding of the molecular mechanisms of TH2-mediated immunopathology. In this study, we found that the Map3 kinase, TPL-2 (Map3k8; Cot) regulated TH2-mediated intestinal, hepatic and pulmonary immunopathology following Schistosoma mansoni infection or S. mansoni egg injection. Elevated inflammation, TH2 cell responses and exacerbated fibrosis in Map3k8-/-mice was observed in mice with myeloid cell-specific (LysM) deletion of Map3k8, but not CD4 cell-specific deletion of Map3k8, indicating that TPL-2 regulated myeloid cell function to limit TH2-mediated immunopathology. Transcriptional and metabolic assays of Map3k8-/-M2 macrophages identified that TPL-2 was required for lipolysis, M2 macrophage activation and the expression of a variety of genes involved in immuno-regulatory and pro-fibrotic pathways. Taken together this study identified that TPL-2 regulated TH2-mediated inflammation by supporting lipolysis and M2 macrophage activation, preventing TH2 cell expansion and downstream immunopathology and fibrosis. PMID:27487182

  13. Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants.

    PubMed

    Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo; Liu, Yule

    2016-07-01

    Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. PMID:27225900

  14. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    PubMed Central

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  15. Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

    PubMed Central

    Whilding, Lynsey M; Archibald, Kyra M; Kulbe, Hagen; Balkwill, Frances R; Öberg, Daniel; McNeish, Iain A

    2013-01-01

    The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events. PMID:23985697

  16. Virus-induced gene silencing in eggplant (Solanum melongena).

    PubMed

    Liu, Haiping; Fu, Daqi; Zhu, Benzhong; Yan, Huaxue; Shen, Xiaoying; Zuo, Jinhua; Zhu, Yi; Luo, Yunbo

    2012-06-01

    Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su (Sulfur), CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function. PMID:22268843

  17. Virus-Induced Dormancy in the Archaeon Sulfolobus islandicus

    PubMed Central

    Bautista, Maria A.; Zhang, Changyi

    2015-01-01

    ABSTRACT We investigated the interaction between Sulfolobus spindle-shaped virus (SSV9) and its native archaeal host Sulfolobus islandicus. We show that upon exposure to SSV9, S. islandicus strain RJW002 has a significant growth delay where the majority of cells are dormant (viable but not growing) for 24 to 48 hours postinfection (hpi) compared to the growth of controls without virus. We demonstrate that in this system, dormancy (i) is induced by both active and inactive virus particles at a low multiplicity of infection (MOI), (ii) is reversible in strains with active CRISPR-Cas immunity that prevents the establishment of productive infections, and (iii) results in dramatic and rapid host death if virus persists in the culture even at low levels. Our results add a new dimension to evolutionary models of virus-host interactions, showing that the mere presence of a virus induces host cell stasis and death independent of infection. This novel, highly sensitive, and risky bet-hedging antiviral response must be integrated into models of virus-host interactions in this system so that the true ecological impact of viruses can be predicted and understood. PMID:25827422

  18. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    PubMed

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  19. Hemorrhagic Fever Occurs After Intravenous, But Not After Intragastric, Inoculation of Rhesus Macaques With Lymphocytic Choriomeningitis Virus

    PubMed Central

    Lukashevich, Igor S.; Djavani, Mahmoud; Rodas, Juan D.; Zapata, Juan C.; Usborne, Amy; Emerson, Carol; Mitchen, Jacque; Jahrling, Peter B.; Salvato, Maria S.

    2008-01-01

    Arenaviruses can cause hemorrhagic fever and death in primates and guinea pigs, but these viruses are not highly pathogenic for most rodent carriers. In the United States, arenaviruses precipitated outbreaks of hepatitis in captive monkeys, and they present an emerging health threat in the tropical areas of Africa and South America. We describe infection of rhesus macaques with the prototype arenavirus, lymphocytic choriome-ningitis virus (LCMV), using the WE strain that has been known to cause both encephalopathy and multifocal hemorrhage. Five macaques were inoculated: two by the intravenous (i.v.) and three by the intragastric (i.g.) route. Whereas the two i.v.-inoculated monkeys developed signs and lesions consistent with fatal hemorrhagic fever, the i.g.-inoculated monkeys had an attenuated infection with no disease. Pathological signs of the primate i.v. infection differ significantly from guinea pig arenavirus infections and make this a superior model for human viral hemorrhagic disease. PMID:11992578

  20. Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

    PubMed

    Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

    2016-01-01

    Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. PMID:26643933

  1. Macrophages in Immunopathology of Atherosclerosis: A Target for Diagnostics and Therapy

    PubMed Central

    Orekhov, Alexander N; Sobenin, Igor A; Gavrilin, Mikhail A; Gratchev, Alexei; Kotyashova, Svetlana Y; Nikiforov, Nikita G; Kzhyshkowska, Julia

    2015-01-01

    Immunopathology plays important roles in the development of different life-threatening diseases, such as atherosclerosis and its consequences (acute myocardial infarction and stroke), cancer, chronic inflammatory diseases. Effective modulation of the immune system may significantly increase the efficacy of prevention and therapy efforts. Currently there are no marketed drugs capable of normalizing immune system function in an intrinsic and comprehensive way. Here, we describe a test system designed for complex analysis of monocyte activity in individuals to diagnose immunopathology and monitor treatment efficacy. This cell-based test system may also be useful for screening compounds with an immune-correcting effects. Both diagnostic and screening systems are based on primary culture of human monocytes and/or monocyte-derived macrophages. This is the first step in creating a method for assessment of macrophage activity, which is required for further development of immune-correcting drugs. The existing preliminary data provide the basis for realization of this idea. PMID:25312739

  2. Nitric oxide controls tuberculosis immunopathology by inhibiting NLRP3 inflammasome-dependent IL-1β processing

    PubMed Central

    Mishra, Bibhuti B.; Rathinam, Vijay A. K.; Martens, Gregory W.; Martinot, Amanda J.; Kornfeld, Hardy; Fitzgerald, Katherine A.; Sassetti, Christopher M.

    2013-01-01

    Interleukin-1 (IL-1) is an important mediator of innate immunity, but can also promote inflammatory tissue damage. During chronic infections, such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo, we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-γ (IFN-γ). This effect was mediated by nitric oxide (NO), which we found to specifically inhibit the assembly of the NLRP3 inflammasome via thiol nitrosylation. These data suggest that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses that are elicited during persistent infections. PMID:23160153

  3. Metabolic regulation of hepatitis B immunopathology by myeloid-derived suppressor cells

    PubMed Central

    Pallett, Laura J.; Gill, Upkar S.; Quaglia, Alberto; Sinclair, Linda V.; Jover-Cobos, Maria; Schurich, Anna; Singh, Kasha P.; Thomas, Niclas; Das, Abhishek; Chen, Antony; Fusai, Giuseppe; Bertoletti, Antonio; Cantrell, Doreen A.; Kennedy, Patrick T.; Davies, Nathan A.; Haniffa, Muzlifah; Maini, Mala K.

    2015-01-01

    Infection with hepatitis B virus (HBV) results in disparate degrees of tissue injury: it can replicate without pathological consequences or trigger immune-mediated necroinflammatory liver damage. We investigated the potential for myeloid-derived suppressor cells (MDSC) to suppress T cell-mediated immunopathology in this setting. Granulocytic MDSC (gMDSC) expanded transiently in acute resolving HBV, decreasing before peak hepatic injury. In persistent infection, arginase-expressing gMDSC (and circulating arginase) increased most in phases characterized by HBV replication without immunopathology, whilst L-arginine decreased. gMDSC expressed liver-homing chemokine receptors and accumulated in the liver, their expansion being supported by hepatic stellate cells. We provide in vitro and ex vivo evidence that gMDSC potently inhibited T cells in a partially arginase-dependent manner. L-arginine-deprived T cells upregulated system-L amino acid transporters to increase uptake of essential nutrients and attempt metabolic reprogramming. These data demonstrate the capacity of expanded arginase-expressing gMDSC to regulate liver immunopathology in HBV infection. PMID:25962123

  4. A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death.

    PubMed

    Hatsugai, Noriyuki; Kuroyanagi, Miwa; Yamada, Kenji; Meshi, Tetsuo; Tsuda, Shinya; Kondo, Maki; Nishimura, Mikio; Hara-Nishimura, Ikuko

    2004-08-01

    Programmed cell death (PCD) in animals depends on caspase protease activity. Plants also exhibit PCD, for example as a response to pathogens, although a plant caspase remains elusive. Here we show that vacuolar processing enzyme (VPE) is a protease essential for a virus-induced hypersensitive response that involves PCD. VPE deficiency prevented virus-induced hypersensitive cell death in tobacco plants. VPE is structurally unrelated to caspases, although VPE has a caspase-1 activity. Thus, plants have evolved a regulated cellular suicide strategy that, unlike PCD of animals, is mediated by VPE and the cellular vacuole. PMID:15297671

  5. Inhibition of the Type I Interferon Response by the Nucleoprotein of the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus

    PubMed Central

    Martínez-Sobrido, Luis; Zúñiga, Elina I.; Rosario, Debralee; García-Sastre, Adolfo; de la Torre, Juan Carlos

    2006-01-01

    The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a formidable battle horse for the study of viral immunology, as well as viral persistence and associated diseases. Investigations with LCMV have uncovered basic mechanisms by which viruses avoid elimination by the host adaptive immune response. In this study we show that LCMV also disables the host innate defense by interfering with beta interferon (IFN-β) production in response to different stimuli, including infection with Sendai virus and liposome-mediated DNA transfection. Inhibition of IFN production in LCMV-infected cells was caused by an early block in the IFN regulatory factor 3 (IRF-3) activation pathway. This defect was restored in cells cured of LCMV, indicating that one or more LCMV products are responsible for the inhibition of IRF-3 activation. Using expression plasmids encoding individual LCMV proteins, we found that expression of the LCMV nucleoprotein (NP) was sufficient to inhibit both IFN production and nuclear translocation of IRF-3. To our knowledge, this is the first evidence of an IFN-counteracting viral protein in the Arenaviridae family. Inhibition of IFN production by the arenavirus NP is likely to be a determinant of virulence in vivo. PMID:16940530

  6. Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice.

    PubMed

    Park, Hyo Jin; Oh, Ji Hoon; Ha, Sang-Jun

    2016-01-01

    Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4(+) T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-γ, TNF-α, and IL-2. It has been demonstrated that Treg cells' ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from naïve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells. PMID:27404802

  7. Methods for Virus-Induced Gene Silencing in Hexaploid Wheat using barley stripe mosaic virus vectors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a useful functional genomics tool for rapidly creating gene knockout phenotypes that can be used to infer gene function. Until recently, VIGS has only been possible in dicotyledonous plants. However, the development of vectors based on barley stripe mosaic vi...

  8. Gene expression profiling reveals insight into how distinct viruses induce symptoms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant viruses induce a wide array of disease symptoms and cytopathic effects including alterations of chloroplasts, ribosomes, and cellular architecture. While some of these changes are virus specific, many are common even among diverse viruses, and in most cases, the molecular determinants respons...

  9. Co-silencing the mirabilis antiviral protein permits virus-induced gene silencing in Mirabilis jalapa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an attractive and rapid technique for loss of function assay that can reveal the phenotype of embryo-lethal sequences and avoids the need for time consuming transformation and regeneration processes. Among various VIGS vectors that have been explored, the tobac...

  10. Virus induced gene silencing of Arabidopsis gene homologues in wheat identify genes conferring improved drought tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a non-model staple crop like wheat, functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for wheat breeding. Virus induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited tra...

  11. Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology

    PubMed Central

    Brucklacher-Waldert, Verena; Ferreira, Cristina; Innocentin, Silvia; Kamdar, Shraddha; Withers, David R.; Kullberg, Marika C.

    2016-01-01

    The discovery of Th17 cell plasticity, in which CD4+ IL-17–producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ+ ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17ACre– or Rag1Cre-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ+ ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17–producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17ACre–mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ+ ex-Th17 cells. However, IL-17ACre–mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17ACre–mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity. PMID:27183623

  12. Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology.

    PubMed

    Brucklacher-Waldert, Verena; Ferreira, Cristina; Innocentin, Silvia; Kamdar, Shraddha; Withers, David R; Kullberg, Marika C; Veldhoen, Marc

    2016-06-15

    The discovery of Th17 cell plasticity, in which CD4(+) IL-17-producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ(+) ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17A(Cre)- or Rag1(Cre)-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ(+) ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17-producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17A(Cre)-mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ(+) ex-Th17 cells. However, IL-17A(Cre)-mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17A(Cre)-mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity. PMID:27183623

  13. Thymic stromal lymphopoietin as a novel mediator amplifying immunopathology in rheumatic disease.

    PubMed

    Hillen, Maarten R; Radstake, Timothy R D J; Hack, Cornelis E; van Roon, Joel A G

    2015-10-01

    Thymic stromal lymphopoietin (TSLP) is an IL-7-related cytokine that has been studied extensively in atopic diseases and more recently in various rheumatic disorders. It is involved in T cell development in the thymus and promotes homeostatic T cell expansion by classical dendritic cells. However, deregulated TSLP expression in various rheumatic diseases has implicated this cytokine as a strong mediator in immunopathology. Overexpressed TSLP induces strong T cell activation and production of pro-inflammatory cytokines in human cells and animal models for RA, SSc and LN, underscoring the therapeutic potential of targeting the TSLP-TSLP receptor axis. PMID:26163286

  14. The contribution of the sympathetic nervous system to the immunopathology of experimental pulmonary tuberculosis.

    PubMed

    Barrios-Payán, Jorge; Revuelta, Alberto; Mata-Espinosa, Dulce; Marquina-Castillo, Brenda; Villanueva, Enrique Becerril; Gutiérrez, María Eugenia Hernández; Pérez-Sánchez, Gilberto; Pavón, Lenin; Hernandez-Pando, Rogelio

    2016-09-15

    The role of norepinephrine (NE) in the immunopathology of experimental tuberculosis (TB) was studied by measuring pulmonary NE and determining its cellular sources and targets. Functional studies were performed administrating adrenergic and anti-adrenergic drugs at different TB phases. Results showed high production of NE during early infection by adrenergic nerve terminals and lymphocytes located in the lungs and mediastinal lymph nodes, these cells highly expressed β2 adreno-receptors (β2AR) which by an autocrine mechanism promote Th-1 cell differentiation favoring protection. During advanced infection, the production of NE and β2AR sharply decreased, suggesting that adrenergic activity is less important during late TB. PMID:27609282

  15. Cutaneous Manifestations of Non-Celiac Gluten Sensitivity: Clinical Histological and Immunopathological Features

    PubMed Central

    Bonciolini, Veronica; Bianchi, Beatrice; Del Bianco, Elena; Verdelli, Alice; Caproni, Marzia

    2015-01-01

    Background: The dermatological manifestations associated with intestinal diseases are becoming more frequent, especially now when new clinical entities, such as Non-Celiac Gluten Sensitivity (NCGS), are identified. The existence of this new entity is still debated. However, many patients with diagnosed NCGS that present intestinal manifestations have skin lesions that need appropriate characterization. Methods: We involved 17 patients affected by NCGS with non-specific cutaneous manifestations who got much better after a gluten free diet. For a histopathological and immunopathological evaluation, two skin samples from each patient and their clinical data were collected. Results: The median age of the 17 enrolled patients affected by NCGS was 36 years and 76% of them were females. On the extensor surfaces of upper and lower limbs in particular, they all presented very itchy dermatological manifestations morphologically similar to eczema, psoriasis or dermatitis herpetiformis. This similarity was also confirmed histologically, but the immunopathological analysis showed the prevalence of deposits of C3 along the dermo-epidermal junction with a microgranular/granular pattern (82%). Conclusions: The exact characterization of new clinical entities such as Cutaneous Gluten Sensitivity and NCGS is an important objective both for diagnostic and therapeutic purposes, since these are patients who actually benefit from a GFD (Gluten Free Diet) and who do not adopt it only for fashion. PMID:26389946

  16. Host Transcriptional Profiles and Immunopathologic Response following Mycobacterium avium subsp. paratuberculosis Infection in Mice

    PubMed Central

    Shin, Min-Kyoung; Park, Hongtae; Shin, Seung Won; Jung, Myunghwan; Lee, Su-Hyung; Kim, Dae-Yong; Yoo, Han Sang

    2015-01-01

    Paratuberculosis or Johne’s disease is a chronic granulomatous enteropathy in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. In the present study, we examined the host response to MAP infection in spleens of mice in order to investigate the host immunopathology accompanying host-pathogen interaction. Transcriptional profiles of the MAP-infected mice at 3 and 6 weeks p.i. showed severe histopathological changes, whereas those at 12 weeks p.i. displayed reduced lesion severity in the spleen and liver. MAP-infected mice at 3 and 6 weeks p.i. showed up-regulation of interferon-related genes, scavenger receptor, and complement components, suggesting an initial innate immune reaction, such as macrophage activation, bactericidal activity, and macrophage invasion of MAP. Concurrently, MAP-infected mice at 3 and 6 weeks p.i. were also suggested to express M2 macrophage phenotype with up-regulation of Mrc1, and Marco and down-regulation of MHC class II, Ccr7, and Irf5, and canonical pathways related to the T cell response including ICOS-ICOSL signaling in T helper cells, calcium-induced T lymphocyte apoptosis, and CD28 signaling in T helper cell. These results provide information which furthers the understanding of the immunopathologic response to MAP infection in mice, thereby providing insights valuable for research into the pathogenesis for MAP infection. PMID:26439498

  17. Monocyte Activation in Immunopathology: Cellular Test for Development of Diagnostics and Therapy

    PubMed Central

    Ivanova, Ekaterina A.; Orekhov, Alexander N.

    2016-01-01

    Several highly prevalent human diseases are associated with immunopathology. Alterations in the immune system are found in such life-threatening disorders as cancer and atherosclerosis. Monocyte activation followed by macrophage polarization is an important step in normal immune response to pathogens and other relevant stimuli. Depending on the nature of the activation signal, macrophages can acquire pro- or anti-inflammatory phenotypes that are characterized by the expression of distinct patterns of secreted cytokines and surface antigens. This process is disturbed in immunopathologies resulting in abnormal monocyte activation and/or bias of macrophage polarization towards one or the other phenotype. Such alterations could be used as important diagnostic markers and also as possible targets for the development of immunomodulating therapy. Recently developed cellular tests are designed to analyze the phenotype and activity of living cells circulating in patient's bloodstream. Monocyte/macrophage activation test is a successful example of cellular test relevant for atherosclerosis and oncopathology. This test demonstrated changes in macrophage activation in subclinical atherosclerosis and breast cancer and could also be used for screening a panel of natural agents with immunomodulatory activity. Further development of cellular tests will allow broadening the scope of their clinical implication. Such tests may become useful tools for drug research and therapy optimization. PMID:26885534

  18. Probiotics, prebiotics and immunomodulation of gut mucosal defences: homeostasis and immunopathology.

    PubMed

    Hardy, Holly; Harris, Jennifer; Lyon, Eleanor; Beal, Jane; Foey, Andrew D

    2013-06-01

    Probiotics are beneficial microbes that confer a realistic health benefit on the host, which in combination with prebiotics, (indigestible dietary fibre/carbohydrate), also confer a health benefit on the host via products resulting from anaerobic fermentation. There is a growing body of evidence documenting the immune-modulatory ability of probiotic bacteria, it is therefore reasonable to suggest that this is potentiated via a combination of prebiotics and probiotics as a symbiotic mix. The need for probiotic formulations has been appreciated for the health benefits in "topping up your good bacteria" or indeed in an attempt to normalise the dysbiotic microbiota associated with immunopathology. This review will focus on the immunomodulatory role of probiotics and prebiotics on the cells, molecules and immune responses in the gut mucosae, from epithelial barrier to priming of adaptive responses by antigen presenting cells: immune fate decision-tolerance or activation? Modulation of normal homeostatic mechanisms, coupled with findings from probiotic and prebiotic delivery in pathological studies, will highlight the role for these xenobiotics in dysbiosis associated with immunopathology in the context of inflammatory bowel disease, colorectal cancer and hypersensitivity. PMID:23760057

  19. Probiotics, Prebiotics and Immunomodulation of Gut Mucosal Defences: Homeostasis and Immunopathology

    PubMed Central

    Hardy, Holly; Harris, Jennifer; Lyon, Eleanor; Beal, Jane; Foey, Andrew D.

    2013-01-01

    Probiotics are beneficial microbes that confer a realistic health benefit on the host, which in combination with prebiotics, (indigestible dietary fibre/carbohydrate), also confer a health benefit on the host via products resulting from anaerobic fermentation. There is a growing body of evidence documenting the immune-modulatory ability of probiotic bacteria, it is therefore reasonable to suggest that this is potentiated via a combination of prebiotics and probiotics as a symbiotic mix. The need for probiotic formulations has been appreciated for the health benefits in “topping up your good bacteria” or indeed in an attempt to normalise the dysbiotic microbiota associated with immunopathology. This review will focus on the immunomodulatory role of probiotics and prebiotics on the cells, molecules and immune responses in the gut mucosae, from epithelial barrier to priming of adaptive responses by antigen presenting cells: immune fate decision—tolerance or activation? Modulation of normal homeostatic mechanisms, coupled with findings from probiotic and prebiotic delivery in pathological studies, will highlight the role for these xenobiotics in dysbiosis associated with immunopathology in the context of inflammatory bowel disease, colorectal cancer and hypersensitivity. PMID:23760057

  20. Multiple mechanisms contribute to impairment of type 1 interferon production during chronic lymphocytic choriomeningitis virus infection of mice.

    PubMed

    Lee, Lian Ni; Burke, Shannon; Montoya, Maria; Borrow, Persephone

    2009-06-01

    Type 1 IFNs, innate cytokines with important effector and immunomodulatory properties, are rapidly induced in the acute phase of many virus infections; however, this is generally a transient response that is not sustained during virus persistence. To gain insight into mechanisms that can contribute to down-regulation of type 1 IFN production during virus persistence, we analyzed type 1 IFN production during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. High-level type 1 IFN production was transiently up-regulated in cells including plasmacytoid and conventional dendritic cells (DCs) following LCMV infection of mice, but LCMV persistence was associated with only low-level type 1 IFN production. Nonetheless, chronically infected mice were able to up-regulate type 1 IFN production in response to TLR3, 7, and 9 ligands, albeit less efficiently than uninfected mice. Splenic DC numbers in mice chronically infected with LCMV were decreased, and the remaining cells exhibited a reduced response to TLR stimulation. LCMV-infected cell lines efficiently up-regulated type 1 IFN production following TLR ligation and infection with a DNA virus, but exhibited a defect in type 1 IFN induction following infection with Sendai, an RNA virus. This block in type 1 IFN production by infected cells, together with abnormalities in DC numbers and functions, likely contribute to the low-level type 1 IFN production in mice chronically infected with LCMV. Impairment of type 1 IFN production may both promote virus persistence and impact on host immunocompetence. Understanding the mechanisms involved may assist in development of strategies for control of virus persistence and superinfection. PMID:19454715

  1. Uncovering subdominant cytotoxic T-lymphocyte responses in lymphocytic choriomeningitis virus-infected BALB/c mice.

    PubMed Central

    van der Most, R G; Concepcion, R J; Oseroff, C; Alexander, J; Southwood, S; Sidney, J; Chesnut, R W; Ahmed, R; Sette, A

    1997-01-01

    The cytotoxic T-lymphocyte response against lymphocytic choriomeningitis virus (LCMV) in BALB/c mice is predominantly directed against a single, Ld-restricted epitope in the viral nucleoprotein (residues 118 to 126). To investigate whether any Kd/Dd-restricted responses were activated but did not expand during the primary response, we used a BALB/c mutant, BALB/c-H-2dm2, which does not express the Ld molecule. Splenocytes from LCMV-infected BALB/c mice were transferred into irradiated BALB/c-H-2dm2 mice and rechallenged with LCMV. Thus, they were exposed to an antigenic stimulus without the involvement of the immunodominant Ld-restricted epitope. In this adoptive transfer model, the donor splenocytes protected the recipient mice against chronic LCMV infection by mounting a potent Kd- and/or Dd-restricted secondary antiviral response. Analysis of a panel of Kd binding LCMV peptides revealed that residues 283 to 291 from the viral glycoprotein (GP(283-291)) comprise a major new epitope in the adoptive transfer model. Because the donor splenocytes were first activated during the primary infection in BALB/c mice, the GP(283-291) epitope is a subdominant epitope in BALB/c mice that becomes dominant after rechallenge in BALB/c-H-2dm2 mice. This study makes two points. First, it shows that subdominant CTL responses can be protective, and second, it provides a general experimental approach for uncovering subdominant CTL responses in vivo. This strategy can be used to identify subdominant T-cell responses in other systems. PMID:9188577

  2. The role of arachidonic acid metabolism in virus-induced alveolar macrophage dysfunction

    SciTech Connect

    Laegreid, W.W.

    1988-01-01

    Alveolar macrophages (AM) recovered from virus-infected lungs have decreased phagocytic, respiratory burst and bactericidal activities. The studies described below investigated the role of eicosanoids in virus induced AM bactericidal dysfunction. The spectrum of eicosanoid metabolites which bovine AM are capable of producing was determined. Cultured AM were exposed to {sup 3}H-arachidonate for 1 hour, stimulated for 4 hours with A23187, phorbol myristate acetate or zymosan and the supernatants extracted and analyzed by HPLC. All stimuli tested caused the release of these cyclooxygenase metabolites: thromboxane B{sub 2}, PGF{sub 2}, PGE{sub 2}, PGD{sub 2} and HHT. The effect of this enhanced release of arachidonate metabolites on the ability of AM to kill bacteria was evaluated. Preincubation with cyclooxygenase inhibitors or dual cyclooxygenase and lipoxygenase inhibitors resulted in partial reversal of the virus-induced bactericidal deficit in PI3 infected AM.

  3. Virus-induced gene silencing in the rapid cycling columbine Aquilegia coerulea "Origami".

    PubMed

    Sharma, Bharti; Kramer, Elena M

    2013-01-01

    Aquilegia Origami is an emerging model system for ecology and evolution, which has numerous genetic and genomic tools. Virus-induced gene silencing (VIGS) has been established as an effective approach to study gene function in Aquilegia. In the current protocol, we demonstrate VIGS using Agrobacterium strain GV3101 carrying tobacco rattle virus (TRV)-based constructs to infect Aquilegia coerulea "Origami" plants via vacuum infiltration. PMID:23386296

  4. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication.

    PubMed

    Wang, Gefei; Li, Rui; Jiang, Zhiwu; Gu, Liming; Chen, Yanxia; Dai, Jianping; Li, Kangsheng

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy. PMID:27525278

  5. Comparative analysis of radiation- and virus-induced leukemias in BALB/c mice

    SciTech Connect

    Newcomb, E.W.; Binari, R.; Fleissner, E.

    1985-01-15

    Endogenous murine leukemia virus (MuLV) proviral copies were analyzed in thymomas induced in normal BALB/c (Fv-1b) and in Fv-1n congenic mice by X-irradiation. Both strains of mice developed leukemia with similar kinetics, indicating that N-tropism of endogenous MuLV was not a rate-limiting factor in development of disease. Southern blot analysis, using a probe specific for ecotropic virus and for ecotropic-specific sequences retained in pathogenic, env-recombinant viruses, showed that the majority of radiation leukemias lacked newly acquired, clonally integrated, proviruses. This was in contrast to virus-induced leukemias, which routinely exhibited several new proviral integration sites. When an internal proviral DNA restriction fragment was monitored, some radiation leukemias showed evidence of nonclonal infection, accounting for more frequent isolation of infectious virus from such leukemias. Differences in expression of T-cell surface antigens were found in X-ray-induced and virus-induced leukemias. All radiation leukemias were TL positive, whereas virus-induced leukemias were primarily negative for TL. Some differences were also found in Lyt-1 and Lyt-2 expression. The data as a whole suggest that, in the majority of cases, radiation leukemogenesis is not initiated by a viral route--that is, the sort of viral mechanism for which exogenous infection by known pathogenic MuLV is the paradigm.

  6. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication

    PubMed Central

    Jiang, Zhiwu; Gu, Liming; Chen, Yanxia

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy. PMID:27525278

  7. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    SciTech Connect

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  8. Reduced Tyk2 gene expression in β-cells due to natural mutation determines susceptibility to virus-induced diabetes.

    PubMed

    Izumi, Kenichi; Mine, Keiichiro; Inoue, Yoshitaka; Teshima, Miho; Ogawa, Shuichiro; Kai, Yuji; Kurafuji, Toshinobu; Hirakawa, Kanako; Miyakawa, Daiki; Ikeda, Haruka; Inada, Akari; Hara, Manami; Yamada, Hisakata; Akashi, Koichi; Niho, Yoshiyuki; Ina, Keisuke; Kobayashi, Takashi; Yoshikai, Yasunobu; Anzai, Keizo; Yamashita, Teruo; Minagawa, Hiroko; Fujimoto, Shuji; Kurisaki, Hironori; Shimoda, Kazuya; Katsuta, Hitoshi; Nagafuchi, Seiho

    2015-01-01

    Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes. PMID:25849081

  9. Reduced Tyk2 gene expression in β-cells due to natural mutation determines susceptibility to virus-induced diabetes

    PubMed Central

    Izumi, Kenichi; Mine, Keiichiro; Inoue, Yoshitaka; Teshima, Miho; Ogawa, Shuichiro; Kai, Yuji; Kurafuji, Toshinobu; Hirakawa, Kanako; Miyakawa, Daiki; Ikeda, Haruka; Inada, Akari; Hara, Manami; Yamada, Hisakata; Akashi, Koichi; Niho, Yoshiyuki; Ina, Keisuke; Kobayashi, Takashi; Yoshikai, Yasunobu; Anzai, Keizo; Yamashita, Teruo; Minagawa, Hiroko; Fujimoto, Shuji; Kurisaki, Hironori; Shimoda, Kazuya; Katsuta, Hitoshi; Nagafuchi, Seiho

    2015-01-01

    Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes. PMID:25849081

  10. Immunopathologic differences of Sjögren's syndrome versus sicca syndrome in HCV and HIV infection

    PubMed Central

    2011-01-01

    A clinical picture of dry eye and dry mouth with the histological counterpart of focal lymphocytic sialoadenitis, usually detected in minor salivary glands, is considered the hallmark of Sjögren's syndrome. The association of sicca complaints and focal sialoadenitis can be also found in a number of other diseases, including some systemic viral infections. Among these conditions, chronic hepatitis C virus infection, associated with mixed cryoglobulinaemia and extra-hepatic manifestations, and HIV infection, particularly in the phase of diffuse interstitial lymphocytic infiltration, may mimic the clinical and histological aspects of Sjögren's syndrome. However, each disorder is characterised by specific, disease-related immunopathological aspects. Besides sicca complaints, the various disorders may also share a number of systemic extra-glandular features and the possible development of mucosa-associated lymphoid tissue lymphomas. This latter event represents in all of these diseases the final result of an antigen-driven chronic stimulation of B lymphocytes. PMID:21888688

  11. Subgingival Microbial Communities in Leukocyte Adhesion Deficiency and Their Relationship with Local Immunopathology

    PubMed Central

    Moutsopoulos, Niki M.; Abusleme, Loreto; Greenwell-Wild, Teresa; Dutzan, Nicolas; Paster, Bruce J.; Munson, Peter J.; Fine, Daniel H.; Uzel, Gulbu; Holland, Steven M.

    2015-01-01

    Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of β2 integrins which result in defective transmigration of neutrophils into the tissues. Affected patients suffer from recurrent life threatening infections and severe oral disease (periodontitis). Microbial communities in the local environment (subgingival plaque) are thought to be the triggers for inflammatory periodontitis, yet little is known regarding the microbial communities associated with LAD-I periodontitis. Here we present the first comprehensive characterization of the subgingival communities in LAD-I, using a 16S rRNA gene-based microarray, and investigate the relationship of this tooth adherent microbiome to the local immunopathology of periodontitis. We show that the LAD subgingival microbiome is distinct from that of health and Localized Aggressive Periodontitits. Select periodontitis-associated species in the LAD microbiome included Parvimonas micra, Porphyromonas endodontalis, Eubacterium brachy and Treponema species. Pseudomonas aeruginosa, a bacterium not typically found in subgingival plaque is detected in LAD-I. We suggest that microbial products from LAD-associated communities may have a role in stimulating the local inflammatory response. We demonstrate that bacterial LPS translocates into the lesions of LAD-periodontitis potentially triggering immunopathology. We also show in in vitro assays with human macrophages and in vivo in animal models that microbial products from LAD-associated subgingival plaque trigger IL-23-related immune responses, which have been shown to dominate in patient lesions. In conclusion, our current study characterizes the subgingival microbial communities in LAD-periodontitis and supports their role as triggers of disease pathogenesis. PMID:25741691

  12. Virus-triggered acquired immunodeficiency by cytotoxic T-cell-dependent destruction of antigen-presenting cells and lymph follicle structure.

    PubMed Central

    Odermatt, B; Eppler, M; Leist, T P; Hengartner, H; Zinkernagel, R M

    1991-01-01

    Virus-induced acquired immune suppression in mice infected with lymphocytic choriomeningitis virus is shown here to be caused by the CD8+-T-cell-dependent elimination of macrophages/antigen-presenting cells. Surprisingly, this is associated with severe destruction of the follicular organization of lymphoid organs, indicating a crucial role for dendritic cells and marginal zone macrophages in maintaining follicular structure. Once established, this immunopathology cannot be readily reversed by the elimination of CD8+ effector cells. Such a T-cell-mediated pathogenesis may play a pivotal role in acquired virus-induced immunosuppression and may represent one strategy by which virus escapes immune surveillance and establishes persistent infections in initially immunocompetent hosts. Images PMID:1910175

  13. Lessons from T cell responses to virus induced tumours for cancer eradication in general.

    PubMed

    Melief, C J; Kast, W M

    1992-01-01

    Immunotherapy of virus induced tumours by adoptive transfer of virus specific cytotoxic T cells (CTL) is now feasible in experimental murine systems. These CTL recognize viral peptide sequences of defined length presented in the groove of MHC class I molecules. Effective eradication of large tumour masses requires coadministration of IL-2. In essence, T cell immunity against virus induced tumours does not differ from anti-viral T cell immunity in general. Tumour escape strategies are numerous but, in various instances, can be counteracted by defined measures. Initiation of CTL responses against poorly immunogenic non-virus induced tumours (the majority of human cancer) requires novel strategies to overcome T cell inertia. Rather than waiting to see whether tumour specific CTL (against unknown antigens) can be cultured from TIL, we propose an alternative strategy in which CTL are raised against target molecules of choice, including differentiation antigens of restricted tissue distribution (autoantigens) or mutated/overexpressed oncogene products. The various steps proposed include: (a) identification of target molecules of choice; (b) identification in these target molecules of MHC allele specific peptide motifs involved in peptide binding to MHC molecules; (c) evaluation of actual binding of such peptides to specific MHC class I molecules; (d) in vitro CTL response induction by such peptides, presented either by highly efficient antigen presenting cells (such as processing defective cells, which carry empty MHC class I molecules) loaded with a single peptide or by dendritic cells, both cell types being capable of primary CTL response induction in vitro and (e) adoptive transfer of tumour specific CTL generated in vivo or, more conveniently, vaccination with immunodominant peptides. The latter possibility seems to be feasible because peptide vaccination with a single immunodominant viral peptide can install CTL memory and confer protection against lethal virus

  14. RIG-I Signaling Is Essential for Influenza B Virus-Induced Rapid Interferon Gene Expression

    PubMed Central

    Österlund, Pamela; Westenius, Veera; Latvala, Sinikka; Diamond, Michael S.; Gale, Michael; Julkunen, Ilkka

    2015-01-01

    ABSTRACT Influenza B virus causes annual epidemics and, along with influenza A virus, accounts for substantial disease and economic burden throughout the world. Influenza B virus infects only humans and some marine mammals and is not responsible for pandemics, possibly due to a very low frequency of reassortment and a lower evolutionary rate than that of influenza A virus. Influenza B virus has been less studied than influenza A virus, and thus, a comparison of influenza A and B virus infection mechanisms may provide new insight into virus-host interactions. Here we analyzed the early events in influenza B virus infection and interferon (IFN) gene expression in human monocyte-derived macrophages and dendritic cells. We show that influenza B virus induces IFN regulatory factor 3 (IRF3) activation and IFN-λ1 gene expression with faster kinetics than does influenza A virus, without a requirement for viral protein synthesis or replication. Influenza B virus-induced activation of IRF3 required the fusion of viral and endosomal membranes, and nuclear accumulation of IRF3 and viral NP occurred concurrently. In comparison, immediate early IRF3 activation was not observed in influenza A virus-infected macrophages. Experiments with RIG-I-, MDA5-, and RIG-I/MDA5-deficient mouse fibroblasts showed that RIG-I is the critical pattern recognition receptor needed for the influenza B virus-induced activation of IRF3. Our results show that innate immune mechanisms are activated immediately after influenza B virus entry through the endocytic pathway, whereas influenza A virus avoids early IRF3 activation and IFN gene induction. IMPORTANCE Recently, a great deal of interest has been paid to identifying the ligands for RIG-I under conditions of natural infection, as many previous studies have been based on transfection of cells with different types of viral or synthetic RNA structures. We shed light on this question by analyzing the earliest step in innate immune recognition of

  15. End products of glutamine oxidation in MC-29 virus-induced chicken hepatoma mitochondria.

    PubMed

    Matsuno, T

    1989-10-01

    Products of glutamine metabolism were examined in the MC-29 virus-induced chicken hepatoma mitochondria incubated in vitro. Glutamine oxidation proceeded in the tumor mitochondria exclusively via a pathway involving glutamic-oxalacetic transaminase. Malate stimulated aspartate production from glutamine, while pyruvate exerted suppressive effect on aspartate production with little alanine formation. The mitochondria of this hepatoma are unique in that the metabolic pattern and response to malate and pyruvate are essentially inconsistent with those reported in normal cells as well as those proposed by Moreadith and Lehninger in various tumor cells. PMID:2571353

  16. MR VIGS: microRNA-based virus-induced gene silencing in plants.

    PubMed

    Chen, Weiwei; Zhang, Qi; Kong, Junhua; Hu, Feng; Li, Bin; Wu, Chaoqun; Qin, Cheng; Zhang, Pengcheng; Shi, Nongnong; Hong, Yiguo

    2015-01-01

    In plants, microRNA (miRNA)-based virus-induced gene silencing, dubbed MR VIGS, is a powerful technique to delineate the biological functions of genes. By targeting to a specific sequence, miRNAs can knock down expression of genes with fewer off-target effects. Here, using a modified Cabbage leaf curling virus (CaLCuV) and Tobacco rattle virus (TRV) as vectors, we describe two virus-based miRNA expression systems to perform MR VIGS for plant functional genomics assays. PMID:25740363

  17. Comorbidity of Narcolepsy Type 1 With Autoimmune Diseases and Other Immunopathological Disorders: A Case-Control Study

    PubMed Central

    Martinez-Orozco, Francisco Javier; Vicario, Jose Luis; De Andres, Clara; Fernandez-Arquero, Miguel; Peraita-Adrados, Rosa

    2016-01-01

    Background Several evidences suggest that autoimmune diseases (ADs) tend to co-occur in an individual and within the same family. Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a selective loss of hypocretin-producing neurons due to a mechanism of neural destruction that indicates an autoimmune pathogenesis, although no evidence is available. We report on the comorbidity of ADs and other immunopathological diseases (including allergy diseases) in narcolepsy. Methods We studied 158 Caucasian NT1 patients (60.7% male; mean age 49.4 ± 19.7 years), in whom the diagnosis was confirmed by polysomnography followed by a multiple sleep latency test, or by hypocretin-1 levels measurements. Results Thirty out of 158 patients (18.99%; 53.3% female; 29 sporadic and one familial cases) had one or more immunopathological diseases associated. A control group of 151 subjects were matched by gender and age with the narcolepsy patients. Results demonstrated that there was a higher frequency of ADs in our series of narcolepsy patients compared to the sample of general population (odds ratio: 3.17; 95% confidence interval: 1.01 - 10.07; P = 0.040). A temporal relationship with the age at onset of the diseases was found. Conclusions Cataplexy was significantly more severe in NT1 patients with immunopathological diseases, and immunopathological diseases are a risk factor for severe forms of cataplexy in our series (odds ratio: 23.6; 95% confidence interval: 5.5 - 100.1). PMID:27298657

  18. Age-related Defects in Ocular and Nasal Mucosal Immune System and the Immunopathology of Dry Eye Disease

    PubMed Central

    Farid, Marjan; Agrawal, Anshu; Fremgen, Daniel; Tao, Jeremiah; Chuyi, He; Nesburn, Anthony B.; BenMohamed, Lbachir

    2014-01-01

    Dry eye disease (DED) is a prevalent public health concern that affects up to 30% of adults and is particularly chronic and severe in the elderly. Two interconnected mechanisms cause DED: (1) an age-related dysfunction of lacrimal and meibomian glands, which leads to decreased tear production and/or an increase in tear evaporation; and (2) an age-related uncontrolled inflammation of the surface of the eye triggered by yet-to-be-determined internal immunopathological mechanisms, independent of tear deficiency and evaporation. In this review we summarize current knowledge on animal models that mimic both the severity and chronicity of inflammatory DED and that have been reliably used to provide insights into the immunopathological mechanisms of DED, and we provide an overview of the opportunities and limitations of the rabbit model in investigating the role of both ocular and nasal mucosal immune systems in the immunopathology of inflammatory DED and in testing novel immunotherapies aimed at delaying or reversing the uncontrolled age-related inflammatory DED. PMID:25535823

  19. Age-related Defects in Ocular and Nasal Mucosal Immune System and the Immunopathology of Dry Eye Disease.

    PubMed

    Farid, Marjan; Agrawal, Anshu; Fremgen, Daniel; Tao, Jeremiah; Chuyi, He; Nesburn, Anthony B; BenMohamed, Lbachir

    2016-06-01

    Dry eye disease (DED) is a prevalent public health concern that affects up to 30% of adults and is particularly chronic and severe in the elderly. Two interconnected mechanisms cause DED: (1) an age-related dysfunction of lacrimal and meibomian glands, which leads to decreased tear production and/or an increase in tear evaporation; and (2) an age-related uncontrolled inflammation of the surface of the eye triggered by yet-to-be-determined internal immunopathological mechanisms, independent of tear deficiency and evaporation. In this review we summarize current knowledge on animal models that mimic both the severity and chronicity of inflammatory DED and that have been reliably used to provide insights into the immunopathological mechanisms of DED, and we provide an overview of the opportunities and limitations of the rabbit model in investigating the role of both ocular and nasal mucosal immune systems in the immunopathology of inflammatory DED and in testing novel immunotherapies aimed at delaying or reversing the uncontrolled age-related inflammatory DED. PMID:25535823

  20. Immunization with non-replicating E. coli minicells delivering both protein antigen and DNA protects mice from lethal challenge with lymphocytic choriomeningitis virus

    PubMed Central

    Giacalone, Matthew J.; Zapata, Juan C.; Berkley, Neil L.; Sabbadini, Roger A.; Chu, Yen-Lin; Salvato, Maria S.; McGuire, Kathleen L.

    2008-01-01

    In the midst of new investigations into the mechanisms of both delivery and protection of new vaccines and vaccine carriers, it has become clear that immunization with delivery mechanisms that do not involve living, replicating organisms are vastly preferred. In this report, non-replicating bacterial minicells simultaneously co-delivering the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) and the corresponding DNA vaccine were tested for the ability to generate protective cellular immune responses in mice. It was found that good protection (89%) was achieved after intramuscular administration, moderate protection (31%) was achieved after intranasal administration, and less protection (7%) was achieved following gastric immunization. These results provide a solid foundation on which to pursue the use of bacterial minicells as a non-replicating vaccine delivery platform. PMID:17258845

  1. Virus-induced gene complementation reveals a transcription factor network in modulation of tomato fruit ripening

    PubMed Central

    Zhou, Tao; Zhang, Hang; Lai, Tongfei; Qin, Cheng; Shi, Nongnong; Wang, Huizhong; Jin, Mingfei; Zhong, Silin; Fan, Zaifeng; Liu, Yule; Wu, Zirong; Jackson, Stephen; Giovannoni, James J.; Rolin, Dominique; Gallusci, Philippe; Hong, Yiguo

    2012-01-01

    Plant virus technology, in particular virus-induced gene silencing, is a widely used reverse- and forward-genetics tool in plant functional genomics. However the potential of virus technology to express genes to induce phenotypes or to complement mutants in order to understand the function of plant genes is not well documented. Here we exploit Potato virus X as a tool for virus-induced gene complementation (VIGC). Using VIGC in tomato, we demonstrated that ectopic viral expression of LeMADS-RIN, which encodes a MADS-box transcription factor (TF), resulted in functional complementation of the non-ripening rin mutant phenotype and caused fruits to ripen. Comparative gene expression analysis indicated that LeMADS-RIN up-regulated expression of the SBP-box (SQUAMOSA promoter binding protein-like) gene LeSPL-CNR, but down-regulated the expression of LeHB-1, an HD-Zip homeobox TF gene. Our data support the hypothesis that a transcriptional network may exist among key TFs in the modulation of fruit ripening in tomato. PMID:23150786

  2. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

    PubMed

    Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2016-06-01

    Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. PMID:26979473

  3. Immunopathology of experimental Chagas' disease: binding of T cells to Trypanosoma cruzi-infected heart tissue.

    PubMed Central

    Mortatti, R C; Maia, L C; de Oliveira, A V; Munk, M E

    1990-01-01

    The immunopathology of Chagas' disease was studied in the experimental model of chronic infection in C57BL/10JT or mice. Sublethal infection with Trypanosoma cruzi, Y strain, induced specific antibodies and a delayed hypersensitivity response to parasite antigens. Mice developed chronic chagasic myocarditis but not skeletal muscle myositis. Binding of T cells to infected heart tissue was investigated during short-term cocultivation of lymphocytes with heart cryostat sections. T cells from infected mice and from normal controls bound equally to myocardium and liver sections from both infected and normal mice. A search in depth was attempted with cells heavily tagged with 99mTc. Labeled T cells from chagasic mice bound to both normal and infected myocardium slices. 99mTc-labeled T cells from controls gave the same binding values. Glass-adherent spleen cells behaved identically to T cells. Prior treatment of the tissue with serum from chronically infected mice did not increase the number of binding cells. Peritoneal macrophages tagged with 99mTc-sulfur colloid also bound to infected myocardium slices. The binding of macrophages was not changed by pretreatment of infected tissue with anti-T, cruzi antibodies. In short, this work did not detect any population of T cells or macrophages which could bind specifically to infected heart tissue to initiate an autoreactive process. Images PMID:2228230

  4. Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics

    PubMed Central

    Dorhoi, Anca; Yeremeev, Vladimir; Nouailles, Geraldine; Weiner, January; Jörg, Sabine; Heinemann, Ellen; Oberbeck-Müller, Dagmar; Knaul, Julia K; Vogelzang, Alexis; Reece, Stephen T; Hahnke, Karin; Mollenkopf, Hans-Joachim; Brinkmann, Volker; Kaufmann, Stefan H E

    2014-01-01

    General interest in the biological functions of IFN type I in Mycobacterium tuberculosis (Mtb) infection increased after the recent identification of a distinct IFN gene expression signature in tuberculosis (TB) patients. Here, we demonstrate that TB-susceptible mice lacking the receptor for IFN I (IFNAR1) were protected from death upon aerogenic infection with Mtb. Using this experimental model to mimic primary progressive pulmonary TB, we dissected the immune processes affected by IFN I. IFNAR1 signaling did not affect T-cell responses, but markedly altered migration of inflammatory monocytes and neutrophils to the lung. This process was orchestrated by IFNAR1 expressed on both immune and tissue-resident radioresistant cells. IFNAR1-driven TB susceptibility was initiated by augmented Mtb replication and in situ death events, along with CXCL5/CXCL1-driven accumulation of neutrophils in alveoli, followed by the discrete compartmentalization of Mtb in lung phagocytes. Early depletion of neutrophils rescued TB-susceptible mice to levels observed in mice lacking IFNAR1. We conclude that IFN I alters early innate events at the site of Mtb invasion leading to fatal immunopathology. These data furnish a mechanistic explanation for the detrimental role of IFN I in pulmonary TB and form a basis for understanding the complex roles of IFN I in chronic inflammation. PMID:24782112

  5. Acquired cystic disease-associated renal cell carcinoma: further characterization of the morphologic and immunopathologic features.

    PubMed

    Ahn, Soomin; Kwon, Ghee Young; Cho, Yong Mee; Jun, Sun-Young; Choi, Chan; Kim, Hyun-Jung; Park, Yong Wook; Park, Weon Seo; Shim, Jung Won

    2013-12-01

    Acquired cystic disease-associated renal cell carcinoma (ACD-RCC) is a subtype of renal cell carcinoma (RCC) with unique morphologic features found exclusively in the background of end-stage renal disease. We analyzed the clinicopathologic features and immumoreactive profiles of 12 cases of ACD-RCC to further characterize this recently recognized entity. Review of histologic slides was performed in conjunction with immunohistochemical staining directed to the contemporary diagnostic antibodies and the putative target therapy-related markers. Histologically, the tumors showed characteristic inter-or intracellular microlumens and eosinophilic tumor cells. Intratumoral hemosiderin deposition and degenerating foamy tumor cells were consistent findings which were not previously described. Immunohistochemically, all the tumors were positive for alpha-methylacyl-CoA-racemase, CD10, pan-cytokeratin, PTEN (phosphatase and tensin homolog deleted on chromosome 10) and c-met, while negative for carbonic anhydrase-9, CD57, CD68, c-kit, pax-2, platelet-derived growth factor receptor (PDGFR)-α or vascular endothelial growth factor receptor (VEGFR)-2. Heterogenous staining was found for CK7 and kidney-specific cadherin. Positive reaction to c-met suggests its utility as a plausible therapeutic target in ACD-RCC. Thus, we present the unique morphologic and immunopathologic features of ACD-RCC, which may be helpful in both diagnostic and therapeutic aspects. PMID:23471757

  6. Immunity, immunopathology, and human vaccine development against sexually transmitted Chlamydia trachomatis

    PubMed Central

    Rey-Ladino, Jose; Ross, Allen GP; Cripps, Allan W

    2014-01-01

    This review examines the immunity, immunopathology, and contemporary problems of vaccine development against sexually transmitted Chlamydia trachomatis. Despite improved surveillance and treatment initiatives, the incidence of C. trachomatis infection has increased dramatically over the past 30 years in both the developed and developing world. Studies in animal models have shown that protective immunity to C. trachomatis is largely mediated by Th1 T cells producing IFN-γ which is needed to prevent dissemination of infection. Similar protection appears to develop in humans but in contrast to mice, immunity in humans may take years to develop. Animal studies and evidence from human infection indicate that immunity to C. trachomatis is accompanied by significant pathology in the upper genital tract. Although no credible evidence is currently available to indicate that autoimmunity plays a role, nevertheless, this underscores the necessity to design vaccines strictly based on chlamydial-specific antigens and to avoid those displaying even minimal sequence homologies with host molecules. Current advances in C. trachomatis vaccine development as well as alternatives for designing new vaccines for this disease are discussed. A novel approach for chlamydia vaccine development, based on targeting endogenous dendritic cells, is described. PMID:25483666

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley Stripe Mosaic Virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  9. Virus-induced gene silencing and transient gene expression in soybean using Bean pod mottle virus infectious clones

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200-300 base pair size range. The recombinant viruses are used to ...

  10. Evaluating the Ability of the Barley Stripe Mosaic Virus-Induced Gene Silencing System to Simultaneously Silence Two Wheat Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley stripe mosaic virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  11. Sex-specific quantitative trait loci govern susceptibility to Theiler's murine encephalomyelitis virus-induced demyelination.

    PubMed Central

    Butterfield, Russell J; Roper, Randall J; Rhein, Dominic M; Melvold, Roger W; Haynes, Lia; Ma, Runlin Z; Doerge, R W; Teuscher, Cory

    2003-01-01

    Susceptibility to Theiler's murine encephalomyelitis virus-induced demyelination (TMEVD), a mouse model for multiple sclerosis (MS), is genetically controlled. Through a mouse-human comparative mapping approach, identification of candidate susceptibility loci for MS based on the location of TMEVD susceptibility loci may be possible. Composite interval mapping (CIM) identified quantitative trait loci (QTL) controlling TMEVD severity in male and female backcross populations derived from susceptible DBA/2J and resistant BALBc/ByJ mice. We report QTL on chromosomes 1, 5, 15, and 16 affecting male mice. In addition, we identified two QTL in female mice located on chromosome 1. Our results support the existence of three linked sex-specific QTL on chromosome 1 with opposing effects on the severity of the clinical signs of TMEV-induced disease in male and female mice. PMID:12663542

  12. Persistent virus-induced gene silencing in asymptomatic accessions of Arabidopsis.

    PubMed

    Flores, Miguel A; Reyes, Maria I; Robertson, Dominique Niki; Kjemtrup, Susanne

    2015-01-01

    Coupled with the advantages afforded by the model plant Arabidopsis, virus-induced gene silencing (VIGS) offers a rapid means to assess gene function. The geminivirus vector based on Cabbage leaf curl virus described here has the benefits of small insert size and persistent silencing of the target gene through the life cycle of the plant. Here, we show that genetic variation in the vast collection of Arabidopsis accessions can be leveraged to ameliorate viral symptomology that accompanies the VIGS procedure. The plasticity of phenotypes under different day lengths or temperature conditions can be exploited to achieve maximum silencing efficacy in either vegetative or inflorescence tissue, according to the question being asked. Protocols and vectors for Agro-infiltration of primary leaves, subapical pricking in older plants, and microprojectile bombardment are described. PMID:25757779

  13. Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.

    PubMed Central

    Akaike, T; Noguchi, Y; Ijiri, S; Setoguchi, K; Suga, M; Zheng, Y M; Dietzschold, B; Maeda, H

    1996-01-01

    The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice. Images Fig. 1 Fig. 5 PMID:8637894

  14. Inhibition of Pim1 kinase, new therapeutic approach in virus-induced asthma exacerbations.

    PubMed

    Vries, Maaike de; Bedke, Nicole; Smithers, Natalie P; Loxham, Matthew; Howarth, Peter H; Nawijn, Martijn C; Davies, Donna E

    2016-03-01

    Therapeutic options to treat virus-induced asthma exacerbations are limited and urgently needed. Therefore, we tested Pim1 kinase as potential therapeutic target in human rhinovirus (HRV) infections. We hypothesised that inhibition of Pim1 kinase reduces HRV replication by augmenting the interferon-induced anti-viral response due to increased activity of the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway.Air-liquid interface (ALI) cultures of primary bronchial epithelial cells (PBECs) from healthy individuals and moderate-to-severe asthmatic volunteers were infected with HRV-16 with or without a specific Pim1 inhibitor; viral replication and induction of anti-viral responses were measured using RT-qPCR. Viral titres were measured by 50% tissue culture infective dose and release of interferon-γ-induced protein 10 (IP-10) and RANTES protein assessed by ELISA. Phosphorylation of STAT-1 was determined using western blotting.Viral replication was reduced in ALI cultures of healthy and asthmatic PBECs treated with the Pim1 inhibitor. Using cultures from healthy donors, enhanced STAT-1 phosphorylation upon inhibition of Pim1 kinase activity resulted in increased mRNA expression of interferon-β, interleukin-29, IP-10 and RANTES 12 h after infection and increased protein levels of IP-10 and RANTES 24 h after infection.We have identified Pim1 kinase as novel target to reduce viral replication in ALI cultures of PBECs. This may open new avenues for therapeutic interventions in virus-induced asthma exacerbations. PMID:26869670

  15. Novel Strategy To Protect against Influenza Virus-Induced Pneumococcal Disease without Interfering with Commensal Colonization.

    PubMed

    Greene, Christopher J; Marks, Laura R; Hu, John C; Reddinger, Ryan; Mandell, Lorrie; Roche-Hakansson, Hazeline; King-Lyons, Natalie D; Connell, Terry D; Hakansson, Anders P

    2016-06-01

    Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines, aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype replacement with, so far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virus-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally induced pneumococcal disease can be conferred without disturbing the desirable preexisting commensal colonization of the nasopharynx. PMID:27001538

  16. Pleiotropic Effects of Levofloxacin, Fluoroquinolone Antibiotics, against Influenza Virus-Induced Lung Injury.

    PubMed

    Enoki, Yuki; Ishima, Yu; Tanaka, Ryota; Sato, Keizo; Kimachi, Kazuhiko; Shirai, Tatsuya; Watanabe, Hiroshi; Chuang, Victor T G; Fujiwara, Yukio; Takeya, Motohiro; Otagiri, Masaki; Maruyama, Toru

    2015-01-01

    Reactive oxygen species (ROS) and nitric oxide (NO) are major pathogenic molecules produced during viral lung infections, including influenza. While fluoroquinolones are widely used as antimicrobial agents for treating a variety of bacterial infections, including secondary infections associated with the influenza virus, it has been reported that they also function as anti-oxidants against ROS and as a NO regulator. Therefore, we hypothesized that levofloxacin (LVFX), one of the most frequently used fluoroquinolone derivatives, may attenuate pulmonary injuries associated with influenza virus infections by inhibiting the production of ROS species such as hydroxyl radicals and neutrophil-derived NO that is produced during an influenza viral infection. The therapeutic impact of LVFX was examined in a PR8 (H1N1) influenza virus-induced lung injury mouse model. ESR spin-trapping experiments indicated that LVFX showed scavenging activity against neutrophil-derived hydroxyl radicals. LVFX markedly improved the survival rate of mice that were infected with the influenza virus in a dose-dependent manner. In addition, the LVFX treatment resulted in a dose-dependent decrease in the level of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress) and nitrotyrosine (a nitrative marker) in the lungs of virus-infected mice, and the nitrite/nitrate ratio (NO metabolites) and IFN-γ in BALF. These results indicate that LVFX may be of substantial benefit in the treatment of various acute inflammatory disorders such as influenza virus-induced pneumonia, by inhibiting inflammatory cell responses and suppressing the overproduction of NO in the lungs. PMID:26086073

  17. Pleiotropic Effects of Levofloxacin, Fluoroquinolone Antibiotics, against Influenza Virus-Induced Lung Injury

    PubMed Central

    Enoki, Yuki; Ishima, Yu; Tanaka, Ryota; Sato, Keizo; Kimachi, Kazuhiko; Shirai, Tatsuya; Watanabe, Hiroshi; Chuang, Victor T. G.; Fujiwara, Yukio; Takeya, Motohiro; Otagiri, Masaki; Maruyama, Toru

    2015-01-01

    Reactive oxygen species (ROS) and nitric oxide (NO) are major pathogenic molecules produced during viral lung infections, including influenza. While fluoroquinolones are widely used as antimicrobial agents for treating a variety of bacterial infections, including secondary infections associated with the influenza virus, it has been reported that they also function as anti-oxidants against ROS and as a NO regulator. Therefore, we hypothesized that levofloxacin (LVFX), one of the most frequently used fluoroquinolone derivatives, may attenuate pulmonary injuries associated with influenza virus infections by inhibiting the production of ROS species such as hydroxyl radicals and neutrophil-derived NO that is produced during an influenza viral infection. The therapeutic impact of LVFX was examined in a PR8 (H1N1) influenza virus-induced lung injury mouse model. ESR spin-trapping experiments indicated that LVFX showed scavenging activity against neutrophil-derived hydroxyl radicals. LVFX markedly improved the survival rate of mice that were infected with the influenza virus in a dose-dependent manner. In addition, the LVFX treatment resulted in a dose-dependent decrease in the level of 8-hydroxy-2’-deoxyguanosine (a marker of oxidative stress) and nitrotyrosine (a nitrative marker) in the lungs of virus-infected mice, and the nitrite/nitrate ratio (NO metabolites) and IFN-γ in BALF. These results indicate that LVFX may be of substantial benefit in the treatment of various acute inflammatory disorders such as influenza virus-induced pneumonia, by inhibiting inflammatory cell responses and suppressing the overproduction of NO in the lungs. PMID:26086073

  18. Murine autoimmune oophoritis, epididymoorchitis, and gastritis induced by day 3 thymectomy. Immunopathology.

    PubMed Central

    Tung, K. S.; Smith, S.; Teuscher, C.; Cook, C.; Anderson, R. E.

    1987-01-01

    (C57BL/6 X A/J) F1, (SWR/J X A/J) F1 and BALB/cBy mice, thymectomized on Day 3 (D3TX) or Day 7 (D7TX) or sham thymectomized (STX), were studied for immunopathologic changes in the ovary, testis, and gastric wall. In 95% of B6AF1, 100% of SWRAF1, and 35% of BALB/cBy mice ovarian disease developed, with onset at 5-6 weeks of age. Ovarian disease was not found in D7TX or STX mice. In D3TX mice, it was associated with mononuclear infiltration and ovarian follicle destruction, leading to atrophy. Epididymovasitis was detected in 70-90% of SWRAF1, 50% of B6AF1, and 64% of BALB/cBy mice after D3TX; whereas orchitis occurred in about 20% of SWRAF1 and B6AF1 mice. In some mice epididymovasitis also developed after D7TX, but not after STX. In contrast to the negative immunohistochemical findings in diseased ovaries, typical immune complex-like deposits of mouse IgG were detected by immunofluorescence along the basement membrane of epididymal ducts and seminiferous tubules. However, maximum incidence of epididymitis preceded immune complex detection. Gastritis developed in both female (57%) and male (50%) BALB/cBy mice after D3TX, occasionally after D7TX, but not after STX. Gastric mucosa was hypertrophic with dilated glands and heavy lymphocytic infiltrations throughout all gastric layers. BALB/cBy mice with gastritis usually did not have disease in the gonads, and vice versa. This study, therefore, confirms and extends the findings of Nishizuka and colleagues. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3548402

  19. Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection.

    PubMed

    Kimmey, Jacqueline M; Huynh, Jeremy P; Weiss, Leslie A; Park, Sunmin; Kambal, Amal; Debnath, Jayanta; Virgin, Herbert W; Stallings, Christina L

    2015-12-24

    Mycobacterium tuberculosis, a major global health threat, replicates in macrophages in part by inhibiting phagosome-lysosome fusion, until interferon-γ (IFNγ) activates the macrophage to traffic M. tuberculosis to the lysosome. How IFNγ elicits this effect is unknown, but many studies suggest a role for macroautophagy (herein termed autophagy), a process by which cytoplasmic contents are targeted for lysosomal degradation. The involvement of autophagy has been defined based on studies in cultured cells where M. tuberculosis co-localizes with autophagy factors ATG5, ATG12, ATG16L1, p62, NDP52, BECN1 and LC3 (refs 2-6), stimulation of autophagy increases bacterial killing, and inhibition of autophagy increases bacterial survival. Notably, these studies reveal modest (~1.5-3-fold change) effects on M. tuberculosis replication. By contrast, mice lacking ATG5 in monocyte-derived cells and neutrophils (polymorponuclear cells, PMNs) succumb to M. tuberculosis within 30 days, an extremely severe phenotype similar to mice lacking IFNγ signalling. Importantly, ATG5 is the only autophagy factor that has been studied during M. tuberculosis infection in vivo and autophagy-independent functions of ATG5 have been described. For this reason, we used a genetic approach to elucidate the role for multiple autophagy-related genes and the requirement for autophagy in resistance to M. tuberculosis infection in vivo. Here we show that, contrary to expectation, autophagic capacity does not correlate with the outcome of M. tuberculosis infection. Instead, ATG5 plays a unique role in protection against M. tuberculosis by preventing PMN-mediated immunopathology. Furthermore, while Atg5 is dispensable in alveolar macrophages during M. tuberculosis infection, loss of Atg5 in PMNs can sensitize mice to M. tuberculosis. These findings shift our understanding of the role of ATG5 during M. tuberculosis infection, reveal new outcomes of ATG5 activity, and shed light on early events in innate

  20. Multiple Inhibitory Pathways Contribute to Lung CD8+ T Cell Impairment and Protect against Immunopathology during Acute Viral Respiratory Infection.

    PubMed

    Erickson, John J; Rogers, Meredith C; Tollefson, Sharon J; Boyd, Kelli L; Williams, John V

    2016-07-01

    Viruses are frequent causes of lower respiratory infection (LRI). Programmed cell death-1 (PD-1) signaling contributes to pulmonary CD8(+) T cell (TCD8) functional impairment during acute viral LRI, but the role of TCD8 impairment in viral clearance and immunopathology is unclear. We now find that human metapneumovirus infection induces virus-specific lung TCD8 that fail to produce effector cytokines or degranulate late postinfection, with minimally increased function even in the absence of PD-1 signaling. Impaired lung TCD8 upregulated multiple inhibitory receptors, including PD-1, lymphocyte activation gene 3 (LAG-3), T cell Ig mucin 3, and 2B4. Moreover, coexpression of these receptors continued to increase even after viral clearance, with most virus-specific lung TCD8 expressing three or more inhibitory receptors on day 14 postinfection. Viral infection also increased expression of inhibitory ligands by both airway epithelial cells and APCs, further establishing an inhibitory environment. In vitro Ab blockade revealed that multiple inhibitory receptors contribute to TCD8 impairment induced by either human metapneumovirus or influenza virus infection. In vivo blockade of T cell Ig mucin 3 signaling failed to enhance TCD8 function or reduce viral titers. However, blockade of LAG-3 in PD-1-deficient mice restored TCD8 effector functions but increased lung pathology, indicating that LAG-3 mediates lung TCD8 impairment in vivo and contributes to protection from immunopathology during viral clearance. These results demonstrate that an orchestrated network of pathways modifies lung TCD8 functionality during viral LRI, with PD-1 and LAG-3 serving prominent roles. Lung TCD8 impairment may prevent immunopathology but also contributes to recurrent lung infections. PMID:27259857

  1. Rapid activation of spleen dendritic cell subsets following lymphocytic choriomeningitis virus infection of mice: analysis of the involvement of type 1 IFN.

    PubMed

    Montoya, Maria; Edwards, Matthew J; Reid, Delyth M; Borrow, Persephone

    2005-02-15

    In this study, we report the dynamic changes in activation and functions that occur in spleen dendritic cell (sDC) subsets following infection of mice with a natural murine pathogen, lymphocytic choriomeningitis virus (LCMV). Within 24 h postinfection (pi), sDCs acquired the ability to stimulate naive LCMV-specific CD8+ T cells ex vivo. Conventional (CD11chigh CD8+ and CD4+) sDC subsets rapidly up-regulated expression of costimulatory molecules and began to produce proinflammatory cytokines. Their tendency to undergo apoptosis ex vivo simultaneously increased, and in vivo the number of conventional DCs in the spleen decreased markedly, dropping approximately 2-fold by day 3 pi. Conversely, the number of plasmacytoid (CD11clowB220+) DCs in the spleen increased, so that they constituted almost 40% of sDCs by day 3 pi. Type 1 IFN production was up-regulated in plasmacytoid DCs by 24 h pi. Analysis of DC activation and maturation in mice unable to respond to type 1 IFNs implicated these cytokines in driving infection-associated phenotypic activation of conventional DCs and their enhanced tendency to undergo apoptosis, but also indicated the existence of type 1 IFN-independent pathways for the functional maturation of DCs during LCMV infection. PMID:15699111

  2. Virus-lymphocyte interaction: T cells of the helper subset are infected with lymphocytic choriomeningitis virus during persistent infection in vivo.

    PubMed Central

    Ahmed, R; King, C C; Oldstone, M B

    1987-01-01

    The lifelong persistence of lymphocytic choriomeningitis virus (LCMV) in neonatally or congenitally infected mice is accompanied by a suppression of virus-specific T-cell responses. In this study, we identified the subset of T cells infected with LCMV during persistent infection in vivo. Using specific monoclonal antibodies to separate the different lymphocyte cell populations and employing both an infectious center assay and immunofluorescence to detect the virus, we found that infection is confined primarily to T cells of the helper subset (L3T4+ Lyt2-), with minimal involvement of cytotoxic T cells (Lyt2+ L3T4-) and mature B cells. About 0.54 to 1.1% of L3T4+ T cells were producing the virus, as determined by the infectious center assay. In contrast, 9.1 to 12.2% of these L3T4+ T cells contained viral antigen, as shown by immunofluorescence studies. This finding suggested that, at any given time, a substantial number of infected T cells were not producing infectious virus. This infection of T helper cells may be involved in the suppression of LCMV-specific T-cell responses observed in persistently infected mice. Images PMID:2952807

  3. Lymphocytic Choriomeningitis Virus Expands a Population of NKG2D+CD8+ T Cells That Exacerbates Disease in Mice Coinfected with Leishmania major.

    PubMed

    Crosby, Erika J; Clark, Megan; Novais, Fernanda O; Wherry, E John; Scott, Phillip

    2015-10-01

    Leishmaniasis is a significant neglected tropical disease that is associated with a wide range of clinical presentations and a lifelong persistent infection. Because of the chronic nature of the disease, there is a high risk for coinfection occurring in patients, and how coinfections influence the outcome of leishmaniasis is poorly understood. To address this issue, we infected mice with Leishmania major and 2 wk later with lymphocytic choriomeningitis virus (LCMV) and then monitored the course of infection. Leishmania parasites are controlled by production of IFN-γ, which leads to macrophage-mediated parasite killing. Thus, one might predict that coinfection with LCMV, which induces a strong systemic type 1 response, would accelerate disease resolution. However, we found that infection with LCMV led to significantly enhanced disease in L. major-infected animals. This increased disease correlated with an infiltration into the leishmanial lesions of NKG2D(+) CD8(+) T cells producing granzyme B, but surprisingly little IFN-γ. We found that depletion of CD8 T cells after viral clearance, as well as blockade of NKG2D, reversed the increased pathology seen in coinfected mice. Thus, this work highlights the impact a secondary infection can have on leishmaniasis and demonstrates that even pathogens known to promote a type 1 response may exacerbate leishmanial infections. PMID:26290604

  4. A novel PRD I and TG binding activity involved in virus-induced transcription of IFN-A genes.

    PubMed Central

    Génin, P; Bragança, J; Darracq, N; Doly, J; Civas, A

    1995-01-01

    Comparative analysis of the inducible elements of the mouse interferon A4 and A11 gene promoters (IE-A4 and IE-A11) by transient transfection experiments, DNase 1 footprinting and electrophoretic mobility shift assays resulted in identification of a virus-induced binding activity suggested to be involved in NDV-induced activation of transcription of these genes. The virus-induced factor, termed VIF, is activated early by contact of virions with cells. It specifically recognizes the PRD I-like domain shared by both inducible elements, as well as the TG-like domain of IE-A4. This factor, distinct from the IRF-1, IRF-2 and the alpha F1 binding proteins and presenting a different affinity pattern from that of the TG protein, is proposed as a candidate for IFN-type I gene regulation. Images PMID:8559665

  5. Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

    PubMed Central

    Krawczyk, Adalbert; Dirks, Miriam; Kasper, Maren; Buch, Anna; Dittmer, Ulf; Giebel, Bernd; Wildschütz, Lena; Busch, Martin; Goergens, Andre; Schneweis, Karl E.; Eis-Hübinger, Anna M.; Sodeik, Beate; Heiligenhaus, Arnd; Roggendorf, Michael; Bauer, Dirk

    2015-01-01

    The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans. PMID:25587898

  6. Delineation of autoantibody repertoire through differential proteogenomics in hepatitis C virus-induced cryoglobulinemia.

    PubMed

    Ogishi, Masato; Yotsuyanagi, Hiroshi; Moriya, Kyoji; Koike, Kazuhiko

    2016-01-01

    Antibodies cross-reactive to pathogens and autoantigens are considered pivotal in both infection control and accompanying autoimmunity. However, the pathogenic roles of autoantibodies largely remain elusive without a priori knowledge of disease-specific autoantigens. Here, through a novel quantitative proteogenomics approach, we demonstrated a successful identification of immunoglobulin variable heavy chain (VH) sequences highly enriched in pathological immune complex from clinical specimens obtained from a patient with hepatitis C virus-induced cryoglobulinemia (HCV-CG). Reconstructed single-domain antibodies were reactive to both HCV antigens and potentially liver-derived human proteins. Moreover, over the course of antiviral therapy, a substantial "de-evolution" of a distinct sub-repertoire was discovered, to which proteomically identified cryoprecipitation-prone autoantibodies belonged. This sub-repertoire was characterized by IGHJ6*03-derived, long, hydrophobic complementarity determining region (CDR-H3). This study provides a proof-of-concept of de novo mining of autoantibodies and corresponding autoantigen candidates in a disease-specific context in human, thus facilitating future reverse-translational research for the discovery of novel biomarkers and the development of antigen-specific immunotherapy against various autoantibody-related disorders. PMID:27403724

  7. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    PubMed

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  8. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize1[OPEN

    PubMed Central

    Mei, Yu; Kernodle, Bliss M.; Hill, John H.

    2016-01-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

  9. Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

    PubMed

    Carqueijeiro, I; Masini, E; Foureau, E; Sepúlveda, L J; Marais, E; Lanoue, A; Besseau, S; Papon, N; Clastre, M; Dugé de Bernonville, T; Glévarec, G; Atehortùa, L; Oudin, A; Courdavault, V

    2015-11-01

    Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses. PMID:26284695

  10. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

    PubMed Central

    Liscombe, David K.; O’Connor, Sarah E.

    2011-01-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by Madagascar periwinkle (Catharanthus roseus) plants. Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. We have developed a VIGS method to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro. PMID:21802100

  11. Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus.

    PubMed

    Zhong, Xionghui; Yuan, Xue; Wu, Ze; Khan, Muhammad Ali; Chen, Jin; Li, Xiaoxin; Gong, Benhe; Zhao, Yang; Wu, Jian; Wu, Chenyu; Yi, Mingfang

    2014-02-01

    Functional analysis of genes in gladiolus has previously been impractical due to the lack of an efficient stable genetic transformation method. However, virus-induced gene silencing (VIGS) is effective in some plants which are difficult to transform through other methods. Although the Tobacco rattle virus (TRV)-based VIGS system has been developed and used for verifying gene functions in diverse plants, an appropriate TRV-VIGS approach for gladiolus has not been established yet. In this report we describe the first use of the TRV-VIGS system for gene silencing in gladiolus. Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The reduction in GhPDS expression was tested after TRV-based vector infection using real-time RT-PCR. In addition, the progress of TRV infection was detected by fluorescence visualization using a pTRV2: CP-GFP vector. In conclusion, the TRV-mediated VIGS described here will be an effective gene function analysis mechanism in gladiolus. PMID:24170343

  12. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    PubMed Central

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana. PMID:27493613

  13. Theiler's murine encephalomyelitis virus-induced cardiac and skeletal muscle disease.

    PubMed Central

    Gómez, R M; Rinehart, J E; Wollmann, R; Roos, R P

    1996-01-01

    The DA strain of Theiler's murine encephalomyelitis virus, a member of the cardiovirus genus of picornaviruses, induces a restricted and persistent infection associated with a demyelinating process following intracerebral inoculation of mice; both virus infection and the immune response are believed to contribute to the late white matter disease. We now report that intraperitoneal inoculation with DA produces an acute myositis that progresses to a chronic inflammatory muscle disease in CD-1 mice as well as several inbred mouse strains. Some mouse strains also develop central nervous system white matter disease and a focal myocarditis. Infectious virus in skeletal muscle falls to undetectable levels 3 weeks postinoculation (p.i.), although viral genome persists for at least 12 weeks p.i., the longest period of observation. Severe combined immunodeficient animals have evidence of muscle pathology as long as 5 weeks p.i., suggesting that DA virus is capable of inducing chronic muscle disease in the absence of an immune response. The presence in immunocompetent mice, however, of prominent muscle inflammation in the absence of infectious virus suggests that the immune system also contributes to the pathology. T lymphocytes are the predominant cell type infiltrating the skeletal muscle during the chronic disease. This murine model may further our understanding of virus-induced chronic myositis and help to clarify the pathogenesis of human inflammatory myopathies. PMID:8971022

  14. Non-targeted effects of virus-induced gene silencing vectors on host endogenous gene expression.

    PubMed

    Oláh, Enikő; Pesti, Réka; Taller, Dénes; Havelda, Zoltán; Várallyay, Éva

    2016-09-01

    Virus-induced gene silencing (VIGS) uses recombinant viruses to study gene function; however, the effect of the virus vector itself on the gene expression of the host is not always considered. In our work, we investigated non-targeted gene expression changes of the host in order to see how often these changes appear. Effects of various VIGS vector infections were analysed by monitoring gene expression levels of housekeeping genes by Northern blot analysis in four different hosts. We found that non-targeted changes happens very often. More importantly, these non-targeted effects can cause drastic changes in the gene-expression pattern of host genes that are usually used as references in these studies. We have also found that in a tobacco rattle virus (TRV)-based VIGS, the presence of foreign sequences in the cloning site of the vector can also have a non-targeted effect, and even the use of an internal control can lead to unpredicted changes. Our results show that although VIGS is a very powerful technique, the VIGS vector, as a pathogen of the host, can cause unwanted changes in its gene-expression pattern, highlighting the importance of careful selection of both the genes to be tested and those to be used as references in the planned experiments. PMID:27283101

  15. Inflammatory cytokine-mediated evasion of virus-induced tumors from NK cell control

    PubMed Central

    Mishra, Rabinarayan; Polic, Bojan; Welsh, Raymond M.; Szomolanyi-Tsuda, Eva

    2013-01-01

    Infections with DNA tumor viruses, including members of the polyomavirus family, often result in tumor formation in immune-deficient hosts. The complex control involved in antiviral and antitumor immune responses during these infections can be studied in murine polyomavirus (PyV)-infected mice as a model. We found that NK cells efficiently kill cells derived from PyV-induced salivary gland tumors in vitro in an NKG2D (effector cell) -RAE-1 (target cell) - dependent manner, but in T cell-deficient mice NK cells only delay but do not prevent the development of PyV-induced tumors. Here we show that the PyV-induced tumors have infiltrating functional NK cells. The freshly removed tumors, however, lack surface RAE-1 expression, and the tumor tissues produce soluble factors that down-regulate RAE-1. These factors include the pro-inflammatory cytokines IL-1α, IL-1β, IL-33, and TNF. Each of these cytokines down-regulate RAE-1 expression and susceptibility to NK cell mediated cytotoxicity. CD11b+F4/80+ macrophages infiltrating the PyV-induced tumors produce high amounts of IL-1β and TNF. Thus, our data suggest a new mechanism whereby inflammatory cytokines generated in the tumor environment lead to evasion of NK cell-mediated control of virus-induced tumors. PMID:23772039

  16. Applications and advantages of virus-induced gene silencing for gene function studies in plants.

    PubMed

    Burch-Smith, Tessa M; Anderson, Jeffrey C; Martin, Gregory B; Dinesh-Kumar, S P

    2004-09-01

    Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes. PMID:15315635

  17. Characterization of Chikungunya Virus Induced Host Response in a Mouse Model of Viral Myositis

    PubMed Central

    Dhanwani, Rekha; Khan, Mohsin; Lomash, Vinay; Rao, Putcha Venkata Lakshmana; Ly, Hinh; Parida, Manmohan

    2014-01-01

    While a number of studies have documented the persistent presence of chikungunya virus (CHIKV) in muscle tissue with primary fibroblast as the preferable cell target, little is known regarding the alterations that take place in muscle tissue in response to CHIKV infection. Hence, in the present study a permissive mouse model of CHIKV infection was established and characterized in order to understand the pathophysiology of the disease. The two dimensional electrophoresis of muscle proteome performed for differential analysis indicated a drastic reprogramming of the proteins from various classes like stress, inflammation, cytoskeletal, energy and lipid metabolism. The roles of the affected proteins were explained in relation to virus induced myopathy which was further supported by the histopathological and behavioural experiments proving the lack of hind limb coordination and other loco-motor abnormalities in the infected mice. Also, the level of various pro-inflammatory mediators like IL-6, MCP-1, Rantes and TNF-α was significantly elevated in muscles of infected mice. Altogether this comprehensive study of characterizing CHIKV induced mouse myopathy provides many potential targets for further evaluation and biomarker study. PMID:24667237

  18. A CRISPR-based screen identifies genes essential for West Nile virus-induced cell death

    PubMed Central

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N.; Wu, Haoquan

    2015-01-01

    Summary West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. PMID:26190106

  19. Virus-induced gene silencing of fiber-related genes in cotton.

    PubMed

    Tuttle, John R; Haigler, Candace H; Robertson, Dominique Niki

    2015-01-01

    Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given. PMID:25740368

  20. Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing

    PubMed Central

    Cooper, Bret; Campbell, Kimberly B; McMahon, Michael B; Luster, Douglas G

    2013-01-01

    Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars. PMID:24401541

  1. Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

    PubMed Central

    2012-01-01

    Background We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns. Results The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively. Conclusions These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns. PMID:22853641

  2. Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation from Latency by Preventing Virus-Induced Systemic Inflammation.

    PubMed

    Park, Sunmin; Buck, Michael D; Desai, Chandni; Zhang, Xin; Loginicheva, Ekaterina; Martinez, Jennifer; Freeman, Michael L; Saitoh, Tatsuya; Akira, Shizuo; Guan, Jun-Lin; He, You-Wen; Blackman, Marcia A; Handley, Scott A; Levine, Beth; Green, Douglas R; Reese, Tiffany A; Artyomov, Maxim N; Virgin, Herbert W

    2016-01-13

    Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-γ (IFN-γ). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ, and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency. PMID:26764599

  3. Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas

    PubMed Central

    Justice, James F.; Morgan, Robin W.

    2015-01-01

    ABSTRACT Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC, MYB, Mir-155, and TERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome. PMID:26670384

  4. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

    PubMed

    Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

    2016-06-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

  5. Delineation of autoantibody repertoire through differential proteogenomics in hepatitis C virus-induced cryoglobulinemia

    PubMed Central

    Ogishi, Masato; Yotsuyanagi, Hiroshi; Moriya, Kyoji; Koike, Kazuhiko

    2016-01-01

    Antibodies cross-reactive to pathogens and autoantigens are considered pivotal in both infection control and accompanying autoimmunity. However, the pathogenic roles of autoantibodies largely remain elusive without a priori knowledge of disease-specific autoantigens. Here, through a novel quantitative proteogenomics approach, we demonstrated a successful identification of immunoglobulin variable heavy chain (VH) sequences highly enriched in pathological immune complex from clinical specimens obtained from a patient with hepatitis C virus-induced cryoglobulinemia (HCV-CG). Reconstructed single-domain antibodies were reactive to both HCV antigens and potentially liver-derived human proteins. Moreover, over the course of antiviral therapy, a substantial “de-evolution” of a distinct sub-repertoire was discovered, to which proteomically identified cryoprecipitation-prone autoantibodies belonged. This sub-repertoire was characterized by IGHJ6*03-derived, long, hydrophobic complementarity determining region (CDR-H3). This study provides a proof-of-concept of de novo mining of autoantibodies and corresponding autoantigen candidates in a disease-specific context in human, thus facilitating future reverse-translational research for the discovery of novel biomarkers and the development of antigen-specific immunotherapy against various autoantibody-related disorders. PMID:27403724

  6. Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing.

    PubMed

    Chantreau, Maxime; Chabbert, Brigitte; Billiard, Sylvain; Hawkins, Simon; Neutelings, Godfrey

    2015-12-01

    Flax (Linum usitatissimum) bast fibres are located in the stem cortex where they play an important role in mechanical support. They contain high amounts of cellulose and so are used for linen textiles and in the composite industry. In this study, we screened the annotated flax genome and identified 14 distinct cellulose synthase (CESA) genes using orthologous sequences previously identified. Transcriptomics of 'primary cell wall' and 'secondary cell wall' flax CESA genes showed that some were preferentially expressed in different organs and stem tissues providing clues as to their biological role(s) in planta. The development for the first time in flax of a virus-induced gene silencing (VIGS) approach was used to functionally evaluate the biological role of different CESA genes in stem tissues. Quantification of transcript accumulation showed that in many cases, silencing not only affected targeted CESA clades, but also had an impact on other CESA genes. Whatever the targeted clade, inactivation by VIGS affected plant growth. In contrast, only clade 1- and clade 6-targeted plants showed modifications in outer-stem tissue organization and secondary cell wall formation. In these plants, bast fibre number and structure were severely impacted, suggesting that the targeted genes may play an important role in the establishment of the fibre cell wall. Our results provide new fundamental information about cellulose biosynthesis in flax that should facilitate future plant improvement/engineering. PMID:25688574

  7. Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants1[OPEN

    PubMed Central

    Liu, Na; Xie, Ke; Jia, Qi; Zhao, Jinping; Chen, Tianyuan; Li, Huangai; Wei, Xiang; Diao, Xianmin; Hong, Yiguo

    2016-01-01

    Virus-induced gene silencing (VIGS) is a powerful technique to study gene function in plants. However, very few VIGS vectors are available for monocot plants. Here we report that Foxtail mosaic virus (FoMV) can be engineered as an effective VIGS system to induce efficient silencing of endogenous genes in monocot plants including barley (Hordeum vulgare L.), wheat (Triticum aestivum) and foxtail millet (Setaria italica). This is evidenced by FoMV-based silencing of phytoene desaturase (PDS) and magnesium chelatase in barley, of PDS and Cloroplastos alterados1 in foxtail millet and wheat, and of an additional gene IspH in foxtail millet. Silencing of these genes resulted in photobleached or chlorosis phenotypes in barley, wheat, and foxtail millet. Furthermore, our FoMV-based gene silencing is the first VIGS system reported for foxtail millet, an important C4 model plant. It may provide an efficient toolbox for high-throughput functional genomics in economically important monocot crops. PMID:27225900

  8. A novel virus-inducible enhancer of the interferon-β gene with tightly linked promoter and enhancer activities

    PubMed Central

    Banerjee, A. Raja; Kim, Yoon Jung; Kim, Tae Hoon

    2014-01-01

    Long-range enhancers of transcription are a key component of the genomic regulatory architecture. Recent studies have identified bi-directionally transcribed RNAs emanating from these enhancers known as eRNAs. However, it remains unclear how tightly coupled eRNA production is with enhancer activity. Through our systematic search for long-range elements that interact with the interferon-β gene, a model system for studying inducible transcription, we have identified a novel enhancer, which we have named L2 that regulates the expression of interferon-β. We have demonstrated its virus-inducible enhancer activity by analyzing epigenomic profiles, transcription factor association, nascent RNA production and activity in reporter assays. This enhancer exhibits intimately linked virus-inducible enhancer and bidirectional promoter activity that is largely dependent on a conserved Interferon Stimulated Response Element and robustly generates virus inducible eRNAs. Notably, its enhancer and promoter activities are fully retained in reporter assays even upon a complete elimination of its associated eRNA sequences. Finally, we show that L2 regulates IFNB1 expression by siRNA knockdown of eRNAs, and the deletion of L2 in a BAC transfection assay. Thus, L2 is a novel enhancer that regulates IFNB1 and whose eRNAs exert significant activity in vivo that is distinct from those activities recapitulated in the luciferase reporter assays. PMID:25348400

  9. Toll-Like Receptor 2 Mediates Fatal Immunopathology in Mice During Treatment of Secondary Pneumococcal Pneumonia Following Influenza

    PubMed Central

    Karlström, Åsa; Heston, Sarah M.; Boyd, Kelli L.; Tuomanen, Elaine I.

    2011-01-01

    Host inflammatory responses contribute to the significant immunopathology that occurs during treatment of secondary bacterial pneumonia following influenza. We undertook the present study to determine the mechanisms underlying disparate outcomes in a mouse model with β-lactam and macrolide antibiotics. Lysis of superinfecting bacteria by ampicillin caused an extensive influx of neutrophils into the lungs resulting in a consolidative pneumonia, necrotic lung damage, and significant mortality. This was mediated through Toll-like receptor (TLR) 2 and was independent of TLR4 and the Streptococcus pneumoniae cytotoxin pneumolysin. Treatment with azithromycin prevented neutrophil accumulation and rescued mice from subsequent mortality. This effect was independent of the antibacterial activity of this macrolide since dual therapy with ampicillin and azithromycin against an azithromycin-resistant strain also was able to cure secondary pneumonia. These data suggest that strategies for eliminating bacteria without lysis coupled with immunomodulation of inflammation should be pursued clinically. PMID:21900488

  10. Heterologous virus-induced gene silencing as a promising approach in plant functional genomics.

    PubMed

    Hosseini Tafreshi, Seied Ali; Shariati, Mansour; Mofid, Mohammad Reza; Khayam Nekui, Mojtaba; Esmaeili, Abolghasem

    2012-03-01

    VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death. PMID:21655951

  11. Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke-Exposed Mice.

    PubMed

    Bucher, Hannes; Duechs, Matthias J; Tilp, Cornelia; Jung, Birgit; Erb, Klaus J

    2016-06-01

    Viral infections trigger exacerbations in chronic obstructive pulmonary disease (COPD), and tiotropium, a M3 receptor antagonist, reduces exacerbations in patients by unknown mechanisms. In this report, we investigated whether tiotropium has anti-inflammatory effects in mice exposed to cigarette smoke (CS) and infected with influenza virus A/PR/8/34 (H1N1) or respiratory syncytial virus (RSV) and compared these effects with those of steroid fluticasone and PDE4-inhibitor roflumilast. Mice were exposed to CS; infected with H1N1 or RSV; and treated with tiotropium, fluticasone, or roflumilast. The amount of cells and cytokine levels in the airways, lung function, and viral load was determined. NCI-H292 cells were infected with H1N1 or RSV and treated with the drugs. In CS/H1N1-exposed mice, tiotropium reduced neutrophil and macrophage numbers and levels of interleukin-6 (IL-6) and interferon-γ (IFN-γ) in the airways and improved lung function. In contrast, fluticasone increased the loss of body weight; failed to reduce neutrophil or macrophage numbers; increased IL-6, KC, and tumor necrosis factor-α (TNF-α) in the lungs; and worsened lung function. Treatment with roflumilast reduced macrophage numbers, IL-6, and KC in the lungs but had no effect on neutrophil numbers or lung function. In CS/RSV-exposed mice, treatment with tiotropium, but not fluticasone or roflumilast, reduced neutrophil numbers and IL-6 and TNF-α levels in the lungs. Viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after fluticasone treatment, whereas tiotropium and roflumilast had no effect. In conclusion, tiotropium has anti-inflammatory effects on CS/virus-induced inflammation in mice that are superior to the effects of roflumilast and fluticasone. This finding might help to explain the observed reduction of exacerbation rates in COPD patients. PMID:27016458

  12. Contrasting community versus population-based estimates of grazing and virus-induced mortality of phytoplankton.

    PubMed

    Staniewski, Michael A; Short, Cindy M; Short, Steven M

    2012-07-01

    In this study, grazing and virus-induced mortality of phytoplankton was investigated in a freshwater pond at the University of Toronto Mississauga, Canada, during September 2009. The modified dilution assay, which partitions phytoplankton mortality into virus and grazing-induced fractions, was used along with newly designed, taxon-specific quantitative polymerase chain reaction (qPCR) assays that target psbA gene fragments to estimate growth and mortality rates for both the entire phytoplankton community and four distinct phytoplankton populations. Community mortality was estimated via fluorometric determination of chlorophyll a (Chl a) concentrations, whereas the relative mortality of individual phytoplankton populations was estimated via qPCR. The sources and amounts of mortality for individual phytoplankton populations differed from those of the whole community, as well as from each other. Grazing was found to be the only significant source of mortality for the community (0.32 day(-1)), and the Prymnesiales (1.65 day(-1)) and Chroococcales (2.79 day(-1)) populations studied. On the other hand, the Chlamydomonadales population examined experienced both significant grazing (1.01 day(-1)) and viral lysis (0.96 day(-1)), while the Chlorellales population only experienced significant mortality as a result of viral lysis (1.38 day(-1)). Our results demonstrate that the combination of qPCR and the modified dilution method can be used to estimate both viral lysis and grazing pressure on several individual phytoplankton populations within a community simultaneously. Further, previously noted limitations of the modified dilution method associated with the dilution of specific phytoplankton populations at low abundances can be overcome with the qPCR-based approach. Most importantly, this study demonstrates that when used alone, whole community-based methods of assessing mortality can overlook valuable information about carbon flow in aquatic microbial food webs. PMID

  13. An efficient virus-induced gene silencing vector for maize functional genomics research.

    PubMed

    Wang, Rong; Yang, Xinxin; Wang, Nian; Liu, Xuedong; Nelson, Richard S; Li, Weimin; Fan, Zaifeng; Zhou, Tao

    2016-04-01

    Maize is a major crop whose rich genetic diversity provides an advanced resource for genetic research. However, a tool for rapid transient gene function analysis in maize that may be utilized in most maize cultivars has been lacking, resulting in reliance on time-consuming stable transformation and mutation studies to obtain answers. We developed an efficient virus-induced gene silencing (VIGS) vector for maize based on a naturally maize-infecting cucumber mosaic virus (CMV) strain, ZMBJ-CMV. An infectious clone of ZMBJ-CMV was constructed, and a vascular puncture inoculation method utilizing Agrobacterium was optimized to improve its utility for CMV infection of maize. ZMBJ-CMV was then modified to function as a VIGS vector. The ZMBJ-CMV vector induced mild to moderate symptoms in many maize lines, making it useful for gene function studies in critically important maize cultivars, such as the sequenced reference inbred line B73. Using this CMV VIGS system, expression of two endogenous genes, ZmPDS and ZmIspH, was found to be decreased by 75% and 78%, respectively, compared with non-silenced tissue. Inserts with lengths of 100-300 bp produced the most complete transcriptional and visual silencing phenotypes. Moreover, genes related to autophagy, ZmATG3 and ZmATG8a, were also silenced, and it was found that they function in leaf starch degradation. These results indicate that our ZMBJ-CMV VIGS vector provides a tool for rapid and efficient gene function studies in maize. PMID:26921244

  14. Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence.

    PubMed

    Chen, Jen-Chih; Jiang, Cai-Zhong; Gookin, Timothy E; Hunter, Donald A; Clark, David G; Reid, Michael S

    2004-07-01

    Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes. PMID:15604697

  15. Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

    PubMed Central

    Benned-Jensen, Tau; Smethurst, Christopher; Holst, Peter J.; Page, Kevin R.; Sauls, Howard; Sivertsen, Bjørn; Schwartz, Thue W.; Blanchard, Andy; Jepras, Robert; Rosenkilde, Mette M.

    2011-01-01

    The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding assays, the potency of this compound was 2.6–53.6 nm, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTPγS binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe111 at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC50. In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound. PMID:21673108

  16. Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke–Exposed Mice

    PubMed Central

    Bucher, Hannes; Duechs, Matthias J.; Tilp, Cornelia; Jung, Birgit

    2016-01-01

    Viral infections trigger exacerbations in chronic obstructive pulmonary disease (COPD), and tiotropium, a M3 receptor antagonist, reduces exacerbations in patients by unknown mechanisms. In this report, we investigated whether tiotropium has anti-inflammatory effects in mice exposed to cigarette smoke (CS) and infected with influenza virus A/PR/8/34 (H1N1) or respiratory syncytial virus (RSV) and compared these effects with those of steroid fluticasone and PDE4-inhibitor roflumilast. Mice were exposed to CS; infected with H1N1 or RSV; and treated with tiotropium, fluticasone, or roflumilast. The amount of cells and cytokine levels in the airways, lung function, and viral load was determined. NCI-H292 cells were infected with H1N1 or RSV and treated with the drugs. In CS/H1N1-exposed mice, tiotropium reduced neutrophil and macrophage numbers and levels of interleukin-6 (IL-6) and interferon-γ (IFN-γ) in the airways and improved lung function. In contrast, fluticasone increased the loss of body weight; failed to reduce neutrophil or macrophage numbers; increased IL-6, KC, and tumor necrosis factor-α (TNF-α) in the lungs; and worsened lung function. Treatment with roflumilast reduced macrophage numbers, IL-6, and KC in the lungs but had no effect on neutrophil numbers or lung function. In CS/RSV-exposed mice, treatment with tiotropium, but not fluticasone or roflumilast, reduced neutrophil numbers and IL-6 and TNF-α levels in the lungs. Viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after fluticasone treatment, whereas tiotropium and roflumilast had no effect. In conclusion, tiotropium has anti-inflammatory effects on CS/virus-induced inflammation in mice that are superior to the effects of roflumilast and fluticasone. This finding might help to explain the observed reduction of exacerbation rates in COPD patients. PMID:27016458

  17. Latitudinal variation in virus-induced mortality of phytoplankton across the North Atlantic Ocean.

    PubMed

    Mojica, Kristina D A; Huisman, Jef; Wilhelm, Steven W; Brussaard, Corina P D

    2016-02-01

    Viral lysis of phytoplankton constrains marine primary production, food web dynamics and biogeochemical cycles in the ocean. Yet, little is known about the biogeographical distribution of viral lysis rates across the global ocean. To address this, we investigated phytoplankton group-specific viral lysis rates along a latitudinal gradient within the North Atlantic Ocean. The data show large-scale distribution patterns of different virus groups across the North Atlantic that are associated with the biogeographical distributions of their potential microbial hosts. Average virus-mediated lysis rates of the picocyanobacteria Prochlorococcus and Synechococcus were lower than those of the picoeukaryotic and nanoeukaryotic phytoplankton (that is, 0.14 per day compared with 0.19 and 0.23 per day, respectively). Total phytoplankton mortality (virus plus grazer-mediated) was comparable to the gross growth rate, demonstrating high turnover rates of phytoplankton populations. Virus-induced mortality was an important loss process at low and mid latitudes, whereas phytoplankton mortality was dominated by microzooplankton grazing at higher latitudes (>56°N). This shift from a viral-lysis-dominated to a grazing-dominated phytoplankton community was associated with a decrease in temperature and salinity, and the decrease in viral lysis rates was also associated with increased vertical mixing at higher latitudes. Ocean-climate models predict that surface warming will lead to an expansion of the stratified and oligotrophic regions of the world's oceans. Our findings suggest that these future shifts in the regional climate of the ocean surface layer are likely to increase the contribution of viral lysis to phytoplankton mortality in the higher-latitude waters of the North Atlantic, which may potentially reduce transfer of matter and energy up the food chain and thus affect the capacity of the northern North Atlantic to act as a long-term sink for CO2. PMID:26262815

  18. Immune cells and type 1 IFN in urine of SLE patients correlate with immunopathology in the kidney.

    PubMed

    Scott, Eric; Dooley, Mary Anne; Vilen, Barbara J; Clarke, Stephen H

    2016-07-01

    The immunopathological events in the kidneys of lupus nephritis (LN) patients are poorly understood due in part to the difficulty in acquiring serial biopsies and the inherent limitations in their analysis. To identify a means to circumvent these limitations, we investigated whether immune cells of kidney origin are present in patient urine and whether they correlate with kidney pathology. Flow cytometry analysis was performed on peripheral blood and urine cells of 69 SLE patients, of whom 41 were LN patients. In addition, type I IFN (IFNα/β) levels were determined in plasma and urine by bioassay. Approximately 60% of non-LN patients had urine lymphocytes. In these patients, T cells were always present and predominantly CD8(+), while B cells were either absent or a mixture of naïve and memory B cells. In contrast, >90% of LN patients had urine lymphocytes. In half, the B and T cells resembled those in non-LN patient urine; however, in the remaining patients, the B cells were exclusively Ig-secreting plasmablasts or plasma cells (PB/PCs) and the T cells were predominantly CD4(+). In addition, pDCs and IFNα/β frequently accompanied PB/PCs. The majority of patients with urine PB/PCs presented with proliferative nephritis and a significant loss of kidney function, which in some cases had progressed to end stage renal disease (ESRD). In conclusion, urine can provide access to cells of kidney resident populations for phenotypic and functional characterization. Analysis of these cells provides insight into the kidney immunopathology and may serve as biomarkers to identify patients at risk for developing LN and progressing to ESRD. PMID:27102764

  19. Multiple proto-oncogene activations in avian leukosis virus-induced lymphomas: evidence for stage-specific events.

    PubMed Central

    Clurman, B E; Hayward, W S

    1989-01-01

    We have examined avian leukosis virus-induced B-cell lymphomas for multiple, stage-specific oncogene activations. Three targets for viral integration were identified: c-myb, c-myc, and a newly identified locus termed c-bic. The c-myb and c-myc genes were associated with different lymphoma phenotypes. The c-bic locus was a target for integration in one class of lymphomas, usually in conjunction with c-myc activation. The data indicate that c-myc and c-bic may act synergistically during lymphomagenesis and that c-bic is involved in late stages of tumor progression. Images PMID:2548084

  20. Histone deacetylase inhibitors suppress RSV infection and alleviate virus-induced airway inflammation.

    PubMed

    Feng, Qiuqin; Su, Zhonglan; Song, Shiyu; Χu, Hui; Zhang, Bin; Yi, Long; Tian, Man; Wang, Hongwei

    2016-09-01

    Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children. However, the majority of RSV-infected patients only show mild symptoms. Different severities of infection and responses among the RSV-infected population indicate that epigenetic regulation as well as personal genetic background may affect RSV infectivity. Histone deacetylase (HDAC) is an important epigenetic regulator in lung diseases. The present study aimed to explore the possible connection between HDAC expression and RSV-induced lung inflammation. To address this question, RSV-infected airway epithelial cells (BEAS‑2B) were prepared and a mouse model of RSV infection was established, and then treated with various concentrations of HDAC inhibitors (HDACis), namely trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Viral replication and markers of virus-induced airway inflammation or oxidative stress were assessed. The activation of the nuclear factor-κB (NF-κB), cyclo-oxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling pathways was evaluated by western blot analysis. Our results showed that RSV infection in airway epithelial cells (AECs) significantly decreased histone acetylation levels by altering HDAC2 expression. The treatment of RSV-infected AECs with HDACis significantly restricted RSV replication by upregulating the interferon-α (IFN-α) related signaling pathways. The treatment of RSV-infected AECs with HDACis also significantly inhibited RSV-induced pro-inflammatory cytokine release [interleukin (IL)-6 and IL-8] and oxidative stress-related molecule production [malondialdehyde (MDA), and nitrogen monoxide (NO)]. The activation of NF-κB, COX-2, MAPK and Stat3, which orchestrate pro‑inflammatory gene expression and oxidative stress injury, was also significantly inhibited. Our in vivo study using a mouse model of

  1. Detection of a unique antigen on radiation leukemia virus-induced leukemia B6RV2

    SciTech Connect

    Nakayama, E.; Uenaka, A.; Stockert, E.; Obata, Y.

    1984-11-01

    Radiation leukemia virus-induced leukemia of a male C57BL/6 mouse, B6RV2, is immunogenic to female BALB/c X C57BL/6 F1 mice. In these mice, B6RV2 tumors regressed after initial growth, and after tumor regression the mice were resistant to repeated inocula of up to 10(8) B6RV2 cells. Serum from these mice reacted with B6RV2 in mixed hemadsorption or protein A assays, and absorption analysis indicated that the antigen was restricted to B6RV2; it could not be detected in normal thymocytes or spleen concanavalin A blasts from different inbred strains, nor in 16 C57BL/6 or BALB/c leukemias. Spleen cells from mice in which the tumor had regressed were cytotoxic to B6RV2 after in vitro stimulation with B6RV2, as shown by /sup 51/chromium release assay. This cytotoxicity was eliminated by pretreatment of the cells with anti-Thy-1.2, anti-Lyt-2.2, anti-Lyt-3.2, and complement, indicating that the effector cells were T-cells. The specificity of T-cell killing of B6RV2 was examined by competitive inhibition assays with unlabeled cells; only B6RV2 inhibited killing, while eight other C57BL/6 leukemias did not inhibit. Thus, the antigen on B6RV2 defined serologically and by cytotoxic T-cells is a unique antigen. However, it was not revealed by antibody-blocking test whether the unique determinant defined serologically was related to that recognized by T-cells; B6RV2 antiserum did not block lytic activity in the absence of added complement, irrespective of whether the target cells were untreated or anti-H-2b-treated B6RV2. H-2Kb antisera, but not H-2Db antisera, blocked lysis. This indicated that the H-2Kb molecule was exclusively involved in recognition of B6RV2 by cytotoxic T-cell.

  2. Histone deacetylase inhibitors suppress RSV infection and alleviate virus-induced airway inflammation

    PubMed Central

    Feng, Qiuqin; Su, Zhonglan; Song, Shiyu; Xu, Hui; Zhang, Bin; Yi, Long; Tian, Man; Wang, Hongwei

    2016-01-01

    Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children. However, the majority of RSV-infected patients only show mild symptoms. Different severities of infection and responses among the RSV-infected population indicate that epigenetic regulation as well as personal genetic background may affect RSV infectivity. Histone deacetylase (HDAC) is an important epigenetic regulator in lung diseases. The present study aimed to explore the possible connection between HDAC expression and RSV-induced lung inflammation. To address this question, RSV-infected airway epithelial cells (BEAS-2B) were prepared and a mouse model of RSV infection was established, and then treated with various concentrations of HDAC inhibitors (HDACis), namely trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Viral replication and markers of virus-induced airway inflammation or oxidative stress were assessed. The activation of the nuclear factor-κB (NF-κB), cyclo-oxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling pathways was evaluated by western blot analysis. Our results showed that RSV infection in airway epithelial cells (AECs) significantly decreased histone acetylation levels by altering HDAC2 expression. The treatment of RSV-infected AECs with HDACis significantly restricted RSV replication by upregulating the interferon-α (IFN-α) related signaling pathways. The treatment of RSV-infected AECs with HDACis also significantly inhibited RSV-induced pro-inflammatory cytokine release [interleukin (IL)-6 and IL-8] and oxidative stress-related molecule production [malondialdehyde (MDA), and nitrogen monoxide (NO)]. The activation of NF-κB, COX-2, MAPK and Stat3, which orchestrate pro-inflammatory gene expression and oxidative stress injury, was also significantly inhibited. Our in vivo study using a mouse model of RSV infection

  3. Dietary Vitamin D3 Suppresses Pulmonary Immunopathology Associated with Late-Stage Tuberculosis in C3HeB/FeJ Mice.

    PubMed

    Reeme, Allison E; Robinson, Richard T

    2016-02-01

    Tuberculosis (TB) is a significant human disease caused by inhalation of Mycobacterium tuberculosis. Left untreated, TB mortality is associated with a failure to resolve pulmonary immunopathology. There is currently widespread interest in using vitamin D3 (VitD3) as an adjunct therapy for TB because numerous in vitro studies have shown that VitD3 has direct and indirect mycobactericidal activities. However, to date, there have been no in vivo studies addressing whether VitD3 affects experimental TB outcome. In this study, we used C3HeB/FeJ mice to determine whether dietary VitD3 influences the outcome of experimental TB. We observed that although M. tuberculosis burdens did not differ between mice on a VitD3-replete diet (VitD(HI) mice) and mice on a VitD3-deficient diet (VitD(LO) mice), the inflammatory response in VitD(HI) mice was significantly attenuated relative to VitD(LO) controls. Specifically, the expression of multiple inflammatory pathways was reduced in the lungs at later disease stages as were splenocyte IL12/23p40 and IFN-γ levels following ex vivo restimulation. Dietary VitD3 also suppressed the accumulation of T cells in the mediastinal lymph nodes and lung granulomatous regions while concomitantly accelerating the accumulation of F4/80(+) and Ly6C/Ly6G(+) lineages. The altered inflammatory profile of VitD(HI) mice also associated with reductions in pulmonary immunopathology. VitD receptor-deficient (vdr(-/-)) radiation bone marrow chimeras demonstrate that reductions in pulmonary TB immunopathology are dependent on hematopoietic VitD responsiveness. Collectively, our data support a model wherein the in vivo role of VitD3 during TB is not to promote M. tuberculosis killing but rather to function through hematopoietic cells to reduce M. tuberculosis-elicited immunopathology. PMID:26729807

  4. Functional Interplay between Type I and II Interferons Is Essential to Limit Influenza A Virus-Induced Tissue Inflammation

    PubMed Central

    Stifter, Sebastian A.; Bhattacharyya, Nayan; Pillay, Roman; Flórido, Manuela; Triccas, James A.; Britton, Warwick J.; Feng, Carl G.

    2016-01-01

    Host control of influenza A virus (IAV) is associated with exuberant pulmonary inflammation characterized by the influx of myeloid cells and production of proinflammatory cytokines including interferons (IFNs). It is unclear, however, how the immune system clears the virus without causing lethal immunopathology. Here, we demonstrate that in addition to its known anti-viral activity, STAT1 signaling coordinates host inflammation during IAV infection in mice. This regulatory mechanism is dependent on both type I IFN and IFN-γ receptor signaling and, importantly, requires the functional interplay between the two pathways. The protective function of type I IFNs is associated with not only the recruitment of classical inflammatory Ly6Chi monocytes into IAV-infected lungs, but also the prevention of excessive monocyte activation by IFN-γ. Unexpectedly, type I IFNs preferentially regulate IFN-γ signaling in Ly6Clo rather than inflammatory Ly6Chi mononuclear cell populations. In the absence of type I IFN signaling, Ly6Clo monocytes/macrophages, become phenotypically and functionally more proinflammatory than Ly6Chi cells, revealing an unanticipated function of the Ly6Clo mononuclear cell subset in tissue inflammation. In addition, we show that type I IFNs employ distinct mechanisms to regulate monocyte and neutrophil trafficking. Type I IFN signaling is necessary, but not sufficient, for preventing neutrophil recruitment into the lungs of IAV-infected mice. Instead, the cooperation of type I IFNs and lymphocyte-produced IFN-γ is required to regulate the tissue neutrophilic response to IAV. Our study demonstrates that IFN interplay links innate and adaptive anti-viral immunity to orchestrate tissue inflammation and reveals an additional level of complexity for IFN-dependent regulatory mechanisms that function to prevent excessive immunopathology while preserving anti-microbial functions. PMID:26731100

  5. A conserved virus-induced cytoplasmic TRAMP-like complex recruits the exosome to target viral RNA for degradation.

    PubMed

    Molleston, Jerome M; Sabin, Leah R; Moy, Ryan H; Menghani, Sanjay V; Rausch, Keiko; Gordesky-Gold, Beth; Hopkins, Kaycie C; Zhou, Rui; Jensen, Torben Heick; Wilusz, Jeremy E; Cherry, Sara

    2016-07-15

    RNA degradation is tightly regulated to selectively target aberrant RNAs, including viral RNA, but this regulation is incompletely understood. Through RNAi screening in Drosophila cells, we identified the 3'-to-5' RNA exosome and two components of the exosome cofactor TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex, dMtr4 and dZcchc7, as antiviral against a panel of RNA viruses. We extended our studies to human orthologs and found that the exosome as well as TRAMP components hMTR4 and hZCCHC7 are antiviral. While hMTR4 and hZCCHC7 are normally nuclear, infection by cytoplasmic RNA viruses induces their export, forming a cytoplasmic complex that specifically recognizes and induces degradation of viral mRNAs. Furthermore, the 3' untranslated region (UTR) of bunyaviral mRNA is sufficient to confer virus-induced exosomal degradation. Altogether, our results reveal that signals from viral infection repurpose TRAMP components to a cytoplasmic surveillance role where they selectively engage viral RNAs for degradation to restrict a broad range of viruses. PMID:27474443

  6. The significance of T cells, B cells, antibodies and macrophages against encephalomyocarditis (EMC)-D virus-induced diabetes in mice.

    PubMed

    Kounoue, Etsushi; Izumi, Ken-ichi; Ogawa, Shuichiro; Kondo, Shiori; Katsuta, Hitoshi; Akashi, Tomoyuki; Niho, Yoshiyuki; Harada, Mine; Tamiya, Sadafumi; Kurisaki, Hironori; Nagafuchi, Seiho

    2008-01-01

    In order to clarify the significance of protective mechanisms against encephalomyocarditis (EMC) virus-induced diabetes in mice, we studied the relative importance of T cells, B cells, antibodies and macrophages in the prevention of virus-induced diabetes. Neither T cell-deficient athymic nude mice nor B cell-deficient microMT/microMT mice showed an enhanced clinical course of EMC-D virus-induced diabetes, indicating that neither T cells nor B cells played a major role in the protection against EMC-D-virus-induced diabetes. Transfer of a large amount of antiserum to EMC-D-virus-infected mice protected the development of diabetes only when transferred within 36 h of infection, the timing of which was earlier than that for the production of natural neutralizing antibodied. Since pretreatment of mice with the macrophage-activating immunopotentiator Corynebacterium parvum (CP) completely prevented the development of diabetes, we studied the clinical outcome of EMC-D-virus-infected mice pretreated with CP. Mice treated with CP showed reduced proliferation of EMC-D virus in the affected organs, including the pancreas, while the levels of development of neutralizing antibody and serum interferon were not enhanced compared with the controls. Finally, we studied the macrophages derived from mice pretreated with CP and found that they inhibited the growth of EMC-D virus in vitro more than those derived from non-treated and thioglycolate-treated mice. Taken together, it can be suggested that neither T cells nor B cells, which have to do with adaptive immunity, play a significant role in the pathogenesis of EMC-D-virus-induced diabetes, while innate immunity, which is dependent on activated macrophages, contributes to in vivo resistance against EMC-D-virus-induced diabetes. PMID:18500429

  7. Chronic graft-versus-host disease in the rat radiation chimera. III. Immunology and immunopathology in rapidly induced models

    SciTech Connect

    Beschorner, W.E.; Tutschka, P.J.; Santos, G.W.

    1983-03-01

    Although chronic graft-versus-host disease (GVHD) frequently develops in the long-term rat radiation chimera, we present three additional models in which a histologically similar disease is rapidly induced. These include adoptive transfer of spleen and bone marrow from rats with spontaneous chronic GVHD into lethally irradiated rats of the primary host strain; sublethal irradiation of stable chimeras followed by a booster transplant; and transfer of spleen cells of chimeras recovering from acute GVHD into second-party (primary recipient strain) or third-party hosts. Some immunopathologic and immune abnormalities associated with spontaneous chronic GVHD were not observed in one or more of the induced models. Thus, IgM deposition in the skin, antinuclear antibodies, and vasculitis appear to be paraphenomena. On the other hand, lymphoid hypocellularity of the thymic medulla, immaturity of splenic follicles, and nonspecific suppressor cells were consistently present in the long term chimeras, and in all models. These abnormalities therefore may be pathogenetically important, or closely related to the development of chronic GVHD.

  8. A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology

    PubMed Central

    Liu, Jie; Haddad, Elias K.; Marceau, Joshua; Morabito, Kaitlyn M.; Rao, Srinivas S.; Filali-Mouhim, Ali; Sekaly, Rafick-Pierre; Graham, Barney S.

    2016-01-01

    CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity. PMID:26943673

  9. Evaluation of immunopathologic effects of aqueous extract of Echinacea purpurea in mice after experimental challenge with Pasteurella multocida serotype A

    PubMed Central

    Rezaie, A; Gharibi, D; Ghorbanpoor, M; Anbari, S; Pourmahdi Broojeni, M

    2014-01-01

    In order to assess the immunopathological effects of aqueous Echinacea purpurea extract (EPE) on mice experimentally challenged with Pasteurella multocida serotype A, forty female BALB/c mice were randomly divided into four groups. The groups included a control group (received sterile distilled water 2 times/week for 2 weeks, intraperitoneally and then 100 µl sterile saline intranasally), a PMA group (received sterile distilled water as the control group and after 2 weeks, 5.6 × 103 CFU/ml of P. multocida serotype A, intranasally), an EPE+PMA group (received E. purpurea extract intraperitoneally 2 times/week for 2 weeks and then challenged as the PMA group) and an EPE group (received E. purpurea extract as EPE+PMA group and then 100 µl sterile saline intranasally). After 24 and 48 h post challenge, half of the animals in each group were sacrificed and analyzed for bacterial counts in their lungs and livers, TNFα serum levels and histapathological changes. The results showed significant differences in lung bacterial counts between PMA and EPE+PMA groups. TNFα serum level was significantly higher in the PMA group. Histopathological examination revealed infiltration of neutrophils in alveolar septa and hyperemia in the PMA group. In addition, the criteria of bronchopneumonia were partially recovered in the EPE+PMA compared to the PMA group. According to the results, it seems that E. purpurea extract has an immunomodulatory effect and can be used to prevent or control of pneumonia caused by Pasteurella. PMID:27175135

  10. Comparison of rapid immunodiagnosis assay kit with molecular and immunopathological approaches for diagnosis of rabies in cattle

    PubMed Central

    Ahmad, Ajaz; Singh, C. K.

    2016-01-01

    Aim: Presently, diagnosis of rabies is primarily based on, conventional fluorescent antibody technique (FAT), immunopathological and molecular techniques. Recently, rapid immunodiagnostic assay (RIDA) - A monoclonal antibody-based technique has been introduced for rapid diagnosis of rabies. The present investigation is envisaged to study the efficacy of RIDA kit for the diagnosis of rabies in cattle. Materials and Methods: About 11 brain samples from cattle, clinically suspected for rabies, were screened by the FAT, Heminested reverse transcriptase polymerase chain reaction (HnRT-PCR), Immunohistochemistry (IHC), and RIDA. Results: The sensitivity for detection of rabies from brain tissue by RIDA was 85.7% as compared to 100% by IHC as well as HnRT-PCR. The accuracy of detection of rabies by RIDA was 91.6% as compared to 100% that of IHC and HnRT-PCR, whereas specificity of RIDA was 100% like that of the IHC and HnRT-PCR. Conclusion: Despite a comparatively low-sensitivity and accuracy of RIDA, latter can still be useful in screening a large number of field samples promptly. However, it is recommended that negative results with RIDA in cattle need to be authenticated by suitable alternative diagnostic approaches. PMID:27051193

  11. Dimethyl fumarate suppresses Theiler's murine encephalomyelitis virus-induced demyelinating disease by modifying the Nrf2-Keap1 pathway.

    PubMed

    Kobayashi, Kunitoshi; Tomiki, Hiroki; Inaba, Yuji; Ichikawa, Motoki; Kim, Byung S; Koh, Chang-Sung

    2015-07-01

    Dimethyl fumarate (DMF) is a modifier of the nuclear factor (erythroid-derived 2)-2 (Nrf2)-kelch-like ECH-associated protein 1 (Keap1) pathway. DMF treatment in the effector phase significantly suppressed the development of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) both clinically and histologically. DMF treatment leads to an enhanced Nrf2 antioxidant response in TMEV-IDD mice. DMF treatment in the effector phase significantly suppressed the level of IL-17A mRNA. DMF is known to inhibit differentiation of T helper 17 (Th17) cells via suppressing NF-κB. Taken together, our data suggest that DMF treatment in the effector phase may suppress TMEV-IDD not only via enhancing the antioxidant response but also via suppressing IL-17A. PMID:25721871

  12. Virus induced gene silencing (VIGS) for functional analysis of wheat genes involved in Zymoseptoria tritici susceptibility and resistance

    PubMed Central

    Lee, Wing-Sham; Rudd, Jason J.; Kanyuka, Kostya

    2015-01-01

    Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetic technology in plants supplementary to stable transgenic RNAi and, in certain species, as a viable alternative approach for gene functional analysis. The RNA virus Barley stripe mosaic virus (BSMV) was developed as a VIGS vector in the early 2000s and since then it has been used to study the function of wheat genes. Several variants of BSMV vectors are available, with some requiring in vitro transcription of infectious viral RNA, while others rely on in planta production of viral RNA from DNA-based vectors delivered to plant cells either by particle bombardment or Agrobacterium tumefaciens. We adapted the latest generation of binary BSMV VIGS vectors for the identification and study of wheat genes of interest involved in interactions with Zymoseptoria tritici and here present detailed and the most up-to-date protocols. PMID:26092793

  13. Characterization of Clonality of Epstein-Barr Virus-Induced Human B Lymphoproliferative Disease in Mice with Severe Combined Immunodeficiency

    PubMed Central

    Nakamine, Hirokazu; Masih, Aneal S.; Okano, Motohiko; Taguchi, Yuichi; Pirruccello, Samuel J.; Davis, Jack R.; Mahloch, Mark L.; Beisel, Kirk W.; Kleveland, Kimberly; Sanger, Warren G.; Purtilo, David T.

    1993-01-01

    To improve the diagnostic accuracy and understanding of the pathogenesis of lymphoproliferative diseases (LPDs) occurring in immunosuppressed transplant recipients (post-transplantation LPD), clonality of Epstein-Barr virus-induced human LPDs in mice with severe combined immunodeficiency was examined by analyzing: 1) human immunoglobulin genes and their products, 2) the clonality of Epstein-Barr virus DNA, and 3) genetic alteration of c-myc or bcl-2 genes. A spectrum of clonality was found in the LPDs comparable with that reported for post-transplantation LPDs, although rearrangements of c-myc or bcl-2 genes were not detected. It is confirmed that this system is useful in terms of clonality for understanding the early phases in the pathogenesis of post-transplantation LPD or LPD in immune deficient patients. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5 PMID:8380952

  14. Institutional Animal Care and Use Committee Considerations Regarding the Use of Virus-Induced Carcinogenesis and Oncolytic Viral Models.

    PubMed

    Lewis, Stephanie D; Hickman-Davis, Judy M; Bergdall, Valerie K

    2016-03-31

    The use of virus-induced carcinogenesis and oncologic experimental animal models is essential in understanding the mechanisms of cancer development to advance prevention, diagnosis, and treatment methods. The Institutional Animal Care and Use Committee (IACUC) is responsible for both the complex philosophical and practical considerations associated with animal models of cancer. Animal models of cancer carry their own unique issues that require special consideration from the IACUC. Many of the considerations to be discussed apply to cancer models in general; specific issues related to viral carcinogenesis or oncolytic viruses will be specifically discussed as they arise. Responsible animal use integrates good science, humane care, and regulatory compliance. To meet those standards, the IACUC, in conjunction with the research investigator and attending veterinarian, must address a wide range of issues, including animal model selection, cancer model selection, humane end point considerations, experimental considerations, postapproval monitoring, reporting requirements, and animal management and personnel safety considerations. PMID:27034398

  15. NK Cell Activation in Human Hantavirus Infection Explained by Virus-Induced IL-15/IL15Rα Expression

    PubMed Central

    Braun, Monika; Björkström, Niklas K.; Gupta, Shawon; Sundström, Karin; Ahlm, Clas; Klingström, Jonas; Ljunggren, Hans-Gustaf

    2014-01-01

    Clinical infection with hantaviruses cause two severe acute diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases are characterized by strong immune activation, increased vascular permeability, and up to 50% case-fatality rates. One prominent feature observed in clinical hantavirus infection is rapid expansion of natural killer (NK) cells in peripheral blood of affected individuals. We here describe an unusually high state of activation of such expanding NK cells in the acute phase of clinical Puumala hantavirus infection. Expanding NK cells expressed markedly increased levels of activating NK cell receptors and cytotoxic effector molecules. In search for possible mechanisms behind this NK cell activation, we observed virus-induced IL-15 and IL-15Rα on infected endothelial and epithelial cells. Hantavirus-infected cells were shown to strongly activate NK cells in a cell-cell contact-dependent way, and this response was blocked with anti-IL-15 antibodies. Surprisingly, the strength of the IL-15-dependent NK cell response was such that it led to killing of uninfected endothelial cells despite expression of normal levels of HLA class I. In contrast, hantavirus-infected cells were resistant to NK cell lysis, due to a combination of virus-induced increase in HLA class I expression levels and hantavirus-mediated inhibition of apoptosis induction. In summary, we here describe a possible mechanism explaining the massive NK cell activation and proliferation observed in HFRS patients caused by Puumala hantavirus infection. The results add further insights into mechanisms behind the immunopathogenesis of hantavirus infections in humans and identify new possible targets for intervention. PMID:25412359

  16. Novel Avian Influenza A (H7N9) Virus Induces Impaired Interferon Responses in Human Dendritic Cells

    PubMed Central

    Arilahti, Veera; Mäkelä, Sanna M.; Tynell, Janne; Julkunen, Ilkka; Österlund, Pamela

    2014-01-01

    In March 2013 a new avian influenza A(H7N9) virus emerged in China and infected humans with a case fatality rate of over 30%. Like the highly pathogenic H5N1 virus, H7N9 virus is causing severe respiratory distress syndrome in most patients. Based on genetic analysis this avian influenza A virus shows to some extent adaptation to mammalian host. In the present study, we analyzed the activation of innate immune responses by this novel H7N9 influenza A virus and compared these responses to those induced by the avian H5N1 and seasonal H3N2 viruses in human monocyte-derived dendritic cells (moDCs). We observed that in H7N9 virus-infected cells, interferon (IFN) responses were weak although the virus replicated as well as the H5N1 and H3N2 viruses in moDCs. H7N9 virus-induced expression of pro-inflammatory cytokines remained at a significantly lower level as compared to H5N1 virus-induced “cytokine storm” seen in human moDCs. However, the H7N9 virus was extremely sensitive to the antiviral effects of IFN-α and IFN-β in pretreated cells. Our data indicates that different highly pathogenic avian viruses may show considerable differences in their ability to induce host antiviral responses in human primary cell models such as moDCs. The unexpected appearance of the novel H7N9 virus clearly emphasizes the importance of the global influenza surveillance system. It is, however, equally important to systematically characterize in normal human cells the replication capacity of the new viruses and their ability to induce and respond to natural antiviral substances such as IFNs. PMID:24804732

  17. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing.

    PubMed

    Kon, Tatsuya; Yoshikawa, Nobuyuki

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification. PMID:25426109

  18. Antigen-pulsed bone marrow derived and pulmonary dendritic cells promote Th2 cell responses and immunopathology in lungs during the pathogenesis of murine mycoplasma pneumonia1

    PubMed Central

    Dobbs, Nicole A.; Zhou, Xia; Pulse, Mark; Hodge, Lisa M.; Schoeb, Trenton R.; Simecka, Jerry W.

    2014-01-01

    Mycoplasmas are a common cause of pneumonia in humans and animals, and attempts to create vaccines have not only failed to generate protective host responses, but exacerbated the disease. Mycoplasma pulmonis causes a chronic inflammatory lung disease resulting from a persistent infection, similar to other mycoplasma respiratory diseases. Using this model, Th1 subsets promote resistance to mycoplasma disease and infection, while Th2 responses contribute to immunopathology. The purpose of these studies was to evaluate the capacity of cytokine differentiated dendritic cells (DC) populations to influence the generation of protective and/or pathologic immune responses during M. pulmonis respiratory disease in BALB/c mice. We hypothesized that intratracheal inoculation of mycoplasma antigen-pulsed bone marrow derived dendritic cells (BMDC) could result in the generation of protective T cell responses during mycoplasma infection. However, intratracheal inoculation (priming) of mice with antigen-pulsed DCs resulted enhanced pathology in the recipient mice when challenged with mycoplasma. Inoculation of immunodeficient SCID mice with antigen-pulsed DCs demonstrated that this effect was dependent on lymphocyte responses. Similar results were observed when mice were primed with antigen-pulsed pulmonary, but not splenic, DCs. Lymphocytes generated in uninfected mice after the transfer of either antigen-pulsed BMDCs or pulmonary DCs were shown to be IL13+ Th2 cells, known to be associated with immunopathology. Thus, resident pulmonary DC most likely promote the development of immunopathology in mycoplasma disease through the generation of mycoplasma-specific Th2 responses. Vaccination strategies that disrupt or bypass this process could potentially result in a more effective vaccination. PMID:24973442

  19. Virus-induced airway hyperresponsiveness in the guinea-pig: possible involvement of histamine and inflammatory cells.

    PubMed Central

    Folkerts, G.; De Clerck, F.; Reijnart, I.; Span, P.; Nijkamp, F. P.

    1993-01-01

    1. Guinea-pig tracheal contractions by histamine and by the cholinoceptor agonist, arecoline, are significantly enhanced (30% and 20%, respectively), 96 h after intra-tracheal inoculation with Parainfluenza-3 (PI-3) virus. 2. The airway hyperresponsiveness in animals inoculated with virus coincides with a significant increase in the number of broncho-alveolar cells (82%), and in the albumin concentration (121%) in lung lavage fluid, relative to values obtained in guinea-pigs challenged with control solution. 3. The chemiluminescence production by isolated broncho-alveolar cells, obtained from virus-infected guinea-pigs 96 h after inoculation stimulated with PI-3 virus in vitro, is significantly reduced by 42% relative to broncho-alveolar cells obtained from animals inoculated with control solution. This diminution was not specific for stimulation by PI-3 virus since the chemiluminescence production was also significantly reduced by 30% in response to zymosan. 4. Pretreatment of the guinea-pigs with the anti-allergic drugs, oxatomide (2.5 mg kg-1) or nedocromil (2.5 mg kg-1), or the specific H1-histamine receptor antagonist, levocabastine (0.25 mg kg-1), administered intra-peritoneally twice a day for five successive days, inhibits the virus-induced airway hyperresponsiveness, suppresses the influx of broncho-alveolar cells and increase in albumin content, and corrects the reduced chemiluminescence production by broncho-alveolar cells in response to zymosan. 5. In contrast, the cyclo-oxygenase inhibitor, suprofen (5.0 mg kg-1), the 5-HT2 receptor antagonist, ketanserin (0.63 mg kg-1), or the Ca2+ overload blocker, flunarizine (2.5 mg kg-1) do not modify the above mentioned processes. 6. The platelet-activating factor receptor antagonist, WEB 2170 (10 mg kg-1), reduces virus-induced airway hyperresponsiveness and influx of broncho-alveolar cells into the lungs but does not attenuate the increase of albumin in the bronchial lavage fluid. 7. Guinea-pigs nebulized with

  20. Mechanisms for virus-induced liver disease: tumor necrosis factor-mediated pathology independent of natural killer and T cells during murine cytomegalovirus infection.

    PubMed Central

    Orange, J S; Salazar-Mather, T P; Opal, S M; Biron, C A

    1997-01-01

    The contribution of endogenous NK cells and cytokines to virus-induced liver pathology was evaluated during murine cytomegalovirus infections of mice. In immunocompetent C57BL/6 mice, the virus induced a self-limited liver disease characterized by hepatitis, with focal inflammation, and large grossly visible subcapsular necrotic foci. The inflammatory foci were most numerous and contained the greatest number of cells 3 days after infection; they colocalized with areas of viral antigen expression. The largest number of necrotic foci was found 2 days after infection. Overall hepatic damage, assessed as increased expression of liver enzymes in serum, accompanied the development of inflammatory and necrotic foci. Experiments with neutralizing antibodies demonstrated that although virus-induced tumor necrosis factor (TNF) can have antiviral effects, it also mediated significant liver pathology. TNF was required for development of hepatic necrotic foci and increased levels of liver enzymes in serum but not for increased numbers of inflammatory foci. The necrotic foci and liver enzyme indications of pathology occurred independently of NK and T cells, because mice rendered NK-cell deficient by treatment with antibodies, T- and B-cell-deficient Rag-/- mice, and NK- and T-cell-deficient E26 mice all manifested both parameters of disease. Development of necrotic foci and maximally increased levels of liver enzymes in serum also were TNF dependent in NK-cell-deficient mice. Moreover, in the immunodeficient E26 mice, virus-induced liver disease was progressive, with eventual death of the host, and neutralization of TNF significantly increased longevity. These results establish conditions separating hepatitis from significant liver damage and demonstrate a cytokine-mediated component to viral pathogenesis. PMID:9371583

  1. Decreased Diversity of the Oral Microbiota of Patients with Hepatitis B Virus-Induced Chronic Liver Disease: A Pilot Project

    PubMed Central

    Ling, Zongxin; Liu, Xia; Cheng, Yiwen; Jiang, Xiawei; Jiang, Haiyin; Wang, Yuezhu; Li, Lanjuan

    2015-01-01

    Increasing evidence suggests that altered gut microbiota is implicated in the pathogenesis of hepatitis B virus-induced chronic liver disease (HBV-CLD). However, the structure and composition of the oral microbiota of patients with HBV-CLD remains unclear. High-throughput pyrosequencing showed that decreased oral bacterial diversity was found in patients with HBV-CLD. The Firmicutes/Bacteroidetes ratio was increased significantly, which indicated that dysbiosis of the oral microbiota participated in the process of HBV-CLD development. However, the changing patterns of the oral microbiota in patients with HBV-induced liver cirrhosis (LC) were almost similar to patients with chronic hepatitis B (CHB). HBV infection resulted in an increase in potential H2S- and CH3SH-producing phylotypes such as Fusobacterium, Filifactor, Eubacterium, Parvimonas and Treponema, which might contribute to the increased oral malodor. These key oral-derived phylotypes might invade into the gut as opportunistic pathogens and contribute to altering the composition of the gut microbiota. This study provided important clues that dysbiosis of the oral microbiota might be involved in the development of HBV-CLD. Greater understanding of the relationships between the dysbiosis of oral microbiota and the development of HBV-CLD might facilitate the development of non-invasive differential diagnostic procedures and targeted treatments of HBV-CLD patients harbouring specific oral phylotypes. PMID:26606973

  2. Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

    PubMed Central

    Hsieh, Ming-Hsien; Pan, Zhao-Jun; Lai, Pei-Han; Lu, Hsiang-Chia; Yeh, Hsin-Hung; Hsu, Chia-Chi; Wu, Wan-Lin; Chung, Mei-Chu; Wang, Shyh-Shyan; Chen, Wen-Huei; Chen, Hong-Hwa

    2013-01-01

    Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis. PMID:23956416

  3. Green Tea Phenolic Epicatechins Inhibit Hepatitis C Virus Replication via Cycloxygenase-2 and Attenuate Virus-Induced Inflammation

    PubMed Central

    Lin, Ying-Ting; Wu, Yu-Hsuan; Tseng, Chin-Kai; Lin, Chun-Kuang; Chen, Wei-Chun; Hsu, Yao-Chin; Lee, Jin-Ching

    2013-01-01

    Chronic hepatitis C virus (HCV) infection is the leading risk factor for hepatocellular carcinoma (HCC) and chronic liver disease worldwide. Green tea, in addition to being consumed as a healthy beverage, contains phenolic catechins that have been used as medicinal substances. In the present study, we illustrated that the epicatechin isomers (+)-epicatechin and (−)-epicatechin concentration-dependently inhibited HCV replication at nontoxic concentrations by using in vitro cell-based HCV replicon and JFH-1 infectious systems. In addition to significantly suppressing virus-induced cyclooxygenase-2 (COX-2) expression, our results revealed that the anti-HCV activity of the epicatechin isomers occurred through the down-regulation of COX-2. Furthermore, both the epicatechin isomers additively inhibited HCV replication in combination with either interferon-α or viral enzyme inhibitors [2′-C-methylcytidine (NM-107) or telaprevir]. They also had prominent anti-inflammatory effects by inhibiting the gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and inducible nitrite oxide synthase as well as the COX-2 in viral protein-expressing hepatoma Huh-7 cells. Collectively, (+)-epicatechin and (−)-epicatechin may serve as therapeutic supplements for treating HCV-related diseases. PMID:23365670

  4. Brain Endothelial- and Epithelial-Specific Interferon Receptor Chain 1 Drives Virus-Induced Sickness Behavior and Cognitive Impairment.

    PubMed

    Blank, Thomas; Detje, Claudia N; Spieß, Alena; Hagemeyer, Nora; Brendecke, Stefanie M; Wolfart, Jakob; Staszewski, Ori; Zöller, Tanja; Papageorgiou, Ismini; Schneider, Justus; Paricio-Montesinos, Ricardo; Eisel, Ulrich L M; Manahan-Vaughan, Denise; Jansen, Stephan; Lienenklaus, Stefan; Lu, Bao; Imai, Yumiko; Müller, Marcus; Goelz, Susan E; Baker, Darren P; Schwaninger, Markus; Kann, Oliver; Heikenwalder, Mathias; Kalinke, Ulrich; Prinz, Marco

    2016-04-19

    Sickness behavior and cognitive dysfunction occur frequently by unknown mechanisms in virus-infected individuals with malignancies treated with type I interferons (IFNs) and in patients with autoimmune disorders. We found that during sickness behavior, single-stranded RNA viruses, double-stranded RNA ligands, and IFNs shared pathways involving engagement of melanoma differentiation-associated protein 5 (MDA5), retinoic acid-inducible gene 1 (RIG-I), and mitochondrial antiviral signaling protein (MAVS), and subsequently induced IFN responses specifically in brain endothelia and epithelia of mice. Behavioral alterations were specifically dependent on brain endothelial and epithelial IFN receptor chain 1 (IFNAR). Using gene profiling, we identified that the endothelia-derived chemokine ligand CXCL10 mediated behavioral changes through impairment of synaptic plasticity. These results identified brain endothelial and epithelial cells as natural gatekeepers for virus-induced sickness behavior, demonstrated tissue specific IFNAR engagement, and established the CXCL10-CXCR3 axis as target for the treatment of behavioral changes during virus infection and type I IFN therapy. PMID:27096319

  5. Decreased Diversity of the Oral Microbiota of Patients with Hepatitis B Virus-Induced Chronic Liver Disease: A Pilot Project.

    PubMed

    Ling, Zongxin; Liu, Xia; Cheng, Yiwen; Jiang, Xiawei; Jiang, Haiyin; Wang, Yuezhu; Li, Lanjuan

    2015-01-01

    Increasing evidence suggests that altered gut microbiota is implicated in the pathogenesis of hepatitis B virus-induced chronic liver disease (HBV-CLD). However, the structure and composition of the oral microbiota of patients with HBV-CLD remains unclear. High-throughput pyrosequencing showed that decreased oral bacterial diversity was found in patients with HBV-CLD. The Firmicutes/Bacteroidetes ratio was increased significantly, which indicated that dysbiosis of the oral microbiota participated in the process of HBV-CLD development. However, the changing patterns of the oral microbiota in patients with HBV-induced liver cirrhosis (LC) were almost similar to patients with chronic hepatitis B (CHB). HBV infection resulted in an increase in potential H2S- and CH3SH-producing phylotypes such as Fusobacterium, Filifactor, Eubacterium, Parvimonas and Treponema, which might contribute to the increased oral malodor. These key oral-derived phylotypes might invade into the gut as opportunistic pathogens and contribute to altering the composition of the gut microbiota. This study provided important clues that dysbiosis of the oral microbiota might be involved in the development of HBV-CLD. Greater understanding of the relationships between the dysbiosis of oral microbiota and the development of HBV-CLD might facilitate the development of non-invasive differential diagnostic procedures and targeted treatments of HBV-CLD patients harbouring specific oral phylotypes. PMID:26606973

  6. Virus-induced gene silencing of pea CHLI and CHLD affects tetrapyrrole biosynthesis, chloroplast development and the primary metabolic network.

    PubMed

    Luo, Tao; Luo, Sha; Araújo, Wagner L; Schlicke, Hagen; Rothbart, Maxi; Yu, Jing; Fan, Tingting; Fernie, Alisdair R; Grimm, Bernhard; Luo, Meizhong

    2013-04-01

    The first committed and highly regulated step of chlorophyll biosynthesis is the insertion of Mg(2+) into protoporphyrin IX, which is catalyzed by Mg chelatase that consists of CHLH, CHLD and CHLI subunits. In this study, CHLI and CHLD genes were suppressed by virus-induced gene silencing (VIGS-CHLI and VIGS-CHLD) in pea (Pisum sativum), respectively. VIGS-CHLI and VIGS-CHLD plants both showed yellow leaf phenotypes with the reduced Mg chelatase activity and the inactivated synthesis of 5-aminolevulinic acid. The lower chlorophyll accumulation correlated with undeveloped thylakoid membranes, altered chloroplast nucleoid structure, malformed antenna complexes and compromised photosynthesis capacity in the yellow leaf tissues of the VIGS-CHLI and VIGS-CHLD plants. Non-enzymatic antioxidant contents and the activities of antioxidant enzymes were altered in response to enhanced accumulation of reactive oxygen species (ROS) in the chlorophyll deficient leaves of VIGS-CHLI and VIGS-CHLD plants. Furthermore, the results of metabolite profiling indicate a tight correlation between primary metabolic pathways and Mg chelatase activity. We also found that CHLD induces a feedback-regulated change of the transcription of photosynthesis-associated nuclear genes. CHLD and CHLI silencing resulted in a rapid reduction of photosynthetic proteins. Taken together, Mg chelatase is not only a key regulator of tetrapyrrole biosynthesis but its activity also correlates with ROS homeostasis, primary interorganellar metabolism and retrograde signaling in plant cells. PMID:23416492

  7. A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots

    PubMed Central

    Yan, Lijie; Jackson, Andrew O.; Liu, Zhiyong; Han, Chenggui; Yu, Jialin; Li, Dawei

    2011-01-01

    Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies. PMID:22031834

  8. Virus-induced gene silencing-based functional verification of six genes associated with vernalization in wheat.

    PubMed

    Feng, Ya-Lan; Wang, Ke-Tao; Ma, Chao; Zhao, Yong-Ying; Yin, Jun

    2015-03-20

    Vernalization requirement is an important characteristic in crop breeding. Wheat is a widely grown crop in the world that possesses enormous economic significance. To better understand the gene networks in vernalization process, we performed a high-throughput RNA sequencing analysis comparing the transcriptomes of spring and winter wheat cultivars, with and without vernalization (unpublished data). In this study, we selected six unigenes (CL14010, CL12788, CL176, Unigene 16777, CL8746 and Unigene10196) from our transcriptome analysis based on their expression differences to further characterize their function. Transient silencing of the six unigenes individually were achieved through virus-induced gene silencing (VIGS) using BSMV vector. The period from germination to spike differentiation were recorded and compared between plants underwent VIGS silencing and the control. Our result showed that VIGS of the six unigenes significantly shortened the period from seedling to double ridge (DR) stage. Resulting in SD period ranging from 59.8 ± 0.60 to 65.8 ± 0.48 days, compared to 85.0 ± 0.73 days in the control. The results indicated that these six unigenes function as suppressors in vernalization process and silence or down-regulation of these genes promoted flower development in wheat. Further characterization of these six unigenes and their function in vernalization and flowering control is needed. PMID:25707852

  9. Epstein-Barr virus-induced gene 3 (EBI3) polymorphisms and expression are associated with susceptibility to pulmonary tuberculosis.

    PubMed

    Zheng, Ruijuan; Liu, Haipeng; Song, Peng; Feng, Yonghong; Qin, Lianhua; Huang, Xiaochen; Chen, Jianxia; Yang, Hua; Liu, Zhonghua; Cui, Zhenglin; Hu, Zhongyi; Ge, Baoxue

    2015-07-01

    Tuberculosis (TB) remains a major global health problem and host genetic factors play a critical role in susceptibility and resistance to TB. The aim of this study was to identify novel candidate genes associated with TB susceptibility. We performed a population-based case-control study to genotype 13 tag SNPs spanning Epstein-Barr virus-induced gene 3 (EBI3), colony stimulating factor 2 (CSF2), IL-4, interferon beta 1 (IFNB1), chemokine (C-X-C motif) ligand 14 (CXCL14) and myeloid differentiation primary response gene 88 (Myd88) genes in 435 pulmonary TB patients and 375 health donors from China. We observed that EBI3 gene rs4740 polymorphism was associated with susceptibility to pulmonary tuberculosis (PTB) and the allele G was associated with a protective effect against PTB. Furthermore, EBI3 deficiency led to reduced bacterial burden and histopathological impairment in the lung of mice infected with Mycobacterium bovis BCG. Meanwhile, higher abundance of EBI3 was observed in the granuloma of PTB patients and in the lung tissue of BCG-infected mice. Of note, the expression of EBI3 in macrophages was remarkably induced by mycobacteria infection at both mRNA and protein level. In conclusion, EBI3 gene rs4740 polymorphism is closely associated with susceptibility to PTB and the elevation and enrichment of EBI3 in the lung which at least partially derived from macrophages may contribute to the exacerbation of mycobacterial infection. PMID:25937126

  10. Immunopathology of malaria*

    PubMed Central

    Voller, Alister

    1974-01-01

    Antibodies with different spectra of reactivity are produced during malarial infections and marked changes in IgG and IgM levels occur. In addition malaria elicits serological changes that are usually associated with connective tissue disease. The excessive anaemia associated with malaria may, in part, be an autoimmune phenomenon. Transient nephritis accompanies many plasmodial infections but chronic malarial nephrotic syndrome is specifically associated with quartan malaria. Malarial infection leads to splenomegaly, the most extreme form of which is idiopathic tropical splenomegaly, which probably represents an aberrant immune response to the infection. Malaria can affect the humoral immune response to unrelated antigens and infectious agents. This may be relevant to the etiology of Burkitt's lymphoma. During pregnancy there is some loss of acquired immunity to P. falciparum and the placenta appears to be an immunologically privileged site for the multiplication of this parasite. PMID:4216408

  11. Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance

    PubMed Central

    Lapitan, Nora

    2013-01-01

    In a non-model staple crop like wheat (Triticum aestivumI L.), functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for breeding. Virus-induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited transformation potential that hamper functional validation studies in wheat. In this study, three potential candidate genes shown to be involved in abiotic stress response pathways in Arabidopsis thaliana were selected for VIGS experiments in wheat. These include Era1 (enhanced response to abscisic acid), Cyp707a (ABA 8’-hydroxylase), and Sal1 (inositol polyphosphate 1-phosphatase). Gene homologues for these three genes were identified in wheat and cloned in the viral vector barley stripe mosaic virus (BSMV) in the antisense direction, followed by rub inoculation of BSMV viral RNA transcripts onto wheat plants. Quantitative real-time PCR showed that VIGS-treated wheat plants had significant reductions in target gene transcripts. When VIGS-treated plants generated for Era1 and Sal1 were subjected to limiting water conditions, they showed increased relative water content, improved water use efficiency, reduced gas exchange, and better vigour compared to water-stressed control plants inoculated with RNA from the empty viral vector (BSMV0). In comparison, the Cyp707a-silenced plants showed no improvement over BSMV0-inoculated plants under limited water condition. These results indicate that Era1 and Sal1 play important roles in conferring drought tolerance in wheat. Other traits affected by Era1 silencing were also studied. Delayed seed germination in Era1-silenced plants suggests this gene may be a useful target for developing resistance to pre-harvest sprouting. PMID:23364940

  12. Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum).

    PubMed

    Fragkostefanakis, Sotirios; Sedeek, Khalid E M; Raad, Maya; Zaki, Marwa Samir; Kalaitzis, Panagiotis

    2014-07-01

    Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves. PMID:24803411

  13. Expression of interleukin 6 receptors and interleukin 6 mRNA by bovine leukaemia virus-induced tumour cells.

    PubMed

    Droogmans, L; Cludts, I; Cleuter, Y; Kerkhofs, P; Adam, E; Willems, L; Kettmann, R; Burny, A

    1994-11-01

    Bovine leukaemia virus (BLV) is the aetiologic agent of bovine leucosis. The virus induces malignancies of the B-cell lineage (leukaemia/lymphoma). The role played by interleukin 6 (IL-6) in the BLV-induced leukemogenesis process was evaluated. Six cell lines derived from BLV-induced tumours were tested for the expression of IL-6 receptors. Two cell lines (LB155 and YR2) display 250-300 receptor per cell (kd = 1.7 10(-10) M and 1.4 10(-10) M, respectively) whereas the other four (LB159, LB167, YR1 and M51) do not display detectable amounts of receptors. Very low (if any) expression of IL-6 receptors has been found in the case of the B lymphocytes of animals in persistent lymphocytosis (PL). Despite the presence of IL-6 receptors on the surface of LB155 and YR2 cells, no influence of exogenous IL-6 on their growth has been observed. Northern analyses indicated the presence of IL-6 transcripts only in the case of mRNA isolated from LB155 cells. Since this cell line also expresses receptors for the cytokine, an autocrine loop may exist in these cells. Experiments in which bovine and bovine epithelial cell lines were transfected with a plasmid containing the bovine IL-6 promoter controlling the expression of the reporter cat gene failed to indicate any influence of the viral transactivator p34tax on the activity of this promoter. We conclude that IL-6 receptors and IL-6 mRNA can be found in some BLV-induced tumours, but this does not correlate with viral expression in BLV-induced leukaemia/lymphoma. PMID:7893972

  14. Expression of Epstein–Barr virus-induced gene 3 and other interleukin-12-related molecules by human intestinal epithelium

    PubMed Central

    Maaser, Christian; Egan, Laurence J; Birkenbach, Mark P; Eckmann, Lars; Kagnoff, Martin F

    2004-01-01

    Antigen-presenting cells, including dendritic cells, monocytes and macrophages, produce members of the interleukin-12 (IL-12) family that are important in initiating and maintaining cell-mediated immune responses. These include IL-12p35 and p19 that dimerize with IL-12p40 to form IL-12 (also termed IL-12p75) and IL-23, respectively, and Epstein–Barr virus-induced gene 3 (EBI3) protein (a protein related to IL-12p40), that forms a dimer with p28, termed IL-27. Intestinal epithelial cells, which are the initial site of contact between the host and enteric pathogens, can act as antigen-presenting cells, and are known to express mediators important in inflammatory and immune responses. In the current studies, we hypothesized that intestinal epithelial cells express members of the IL-12 family, which can function as an early signalling system important in mucosal immunity. Using in vitro and in vivo model systems of human intestinal epithelium, we demonstrate the regulated expression of EBI3, IL-12p35 and p19 by human intestinal epithelial cells. However, intestinal epithelial cells do not coexpress IL-12p40 or p28 that are required to generate heterodimeric IL-12p75, IL-23 and IL-27. To the extent that IL-12p35, p19 and EBI3 cannot form IL-12p75, IL-23 or IL-27 heterodimers in intestinal epithelial cells, these data suggest that those cells may express other, currently unknown, molecules that can associate with EBI3, IL-12p35 and/or p19 or, alternatively, intestinal epithelial cells may release IL-12-related molecules that by themselves, or in combination with other molecules in the mucosal microenvironment, mediate biological activities. PMID:15196212

  15. Anti-apoptotic Bcl-XL but not Mcl-1 contributes to protection against virus-induced apoptosis.

    PubMed

    Ohmer, Michaela; Weber, Arnim; Sutter, Gerd; Ehrhardt, Katrin; Zimmermann, Albert; Häcker, Georg

    2016-01-01

    Infection of mammalian cells with viruses often induces apoptosis. How the recognition of viruses leads to apoptosis of the infected cell and which host cell factors regulate this cell death is incompletely understood. In this study, we focussed on two major anti-apoptotic proteins of the host cell, whose abundance and activity are important for cell survival, the Bcl-2-like proteins Mcl-1 and Bcl-XL. During infection of epithelial cells and fibroblasts with modified vaccinia virus Ankara (MVA), Mcl-1 protein levels dropped but the MVA Bcl-2-like protein F1L could replace Mcl-1 functionally; a similar activity was found in vaccinia virus (VACV)-infected cells. During infection with murine cytomegalovirus (MCMV), Mcl-1-levels were not reduced but a viral Mcl-1-like activity was also generated. Infection of mouse macrophages with any of these viruses, on the other hand, induced apoptosis. Virus-induced macrophage apoptosis was unaltered in the absence of Mcl-1. However, apoptosis was substantially increased in infected Bcl-XL-deficient macrophages or macrophages treated with the Bcl-2/Bcl-XL-inhibitor ABT-737. Genetic loss of Bcl-XL or treatment of macrophages with ABT-737 reduced the generation of infectious VACV. These data show that Mcl-1 is dispensable for the regulation of apoptosis during infection with different large DNA viruses, either because the viruses replace its function (in fibroblasts and epithelial cells) or because the pro-apoptotic activity generated by the infection appears not to be blocked by it (in macrophages). Bcl-XL, on the other hand, can be important to maintain survival of virus-infected cells, and its activity can determine outcome of the infection. PMID:27537523

  16. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

    SciTech Connect

    Verma, Saguna; Ziegler, Katja; Ananthula, Praveen; Co, Juliene K.G.; Frisque, Richard J.; Yanagihara, Richard; Nerurkar, Vivek R. . E-mail: nerurkar@pbrc.hawaii.edu

    2006-02-20

    Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

  17. Protective Immunity to Vaccinia Virus Induced by Vaccination with Multiple Recombinant Outer Membrane Proteins of Intracellular and Extracellular Virions

    PubMed Central

    Fogg, Christiana; Lustig, Shlomo; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2004-01-01

    Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-μg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine. PMID:15367588

  18. Anti-inflammatory effects of the novel inhaled phosphodiesterase type 4 inhibitor CHF6001 on virus-inducible cytokines.

    PubMed

    Edwards, Michael R; Facchinetti, Fabrizio; Civelli, Maurizio; Villetti, Gino; Johnston, Sebastian L

    2016-02-01

    Respiratory virus infections precipitate asthma and chronic obstructive pulmonary disease (COPD) exacerbations, with most exacerbations due to rhinovirus infection. Both asthma and COPD exacerbations are not well controlled by steroid therapies, and there is a much research interest in finding improved therapies or combinations of therapies for controlling exacerbations. CHF6001 is a new, inhaled highly potent and selective phosphodiesterase type 4 (PDE4) inhibitor. Using in vitro human bronchial epithelial cells (BEAS-2B), we investigated the potential anti-inflammatory effects of CHF6001 on rhinovirus (RV1B)-induced cytokines. Cytokine mRNA was measured by real-time PCR, while protein release was measured by ELISA. CHF6001 was used in a 7-point dose-response curve (1000-0.001 nmol/L) as a 1.5-h pretreatment prior to infection in comparison with roflumilast. Both roflumilast and CHF6001 reduced RV1B-induced IL-8, IL-29, IP-10, and RANTES mRNA and protein in a concentration-dependent manner. Generally, CHF6001 was 13- to 16-fold more potent (subnanomolar EC 50 values) than roflumilast at reducing IL-8, IL-29, IP-10, and RANTES mRNA and protein release, but had similar efficacies. In combination with the steroid fluticasone propionate (1 nmol/L), CHF6001 had additive effects, significantly reducing RV-induced cytokines when compared with steroid or CHF6001 alone. Combined low-dose steroid and low-dose CHF6001 had a similar efficacy as high-dose steroid or CHF6001 alone, indicating the combination had steroid and PDE4 inhibitor sparing effects. Overall results indicate that PDE4 inhibitors have anti-inflammatory activity against virus-induced inflammatory mediators and that CHF6001 is more potent than roflumilast. PMID:26977295

  19. Revelation of the IFNα, IL-10, IL-8 and IL-1β as promising biomarkers reflecting immuno-pathological mechanisms in porcine Huntington's disease model.

    PubMed

    Valekova, Ivona; Jarkovska, Karla; Kotrcova, Eva; Bucci, John; Ellederova, Zdenka; Juhas, Stefan; Motlik, Jan; Gadher, Suresh Jivan; Kovarova, Hana

    2016-04-15

    Studies on Huntington's disease (HD) demonstrated altered immune response in HD gene carriers. Using multiplexing immunoassay, we simultaneously investigated seven cytokines in secretomes of microglia and blood monocytes, cerebrospinal fluid (CSF) and serum collected from transgenic HD minipigs at pre-symptomatic disease stage. Decline in IFNα and IL-10 was observed in CSF and secretome of microglia whilst elevated IL-8 and IL-1β levels were secreted by microglia. Additionally, IL-8 was increased in serum. The proportion of mutant huntingtin in microglia may have causative impact on cytokine production. IFNα, IL-10, IL-8 and IL-1β represent promising biomarkers reflecting immuno-pathological mechanisms in porcine HD model. PMID:27049565

  20. Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

    PubMed Central

    Gomes, Rafael G. B.; da Silva, Camila T.; Taniguchi, Juliana B.; No, Joo Hwan; Lombardot, Benoit; Schwartz, Olivier; Hansen, Michael A. E.; Freitas-Junior, Lucio H.

    2013-01-01

    antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis. PMID:24205414

  1. Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)*

    PubMed Central

    Benned-Jensen, Tau; Norn, Christoffer; Laurent, Stephane; Madsen, Christian M.; Larsen, Hjalte M.; Arfelt, Kristine N.; Wolf, Romain M.; Frimurer, Thomas; Sailer, Andreas W.; Rosenkilde, Mette M.

    2012-01-01

    Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors. PMID:22875855

  2. Genetic determinants of feline leukemia virus-induced lymphoid tumors: patterns of proviral insertion and gene rearrangement.

    PubMed

    Tsatsanis, C; Fulton, R; Nishigaki, K; Tsujimoto, H; Levy, L; Terry, A; Spandidos, D; Onions, D; Neil, J C

    1994-12-01

    The genetic basis of feline leukemia virus (FeLV)-induced lymphoma was investigated in a series of 63 lymphoid tumors and tumor cell lines of presumptive T-cell origin. These were examined for virus-induced rearrangements of the c-myc, flvi-2 (bmi-1), fit-1, and pim-1 loci, for T-cell receptor (TCR) gene rearrangements, and for the presence of env recombinant FeLV (FeLV-B). The myc locus was most frequently affected in naturally occurring lymphomas (32%; n = 38) either by transduction (21%) or by proviral insertion (11%). Proviral insertions were also common at flvi-2 (24%). The two other loci were occupied in a smaller number of the naturally occurring tumors (fit-1, 8%; pim-1, 5%). Examination of the entire set of tumors showed that significant numbers were affected at two (19%) or three (5%) of the loci. Occupation of the fit-1 locus was observed most frequently in tumors induced by FeLV-myc strains, while flvi-2 insertions occurred with similar frequency in the presence or absence of obvious c-myc activation. These results suggest a hierarchy of mutational events in the genesis of feline T-cell lymphomas by FeLV and implicate insertion at fit-1 as a late progression step. The strongest links observed were with T-cell development, as monitored by rearrangement status of the TCR beta-chain gene, which was positively associated with activation of myc (P < 0.001), and with proviral insertion at flvi-2 (P = 0.02). This analysis also revealed a genetically distinct subset of thymic lymphomas with unrearranged TCR beta-chain genes in which the known target loci were involved very infrequently. The presence of env recombinant FeLV (FeLV-B) showed a negative correlation with proviral insertion at fit-1, possibly due to the rapid onset of these tumors. These results shed further light on the multistep process of FeLV leukemogenesis and the relationships between lymphoid cell maturation and susceptibility to FeLV transformation. PMID:7966623

  3. Involvement of fish signal transducer and activator of transcription 3 (STAT3) in SGIV replication and virus induced paraptosis.

    PubMed

    Huang, Xiaohong; Huang, Youhua; Yang, Ying; Wei, Shina; Qin, Qiwei

    2014-12-01

    Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor which plays crucial roles in immune regulation, inflammation, cell proliferation, transformation, and other physiological processes of the organism. In this study, a novel STAT3 gene from orange spotted grouper (Ec-STAT3) was cloned and characterized. Bioinformatic analysis revealed that full-length of Ec-STAT3 was 3105-bp long and contained a 280-bp 5'UTR, a 470-bp 3'UTR, and a 2355-bp open reading frame (ORF) that encoded a 784-amino acid peptide. The deduced protein of Ec-STAT3 showed 98% identity to that of turbot (Scophthalmus maximus). Amino acid alignment showed that Ec-STAT3 contained four conserved domains, including a protein interaction domain, a coiled coil domain, a DNA binding domain, and an SH2 domain. Quantitative real-time PCR analysis showed that the highest expression level was detected in the liver, followed by skin and spleen. After injection with Singapore grouper iridovirus (SGIV), the transcript of Ec-STAT3 in spleen was increased significantly. To further explore the function of Ec-STAT3, we investigated the roles of Ec-STAT3 in SGIV infection in vitro. Immune fluorescence analysis indicated that SGIV infection altered the distribution of phosphorylated Ec-STAT3 in nucleus, and a small part of phosphorylated Ec-STAT3 was associated with virus assembly sites, suggesting that Ec-STAT3 might be important for SGIV infection. Using STAT3 specific inhibitor, S3I-201, we found that inhibition of Ec-STAT3 activation decreased the SGIV replication significantly. Moreover, inhibition of Ec-STAT3 activation obviously altered SGIV infection induced cell cycle arrest and the expression of pro-survival genes, including Bcl-2, Bcl-xL and Bax inhibitor. Together, our results firstly demonstrated the critical roles of fish STAT3 in DNA virus replication and virus induced paraptosis, but also provided new insights into the mechanism of iridovirus pathogenesis

  4. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the /sup 51/Cr-release assay

    SciTech Connect

    Zinkernagel, R.M.; Haenseler, E.; Leist, T.; Cerny, A.; Hengartner, H.; Althage, A.

    1986-10-01

    A model for immunologically T cell-mediated hepatitis was established in mice infected with lymphocytic choriomeningitis virus (LCMV). The severity of hepatitis was monitored histologically and by determination of changes in serum levels of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), and alkaline phosphatase (AP). Kinetics of histological disease manifestations, increases of liver enzyme levels in the serum, and cytotoxic T cell activities in livers and spleens all correlated and were dependent upon several parameters: LCMV-isolate; LCMV-WE caused extensive hepatitis, LCMV-Armstrong virtually none. Virus dose. Route of infection; i.v. or i.p. infection caused hepatitis, whereas infection into the footpad did not. The general genetic background of the murine host; of the strains tested, Swiss mice and A-strain mice were more susceptible than C57BL or CBA mice; BALB/c and DBA/2 mice were least susceptible. The degree of immunocompetence of the murine host; T cell deficient nu/nu mice never developed hepatitis, whereas nu/+ or +/+ mice always did. B cell-depleted anti-IgM-treated mice developed immune-mediated hepatitis comparably or even more extensively than control mice. Local cytotoxic T cell activity; mononuclear cells isolated from livers during the period of overt hepatitis were two to five times more active than equal numbers of spleen cells. Adoptive transfer of nylon wool-nonadherent anti-Thy-1.2 and anti-Lyt-2 plus C-sensitive, anti-L3T4 plus C-resistant lymphocytes into irradiated mice preinfected with LCMV-WE caused a rapid time- and dose-dependent linear increase of serum enzyme levels. This increase was caused by adoptive transfer of lymphocytes if immune cell donors and recipient mice shared class I, but not when they shared class II histocompatibility antigens.

  5. Analysis of herpes simplex virus-induced mRNA destabilizing activity using an in vitro mRNA decay system.

    PubMed Central

    Sorenson, C M; Hart, P A; Ross, J

    1991-01-01

    Most host mRNAs are degraded soon after infection of cells with herpes simplex virus type 1 (HSV-1). This early shutoff or early destabilization response is induced by a virion component, the virion host shutoff (vhs) protein. HSV-1 mutants, vhs1 and vhs-delta Sma, which produce defective or inactive vhs protein, fail to induce early shutoff. We have used an in vitro mRNA decay system to analyze the destabilization process. Polysomes from uninfected human erythroleukemia cells, used as a source of target mRNAs, were mixed with polysomes or with post-polysomal supernatant (S130) from HSV-1- or mock-infected murine erythroleukemia cells. Normally stable gamma-globin mRNA was destabilized by approximately 15-fold with S130 from wild-type virus-infected cells but was not destabilized with S130 from mock-infected cells or from cells infected with either of the two HSV mutants. The virus-induced destabilizing activity had no significant effect on the in vitro half-lives of two normally unstable mRNAs, histone and c-myc. No destabilizing activity was detected in polysomes from infected cells. We conclude that a virus-induced destabilizer activity can function in vitro, is located in the S130 of infected cells, and accelerates the decay rates of some, but not all, polysome-associated host mRNAs. Images PMID:1653415

  6. Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion

    PubMed Central

    Schmid, Erik; Zurbriggen, Andreas; Gassen, Uta; Rima, Bert; ter Meulen, Volker; Schneider-Schaulies, Jürgen

    2000-01-01

    Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected. PMID:10906209

  7. Peptide vaccination of mice immune to LCMV or vaccinia virus causes serious CD8+ T cell-mediated, TNF-dependent immunopathology

    PubMed Central

    Liu, Fei; Feuer, Ralph; Hassett, Daniel E.; Whitton, J. Lindsay

    2006-01-01

    CD8+ T cells play a key role in clearing primary virus infections and in protecting against subsequent challenge. The potent antiviral effects of these cells make them important components of vaccine-induced immunity and, because of this, peptide vaccines often contain epitopes designed to induce strong CD8+ T cell responses. However, the same effector functions that protect the host also can be harmful if they are not tightly regulated, and virus-specific CD8+ T cells are a frequent cause of immunopathology. Here, we report that the administration of peptide to virus-immune recipient mice can lead to the synchronous activation of preexisting virus-specific CD8+ T cells with serious, and even lethal, consequences. Mice infected with LCMV or vaccinia virus developed rapid and profound hypothermia following injection of cognate synthetic peptides, and LCMV-infected mice frequently died within hours. Detailed analyses of the LCMV infected mice revealed enterocyte apoptosis and implicated TNF produced by peptide-specific CD8+ T cells as the major mediator of disease. The caspase inhibitor zVADfmk had no demonstrable effect on the development of hypothermia, but diminished enterocyte apoptosis and greatly reduced the number of deaths. These findings, if similarly observed in patients, counsel caution when administering powerful immunogens such as peptide vaccines to individuals who may have a large preexisting pool of epitope-specific CD8+ T cells. PMID:16424939

  8. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

    SciTech Connect

    Igarashi, Aki; Yamagata, Kousuke; Sugai, Tomokazu; Takahashi, Yukari; Sugawara, Emiko; Tamura, Akihiro; Yaegashi, Hajime; Yamagishi, Noriko; Takahashi, Tsubasa; Isogai, Masamichi; Takahashi, Hideki; Yoshikawa, Nobuyuki

    2009-04-10

    Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

  9. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

    PubMed Central

    Ink, B S; Gilbert, C S; Evan, G I

    1995-01-01

    The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family. PMID:7815529

  10. The presence of tomato leaf curl Kerala virus AC3 protein enhances viral DNA replication and modulates virus induced gene-silencing mechanism in tomato plants

    PubMed Central

    2011-01-01

    Background Geminiviruses encode few viral proteins. Most of the geminiviral proteins are multifunctional and influence various host cellular processes for the successful viral infection. Though few viral proteins like AC1 and AC2 are well characterized for their multiple functions, role of AC3 in the successful viral infection has not been investigated in detail. Results We performed phage display analysis with the purified recombinant AC3 protein with Maltose Binding Protein as fusion tag (MBP-AC3). Putative AC3 interacting peptides identified through phage display were observed to be homologous to peptides of proteins from various metabolisms. We grouped these putative AC3 interacting peptides according to the known metabolic function of the homologous peptide containing proteins. In order to check if AC3 influences any of these particular metabolic pathways, we designed vectors for assaying DNA replication and virus induced gene-silencing of host gene PCNA. Investigation with these vectors indicated that AC3 enhances viral replication in the host plant tomato. In the PCNA gene-silencing experiment, we observed that the presence of functional AC3 ORF strongly manifested the stunted phenotype associated with the virus induced gene-silencing of PCNA in tomato plants. Conclusions Through the phage display analysis proteins from various metabolic pathways were identified as putative AC3 interacting proteins. By utilizing the vectors developed, we could analyze the role of AC3 in viral DNA replication and host gene-silencing. Our studies indicate that AC3 is also a multifunctional protein. PMID:21496351

  11. Lack of essential role of NF-kappa B p50, RelA, and cRel subunits in virus-induced type 1 IFN expression.

    PubMed

    Wang, Xingyu; Hussain, Sofia; Wang, Emilie-Jeanne; Wang, Xiuyan; Li, Ming O; García-Sastre, Adolfo; Beg, Amer A

    2007-06-01

    Type 1 IFNs (IFN-alphabeta) play pivotal roles in the host antiviral response and in TLR-induced signaling. IFN regulatory factor (IRF) and NF-kappaB transcription factors are thought to be crucial for virus-induced mRNA expression of IFN-beta. Although recent studies have demonstrated essential roles for IRF3 and IRF7, the definitive role of NF-kappaB factors in IFN-beta (or IFN-alpha) expression remains unknown. Using mice deficient in distinct members of the NF-kappaB family, we investigated NF-kappaB function in regulating type 1 IFN expression in response to Sendai virus and Newcastle disease virus infection. Surprisingly, IFN-beta and IFN-alpha expression was strongly induced following virus infection of mouse embryonic fibroblasts (MEFs) from p50(-/-), RelA/p65(-/-), cRel(-/-), p50(-/-)cRel(-/-), and p50(-/-)RelA(-/-) mice. Compared with wild-type MEFs, only RelA(-/-) and p50(-/-)RelA(-/-) MEFs showed a modest reduction in IFN-beta expression. To overcome functional redundancy between different NF-kappaB subunits, we expressed a dominant-negative IkappaBalpha protein in p50(-/-)RelA(-/-) MEFs to inhibit activation of remaining NF-kappaB subunits. Although viral infection of these cells failed to induce detectable NF-kappaB activity, both Sendai virus and Newcastle disease virus infection led to robust IFN-beta expression. Virus infection of dendritic cells or TLR9-ligand CpG-D19 treatment of plasmacytoid dendritic cells from RelA(-/-) or p50(-/-)cRel(-/-) mice also induced robust type 1 IFN expression. Our findings therefore indicate that NF-kappaB subunits p50, RelA, and cRel play a relatively minor role in virus-induced type 1 IFN expression. PMID:17513724

  12. Chronic graft-versus-host disease in the rat radiation chimera: I. clinical features, hematology, histology, and immunopathology in long-term chimeras

    SciTech Connect

    Beschorner, W.E.; Tutschka, P.J.; Santos, G.W.

    1982-04-01

    The clinical features, pathology, and immunopathology of chronic graft-versus-host disease (GVHD) developing in the long-term rat radiation chimera are described. At 6 to 12 months post-transplant, the previously stable ACI/LEW chimeras developed patchy to diffuse severe hair loss and thickened skin folds, and had microscopic features resembling scleroderma, Sjogren's syndrome, and chronic hepatitis. Skin histology showed dermal inflammation and acanthosis with atrophy of the appendages, with progression to dermal sclerosis. The liver revealed chronic hepatitis with bile duct injury and proliferation and periportal piecemeal necrosis. The tongue had considerable submucosal inflammation, muscular necrosis, and atrophy and arteritis. The serous salivary glands, lacrimal glands, and bronchi had lymphocytic inflammation and injury to duct, acinar, and mucosal columnar epithelium. The thymus had lymphocyte depletion of the medulla with prominent epithelium. The spleen and lymph nodes had poorly developed germinal centers but increased numbers of plasma cells. IgM was observed along the basement membrane and around the basal cells of the skin and tongue and along the basement membrane of the bile ducts. IgM was present also in the arteries of the tongue. Immunoglobulins eluted from the skin, cross-reacted with the bile duct epithelium and usually with both ACI and Lewis skin. Increased titers of speckled antinuclear antibodies were present in the serum of rats with chronic (GVHD). Chronic GVHD in the long-term rat radiation chimera is very similar to human chronic GVHD and is a potentially excellent model for autoimmune disorders including scleroderma, Sjorgren's syndrome, and chronic hepatitis.

  13. Helicobacter pylori Induced Gastric Immunopathology Is Associated with Distinct Microbiota Changes in the Large Intestines of Long-Term Infected Mongolian Gerbils

    PubMed Central

    Heimesaat, Markus M.; Fischer, André; Plickert, Rita; Wiedemann, Tobias; Loddenkemper, Christoph; Göbel, Ulf B.

    2014-01-01

    Background Gastrointestinal (GI) inflammation in mice and men are frequently accompanied by distinct changes of the GI microbiota composition at sites of inflammation. Helicobacter (H.) pylori infection results in gastric immunopathology accompanied by colonization of stomachs with bacterial species, which are usually restricted to the lower intestine. Potential microbiota shifts distal to the inflammatory process following long-term H. pylori infection, however, have not been studied so far. Methodology/Principal Findings For the first time, we investigated microbiota changes along the entire GI tract of Mongolian gerbils after 14 months of infection with H. pylori B8 wildtype (WT) or its isogenic ΔcagY mutant (MUT) strain which is defective in the type IV secretion system and thus unable to modulate specific host pathways. Comprehensive cultural analyses revealed that severe gastric diseases such as atrophic pangastritis and precancerous transformations were accompanied by elevated luminal loads of E. coli and enterococci in the caecum and together with Bacteroides/Prevotella spp. in the colon of H. pylori WT, but not MUT infected gerbils as compared to naïve animals. Strikingly, molecular analyses revealed that Akkermansia, an uncultivable species involved in mucus degradation, was exclusively abundant in large intestines of H. pylori WT, but not MUT infected nor naïve gerbils. Conclusion/Significance Taken together, long-term infection of Mongolian gerbils with a H. pylori WT strain displaying an intact type IV secretion system leads to distinct shifts of the microbiota composition in the distal uninflamed, but not proximal inflamed GI tract. Hence, H. pylori induced immunopathogenesis of the stomach, including hypochlorhydria and hypergastrinemia, might trigger large intestinal microbiota changes whereas the exact underlying mechanisms need to be further unraveled. PMID:24941045

  14. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis

    PubMed Central

    Groten, Karin; Pahari, Nabin T.; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T.

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  15. Virus-Induced Gene Silencing Using Tobacco Rattle Virus as a Tool to Study the Interaction between Nicotiana attenuata and Rhizophagus irregularis.

    PubMed

    Groten, Karin; Pahari, Nabin T; Xu, Shuqing; Miloradovic van Doorn, Maja; Baldwin, Ian T

    2015-01-01

    Most land plants live in a symbiotic association with arbuscular mycorrhizal fungi (AMF) that belong to the phylum Glomeromycota. Although a number of plant genes involved in the plant-AMF interactions have been identified by analyzing mutants, the ability to rapidly manipulate gene expression to study the potential functions of new candidate genes remains unrealized. We analyzed changes in gene expression of wild tobacco roots (Nicotiana attenuata) after infection with mycorrhizal fungi (Rhizophagus irregularis) by serial analysis of gene expression (SuperSAGE) combined with next generation sequencing, and established a virus-induced gene-silencing protocol to study the function of candidate genes in the interaction. From 92,434 SuperSAGE Tag sequences, 32,808 (35%) matched with our in-house Nicotiana attenuata transcriptome database and 3,698 (4%) matched to Rhizophagus genes. In total, 11,194 Tags showed a significant change in expression (p<0.05, >2-fold change) after infection. When comparing the functions of highly up-regulated annotated Tags in this study with those of two previous large-scale gene expression studies, 18 gene functions were found to be up-regulated in all three studies mainly playing roles related to phytohormone metabolism, catabolism and defense. To validate the function of identified candidate genes, we used the technique of virus-induced gene silencing (VIGS) to silence the expression of three putative N. attenuata genes: germin-like protein, indole-3-acetic acid-amido synthetase GH3.9 and, as a proof-of-principle, calcium and calmodulin-dependent protein kinase (CCaMK). The silencing of the three plant genes in roots was successful, but only CCaMK silencing had a significant effect on the interaction with R. irregularis. Interestingly, when a highly activated inoculum was used for plant inoculation, the effect of CCaMK silencing on fungal colonization was masked, probably due to trans-complementation. This study demonstrates that large

  16. The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection

    PubMed Central

    2013-01-01

    Background Chlamydia trachomatis infection results in reproductive damage in some women. The process and factors involved in this immunopathology are not well understood. This study aimed to investigate the role of primary human cellular responses to chlamydial stress response proteases and chlamydial infection to further identify the immune processes involved in serious disease sequelae. Results Laboratory cell cultures and primary human reproductive epithelial cultures produced IL-6 in response to chlamydial stress response proteases (CtHtrA and CtTsp), UV inactivated Chlamydia, and live Chlamydia. The magnitude of the IL-6 response varied considerably (up to 1000 pg ml-1) across different primary human reproductive cultures. Thus different levels of IL-6 production by reproductive epithelia may be a determinant in disease outcome. Interestingly, co-culture models with either THP-1 cells or autologous primary human PBMC generally resulted in increased levels of IL-6, except in the case of live Chlamydia where the level of IL-6 was decreased compared to the epithelial cell culture only, suggesting this pathway may be able to be modulated by live Chlamydia. PBMC responses to the stress response proteases (CtTsp and CtHtrA) did not significantly vary for the different participant cohorts. Therefore, these proteases may possess conserved innate PAMPs. MAP kinases appeared to be involved in this IL-6 induction from human cells. Finally, we also demonstrated that IL-6 was induced by these proteins and Chlamydia from mouse primary reproductive cell cultures (BALB/C mice) and mouse laboratory cell models. Conclusions We have demonstrated that IL-6 may be a key factor for the chlamydial disease outcome in humans, given that primary human reproductive epithelial cell culture showed considerable variation in IL-6 response to Chlamydia or chlamydial proteins, and that the presence of live Chlamydia (but not UV killed) during co-culture resulted in a reduced IL-6 response

  17. The Role of T Cell Immunoglobulin Mucin Domains 1 and 4 in a Herpes Simplex Virus-Induced Behçet's Disease Mouse Model

    PubMed Central

    Shim, Ju A.

    2013-01-01

    The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. TIM-1 plays an important role in the regulation of immune responses and the development of autoimmune diseases. TIM-4 is a natural ligand of TIM-1, and the interaction of TIM-1 and TIM-4 is involved in the regulation of T helper (Th) cell responses and modulation of the Th1/Th2 cytokine balance. Behçet's disease (BD) is a chronic, multisystemic inflammatory disorder with arthritic, intestinal, mucocutaneous, ocular, vascular, and central nervous system involvement. Tim-1 expression was lower in a herpes simplex virus-induced BD mouse model compared to that in asymptomatic BD normal (BDN) mice. Tim-4 expression was higher in BD mice than that in BDN mice. In this study, we investigated the Tim expression in a BD mouse model with BD-like symptoms. Tim-1 and Tim-4 expression was regulated by an expression vector or siRNA injected into the BD mouse model. The Tim-1 vector injected into BD mice resulted in changes in BD-like symptoms and decreased the severity score. Treatment with Tim-4 siRNA also improved BD-like symptoms and decreased the severity score accompanied by upregulation of regulatory T cells. We showed that regulating Tim-1 or Tim-4 affected BD-like symptoms in mice. PMID:24453431

  18. Enhancement by tumor necrosis factor alpha of dengue virus-induced endothelial cell production of reactive nitrogen and oxygen species is key to hemorrhage development.

    PubMed

    Yen, Yu-Ting; Chen, Hsuen-Chin; Chen, Hseun-Chin; Lin, Yang-Ding; Shieh, Chi-Chang; Wu-Hsieh, Betty A

    2008-12-01

    Hemorrhage is a severe manifestation of dengue disease. Virus strain and host immune response have been implicated as the risk factors for hemorrhage development. To delineate the complex interplay between the virus and the host, we established a dengue hemorrhage model in immune-competent mice. Mice inoculated intradermally with dengue virus develop hemorrhage within 3 days. In the present study, we showed by the presence of NS1 antigen and viral nuclei acid that dengue virus actively infects the endothelium at 12 h and 24 h after inoculation. Temporal studies showed that beginning at day 2, there was macrophage infiltration into the vicinity of the endothelium, increased tumor necrosis factor alpha (TNF-alpha) production, and endothelial cell apoptosis in the tissues. In the meantime, endothelial cells in the hemorrhage tissues expressed inducible nitric oxide synthase (iNOS) and nitrotyrosine. In vitro studies showed that primary mouse and human endothelial cells were productively infected by dengue virus. Infection by dengue virus induced endothelial cell production of reactive nitrogen and oxygen species and apoptotic cell death, which was greatly enhanced by TNF-alpha. N(G)-nitro-L-arginine methyl ester and N-acetyl cysteine reversed the effects of dengue virus and TNF-alpha on endothelial cells. Importantly, hemorrhage development and the severity of hemorrhage were greatly reduced in mice lacking iNOS or p47(phox) or treatment with oxidase inhibitor, pointing to the critical roles of reactive nitrogen and oxygen species in dengue hemorrhage. PMID:18842737

  19. Targeted activation of human Vγ9Vδ2-T cells controls epstein-barr virus-induced B cell lymphoproliferative disease.

    PubMed

    Xiang, Zheng; Liu, Yinping; Zheng, Jian; Liu, Ming; Lv, Aizhen; Gao, Yulong; Hu, Huaidong; Lam, Kowk-Tai; Chan, Godfrey Chi-Fung; Yang, Yuanzhong; Chen, Honglin; Tsao, George Sai-Wah; Bonneville, Marc; Lau, Yu-Lung; Tu, Wenwei

    2014-10-13

    Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a serious and life-threatening complication. Herein we showed that the aminobisphosphonate pamidronate-expanded human Vγ9Vδ2-T cells efficiently killed EBV-transformed autologous lymphoblastoid B cell lines (EBV-LCL) through γ/δ-TCR and NKG2D receptor triggering and Fas and TRAIL engagement. By inoculation of EBV-LCL in Rag2(-/-)γc(-/-) mice and humanized mice, we established lethal EBV-LPD with characteristics close to those of the human disease. Adoptive transfer of pamidronate-expanded Vγ9Vδ2-T cells alone effectively prevented EBV-LPD in Rag2(-/-)γc(-/-) mice and induced EBV-LPD regression in EBV(+) tumor-bearing Rag2(-/-)γc(-/-) mice. Pamidronate treatment inhibited EBV-LPD development in humanized mice through selective activation and expansion of Vγ9Vδ2-T cells. This study provides proof-of-principle for a therapeutic approach using pamidronate to control EBV-LPD through Vγ9Vδ2-T cell targeting. PMID:25220446

  20. Tumor necrosis factor alpha is reparative via TNFR2 [corrected] in the hippocampus and via TNFR1 [corrected] in the striatum after virus-induced encephalitis.

    PubMed

    Rodriguez, Moses; Zoecklein, Laurie; Papke, Louisa; Gamez, Jeff; Denic, Aleksandar; Macura, Slobodan; Howe, Charles

    2009-01-01

    Differentiating between injurious and reparative factors facilitates appropriate therapeutic intervention. We evaluated the role of tumor necrosis factor alpha (TNFalpha) in parenchymal brain pathology resolution following virus-induced encephalitis from a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). We infected the following animals with TMEV for 7 to 270 days: B6/129 TNF(-/-) mice (without TNFalpha expression), B6/129 TNFR1(-/-) mice (without TNFalpha receptor 1 expression), and B6/129 TNFR2(-/-) mice (without TNFalpha receptor 2 expression). Normal TNFalpha-expressing controls were TMEV-infected B6, 129/J, B6/129F1 and B6/129F2 mice. Whereas all strains developed inflammation and neuronal injury in the hippocampus and striatum 7 to 21 days postinfection (dpi), the control mice resolved the pathology by 45 to 90 dpi. However, parenchymal hippocampal and striatal injury persisted in B6/129 TNF(-/-) mice following infection. Treating virus-infected mice with active recombinant mouse TNFalpha resulted in less hippocampal and striatal pathology, whereas TNFalpha-neutralizing treatment worsened pathology. T1 "black holes" appeared on MRI during early infection in the hippocampus and striatum in all mice but persisted only in TNF(-/-) mice. TNFR2 [corrected] mediated hippocampal pathology resolution whereas TNFR1 [corrected] mediated striatal healing. These findings indicate the role of TNFalpha in resolution of sublethal hippocampal and striatal injury. PMID:18422761

  1. Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.

    PubMed

    Salim, Vonny; Yu, Fang; Altarejos, Joaquín; De Luca, Vincenzo

    2013-12-01

    Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus. PMID:24103035

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

    PubMed Central

    Nemchinov, Lev G.; Boutanaev, Alexander M.; Postnikova, Olga A.

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  3. Dissecting Functions of KATANIN and WRINKLED1 in Cotton Fiber Development by Virus-Induced Gene Silencing1[C][W][OA

    PubMed Central

    Qu, Jing; Ye, Jian; Geng, Yun-Feng; Sun, Yan-Wei; Gao, Shi-Qiang; Zhang, Bi-Pei; Chen, Wen; Chua, Nam-Hai

    2012-01-01

    Most of the world’s natural fiber comes from cotton (Gossypium spp.), which is an important crop worldwide. Characterizing genes that regulate cotton yield and fiber quality is expected to benefit the sustainable production of natural fiber. Although a huge number of expressed sequence tag sequences are now available in the public database, large-scale gene function analysis has been hampered by the low-efficiency process of generating transgenic cotton plants. Tobacco rattle virus (TRV) has recently been reported to trigger virus-induced gene silencing (VIGS) in cotton leaves. Here, we extended the utility of this method by showing that TRV-VIGS can operate in reproductive organs as well. We used this method to investigate the function of KATANIN and WRINKLED1 in cotton plant development. Cotton plants with suppressed KATANIN expression produced shorter fibers and elevated weight ratio of seed oil to endosperm. By contrast, silencing of WRINKLED1 expression resulted in increased fiber length but reduced oil seed content, suggesting the possibility to increase fiber length by repartitioning carbon flow. Our results provide evidence that the TRV-VIGS system can be used for rapid functional analysis of genes involved in cotton fiber development. PMID:22837356

  4. Remodeling the Endoplasmic Reticulum by Poliovirus Infection and by Individual Viral Proteins: an Autophagy-Like Origin for Virus-Induced Vesicles

    PubMed Central

    Suhy, David A.; Giddings, Thomas H.; Kirkegaard, Karla

    2000-01-01

    All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes. PMID:10982339

  5. Activation of the c-H-ras proto-oncogene by retrovirus insertion and chromosomal rearrangement in a Moloney leukemia virus-induced T-cell leukemia.

    PubMed Central

    Ihle, J N; Smith-White, B; Sisson, B; Parker, D; Blair, D G; Schultz, A; Kozak, C; Lunsford, R D; Askew, D; Weinstein, Y

    1989-01-01

    A rearrangement of the c-H-ras locus was detected in a T-cell line (DA-2) established from a Moloney leukemia virus-induced tumor. This rearrangement was associated with the high-level expression of H-ras RNA and the H-ras gene product, p21. DNA from DA-2 cells transformed fibroblasts in DNA transfection experiments, and the transformed fibroblasts contained the rearranged H-ras locus. The rearrangement involved one allele and was present in tissue from the primary tumor from which the cell line was isolated. Cloning and sequencing of the rearranged allele and comparison with the normal allele demonstrated that the rearrangement was complex and probably resulted from the integration of a retrovirus in the H-ras locus between a 5' noncoding exon and the first coding exon and a subsequent homologous recombination between this provirus and another newly acquired provirus also located on chromosome 7. These events resulted in the translocation of the coding exons of the H-ras locus away from the 5' noncoding exon region to a new genomic site on chromosome 7. Sequencing of the coding regions of the gene failed to detect mutations in the 12th, 13th, 59th, or 61st codons. The possible reasons for the complexity of the rearrangement and the significance of the activation of the H-ras locus to T-cell transformation are discussed. Images PMID:2542606

  6. Development of tobacco ringspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants.

    PubMed

    Zhao, Fumei; Lim, Seungmo; Igori, Davaajargal; Yoo, Ran Hee; Kwon, Suk-Yoon; Moon, Jae Sun

    2016-05-01

    We report here the development of tobacco ringspot virus (TRSV)-based vectors for the transient expression of foreign genes and for the analysis of endogenous gene function in plants using virus-induced gene silencing. The jellyfish green fluorescent protein (GFP) gene was inserted between the TRSV movement protein (MP) and coat protein (CP) regions, resulting in high in-frame expression of the RNA2-encoded viral polyprotein. GFP was released from the polyprotein via an N-terminal homologous MP-CP cleavage site and a C-terminal foot-and-mouth disease virus (FMDV) 2 A catalytic peptide in Nicotiana benthamiana. The VIGS target gene was introduced in the sense and antisense orientations into a SnaBI site, which was created by mutating the sequence following the CP stop codon. VIGS of phytoene desaturase (PDS) in N. benthamiana, Arabidopsis ecotype Col-0, cucurbits and legumes led to obvious photo-bleaching phenotypes. A significant reduction in PDS mRNA levels in silenced plants was confirmed by semi-quantitative RT-PCR. PMID:26950504

  7. pol-miR-731, a teleost miRNA upregulated by megalocytivirus, negatively regulates virus-induced type I interferon response, apoptosis, and cell cycle arrest.

    PubMed

    Zhang, Bao-Cun; Zhou, Ze-Jun; Sun, Li

    2016-01-01

    Megalocytivirus is a DNA virus that is highly infectious in a wide variety of marine and freshwater fish, including Japanese flounder (Paralichthys olivaceus), a flatfish that is farmed worldwide. However, the infection mechanism of megalocytivirus remains largely unknown. In this study, we investigated the function of a flounder microRNA, pol-miR-731, in virus-host interaction. We found that pol-miR-731 was induced in expression by megalocytivirus and promoted viral replication at the early infection stage. In vivo and in vitro studies revealed that pol-miR-731 (i) specifically suppresses the expression of interferon regulatory factor 7 (IRF7) and cellular tumor antigen p53 in a manner that depended on the integrity of the pol-miR-731 complementary sequences in the 3' untranslated regions of IRF7 and p53, (ii) disrupts megalocytivirus-induced Type I interferon response through IRF7, (iii) inhibits megalocytivirus-induced splenocyte apoptosis and cell cycle arrest through p53. Furthermore, overexpression of IRF7 and p53 abolished both the inhibitory effects of pol-miR-731 on these biological processes and its stimulatory effect on viral replication. These results disclosed a novel evasion mechanism of megalocytivirus mediated by a host miRNA. This study also provides the first evidence that a virus-induced host miRNA can facilitate viral infection by simultaneously suppressing several antiviral pathways. PMID:27311682

  8. Association between susceptibility to Theiler's virus-induced demyelination and T-cell receptor Jbeta1-Cbeta1 polymorphism rather than Vbeta deletion.

    PubMed Central

    Bahk, Y Y; Kappel, C A; Rasmussen, G; Kim, B S

    1997-01-01

    Theiler's murine encephalomyelitis virus (TMEV) induces demyelinating disease in susceptible mouse strains after intracerebral inoculation. The clinical symptoms and histopathology of the central nervous system appear to be similar to those of human multiple sclerosis (MS), and thus, this system provides an excellent infectious animal model for studying MS. The virus-induced demyelination is immune mediated, and the genes involved in the immune response such as those for the T-cell receptor beta-chain and major histocompatibility complex (MHC) haplotypes are known to influence disease susceptibility. To define whether the T-cell receptor Jbeta-Cbeta or Vbeta genes are associated with susceptibility, we have analyzed F2 mice from crosses of susceptible SJL/J (Vbeta(a)-JCbeta(b)) mice and resistant C57L (Vbeta(a)-JCbeta(a)) mice. Our results indicate that susceptibility to TMEV-induced demyelination is associated with restriction fragment length polymorphism reflecting the T-cell receptor Jbeta1-Cbeta1 region rather than the Vbeta polymorphism. This association becomes stronger when the MHC haplotype is considered in the linkage analysis. However, differences in the T-cell receptor alpha-chain haplotype have no significant influence on the pathogenesis of TMEV-induced demyelination. PMID:9094705

  9. A new mechanism of vitamin C effects on A/FM/1/47(H1N1) virus-induced pneumonia in restraint-stressed mice.

    PubMed

    Cai, Ying; Li, Yi-Fang; Tang, Lu-Ping; Tsoi, Bun; Chen, Min; Chen, Huan; Chen, Xiao-Mei; Tan, Rui-Rong; Kurihara, Hiroshi; He, Rong-Rong

    2015-01-01

    It is well known that vitamin C could protect against influenza infection, but little is known about the mechanisms. This study aimed to investigate the influence and possible mechanisms of vitamin C on pneumonia induced by influenza virus in stressed mice. Results showed that restraint stress significantly increased the mortality and the severity of pneumonia in mice caused by A/FM/1/47(H1N1) virus infection, which was attenuated by oral administration of vitamin C (125 and 250 mg/kg). Moreover, vitamin C administration significantly decreased expression of susceptibility genes, including mitochondrial antiviral signaling (MAVS) and interferon regulatory factor 3 (IRF3), and increased expression of NF-κB. These work in conjunction to induce type I interferons (IFNs) and elicit innate antiviral response as key factors in RIG-I-mediated signal transduction pathway. The above effects of vitamin C were further found to relate with inhibition of excess CORT synthesis by regulating steroid hydroxylating enzymes in adrenal gland. In conclusion, the protective effects of vitamin C on influenza virus-caused pneumonia might be related to its inhibition of CORT synthesis, which reduces the susceptibility to influenza viral infection in restraint-stressed mice. These findings provide a new mechanism for the effects of vitamin C on influenza virus-induced pneumonia in restraint-stressed mice. PMID:25710018

  10. Ubiquitin-specific Protease 15 Negatively Regulates Virus-induced Type I Interferon Signaling via Catalytically-dependent and -independent Mechanisms

    PubMed Central

    Zhang, Huan; Wang, Dang; Zhong, Huijuan; Luo, Rui; Shang, Min; Liu, Dezhi; Chen, Huanchun; Fang, Liurong; Xiao, Shaobo

    2015-01-01

    Viral infection triggers a series of signaling cascades, which converge to activate the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3), thereby inducing the transcription of type I interferons (IFNs). Although not fully characterized, these innate antiviral responses are fine-tuned by dynamic ubiquitination and deubiquitination processes. In this study, we report ubiquitin-specific protease (USP) 15 is involved in regulation of the retinoic acid-inducible gene I (RIG-I)-dependent type I IFN induction pathway. Knockdown of endogenous USP15 augmented cellular antiviral responses. Overexpression of USP15 inhibited the transcription of IFN-β. Further analyses identified histidine 862 as a critical residue for USP15’s catalytic activity. Interestingly, USP15 specifically removed lysine 63-linked polyubiquitin chains from RIG-I among the essential components in RIG-I-like receptor-dependent pathway. In addition, we demonstrated that in contrast to USP15 de-ubiquitinating (DUB) activity, USP15-mediated inhibition of IFN signaling was not abolished by mutations eliminating the catalytic activity, indicating that a fraction of USP15-mediated IFN antagonism was independent of the DUB activity. Catalytically inactive USP15 mutants, as did the wild-type protein, disrupted virus-induced interaction of RIG-I and IFN-β promoter stimulator 1. Taken together, our data demonstrate that USP15 acts as a negative regulator of RIG-I signaling via DUB-dependent and independent mechanisms. PMID:26061460

  11. pol-miR-731, a teleost miRNA upregulated by megalocytivirus, negatively regulates virus-induced type I interferon response, apoptosis, and cell cycle arrest

    PubMed Central

    Zhang, Bao-cun; Zhou, Ze-jun; Sun, Li

    2016-01-01

    Megalocytivirus is a DNA virus that is highly infectious in a wide variety of marine and freshwater fish, including Japanese flounder (Paralichthys olivaceus), a flatfish that is farmed worldwide. However, the infection mechanism of megalocytivirus remains largely unknown. In this study, we investigated the function of a flounder microRNA, pol-miR-731, in virus-host interaction. We found that pol-miR-731 was induced in expression by megalocytivirus and promoted viral replication at the early infection stage. In vivo and in vitro studies revealed that pol-miR-731 (i) specifically suppresses the expression of interferon regulatory factor 7 (IRF7) and cellular tumor antigen p53 in a manner that depended on the integrity of the pol-miR-731 complementary sequences in the 3′ untranslated regions of IRF7 and p53, (ii) disrupts megalocytivirus-induced Type I interferon response through IRF7, (iii) inhibits megalocytivirus-induced splenocyte apoptosis and cell cycle arrest through p53. Furthermore, overexpression of IRF7 and p53 abolished both the inhibitory effects of pol-miR-731 on these biological processes and its stimulatory effect on viral replication. These results disclosed a novel evasion mechanism of megalocytivirus mediated by a host miRNA. This study also provides the first evidence that a virus-induced host miRNA can facilitate viral infection by simultaneously suppressing several antiviral pathways. PMID:27311682

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

    PubMed

    Nemchinov, Lev G; Boutanaev, Alexander M; Postnikova, Olga A

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  13. Inflammatory and oncogenic roles of a tumor stem cell marker doublecortin-like kinase (DCLK1) in virus-induced chronic liver diseases.

    PubMed

    Ali, Naushad; Chandrakesan, Parthasarathy; Nguyen, Charles B; Husain, Sanam; Gillaspy, Allison F; Huycke, Mark; Berry, William L; May, Randal; Qu, Dongfeng; Weygant, Nathaniel; Sureban, Sripathi M; Bronze, Michael S; Dhanasekaran, Danny N; Houchen, Courtney W

    2015-08-21

    Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. We previously showed that a tumor/cancer stem cell (CSC) marker, doublecortin-like kinase (DCLK1) positively regulates hepatitis C virus (HCV) replication, and promotes tumor growth in colon and pancreas. Here, we employed transcriptome analysis, RNA interference, tumor xenografts, patient's liver tissues and hepatospheroids to investigate DCLK1-regulated inflammation and tumorigenesis in the liver. Our studies unveiled novel DCLK1-controlled feed-forward signaling cascades involving calprotectin subunit S100A9 and NFκB activation as a driver of inflammation. Validation of transcriptome data suggests that DCLK1 co-expression with HCV induces BRM/SMARCA2 of SW1/SNF1 chromatin remodeling complexes. Frequently observed lymphoid aggregates including hepatic epithelial and stromal cells of internodular septa extensively express DCLK1 and S100A9. The DCLK1 overexpression also correlates with increased levels of S100A9, c-Myc, and BRM levels in HCV/HBV-positive patients with cirrhosis and HCC. DCLK1 silencing inhibits S100A9 expression and hepatoma cell migration. Normal human hepatocytes (NHH)-derived spheroids exhibit CSC properties. These results provide new insights into the molecular mechanism of the hepatitis B/C-virus induced liver inflammation and tumorigenesis via DCLK1-controlled networks. Thus, DCLK1 appears to be a novel therapeutic target for the treatment of inflammatory diseases and HCC. PMID:25948779

  14. Virus-induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ-aminobutyric acid metabolic pathway.

    PubMed

    Bao, Hexigeduleng; Chen, Xianyang; Lv, Sulian; Jiang, Ping; Feng, Juanjuan; Fan, Pengxiang; Nie, Lingling; Li, Yinxin

    2015-03-01

    γ-Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABA transaminases (SlGABA-Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus-induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA-Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200 mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs- and SlGABA-Ts-silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH-silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ-hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ-hydroxybutyrate and subsequent ROS accumulation under salt stress. PMID:25074245

  15. Aquaporin-mediated long-distance polyphosphate translocation directed towards the host in arbuscular mycorrhizal symbiosis: application of virus-induced gene silencing.

    PubMed

    Kikuchi, Yusuke; Hijikata, Nowaki; Ohtomo, Ryo; Handa, Yoshihiro; Kawaguchi, Masayoshi; Saito, Katsuharu; Masuta, Chikara; Ezawa, Tatsuhiro

    2016-09-01

    Arbuscular mycorrhizal fungi translocate polyphosphate through hyphae over a long distance to deliver to the host. More than three decades ago, suppression of host transpiration was found to decelerate phosphate delivery of the fungal symbiont, leading us to hypothesize that transpiration provides a primary driving force for polyphosphate translocation, probably via creating hyphal water flow in which fungal aquaporin(s) may be involved. The impact of transpiration suppression on polyphosphate translocation through hyphae of Rhizophagus clarus was evaluated. An aquaporin gene expressed in intraradical mycelia was characterized and knocked down by virus-induced gene silencing to investigate the involvement of the gene in polyphosphate translocation. Rhizophagus clarus aquaporin 3 (RcAQP3) that was most highly expressed in intraradical mycelia encodes an aquaglyceroporin responsible for water transport across the plasma membrane. Knockdown of RcAQP3 as well as the suppression of host transpiration decelerated polyphosphate translocation in proportion to the levels of knockdown and suppression, respectively. These results provide the first insight into the mechanism underlying long-distance polyphosphate translocation in mycorrhizal associations at the molecular level, in which host transpiration and the fungal aquaporin play key roles. A hypothetical model of the translocation is proposed for further elucidation of the mechanism. PMID:27136716

  16. Optimization of automated segmentation of monkeypox virus-induced lung lesions from normal lung CT images using hard C-means algorithm

    NASA Astrophysics Data System (ADS)

    Castro, Marcelo A.; Thomasson, David; Avila, Nilo A.; Hufton, Jennifer; Senseney, Justin; Johnson, Reed F.; Dyall, Julie

    2013-03-01

    Monkeypox virus is an emerging zoonotic pathogen that results in up to 10% mortality in humans. Knowledge of clinical manifestations and temporal progression of monkeypox disease is limited to data collected from rare outbreaks in remote regions of Central and West Africa. Clinical observations show that monkeypox infection resembles variola infection. Given the limited capability to study monkeypox disease in humans, characterization of the disease in animal models is required. A previous work focused on the identification of inflammatory patterns using PET/CT image modality in two non-human primates previously inoculated with the virus. In this work we extended techniques used in computer-aided detection of lung tumors to identify inflammatory lesions from monkeypox virus infection and their progression using CT images. Accurate estimation of partial volumes of lung lesions via segmentation is difficult because of poor discrimination between blood vessels, diseased regions, and outer structures. We used hard C-means algorithm in conjunction with landmark based registration to estimate the extent of monkeypox virus induced disease before inoculation and after disease progression. Automated estimation is in close agreement with manual segmentation.

  17. Tobacco mosaic virus 126-kDa protein increases the susceptibility of Nicotiana tabacum to other viruses and its dosage affects virus-induced gene silencing.

    PubMed

    Harries, Phillip A; Palanichelvam, Karuppaiah; Bhat, Sumana; Nelson, Richard S

    2008-12-01

    The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein-green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS. PMID:18986250

  18. Development of an Efficient Virus Induced Gene Silencing Strategy in the Non-Model Wild Ginger-Zingiber zerumbet and Investigation of Associated Proteome Changes

    PubMed Central

    Mahadevan, Chidambareswaren; Jaleel, Abdul; Deb, Lokesh; Thomas, George; Sakuntala, Manjula

    2015-01-01

    Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS. PMID:25918840

  19. Characterization of the Rana grylio virus 3{beta}-hydroxysteroid dehydrogenase and its novel role in suppressing virus-induced cytopathic effect

    SciTech Connect

    Sun Wei; Huang Youhua; Zhao Zhe; Gui Jianfang; Zhang Qiya . E-mail: zhangqy@ihb.ac.cn

    2006-12-08

    The 3{beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Here, a 3{beta}-HSD gene homolog was cloned from Rana grylio virus (RGV), a member of family Iridoviridae. RGV 3{beta}-HSD gene has 1068 bp, encoding a 355 aa predicted protein. Transcription analyses showed that RGV 3{beta}-HSD gene was transcribed immediate-early during infection from an initiation site 19 nucleotides upstream of the translation start site. Confocal microscopy revealed that the 3{beta}-HSD-EGFP fusion protein was exclusively colocalized with the mitochondria marker (pDsRed2-Mito) in EPC cells. Upon morphological observation and MTT assay, it was revealed that overexpression of RGV 3{beta}-HSD in EPC cells could apparently suppress RGV-induced cytopathic effect (CPE). The present studies indicate that the RGV immediate-early 3{beta}-HSD gene encodes a mitochondria-localized protein, which has a novel role in suppressing virus-induced CPE. All these suggest that RGV 3{beta}-HSD might be a protein involved in host-virus interaction.

  20. A New Mechanism of Vitamin C Effects on A/FM/1/47(H1N1) Virus-Induced Pneumonia in Restraint-Stressed Mice

    PubMed Central

    Cai, Ying; Li, Yi-Fang; Tang, Lu-Ping; Tsoi, Bun; Chen, Min; Chen, Huan; Chen, Xiao-Mei; Tan, Rui-Rong; Kurihara, Hiroshi; He, Rong-Rong

    2015-01-01

    It is well known that vitamin C could protect against influenza infection, but little is known about the mechanisms. This study aimed to investigate the influence and possible mechanisms of vitamin C on pneumonia induced by influenza virus in stressed mice. Results showed that restraint stress significantly increased the mortality and the severity of pneumonia in mice caused by A/FM/1/47(H1N1) virus infection, which was attenuated by oral administration of vitamin C (125 and 250 mg/kg). Moreover, vitamin C administration significantly decreased expression of susceptibility genes, including mitochondrial antiviral signaling (MAVS) and interferon regulatory factor 3 (IRF3), and increased expression of NF-κB. These work in conjunction to induce type I interferons (IFNs) and elicit innate antiviral response as key factors in RIG-I-mediated signal transduction pathway. The above effects of vitamin C were further found to relate with inhibition of excess CORT synthesis by regulating steroid hydroxylating enzymes in adrenal gland. In conclusion, the protective effects of vitamin C on influenza virus-caused pneumonia might be related to its inhibition of CORT synthesis, which reduces the susceptibility to influenza viral infection in restraint-stressed mice. These findings provide a new mechanism for the effects of vitamin C on influenza virus-induced pneumonia in restraint-stressed mice. PMID:25710018

  1. β-Catenin Upregulates the Constitutive and Virus-Induced Transcriptional Capacity of the Interferon Beta Promoter through T-Cell Factor Binding Sites

    PubMed Central

    Marcato, Vasco; Luron, Lionel; Laqueuvre, Lucie M.; Simon, Dominique; Mansuroglu, Zeyni; Flamand, Marie; Panthier, Jean-Jacques; Souès, Sylvie; Massaad, Charbel

    2015-01-01

    Rapid upregulation of interferon beta (IFN-β) expression following virus infection is essential to set up an efficient innate antiviral response. Biological roles related to the antiviral and immune response have also been associated with the constitutive production of IFN-β in naive cells. However, the mechanisms capable of modulating constitutive IFN-β expression in the absence of infection remain largely unknown. In this work, we demonstrate that inhibition of the kinase glycogen synthase kinase 3 (GSK-3) leads to the upregulation of the constitutive level of IFN-β expression in noninfected cells, provided that GSK-3 inhibition is correlated with the binding of β-catenin to the IFN-β promoter. Under these conditions, IFN-β expression occurred through the T-cell factor (TCF) binding sites present on the IFN-β promoter independently of interferon regulatory factor 3 (IRF3). Enhancement of the constitutive level of IFN-β per se was able to confer an efficient antiviral state to naive cells and acted in synergy with virus infection to stimulate virus-induced IFN-β expression. Further emphasizing the role of β-catenin in the innate antiviral response, we show here that highly pathogenic Rift Valley fever virus (RVFV) targets the Wnt/β-catenin pathway and the formation of active TCF/β-catenin complexes at the transcriptional and protein level in RVFV-infected cells and mice. PMID:26459757

  2. Immunopathologic aspects of woodchuck hepatitis.

    PubMed Central

    Frommel, D.; Crevat, D.; Vitvitsky, L.; Pichoud, C.; Hantz, O.; Chevalier, M.; Grimaud, J. A.; Lindberg, J.; Trépo, C. G.

    1984-01-01

    The natural history of infection with woodchuck hepatitis virus (WHV) has been studied in a colony of 38 Marmota monax. Besides serologic assessment for WHV markers, light-microscopic findings of 61 liver biopsies were correlated with the results of immunofluorescence analysis for nucleocapsid (WHcAg) and surface (WHsAg) antigens. Twenty-four chronic WHsAg carriers all featured signs of continuous viral replication. Two major immunomorphologic patterns were observed in their livers: 1) portal hepatitis in which WHcAg accumulated in the cytoplasm and WHsAg was associated with the hepatocyte membrane and 2) periportal hepatitis in which WHcAg shifted toward nuclear localization and WHsAg became mostly intracytoplasmic. Progression from portal to periportal hepatitis, observed in 7 woodchucks, appeared to be induced by a partial recovery of specific immune reactivity to WHV, insufficient, however, to interrupt WHV replication. Deposits of WHsAg and immunoglobulins were present in the kidney and spleen of animals with severe hepatitis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:6324594

  3. Immunopathology in Taenia solium neurocysticercosis.

    PubMed

    Fleury, A; Cardenas, G; Adalid-Peralta, L; Fragoso, G; Sciutto, E

    2016-03-01

    Neurocysticercosis is a clinically and radiologically heterogeneous disease, ranging from asymptomatic infection to a severe, potentially fatal clinical picture. The intensity and extension of the parasite-elicited inflammatory reaction is a key factor for such variability. The main features of the inflammatory process found in the brain and in the peripheral blood of neurocysticercosis patients will be discussed in this review, and the factors involved in its modulation will be herein presented. PMID:26667781

  4. Immunopathology of experimental cutaneous leishmaniasis.

    PubMed Central

    Andrade, Z. A.; Reed, S. G.; Roters, S. B.; Sadigursky, M.

    1984-01-01

    Relatively susceptible BALB/c and relatively resistant A/J mice were infected subcutaneously in the right hind footpad with promastigotes of Leishmania mexicana amazonensis. A large localized lesion developed within 2 months after infection in the BALB/c mice, while A/J mice exhibited a small discrete fibrotic nodule. Sequential immunologic and histologic examination demonstrated that BALB/c mice developed a nodular foam-cell type of lesion and progressive depression of a delayed-type hypersensitivity (DTH) response to leishmania antigen, while the A/J mice had a mixed cellular fibrosing and encapsulating reaction and developed and maintained positive DTH responses to leishmania antigen. Anti-leishmania antibody responses were positive at similar levels in both strains. The lesions in BALB/c mice were found in bone marrow, tendon, skin appendages, and regional lymph nodes, with a tendency toward cutaneous metastases. Lesions in A/J mice remained localized. Fibrosis, focal fibrinoid necrosis, and lymphocytic and macrophagic infiltration were the outstanding features. Light and transmission electron microscopic studies indicated that no outstanding destruction of leishmanias seemed to occur within macrophages of either mouse strain. In the more resistant A/J mice, however, parasitized macrophages were frequently necrotic, and degenerating leishmanias were often seen free in the interstitial tissue. These observations help the interpretation of the histologic features, as well as the pathogenesis, of cutaneous and mucocutaneous leishmaniasis in man. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:6691411

  5. Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum

    PubMed Central

    Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

    2014-01-01

    Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. PMID:25153631

  6. Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans

    PubMed Central

    Kim, Kil Hyun; Lim, Seungmo; Kang, Yang Jae; Yoon, Min Young; Nam, Moon; Jun, Tae Hwan; Seo, Min-Jung; Baek, Seong-Bum; Lee, Jeom-Ho; Moon, Jung-Kyung; Lee, Suk-Ha; Lee, Su-Heon; Lim, Hyoun-Sub; Moon, Jae Sun; Park, Chang-Hwan

    2016-01-01

    Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments. PMID:27147931

  7. Integrated Clinical, Pathologic, Virologic, and Transcriptomic Analysis of H5N1 Influenza Virus-Induced Viral Pneumonia in the Rhesus Macaque

    PubMed Central

    Shinya, Kyoko; Gao, Yuwei; Cilloniz, Cristian; Suzuki, Yasuhiro; Fujie, Masahiro; Deng, Guohua; Zhu, Qiyun; Fan, Shufang; Makino, Akiko; Muramoto, Yukiko; Fukuyama, Satoshi; Tamura, Daisuke; Noda, Takeshi; Eisfeld, Amie J.; Katze, Michael G.

    2012-01-01

    Viral pneumonia has been frequently reported during early stages of influenza virus pandemics and in many human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection. To better understand the pathogenesis of this disease, we produced nonlethal viral pneumonia in rhesus macaques by using an HPAI H5N1 virus (A/Anhui/2/2005; referred to as Anhui/2). Infected macaques were monitored for 14 days, and tissue samples were collected at 6 time points for virologic, histopathologic, and transcriptomic analyses. Anhui/2 efficiently replicated in the lung from 12 h to 3 days postinfection (p.i.) and caused temporal but severe pneumonia that began to resolve by day 14. Lung transcriptional changes were first observed at 6 h, and increased expression of vascular permeability regulators and neutrophil chemoattractants correlated with increased serum leakage and neutrophil infiltration in situ. Additional inflammatory, antiviral, and apoptotic genes were upregulated from 12 h, concurrent with viral antigen detection and increasing immune cell populations. A shift toward upregulation of acquired immunity was apparent after day 6. Expression levels of established immune cell molecular markers revealed remarkable similarity with pathological findings, indicating early and robust neutrophil infiltration, a slight delay in macrophage accumulation, and abundant late populations of T lymphocytes. We also characterized the putative mechanisms regulating a unique, pneumonia-associated biphasic fever pattern. Thus, this study is the first to use a comprehensive and integrative approach to delineate specific molecular mechanisms regulating influenza virus-induced pneumonia in nonhuman primates, an important first step toward better management of human influenza virus disease. PMID:22491448

  8. Characterization of a Previously Unidentified Viral Protein in Porcine Circovirus Type 2-Infected Cells and Its Role in Virus-Induced Apoptosis

    PubMed Central

    Liu, Jue; Chen, Isabelle; Kwang, Jimmy

    2005-01-01

    Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs. In this study, transcription and translation of a novel viral gene (termed ORF3 here) was detected during productive infection of PCV2 in PK15 cells. The results of infection with ORF3-deficient PCV2 by site-directed mutagenesis indicated that the protein is not essential for viral replication. To investigate the underlying mechanism of cell death caused by replication of PCV2, apoptosis characterized by chromosomal condensation and fragmentation, formation of apoptotic bodies, and significant increase in hypodiploids were detected in infected cells. We further demonstrated that PCV2-induced apoptosis required the activation of caspase-8 but not caspase-9. The activation of caspase-8 results in the activation of caspase-3 as shown by an increase in the cleavage of the caspase substrate in the infected cells. To determine whether ORF3 protein could trigger apoptosis, ORF3 as well as ORF1 and ORF2 genes were transiently expressed in PK15 and Cos-7 cells for apoptotic activity assay. Transfection of cells with the ORF3 alone induced apoptosis using a pathway similar to that described in the context of viral infection. This is further confirmed by a significant decrease in apoptotic activity of infected cells in the absence of the ORF3 expression, suggesting that the protein plays a major role in the induction of virus-induced apoptosis. Altogether, these results indicate that ORF3 is a novel PCV2 protein that is not essential for viral replication in cultured cells but is involved in PCV2-induced apoptosis by activating caspase-8 and caspase-3 pathways. PMID:15956572

  9. Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

    PubMed

    Singh, Anup Kumar; Dwivedi, Varun; Rai, Avanish; Pal, Shaifali; Reddy, Sajjalavarahalli Gangireddy Eswara; Rao, Dodaghatta Krishnarao Venkata; Shasany, Ajit Kumar; Nagegowda, Dinesh A

    2015-12-01

    Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides

  10. Antibody response is required for protection from Theiler's virus-induced encephalitis in C57BL/6 mice in the absence of CD8{sup +} T cells

    SciTech Connect

    Kang, B.-S.; Palma, Joann P.; Lyman, Michael A.; Dal Canto, Mauro; Kim, Byung S. . E-mail: bskim@northwestern.edu

    2005-09-15

    Intracerebral infection of susceptible mice with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease and this system serves as a relevant infectious model for human multiple sclerosis. It was previously shown that {beta}{sub 2}M-deficient C57BL/6 mice lacking functional CD8{sup +} T cells display increased viral persistence and enhanced susceptibility to TMEV-induced demyelination, and yet the majority of mice are free of clinical signs. To understand the mechanisms involved in this general resistance of C57BL/6 mice in the absence of CTL responses, mice ({mu}MT) deficient in the B-cell compartment lacking membrane IgM molecules were treated with anti-CD8 antibody and then infected with TMEV. Although little difference in the proliferative responses of peripheral T cells to UV-inactivated TMEV and the resistance to demyelinating disease was observed between virus-infected {mu}MT and control B6 mice, the levels of CD4{sup +} T cells were higher in the CNS of {mu}MT mice. However, after treatment with anti-CD8 antibody, 100% of the mice displayed clinical gray matter disease and prolonged viral persistence in {mu}MT mice, while only 10% of B6 mice showed clinical symptoms and very low viral persistence. Transfusion of sera from TMEV-infected B6 mice into anti-CD8 antibody-treated {mu}MT mice partially restored resistance to virus-induced encephalitis. These results indicate that the early anti-viral antibody response is also important in the protection from TMEV-induced encephalitis particularly in the absence of CD8{sup +} T cells.