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Sample records for revisionist replicon model

  1. Revisionists Respond to Ravitch.

    ERIC Educational Resources Information Center

    Feinberg, Walter; And Others

    Responses of four revisionist historians and economists to Diane Ravitch's review of their works are presented. Those defending the revisionist viewpoint are: Walter Feinberg, Harvey Kantor, Michael Katz, and Paul Violas. (JD)

  2. Remapping Revisionist Historiography

    ERIC Educational Resources Information Center

    Gold, David

    2012-01-01

    Rhetoric and composition historiography has recently undergone a rapid transformation as scholars have complicated and challenged earlier narratives by examining diverse local histories and alternative rhetorical traditions. This revisionist scholarship has in turn created new research challenges, as scholars must now demonstrate connections…

  3. Brecht's Galileo: A revisionist view

    NASA Astrophysics Data System (ADS)

    Schroeer, Dietrich

    1980-02-01

    Galileo is often claimed by scientists to be the first modern physicist, and because of his conflict with the Catholic Church is seen as a heroic figure fighting for the independence of pure science. Brecht, in his play Galileo, has presented a revisionist view of Galileo. This view developed over several versions of the play, and finally used him as a symbol for all scientists who reject social responsibility for their work. Is this revisionist view of Galileo any more distorted than the portrayal of him as the patron saint of modern science?

  4. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

    PubMed Central

    diCenzo, George C.; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M.; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  5. Teaching the Revisionist Interpretation of the Cold War.

    ERIC Educational Resources Information Center

    O'Reilly, Kevin

    1986-01-01

    Provides a lesson plan, complete with readings and teaching procedures, for teaching students about the revisionist interpretations of Cold War history. Students are challenged to think critically about the differences in the traditional and revisionist interpretations. (JDH)

  6. Replicon particles of Venezuelan equine encephalitis virus as a reductionist murine model for encephalitis.

    PubMed

    Schäfer, Alexandra; Whitmore, Alan C; Konopka, Jennifer L; Johnston, Robert E

    2009-05-01

    Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) were used to model the initial phase of VEE-induced encephalitis in the mouse brain. VRP can target and infect cells as VEE, but VRP do not propagate beyond the first infected cell due to the absence of the structural genes. Direct intracranial inoculation of VRP into mice induced acute encephalitis with signs similar to the neuronal phase of wild-type VEE infection and other models of virus-induced encephalitis. Using the previously established VRP-mRNP tagging system, a new method to distinguish the host responses in infected cells from those in uninfected bystander cell populations, we detected a robust and rapid innate immune response in the central nervous system (CNS) by infected neurons and uninfected bystander cells. Moreover, this innate immune response in the CNS compromised blood-brain barrier integrity, created an inflammatory response, and directed an adaptive immune response characterized by proliferation and activation of microglia cells and infiltration of inflammatory monocytes, in addition to CD4(+) and CD8(+) T lymphocytes. Taken together, these data suggest that a naïve CNS has an intrinsic potential to induce an innate immune response that could be crucial to the outcome of the infection by determining the composition and dynamics of the adaptive immune response. Furthermore, these results establish a model for neurotropic virus infection to identify host and viral factors that contribute to invasion of the brain, the mechanism(s) whereby the adaptive immune response can clear the infection, and the role of the host innate response in these processes.

  7. Replicon Particles of Venezuelan Equine Encephalitis Virus as a Reductionist Murine Model for Encephalitis▿

    PubMed Central

    Schäfer, Alexandra; Whitmore, Alan C.; Konopka, Jennifer L.; Johnston, Robert E.

    2009-01-01

    Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) were used to model the initial phase of VEE-induced encephalitis in the mouse brain. VRP can target and infect cells as VEE, but VRP do not propagate beyond the first infected cell due to the absence of the structural genes. Direct intracranial inoculation of VRP into mice induced acute encephalitis with signs similar to the neuronal phase of wild-type VEE infection and other models of virus-induced encephalitis. Using the previously established VRP-mRNP tagging system, a new method to distinguish the host responses in infected cells from those in uninfected bystander cell populations, we detected a robust and rapid innate immune response in the central nervous system (CNS) by infected neurons and uninfected bystander cells. Moreover, this innate immune response in the CNS compromised blood-brain barrier integrity, created an inflammatory response, and directed an adaptive immune response characterized by proliferation and activation of microglia cells and infiltration of inflammatory monocytes, in addition to CD4+ and CD8+ T lymphocytes. Taken together, these data suggest that a naïve CNS has an intrinsic potential to induce an innate immune response that could be crucial to the outcome of the infection by determining the composition and dynamics of the adaptive immune response. Furthermore, these results establish a model for neurotropic virus infection to identify host and viral factors that contribute to invasion of the brain, the mechanism(s) whereby the adaptive immune response can clear the infection, and the role of the host innate response in these processes. PMID:19225006

  8. Dream experience and a revisionist account of delusions of misidentification.

    PubMed

    Gerrans, Philip

    2012-03-01

    Standard accounts of delusion explain them as responses to experience. Cognitive models of feature binding in the face recognition systems explain how experiences of mismatch between feelings of "familiarity" and faces can arise. Similar mismatches arise in phenomena such as déjà and jamais vu in which places and scenes are mismatched to feelings of familiarity. These cognitive models also explain similarities between the phenomenology of these delusions and some dream states which involve mismatch between faces, feelings of familiarity and identities. Given these similarities it makes sense to retain that aspect of the standard account in the face of revisionist arguments that feature binding anomalies which lead to delusions of misidentification are not consciously experienced. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Interdisciplinary team leadership: a revisionist approach for an old problem?

    PubMed

    McCallin, Antoinette

    2003-11-01

    Understanding of interdisciplinary teamwork is evolving. During health care restructuring, leaders across organizations have challenging responsibilities when work groups must integrate changing organizational values with new modes of service delivery. In this environment, a well-functioning interdisciplinary team in which clinicians work as member-leaders has the potential to further organizational change and foster improvements in patient outcomes. In this paper it is argued that the term interdisciplinary team leadership should be embraced cautiously as it may be a revisionist approach to an old problem, namely a means to modify existing theories of leadership that have been vague and continue to be poorly understood despite considerable effort to explicate knowledge over several decades. Preliminary research suggests that interdisciplinary team leadership is a model of shared leadership that requires more development if it is to become the cornerstone of interdisciplinary team practice in a radically reforming health sector. Stewardship is proposed as a potential philosophy for interdisciplinary team leadership, and a new, shared leadership role of practice leader is suggested.

  10. Replicon RNA Viral Vectors as Vaccines

    PubMed Central

    Lundstrom, Kenneth

    2016-01-01

    Single-stranded RNA viruses of both positive and negative polarity have been used as vectors for vaccine development. In this context, alphaviruses, flaviviruses, measles virus and rhabdoviruses have been engineered for expression of surface protein genes and antigens. Administration of replicon RNA vectors has resulted in strong immune responses and generation of neutralizing antibodies in various animal models. Immunization of mice, chicken, pigs and primates with virus-like particles, naked RNA or layered DNA/RNA plasmids has provided protection against challenges with lethal doses of infectious agents and administered tumor cells. Both prophylactic and therapeutic efficacy has been achieved in cancer immunotherapy. Moreover, recombinant particles and replicon RNAs have been encapsulated by liposomes to improve delivery and targeting. Replicon RNA vectors have also been subjected to clinical trials. Overall, immunization with self-replicating RNA viruses provides high transient expression levels of antigens resulting in generation of neutralizing antibody responses and protection against lethal challenges under safe conditions. PMID:27827980

  11. Chromosomal replicons of higher plants

    SciTech Connect

    Van't Hof, J.

    1987-03-16

    This brief discussion of replicons of higher plants offers a glimpse into the properties of chromosomal DNA replication. It gives evidence that the S phase of unrelated plant species is comprised of temporally ordered replicon families that increase in number with genome size. This orderly process, which assures a normal inheritance of genetic material to recipient daughter cells, is maintained at the level of replicon clusters by two mutually exclusive mechanisms, one involving the rate at which single replicons replicate their allotment of DNA, and another by means of the tempo-pause. The same two mechanisms are used by cells to alter the pattern of chromosomal DNA replication just prior to and during normal development. Both mechanisms are genetically determined and produce genetic effects when disturbed of disrupted by additional non-conforming DNAs. Further insight into how these two mechanisms operate requires more molecular information about the nature of replicons and the factors that govern when a replicon family replicates. Plant material is a rich and ideal source for this information just awaiting exploitation. 63 refs.

  12. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons

    PubMed Central

    Chagin, V. O.; Casas-Delucchi, C. S.; Reinhart, M.; Schermelleh, L.; Markaki, Y.; Maiser, A.; Bolius, J. J.; Bensimon, A.; Fillies, M.; Domaing, P.; Rozanov, Y. M.; Leonhardt, H.; Cardoso, M. C.

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  13. Construction and applications of rabbit hemorrhagic disease virus replicon.

    PubMed

    Wang, Binbin; Zhe, Mingjia; Chen, Zongyan; Li, Chuanfeng; Meng, Chunchun; Zhang, Miaotao; Liu, Guangqing

    2013-01-01

    The study of rabbit hemorrhagic disease virus (RHDV) has long been hindered by the absence of an in vitro culture system. In this study, using RHDV as a model, a series of DNA-based reporter replicons were constructed in which the firefly luciferase (Fluc) gene was fused in-frame with the open reading frame of the replicon. In this construct, the Fluc gene was inserted where the coding region of viral structural protein was deleted and was under the control of a minimal cytomegalovirus (CMV) immediate-early promoter. Fluc activity analysis showed that these reporter replicons replicate efficiently in mammalian cells. On the basis of the replicon, 5'non-coding regions (5'NCR) and genome-linked protein (VPg) were deleted, and the effect on the expression of replicon was analyzed. The results showed that the expression level of Fluc was reduced in the absence of 5'NCR and VPg, suggesting that the 5'NCR and VPg may play an important role in replication and/or translation of RHDV. To further verify the speculation, we also constructed a replication deficient mutant (pRHDV-luc/Δ3D), and the impact of 5'NCR and VPg deletion on viral translation efficiency was analyzed, our results indicated that both VPg and 5'NCR were involved in RHDV translation.

  14. Replicon system for Lassa virus.

    PubMed

    Hass, Meike; Gölnitz, Uta; Müller, Stefanie; Becker-Ziaja, Beate; Günther, Stephan

    2004-12-01

    Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5' and 3' untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5' end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

  15. Combination of Alphavirus replicon particle-based vaccination with immunomodulatory antibodies: therapeutic activity in the B16 melanoma mouse model and immune correlates

    PubMed Central

    Avogadri, Francesca; Zappasodi, Roberta; Yang, Arvin; Budhu, Sadna; Malandro, Nicole; Hirshhorn-Cymerman, Daniel; Tiwari, Shakuntala; Maughan, Maureen F.; Olmsted, Robert; Wolchok, Jedd D; Merghoub, Taha

    2015-01-01

    Induction of potent immune responses to self-antigens remains a major challenge in tumor immunology. We have shown that a vaccine based on alphavirus replicon particles (VRP) activates strong cellular and humoral immunity to tyrosinase related protein-2 (TRP2) melanoma antigen, providing prophylactic and therapeutic effects in stringent mouse models. Here we report that the immunogenicity and efficacy of this vaccine is increased in combination with either antagonist anti-CTLA-4 or agonist anti-GITR immunomodulatory monoclonal antibodies (mAbs). In the challenging therapeutic setting, VRP-TRP2 plus anti-GITR or anti-CTLA-4 mAb induced complete tumor regression respectively in 90% and 50% of mice. These mAbs had similar adjuvant effects in priming an adaptive immune response against the vaccine-encoded antigen, augmenting respectively ~4- and 2-fold the TRP2-specific CD8+ T-cell response and circulating Abs, compared to the vaccine alone. Furthermore, while both mAbs increased the frequency of tumor-infiltrating CD8+ T cells, anti-CTLA-4 mAb also increased the quantity of intra-tumor CD4+Foxp3− T cells expressing the negative co-stimulatory molecule programmed death-1 (PD-1). Concurrent GITR expression on these cells suggests that they might be controlled by anti-GITR mAbs, thus potentially explaining their differential accumulation under the two treatment conditions. These findings indicate that combining immunomodulatory mAbs with alphavirus-based anti-cancer vaccines can provide therapeutic anti-tumor immune responses in a stringent mouse model, suggesting potential utility in clinical trials. They also indicate that tumor-infiltrating CD4+Foxp3−PD-1+ T cells may affect the outcome of immunomodulatory treatments. PMID:24795357

  16. Venezuelan equine encephalitis replicon immunization overcomes intrinsic tolerance and elicits effective anti-tumor immunity to the 'self' tumor-associated antigen, neu in a rat mammary tumor model.

    PubMed

    Nelson, Edward L; Prieto, Darue; Alexander, Terri G; Pushko, Peter; Lofts, Loreen A; Rayner, Jonathan O; Kamrud, Kurt I; Fralish, Bolyn; Smith, Jonathan F

    2003-12-01

    Many tumor-associated antigens (TAAs) represent 'self' antigens and as such, are subject to the constraints of immunologic tolerance. There are significant barriers to eliciting anti-tumor immune responses of sufficient magnitude. We have taken advantage of a Venezuelan equine encephalitis-derived alphavirus replicon vector system with documented in vivo tropism for immune system dendritic cells. We have overcome the intrinsic tolerance to the 'self' TAA rat neu and elicited an effective anti-tumor immune response using this alphavirus replicon vector system and a designed target antigen in a rigorous rat mammary tumor model. We have demonstrated the capacity to generate 50% protection in tumor challenge experiments (p = 0.004) and we have confirmed the establishment of immunologic memory by both second tumor challenge and Winn Assay (p = 0.009). Minor antibody responses were identified and supported the establishment of T helper type 1 (Th1) anti-tumor immune responses by isotype. Animals surviving in excess of 300 days with established effective anti-tumor immunity showed no signs of autoimmune phenomena. Together these experiments support the establishment of T lymphocyte dependent, Th1-biased anti-tumor immune responses to a non-mutated 'self' TAA in an aggressive tumor model. Importantly, this tumor model is subject to the constraints of immunologic tolerance present in animals with normal developmental, temporal, and anatomical expression of a non-mutated TAA. These data support the continued development and potential clinical application of this alphaviral replicon vector system and the use of appropriately designed target antigen sequences for anti-tumor immunotherapy.

  17. The Assessor Assessed: A "Revisionist" Looks at a Critique of the Sandia Report.

    ERIC Educational Resources Information Center

    Bracey, Gerald W.

    1995-01-01

    In critiquing the Sandia Report, Lawrence Stedman faulted it and other revisionist reports for emphasizing trend data rather than quality and not acknowledging educational requirements within a democratic society. The article addresses the issues, presenting evidence from additional studies to argue that none of Stedman's general contentions…

  18. Revisionist History in the Library: To Facilitate Access or Not to Facilitate Access?

    ERIC Educational Resources Information Center

    Katz, Jeffrey

    1991-01-01

    Discussion of the acquisition and dissemination of revisionist history materials by public libraries focuses on revisionism related to the Jewish Holocaust. Intellectual freedom is addressed, the role of the public library is considered, handling holocaust-denial literature is described, and an example from the Edmonton Public Library is…

  19. Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors.

    PubMed

    Yu, Yun-Zhou; Guo, Jin-Peng; An, Huai-Jie; Zhang, Shu-Ming; Wang, Shuang; Yu, Wei-Yuan; Sun, Zhi-Wei

    2013-05-07

    Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.

  20. Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based Outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease.

    PubMed

    Gipson, Clay L; Davis, Nancy L; Johnston, Robert E; de Silva, Aravinda M

    2003-09-08

    Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less immunogenic. The VRP method of delivery induced a Th1 type immune response unlike the recombinant OspA protein in Freund's adjuvant, which induced a mixed (Th1 and Th2) immune response. The VRP tPA-OspA construct induced an immune response that reduced the bacterial load in feeding Ixodes scapularis and blocked transmission to the host. These results indicate that VRPs are capable of providing protection against tick-borne B. burgdorferi, and potentially can be used for developing improved vaccines against Lyme disease.

  1. Enhanced potency of replicon vaccine using one vector to simultaneously co-express antigen and interleukin-4 molecular adjuvant

    PubMed Central

    Ma, Yao; An, Huai-Jie; Wei, Xiao-Qi; Xu, Qing; Yu, Yun-Zhou; Sun, Zhi-Wei

    2013-01-01

    We evaluated the utility of interleukin-4 (IL-4) as molecular adjuvant of replicon vaccines for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In both Balb/c and C57/BL6 mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) encoding the Hc gene of BoNT/A (AHc), the immunogenicity was significantly modulated and enhanced by co-delivery or co-express of the IL-4 molecular adjuvant. The enhanced potencies were also produced by co-delivery or co-expression of the IL-4 molecular adjuvant in mice immunized with the recombinant SFV replicon particles (VRP) vaccines. In particular, when AHc and IL-4 were co-expressed within the same replicon vaccine vector using dual-expression or bicistronic IRES, the anti-AHc antibody titers, serum neutralization titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased. These results indicate IL-4 is an effective Th2-type adjuvant for the replicon vaccines in both strain mice, and the co-expression replicon vaccines described here may be an excellent candidate for further vaccine development in other animals or humans. Thus, we described a strategy to design and develop efficient vaccines against BoNT/A or other pathogens using one replicon vector to simultaneously co-express antigen and molecular adjuvant. PMID:23291932

  2. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice

    PubMed Central

    Boylan, Brendan T.; Moreira, Fernando R.; Carlson, Tim W.

    2017-01-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses. PMID:28187142

  3. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

    PubMed

    Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

    2017-02-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

  4. Genome-wide profiling of S/MAR-based replicon contact sites

    PubMed Central

    Gogol-Döring, Andreas; Schreiber, Sabrina; Epplen, Jörg T.; Lipps, Hans J.

    2017-01-01

    Abstract Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites. PMID:28609784

  5. Genome-wide profiling of S/MAR-based replicon contact sites.

    PubMed

    Hagedorn, Claudia; Gogol-Döring, Andreas; Schreiber, Sabrina; Epplen, Jörg T; Lipps, Hans J

    2017-07-27

    Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Stochastic association of neighboring replicons creates replication factories in budding yeast

    PubMed Central

    Saner, Nazan; Karschau, Jens; Natsume, Toyoaki; Gierliński, Marek; Retkute, Renata; Hawkins, Michelle; Nieduszynski, Conrad A.; Blow, J. Julian; de Moura, Alessandro P.S.

    2013-01-01

    Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures. PMID:24062338

  7. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  8. Revisionist or simply wrong? A response to Armstrong's article on chronic illness

    PubMed Central

    Gilleard, Chris; Higgs, Paul

    2014-01-01

    This article is a response to David Armstrong's recent, revisionist account of the epidemiological transition which he claims replaced earlier discourses of ageing with new discourses of chronic disease. We argue (i) that he misrepresents a key element in Omran's account of the epidemiological transition, namely the decline in infant, child and maternal mortality; (ii) that he fails to acknowledge debates going back centuries in Western medicine over the distinctions between natural and accidental death and between endogenous and extrinsic causes of ageing and (iii) that he misrepresents the growth of medical interest in the everyday illnesses of old age over the course of the 20th century as a discourse of suppression rather than a process of inclusion. While we would acknowledge that the chronic illnesses of today are different from those of the past, this amounts to something more than the changing semantics of senility. PMID:25155775

  9. Venezuelan Equine Encephalitis Replicon Immunization Overcomes Intrinsic Tolerance and Elicits Effective Anti-Tumor Immunity to the ’Self’ Tumor-Associated Antigen, neu in a Rat Mammary Tumor Model

    DTIC Science & Technology

    2003-01-01

    Williamson C, Suarez DL, Johnston R, Perdue ML: Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens...Immunol Immunother 50: 615–624, 2002 75. Rongcun Y, Salazar -Onfray F, Charo J, Malmberg KJ, Evrin K, Maes H, Kono K, Hising C, Petersson M, Larsson O

  10. An Alphavirus Replicon Particle Chimera Derived from Venezuelan Equine Encephalitis and Sindbis Viruses Is a Potent Gene-Based Vaccine Delivery Vector

    PubMed Central

    Perri, Silvia; Greer, Catherine E.; Thudium, Kent; Doe, Barbara; Legg, Harold; Liu, Hong; Romero, Raul E.; Tang, Zequn; Bin, Qian; Dubensky, Thomas W.; Vajdy, Michael; Otten, Gillis R.; Polo, John M.

    2003-01-01

    Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55Gag antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55gag replicon RNA packaged within SIN envelope glycoproteins and SIN-p55gag replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8+ T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety. PMID:12970424

  11. Synchronization of replicons in Ehrlich ascites cells

    SciTech Connect

    Gekeler, V.; Probst, H. )

    1988-03-01

    Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G{sub 1} cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 {mu}M) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G{sub 1} cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles high synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.

  12. RNA Replicons - A New Approach for Influenza Virus Immunoprophylaxis

    PubMed Central

    Zimmer, Gert

    2010-01-01

    RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed. PMID:21994644

  13. RNA replicons - a new approach for influenza virus immunoprophylaxis.

    PubMed

    Zimmer, Gert

    2010-02-01

    RNA replicons are derived from either positive- or negative-strand RNA viruses. They represent disabled virus vectors that are not only avirulent, but also unable to revert to virulence. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of these vaccine strategies will be discussed and future perspectives disclosed.

  14. Pentavalent replicon vaccines against botulinum neurotoxins and tetanus toxin using DNA-based Semliki Forest virus replicon vectors

    PubMed Central

    Yu, YunZhou; Liu, Si; Ma, Yao; Gong, Zheng-Wei; Wang, Shuang; Sun, Zhi-Wei

    2014-01-01

    The clostridial neurotoxin (CNT) family includes botulinum neurotoxin (BoNT), serotypes A, B, E, and F of which can cause human botulism, and tetanus neurotoxin (TeNT), which is the causative agent of tetanus. This suggests that the greatest need is for a multivalent or multiagent vaccine that provides protection against all 5 agents. In this study, we investigated the feasibility of generating several pentavalent replicon vaccines that protected mice against BoNTs and TeNT. First, we evaluated the potency of individual replicon DNA or particle vaccine against TeNT, which induced strong antibody and protective responses in BALB/c mice following 2 or 3 immunizations. Then, the individual replicon TeNT vaccines were combined with tetravalent BoNTs vaccines to prepare 4 types of pentavalent replicon vaccines. These replicon DNA or particle pentavalent vaccines could simultaneously and effectively induce antibody responses and protect effects against the 5 agents. Finally, a solid-phase assay showed that the sera of pentavalent replicon formulations-immunized mice inhibited the binding of THc to the ganglioside GT1b as the sera of individual replicon DNA or particle-immunized mice. These results indicated these pentavalent replicon vaccines could protect against the 4 BoNT serotypes and effectively neutralize and protect the TeNT. Therefore, our studies demonstrate the utility of combining replicon DNA or particle vaccines into multi-agent formulations as potent pentavalent vaccines for eliciting protective responses against BoNTs and TeNT. PMID:25424795

  15. Kunjin virus replicon-based vaccines expressing Ebola virus glycoprotein GP protect the guinea pig against lethal Ebola virus infection.

    PubMed

    Reynard, O; Mokhonov, V; Mokhonova, E; Leung, J; Page, A; Mateo, M; Pyankova, O; Georges-Courbot, M C; Raoul, H; Khromykh, A A; Volchkov, V E

    2011-11-01

    Pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. We evaluated, in a guinea pig model, the immunogenic potential of Kunjin virus (KUN)-derived replicons as a vaccine candidate against Ebola virus (EBOV). Virus like particles (VLPs) containing KUN replicons expressing EBOV wild-type glycoprotein GP, membrane anchor-truncated GP (GP/Ctr), and mutated GP (D637L) with enhanced shedding capacity were generated and assayed for their protective efficacy. Immunization with KUN VLPs expressing full-length wild-type and D637L-mutated GPs but not membrane anchor-truncated GP induced dose-dependent protection against a challenge of a lethal dose of recombinant guinea pig-adapted EBOV. The surviving animals showed complete clearance of the virus. Our results demonstrate the potential for KUN replicon vectors as vaccine candidates against EBOV infection.

  16. Lack of Interference with Immunogenicity of a Chimeric Alphavirus Replicon Particle-Based Influenza Vaccine by Preexisting Antivector Immunity

    PubMed Central

    Vajdy, Michael; Lian, Ying; Perri, Silvia; Greer, Catherine E.; Legg, Harold S.; Galli, Grazia; Saletti, Giulietta; Otten, Gillis R.; Rappuoli, Rino; Barnett, Susan W.; Polo, John M.

    2012-01-01

    Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens. PMID:22623651

  17. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays.

    PubMed

    Pohjala, Leena; Utt, Age; Varjak, Margus; Lulla, Aleksei; Merits, Andres; Ahola, Tero; Tammela, Päivi

    2011-01-01

    Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti

  18. Preconceptual administration of an alphavirus replicon UL83 (pp65 homolog) vaccine induces humoral and cellular immunity and improves pregnancy outcome in the guinea pig model of congenital cytomegalovirus infection.

    PubMed

    Schleiss, Mark R; Lacayo, Juan C; Belkaid, Yasmine; McGregor, Alistair; Stroup, Greg; Rayner, Jon; Alterson, Kimberly; Chulay, Jeffrey D; Smith, Jonathan F

    2007-03-15

    Development of a vaccine against congenital cytomegalovirus (CMV) infection is a major public health priority. We report the use of a propagation-defective, single-cycle, RNA replicon vector system, derived from an attenuated strain of the alphavirus Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRPs) expressing GP83, the guinea pig CMV (GPCMV) homolog of the human CMV pp65 phosphoprotein. Vaccination with VRP-GP83 induced antibodies and CD4(+) and CD8(+) T cell responses in GPCMV-seronegative female guinea pigs. Guinea pigs immunized with VRP-GP83 vaccine or with a VRP vaccine expressing influenza hemagglutinin (VRP-HA) were bred for pregnancy and subsequent GPCMV challenge during the early third trimester. Dams vaccinated with VRP-GP83 had improved pregnancy outcomes, compared with dams vaccinated with the VRP-HA control. For VRP-GP83-vaccinated dams, there were 28 live pups and 4 dead pups (13% mortality) among 10 evaluable litters, compared with 9 live pups and 12 dead pups (57% mortality) among 8 evaluable litters in the VRP-HA-vaccinated group (P<.001, Fisher's exact test). Improved pregnancy outcome was accompanied by reductions in maternal blood viral load, measured by real-time polymerase chain reaction. These results indicate that cell-mediated immune responses directed against a CMV matrix protein can protect against congenital CMV infection and disease.

  19. Multiple replicons constituting the genome of Pseudomonas cepacia 17616.

    PubMed Central

    Cheng, H P; Lessie, T G

    1994-01-01

    Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates. Images PMID:7517389

  20. Construction and characterization of poliovirus subgenomic replicons

    SciTech Connect

    Kaplan, G.; Racaniello, V.R. )

    1988-05-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3,064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of ({sup 35}S-labeled) viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2A{sup pro}, 2C, and 3D{sup pol} and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs.

  1. FMDV replicons encoding green fluorescent protein are replication competent.

    PubMed

    Tulloch, Fiona; Pathania, Uday; Luke, Garry A; Nicholson, John; Stonehouse, Nicola J; Rowlands, David J; Jackson, Terry; Tuthill, Toby; Haas, Juergen; Lamond, Angus I; Ryan, Martin D

    2014-12-01

    The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (L(pro)) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the L(pro) showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.

  2. Identification of two replicons in phage-plasmid P4.

    PubMed

    Tocchetti, A; Serina, S; Terzano, S; Dehò, G; Ghisotti, D

    1998-06-05

    DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting region, crr. Replication depends on the product of the P4 alpha gene, a protein with primase and helicase activity, that binds both ori1 and crr. A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4. Using a plasmid complementation test for replication, we found that two replicons, both dependent on the alpha gene product, coexist in P4. The first replicon is made by the cnr and alpha genes and the ori1 and crr sites. The second is limited to the alpha and crr region. Thus, in the absence of the ori1 region, replication can initiate at a different site. By deletion mapping, a cis-acting region, ori2, essential for replication of the alpha-crr replicon was mapped within a 270-bp fragment in the first half of the alpha gene. The ori2 site was found to be dispensable in a replicon that contains ori1. A construct that besides crr and alpha carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2.

  3. The Father Speaks, the Mother Talks Back: Revisionist, Rebellious Models for the Creative Writing Classroom.

    ERIC Educational Resources Information Center

    Brown, Julie; Brown, Robert

    The "writing workshop" approach to teaching creative writing, virtually unchallenged throughout the United States, has recently come under fire. Two schools of thought, while agreeing that the traditional workshop needs a thorough overhaul, differ in approaches to that overhaul. One approach, using the theories of Harold Bloom, argues…

  4. Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle.

    PubMed

    Kamrud, K I; Alterson, K; Custer, M; Dudek, J; Goodman, C; Owens, G; Smith, J F

    2010-07-01

    Alphavirus-based replicon systems are frequently used as preclinical vectors and as antigen discovery tools, and they have recently been assessed in clinical vaccine trials. Typically, alphavirus replicon RNAs are delivered within virus-like replicon particles (VRP) that are produced following transfection of replicon RNA and two helper RNAs into permissive cells in vitro. The non-structural proteins expressed from the replicon RNA amplify the replicon RNA in cis and the helper RNAs in trans, the latter providing the viral structural proteins necessary to package the replicon RNA into VRP. Current helper RNA designs incorporate the alphavirus 26S promoter to direct the transcription of high levels of structural gene mRNAs. We demonstrate here that the 26S promoter is not required on helper RNAs to produce VRP and propose that such promoterless helper RNAs, by design, reduce the probability of generating replication-competent virus that may otherwise result from RNA recombination.

  5. CD8+ T-cell interaction with HCV replicon cells: evidence for both cytokine- and cell-mediated antiviral activity.

    PubMed

    Liu, Chen; Zhu, Haizhen; Tu, Zhengkun; Xu, Yi-Ling; Nelson, David R

    2003-06-01

    The interaction between the host immune response and infected hepatocytes plays a central role in the pathogenesis of hepatitis C virus (HCV). The lack of a suitable animal or in vitro model has hindered our understanding of the host T-cell/HCV interaction. Our aim was to develop an in vitro model to study the mechanisms of HCV-specific T-cell-mediated antiviral and cytolytic function. The HCV replicon was HLA typed and lymphocytes were obtained from an HLA class I-matched subject. CD8(+) T cells were expanded with 2 HCV-specific/HLA-restricted peptides for NS3. Lymphocyte preparations were cocultured with HCV replicon (FCA1) and control (Huh7) cells labeled with (51)Cr. After a 48-hour incubation, the cells were harvested for RNA extraction. Standard blocking assays were performed in the presence of anti-interferon gamma (IFN-gamma), anti-tumor necrosis factor alpha (TNF-alpha), and anti-FasL. Cytolytic activity was measured by (51)Cr release. HCV replicon cells express homozygous HLA-A11 alleles and present HCV nonstructural proteins. HCV-specific expansion of CD8(+) cells led to a 10-fold decrease in HCV replication by Northern blot analysis and 21% specific lysis of FCA1 cells (compared with 2% of control Huh7 cells). Twenty percent of this antiviral activity was independent of T-cell binding, suggesting cytokine-mediated antiviral activity. The CD8(+) antiviral effect was markedly reduced by blocking either IFN-gamma or FasL but was unaffected by blocking TNF-alpha. In conclusion, HCV-specific CD8(+) cells inhibit viral RNA replication by cytokine-mediated and direct cytolytic effects. This T-cell/HCV subgenomic replicon system represents a model for the investigation of CD8 cell interaction with HCV-infected hepatocytes.

  6. The dynamic replicon: adapting to a changing cellular environment.

    PubMed

    Herrick, John

    2010-02-01

    Eukaryotic cells are often exposed to fluctuations in growth conditions as well as endogenous and exogenous stress-related agents. During development, global patterns of gene transcription change substantially, and these changes are associated with altered patterns of DNA replication and larger distances between replication origins in somatic cells compared to embryos. Conversely, when cells experience difficulties while replicating DNA, the replication program is dramatically altered and distances between replication origins decrease. Recent evidence indicates that each unit of replication, or replicon, can correspond to one or more potential replication origins, but in the case of multiple potential origins, only one is selected to initiate replication of the replicon. How one origin is selected from multiple potential origins and how origin densities are regulated during genome duplication remains unclear. The following review addresses some of the mechanisms involved in regulating replication origins during both a normal and perturbed eukaryotic cell cycle.

  7. Engineered alphavirus replicon vaccines based on known attenuated viral mutants show limited effects on immunogenicity.

    PubMed

    Maruggi, Giulietta; Shaw, Christine A; Otten, Gillis R; Mason, Peter W; Beard, Clayton W

    2013-12-01

    The immunogenicity of alphavirus replicon vaccines is determined by many factors including the level of antigen expression and induction of innate immune responses. Characterized attenuated alphavirus mutants contain changes to the genomic 5' UTR and mutations that result in altered non-structural protein cleavage timing leading to altered levels of antigen expression and interferon (IFN) induction. In an attempt to create more potent replicon vaccines, we engineered a panel of Venezuelan equine encephalitis-Sindbis virus chimeric replicons that contained these attenuating mutations. Modified replicons were ranked for antigen expression and IFN induction levels in cell culture and then evaluated in mice. The results of these studies showed that differences in antigen production and IFN induction in vitro did not correlate with large changes in immunogenicity in vivo. These findings indicate that the complex interactions between innate immune response and the replicon's ability to express antigen complicate rational design of more potent alphavirus replicons.

  8. Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture.

    PubMed

    Yu, Mei; Peng, Betty; Chan, Katie; Gong, Ruoyu; Yang, Huiling; Delaney, William; Cheng, Guofeng

    2014-05-01

    Genotype 6 (GT6) hepatitis C virus (HCV) is prevalent in Southeast Asia and southern China, where it can constitute up to 50% of HCV infections. Despite this, no direct-acting antivirals are approved to treat GT6 HCV infection, and no cell culture systems have been described. In this study, we aimed to develop a GT6 HCV subgenomic replicon to facilitate the identification and development of new HCV therapies with pan-genotype activity. A subgenomic replicon cDNA encoding a GT6a consensus sequence plus an NS5A amino acid substitution (S232I) was synthesized. Electroporation of RNA encoding the GT6a replicon into Huh-7-derived cells consistently yielded 20 to 100 stable replicon colonies. Genotypic analyses of individual replicon colonies revealed new adaptive mutations across multiple viral nonstructural proteins. The E30V and K272R mutations in NS3 and the K34R mutation in NS4A were observed most frequently and were confirmed to enhance GT6a replicon replication in the presence of the NS5A amino acid substitution S232I. These new adaptive mutations allowed establishment of robust luciferase-encoding GT6a replicons for reproducible quantification of HCV replication, and the luciferase-encoding replicons enabled efficient determinations of antiviral activity for HCV inhibitors in a 384-well assay format. While nucleoside/nucleotide NS5B inhibitors and cyclophilin A inhibitors had similar antiviral activities against both GT6a and GT1b replicons, some nonnucleoside NS5B inhibitors, NS3 protease inhibitors, and NS5A inhibitors had less antiviral activity against GT6a replicons. In conjunction with other genotype replicons, this robust GT6a replicon system will aid in the development of pan-genotypic HCV regimens.

  9. Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules.

    PubMed

    Bardaji, Leire; Añorga, Maite; Ruiz-Masó, José A; Del Solar, Gloria; Murillo, Jesús

    2017-01-01

    Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.

  10. Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules

    PubMed Central

    Bardaji, Leire; Añorga, Maite; Ruiz-Masó, José A.; del Solar, Gloria; Murillo, Jesús

    2017-01-01

    Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities. PMID:28243228

  11. Mucosal and systemic adjuvant activity of alphavirus replicon particles

    NASA Astrophysics Data System (ADS)

    Thompson, Joseph M.; Whitmore, Alan C.; Konopka, Jennifer L.; Collier, Martha L.; Richmond, Erin M. B.; Davis, Nancy L.; Staats, Herman F.; Johnston, Robert E.

    2006-03-01

    Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines. vaccine vector | Venezuelan equine encephalitis virus | viral immunology | RNA virus

  12. Novel hepatitis C virus reporter replicon cell lines enable efficient antiviral screening against genotype 1a.

    PubMed

    Robinson, Margaret; Yang, Huiling; Sun, Siu-Chi; Peng, Betty; Tian, Yang; Pagratis, Nikos; Greenstein, Andrew E; Delaney, William E

    2010-08-01

    The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (approximately 70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.

  13. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    PubMed

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax.

  14. Antimicrobial susceptibility and plasmid replicon typing of Salmonella enterica serovar Kentucky isolates recovered from broilers

    USDA-ARS?s Scientific Manuscript database

    Salmonella Kentucky has become the predominate serotype recovered from broiler slaughter in the United States and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serotype. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 ...

  15. Single-step construction of a picornavirus replicon RNA with precise ends.

    PubMed

    Xiong, Qing; Wang, Yuya; Xie, Bingyu; Pei, Xinyi; Peng, Yihong

    2017-10-01

    A versatile single-step method is described for constructing a picornavirus replicon RNA with precise ends to facilitate improved understanding of viral genome function and mimic native virus replication in host cells as far as possible. The key innovation in this new approach is the use of a bridge primer to both introduce a ribozyme sequence for cis-cleavage of RNA to generate precise 5' ends of EV71 RNA and also mediate overlapping assembly of two fragments. Using an EV71 replicon as a test case, precise ends for the viral replicon were shown to be important for efficient virus replication. Thus, our work provides a novel efficient way to generating higher efficient viral replicon with precise ends and this novel method can be applied to other picornaviruses' research. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus.

    PubMed

    Reap, Elizabeth A; Morris, John; Dryga, Sergey A; Maughan, Maureen; Talarico, Todd; Esch, Robert E; Negri, Sarah; Burnett, Bruce; Graham, Andrew; Olmsted, Robert A; Chulay, Jeffrey D

    2007-10-16

    We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.

  17. Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

    PubMed Central

    Kaufmann, W K; Boyer, J C; Estabrooks, L L; Wilson, S J

    1991-01-01

    Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes. PMID:1646393

  18. Proteome Analysis of Liver Cells Expressing a Full- Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

    SciTech Connect

    Jacobs, Jon M.; Diamond, Deborah L.; Chan, Eric Y.; Gritsenko, Marina A.; Qian, Weijun; Stastna, Miroslava; Baas, Tracey; Camp, David G.; Carithers, Jr., Robert L.; Smith, Richard D.; Katze, Michael G.

    2005-06-01

    The development of a reproducible model system for the study of Hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full length HCV replicon. We detected > 4,400 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry (MS). The set of Huh-7.5 proteins confidently identified is, to our knowledge, the most comprehensive yet reported for a human cell line. Consistent with the literature, a comparison of Huh-7.5 cells (+) and (-) the HCV replicon identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where > 1,500 proteins were detected from 2 {micro}g protein lysate using the Huh-7.5 protein database and the accurate mass and time (AMT) tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.

  19. Enhancement of the immunogenicity of DNA replicon vaccine of Clostridium botulinum neurotoxin serotype A by GM-CSF gene adjuvant.

    PubMed

    Li, Na; Yu, Yun-Zhou; Yu, Wei-Yuan; Sun, Zhi-Wei

    2011-03-01

    Granulocyte-macrophage clony-stimulating factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells to the site of antigen synthesis as well as stimulate the maturation of dendritic cells.This study evaluated the utility of GM-CSF as a plasmid DNA replicon vaccine adjuvants for botulinum neurotoxin serotype A (BoNT/A) in mouse model. In balb/c mice that received the plasmid DNA replicon vaccines derived from Semliki Forest virus (SFV) carrying the Hc gene of BoNT/A (AHc), both antibody and lymphoproliferative response specific to AHc were induced, the immunogenicity was enhanced by co-delivery or coexpress of the GM-CSF gene. In particular, when AHc and GM-CSF were coexpressed within the SFV based DNA vaccine, the anti-AHc antibody titers and survival rates of immunized mice after challenged with BoNT/A were significantly increased, and further enhanced by coimmunization with aluminum phosphate adjuvant.

  20. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.

    PubMed

    Gil-Humanes, Javier; Wang, Yanpeng; Liang, Zhen; Shan, Qiwei; Ozuna, Carmen V; Sánchez-León, Susana; Baltes, Nicholas J; Starker, Colby; Barro, Francisco; Gao, Caixia; Voytas, Daniel F

    2017-03-01

    The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

  1. In vitro and in vivo characterization of microRNA-targeted alphavirus replicon and helper RNAs.

    PubMed

    Kamrud, Kurt I; Coffield, V McNeil; Owens, Gary; Goodman, Christin; Alterson, Kim; Custer, Max; Murphy, Michael A; Lewis, Whitney; Timberlake, Sarah; Wansley, Elizabeth K; Berglund, Peter; Smith, Jonathan

    2010-08-01

    Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.

  2. Comparative analyses of extrachromosomal bacterial replicons, identification of chromids, and experimental evaluation of their indispensability.

    PubMed

    Dziewit, Lukasz; Bartosik, Dariusz

    2015-01-01

    Bacterial genomic information can be divided between various replicons, including chromosomes, plasmids, and chromids (essential plasmid-like replicons with properties of both chromosomes and plasmids). Comparative analyses of bacterial plasmids, including homology searches, phylogenetic and phylogenomic analyses, as well as network construction for the characterization of their relationships, are good starting points for the identification of chromids. Chromids possess several chromosome-like genetic features (e.g., codon usage, GC content), but most significantly, they carry housekeeping genes, which make them indispensable for cell viability. However, it is important to confirm in silico predictions experimentally. The essential nature of a predicted chromid is usually verified by the application of a target-oriented replicon curing technique, based on the incompatibility phenomenon. Further tests examining growth in various media are used to distinguish secondary chromids from plasmids, and mutational analysis (e.g., using the yeast FLP/FRT recombination system) is employed to identify essential genes carried by particular chromids.

  3. Development and preclinical evaluation of an alphavirus replicon vaccine for influenza.

    PubMed

    Hubby, Bolyn; Talarico, Todd; Maughan, Maureen; Reap, Elizabeth A; Berglund, Peter; Kamrud, Kurt I; Copp, Laura; Lewis, Whitney; Cecil, Chad; Norberg, Pamela; Wagner, Jordan; Watson, Aubrey; Negri, Sarah; Burnett, Bruce K; Graham, Andrew; Smith, Jonathan F; Chulay, Jeffrey D

    2007-11-23

    We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-gamma ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.

  4. A cloned replicon of Saccharopolyspora phages JHJ-1 and JHJ-3 is stably maintained as a plasmid in various actinomycetes.

    PubMed

    Gaudreau, L R; Déry, C V

    1993-04-15

    A replicon of phage JHJ-1 (and JHJ-3) was cloned. The autonomously replicating phage element was maintained as a medium-copy-number shuttle plasmid in many actinomycetes, and was efficiently transmitted to spores without antibiotic selection. One gene was shown to be expressed in a vector containing the JHJ-3 replicon.

  5. Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

    PubMed

    Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

    2016-04-01

    Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    PubMed

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

  7. Conserved aspartic acid 233 and alanine 231 are not required for poliovirus polymerase function in replicons

    PubMed Central

    Freistadt, Marion S; Eberle, Karen E

    2007-01-01

    Nucleic acid polymerases have similar structures and motifs. The function of an aspartic acid (conserved in all classes of nucleic acid polymerases) in motif A remains poorly understood in RNA-dependent RNA polymerases. We mutated this residue to alanine in a poliovirus replicon. The resulting mutant could still replicate, although at a reduced level. In addition, mutation A231C (also in motif A) yielded high levels of replication. Taken together these results show that poliovirus polymerase conserved residues D233 and A231 are not essential to poliovirus replicon function. PMID:17352827

  8. [The vaccines based on the replicon of the venezuelan equine encephalomyelitis virus against viral hemorrhagic fevers].

    PubMed

    Petrov, A A; Plekhanova, T M; Sidorova, O N; Borisevich, S V; Makhlay, A A

    2015-01-01

    The status of the various recombinant DNA and RNA-derived candidate vaccines, as well as the Venezuelan equine encephalomyelitis virus (VEEV) replicon vaccine system against extremely hazardous viral hemorrhagic fevers, were reviewed. The VEEV-based replication-incompetent vectors offer attractive features in terms of safety, high expression levels of the heterologous viral antigen, tropism to dendritic cells, robust immune responses, protection efficacy, low potential for pre-existing anti-vector immunity and possibility of engineering multivalent vaccines were tested. These features of the VEEV replicon system hold much promise for the development of new generation vaccine candidates against viral hemorrhagic fevers.

  9. Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.

    PubMed

    Manzano, Mark; Padmanabhan, Radhakrishnan

    2014-01-01

    The use of cDNA infectious clones or subgenomic replicons is indispensable in studying flavivirus biology. Mutating nucleotides or amino acid residues gives important clues to their function in the viral life cycle. However, a major challenge to the establishment of a reverse genetics system for flaviviruses is the instability of their nucleotide sequences in Escherichia coli. Thus, direct cloning using conventional restriction enzyme-based procedures usually leads to unwanted rearrangements of the construct. In this chapter, we discuss a cloning strategy that bypasses traditional cloning procedures. We take advantage of the observations from previous studies that (1) unstable sequences in bacteria can be cloned in eukaryotic systems and (2) Saccharomyces cerevisiae has a well-studied genetics system to introduce sequences using homologous recombination. We describe a protocol to perform targeted mutagenesis in a subgenomic dengue virus 2 replicon. Our method makes use of homologous recombination in yeast using a linearized replicon and a PCR product containing the desired mutation. Constructs derived from this method can be propagated in E. coli with improved stability. Thus, yeast in vivo recombination provides an excellent strategy to genetically engineer flavivirus infectious clones or replicons because this system is compatible with inherently unstable sequences of flaviviruses and is not restricted by the limitations of traditional cloning procedures.

  10. Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

    PubMed Central

    Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M.; Rossolini, Gian Maria

    2006-01-01

    Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  11. Replicon typing of plasmids encoding resistance to newer beta-lactams.

    PubMed

    Carattoli, Alessandra; Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M; Rossolini, Gian Maria

    2006-07-01

    Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems.

  12. Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

    PubMed Central

    Akhtar, Parvez; Khan, Saleem A.

    2014-01-01

    The large pXO1 plasmid (181.6 kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid. PMID:22239982

  13. Single-Dose Immunization with Virus Replicon Particles Confers Rapid Robust Protection against Rift Valley Fever Virus Challenge

    PubMed Central

    Dodd, Kimberly A.; Metcalfe, Maureen G.; Nichol, Stuart T.; Albariño, César G.

    2012-01-01

    Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRPRVF) vaccine candidate. Using a mouse model, we show that VRPRVF immunization provides the optimal balance of safety and single-dose robust efficacy. VRPRVF can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRPRVF proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRPRVF, although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD50). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRPRVF immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection. PMID:22345465

  14. An immunogenic and protective alphavirus replicon particle-based dengue vaccine overcomes maternal antibody interference in weanling mice.

    PubMed

    White, Laura J; Parsons, Melissa M; Whitmore, Alan C; Williams, Brandon M; de Silva, Aravinda; Johnston, Robert E

    2007-10-01

    A candidate pediatric dengue virus (DENV) vaccine based on nonpropagating Venezuelan equine encephalitis virus replicon particles (VRP) was tested for immunogenicity and protective efficacy in weanling mice in the presence and absence of potentially interfering maternal antibodies. A gene cassette encoding envelope proteins prM and E from mouse-adapted DENV type 2 (DENV2) strain NGC was cloned into a VEE replicon vector and packaged into VRP, which programmed proper in vitro expression and processing of DENV2 envelope proteins upon infection of Vero cells. Primary immunization of 3-week-old weanling BALB/c mice in the footpad with DENV2 VRP resulted in high levels of DENV-specific serum immunoglobulin G antibodies and significant titers of neutralizing antibodies in all vaccinates. A booster immunization 12 weeks after the prime immunization resulted in increased neutralizing antibodies that were sustained for at least 30 weeks. Immunization at a range of doses of DENV2 VRP protected mice from an otherwise-lethal intracranial DENV2 challenge. To model vaccination in the presence of maternal antibodies, weanling pups born to DENV2-immune or DENV2-naïve dams were immunized with either DENV2 VRP or live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the utility of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic.

  15. An Immunogenic and Protective Alphavirus Replicon Particle-Based Dengue Vaccine Overcomes Maternal Antibody Interference in Weanling Mice▿

    PubMed Central

    White, Laura J.; Parsons, Melissa M.; Whitmore, Alan C.; Williams, Brandon M.; de Silva, Aravinda; Johnston, Robert E.

    2007-01-01

    A candidate pediatric dengue virus (DENV) vaccine based on nonpropagating Venezuelan equine encephalitis virus replicon particles (VRP) was tested for immunogenicity and protective efficacy in weanling mice in the presence and absence of potentially interfering maternal antibodies. A gene cassette encoding envelope proteins prM and E from mouse-adapted DENV type 2 (DENV2) strain NGC was cloned into a VEE replicon vector and packaged into VRP, which programmed proper in vitro expression and processing of DENV2 envelope proteins upon infection of Vero cells. Primary immunization of 3-week-old weanling BALB/c mice in the footpad with DENV2 VRP resulted in high levels of DENV-specific serum immunoglobulin G antibodies and significant titers of neutralizing antibodies in all vaccinates. A booster immunization 12 weeks after the prime immunization resulted in increased neutralizing antibodies that were sustained for at least 30 weeks. Immunization at a range of doses of DENV2 VRP protected mice from an otherwise-lethal intracranial DENV2 challenge. To model vaccination in the presence of maternal antibodies, weanling pups born to DENV2-immune or DENV2-naïve dams were immunized with either DENV2 VRP or live DENV2 given peripherally. The DENV2 VRP vaccine induced neutralizing-antibody responses in young mice regardless of the maternal immune status. In contrast, live-DENV2 vaccination performed poorly in the presence of preexisting anti-DENV2 antibodies. This study demonstrates the feasibility of a VRP vaccine approach as an early-life DENV vaccine in populations with high levels of circulating DENV antibodies and suggests the utility of VRP-based vaccines in other instances where maternal antibodies make early vaccination problematic. PMID:17652394

  16. Single-dose immunization with virus replicon particles confers rapid robust protection against Rift Valley fever virus challenge.

    PubMed

    Dodd, Kimberly A; Bird, Brian H; Metcalfe, Maureen G; Nichol, Stuart T; Albariño, César G

    2012-04-01

    Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRP(RVF) proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRP(RVF), although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD(50)). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRP(RVF) immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection.

  17. Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

    PubMed

    Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

    2017-05-01

    Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. The lytic replicon of bacteriophage P1 is controlled by an antisense RNA.

    PubMed Central

    Heinrich, J; Riedel, H D; Rückert, B; Lurz, R; Schuster, H

    1995-01-01

    The lytic replicon of phage P1 is used for DNA replication during the lytic cycle. It comprises about 2% of the P1 genome and contains the P1 C1 repressor-controlled operator-promoter element Op53.P53 and the kilA and the repL genes, in that order. Transcription of the lytic replicon of P53 and synthesis of the product of repL, but not kilA, are required for replicon function. We have identified an additional promoter, termed P53as (antisense), at the 5'-end of the kilA gene from which a 180 base transcript is constitutively synthesized and in the opposite direction to the P53 transcript. By using a promoter probe plasmid we show that transcription from P53 is strongly repressed by the C1 repressor, whereas that of P53as remains unaffected. Accordingly, the C1 repressor inhibits binding of Escherichia coli RNA polymerase to P53, but not to P53as, as shown by electron microscopy. Under non-repressed conditions transcription from P53 appears to be inhibited by P53as activity and vice versa. An inhibitory effect of P53as on the P1 lytic replicon was revealed by the construction and characterization of a P53as promoter-down mutant. Under non-repressed conditions transcription of repL and, as a consequence, replication of the plasmid is strongly enhanced when P53as is inactive. The results suggest a regulatory role for P53as on the P1 lytic replicon. Images PMID:7784198

  19. Pea (Pisum sativum) cells arrested in G2 have nascent DNA with breaks between replicons and replication clusters

    SciTech Connect

    Van't Hof, J.

    1980-01-01

    DNA fiber autoradiography and alkaline sucrose sedimentation of DNA of cultured pea-root cells (Pisum sativum) arrested in G2 by carbohydrate starvation demonstrated that nascent DNA molecules of replicon (16 to 27 x 10/sup 6/D) and apparent cluster (approx. 330 x 10/sup 6/D) size were not joined. That the arrested cells were in G2 was confirmed by single-cell autoradiography and cytophotometry. In pea there are about 18 replicons per average cluster, 4.2 x 10/sup 3/ clusters, and 7.7 x 10/sup 4/ replicons per genome.

  20. The contribution of type I interferon signaling to immunity induced by alphavirus replicon vaccines.

    PubMed

    Thompson, Joseph M; Whitmore, Alan C; Staats, Herman F; Johnston, Robert

    2008-09-15

    The type I interferon (IFN) system is critical for protecting the mammalian host from numerous virus infections and plays a key role in shaping the antiviral adaptive immune response. In this report, the importance of type I IFN signaling was assessed in a mouse model of alphavirus-induced humoral immune induction. Venezuelan equine encephalitis virus replicon particles (VRP) expressing the hemagglutinin (HA) gene from influenza virus (HA-VRP) were used to vaccinate both wildtype (wt) and IFN alpha/beta receptor knockout (RKO) mice. HA-VRP vaccination induced equivalent levels of flu-specific systemic IgG, mucosal IgG, and systemic IgA antibodies in both wt and IFN RKO mice. In contrast, HA-VRP vaccination of IFN RKO mice failed to induce significant levels of flu-specific mucosal IgA antibodies at multiple mucosal surfaces. In the VRP adjuvant system, co-delivery of null VRP with ovalbumin (OVA) protein significantly increased the levels of OVA-specific serum IgG, fecal IgG, and fecal IgA antibodies in both wt and RKO mice, suggesting that type I IFN signaling plays a less significant role in the VRP adjuvant effect. Taken together, these results suggest that (1) at least in regard to IFN signaling, the mechanisms which regulate alphavirus-induced immunity differ when VRP are utilized as expression vectors as opposed to adjuvants, and (2) type I IFN signaling is required for the induction of mucosal IgA antibodies directed against VRP-expressed antigen. These results shed new light on the regulatory networks which promote immune induction, and specifically mucosal immune induction, with alphavirus vaccine vectors.

  1. THE CONTRIBUTION OF TYPE I INTERFERON SIGNALING TO IMMUNITY INDUCED BY ALPHAVIRUS REPLICON VACCINES

    PubMed Central

    Thompson, Joseph M.; Whitmore, Alan C.; Staats, Herman F.; Johnston, Robert

    2013-01-01

    The type I interferon (IFN) system is critical for protecting the mammalian host from numerous virus infections and plays a key role in shaping the anti-viral adaptive immune response. In this report, the importance of type I IFN signaling was assessed in a mouse model of alphavirus-induced humoral immune induction. Venezuelan equine encephalitis virus replicon particles (VRP) expressing the hemagglutinin (HA) gene from influenza virus (HA-VRP) were used to vaccinate both wildtype (wt) and IFN α/β receptor knockout (RKO) mice. HA-VRP vaccination induced equivalent levels of flu-specific systemic IgG, mucosal IgG, and systemic IgA antibodies in both wt and IFN RKO mice. In contrast, HA-VRP vaccination of IFN RKO mice failed to induce significant levels of flu-specific mucosal IgA antibodies at multiple mucosal surfaces. In the VRP adjuvant system, co-delivery of null VRP with ovalbumin (OVA) protein significantly increased the levels of OVA-specific serum IgG, fecal IgG, and fecal IgA antibodies in both wt and RKO mice, suggesting that type I IFN signaling plays a less significant role in the VRP adjuvant effect. Taken together, these results suggest that, 1) at least in regard to IFN signaling, the mechanisms which regulate VRP-induced immunity differ when VRP are utilized as expression vectors as opposed to adjuvants, and 2) type I IFN signaling is required for the induction of mucosal IgA antibodies directed against VRP-expressed antigen. These results potentially shed new light on the regulatory networks which promote immune induction, and specifically mucosal immune induction, with alphavirus vaccine vectors. PMID:18656518

  2. The Third Replicon of Members of the Burkholderia cepacia Complex, Plasmid pC3, Plays a Role in Stress Tolerance

    PubMed Central

    Agnoli, Kirsty; Frauenknecht, Carmen; Freitag, Roman; Schwager, Stephan; Jenul, Christian; Vergunst, Annette; Carlier, Aurelien

    2014-01-01

    The metabolically versatile Burkholderia cepacia complex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined. B. cenocepacia strains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and the Caenorhabditis elegans model of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance of B. cenocepacia H111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc. PMID:24334662

  3. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315

    PubMed Central

    Kamgoué, Alain; Murray, Heath; Pasta, Franck

    2016-01-01

    Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of

  4. Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1

    PubMed Central

    Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart

    2016-01-01

    ABSTRACT The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980T, suggesting their broad applicability as genetic tools for Natrinema species. IMPORTANCE Archaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980T. This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of

  5. Gene expression in the muscle and central nervous system following intramuscular inoculation of encapsidated or naked poliovirus replicons.

    PubMed

    Jackson, Cheryl A; Messinger, Jeff; Palmer, Matthew T; Peduzzi, Jean D; Morrow, Casey D

    2003-09-15

    The spread of intramuscularly inoculated poliovirus to the central nervous system (CNS) has been documented in humans, monkeys, and mice transgenic for the human poliovirus receptor. Poliovirus spread is thought to be due to infection of the peripheral nerve and retrograde transport of poliovirus through the axon to the neuron cell body, where final virus uncoating occurs and translation/replication ensues. In previous studies, we have shown that polio-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. Using a replicon that encodes green fluorescent protein (GFP), we found that following intrathecal inoculation, GFP expression was confined to motorneurons of the spinal cord. To further characterize the gene expression of poliovirus in the periphery and CNS, we have intramuscularly inoculated transgenic mice with poliovirus replicons encoding GFP. Expression of GFP was demonstrated in the muscle, sciatic nerve, dorsal root ganglion, and the ventral horn motorneurons following intramuscular inoculation. There was no evidence of paralysis or behavioral abnormalities in the mice following intramuscular inoculation of the replicon encoding GFP. Injection of replicon RNA alone (naked RNA) into the muscle of transgenic mice or rats, which do not express the poliovirus receptor, also resulted in expression of GFP in the muscle, sciatic nerve, dorsal root ganglion, and ventral horn motorneurons, indicating that transport of the replicon RNA from the periphery to CNS had occurred. GFP expression was found in the muscles and sciatic nerve as early as 6 h after injection of replicons or replicon RNA, even after sciatic nerve section. Analysis at longer times postinjection revealed GFP expression similar to 6 h levels in the cut sciatic nerves and robust expression in the nerves of uncut animals. The infection and expression of GFP in the CNS following intramuscular inoculation of encapsidated replicons encoding GFP occurred in juvenile or

  6. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis.

    PubMed

    Lindsey, Rebecca L; Frye, Jonathan G; Thitaram, Sutawee N; Meinersmann, Richard J; Fedorka-Cray, Paula J; Englen, Mark D

    2011-06-01

    The objective of this study was to examine the distribution of multidrug resistance in Escherichia coli in relation to plasmid replicon types, animal sources, and genotypes. E. coli isolates (n = 35) from seven different animal sources were selected and tested for susceptibility to 15 antimicrobials; pulsed-field gel electrophoresis was used to determine genetic relationships among the E. coli isolates. Plasmid types based on their incompatibility (Inc) replicon types were determined, and linkage disequilibrium analysis was performed for antimicrobial resistance profiles, replicon types, and animal source. A high degree of genotypic diversity was observed: 34 different pulsed-field gel electrophoresis types among the 35 isolates examined. Twelve different plasmid Inc types were detected, and all isolates carried at least one replicon type. IncF (n = 25; 71.4%) and IncFIB (n = 19; 54.3%) were the most common replicon types identified. Chloramphenicol resistance was significantly linked with four Inc types (A/C, FIIA, F, and Y), and amoxicillin/clavulanic acid was linked with three Inc types (B/O, P and Y). Resistance to any other antimicrobial was linked to two or fewer replicon types. The isolate source was linked with resistance to seven antimicrobials and IncI1. We conclude that commensal E. coli from animal sources are highly variable genotypically and are reservoirs of a diverse array of plasmids carrying antimicrobial resistance.

  7. Salmonid alphavirus replicon is functional in fish, mammalian and insect cells and in vivo in shrimps (Litopenaeus vannamei).

    PubMed

    Olsen, Christel M; Pemula, Anand Kumar; Braaen, Stine; Sankaran, Krishnan; Rimstad, Espen

    2013-11-19

    The Salmonid alphavirus (SAV) is the etiological agent of pancreas disease in Atlantic salmon (Salmo salar) and Sleeping disease in rainbow trout (Oncorhynchus mykiss). SAV differs from alphaviruses infecting terrestrial animals in that it infects salmonid fish at low temperatures and does not use an arthropod vector for transmission. In this study we have shown that a SAVbased replicon could express proteins when driven by the subgenomic promoter in vitro in cells from fish, mammals and insects, as well as in vivo in shrimps (Litopanaeus vannamei). The SAV-replicon was found to be functional at temperatures ranging from 4 to 37°C. Protein expression was slow and moderate compared to that reported from terrestrial alphavirus replicons or from vectors where protein expression was under control of the immediate early CMV-promoter. No cytopathic effect was visually observable in cells transfected with SAV-replicon vectors. Double stranded RNA was present for several days after transfection of the SAV-replicon in fish cell lines and its presence was indicated also in shrimp. The combination of prolonged dsRNA production, low toxicity, and wide temperature range for expression, may potentially be advantageous for the use of the SAV replicon to induce immune responses in aquaculture of fish and shrimp.

  8. A topoisomerase II-dependent mechanism for resetting replicons at the S–M-phase transition

    PubMed Central

    Cuvier, Olivier; Stanojcic, Slavica; Lemaitre, Jean-Marc; Mechali, Marcel

    2008-01-01

    Topoisomerase II (topo II) is required for chromosome segregation and for reprogramming replicons. Here, we show that topo II couples DNA replication termination with the clearing of replication complexes for resetting replicons at mitosis. Topo II inhibition impairs completion of DNA replication, accounting for replication protein A (RPA) stabilization onto ssDNA. Topo II inhibition does not affect the caffeine-sensitive ORC1 degradation found upon origin firing, but it impairs the cdk-dependent degradation/chromatin dissociation of an ORC1/2 reservoir at mitosis. Our results show that ORC1 degradation is rescued by Pin1 depletion and that this topo II-dependent clearing of ORC1/2 from chromatin involves the APC. PMID:18381889

  9. Mapping of active replication origins in vivo in thaum- and euryarchaeal replicons.

    PubMed

    Pelve, Erik A; Martens-Habbena, Willm; Stahl, David A; Bernander, Rolf

    2013-11-01

    We report mapping of active replication origins in thaum- and euryarchaeal replicons using high-throughput sequencing-based marker frequency analysis. The chromosome of the thaumarchaeon Nitrosopumilus maritimus is shown to contain a single origin of replication, whereas the main chromosome in the halophilic euryarchaea Haloferax mediterranei and Haloferax volcanii each contains two origins. All replication origins specified bidirectional replication, and the two origins in the halophiles were initiated in synchrony. The pHM500 plasmid of H. mediterranei is shown to contain a single origin, and the copy numbers of five plasmid replicons in the two halophiles were inferred to be close to that of the main chromosome. Origin recognition boxes (ORBs) that provide binding sites for Orc1/Cdc6 replication initiator proteins are identified at all chromosomal origins, as well as in a range of additional thaumarchaeal species. An annotation update is provided for all three species. © 2013 John Wiley & Sons Ltd.

  10. Replicon-dependent differentiation of symbiosis-related genes in Sinorhizobium strains nodulating Glycine max.

    PubMed

    Guo, Hui Juan; Wang, En Tao; Zhang, Xing Xing; Li, Qin Qin; Zhang, Yan Ming; Tian, Chang Fu; Chen, Wen Xin

    2014-02-01

    In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.

  11. Three replicons of Rhizobium sp. Strain NGR234 harbor symbiotic gene sequences.

    PubMed

    Flores, M; Mavingui, P; Girard, L; Perret, X; Broughton, W J; Martínez-Romero, E; Dávila, G; Palacios, R

    1998-11-01

    Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b.

  12. Replicon-Dependent Differentiation of Symbiosis-Related Genes in Sinorhizobium Strains Nodulating Glycine max

    PubMed Central

    Guo, Hui Juan; Wang, En Tao; Zhang, Xing Xing; Li, Qin Qin; Zhang, Yan Ming; Chen, Wen Xin

    2014-01-01

    In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons. PMID:24317084

  13. Three Replicons of Rhizobium sp. Strain NGR234 Harbor Symbiotic Gene Sequences

    PubMed Central

    Flores, Margarita; Mavingui, Patrick; Girard, Lourdes; Perret, Xavier; Broughton, William J.; Martínez-Romero, Esperanza; Dávila, Guillermo; Palacios, Rafael

    1998-01-01

    Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b. PMID:9811668

  14. Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs.

    PubMed

    Kim, Dal Young; Atasheva, Svetlana; McAuley, Alexander J; Plante, Jessica A; Frolova, Elena I; Beasley, David W C; Frolov, Ilya

    2014-07-22

    Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

  15. Noncytopathic Replication of Venezuelan Equine Encephalitis Virus and Eastern Equine Encephalitis Virus Replicons in Mammalian Cells

    PubMed Central

    Petrakova, Olga; Volkova, Eugenia; Gorchakov, Rodion; Paessler, Slobodan; Kinney, Richard M.; Frolov, Ilya

    2005-01-01

    Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5′ untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins. PMID:15919912

  16. Selection of RNA Replicons Capable of Persistent Noncytopathic Replication in Mammalian Cells

    PubMed Central

    Frolov, Ilya; Agapov, Eugene; Hoffman, Thomas A.; Prágai, Béla M.; Lippa, Mara; Schlesinger, Sondra; Rice, Charles M.

    1999-01-01

    The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989–12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments. PMID:10196280

  17. Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

    PubMed Central

    Forrest, Sophie; Lear, Zoe; Herod, Morgan R.; Ryan, Martin; Rowlands, David J.

    2014-01-01

    We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose dependent. The 3Dpol aptamers appeared to be more effective than 2′-C-methylcytidine (2′CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future. PMID:25096816

  18. Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells.

    PubMed

    Petrakova, Olga; Volkova, Eugenia; Gorchakov, Rodion; Paessler, Slobodan; Kinney, Richard M; Frolov, Ilya

    2005-06-01

    Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.

  19. Three types of human CpG motifs differentially modulate and augment immunogenicity of nonviral and viral replicon DNA vaccines as built-in adjuvants.

    PubMed

    Yu, Yun-Zhou; Li, Na; Ma, Yao; Wang, Shuang; Yu, Wei-Yuan; Sun, Zhi-Wei

    2013-01-01

    NakedDNA vaccines given by intramuscular injection are efficient in mouse models, but they require improvement for human use. As the immunogenicity of DNA vaccines depends, to a large extent, on the presence of CpG motifs as built-in adjuvants, we addressed this issue by inserting three types of human CpG motifs (A-type, B-type, and C-type) into the backbone of nonviral DNA and viral DNA replicon vectors with distinct immunostimulatory activities on human PBMCs. The adjuvant effects of CpG modifications in DNA vaccines expressing three types of antigens (β-Gal, AHc, or PA4) were then characterized in mice and found to significantly enhance antigen-specific humoral and cell-mediated immune responses. The three types of CpG motifs also differentially affected and modulated immune responses and protective potency against botulinum neurotoxin serotype A and Bacillus anthracis A16R challenge. Taken together, these results demonstrate that insertion of human CpG motifs can differentially modulate the immunogenicity of nonviral DNA vaccines as well as viral DNA replicon vaccines. Our study provides not only a better understanding of the in vivo activities of CpG motif adjuvants but implications for the rational design of such motifs as built-in adjuvants for DNA vectors targeting specific antigens.

  20. Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins.

    PubMed

    Zhang, Zhaoduo; Cavalier-Smith, Thomas; Green, Beverley R

    2002-04-01

    Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and

  1. A Novel Adenoviral Hybrid-vector System Carrying a Plasmid Replicon for Safe and Efficient Cell and Gene Therapeutic Applications.

    PubMed

    Voigtlander, Richard; Haase, Rudolf; Mück-Hausl, Martin; Zhang, Wenli; Boehme, Philip; Lipps, Hans-Joachim; Schulz, Eric; Baiker, Armin; Ehrhardt, Anja

    2013-04-02

    In dividing cells, the two aims a gene therapeutic approach should accomplish are efficient nuclear delivery and retention of therapeutic DNA. For stable transgene expression, therapeutic DNA can either be maintained by somatic integration or episomal persistence of which the latter approach would diminish the risk of insertional mutagenesis. As most monosystems fail to fulfill both tasks with equal efficiency, hybrid-vector systems represent promising alternatives. Our hybrid-vector system synergizes high-capacity adenoviral vectors (HCAdV) for efficient delivery and the scaffold/matrix attachment region (S/MAR)-based pEPito plasmid replicon for episomal persistence. After proving that this plasmid replicon can be excised from adenovirus in vitro, colony forming assays were performed. We found an increased number of colonies of up to sevenfold in cells that received the functional plasmid replicon proving that the hybrid-vector system is functional. Transgene expression could be maintained for 6 weeks and the extrachromosomal plasmid replicon was rescued. To show efficacy in vivo, the adenoviral hybrid-vector system was injected into C57Bl/6 mice. We found that the plasmid replicon can be released from adenoviral DNA in murine liver resulting in long-term transgene expression. In conclusion, we demonstrate the efficacy of our novel HCAdV-pEPito hybrid-vector system in vitro and in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e83; doi:10.1038/mtna.2013.11; published online 2 April 2013.

  2. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout

    PubMed Central

    2013-01-01

    Background Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). Methods VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Results Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. Conclusions A VRP based CHIKV neutralization assay using Gluc as readout

  3. Interferons and Ribavirin Effectively Inhibit Norwalk Virus Replication in Replicon-Bearing Cells▿

    PubMed Central

    Chang, Kyeong-Ok; George, David W.

    2007-01-01

    The development of effective therapies for noroviral gastroenteritis has been hampered by the absence of a cell culture system. Recently, we reported the generation of Norwalk virus (NV) replicon-bearing cells in BHK21 and Huh-7 cells and demonstrated that alpha interferon (IFN-α) effectively inhibited the replication of NV in these cells. In continuing studies for screening potential antinoroviral agents, we tested IFN-γ and ribavirin for their effects on NV replication in the cells. Like IFN-α, IFN-γ inhibited the replication of NV in the replicon-bearing cells, showing the reduction of the NV genome and proteins in a dose-dependent manner. The effective dose for reducing 50% (ED50) of the NV genome and protein was calculated to be approximately 40 units/ml. When ribavirin was applied to the cells, it effectively reduced the NV genome and protein with the ED50 calculated as approximately 40 μM. The combination of IFN-α and ribavirin showed additive effects on the inhibition of NV replication. With the addition of guanosine to the ribavirin treatment, moderately reversed antiviral effects were observed, suggesting that the ribavirin effect may be associated with the depletion of GTP in the cells. Sequencing analysis of the conserved polymerase regions of NV in the ribavirin-treated (100 μM) and nontreated groups showed that the mutation rates were similar and indicated that ribavirin did not induce catastrophic mutations. The NV replicon-bearing cells provide an excellent tool for screening potential antinoroviral agents, and our results indicated that IFNs and ribavirin may be good therapeutic options for noroviral gastroenteritis. PMID:17855555

  4. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout.

    PubMed

    Gläsker, Sabine; Lulla, Aleksei; Lulla, Valeria; Couderc, Therese; Drexler, Jan Felix; Liljeström, Peter; Lecuit, Marc; Drosten, Christian; Merits, Andres; Kümmerer, Beate Mareike

    2013-07-15

    Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. A VRP based CHIKV neutralization assay using Gluc as readout represents a fast and useful method to

  5. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia.

    PubMed

    Althabegoiti, María Julia; Ormeño-Orrillo, Ernesto; Lozano, Luis; Torres Tejerizo, Gonzalo; Rogel, Marco Antonio; Mora, Jaime; Martínez-Romero, Esperanza

    2014-01-08

    Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.

  6. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

    PubMed Central

    2014-01-01

    Background Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Results Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Conclusion Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization

  7. Evaluation of neurovirulence and biodistribution of Venezuelan equine encephalitis replicon particles expressing herpes simplex virus type 2 glycoprotein D.

    PubMed

    Kowalski, Jacek; Adkins, Karissa; Gangolli, Seema; Ren, Jian; Arendt, Heather; DeStefano, Joanne; Obregon, Jennifer; Tummolo, Donna; Natuk, Robert J; Brown, Tom P; Parks, Christopher L; Udem, Stephen A; Long, Deborah

    2007-03-08

    The safety of a propagation-defective Venezuelan equine encephalitis virus (VEEV) replicon particle vaccine was examined in mice. After intracranial inoculation we observed approximately 5% body weight loss, modest inflammatory changes in the brain, genome replication, and foreign gene expression. These changes were transient and significantly less severe than those caused by TC-83, a live-attenuated vaccinal strain of VEEV that has been safely used to immunize military personnel and laboratory workers. Replicon particles injected intramuscularly or intravenously were detected at limited sites 3 days post-administration, and were undetectable by day 22. There was no evidence of dissemination to spinal cord or brain after systemic administration. These results demonstrate that propagation-defective VEEV replicon particles are minimally neurovirulent and lack neuroinvasive potential.

  8. Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle

    USDA-ARS?s Scientific Manuscript database

    Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating...

  9. Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

    PubMed Central

    Porter, D C; Ansardi, D C; Morrow, C D

    1995-01-01

    Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated

  10. Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences.

    PubMed

    Yu, Yun-Zhou; Li, Na; Wang, Wen-Bin; Wang, Shuang; Ma, Yao; Yu, Wei-Yuan; Sun, Zhi-Wei

    2010-11-03

    Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.

  11. Utilization of Two Distinct Modes of Replication by a Hybrid Plasmid Constructed In Vitro from Separate Replicons

    PubMed Central

    Timmis, Kenneth; Cabello, Felipe; Cohen, Stanley N.

    1974-01-01

    A hybrid plasmid, pSC134, that codes for two distinct sets of replication functions has been constructed in vitro by ligation of EcoRI endonuclease-cleaved pSC101 and Col E1 plasmid replicons, and has been introduced into Escherichia coli by transformation. The replication properties of the pSC134 plasmid in DNA polymerase I-defective mutants or in the presence of chloramphenicol indicate that this hybrid plasmid can utilize the functionally distinct modes of replication specified by both of its parent replicons. Images PMID:4612523

  12. Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.

    PubMed

    Date, Tomoko; Kato, Takanobu; Miyamoto, Michiko; Zhao, Zijiang; Yasui, Kotaro; Mizokami, Masashi; Wakita, Takaji

    2004-05-21

    A hepatitis C virus genotype 2a subgenomic replicon, JFH-1 replicon, was previously established using the consensus sequence of clone JFH-1 from a patient with fulminant hepatitis and, in a previous report, was indicated to replicate efficiently in Huh7. Here the replication of JFH-1 replicon was tested in HepG2, a human hepatocyte-derived cell line, and in IMY-N9, a cell line developed by fusing human hepatocytes and HepG2 cells. Following transfection with in vitro transcribed replicon RNA and selection by cultivation with G418, colonies formed in both cell lines although at efficiencies substantially lower than those of Huh7. The H2476L mutation identified in the Huh7 replicon in our previous study increased the colony formation efficiencies of the JFH-1 replicon in HepG2 and IMY-N9 cells. Higher amounts of replicon RNA were detected in IMY-N9 clones than in HepG2 clones by real time detection reverse transcription-PCR, and replicon RNA replication and viral protein expression were confirmed by Northern and Western blotting in isolated clones. Sequencing of replicon RNAs revealed that mutations found in hepatitis C virus-derived regions were not identical and that two of nine HepG2 clones and three of nine IMY-N9 clones had no or one synonymous mutation. This system with the JFH-1 replicon and three cell lines is useful not only for estimating the cellular factors affecting viral activity but also for clarifying the common gene response of the host.

  13. Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

    PubMed

    Kamrud, Kurt I; Alterson, Kim D; Andrews, Chasity; Copp, Laura O; Lewis, Whitney C; Hubby, Bolyn; Patel, Deepa; Rayner, Jonathan O; Talarico, Todd; Smith, Jonathan F

    2008-07-16

    The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

  14. Comparison of two cancer vaccines targeting tyrosinase: plasmid DNA and recombinant alphavirus replicon particles.

    PubMed

    Goldberg, Stacie M; Bartido, Shirley M; Gardner, Jason P; Guevara-Patiño, José A; Montgomery, Stephanie C; Perales, Miguel-Angel; Maughan, Maureen F; Dempsey, JoAnn; Donovan, Gerald P; Olson, William C; Houghton, Alan N; Wolchok, Jedd D

    2005-11-15

    Immunization of mice with xenogeneic DNA encoding human tyrosinase-related proteins 1 and 2 breaks tolerance to these self-antigens and leads to tumor rejection. Viral vectors used alone or in heterologous DNA prime/viral boost combinations have shown improved responses to certain infectious diseases. The purpose of this study was to compare viral and plasmid DNA in combination vaccination strategies in the context of a tumor antigen. Using tyrosinase as a prototypical differentiation antigen, we determined the optimal regimen for immunization with plasmid DNA. Then, using propagation-incompetent alphavirus vectors (virus-like replicon particles, VRP) encoding tyrosinase, we tested different combinations of priming with DNA or VRP followed by boosting with VRP. We subsequently followed antibody production, T-cell response, and tumor rejection. T-cell responses to newly identified mouse tyrosinase epitopes were generated in mice immunized with plasmid DNA encoding human (xenogeneic) tyrosinase. In contrast, when VRP encoding either mouse or human tyrosinase were used as single agents, antibody and T-cell responses and a significant delay in tumor growth in vivo were observed. Similarly, a heterologous vaccine regimen using DNA prime and VRP boost showed a markedly stronger response than DNA vaccination alone. Alphavirus replicon particle vectors encoding the melanoma antigen tyrosinase (self or xenogeneic) induce immune responses and tumor protection when administered either alone or in the heterologous DNA prime/VRP boost approaches that are superior to the use of plasmid DNA alone.

  15. Novel replicons and trans-encapsidation systems for Hepatitis C Virus proteins live imaging and virus-host interaction proteomics.

    PubMed

    Vlaicu, Ovidiu; Selescu, Tudor; Pastrama, Florin; Munteanu, Cristian; Riva, Laura; Dubuisson, Jean; Rouille, Yves; Popescu, Costin-Ioan

    2017-08-01

    Proteomics and imaging techniques are used more and more in tandem to investigate the virus-host interaction. Herein we present novel replicons, methods and trans-encapsidation systems suitable for determination of Hepatitis C Virus (HCV) proteins interactomes and live imaging of viral proteins dynamics in HCV cell culture (HCVcc) system. To identify endogenous factors involved in the HCV life cycle, we constructed full-length functional replicons with affinity purification (AP) tags fused to NS2 and NS5A proteins. Viral-host interactomes were determined and validated in HCVcc system. To investigate the dynamics of viral-host interactions, we developed a core-inducible packaging cell line which trans-encapsidates various subgenomic replicons suitable for AP in replication and assembly stages. Further, a transient trans-encapsidation system was developed for live imaging of the NS5A viral protein in replication and assembly steps, respectively. The NS5A dynamics was determined also in the full-length HCV replicon system. The analysis of NS5A dynamics showed a decreased mobility of the protein in assembly versus the replication step. The tools presented herein will allow the investigation of HCV-host interaction with improved biological relevance and biosafety. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus.

    PubMed

    Pushko, P; Bray, M; Ludwig, G V; Parker, M; Schmaljohn, A; Sanchez, A; Jahrling, P B; Smith, J F

    2000-08-15

    RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.

  17. A polyprotein-expressing salmonid alphavirus replicon induces modest protection in atlantic salmon (Salmo salar) against infectious pancreatic necrosis.

    PubMed

    Abdullah, Azila; Olsen, Christel M; Hodneland, Kjartan; Rimstad, Espen

    2015-01-19

    Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN) in farmed Atlantic salmon (Salmo salar) in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV), was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214) and epithelioma papulosum cyprini (EPC) cells. The replicon constructs pSAV/polyprotein (pSAV/PP) and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar) by a single intramuscular injection and tested in a subsequent IPN virus (IPNV) challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection.

  18. Combined Alphavirus Replicon Particle Vaccine Induces Durable and Cross-Protective Immune Responses against Equine Encephalitis Viruses

    PubMed Central

    Glass, Pamela J.; Bakken, Russell R.; Barth, James F.; Lind, Cathleen M.; da Silva, Luis; Hart, Mary Kate; Rayner, Jonathan; Alterson, Kim; Custer, Max; Dudek, Jeanne; Owens, Gary; Kamrud, Kurt I.; Parker, Michael D.; Smith, Jonathan

    2014-01-01

    ABSTRACT Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV, or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV-IAB virus, Trinidad donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American variety of EEEV. Both the WEEV replicon and combination V/W/E vaccination, however, elicited poor neutralizing antibodies to WEEV in macaques, and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal. IMPORTANCE Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV). Normally transmitted by mosquitoes, these viruses can cause disease when inhaled, so there is concern that these viruses could be used as biological weapons. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis expressing the

  19. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections.

    PubMed

    Yang, Zhenhua; Wu, Rui; Li, Robert W; Li, Ling; Xiong, Zhongliang; Zhao, Haizhong; Guo, Deyin; Pan, Zishu

    2012-04-01

    A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.

  20. In Vitro and In Vivo Characterization of MicroRNA-Targeted Alphavirus Replicon and Helper RNAs ▿ ‡

    PubMed Central

    Kamrud, Kurt I.; Coffield, V. McNeil; Owens, Gary; Goodman, Christin; Alterson, Kim; Custer, Max; Murphy, Michael A.; Lewis, Whitney; Timberlake, Sarah; Wansley, Elizabeth K.; Berglund, Peter; Smith, Jonathan

    2010-01-01

    Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans. PMID:20504925

  1. Effects of activated aflatoxin B/sub 1/ and caffeine on DNA replicon initiation in HeLa cells

    SciTech Connect

    Cramer, P.; Painter, R.B.

    1981-01-01

    Afatoxin B/sub 1/ (AFB/sub 1/) is activated by a rat microsomal extract (S-9) to form a product that inhibits DNA synthesis in HeLa cells. At 10/sup -7/ M, AFB/sub 1/ inhibited initiation of replicons, as shown in alkaline sucrose gradient profiles 30 min after incubation with the drug. Ninety minutes later, the profile of treated cells was similar to that of control, but 4 h later there was another effect on replicon initiation. At 10/sup -6/ M, the inhibition of initiation was greater than at 10/sup -7/ M and increased progressively. Four hours after removal of the drug, the gradient profile showed low amounts of radioactivity in all size classes of DNA. When cells were incubated in medium containing caffeine (2 mM) even as late as 60 min after incubation with AFB/sub 1/, the inhibition of replicon initiation was prevented. If caffeine was later removed from the medium, replicon initiation was then inhibited. At 10/sup -7/ M or 10/sup -6/ M, AFB/sub 1/ had little immediate effect on chain elongation, but at 10/sup -5/ M, the gradient profiles showed an accumulation of low molecular weight DNA molecules, with no radioactivity in the region of high molecular weight DNA, owing to a block to chain elongation; this was not affected by caffeine. These results suggest that AFB/sub 1/ induces damage that changes the fonformation of chromatin so that initiation of new replicons cannot occur; in the presence of caffeine this change does not occur and DNA replication is not inhibited.

  2. Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses.

    PubMed

    Pushko, P; Geisbert, J; Parker, M; Jahrling, P; Smith, J

    2001-12-01

    Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.

  3. Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    PubMed Central

    Utt, Age; Das, Pratyush Kumar; Varjak, Margus; Lulla, Valeria; Lulla, Aleksei

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the

  4. Development and application of West Nile virus subgenomic replicon RNA expressing secreted alkaline phosphatase.

    PubMed

    Moritoh, Kanako; Maeda, Akihiko; Nishino, Tomohiro; Sasaki, Nobuya; Agui, Takashi

    2011-05-01

    We have developed a West Nile virus (WNV) subgenomic replicon harboring the secreted alkaline phosphatase (SEAP) reporter gene instead of viral structural genes (designated repWNV/SEAP). The repWNV/SEAP allowed easy evaluation of viral replication efficiency by direct measurement of SEAP secretion in the cell culture medium in physical containment level 2 facilities. Furthermore, we validated the availability of this system using a known anti-flavivirus gene, mouse oligoadenylate synthetase 1b (Oas1b). The Oas1b-transfected cells were more resistant to repWNV/SEAP replication than the original cells. Thus, this system not only affords a useful tool for identification/evaluation of anti-flavivirus genes/drugs in terms of safety, ease of use and reliability, but should be able to reduce or replace the bioassay using laboratory animals.

  5. Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes.

    PubMed

    Cassetti, M Cristina; McElhiney, Sue P; Shahabi, Vafa; Pullen, Jeffrey K; Le Poole, I Caroline; Eiben, Gretchen L; Smith, Larry R; Kast, W Martin

    2004-01-02

    An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7(49-57) epitope and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7(49-57) epitope was observed in 90% of human leukocyte antigen (HLA)-A(*)0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.

  6. Prime-boost vaccinations using recombinant flavivirus replicon and vaccinia virus vaccines: an ELISPOT analysis.

    PubMed

    Rattanasena, Paweena; Anraku, Itaru; Gardner, Joy; Le, Thuy T; Wang, Xiang Ju; Khromykh, Alexander A; Suhrbier, Andreas

    2011-03-01

    Recombinant Kunjin replicon virus-like particle (VLP), vaccinia virus (rVV) and DNA vaccines were tested in a large series of prime-boost vaccinations using interferon (IFN)γ ELISPOT assays that reflected effector (E), effector memory (EM) and central memory (CM) responses. All vaccine constructs encoded the murine polytope immunogen and responses to four CD8 T-cell epitopes (TYQRTRALV, SYIPSAEKI, YPHFMPTNL and RPQASGVYM) were measured. VLP/rVV out performed (by 14- to 20-fold) DNA/rVV for induction of CM responses, whereas EM responses were only marginally increased. DNA/VLP induced more EM, but not CM responses, than VLP alone, illustrating that DNA priming is not universally beneficial. rVV/VLP gave comparable results to VLP/rVV combinations, although the former induced approximately threefold more E responses, illustrating the utility of poxvirus priming in this setting. Although higher doses of VLP and rVV increased responses after single immunizations, such dose increases provided only marginal benefit in heterologous prime-boost settings. Triple combinations also provided no benefit over two vaccinations. DNA vaccination was associated with broad CM, but not EM responses, and the breadth of EM and E responses was significantly improved by increasing viral vector dose. VLP/rVV, rather than DNA priming, induced T cells with consistently high IFNγ secretion profiles across all ELISPOT measures. Vector-specific CD8 T-cell responses generally correlated well with immunogen-specific responses, although, as expected, single use of each vector reduced the relative levels of vector-specific responses. These experiments illustrate the utility of replicons in heterologous prime-boost vaccinations, and illustrate the diversity of data that can be obtained from ELISPOT analyses.

  7. Zebrafish as a Potential Model Organism for Drug Test Against Hepatitis C Virus

    PubMed Central

    Ding, Cun-Bao; Zhang, Jing-Pu; Zhao, Ye; Peng, Zong-Gen; Song, Dan-Qing; Jiang, Jian-Dong

    2011-01-01

    Screening and evaluating anti- hepatitis C virus (HCV) drugs in vivo is difficult worldwide, mainly because of the lack of suitable small animal models. We investigate whether zebrafish could be a model organism for HCV replication. To achieve NS5B-dependent replication an HCV sub-replicon was designed and created with two vectors, one with HCV ns5b and fluorescent rfp genes, and the other containing HCV's 5′UTR, core, 3′UTR and fluorescent gfp genes. The vectors containing sub-replicons were co-injected into zebrafish zygotes. The sub-replicon amplified in liver showing a significant expression of HCV core RNA and protein. The sub-replicon amplification caused no abnormality in development and growth of zebrafish larvae, but induced gene expression change similar to that in human hepatocytes. As the amplified core fluorescence in live zebrafish was detectable microscopically, it rendered us an advantage to select those with replicating sub-replicon for drug experiments. Ribavirin and oxymatrine, two known anti-HCV drugs, inhibited sub-replicon amplification in this model showing reduced levels of HCV core RNA and protein. Technically, this method had a good reproducibility and is easy to operate. Thus, zebrafish might be a model organism to host HCV, and this zebrafish/HCV (sub-replicon) system could be an animal model for anti-HCV drug screening and evaluation. PMID:21857967

  8. Multiagent Vaccines Vectored by Venezuelan Equine Encephalitis Virus Replicon Elicits Immune Responses to Marburg Virus and Protection Against Anthrax and Botulinum Neurotoxin in Mice

    DTIC Science & Technology

    2006-01-01

    formulations of individual Venezuelan equine encephalitis (VEE) virus replicon- vectored vaccines against a bacterial disease, anthrax; a viral disease...here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease...on days 0, 35, and 70 with the indicated vaccines. Ne b Infectious units were used to measure VRP and milliliters were used to measur c The

  9. VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells

    PubMed Central

    Lin, Kai; Perni, Robert B.; Kwong, Ann D.; Lin, Chao

    2006-01-01

    The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log10 was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log10 reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells. PMID:16641454

  10. In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons.

    PubMed

    Graff, Judith; Nguyen, Hanh; Kasorndorkbua, Chaiyan; Halbur, Patrick G; St Claire, Marisa; Purcell, Robert H; Emerson, Suzanne U

    2005-01-01

    Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

  11. The combined use of alphavirus replicons and pseudoinfectious particles for the discovery of antivirals derived from natural products.

    PubMed

    Delekta, Phillip C; Raveh, Avi; Larsen, Martha J; Schultz, Pamela J; Tamayo-Castillo, Giselle; Sherman, David H; Miller, David J

    2015-06-01

    Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To address this limitation, we developed a western equine encephalitis virus pseudoinfectious particle system that reproduces several crucial viral life cycle steps in addition to genome replication. We used this system to screen a library containing ~26,000 extracts derived from marine microbes, and we identified multiple bacterial strains that produce compounds with potential antiviral activity. We subsequently used pseudoinfectious particle and replicon assays in parallel to counterscreen candidate extracts, and followed antiviral activity during biochemical fractionation and purification to differentiate between inhibitors of viral entry and genome replication. This novel process led to the isolation of a known alphavirus entry inhibitor, bafilomycin, thereby validating the approach for the screening and identification of potential antiviral compounds.

  12. Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles.

    PubMed

    McCullough, Kenneth C; Milona, Panagiota; Thomann-Harwood, Lisa; Démoulins, Thomas; Englezou, Pavlos; Suter, Rolf; Ruggli, Nicolas

    2014-10-16

    Dendritic cells (DC) play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA) carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.

  13. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

    PubMed Central

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo

    2000-01-01

    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  14. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

    PubMed Central

    Lai, Huafang; He, Junyun; Engle, Michael; Diamond, Michael S.; Chen, Qiang

    2011-01-01

    Summary Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties due to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites, and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that is effective, safe, low-cost, and amenable to large-scale manufacturing. PMID:21883868

  15. Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1 replicon.

    PubMed

    Alvarez-Martín, Pablo; Belén Flórez, Ana; Margolles, Abelardo; del Solar, Gloria; Mayo, Baltasar

    2008-08-01

    This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 x 10(1) to 1.0 x 10(5) transformants microg(-1) but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZalpha peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an alpha-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in alpha-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.

  16. Comparison of HCV NS3 protease and NS5B polymerase inhibitor activity in 1a, 1b and 2a replicons and 2a infectious virus.

    PubMed

    Paulson, Matthew S; Yang, Huiling; Shih, I-hung; Feng, Joy Y; Mabery, Eric M; Robinson, Margaret F; Zhong, Weidong; Delaney, William E

    2009-08-01

    The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2'-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61-109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (<3-fold changes in EC(50)) regardless of genotype or subtype. Importantly, we observed a significant correlation (p<0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested.

  17. Venezuelan Equine Encephalitis Replicon Particles Can Induce Rapid Protection against Foot-and-Mouth Disease Virus

    PubMed Central

    Diaz-San Segundo, Fayna; Dias, Camila C. A.; Moraes, Mauro P.; Weiss, Marcelo; Perez-Martin, Eva; Owens, Gary; Custer, Max; Kamrud, Kurt; de los Santos, Teresa

    2013-01-01

    We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/β) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice against either homologous or, in some cases, heterologous virus challenge. As an alternative approach to induce rapid protection against FMDV, we have examined the ability of VRPs containing either the gene for green fluorescent protein (VRP-GFP) or poIFN-α (VRP-poIFN-α) to block FMDV replication in vitro and in vivo. Pretreatment of swine or bovine cell lines with either VRP significantly inhibited subsequent infection with FMDV as early as 6 h after treatment and for at least 120 h posttreatment. Furthermore, mice pretreated with either 107 or 108 infectious units of VRP-GFP and challenged with a lethal dose of FMDV 24 h later were protected from death. Protection was induced as early as 6 h after treatment and lasted for at least 48 h and correlated with induction of an antiviral response and production of IFN-α. By 6 h after treatment several genes were upregulated, and the number of genes and the level of induction increased at 24 h. Finally, we demonstrated that the chemokine IP-10, which is induced by IFN-α and VRP-GFP, is directly involved in protection against FMDV. PMID:23468490

  18. Venezuelan equine encephalitis replicon particles can induce rapid protection against foot-and-mouth disease virus.

    PubMed

    Diaz-San Segundo, Fayna; Dias, Camila C A; Moraes, Mauro P; Weiss, Marcelo; Perez-Martin, Eva; Owens, Gary; Custer, Max; Kamrud, Kurt; de los Santos, Teresa; Grubman, Marvin J

    2013-05-01

    We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/β) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice against either homologous or, in some cases, heterologous virus challenge. As an alternative approach to induce rapid protection against FMDV, we have examined the ability of VRPs containing either the gene for green fluorescent protein (VRP-GFP) or poIFN-α (VRP-poIFN-α) to block FMDV replication in vitro and in vivo. Pretreatment of swine or bovine cell lines with either VRP significantly inhibited subsequent infection with FMDV as early as 6 h after treatment and for at least 120 h posttreatment. Furthermore, mice pretreated with either 10(7) or 10(8) infectious units of VRP-GFP and challenged with a lethal dose of FMDV 24 h later were protected from death. Protection was induced as early as 6 h after treatment and lasted for at least 48 h and correlated with induction of an antiviral response and production of IFN-α. By 6 h after treatment several genes were upregulated, and the number of genes and the level of induction increased at 24 h. Finally, we demonstrated that the chemokine IP-10, which is induced by IFN-α and VRP-GFP, is directly involved in protection against FMDV.

  19. A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells.

    PubMed

    Lee, Jin-Ching; Chang, Ching-Fung; Chi, Ya-Hui; Hwang, Der-Ren; Hsu, John T A

    2004-03-01

    Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.

  20. ParABS Systems of the Four Replicons of Burkholderia cenocepacia: New Chromosome Centromeres Confer Partition Specificity†

    PubMed Central

    Dubarry, Nelly; Pasta, Franck; Lane, David

    2006-01-01

    Most bacterial chromosomes carry an analogue of the parABS systems that govern plasmid partition, but their role in chromosome partition is ambiguous. parABS systems might be particularly important for orderly segregation of multipartite genomes, where their role may thus be easier to evaluate. We have characterized parABS systems in Burkholderia cenocepacia, whose genome comprises three chromosomes and one low-copy-number plasmid. A single parAB locus and a set of ParB-binding (parS) centromere sites are located near the origin of each replicon. ParA and ParB of the longest chromosome are phylogenetically similar to analogues in other multichromosome and monochromosome bacteria but are distinct from those of smaller chromosomes. The latter form subgroups that correspond to the taxa of their hosts, indicating evolution from plasmids. The parS sites on the smaller chromosomes and the plasmid are similar to the “universal” parS of the main chromosome but with a sequence specific to their replicon. In an Escherichia coli plasmid stabilization test, each parAB exhibits partition activity only with the parS of its own replicon. Hence, parABS function is based on the independent partition of individual chromosomes rather than on a single communal system or network of interacting systems. Stabilization by the smaller chromosome and plasmid systems was enhanced by mutation of parS sites and a promoter internal to their parAB operons, suggesting autoregulatory mechanisms. The small chromosome ParBs were found to silence transcription, a property relevant to autoregulation. PMID:16452432

  1. Development and characterization of a replicon-based phenotypic assay for assessing HCV NS4B from clinical isolates.

    PubMed

    Rajyaguru, Sonal; Yang, Huiling; Martin, Ross; Miller, Michael D; Mo, Hongmei

    2013-11-01

    The hepatitis C virus (HCV) NS4B inhibitors have shown potent inhibition of HCV replication in vitro. To assess the effect of viral diversity on the susceptibility to NS4B inhibitors, genotype (GT)-specific GT1a and GT1b replicon shuttle vectors were designed and created for cloning HCV NS4B genes from clinical isolates. For the GT1b NS4B shuttle vector, the S2204I adaptive mutation was introduced in NS5A to improve replication due to the replacement of the K1846T adaptive mutation in NS4B with NS4B from the clinical isolates. In addition to the adaptive mutations, a newly identified Huh-7 cell line, Huh-7-1C, which is highly permissive for both GT1a and GT1b replication, was used to further enhance the replication levels. HCV NS4B gene from clinical isolates was amplified and inserted into the corresponding GT1a and GT1b modified lab strain chimeric replicons. GT1a and GT1b chimeric replicons expressing diverse NS4B genes from corresponding subtypes of clinical isolates replicated at highly efficient levels for phenotypic analysis. Due to natural variation in their amino acid residues in NS4B, these isolates displayed varying drug susceptibilities to an NS4B inhibitor. In mixed populations with wild-type, the sensitivity of resistance detection of NS4B resistant mutants H94R and V105M was between 20% and 80%. The chimeric shuttle vectors can be used to characterize the activity of antiviral drugs targeting NS4B from diverse natural clinical isolates and aid in the development of novel compounds against HCV NS4B.

  2. A chimeric alphavirus RNA replicon gene-based vaccine for human parainfluenza virus type 3 induces protective immunity against intranasal virus challenge.

    PubMed

    Greer, Catherine E; Zhou, Fengmin; Legg, Harold S; Tang, Zequn; Perri, Silvia; Sloan, Barbara A; Megede, Jan Zur; Uematsu, Yasushi; Vajdy, Michael; Polo, John M

    2007-01-05

    Parainfluenza virus type 3 (PIV3) infections continue to be a significant health risk for infants, young children, and immunocompromised adults. We describe a gene-based vaccine strategy against PIV3 using replication-defective alphavirus vectors. These RNA replicon vectors, delivered as virus-like particles and expressing the PIV3 hemagglutinin-neuraminidase glycoprotein, were shown to be highly immunogenic in mice and hamsters, inducing PIV3-specific neutralizing antibody responses. Importantly, the replicon particle-based vaccine administered intramuscularly or intranasally protected against mucosal PIV3 challenge in hamsters, preventing virus replication in both nasal turbinates and lungs. These data suggest that the alphavirus replicon platform can be useful for a PIV3 vaccine and possibly other respiratory viruses.

  3. Replication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis.

    PubMed

    Wilcks, A; Smidt, L; Okstad, O A; Kolsto, A B; Mahillon, J; Andrup, L

    1999-05-01

    A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.

  4. A Vaccinia Virus Recombinant Transcribing an Alphavirus Replicon and Expressing Alphavirus Structural Proteins Leads to Packaging of Alphavirus Infectious Single Cycle Particles

    PubMed Central

    Sánchez-Puig, Juana M.; Lorenzo, María M.; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses. PMID:24130722

  5. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    PubMed

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  6. Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model

    PubMed Central

    Fu, Qiu-Xia; Wang, Li-Cui; Jia, Shuai-Zheng; Gao, Bo; Zhou, Yong; Du, Juan; Wang, Ying-Li; Wang, Xiao-Hui; Peng, Jian-Chun; Zhan, Lin-Sheng

    2010-01-01

    AIM: To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). METHODS: The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting. RESULTS: HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-β promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A. CONCLUSION: Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors. PMID:21105190

  7. Alphavirus replicon-based adjuvants enhance the immunogenicity and effectiveness of Fluzone ® in rhesus macaques.

    PubMed

    Carroll, Timothy D; Matzinger, Shannon R; Barro, Mario; Fritts, Linda; McChesney, Michael B; Miller, Christopher J; Johnston, Robert E

    2011-01-29

    Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone(®) alone or Fluzone(®) mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone(®) alone, Fluzone(®)+null VRP immunized animals had stronger influenza-specific CD4(+) T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone(®)+null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p<0.0001, 1.3 log) and IgA (p<0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone(®)+null VRP immunization was 1.2 log greater (p<0.04) than after two immunizations with Fluzone(®) alone. After virus challenge, only Fluzone(®)+null VRP immunized monkeys had a significantly lower level of viral replication (p<0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone(®)+null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine. Copyright © 2010 Elsevier Ltd. All rights reserved.

  8. Incompatibility and the partitioning site of the repABC basic replicon of the symbiotic plasmid from Rhizobium etli.

    PubMed

    Soberón, Nora; Venkova-Canova, Tatiana; Ramírez-Romero, Miguel A; Téllez-Sosa, Juan; Cevallos, Miguel A

    2004-05-01

    The basic replicon of the symbiotic plasmid (p42d) of Rhizobium etli CE3 is constituted by the repABC operon. Whereas RepC is essential for plasmid replication, RepA and RepB are involved in plasmid partitioning. Three incompatibility regions have been previously identified in this plasmid: the first one encodes RepA, a partitioning protein that also down-regulates the repABC transcription. The second region is situated within the repB-repC intergenic sequence (inc(alpha)), and the last one, inc(beta), is located in a 502 bp EcoRI fragment spanning the last 72-bp of the coding region of repC and the following downstream sequence. In this paper we show that: (1) The inc(beta) region is required for plasmid partitioning. (2) A 16-bp palindrome sequence, located 40 bp downstream of the repC gene of plasmid p42d, is necessary and sufficient to induce incompatibility towards the parental plasmid, and accounts for all the incompatibility properties of this region (inc(beta)). (3). The palindrome is the DNA target site for RepB binding. With these findings we propose that inc(beta) contains the partitioning site (par site) of the basic replicon of plasmid p42d, and that the 16-bp palindrome is the core sequence to nucleate the RepB binding. Copyright 2004 Elsevier Inc.

  9. Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

    PubMed

    Herbert, Andrew S; Kuehne, Ana I; Barth, James F; Ortiz, Ramon A; Nichols, Donald K; Zak, Samantha E; Stonier, Spencer W; Muhammad, Majidat A; Bakken, Russell R; Prugar, Laura I; Olinger, Gene G; Groebner, Jennifer L; Lee, John S; Pratt, William D; Custer, Max; Kamrud, Kurt I; Smith, Jonathan F; Hart, Mary Kate; Dye, John M

    2013-05-01

    There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

  10. ALPHAVIRUS REPLICON PARTICLES ACTING AS ADJUVANTS PROMOTE CD8+ T CELL RESPONSES TO CO-DELIVERED ANTIGEN

    PubMed Central

    Thompson, Joseph M.; Whitmore, Alan C.; Staats, Herman F.; Johnston, Robert E.

    2013-01-01

    Alphavirus replicon particles induce strong antibody and CD8+ T cell responses to expressed antigens in numerous experimental systems. We have recently demonstrated that Venezuelan equine encephalitis virus replicon particles (VRP) possess adjuvant activity for systemic and mucosal antibody responses. In this report, we demonstrate that VRP induced an increased and balanced serum IgG subtype response to co-delivered antigen, with simultaneous induction of antigen-specific IgG1 and IgG2a antibodies, and increased both systemic and mucosal antigen-specific CD8+ T cell responses, as measured by an IFN-γ ELISPOT assay. Additionally, VRP further increased antigen-specific T cell immunity in an additive fashion following co-delivery with the TLR ligand, CpG DNA. VRP infection led to recruitment of CD8+ T cells into the mucosal compartment, possibly utilizing the mucosal homing receptor, as this integrin was upregulated on CD8+ T cells in the draining lymph node of VRP-infected animals, where VRP-infected dendritic cells reside. This newly recognized ability of VRP to mediate increased T cell response towards co-delivered antigen provides the potential to both define the molecular basis of alphavirus-induced immunity, and improve alphavirus-based vaccines. PMID:18582997

  11. Alphavirus replicon particles acting as adjuvants promote CD8+ T cell responses to co-delivered antigen.

    PubMed

    Thompson, Joseph M; Whitmore, Alan C; Staats, Herman F; Johnston, Robert E

    2008-08-05

    Alphavirus replicon particles induce strong antibody and CD8+ T cell responses to expressed antigens in numerous experimental systems. We have recently demonstrated that Venezuelan equine encephalitis virus replicon particles (VRP) possess adjuvant activity for systemic and mucosal antibody responses. In this report, we demonstrate that VRP induced an increased and balanced serum IgG subtype response to co-delivered antigen, with simultaneous induction of antigen-specific IgG1 and IgG2a antibodies, and increased both systemic and mucosal antigen-specific CD8+ T cell responses, as measured by an IFN-gamma ELISPOT assay. Additionally, VRP further increased antigen-specific T cell immunity in an additive fashion following co-delivery with the TLR ligand, CpG DNA. VRP infection led to recruitment of CD8+ T cells into the mucosal compartment, possibly utilizing the mucosal homing receptor, as this integrin was upregulated on CD8+ T cells in the draining lymph node of VRP-infected animals, where VRP-infected dendritic cells reside. This newly recognized ability of VRP to mediate increased T cell response towards co-delivered antigen provides the potential to both define the molecular basis of alphavirus-induced immunity, and improve alphavirus-based vaccines.

  12. Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus

    PubMed Central

    Herbert, Andrew S.; Kuehne, Ana I.; Barth, James F.; Ortiz, Ramon A.; Nichols, Donald K.; Zak, Samantha E.; Stonier, Spencer W.; Muhammad, Majidat A.; Bakken, Russell R.; Prugar, Laura I.; Olinger, Gene G.; Groebner, Jennifer L.; Lee, John S.; Pratt, William D.; Custer, Max; Kamrud, Kurt I.; Smith, Jonathan F.; Hart, Mary Kate

    2013-01-01

    There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine. PMID:23408633

  13. Restricted and selective tropism of a Venezuelan equine encephalitis virus-derived replicon vector for human dendritic cells.

    PubMed

    Nishimoto, Kevin P; Laust, Amanda K; Wang, Kehui; Kamrud, Kurt I; Hubby, Bolyn; Smith, Jonathan F; Nelson, Edward L

    2007-01-01

    Dendritic cells (DCs) consist of heterogeneous phenotypic populations and have diverse immunostimulatory functions dependent on both lineage and functional phenotype, but as exceptionally potent antigen-presenting cells, they are targets for generating effective antigen-specific immune responses. A promising replicon particle vector derived from Venezuelan equine encephalitis virus (VEE) has been reported to transduce murine footpad DCs. However, the receptive DC subset, the degree of restriction for this tropism, and the extent of conservation between rodents and humans have not been well characterized. Using fresh peripheral blood DCs, mononuclear cells, monocyte-derived macrophages, and monocyte-derived DCs, our results demonstrate conservation of VEE replicon particle (VRP) tropism for DCs between humans and rodents. We observed that a subset of immature myeloid DCs is the target population, and that VRP-transduced immature DCs retain intact functional capacity, for example, the ability to resist the cytopathic effects of VRP transduction and the capacity to acquire the mature phenotype. These studies support the demonstration of selective VRP tropism for human DCs and provide further insight into the biology of the VRP vector, its parent virus, and human DCs.

  14. Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1

    PubMed Central

    Berkner, Silvia; Hinojosa, Mery Pina; Prangishvili, David; Lipps, Georg

    2014-01-01

    We have determined the minimal replicon of the crenarchaeal plasmid pRN1. It consists of 3097 base pairs amounting to 58% of the genome of pRN1. The minimal replicon comprises replication operon orf56/orf904 coding for a transcriptional repressor and the replication protein of pRN1. An upstream region of 64 bp that contains the promoter of the replication operon is essential as well as 166 bp of sequence downstream of the orf904 gene. This region contains a putative transcriptional terminator and a 100 nucleotides long stem–loop structure. Only the latter structure was shown to be required for replication. In addition replication was sustained when the stem–loop was displaced to another part of the pRN1 sequence. By mutational analysis we also find that the integrity of the stem–loop structure is required to maintain the replication of pRN1-derived constructs. As similar stem–loop structures are also present in other members of the pRN family, we suggest that this conserved structural element could be the origin of replication for the pRN plasmids. Further bioinformatic analysis revealed that the domain structure of the replication protein and the presence of a similar stem–loop structure as the putative replication origin are also found in several bacteriophages. PMID:25060695

  15. Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system.

    PubMed

    Bourne, Nigel; Pyles, Richard B; Yi, MinKyung; Veselenak, Ronald L; Davis, Melissa M; Lemon, Stanley M

    2005-08-01

    We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.

  16. Large plasmids of Escherichia coli and Salmonella encode highly diverse arrays of accessory genes on common replicon families.

    PubMed

    Williams, Laura E; Wireman, Joy; Hilliard, Valda C; Summers, Anne O

    2013-01-01

    Plasmids are important in evolution and adaptation of host bacteria, yet we lack a comprehensive picture of their own natural variation. We used replicon typing and RFLP analysis to assess diversity and distribution of plasmids in the ECOR, SARA, SARB and SARC reference collections of Escherichia coli and Salmonella. Plasmids, especially large (≥30 kb) plasmids, are abundant in these collections. Host species and genotype clearly impact plasmid prevalence; plasmids are more abundant in ECOR than SAR, but, within ECOR, subgroup B2 strains have the fewest large plasmids. The majority of large plasmids have unique RFLP patterns, suggesting high variation, even within dominant replicon families IncF and IncI1. We found only four conserved plasmid types within ECOR, none of which are widely distributed. Within SAR, conserved plasmid types are primarily serovar-specific, including a pSLT-like plasmid in 13 Typhimurium strains. Conservation of pSLT contrasts with variability of other plasmids, suggesting evolution of serovar-specific virulence plasmids is distinct from that of most enterobacterial plasmids. We sequenced a conserved serovar Heidelberg plasmid but did not detect virulence or antibiotic resistance genes. Our data illustrate the high degree of natural variation in large plasmids of E. coli and Salmonella, even among plasmids sharing backbone genes.

  17. The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids.

    PubMed

    Weaver, Keith E; Kwong, Stephen M; Firth, Neville; Francia, Maria Victoria

    2009-03-01

    The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

  18. High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system.

    PubMed

    Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang; Piensook, Khanrat; Cardineau, Guy; Zeitlin, Larry; Whaley, Kevin J; Arntzen, Charles J; Mason, Hugh S; Chen, Qiang

    2010-05-01

    Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of hetero-oligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the "competing" nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al., 2009. Biotechnol Bioeng 103: 706-714). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al., 2000. Science 287: 1664-1666) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post-infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for hetero-oligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a

  19. Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates

    PubMed Central

    Bates, John T.; Pickens, Jennifer A.; Schuster, Jennifer E.; Johnson, Monika; Tollefson, Sharon J.; Williams, John V.; Davis, Nancy L.; Johnston, Robert E.; Schultz-Darken, Nancy; Slaughter, James C.; Smith-House, Frances; Crowe, James E.

    2016-01-01

    Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development. PMID:26772634

  20. A Kunjin Replicon Virus-like Particle Vaccine Provides Protection Against Ebola Virus Infection in Nonhuman Primates

    PubMed Central

    Pyankov, Oleg V.; Bodnev, Sergey A.; Pyankova, Olga G.; Solodkyi, Vladislav V.; Pyankov, Stepan A.; Setoh, Yin Xiang; Volchkova, Valentina A.; Suhrbier, Andreas; Volchkov, Viktor V.; Agafonov, Alexander A.; Khromykh, Alexander A.

    2015-01-01

    The current unprecedented outbreak of Ebola virus (EBOV) disease in West Africa has demonstrated the urgent need for a vaccine. Here, we describe the evaluation of an EBOV vaccine candidate based on Kunjin replicon virus-like particles (KUN VLPs) encoding EBOV glycoprotein with a D637L mutation (GP/D637L) in nonhuman primates. Four African green monkeys (Cercopithecus aethiops) were injected subcutaneously with a dose of 109 KUN VLPs per animal twice with an interval of 4 weeks, and animals were challenged 3 weeks later intramuscularly with 600 plaque-forming units of Zaire EBOV. Three animals were completely protected against EBOV challenge, while one vaccinated animal and the control animal died from infection. We suggest that KUN VLPs encoding GP/D637L represent a viable EBOV vaccine candidate. PMID:25732811

  1. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies

    PubMed Central

    Ricklin, Meret E.; Vielle, Nathalie J.; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4+ T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value. PMID:27446083

  2. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies.

    PubMed

    Ricklin, Meret E; Vielle, Nathalie J; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4(+) T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value.

  3. Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

    PubMed Central

    Chain, Patrick S. G.; Denef, Vincent J.; Konstantinidis, Konstantinos T.; Vergez, Lisa M.; Agulló, Loreine; Reyes, Valeria Latorre; Hauser, Loren; Córdova, Macarena; Gómez, Luis; González, Myriam; Land, Miriam; Lao, Victoria; Larimer, Frank; LiPuma, John J.; Mahenthiralingam, Eshwar; Malfatti, Stephanie A.; Marx, Christopher J.; Parnell, J. Jacob; Ramette, Alban; Richardson, Paul; Seeger, Michael; Smith, Daryl; Spilker, Theodore; Sul, Woo Jun; Tsoi, Tamara V.; Ulrich, Luke E.; Zhulin, Igor B.; Tiedje, James M.

    2006-01-01

    Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven “central aromatic” and twenty “peripheral aromatic” pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes. PMID:17030797

  4. Molecular Smallpox Vaccine Delivered by Alphavirus Replicons Elicits Protective Immunity in Mice and Non-human Primates

    PubMed Central

    Hooper, Jay W.; Ferro, Anthony M.; Golden, Joseph W.; Silvera, Peter; Dudek, Jeanne; Alterson, Kim; Custer, Max; Rivers, Bryan; Morris, John; Owens, Gary; Smith, Jonathan F.; Kamrud, Kurt I.

    2009-01-01

    Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 70s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRP) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 106 PFU of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine. PMID:19833247

  5. Molecular smallpox vaccine delivered by alphavirus replicons elicits protective immunity in mice and non-human primates.

    PubMed

    Hooper, Jay W; Ferro, Anthony M; Golden, Joseph W; Silvera, Peter; Dudek, Jeanne; Alterson, Kim; Custer, Max; Rivers, Bryan; Morris, John; Owens, Gary; Smith, Jonathan F; Kamrud, Kurt I

    2009-12-11

    Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 1970s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRPs) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 x 10(6)pfu of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine.

  6. Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles

    PubMed Central

    Tonkin, Daniel R; Whitmore, Alan; Johnston, Robert E; Barro, Mario

    2012-01-01

    Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity. PMID:22531556

  7. Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles.

    PubMed

    Tonkin, Daniel R; Whitmore, Alan; Johnston, Robert E; Barro, Mario

    2012-06-22

    Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity. Published by Elsevier Ltd.

  8. Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility.

    PubMed

    Chain, Patrick S G; Denef, Vincent J; Konstantinidis, Konstantinos T; Vergez, Lisa M; Agulló, Loreine; Reyes, Valeria Latorre; Hauser, Loren; Córdova, Macarena; Gómez, Luis; González, Myriam; Land, Miriam; Lao, Victoria; Larimer, Frank; LiPuma, John J; Mahenthiralingam, Eshwar; Malfatti, Stephanie A; Marx, Christopher J; Parnell, J Jacob; Ramette, Alban; Richardson, Paul; Seeger, Michael; Smith, Daryl; Spilker, Theodore; Sul, Woo Jun; Tsoi, Tamara V; Ulrich, Luke E; Zhulin, Igor B; Tiedje, James M

    2006-10-17

    Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven "central aromatic" and twenty "peripheral aromatic" pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

  9. 5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

    PubMed Central

    Diamos, Andrew G.; Rosenthal, Sun H.; Mason, Hugh S.

    2016-01-01

    We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody

  10. A Revisionist Look at Population and Growth.

    ERIC Educational Resources Information Center

    Holden, Constance

    1986-01-01

    Presents highlights from a National Research Council report titled "Population Growth and Economic Questions: Policy Questions." Includes brief comments on the report's conclusions related to exhaustible resources, renewable resources, pollution, work productivity, economics of scale and technological innovation, schooling, income…

  11. A Revisionist Look at Population and Growth.

    ERIC Educational Resources Information Center

    Holden, Constance

    1986-01-01

    Presents highlights from a National Research Council report titled "Population Growth and Economic Questions: Policy Questions." Includes brief comments on the report's conclusions related to exhaustible resources, renewable resources, pollution, work productivity, economics of scale and technological innovation, schooling, income…

  12. A revisionist timetable for the ice ages

    SciTech Connect

    Kerr, R.A.

    1992-10-09

    In terms of sheer mass, there's no contest. In one corner, there's a land-based record of ice age climates that takes the form of a single carbonate cylinder about the size of the cardboard tube in a roll of paper towels. In the other corner, there's the marine record, which draws on the tons of deep-sea mud cored around the world during the past 20 years. But a group of researchers argues that the lone continental record, drilled from a wall of calcite in Devil's Hole, Nevada, is enough to unseat the conventional wisdom about the causes of the ice ages. The reason a single stick of carbonate has received all this attention is the unique resource it contains: a precisely dated continental climate record of the past 600,000 years. The record was deposited from ground water, which carried a measure of air temperature in the form of the water's oxygen isotope composition. As the water seeped into Devil's Hole - an open, water-filled fault zone - carbonate crystallized out, locking up some of the water's oxygen and building up a climate record layer by layer. Drilling into the walls of the fault, a core was retrieved spanning layers formed between 60,000 and 560,000 years ago, as measured by high-precision uranium thorium dating.

  13. An integrated approach identifies IFN-regulated microRNAs and targeted mRNAs modulated by different HCV replicon clones

    PubMed Central

    2011-01-01

    Background Infections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-β (IFN-β) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system). Methods The expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction. Results The expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection. Conclusions The present findings reveal that 3 IFN-β-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use

  14. The alien replicon: Artificial genetic constructs to direct the synthesis of transmissible self-replicating RNAs: In vivo synthesised heterologous (alien) RNA constructs are capable of initiating self-replication following transmission to the host organism.

    PubMed

    Kochetov, Alex V

    2014-12-01

    Artificial genetic constructs that direct the synthesis of self-replicating RNA molecules are used widely to induce gene silencing, for bioproduction, and for vaccination. Interestingly, one variant of the self-replicon has not been discussed in the literature: namely, transgenic organisms that synthesise alien replicons. For example, plant cells may be easily genetically modified to produce bacteriophages or insect viruses. Alien replicon-producing organisms (ARPOs) may serve as a unique tool for biocontrol or to selectively influence the characteristics of a target organism. The ARPO approach would have to meet strict biosafety criteria, and its practical applications are problematic. However, a discussion on ARPO applicability would be valuable to outline the full set of options available in the bioengineering toolbox. In this paper, RNA replicons for bioengineering are reviewed briefly, and the ARPO approach is discussed.

  15. Assessment of the effectiveness of a nuclear-launched TMV-based replicon as a tool for foreign gene expression in plants in comparison to direct gene expression from a nuclear promoter.

    PubMed

    Man, Michal; Epel, Bernard L

    2006-02-01

    An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.

  16. Occurrence of 20S RNA and 23S RNA replicons in industrial yeast strains and their variation under nutritional stress conditions.

    PubMed

    López, Victoria; Gil, Rosario; Vicente Carbonell, José; Navarro, Alfonso

    2002-04-01

    We have characterized industrial yeast strains used in the brewing, baking, and winemaking industries for the presence or absence of cytoplasmic single-stranded 20S and 23S RNAs. Furthermore, the variation of intracellular concentrations of these replicons in brewing and laboratory strains under nutritional stress conditions was determined. Our results show a correlation between the relative abundance of these replicons and exposure of yeast to nutritionally stressful conditions, indicating that these RNAs could be employed as molecular probes to evaluate the exposure of 20S(+) and/or 23S(+) yeast strains to stress situations during industrial manipulation. During this study, several 20S(-)23S(+) Saccharomyces cerevisiae strains were isolated and identified. This is the first time that a yeast strain containing only 23S RNA has been reported, demonstrating that 20S RNA is not required for 23S RNA replication. Copyright 2002 John Wiley & Sons, Ltd.

  17. Construction of a shuttle lacZ alpha-based Escherichia coli-actinomycetes vector containing the phage JHJ-3 replicon.

    PubMed

    Gaudreau, L R; Le Gouill, C; Déry, C V

    1992-12-01

    Using the broad replicating range JHJ-3 phage replicon, a shuttle vector for Escherichia coli and actinomycetes has been constructed. The vector, pOJ31, bears the lacZ alpha fragment allowing a blue/white gene cloning system. pOJ31 also contains a polylinker of 15 unique cloning sites and the phage T7 promoter. The vector has been used to stably express the mel gene from plasmid pIJ702 in Streptomyces lividans.

  18. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    PubMed

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines.

  19. Induction and Characterization of Immune Responses in Small Animals Using a Venezuelan Equine Encephalitis Virus (VEE) Replicon System, Expressing Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Genes

    DTIC Science & Technology

    2003-01-01

    Montelaro, and C. J. Issel. 1995. Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid...371-8. 64 36. Davis, N. L., L. V. Willis, J. F. Smith, and R. E. Johnston. 1989. In vitro synthesis of infectious venezuelan equine encephalitis...Characterization of Immune Responses in small animals using a Venezuelan Equine Encephalitis Virus (VEE) Replicon System, Expressing Human

  20. Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection

    PubMed Central

    2014-01-01

    Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial. PMID:24928313

  1. Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

    DOE PAGES

    Groom, Joseph; Chung, Daehwan; Olson, Daniel G.; ...

    2016-01-29

    Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproduciblymore » transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.« less

  2. Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

    SciTech Connect

    Groom, Joseph; Chung, Daehwan; Olson, Daniel G.; Lynd, Lee R.; Guss, Adam M.; Westpheling, Janet

    2016-01-29

    Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.

  3. The tra locus of streptomycete plasmid pIJ101 mediates efficient transfer of a circular but not a linear version of the same replicon.

    PubMed

    Wang, Jing; Pettis, Gregg S

    2010-09-01

    Conjugal transfer of circular plasmids in Streptomyces involves a unique mechanism employing few plasmid-encoded loci and the transfer of double-stranded DNA by an as yet uncharacterized intercellular route. Efficient transfer of the circular streptomycete plasmid pIJ101 requires only two plasmid loci: the pIJ101 tra gene, and as a cis-acting function known as clt. Here, we compared the ability of the pIJ101 transfer apparatus to promote conjugal transfer of circular versus linear versions of the same replicon. While the pIJ101 tra locus readily transferred the circular form of the replicon, the linear version was transferred orders of magnitude less efficiently and all plasmids isolated from the transconjugants were circular, regardless of their original configuration in the donor. Additionally, relatively rare circularization of linear plasmids was detectable in the donor cells, which is consistent with the notion that this event was a prerequisite for transfer by TraB(pIJ101). Linear versions of this same replicon did transfer efficiently, in that configuration, from strains containing the conjugative linear plasmid SLP2. Our data indicate that functions necessary and sufficient for transfer of circular DNA were insufficient for transfer of a related linear DNA molecule. The results here suggest that the conjugation mechanisms of linear versus circular DNA in Streptomyces spp. are inherently different and/or that efficient transfer of linear DNA requires additional components.

  4. Rapid screening for dominant negative mutations in the beet necrotic yellow vein virus triple gene block proteins P13 and P15 using a viral replicon.

    PubMed

    Lauber, E; Janssens, L; Weyens, G; Jonard, G; Richards, K E; Lefèbvre, M; Guilley, H

    2001-08-01

    Point mutations were introduced into the genes encoding the triple gene bock movement proteins P13 and P15 of beet necrotic yellow vein virus (BNYVV). Mutations which disabled viral cell-to-cell movement in Chenopodium quinoa were then tested for their ability to act as dominant negative inhibiters of movement of wild-type BNYVV when expressed from a co-inoculated BNYVV RNA 3-based replicon. For P13, three types of mutation inhibited the movement function: non-synomynous mutations in the N- and C-terminal hydrophobic domains, a mutation at the boundary between the N-terminal hydrophobic domain and the central hydrophilic domain (mutant P13-A12), and mutations in the conserved sequence motif in the central hydrophilic domain. However, only the 'boundary' mutant P13-A12 strongly inhibited movement of wild-type virus when expressed from the co-inoculated replicon. Similar experiments with P15 detected four movement-defective mutants which strongly inhibited cell-to-cell movement of wild-type BNYVV when the mutants were expressed from a co-inoculated replicon. Beta vulgaris transformed with two of these P15 mutants were highly resistant to fungus-mediated infection with BNYVV.

  5. Dual Roles of Two Isoforms of Autophagy-related Gene ATG10 in HCV-Subgenomic replicon Mediated Autophagy Flux and Innate Immunity.

    PubMed

    Zhao, Qiong; Hu, Zhan-Ying; Zhang, Jing-Pu; Jiang, Jian-Dong; Ma, Yuan-Yuan; Li, Jian-Rui; Peng, Zong-Gen; Chen, Jin-Hua

    2017-09-12

    Autophagy and immune response are two defense systems that human-body uses against viral infection. Previous studies documented that some viral mechanisms circumvented host immunity mechanisms and hijacked autophagy for its replication and survival. Here, we focus on interactions between autophagy mechanism and innate-immune-response in HCV-subgenomic replicon cells to find a mechanism linking the two pathways. We report distinct effects of two autophagy-related protein ATG10s on HCV-subgenomic replication. ATG10, a canonical long isoform in autophagy process, can facilitate HCV-subgenomic replicon amplification by promoting autophagosome formation and by combining with and detaining autophagosomes in cellular periphery, causing impaired autophagy flux. ATG10S, a non-canonical short isoform of ATG10 proteins, can activate expression of IL28A/B and immunity genes related to viral ds-RNA including ddx-58, tlr-3, tlr-7, irf-3 and irf-7, and promote autophagolysosome formation by directly combining and driving autophagosomes to perinuclear region where lysosomes gather, leading to lysosomal degradation of HCV-subgenomic replicon in HepG2 cells. ATG10S also can suppress infectious HCV virion replication in Huh7.5 cells. Another finding is that IL28A protein directly conjugates ATG10S and helps autophagosome docking to lysosomes. ATG10S might be a new host factor against HCV replication, and as a target for screening chemicals with new anti-virus mechanisms.

  6. The use of synthetic oligonucleotides with universal templates for rapid DNA sequencing: results with staphylococcal replicon pC221.

    PubMed

    Brenner, D G; Shaw, W V

    1985-02-01

    A rapid sequencing strategy has been devised and applied to determine the complete nucleotide sequence (4555 bp) of Staphylococcus aureus plasmid pC221. The entire replicon was cloned into phage M13mp8 in both orientations to provide 'universal templates' for primed DNA synthesis from internally-sited oligonucleotide primers. The latter were synthesized by a modification of a recently described paper disc method which employs phosphotriester chemistry. Less than 4 weeks was required for the synthesis of the required primers and for the sequencing experiments. Plasmid pC221 bears a substrate-inducible chloramphenicol acetyltransferase (CAT) gene that shares much homology with its counterparts in pC194 (S. aureus) and the chromosomal cat-86 gene of Bacillus pumilus, both in coding regions and upstream sequences believed to be involved in the induction phenomenon. A second plasmid-specified protein, REP D, has an 81% identity in the REP C polypeptide that has been shown to be essential for the replication of staphylococcal plasmid pT181. The 5' flanking region of rep D shows striking similarities with its counterpart in rep C that determines copy number and incompatibility. The nucleotide sequence reveals two additional and overlapping open reading frames that may specify proteins that play roles in plasmid relaxation and transfer.

  7. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  8. Profiling of antimicrobial resistance and plasmid replicon types in β-lactamase producing Escherichia coli isolated from Korean beef cattle

    PubMed Central

    Shin, Seung Won; Jung, Myunghwan; Shin, Min-Kyung

    2015-01-01

    In this study, 78 isolates of Escherichia coli isolated from Korean beef cattle farms were investigated for the production of extended-spectrum β-lactamase (ESBL) and/or AmpC β-lactamase. In the disc diffusion test with ampicillin, amoxicillin, cephalothin, ceftiofur, cefotaxime, ceftazidime, and cefoxitin, 38.5% of the isolates showed resistance to all of ampicillin, amoxicillin, and cephalothin. The double disc synergy method revealed that none of the isolates produced ESBL or AmpC β-lactamases. DNA sequencing showed that all isolates encoded genes for TEM-1-type β-lactamase. Moreover, 78.2% of the isolates transferred the TEM-1-type β-lactamase gene via conjugation. In plasmid replicon typing of all donors, IncFIB and IncFIA were identified in 71.4% and 41.0% of plasmids, respectively. In transconjugants, IncFIB and IncFIA were the most frequent types detected (61.5% and 41.0%, respectively). Overall, the present study indicates that selection pressures of antimicrobials on β-lactamases in beef cattle may be low relative to other livestock animals in Korea. Moreover, to reduce selection pressure and dissemination of β-lactamase, the long-term surveillance of antimicrobial use in domestic beef cattle should be established. PMID:26119172

  9. Alphavirus replicon-based enhancement of mucosal and systemic immunity is linked to the innate response generated by primary immunization

    PubMed Central

    Tonkin, Daniel R; Jorquera, Patricia; Todd, Tracie; Beard, Clayton W; Johnston, Robert E; Barro, Mario

    2010-01-01

    Venezuelan equine encephalitis virus replicon particles (VRP) function as an effective systemic, cellular and mucosal adjuvant when codelivered with antigen, and show promise for use as a component in new and existing human vaccine formulations. We show here that VRP are effective at low dose and by intramuscular delivery, two useful features for implementation of VRP as a vaccine adjuvant. In mice receiving a prime and boost with antigen, we found that VRP are required in prime only to produce a full adjuvant effect. This outcome indicates that the events triggered during prime with VRP are sufficient to establish the nature and magnitude of the immune response to a second exposure to antigen. Events induced by VRP in the draining lymph node after prime include robust secretion of many inflammatory cytokines, upregulation of CD69 on leukocytes, and increased cellularity, with a disproportionate increase of a cell population expressing CD11c, CD11b, and F4/80. We show that antigen delivered 24 hours after administration of VRP does not benefit from an adjuvant effect, indicating that the events which are critical to VRP-mediated adjuvant activity occur within the first 24 hours. Further studies of the events induced by VRP will help elucidate the mechanism of VRP adjuvant activity and will advance the safe implementation of this adjuvant in human vaccines. PMID:20184975

  10. Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge.

    PubMed

    Thornburg, Natalie J; Ray, Caroline A; Collier, Martha L; Liao, Hua-Xin; Pickup, David J; Johnston, Robert E

    2007-06-05

    An anti-poxvirus vaccine based on replicon particles of Venezuelan equine encephalitis virus (VRP) is being developed. The cowpox virus genes encoding structural proteins corresponding to vaccinia virus proteins A33, B5, and A27 were each expressed from VRP. High serum IgG titers against these proteins were generated in BALB/c mice vaccinated with each of these VRP. VRP induced both IgG1 and IgG2a with a strong predominance of IgG2a production. The response is long-lasting, as evidenced by the retention of high anti-B5 serum IgG titers through at least 50 weeks after priming immunization. Mice vaccinated with B5-, A33- or A27-VRP individually or together survived intranasal challenge with cowpox virus, with the multivalent vaccine formulation providing more effective protection from weight loss and clinical signs of illness than the monovalent vaccines. These results demonstrate that VRP may provide an effective alternative to vaccinia virus vaccines against poxvirus infection.

  11. Effects of rapid antigen degradation and VEE glycoprotein specificity on immune responses induced by a VEE replicon vaccine.

    PubMed

    Fluet, M E; Whitmore, A C; Moshkoff, D A; Fu, K; Tang, Y; Collier, M L; West, A; Moore, D T; Swanstrom, R; Johnston, R E; Davis, N L

    2008-01-05

    Genetic vaccines are engineered to produce immunogens de novo in the cells of the host for stimulation of a protective immune response. In some of these systems, antigens engineered for rapid degradation have produced an enhanced cellular immune response by more efficient entry into pathways for processing and presentation of MHC class I peptides. VEE replicon particles (VRP), single cycle vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), are examined here for the effect of an increased rate of immunogen degradation on VRP vaccine efficacy. VRP expressing the matrix capsid (MA/CA) portion of SIV Gag were altered to promote rapid degradation of MA/CA by various linkages to co-translated ubiquitin or by destabilizing mutations and were used to immunize BALB/c mice for quantitation of anti-MA/CA cellular and humoral immune responses. Rapid degradation by the N-end rule correlated with a dampened immune response relative to unmodified MA/CA when the VRP carried a glycoprotein spike from an attenuated strain of VEE. In contrast, statistically equivalent numbers of IFNgamma(+)T-cells resulted when VRP expressing unstable MA/CA were packaged with the wild-type VEE glycoproteins. These results suggest that the cell types targeted in vivo by VRP carrying mutant or wild type glycoprotein spikes are functionally different, and are consistent with previous findings suggesting that wild-type VEE glycoproteins preferentially target professional antigen presenting cells that use peptides generated from the degraded antigen for direct presentation on MHC.

  12. Acute infection with venezuelan equine encephalitis virus replicon particles catalyzes a systemic antiviral state and protects from lethal virus challenge.

    PubMed

    Konopka, Jennifer L; Thompson, Joseph M; Whitmore, Alan C; Webb, Drue L; Johnston, Robert E

    2009-12-01

    The host innate immune response provides a critical first line of defense against invading pathogens, inducing an antiviral state to impede the spread of infection. While numerous studies have documented antiviral responses within actively infected tissues, few have described the earliest innate response induced systemically by infection. Here, utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) to limit infection to the initially infected cells in vivo, a rapid activation of the antiviral response was demonstrated not only within the murine draining lymph node, where replication was confined, but also within distal tissues. In the liver and brain, expression of interferon-stimulated genes was detected by 1 to 3 h following VRP footpad inoculation, reaching peak expression of >100-fold over that in mock-infected animals. Moreover, mice receiving a VRP footpad inoculation 6, 12, or 24 h prior to an otherwise lethal VEE footpad challenge were completely protected from death, including a drastic reduction in challenge virus titers. VRP pretreatment also provided protection from intranasal VEE challenge and extended the average survival time following intracranial challenge. Signaling through the interferon receptor was necessary for antiviral gene induction and protection from VEE challenge. However, VRP pretreatment failed to protect mice from a heterologous, lethal challenge with vesicular stomatitis virus, yet conferred protection following challenge with influenza virus. Collectively, these results document a rapid modulation of the host innate response within hours of infection, capable of rapidly alerting the entire animal to pathogen invasion and leading to protection from viral disease.

  13. Alphavirus replicon-based enhancement of mucosal and systemic immunity is linked to the innate response generated by primary immunization.

    PubMed

    Tonkin, Daniel R; Jorquera, Patricia; Todd, Tracie; Beard, Clayton W; Johnston, Robert E; Barro, Mario

    2010-04-19

    Venezuelan equine encephalitis virus replicon particles (VRP) function as an effective systemic, cellular and mucosal adjuvant when codelivered with antigen, and show promise for use as a component in new and existing human vaccine formulations. We show here that VRP are effective at low dose and by intramuscular delivery, two useful features for implementation of VRP as a vaccine adjuvant. In mice receiving a prime and boost with antigen, we found that VRP are required in prime only to produce a full adjuvant effect. This outcome indicates that the events triggered during prime with VRP are sufficient to establish the nature and magnitude of the immune response to a second exposure to antigen. Events induced by VRP in the draining lymph node after prime include robust secretion of many inflammatory cytokines, upregulation of CD69 on leukocytes, and increased cellularity, with a disproportionate increase of a cell population expressing CD11c, CD11b, and F4/80. We show that antigen delivered 24h after administration of VRP does not benefit from an adjuvant effect, indicating that the events which are critical to VRP-mediated adjuvant activity occur within the first 24h. Further studies of the events induced by VRP will help elucidate the mechanism of VRP adjuvant activity and will advance the safe implementation of this adjuvant in human vaccines. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system.

    PubMed

    Guglielmetti, Simone; Karp, Matti; Mora, Diego; Tamagnini, Isabella; Parini, Carlo

    2007-04-01

    In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids.

  15. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins.

    PubMed

    Zheng, Min; Jin, Ningyi; Liu, Qi; Huo, Xiaowei; Li, Yang; Hu, Bo; Ma, Haili; Zhu, Zhanbo; Cong, Yanzhao; Li, Xiao; Jin, Minglan; Zhu, Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  16. Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon.

    PubMed

    Karlsen, Marius; Villoing, Stephane; Rimstad, Espen; Nylund, Are

    2009-10-27

    Salmonid alphavirus (SAV) causes disease in farmed salmonid fish and is divided into different genetic subtypes (SAV1-6). Here we report the cloning and characterization of the 5'- and 3'- untranslated regions (UTR) of a SAV3 isolated from Atlantic salmon in Norway. The sequences of the UTRs are very similar to those of SAV1 and SAV2, but single nucleotide polymorphisms are present, also in the 3' - conserved sequence element (3'-CSE). Prediction of the RNA secondary structure suggested putative stem-loop structures in both the 5'- and 3'-ends, similar to those of alphaviruses from the terrestrial environment, indicating that the general genome replication initiation strategy for alphaviruses is also utilized by SAV. A DNA replicon vector, pmSAV3, based upon a pVAX1 backbone and the SAV3 genome was constructed, and the SAV3 non-structural proteins were used to express a reporter gene controlled by the SAV3 subgenomic promoter. Transfection of pmSAV3 into CHSE and BF2 cell lines resulted in expression of the reporter protein, confirming that the cloned SAV3 replication apparatus and UTRs are functional in fish cells.

  17. 3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

    PubMed

    Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

    2016-04-07

    DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

  18. 3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

    PubMed Central

    Löb, D.; Lengert, N.; Chagin, V. O.; Reinhart, M.; Casas-Delucchi, C. S.; Cardoso, M. C.; Drossel, B.

    2016-01-01

    DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

  19. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    SciTech Connect

    Filone, Claire Marie; Heise, Mark; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Bertolotti-Ciarlet, Andrea . E-mail: aciarlet@mail.med.upenn.edu

    2006-12-20

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

  20. Alphavirus replicon particles expressing TRP-2 provide potent therapeutic effect on melanoma through activation of humoral and cellular immunity.

    PubMed

    Avogadri, Francesca; Merghoub, Taha; Maughan, Maureen F; Hirschhorn-Cymerman, Daniel; Morris, John; Ritter, Erika; Olmsted, Robert; Houghton, Alan N; Wolchok, Jedd D

    2010-09-10

    Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8+ T cells. Activation of effector CD8+ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs. VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8+ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8+ T cell effector responses, and depends on signaling through activating Fcγ receptors. This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8+ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.

  1. Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

    PubMed Central

    Avogadri, Francesca; Merghoub, Taha; Maughan, Maureen F.; Hirschhorn-Cymerman, Daniel; Morris, John; Ritter, Erika; Olmsted, Robert; Houghton, Alan N.; Wolchok, Jedd D.

    2010-01-01

    Background Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8+ T cells. Activation of effector CD8+ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs. Methodology/Principal Findings VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8+ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8+ T cell effector responses, and depends on signaling through activating Fcγ receptors. Conclusions/Significance This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8+ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials. PMID:20844763

  2. Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

    PubMed

    Kobayashi, Jyumpei; Tanabiki, Misaki; Doi, Shohei; Kondo, Akihiko; Ohshiro, Takashi; Suzuki, Hirokazu

    2015-11-01

    The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75(αβ)-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75(αβ)-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(αβ)-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(αβ)-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(αβ)-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Multiagent vaccines vectored by Venezuelan equine encephalitis virus replicon elicits immune responses to Marburg virus and protection against anthrax and botulinum neurotoxin in mice.

    PubMed

    Lee, John S; Groebner, Jennifer L; Hadjipanayis, Angela G; Negley, Diane L; Schmaljohn, Alan L; Welkos, Susan L; Smith, Leonard A; Smith, Jonathan F

    2006-11-17

    The development of multiagent vaccines offers the advantage of eliciting protection against multiple diseases with minimal inoculations over a shorter time span. We report here the results of using formulations of individual Venezuelan equine encephalitis (VEE) virus replicon-vectored vaccines against a bacterial disease, anthrax; a viral disease, Marburg fever; and against a toxin-mediated disease, botulism. The individual VEE replicon particles (VRP) expressed mature 83-kDa protective antigen (MAT-PA) from Bacillus anthracis, the glycoprotein (GP) from Marburg virus (MBGV), or the H(C) fragment from botulinum neurotoxin (BoNT H(C)). CBA/J mice inoculated with a mixture of VRP expressing BoNT H(C) serotype C (BoNT/C H(C)) and MAT-PA were 80% protected from a B. anthracis (Sterne strain) challenge and then 100% protected from a sequential BoNT/C challenge. Swiss mice inoculated with individual VRP or with mixtures of VRP vaccines expressing BoNT H(C) serotype A (BoNT/A H(C)), MAT-PA, and MBGV-GP produced antibody responses specific to the corresponding replicon-expressed protein. Combination of the different VRP vaccines did not diminish the antibody responses measured for Swiss mice inoculated with formulations of two or three VRP vaccines as compared to mice that received only one VRP vaccine. Swiss mice inoculated with VRP expressing BoNT/A H(C) alone or in combination with VRP expressing MAT-PA and MBGV GP, were completely protected from a BoNT/A challenge. These studies demonstrate the utility of combining individual VRP vaccines into multiagent formulations for eliciting protective immune responses to various types of diseases.

  4. Inhibition of Hepatitis C Virus Replicon RNA Synthesis by PSI-352938, a Cyclic Phosphate Prodrug of β-d-2′-Deoxy-2′-α-Fluoro-2′-β-C-Methylguanosine▿†

    PubMed Central

    Lam, Angela M.; Espiritu, Christine; Murakami, Eisuke; Zennou, Veronique; Bansal, Shalini; Micolochick Steuer, Holly M.; Niu, Congrong; Keilman, Meg; Bao, Haiying; Bourne, Nigel; Veselenak, Ronald L.; Reddy, P. Ganapati; Chang, Wonsuk; Du, Jinfa; Nagarathnam, Dhanapalan; Sofia, Michael J.; Otto, Michael J.; Furman, Phillip A.

    2011-01-01

    PSI-352938 is a novel cyclic phosphate prodrug of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methylguanosine 5′-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 μM, respectively. The active 5′-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. In contrast, PSI-352938 did not inhibit the replication of hepatitis B virus or human immunodeficiency virus in vitro. PSI-352666 did not significantly affect the activity of human DNA and RNA polymerases. PSI-352938 and its cyclic phosphate metabolites did not affect the cyclic GMP-mediated activation of protein kinase G. Clearance studies using replicon cells demonstrated that PSI-352938 cleared cells of HCV replicon RNA and prevented replicon rebound. An additive to synergistic effect was observed when PSI-352938 was combined with other classes of HCV inhibitors, including alpha interferon, ribavirin, NS3/4A inhibitors, an NS5A inhibitor, and nucleoside/nucleotide and nonnucleoside inhibitors. Cross-resistance studies showed that PSI-352938 remained fully active against replicons containing the S282T or the S96T/N142T amino acid alteration. Replicons that contain mutations conferring resistance to various classes of nonnucleoside inhibitors also remained sensitive to inhibition by PSI-352938. PSI-352938 is currently being evaluated in a phase I clinical study in genotype 1-infected individuals. PMID:21444700

  5. In Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular growth require interaction among different replicons.

    PubMed

    Brom, S; García-de los Santos, A; Cervantes, L; Palacios, R; Romero, D

    2000-07-01

    Bacteria belonging to the genus Rhizobium are able to develop two different lifestyles, in symbiotic association with plant roots or through saprophytic growth. The genome of Rhizobium strains is constituted by a chromosome and several large plasmids, one of them containing most of the genes involved in symbiosis (symbiotic plasmid or pSym). Our model strain Rhizobium etli CFN42 contains six plasmids. We have constructed multiple plasmid-cured derivatives of this strain and used them to analyze the contribution of these plasmids to free-living cellular viability, competitivity for nodulation, plasmid transfer, and utilization of diverse carbon sources. Our results show that the transfer of the pSym is strictly dependent on the presence of another plasmid; consequently under conditions where pSym transfer is required, nodulation relies on the presence of a plasmid devoid of nodulation genes. We also found a drastic decrease in competitivity for nodulation in multiple plasmid-cured derivatives when compared with single plasmid-cured strains. Cellular growth and viability were greatly diminished in some multiple plasmid-cured strains. The utilization of a number of carbon sources depends on the presence of specific plasmids. The results presented in this work indicate that functional interactions among sequences scattered in the different plasmids are required for successful completion of both lifestyles. Copyright 2000 Academic Press.

  6. Alphavirus Replicon-based Adjuvants Enhance the Immunogenicity and Effectiveness of Fluzone® in Rhesus Macaques

    PubMed Central

    Carroll, Timothy D; Matzinger, Shannon R; Barro, Mario; Fritts, Linda; McChesney, Michael B; Miller, Christopher J; Johnston, Robert E

    2010-01-01

    Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone® alone or Fluzone® mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone® alone, Fluzone®+null VRP immunized animals had stronger influenza-specific CD4+ T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone®+null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p<0.0001, 1.3 log) and IgA (p<0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone®+null VRP immunization was 1.2 log greater (p<0.04) than after two immunizations with Fluzone® alone. After virus challenge, only Fluzone®+null VRP immunized monkeys had a significantly lower level of viral replication (p<0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone®+null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine. PMID:21111777

  7. Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo

    PubMed Central

    McCullough, Kenneth C; Bassi, Isabelle; Milona, Panagiota; Suter, Rolf; Thomann-Harwood, Lisa; Englezou, Pavlos; Démoulins, Thomas; Ruggli, Nicolas

    2014-01-01

    Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements—slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein. PMID:25004099

  8. Enhancement of the immunogenicity of an alphavirus replicon-based DNA vaccine against classical swine fever by electroporation and coinjection with a plasmid expressing porcine interleukin 2.

    PubMed

    Tian, Da-Yong; Sun, Yuan; Wai, Sing Fai; Lee, Fuk Ki; Meng, Qi-Lin; Suen, Kar Man; Wang, Nan; Han, Wen; Li, Su; Li, Yong-Feng; Li, Dan; Ling, Li-Jun; Liao, Ya-Jin; Qiu, Hua-Ji

    2012-05-21

    Alphavirus replicon-based DNA vaccines have emerged as a promising approach to generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for other approaches to enhance the vaccine potency. In this study, electroporation (EP) and a plasmid expressing porcine interleukin 2 (IL-2) were used to improve the immunogenicity of an alphavirus replicon-based DNA vaccine pSFV1CS-E2 against classical swine fever (CSF). Pigs were immunized with pSFV1CS-E2 alone or together with IL-2 by EP or by simple intramuscular injection. The results showed that EP combined with IL-2 resulted in marked enhancement of E2-specific antibody responses. Moreover, CSFV-specific lymphocyte proliferation, IFN-γ and IL-4 responses were increased significantly in the pSFV1CS-E2+IL-2/EP group. Pigs immunized with pSFV1CS-E2 plus IL-2 by EP were completely protected from lethal challenge, which is comparable to the sterilizing immunity and full protection offered by the live attenuated vaccine C-strain and in contrast with the incomplete protection conferred by pSFV1CS-E2 without or with IL-2 or EP alone, as demonstrated by the presence of pathological changes or/and viral loads. We conclude that EP in combination with IL-2 can significantly improve the immunogenicity of the plasmid DNA vaccine.

  9. Relicts and models of the RNA world

    NASA Astrophysics Data System (ADS)

    Lehto, Kirsi; Karetnikov, Alexey

    2005-01-01

    It is widely believed that the current DNA-RNA-protein-based life forms have evolved from preceding RNA-protein-based life forms, and these again, from mere RNA replicons. By rationale, it can be assumed that the early RNA replicons were fully heterotrophic in terms of obtaining all their building blocks from their environment. In the absence of protein catalysts, their essential life functions had to be mediated by simple functional structures and mechanisms, such as RNA secondary structures, RNA-RNA interactions and RNA-mediated catalysis, and possibly by catalytic minerals or clays. The central role of RNA catalysts in early life forms is supported by the fact that several catalytic RNAs still perform central biological functions in current life forms, and at least some of these may be derived as molecular relicts from the early RNA-based life. The RNA-catalysed metabolic reactions and molecular fossils are more conserved in the eukaryotic life forms than in the prokaryotes, suggesting that the linear eukaryote genomes may more closely resemble the structure and function of the early RNA replicons, than what do the circular prokaryote genomes. Present-day RNA viruses and viroids utilize ultimately simple life strategies, which may be similar to those used by the early RNA replicons. Thus, molecular and functional properties of viruses and viroids may be considered as examples or models of the structures and replication mechanisms, which might have been used for the replication of the early biopolymers.

  10. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons

    PubMed Central

    Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y. M.; Asante-Appiah, Ernest

    2016-01-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  11. RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon.

    PubMed

    Ramírez-Romero, M A; Téllez-Sosa, J; Barrios, H; Pérez-Oseguera, A; Rosas, V; Cevallos, M A

    2001-10-01

    The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon. RepC is essential for replication, and RepA and RepB play a role in plasmid segregation. It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number. RepA is also a trans-incompatibility factor. To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site-directed mutagenesis of the putative -35 and -10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon.

  12. Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

    PubMed Central

    Hoflack, L; Seurinck, J; Mahillon, J

    1997-01-01

    The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C). PMID:9260939

  13. Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

    PubMed

    Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

    2016-02-19

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

  14. Antiviral activity of CHO-SS cell-derived human omega interferon and other human interferons against HCV RNA replicons and related viruses.

    PubMed

    Buckwold, Victor E; Wei, Jiayi; Huang, Zhuhui; Huang, Chunsheng; Nalca, Aysegul; Wells, Jay; Russell, Julie; Collins, Barbara; Ptak, Roger; Lang, William; Scribner, Curtis; Blanchett, Dennis; Alessi, Tom; Langecker, Peter

    2007-02-01

    The fully glycosylated human omega interferon produced from CHO-SS cells (glycosylated IFN-omega) has been shown to be well-tolerated in man and to induce a sustained virologic response in patients infected with hepatitis C virus (HCV). We examined the antiviral activity of glycosylated IFN-omega and various human IFNs (IFN-alpha, -beta, -gamma and non-glycosylated bacterial (Escherichia coli) recombinant IFN-omega (non-glycosylated IFN-omega)) against HCV RNA replicons and several viruses related to HCV. Since none of the IFNs displayed cytotoxicity we compared their activities based on the effective concentration of the IFN that inhibited virus growth by 50% (EC50). Glycosylated IFN-omega was found to be the most potent antiviral agent of all the IFNs tested against bovine viral diarrhea virus (BVDV), yellow fever virus and West Nile virus. With HCV RNA replicons, non-glycosylated IFN-omega was comparable in activity to IFN-alpha while glycosylated IFN-omega was markedly more potent, indicating that glycosylation has an important effect on its activity. Drug combination analysis of glycosylated IFN-omega+ribavirin (RBV) in BVDV showed a synergy of antiviral effects similar to IFN-alpha+RBV, as well as a unique antagonism of RBV cytotoxic effects by glycosylated IFN-omega. Transcription factor (TF) profiling indicated that IFN-alpha or glycosylated IFN-omega treatment upregulated the same 17 TFs. IFN-alpha and glycosylated IFN-omega also upregulated 9 and 40 additional unique TFs, respectively. The differences in the expression of these TFs were modest, but statistically significantly different for eight of the TFs that were upregulated exclusively by glycosylated IFN-omega. The activation of these additional TFs by glycosylated IFN-omega might contribute to its high potency.

  15. Plasmid curing and the loss of grip--the 65-kb replicon of Phaeobacter inhibens DSM 17395 is required for biofilm formation, motility and the colonization of marine algae.

    PubMed

    Frank, Oliver; Michael, Victoria; Päuker, Orsola; Boedeker, Christian; Jogler, Christian; Rohde, Manfred; Petersen, Jörn

    2015-03-01

    Surface colonization is characteristic for a broad range of marine roseobacters and many strains have been isolated from biofilms, microbial mats and dinoflagellates. Phaeobacter inhibens DSM 17395, one of the best-studied representatives of the Roseobacter group, is an effective colonizer of marine surfaces, but the genetic basis of this trait is unknown. Based on the composition of its 65-kb RepA-I type plasmid that contains more than 20 genes for polysaccharide metabolism, including a rhamnose operon, which is required for O-antigen formation in Escherichia coli, it was hypothesized that this replicon was essential for surface attachment. Accordingly, a holistic approach was taken and the functional role of this extrachromosomal element in P. inhibens was investigated. Plasmid curing was performed with the homologous RepA-I replication system of Dinoroseobacter shibae DSM 16493(T). The Δ65-kb mutant completely lost its stickiness and could neither attach to artificial (glass, polystyrene) nor to natural surfaces (algae) and, consequently, its ability to form biofilms was impaired. Surprisingly, the mutant also lost the capacity for flagellar swimming motility required for surface colonization and the dispersal of biofilms. The data clearly showed that the 65-kb replicon of P. inhibens DSM 17395 was a genuine biofilm plasmid-mediating surface attachment. Homologous replicons are widely distributed among Rhodobacterales thus indicating the general importance of extrachromosomal elements for biofilm formation.

  16. RNA-Seq analysis of the multipartite genome of Rhizobium etli CE3 shows different replicon contributions under heat and saline shock.

    PubMed

    López-Leal, Gamaliel; Tabche, Maria Luisa; Castillo-Ramírez, Santiago; Mendoza-Vargas, Alfredo; Ramírez-Romero, Miguel A; Dávila, Guillermo

    2014-09-08

    Regulation of transcription is essential for any organism and Rhizobium etli (a multi-replicon, nitrogen-fixing symbiotic bacterium) is no exception. This bacterium is commonly found in the rhizosphere (free-living) or inside of root-nodules of the common bean (Phaseolus vulgaris) in a symbiotic relationship. Abiotic stresses, such as high soil temperatures and salinity, compromise the genetic stability of R. etli and therefore its symbiotic interaction with P. vulgaris. However, it is still unclear which genes are up- or down-regulated to cope with these stress conditions. The aim of this study was to identify the genes and non-coding RNAs (ncRNAs) that are differentially expressed under heat and saline shock, as well as the promoter regions of the up-regulated loci. Analysing the heat and saline shock responses of R. etli CE3 through RNA-Seq, we identified 756 and 392 differentially expressed genes, respectively, and 106 were up-regulated under both conditions. Notably, the set of genes over-expressed under either condition was preferentially encoded on plasmids, although this observation was more significant for the heat shock response. In contrast, during either saline shock or heat shock, the down-regulated genes were principally chromosomally encoded. Our functional analysis shows that genes encoding chaperone proteins were up-regulated during the heat shock response, whereas genes involved in the metabolism of compatible solutes were up-regulated following saline shock. Furthermore, we identified thirteen and nine ncRNAs that were differentially expressed under heat and saline shock, respectively, as well as eleven ncRNAs that had not been previously identified. Finally, using an in silico analysis, we studied the promoter motifs in all of the non-coding regions associated with the genes and ncRNAs up-regulated under both conditions. Our data suggest that the replicon contribution is different for different stress responses and that the heat shock response

  17. Transient Expression of Lumbrokinase (PI239) in Tobacco (Nicotiana tabacum) Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots

    PubMed Central

    Dickey, Alexia; Wang, Nan; Breedlove, Drew

    2017-01-01

    Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239. PMID:28932252

  18. PTC725, an NS4B-Targeting Compound, Inhibits a Hepatitis C Virus Genotype 3 Replicon, as Predicted by Genome Sequence Analysis and Determined Experimentally

    PubMed Central

    Graci, Jason D.; Jung, Stephen P.; Pichardo, John; Tong, Xiao; Gu, Zhengxian

    2016-01-01

    PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of ∼5 nM. De novo resistance selection identified mutations encoding amino acid substitutions mapping to the first predicted transmembrane region of NS4B, a finding consistent with results for PTC725 and other NS4B-targeting compounds against HCV gt1. This is the first report of the activity of an NS4B targeting compound against HCV gt3. In addition, we have identified previously unreported amino acid substitutions selected by PTC725 treatment which further demonstrate that these compounds target the NS4B first transmembrane region. PMID:27620477

  19. Structure and immunogenicity of alternative forms of the simian immunodeficiency virus gag protein expressed using Venezuelan equine encephalitis virus replicon particles.

    PubMed

    Cecil, Chad; West, Ande; Collier, Martha; Jurgens, Christy; Madden, Victoria; Whitmore, Alan; Johnston, Robert; Moore, Dominic T; Swanstrom, Ronald; Davis, Nancy L

    2007-06-05

    Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-) or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gagmyr+) and distinctive cytoplasmic aggregates (VRP-Gagmyr-) with each cell type. VRP were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-gamma ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-gamma-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gagmyr+-VRP. Antibody responses to MA/CA-VRP and Gagmyr+-VRP were not significantly different.

  20. Characterization of the basic replicon of pCM1, a narrow-host-range plasmid from the moderate halophile Chromohalobacter marismortui.

    PubMed Central

    Mellado, E; Asturias, J A; Nieto, J J; Timmis, K N; Ventosa, A

    1995-01-01

    The moderately halophilic bacterium Chromohalobacter marismortui contains a 17.5-kb narrow-host-range plasmid, pCM1, which shows interesting properties for the development of cloning vectors for the genetic manipulation of this important group of extremophiles. Plasmid pCM1 can stably replicate and is maintained in most gram-negative moderate halophiles tested. The replication origin has been identified and sequenced, and the minimal pCM1 replicon has been localized to a 1,600-bp region which includes two functionally discrete regions, the oriV region and the repA gene. oriV, located on a 700-bp fragment, contains four iterons 20 bp in length adjacent to a DnaA box that is dispensable but required for efficient replication of pCM1, and it requires trans-acting functions. The repA gene, which encodes a replication protein of 289 residues, is similar to the replication proteins of other gram-negative bacteria. PMID:7768853

  1. A heterologous DNA prime-Venezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus challenge in cynomolgus macaques.

    PubMed

    Chen, Lan; Ewing, Dan; Subramanian, Hemavathy; Block, Karla; Rayner, Jonathan; Alterson, Kimberly D; Sedegah, Martha; Hayes, Curtis; Porter, Kevin; Raviprakash, Kanakatte

    2007-11-01

    A candidate vaccine (D1ME-VRP) expressing dengue virus type 1 premembrane and envelope proteins in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) system was constructed and tested in conjunction with a plasmid DNA vaccine (D1ME-DNA) expressing identical dengue virus sequences. Cynomolgus macaques were vaccinated with three doses of DNA (DDD), three doses of VRP (VVV group), or a heterologous DNA prime-VRP boost regimen (DDV) using two doses of DNA vaccine and a third dose of VRP vaccine. Four weeks after the final immunization, the DDV group produced the highest dengue virus type 1-specific immunoglobulin G antibody responses and virus-neutralizing antibody titers. Moderate T-cell responses were demonstrated only in DDD- and DDV-vaccinated animals. When vaccinated animals were challenged with live virus, all vaccination regimens showed significant protection from viremia. DDV-immunized animals were completely protected from viremia (mean time of viremia = 0 days), whereas DDD- and VVV-vaccinated animals had mean times of viremia of 0.66 and 0.75 day, respectively, compared to 6.33 days for the control group of animals.

  2. Immunogenic and replicative properties of classical swine fever virus replicon particles modified to induce IFN-α/β and carry foreign genes.

    PubMed

    Suter, Rolf; Summerfield, Artur; Thomann-Harwood, Lisa J; McCullough, Kenneth C; Tratschin, Jon-Duri; Ruggli, Nicolas

    2011-02-04

    Virus replicon particles (VRP) are genetically engineered infectious virions incapable of generating progeny virus due to partial or complete deletion of at least one structural gene. VRP fulfil the criteria of a safe vaccine and gene delivery system. With VRP derived from classical swine fever virus (CSF-VRP), a single intradermal vaccination protects from disease. Spreading of the challenge virus in the host is however not completely abolished. Parameters that are critical for immunogenicity of CSF-VRP are not well characterized. Considering the importance of type I interferon (IFN-α/β) to immune defence development, we generated IFN-α/β-inducing VRP to determine how this would influence vaccine efficacy. We also evaluated the effect of co-expressing granulocyte macrophage colony-stimulating factor (GM-CSF) in the vaccine context. The VRP were capable of long-term replication in cell culture despite the presence of IFN-α/β. In vivo, RNA replication was essential for the induction of an immune response. IFN-α/β-inducing and GM-CSF-expressing CSF-VRP were similar to unmodified VRP in terms of antibody and peripheral T-cell responses, and in reducing the blood levels of challenge virus RNA. Importantly, the IFN-α/β-inducing VRP did show increased efficacy over the unmodified VRP in terms of B-cell and T-cell responses, when tested with secondary immune responses by in vitro restimulation assay. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. A phase I dose escalation trial of vaccine replicon particles (VRP) expressing prostate-specific membrane antigen (PSMA) in subjects with prostate cancer.

    PubMed

    Slovin, Susan F; Kehoe, Marissa; Durso, Robert; Fernandez, Celina; Olson, William; Gao, Jian P; Israel, Robert; Scher, Howard I; Morris, Stephen

    2013-01-30

    PSMA-VRP is a propagation defective, viral replicon vector system encoding PSMA under phase I evaluation for patients with castration resistant metastatic prostate cancer (CRPC). The product is derived from an attenuated strain of the alphavirus, Venezuelan Equine Encephalitis (VEE) virus, and incorporates multiple redundant safety features. In this first in human trial, two cohorts of 3 patients with CRPC metastatic to bone were treated with up to five doses of either 0.9×10(7)IU or 0.36×10(8)IU of PSMA-VRP at weeks 1, 4, 7, 10 and 18, followed by an expansion cohort of 6 patients treated with 0.36×10(8)IU of PSMA-VRP at weeks 1, 4, 7, 10 and 18. No toxicities were observed. In the first dose cohort, no PSMA specific cellular immune responses were seen but weak PSMA-specific signals were observed by ELISA. The remaining 9 patients, which included the higher cohort and the extension cohort, had no PSMA specific cellular responses. PSMA-VRP was well-tolerated at both doses. While there did not appear to be clinical benefit nor robust immune signals at the two doses studied, neutralizing antibodies were produced by both cohorts suggesting that dosing was suboptimal. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

    PubMed Central

    Aiyar, A; Tyree, C; Sugden, B

    1998-01-01

    Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences. PMID:9799247

  5. Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses.

    PubMed

    Yamanaka, Atsushi; Moi, Meng Ling; Takasaki, Tomohiko; Kurane, Ichiro; Matsuda, Mami; Suzuki, Ryosuke; Konishi, Eiji

    2017-05-01

    The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections.

  6. oriGNAI3: a narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques.

    PubMed Central

    Toledo, F; Baron, B; Fernandez, M A; Lachagès, A M; Mayau, V; Buttin, G; Debatisse, M

    1998-01-01

    The nature of mammalian origins of DNA replication remains controversial and this is primarily because two-dimensional gel replicon mapping techniques have identified broad zones of replication initiation whereas several other techniques, such as quantitative PCR, have disclosed more discrete sites of initiation at the same chromosomal loci. In this report we analyze the replication of an amplified genomic region encompassing the 3'-end of the GNAI3 gene, the entire GNAT2 gene and the intergenic region between them in exponentially growing Chinese hamster fibroblasts. These cells express GNAI3 but not GNAT2 . The replication pattern was first analyzed by two-dimensional neutral-alkaline gel electrophoresis. Surprisingly, the results revealed a small preferential zone of replication initiation, of at most 1.7 kb, located in a limited part of the GNAI3 - GNAT2 intergenic region. Mapping of this initiation zone was then confirmed by quantitative PCR. The agreement between the two techniques exploited here strengthens the hypothesis that preferred sites of replication initiation do exist in mammalian genomes. PMID:9580680

  7. Phase I safety and immunogenicity evaluations of an alphavirus replicon HIV-1 subtype C gag vaccine in healthy HIV-1-uninfected adults.

    PubMed

    Wecker, M; Gilbert, P; Russell, N; Hural, J; Allen, M; Pensiero, M; Chulay, J; Chiu, Ya-Lin; Abdool Karim, S S; Burke, D S

    2012-10-01

    On the basis of positive preclinical data, we evaluated the safety and immunogenicity of an alphavirus replicon HIV-1 subtype C gag vaccine (AVX101), expressing a nonmyristoylated form of Gag, in two double-blind, randomized, placebo-controlled clinical trials in healthy HIV-1-uninfected adults. Escalating doses of AVX101 or placebo were administered subcutaneously to participants in the United States and Southern Africa. Because of vaccine stability issues, the first trial was halted prior to completion of all dose levels and a second trial was implemented. The second trial was also stopped prematurely due to documentation issues with the contract manufacturer. Safety and immunogenicity were evaluated through assessments of reactogenicity, reports of adverse events, and assessment of replication-competent and Venezuelan equine encephalitis (VEE) viremia. Immunogenicity was measured using the following assays: enzyme-linked immunosorbent assay (ELISA), chromium 51 ((51)Cr)-release cytotoxic T lymphocyte (CTL), gamma interferon (IFN-γ) ELISpot, intracellular cytokine staining (ICS), and lymphoproliferation assay (LPA). Anti-vector antibodies were also measured. AVX101 was well tolerated and exhibited only modest local reactogenicity. There were 5 serious adverse events reported during the trials; none were considered related to the study vaccine. In contrast to the preclinical data, immune responses in humans were limited. Only low levels of binding antibodies and T-cell responses were seen at the highest doses. This trial also highlighted the difficulties in developing a novel vector for HIV.

  8. Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae.

    PubMed

    Ruiz-Masó, José A; López-Aguilar, Celeste; Nieto, Concha; Sanz, Marta; Burón, Patricia; Espinosa, Manuel; del Solar, Gloria

    2012-01-01

    We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter P(M) of the S. pneumoniaemalMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from P(M) is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive P(tet) promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniae. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Whence and whither health insurance? A revisionist history.

    PubMed

    Moran, Donald W

    2005-01-01

    Throughout the postwar era in federal health policy, policymakers have sought to expand both public and private insurance coverage, while wrestling with the cost consequences of the demand generated by the insurance-financing mechanisms thus created. This essay advances the view that the limits to insurance expansion have been reached and that public and private plan sponsors will henceforth continually "thin out" the coverage they offer. In this environment, policymakers seeking to mitigate access concerns may need to consider strategies that promote direct service delivery. This emerging regime, it is argued, will have important implications for the future of innovation in health care.

  10. The Indecisive Feminist: Study of Anne Sexton's Revisionist Fairy Tales

    ERIC Educational Resources Information Center

    Mohammed, Nadia Fayidh

    2015-01-01

    Fairy tales to female writers are major resource for their abundant writings, but for the feminist poets since 1960s, they become essential subject matter to often deal with in their literary production. With the motivation to address the conventional tradition of patriarchal society, and re-address the stereotype females inhabiting these tales,…

  11. Characterization of the Minimal Replicon of a Cryptic Deinococcus radiodurans SARK Plasmid and Development of Versatile Escherichia coli-D. radiodurans Shuttle Vectors

    PubMed Central

    Meima, Rob; Lidstrom, Mary E.

    2000-01-01

    The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ. PMID:10966401

  12. Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection.

    PubMed

    Flores, R; Minoia, S; Carbonell, A; Gisel, A; Delgado, S; López-Carrasco, A; Navarro, B; Di Serio, F

    2015-11-02

    The discovery of viroids about 45 years ago heralded a revolution in Biology: small RNAs comprising around 350 nt were found to be able to replicate autonomously-and to incite diseases in certain plants-without encoding proteins, fundamental properties discriminating these infectious agents from viruses. The initial focus on the pathological effects usually accompanying infection by viroids soon shifted to their molecular features-they are circular molecules that fold upon themselves adopting compact secondary conformations-and then to how they manipulate their hosts to be propagated. Replication of viroids-in the nucleus or chloroplasts through a rolling-circle mechanism involving polymerization, cleavage and circularization of RNA strands-dealt three surprises: (i) certain RNA polymerases are redirected to accept RNA instead of their DNA templates, (ii) cleavage in chloroplastic viroids is not mediated by host enzymes but by hammerhead ribozymes, and (iii) circularization in nuclear viroids is catalyzed by a DNA ligase redirected to act upon RNA substrates. These enzymes (and ribozymes) are most probably assisted by host proteins, including transcription factors and RNA chaperones. Movement of viroids, first intracellularly and then to adjacent cells and distal plant parts, has turned out to be a tightly regulated process in which specific RNA structural motifs play a crucial role. More recently, the advent of RNA silencing has brought new views on how viroids may cause disease and on how their hosts react to contain the infection; additionally, viroid infection may be restricted by other mechanisms. Representing the lowest step on the biological size scale, viroids have also attracted considerable interest to get a tentative picture of the essential characteristics of the primitive replicons that populated the postulated RNA world. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Alphavirus Replicon DNA Expressing HIV Antigens Is an Excellent Prime for Boosting with Recombinant Modified Vaccinia Ankara (MVA) or with HIV gp140 Protein Antigen

    PubMed Central

    Knudsen, Maria L.; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose. PMID:25643354

  14. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

    PubMed

    Osada, Takuya; Berglund, Peter; Morse, Michael A; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Yang, Xiao Yi; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R; Clay, Timothy M; Smith, Jonathan; Kim Lyerly, H

    2012-11-01

    We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.

  15. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and anti-tumor effects

    PubMed Central

    Osada, Takuya; Berglund, Peter; Morse, Michael A.; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R.; Clay, Timothy M.; Smith, Jonathan; Lyerly, H. Kim

    2013-01-01

    We recently demonstrated that Venezuelan equine encephalitis (VEE) virus-based replicon particles (VRP) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP expressing Interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and anti-tumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)) and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12 and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing anti-tumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted. PMID:22488274

  16. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA) or with HIV gp140 protein antigen.

    PubMed

    Knudsen, Maria L; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  17. Hepatitis C virus NS5A replication complex inhibitors. Part 6: Discovery of a novel and highly potent biarylimidazole chemotype with inhibitory activity toward genotypes 1a and 1b replicons.

    PubMed

    Belema, Makonen; Nguyen, Van N; Romine, Jeffrey L; St Laurent, Denis R; Lopez, Omar D; Goodrich, Jason T; Nower, Peter T; O'Boyle, Donald R; Lemm, Julie A; Fridell, Robert A; Gao, Min; Fang, Hua; Krause, Rudolph G; Wang, Ying-Kai; Oliver, A Jayne; Good, Andrew C; Knipe, Jay O; Meanwell, Nicholas A; Snyder, Lawrence B

    2014-03-13

    A medicinal chemistry campaign that was conducted to address a potential genotoxic liability associated with an aniline-derived scaffold in a series of HCV NS5A inhibitors with dual GT-1a/-1b inhibitory activity is described. Anilides 3b and 3c were used as vehicles to explore structural modifications that retained antiviral potency while removing the potential for metabolism-based unmasking of the embedded aniline. This effort resulted in the discovery of a highly potent biarylimidazole chemotype that established a potency benchmark in replicon assays, particularly toward HCV GT-1a, a strain with significant clinical importance. Securing potent GT-1a activity in a chemotype class lacking overt structural liabilities was a critical milestone in the effort to realize the full clinical potential of targeting the HCV NS5A protein.

  18. Antibody-Mediated Protection against Mucosal Simian-Human Immunodeficiency Virus Challenge of Macaques Immunized with Alphavirus Replicon Particles and Boosted with Trimeric Envelope Glycoprotein in MF59 Adjuvant▿

    PubMed Central

    Barnett, Susan W.; Burke, Brian; Sun, Yide; Kan, Elaine; Legg, Harold; Lian, Ying; Bost, Kristen; Zhou, Fengmin; Goodsell, Amanda; zur Megede, Jan; Polo, John; Donnelly, John; Ulmer, Jeffrey; Otten, Gillis R.; Miller, Christopher J.; Vajdy, Michael; Srivastava, Indresh K.

    2010-01-01

    We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIVSF162P4 following sequential immunization with alphavirus replicon particles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1SF162 gp140ΔV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIVSF162P4 (intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against HIV

  19. Antibody-mediated protection against mucosal simian-human immunodeficiency virus challenge of macaques immunized with alphavirus replicon particles and boosted with trimeric envelope glycoprotein in MF59 adjuvant.

    PubMed

    Barnett, Susan W; Burke, Brian; Sun, Yide; Kan, Elaine; Legg, Harold; Lian, Ying; Bost, Kristen; Zhou, Fengmin; Goodsell, Amanda; Zur Megede, Jan; Polo, John; Donnelly, John; Ulmer, Jeffrey; Otten, Gillis R; Miller, Christopher J; Vajdy, Michael; Srivastava, Indresh K

    2010-06-01

    We have previously shown that rhesus macaques were partially protected against high-dose intravenous challenge with simian-human immunodeficiency virus SHIV(SF162P4) following sequential immunization with alphavirus replicon particles (VRP) of a chimeric recombinant VEE/SIN alphavirus (derived from Venezuelan equine encephalitis virus [VEE] and the Sindbis virus [SIN]) encoding human immunodeficiency virus type 1 HIV-1(SF162) gp140DeltaV2 envelope (Env) and trimeric Env protein in MF59 adjuvant (R. Xu, I. K. Srivastava, C. E. Greer, I. Zarkikh, Z. Kraft, L. Kuller, J. M. Polo, S. W. Barnett, and L. Stamatatos, AIDS Res. Hum. Retroviruses 22:1022-1030, 2006). The protection did not require T-cell immune responses directed toward simian immunodeficiency virus (SIV) Gag. We extend those findings here to demonstrate antibody-mediated protection against mucosal challenge in macaques using prime-boost regimens incorporating both intramuscular and mucosal routes of delivery. The macaques in the vaccination groups were primed with VRP and then boosted with Env protein in MF59 adjuvant, or they were given VRP intramuscular immunizations alone and then challenged with SHIV(SF162P4) (intrarectal challenge). The results demonstrated that these vaccines were able to effectively protect the macaques to different degrees against subsequent mucosal SHIV challenge, but most noteworthy, all macaques that received the intramuscular VRP prime plus Env protein boost were completely protected. A statistically significant association was observed between the titer of virus neutralizing and binding antibodies as well as the avidity of anti-Env antibodies measured prechallenge and protection from infection. These results highlight the merit of the alphavirus replicon vector prime plus Env protein boost vaccine approach for the induction of protective antibody responses and are of particular relevance to advancing our understanding of the potential correlates of immune protection against

  20. Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1

    PubMed Central

    Ku, Hye-Jin; Park, Myeong Soo; Lee, Ju-Hoon

    2015-01-01

    A 2.1-kb plasmid was previously isolated from Weissella cibaria KLC140 in kimchi and cloned into pUC19 along with the slpA and gfp genes, resulting in an 8.6-kb pKWCSLGFP construct for use as a novel surface display vector. To reduce the size of the vector, the minimal replicon of pKW2124 was determined. The pKW2124 plasmid contains a putative origin of replication (ori), a potential ribosomal binding site (RBS), and the repA gene encoding a plasmid replication protein. To conduct the minimal replicon experiment, four different PCR products (MR1, ori+RBS+repA; MR2, RBS+repA; MR2’, repA; MR3, fragment of repA) were obtained and cloned into pUC19 (pKUCm1, pKUCm2, pKUCm2’, and pKUCm3, respectively) containing the chloramphenicol acetyltransferase (CAT) gene. These constructed vectors were electroporated into W. confusa ATCC 10881 with different transformation efficiencies of 1.5 × 105 CFU/μg, 1.3 × 101 CFU/μg, and no transformation, respectively, suggesting that the putative ori, RBS, and repA gene are essential for optimum plasmid replication. Subsequent segregational plasmid stability testing of pKUCm1 and pKUCm2 showed that the vector pKUCm1 is highly stable up to 100 generations but pKUCm2 was completely lost after 60 generations, suggesting that the putative ori may be important for plasmid stability in the host strain. In addition, a host range test of pKUCm1 revealed that it has a broad host range spectrum including Weissella, Lactococcus, Leuconostoc, and even Lactobacillus. To verify the application of pKUCm1, the β-galactosidase gene and its promoter region from W. cibaria KSD1 were cloned in the vector, resulting in pKUGal. Expression of the β-galactosidase gene was confirmed using blue-white screening after IPTG induction. The small and stable pKUGal vector will be useful for gene transfer, expression, and manipulation in the Weissella genome and in other lactic acid bacteria. PMID:25691882

  1. A rat model for hepatitis E virus

    PubMed Central

    Mishra, Niraj; Verbeken, Erik; Ramaekers, Kaat; Dallmeier, Kai

    2016-01-01

    ABSTRACT Hepatitis E virus (HEV) is one of the prime causes of acute viral hepatitis, and chronic hepatitis E is increasingly recognized as an important problem in the transplant setting. Nevertheless, the fundamental understanding of the biology of HEV replication is limited and there are few therapeutic options. The development of such therapies is partially hindered by the lack of a robust and convenient animal model. We propose the infection of athymic nude rats with the rat HEV strain LA-B350 as such a model. A cDNA clone, pLA-B350, was constructed and the infectivity of its capped RNA transcripts was confirmed in vitro and in vivo. Furthermore, a subgenomic replicon, pLA-B350/luc, was constructed and validated for in vitro antiviral studies. Interestingly, rat HEV proved to be less sensitive to the antiviral activity of α-interferon, ribavirin and mycophenolic acid than genotype 3 HEV (a strain that infects humans). As a proof-of-concept, part of the C-terminal polymerase sequence of pLA-B350/luc was swapped with its genotype 3 HEV counterpart: the resulting chimeric replicon replicated with comparable efficiency as the wild-type construct, confirming that LA-B350 strain is amenable to humanization (replacement of certain sequences or motifs by their counterparts from human HEV strains). Finally, ribavirin effectively inhibited LA-B350 replication in athymic nude rats, confirming the suitability of the rat model for antiviral studies. PMID:27483350

  2. Lifestyle-related diseases of the digestive system: a new in vitro model of hepatitis C virion production: application of basic research on hepatitis C virus to clinical medicine.

    PubMed

    Saito, Satoru; Heller, Theo; Yoneda, Masato; Takahashi, Hirokazu; Nakajima, Atsushi; Liang, Jake T

    2007-10-01

    The hepatitis C virus (HCV) is an enveloped virus with a single positive-strand RNA genome of about 9.6 kb. It is a major cause of liver disease worldwide. Clear understanding of the viral life cycle has been hampered by the lack of a robust cell culture system. While the development of the HCV replicon system was a major breakthrough, infectious virions could not be produced with the replicon system. Recently, several groups have reported producing HCV virions using in vitro systems. One of these is a replicon system, but with the special genotype 2a strain JFH-1. Another is a DNA transfection system, with the construct containing the cDNA of the known infectious HCV genotype 1b flanked by two ribozymes. The development of these models further extends the repertoire of tools available for the study of HCV biology, and in particular, they may help to elucidate the molecular details of hepatitis C viral assembly and release. This review discusses the progression of experimental strategies related to HCV and how these strategies may be applied to clinical medicine.

  3. Coupling between the basic replicon and the Kis-Kid maintenance system of plasmid R1: modulation by Kis antitoxin levels and involvement in control of plasmid replication.

    PubMed

    López-Villarejo, Juan; Lobato-Márquez, Damián; Díaz-Orejas, Ramón

    2015-02-05

    kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

  4. The Great School Legend: A Revisionist Interpretation of American Public Education.

    ERIC Educational Resources Information Center

    Greer, Colin

    American schools have been credited with building American democracy. This is a myth which has been largely responsible for the resistance of today's schools to needed change. Exposing this myth are records of several major urban school systems which show the high rate of school failure among the urban poor since before 1900. These statistics have…

  5. Living in the Past: Some Revisionist Thoughts on the Historiography of Art and Design Education

    ERIC Educational Resources Information Center

    Romans, Mervyn

    2004-01-01

    There is a dominant history of art and design education in Britain. This has been established by five books published in the 1960s and 1970s. They are Quentin Bell's The Schools of Design (1963), Gordon Sutton's Artisan or Artist(1967), Richard Carline's Draw They Must(1968), Stuart Macdonald's The History and Philosophy of Art Education (1970),…

  6. The medium and the message: a revisionist view of image quality

    NASA Astrophysics Data System (ADS)

    Ferwerda, James A.

    2010-02-01

    In his book "Understanding Media" social theorist Marshall McLuhan declared: "The medium is the message." The thesis of this paper is that with respect to image quality, imaging system developers have taken McLuhan's dictum too much to heart. Efforts focus on improving the technical specifications of the media (e.g. dynamic range, color gamut, resolution, temporal response) with little regard for the visual messages the media will be used to communicate. We present a series of psychophysical studies that investigate the visual system's ability to "see through" the limitations of imaging media to perceive the messages (object and scene properties) the images represent. The purpose of these studies is to understand the relationships between the signal characteristics of an image and the fidelity of the visual information the image conveys. The results of these studies provide a new perspective on image quality that shows that images that may be very different in "quality", can be visually equivalent as realistic representations of objects and scenes.

  7. The Art of Wondering: A Revisionist Return to the History of Rhetoric.

    ERIC Educational Resources Information Center

    Covino, William A.

    Reacting to the tradition which has reduced rhetorics to summaries of rules and principles, this book presupposes that Plato's "Phaedrus," Aristotle's "Rhetoric," and Cicero's "De Oratore" cannot be reduced to summary information or pedagogical advice. The book considers that these works, on the contrary, along with…

  8. Transforming practice knowledge into nursing knowledge--a revisionist analysis of Peplau.

    PubMed

    Reed, P G

    1996-01-01

    Nursing practice typically has been viewed as applying knowledge. However, currently, there is increasing awareness that nursing practice is also a process of knowledge development. Still, research and practice are not always connected. Analysis of Peplau's works illuminates a scholarship of nursing practice that is relevant today. This paper focuses on a specific strategy and philosophic perspective, as derived from Peplau, for integrating nursing practice more fully into today's knowledge development. Emphasis is on the need for nursing practice-based theory, as well as nursing theory-based practice.

  9. O'Connor v. Donaldson: retelling a classic and finding some revisionist history.

    PubMed

    Behnke, S H

    1999-01-01

    This article discusses the 1975 Supreme Court opinion O'Connor v. Donaldson. The article first examines the due process clause of the Fourteenth Amendment, the basis for the O'Connor ruling. It then looks carefully at the Court's conclusions, with particular attention to the Court's reasoning and analysis. The article goes on to look at how the Supreme Court has interpreted O'Connor on subsequent occasions and suggests that the Court has often misconstrued its own decision. The article concludes by offering thoughts and commentary on the O'Connor opinion and its progeny.

  10. John F. Kennedy and Vietnam: The Historical Record Versus the Revisionists.

    ERIC Educational Resources Information Center

    Turner, Thomas R.

    1995-01-01

    Criticizes the current historical interpretation (promoted by Arthur Schlesinger, Jr. and Oliver Stone) that President John F. Kennedy intended to withdraw from Vietnam if elected to a second term. Maintains that the preponderance of historical evidence suggests that Kennedy intended to exit Vietnam only after a military victory. (MJP)

  11. The Social Scientists' Role in Brown vs. Board of Education: A Non-Revisionist Appraisal.

    ERIC Educational Resources Information Center

    Smith, M. Brewster

    The social scientist has a public responsibility which deserves as much attention as scientific and professional concerns. Some social scientists, retreating from this ideal, are now rewriting the history of the liberal movements of the 1960s. This revisionism, a product of contemporary neoconservatism, hinders the continuing efforts that social…

  12. The Art of Wondering: A Revisionist Return to the History of Rhetoric.

    ERIC Educational Resources Information Center

    Covino, William A.

    Reacting to the tradition which has reduced rhetorics to summaries of rules and principles, this book presupposes that Plato's "Phaedrus," Aristotle's "Rhetoric," and Cicero's "De Oratore" cannot be reduced to summary information or pedagogical advice. The book considers that these works, on the contrary, along with…

  13. Romantic Rhetoric, Revisionist Reality: The Effectiveness of Regulation in Maritime Education and Training

    ERIC Educational Resources Information Center

    Sampson, Helen

    2004-01-01

    This article offers an insight into the regulation and conduct of training and education in a globalised industry, and across an international labour market. Focusing on the cargo shipping sector of the maritime industry, it considers the provision of training and education for modern merchant officers within the context of an international…

  14. The Great School Legend: A Revisionist Interpretation of American Public Education.

    ERIC Educational Resources Information Center

    Greer, Colin

    American schools have been credited with building American democracy. This is a myth which has been largely responsible for the resistance of today's schools to needed change. Exposing this myth are records of several major urban school systems which show the high rate of school failure among the urban poor since before 1900. These statistics have…

  15. Grease, anthraxgate, and kennel cough: a revisionist history of early veterinary vaccines.

    PubMed

    Tizard, I

    1999-01-01

    In conclusion, it is remarkable just how farsighted many of the early vaccine investigators were. Jenner was apparently very comfortable with contagion and even recognized that infectious agents could gradually change and adapt to a new species. Pasteur, long before his fowl cholera experiment, dreamed that attenuation could yield safe vaccines and it took him no time at all therefore to recognize the significance of that serendipitous experiment. The fact that two other investigators were also developing anthrax vaccines simultaneously is yet another example of how the times favor certain discoveries. Finally Ferry, while constrained by the fact that he had no idea that distemper was caused by a virus, recognized well the concept of secondary infection and rationalized, not unreasonably, that his vaccine might assist in controlling this. It is also clear that we must look skeptically at the accepted historical record. Thus, it is clear that Jenner used horse-derived material as a source of vaccine material and that vaccinia may in fact be the long-lost agent of horsepox. Certainly this would not be news to many nineteenth-century investigators and veterinarians. Individuals planning to use live vaccinia in recombinant vaccines may wish to keep this in mind. Who discovered anthrax vaccine? Burdon-Sanderson clearly recognized that he could attenuate the organism. Greenfield showed that this could protect against disease although he was far from developing an effective vaccine. Poor Henri Toussaint was probably the first to develop an effective product but did not publicize his results widely. It was left to Louis Pasteur to take the risks inherent in a high-profile public experiment and win. I believe that he richly deserves the prize. Finally, who deserves the credit for distemper vaccine? First, Carré deserves much more credit than hitherto for discovering that distemper was caused by a virus. Second, Ferry, although misled by his identification of B. bronchiseptica deserves credit for realizing that his vaccine could play a role in controlling secondary infections. The true discoverer of an effective distemper vaccine was the Italian, Puntoni, but once again the publicity went to others, Laidlaw and Dunkin. Thus a pattern emerges that prior discovery matters little in the face of aggressive publicity. If nobody knows you did the experiment you might as well have never done it in the first place. Publish or perish is by no means a new phenomenon.

  16. A revisionist history of adult marrow stem cell biology or 'they forgot about the discard'.

    PubMed

    Quesenberry, P; Goldberg, L

    2017-08-01

    The adult marrow hematopoietic stem cell biology has largely been based on studies of highly purified stem cells. This is unfortunate because during the stem cell purification the great bulk of stem cells are discarded. These cells are actively proliferating. The final purified stem cell is dormant and not representative of the whole stem cell compartment. Thus, a large number of studies on the cellular characteristics, regulators and molecular details of stem cells have been carried on out of non-represented cells. Niche studies have largely pursued using these purified stem cells and these are largely un-interpretable. Other considerations include the distinction between baseline and transplant stem cells and the modulation of stem cell phenotype by extracellular vesicles, to cite a non-inclusive list. Work needs to proceed on characterizing the true stem cell population.

  17. Divergent Evolution of the repFII Replicon of IncF Plasmids Carrying Cytotoxic Necrotizing Factor cnf2, Cytolethal Distending Toxin cdtIII, and f17Ae Fimbrial Variant Genes in Type 2 Necrotoxigenic Escherichia coli Isolates from Calves

    PubMed Central

    Bihannic, Morgan; Haenni, Marisa; Oswald, Eric

    2015-01-01

    Among the pathovars of Escherichia coli in cattle, necrotoxigenic E. coli (NTEC) is defined by the production of cytotoxic necrotizing factors (CNFs). In particular, type 2 NTEC (NTEC2) strains are frequent in diarrheic and septicemic calves and usually coproduce CNF type 2 (CNF2), cytolethal distending toxin type III (CDTIII), and fimbrial adhesins of the F17 family, whose genetic determinants have frequently been reported on the same Vir-like plasmid. In this study, we investigated the genetic environment of the cnf2, f17Ae, and cdtIII genes in a collection of fecal E. coli isolates recovered from 484 French and 58 Iranian calves. In particular, we highlighted the spread of cnf2, f17Ae, and cdtIII on similar 150-kb IncF plasmids harboring the newly assigned repFII replicon allele F74 in NTEC2 isolates. Interestingly, this 150-kb IncF plasmid differed from the 140-kb IncF plasmid harboring the newly assigned repFII replicon allele F75 and carrying cnf2 alone. These results suggest two divergent lineages of cnf2-carrying IncF plasmids depending on the presence of the f17Ae and cdtIII genes. This partition was observed in E. coli strains of unrelated backgrounds, suggesting two different evolutionary paths of cnf2-carrying IncF plasmids rather than divergent evolutions of NTEC2 clones. The driving forces for such divergent evolutions are not known, and further studies are required to clarify the selection of plasmid subtypes spreading virulence determinants in E. coli, in particular, plasmids of the IncF family. PMID:26546422

  18. Spin-glass phase transition and behavior of nonlinear susceptibility in the Sherrington-Kirkpatrick model with random fields

    NASA Astrophysics Data System (ADS)

    Morais, C. V.; Zimmer, F. M.; Lazo, M. J.; Magalhães, S. G.; Nobre, F. D.

    2016-06-01

    The behavior of the nonlinear susceptibility χ3 and its relation to the spin-glass transition temperature Tf in the presence of random fields are investigated. To accomplish this task, the Sherrington-Kirkpatrick model is studied through the replica formalism, within a one-step replica-symmetry-breaking procedure. In addition, the dependence of the Almeida-Thouless eigenvalue λAT (replicon) on the random fields is analyzed. Particularly, in the absence of random fields, the temperature Tf can be traced by a divergence in the spin-glass susceptibility χSG, which presents a term inversely proportional to the replicon λAT. As a result of a relation between χSG and χ3, the latter also presents a divergence at Tf, which comes as a direct consequence of λAT=0 at Tf. However, our results show that, in the presence of random fields, χ3 presents a rounded maximum at a temperature T* which does not coincide with the spin-glass transition temperature Tf (i.e., T*>Tf for a given applied random field). Thus, the maximum value of χ3 at T* reflects the effects of the random fields in the paramagnetic phase instead of the nontrivial ergodicity breaking associated with the spin-glass phase transition. It is also shown that χ3 still maintains a dependence on the replicon λAT, although in a more complicated way as compared with the case without random fields. These results are discussed in view of recent observations in the LiHoxY1 -xF4 compound.

  19. Arabidopsis thaliana and Pisum sativum models demonstrate that root colonization is an intrinsic trait of Burkholderia cepacia complex bacteria.

    PubMed

    Vidal-Quist, J Cristian; O'Sullivan, Louise A; Desert, Annaëlle; Fivian-Hughes, Amanda S; Millet, Coralie; Jones, T Hefin; Weightman, Andrew J; Rogers, Hilary J; Berry, Colin; Mahenthiralingam, Eshwar

    2014-02-01

    Burkholderia cepacia complex (Bcc) bacteria possess biotechnologically useful properties that contrast with their opportunistic pathogenicity. The rhizosphere fitness of Bcc bacteria is central to their biocontrol and bioremediation activities. However, it is not known whether this differs between species or between environmental and clinical strains. We investigated the ability of 26 Bcc strains representing nine different species to colonize the roots of Arabidopsis thaliana and Pisum sativum (pea). Viable counts, scanning electron microscopy and bioluminescence imaging were used to assess root colonization, with Bcc bacteria achieving mean (±sem) levels of 2.49±0.23×10(6) and 5.16±1.87×10(6) c.f.u. per centimetre of root on the A. thaliana and P. sativum models, respectively. The A. thaliana rhizocompetence model was able to reveal loss of colonization phenotypes in Burkholderia vietnamiensis G4 transposon mutants that had only previously been observed in competition experiments on the P. sativum model. Different Bcc species colonized each plant model at different rates, and no statistical difference in root colonization was observed between isolates of clinical or environmental origin. Loss of the virulence-associated third chromosomal replicon (>1 Mb DNA) did not alter Bcc root colonization on A. thaliana. In summary, Bcc bacteria possess intrinsic root colonization abilities irrespective of their species or source. As Bcc rhizocompetence does not require their third chromosomal replicon, the possibility of using synthetic biology approaches to engineer virulence-attenuated biotechnological strains is tractable.

  20. A putative multi-replicon plasmid co-harboring beta-lactamase genes blaKPC-2, blaCTX-M-14 and blaTEM-1 and trimethoprim resistance gene dfrA25 from a Klebsiella pneumoniae sequence type (ST) 11 strain in China.

    PubMed

    Tang, Yu; Shen, Pinghua; Liang, Wei; Jin, Jialin; Jiang, Xiaofei

    2017-01-01

    The global emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae poses a major public health threat requiring immediate and aggressive action. Some older generation antibiotics, such as trimethoprim, serve as alternatives for treatment of infections. Here, we determined the complete nucleotide sequence of plasmid pHS091147, which co-harbored the carbapenemase (blaKPC-2) and trimethoprim resistance genes (dfrA25) from a Klebsiella pneumoniae sequence type (ST) 11 clone recovered in Shanghai, China. pHS091147 had three replication genes, several plasmid-stability genes and an intact type IV secretion system gene cluster. Besides blaKPC-2 and dfrA25, pHS091147 carried several other resistance genes, including β-lactamase genes blaTEM-1 and blaCTX-M-14, sulphonamide resistance gene sul1, a quinolone resistance gene remnant (ΔqnrB2), and virulence associated gene iroN. Notably, the multidrug-resistance region was a chimeric structure composed of three subregions, which shared strong sequence homology with several plasmids previously assigned in Genbank. To our knowledge, this is the first report of the co-localization of blaKPC-2 and dfrA25 on a novel putative multi-replicon plasmid in a Klebsiella pneumoniae ST11 clone.

  1. A putative multi-replicon plasmid co-harboring beta-lactamase genes blaKPC-2, blaCTX-M-14 and blaTEM-1 and trimethoprim resistance gene dfrA25 from a Klebsiella pneumoniae sequence type (ST) 11 strain in China

    PubMed Central

    Tang, Yu; Shen, Pinghua; Liang, Wei; Jin, Jialin; Jiang, Xiaofei

    2017-01-01

    The global emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae poses a major public health threat requiring immediate and aggressive action. Some older generation antibiotics, such as trimethoprim, serve as alternatives for treatment of infections. Here, we determined the complete nucleotide sequence of plasmid pHS091147, which co-harbored the carbapenemase (blaKPC-2) and trimethoprim resistance genes (dfrA25) from a Klebsiella pneumoniae sequence type (ST) 11 clone recovered in Shanghai, China. pHS091147 had three replication genes, several plasmid-stability genes and an intact type IV secretion system gene cluster. Besides blaKPC-2 and dfrA25, pHS091147 carried several other resistance genes, including β-lactamase genes blaTEM-1 and blaCTX-M-14, sulphonamide resistance gene sul1, a quinolone resistance gene remnant (ΔqnrB2), and virulence associated gene iroN. Notably, the multidrug-resistance region was a chimeric structure composed of three subregions, which shared strong sequence homology with several plasmids previously assigned in Genbank. To our knowledge, this is the first report of the co-localization of blaKPC-2 and dfrA25 on a novel putative multi-replicon plasmid in a Klebsiella pneumoniae ST11 clone. PMID:28152085

  2. A Multi-Variant, Viral Dynamic Model of Genotype 1 HCV to Assess the in vivo Evolution of Protease-Inhibitor Resistant Variants

    PubMed Central

    Adiwijaya, Bambang S.; Herrmann, Eva; Hare, Brian; Kieffer, Tara; Lin, Chao; Kwong, Ann D.; Garg, Varun; Randle, John C. R.; Sarrazin, Christoph; Zeuzem, Stefan; Caron, Paul R.

    2010-01-01

    Variants resistant to compounds specifically targeting HCV are observed in clinical trials. A multi-variant viral dynamic model was developed to quantify the evolution and in vivo fitness of variants in subjects dosed with monotherapy of an HCV protease inhibitor, telaprevir. Variant fitness was estimated using a model in which variants were selected by competition for shared limited replication space. Fitness was represented in the absence of telaprevir by different variant production rate constants and in the presence of telaprevir by additional antiviral blockage by telaprevir. Model parameters, including rate constants for viral production, clearance, and effective telaprevir concentration, were estimated from 1) plasma HCV RNA levels of subjects before, during, and after dosing, 2) post-dosing prevalence of plasma variants from subjects, and 3) sensitivity of variants to telaprevir in the HCV replicon. The model provided a good fit to plasma HCV RNA levels observed both during and after telaprevir dosing, as well as to variant prevalence observed after telaprevir dosing. After an initial sharp decline in HCV RNA levels during dosing with telaprevir, HCV RNA levels increased in some subjects. The model predicted this increase to be caused by pre-existing variants with sufficient fitness to expand once available replication space increased due to rapid clearance of wild-type (WT) virus. The average replicative fitness estimates in the absence of telaprevir ranged from 1% to 68% of WT fitness. Compared to the relative fitness method, the in vivo estimates from the viral dynamic model corresponded more closely to in vitro replicon data, as well as to qualitative behaviors observed in both on-dosing and long-term post-dosing clinical data. The modeling fitness estimates were robust in sensitivity analyses in which the restoration dynamics of replication space and assumptions of HCV mutation rates were varied. PMID:20419154

  3. The Rationalization of Unethical Research: Revisionist Accounts of the Tuskegee Syphilis Study and the New Zealand "Unfortunate Experiment".

    PubMed

    Paul, Charlotte; Brookes, Barbara

    2015-10-01

    Two studies, widely condemned in the 1970s and 1980s-the Tuskegee study of men with untreated syphilis and the New Zealand study of women with untreated carcinoma in situ of the cervix-received new defenses in the 21st century. We noted remarkable similarities in both the studies and their defenses. Here we evaluate the scientific, political, and moral claims of the defenders. The scientific claims are largely based on incomplete or misinterpreted evidence and exaggeration of the uncertainties of science. The defenders' political arguments mistakenly claim that identity politics clouded the original critiques; in fact such politics opened the eyes of the public to exploitation. The moral defenses demonstrate an overreliance on codes of conduct and have implications for research ethics today.

  4. Novel robust hepatitis C virus mouse efficacy model.

    PubMed

    Zhu, Qing; Oei, Yoko; Mendel, Dirk B; Garrett, Evelyn N; Patawaran, Montesa B; Hollenbach, Paul W; Aukerman, Sharon L; Weiner, Amy J

    2006-10-01

    The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-alpha) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-alpha combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

  5. Novel Robust Hepatitis C Virus Mouse Efficacy Model

    PubMed Central

    Zhu, Qing; Oei, Yoko; Mendel, Dirk B.; Garrett, Evelyn N.; Patawaran, Montesa B.; Hollenbach, Paul W.; Aukerman, Sharon L.; Weiner, Amy J.

    2006-01-01

    The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-α) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-α combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts. PMID:17005803

  6. Modeling

    SciTech Connect

    Loth, E.; Tryggvason, G.; Tsuji, Y.; Elghobashi, S. E.; Crowe, Clayton T.; Berlemont, A.; Reeks, M.; Simonin, O.; Frank, Th; Onishi, Yasuo; Van Wachem, B.

    2005-09-01

    Slurry flows occur in many circumstances, including chemical manufacturing processes, pipeline transfer of coal, sand, and minerals; mud flows; and disposal of dredged materials. In this section we discuss slurry flow applications related to radioactive waste management. The Hanford tank waste solids and interstitial liquids will be mixed to form a slurry so it can be pumped out for retrieval and treatment. The waste is very complex chemically and physically. The ARIEL code is used to model the chemical interactions and fluid dynamics of the waste.

  7. Modeling

    NASA Technical Reports Server (NTRS)

    Zook, H. A.

    1985-01-01

    A prediction of the future population of satellites, satellite fragments, and assorted spacecraft debris in Earth orbit can be reliably made only after three conditions are satisfied: (1) the size and spatial distributions of these Earth-orbiting objects are established at some present-day time; (2) the processes of orbital evolution, explosions, hypervelocity impact fragmentation, and atmospheric drag are understood; and (3) a reasonable traffic model for the future launch rate of Earth-orbiting objects is assumed. The theoretician will then take these three quantities as input data and will carry through the necessary mathematica and numerical analyses to project the present-day orbital population into the future.

  8. Interferon α/β Receptor-Deficient Mice as a Model for Ebola Virus Disease.

    PubMed

    Brannan, Jennifer M; Froude, Jeffery W; Prugar, Laura I; Bakken, Russell R; Zak, Samantha E; Daye, Sharon P; Wilhelmsen, Catherine E; Dye, John M

    2015-10-01

    A major obstacle in ebolavirus research is the lack of a small-animal model for Sudan virus (SUDV), as well as other wild-type (WT) ebolaviruses. Here, we expand on research by Bray and by Lever et al suggesting that WT ebolaviruses are pathogenic in mice deficient for the type 1 interferon (IFN) α/β receptor (IFNα/βR-/-). We examined the disease course of several WT ebolaviruses: Boneface (SUDV/Bon) and Gulu variants of SUDV, Ebola virus (EBOV), Bundibugyo virus (BDBV), Taï Forest virus, and Reston virus (RESTV). We determined that exposure to WT SUDV or EBOV results in reproducible signs of disease in IFNα/βR-/- mice, as measured by weight loss and partial lethality. Vaccination with the SUDV or EBOV glycoprotein (GP)-expressing Venezuelan equine encephalitis viral replicon particle vaccine protected these mice from SUDV/Bon and EBOV challenge, respectively. Treatment with SUDV- or EBOV-specific anti-GP antibodies protected mice from challenge when delivered 1-3 days after infection. Serial sampling experiments revealed evidence of disseminated intravascular coagulation in the livers of mice infected with the Boneface variant of SUDV, EBOV, and BDBV. Taken together, these data solidify the IFNα/βR-/- mouse as an important and useful model for the study of WT EBOV disease.

  9. Illegal, Unethical or Just Fattening? A Revisionist Look at the FTC Hearings on Electric Utility Public Relations and Franklin Roosevelt's 1932 Public Power Pledge.

    ERIC Educational Resources Information Center

    Jordan, Myron K.

    Did President Franklin D. Roosevelt's condemnation of electric utility public relations represent a fair interpretation of the findings of the Federal Trade Commission (FTC) investigation into the electric utility industry as authorized by Senate Resolution 83 in February, 1928, or were Roosevelt's statements simply campaign hyperbole that met the…

  10. Coenzyme world model of the origin of life.

    PubMed

    Sharov, Alexei A

    2016-06-01

    The origin of life means the emergence of heritable and evolvable self-reproduction. However the mechanisms of primordial heredity were different from those in contemporary cells. Here I argue that primordial life had no nucleic acids; instead heritable signs were represented by isolated catalytically active self-reproducing molecules, similar to extant coenzymes, which presumably colonized surfaces of oil droplets in water. The model further assumes that coenzyme-like molecules (CLMs) changed surface properties of oil droplets (e.g., by oxidizing terminal carbons), and in this way created and sustained favorable conditions for their own self-reproduction. Such niche-dependent self-reproduction is a necessary condition for cooperation between different kinds of CLMs because they have to coexist in the same oil droplets and either succeed or perish together. Additional kinds of hereditary molecules were acquired via coalescence of oil droplets carrying different kinds of CLMs or via modification of already existing CLMs. Eventually, polymerization of CLMs became controlled by other polymers used as templates; and this kind of template-based synthesis eventually resulted in the emergence of RNA-like replicons. Apparently, oil droplets transformed into the outer membrane of cells via engulfing water, stabilization of the surface, and osmoregulation. In result, the metabolism was internalized allowing cells to accumulate free-floating resources (e.g., animoacids, ATP), which was a necessary condition for the development of protein synthesis. Thus, life originated from simple but already functional molecules, and its gradual evolution towards higher complexity was driven by cooperation and natural selection. Published by Elsevier Ireland Ltd.

  11. Mosaic gene network modelling identified new regulatory mechanisms in HCV infection.

    PubMed

    Popik, Olga V; Petrovskiy, Evgeny D; Mishchenko, Elena L; Lavrik, Inna N; Ivanisenko, Vladimir A

    2016-06-15

    , the mosaic model integrates the model of HCV subgenomic replicon replication with the model of TNF-induced apoptosis and NF-κB induction. Analysis of the mosaic model revealed that the regulation of TNF-induced signaling by the HCV network is crucially dependent on the RIP1, TRADD, TRAF2, FADD, IKK, IκBα, c-FLIP, and BAR genes. Overall, the developed mosaic gene network modelling approach demonstrated good predictive power and allowed the prediction of new regulatory nodes in HCV action on apoptosis and the NF-κB pathway. Those theoretical predictions could be a basis for further experimental verification.

  12. In vivo validation of predicted and conserved T cell epitopes in a swine influenza model

    USDA-ARS?s Scientific Manuscript database

    Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection using inactivated whole-virus, subunit and alpha replicon-based vaccines do not provide broad prot...

  13. VRP immunotherapy targeting neu: treatment efficacy and evidence for immunoediting in a stringent rat mammary tumor model.

    PubMed

    Laust, Amanda K; Sur, Brandon W; Wang, Kehui; Hubby, Bolyn; Smith, Jonathan F; Nelson, Edward L

    2007-12-01

    The ability to overcome intrinsic tolerance to a strict "self" tumor-associated antigen (TAA) and successfully treat pre-existing tumor is the most stringent test for anti-tumor immunotherapeutic strategies. Although this capacity has been demonstrated in various models using complicated strategies that may not be readily translated into the clinical arena, straightforward antigen-specific immunotherapeutic strategies in the most stringent models of common epithelial cancers have largely failed to meet this standard. We employed an immunotherapeutic strategy using an alphavirus-based, virus-like replicon particle (VRP), which has in vivo tropism for dendritic cells, to elicit immune responses to the non-mutated TAA rat neu in an aggressive rat mammary tumor model. Using this VRP-based immunotherapeutic strategy targeting a single TAA, we generated effective anti-tumor immunity in the setting of pre-existing tumor resulting in the cure of 36% of rats over multiple experiments, P = 0.002. We also observed down-regulation of rat neu expression in tumors that showed initial responses followed by tumor escape with resumption of rapid tumor growth. These responses were accompanied by significant anti-tumor proliferative responses and CD8+ cellular tumor infiltrates, all of which were restricted to animals receiving the anti-neu immunotherapy. Together these data, obtained in a stringent "self" TAA model, indicate that the VRP-based antigen-specific immunotherapy elicits sufficiently potent immune responses to exert immunologic pressure, selection, and editing of the growing tumors, thus supporting the activity of this straightforward immunotherapy and suggesting that it is a promising platform upon which to build even more potent strategies.

  14. Mathematical Modelling of DNA Replication Reveals a Trade-off between Coherence of Origin Activation and Robustness against Rereplication

    PubMed Central

    Brümmer, Anneke; Salazar, Carlos; Zinzalla, Vittoria; Alberghina, Lilia; Höfer, Thomas

    2010-01-01

    Eukaryotic genomes are duplicated from multiple replication origins exactly once per cell cycle. In Saccharomyces cerevisiae, a complex molecular network has been identified that governs the assembly of the replication machinery. Here we develop a mathematical model that links the dynamics of this network to its performance in terms of rate and coherence of origin activation events, number of activated origins, the resulting distribution of replicon sizes and robustness against DNA rereplication. To parameterize the model, we use measured protein expression data and systematically generate kinetic parameter sets by optimizing the coherence of origin firing. While randomly parameterized networks yield unrealistically slow kinetics of replication initiation, networks with optimized parameters account for the experimentally observed distribution of origin firing times. Efficient inhibition of DNA rereplication emerges as a constraint that limits the rate at which replication can be initiated. In addition to the separation between origin licensing and firing, a time delay between the activation of S phase cyclin-dependent kinase (S-Cdk) and the initiation of DNA replication is required for preventing rereplication. Our analysis suggests that distributive multisite phosphorylation of the S-Cdk targets Sld2 and Sld3 can generate both a robust time delay and contribute to switch-like, coherent activation of replication origins. The proposed catalytic function of the complex formed by Dpb11, Sld3 and Sld2 strongly enhances coherence and robustness of origin firing. The model rationalizes how experimentally observed inefficient replication from fewer origins is caused by premature activation of S-Cdk, while premature activity of the S-Cdk targets Sld2 and Sld3 results in DNA rereplication. Thus the model demonstrates how kinetic deregulation of the molecular network governing DNA replication may result in genomic instability. PMID:20485558

  15. A Nonhuman Primate Scrub Typhus Model: Protective Immune Responses Induced by pKarp47 DNA Vaccination in Cynomolgus Macaques

    PubMed Central

    Chattopadhyay, Suchismita; Jiang, Ju; Nawtaisong, Pruksa; Lee, John S.; Tan, Esterlina; Dela Cruz, Eduardo; Burgos, Jasmin; Abalos, Rodolfo; Blacksell, Stuart D.; Lombardini, Eric; Turner, Gareth D.; Day, Nicholas P. J.; Richards, Allen L.

    2015-01-01

    We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi–specific, IFN-γ–producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine

  16. Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens.

    PubMed

    Balsiger, Sylvia; Ragaz, Curdin; Baron, Christian; Narberhaus, Franz

    2004-10-01

    Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (sigma32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5' untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive.

  17. Replicon-Specific Regulation of Small Heat Shock Genes in Agrobacterium tumefaciens

    PubMed Central

    Balsiger, Sylvia; Ragaz, Curdin; Baron, Christian; Narberhaus, Franz

    2004-01-01

    Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (σ32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5′ untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive. PMID:15466035

  18. A Brevibacterium linens pRBL1 replicon functional in Corynebacterium glutamicum.

    PubMed

    Ankri, S; Bouvier, I; Reyes, O; Predali, F; Leblon, G

    1996-07-01

    Brevibacterium linens RBL strain cryptic plasmid pRBL1 (8.0 kb) is described. A region involved in pRBL1 autonomous replication in Corynebacterium glutamicum was identified by insertion and deletion mapping and partially sequenced. This region encodes for a hypothetical 310-amino acid (aa) protein closely related to the replicases of plasmids pXZ10142 (C. glutamicum) and pAL5000 (Mycobacterium fortuitum). The 310-aa protein also shows significant homology to proteins of pColE5-099 (Shigella sonnei) and pJD1 (Neisseria gonorrhoea). At least one of these proteins, the Rep protein of pColE5-099, is known to be involved in theta replication.

  19. Application of a plasmid classification system to determine prevalence of replicon families among multidrug resistant enterococci

    USDA-ARS?s Scientific Manuscript database

    The presence and transfer of plasmids from commensal bacteria to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. However, prevalence of plasmids from commensal bacteria in food animals such as the enterococci remains largely unknown. In this study, the prevale...

  20. Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon

    PubMed Central

    Silo-Suh, Laura A.; Elmore, Brett; Ohman, Dennis E.; Suh, Sang-Jin

    2009-01-01

    In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSFts1. The mutations that resulted in a temperature-sensitive phenotype in mSFts1 were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42°C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 hours of growth. In order to facilitate its utilization, the mSFts1 was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSFts1 for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research. PMID:19615413

  1. The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties.

    PubMed

    Fukuhara, T; Koga, R; Aoki, N; Yuki, C; Yamamoto, N; Oyama, N; Udagawa, T; Horiuchi, H; Miyazaki, S; Higashi, Y; Takeshita, M; Ikeda, K; Arakawa, M; Matsumoto, N; Moriyama, H

    2006-05-01

    The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms.

  2. Antimicrobial susceptibility and plasmid replicon typing of Salmonella enterica serovar Kentucky isolates recovered from broilers

    USDA-ARS?s Scientific Manuscript database

    In the United States, Salmonella enterica serotype Kentucky has become the predominate serotype recovered from broiler slaughter samples and the prevalence of resistance to streptomycin and tetracycline has increased dramatically in this serotype. To characterize the relationships between antimicro...

  3. Toward Viral Vaccine Development: A Modified Venezuelan Equine Encephalitis Replicon as Strategy for Optimizing Immunogenicity

    DTIC Science & Technology

    2007-04-13

    and termination are indicated by triangles and diamonds , respectively. Adapted from Rice and Frolov 1996. 9 10 Figure 1.3: Schematic...2001 to 2005 and were associated with increased incidence of acute respiratory distress syndrome in conjunction with encephalitis (5-7, 80). Higher...Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome

  4. A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast.

    PubMed

    Ishibashi, Kazuhiro; Matsumoto-Yokoyama, Eiko; Ishikawa, Masayuki

    2017-10-04

    Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus of the order Bunyavirales, family Tospoviridae, genus Orthotospovirus. TSWV infects a broad range of plant species, causing serious economic losses. Despite its agronomic importance, molecular biological understanding of TSWV has been limited, partly due to the lack of a reverse genetics system, which would enable genetic manipulation of the virus. Here, we report that RNA synthesis by TSWV RNA polymerase occurs in the yeast Saccharomyces cerevisiae using a segment of the TSWV genome, S RNA expressed from cloned cDNA, as a template. Viral nucleocapsid protein was required for RNA synthesis. Replacement of the protein-coding and intergenic regions of TSWV S RNA by a yellow fluorescent protein (YFP)-coding sequence drastically increased the accumulation of both sense and antisense strands of the RNA, showing that this RNA was replicated. Using this system, we revealed that efficient RNA synthesis by TSWV RNA polymerase in yeast requires the 5'-terminal 17-nt and 3'-terminal ~50-nt regions of the TSWV S cRNA (complementary RNA to the genomic RNA) template.

  5. Excision and integration of a self-transmissible replicon of Streptomyces ambofaciens.

    PubMed

    Simonet, J M; Boccard, F; Pernodet, J L; Gagnat, J; Guérineau, M

    1987-01-01

    When Streptomyces ambofaciens OSF was crossed with the plasmid-free Streptomyces lividans TK24, almost all S. lividans exconjugants contained the free 11.1-kb plasmid pOS1. Southern hybridizations showed that pOS1 was derived from the integrated copy of previously recognized plasmid pSAM2 present in strain OSF. A shorter derivative of pOS1 was constructed carrying the tsr gene in a non-essential region, and this pOS7 plasmid was used in transformation experiments with protoplasts of S. ambofaciens ATCC23877 (containing pSAM2 only as an integrated sequence) and S. ambofaciens DSM40697 (devoid of pSAM2-related forms). In both cases, some clones carrying pOS7 in an integrated state were found. Integration into strain ATCC23877 was into the pre-existing integrated copy of pSAM2. In contrast, plasmid pOS7 integrated through specific plasmidic and chromosomal sites into strain DSM40697. Thus it is probable that pSAM2 integrates by interaction between preferred regions of the plasmid and host genomes.

  6. Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

    PubMed Central

    Mohr, Hermine; Mohr, Christian A.; Schneider, Marlon R.; Scrivano, Laura; Adler, Barbara; Kraner-Schreiber, Simone; Schnieke, Angelika; Dahlhoff, Maik; Wolf, Eckhard; Koszinowski, Ulrich H.; Ruzsics, Zsolt

    2012-01-01

    There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed. PMID:22685399

  7. The Plasmid Mobilome of the Model Plant-Symbiont Sinorhizobium meliloti: Coming up with New Questions and Answers.

    PubMed

    Lagares, Antonio; Sanjuán, Juan; Pistorio, Mariano

    2014-10-01

    Rhizobia are Gram-negative Alpha- and Betaproteobacteria living in the underground which have the ability to associate with legumes for the establishment of nitrogen-fixing symbioses. Sinorhizobium meliloti in particular-the symbiont of Medicago, Melilotus, and Trigonella spp.-has for the past decades served as a model organism for investigating, at the molecular level, the biology, biochemistry, and genetics of a free-living and symbiotic soil bacterium of agricultural relevance. To date, the genomes of seven different S. meliloti strains have been fully sequenced and annotated, and several other draft genomic sequences are also available. The vast amount of plasmid DNA that S. meliloti frequently bears (up to 45% of its total genome), the conjugative ability of some of those plasmids, and the extent of the plasmid diversity has provided researchers with an extraordinary system to investigate functional and structural plasmid molecular biology within the evolutionary context surrounding a plant-associated model bacterium. Current evidence indicates that the plasmid mobilome in S. meliloti is composed of replicons varying greatly in size and having diverse conjugative systems and properties along with different evolutionary stabilities and biological roles. While plasmids carrying symbiotic functions (pSyms) are known to have high structural stability (approaching that of chromosomes), the remaining plasmid mobilome (referred to as the non-pSym, functionally cryptic, or accessory compartment) has been shown to possess remarkable diversity and to be highly active in conjugation. In light of the modern genomic and current biochemical data on the plasmids of S. meliloti, the current article revises their main structural components, their transfer and regulatory mechanisms, and their potential as vehicles in shaping the evolution of the rhizobial genome.

  8. A tetravalent alphavirus-vector based dengue vaccine provides effective immunity in an early life mouse model.

    PubMed

    Khalil, Syed Muaz; Tonkin, Daniel R; Mattocks, Melissa D; Snead, Andrew T; Johnston, Robert E; White, Laura J

    2014-07-07

    Dengue viruses (DENV1-4) cause 390 million clinical infections every year, several hundred thousand of which progress to severe hemorrhagic and shock syndromes. Preexisting immunity resulting from a previous DENV infection is the major risk factor for severe dengue during secondary heterologous infections. During primary infections in infants, maternal antibodies pose an analogous risk. At the same time, maternal antibodies are likely to prevent induction of endogenous anti-DENV antibodies in response to current live, attenuated virus (LAV) vaccine candidates. Any effective early life dengue vaccine has to overcome maternal antibody interference (leading to ineffective vaccination) and poor induction of antibody responses (increasing the risk of severe dengue disease upon primary infection). In a previous study, we demonstrated that a non-propagating Venezuelan equine encephalitis virus replicon expression vector (VRP), expressing the ectodomain of DENV E protein (E85), overcomes maternal interference in a BALB/c mouse model. We report here that a single immunization with a tetravalent VRP vaccine induced NAb and T-cell responses to each serotype at a level equivalent to the monovalent vaccine components, suggesting that this vaccine modality can overcome serotype interference. Furthermore, neonatal immunization was durable and could be boosted later in life to further increase NAb and T-cell responses. Although the neonatal immune response was lower in magnitude than responses in adult BALB/c mice, we demonstrate that VRP vaccines generated protective immunity from a lethal challenge after a single neonatal immunization. In summary, VRP vaccines expressing DENV antigens were immunogenic and protective in neonates, and hence are promising candidates for safe and effective vaccination in early life.

  9. A tetravalent alphavirus-vector based Dengue vaccine provides effective immunity in an early life mouse model

    PubMed Central

    Khalil, Syed Muaz; Tonkin, Daniel R.; Mattocks, Melissa D.; Snead, Andrew T.; Johnston, Robert E.; White, Laura J.

    2014-01-01

    Dengue viruses (DENV1-4) cause 390 million clinical infections every year, several hundred thousand of which progress to severe hemorrhagic and shock syndromes. Preexisting immunity resulting from a previous DENV infection is the major risk factor for severe dengue during secondary heterologous infections. During primary infections in infants, maternal antibodies pose an analogous risk. At the same time, maternal antibodies are likely to prevent induction of endogenous anti-DENV antibodies in response to current live, attenuated virus (LAV) vaccine candidates. Any effective early life dengue vaccine has to overcome maternal antibody interference (leading to ineffective vaccination) and poor induction of antibody responses (increasing the risk of severe dengue disease upon primary infection). In a previous study, we demonstrated that a non-propagating Venezuelan equine encephalitis virus replicon expression vector (VRP), expressing the ectodomain of DENV E protein (E85), overcomes maternal interference in a BALB/c mouse model. We report here that a single immunization with a tetravalent VRP vaccine induced NAb and T-cell responses to each serotype at a level equivalent to the monovalent vaccine components, suggesting that this vaccine modality can overcome serotype interference. Furthermore, neonatal immunization was durable and could be boosted later in life to further increase NAb and T-cell responses. Although the neonatal immune response was lower in magnitude than responses in adult BALB/c mice, we demonstrate that VRP vaccines generated protective immunity from a lethal challenge after a single neonatal immunization. In summary, VRP vaccines expressing DENV antigens were immunogenic and protective in neonates, and hence are promising candidates for safe and effective vaccination in early life. PMID:24882043

  10. Models and role models.

    PubMed

    ten Cate, Jacob M

    2015-01-01

    Developing experimental models to understand dental caries has been the theme in our research group. Our first, the pH-cycling model, was developed to investigate the chemical reactions in enamel or dentine, which lead to dental caries. It aimed to leverage our understanding of the fluoride mode of action and was also utilized for the formulation of oral care products. In addition, we made use of intra-oral (in situ) models to study other features of the oral environment that drive the de/remineralization balance in individual patients. This model addressed basic questions, such as how enamel and dentine are affected by challenges in the oral cavity, as well as practical issues related to fluoride toothpaste efficacy. The observation that perhaps fluoride is not sufficiently potent to reduce dental caries in the present-day society triggered us to expand our knowledge in the bacterial aetiology of dental caries. For this we developed the Amsterdam Active Attachment biofilm model. Different from studies on planktonic ('single') bacteria, this biofilm model captures bacteria in a habitat similar to dental plaque. With data from the combination of these models, it should be possible to study separate processes which together may lead to dental caries. Also products and novel agents could be evaluated that interfere with either of the processes. Having these separate models in place, a suggestion is made to design computer models to encompass the available information. Models but also role models are of the utmost importance in bringing and guiding research and researchers.

  11. The effect of chemical carcinogens on DNA bypass replication and the development of in vitro and in vivo models for chemical mutagenesis

    SciTech Connect

    Yamanishi, D.T.

    1989-01-01

    This study with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with benzo(a)pyrene, 7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDEI). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-nitroso-N{prime}-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines.

  12. Leadership Models.

    ERIC Educational Resources Information Center

    Freeman, Thomas J.

    This paper discusses six different models of organizational structure and leadership, including the scalar chain or pyramid model, the continuum model, the grid model, the linking pin model, the contingency model, and the circle or democratic model. Each model is examined in a separate section that describes the model and its development, lists…

  13. Bench-to-bedside review: Fundamental principles of acid-base physiology

    PubMed Central

    Corey, Howard E

    2005-01-01

    Complex acid–base disorders arise frequently in critically ill patients, especially in those with multiorgan failure. In order to diagnose and treat these disorders better, some intensivists have abandoned traditional theories in favor of revisionist models of acid–base balance. With claimed superiority over the traditional approach, the new methods have rekindled debate over the fundmental principles of acid–base physiology. In order to shed light on this controversy, we review the derivation and application of new models of acid–base balance. PMID:15774076

  14. Models, Fiction, and Fictional Models

    NASA Astrophysics Data System (ADS)

    Liu, Chuang

    2014-03-01

    The following sections are included: * Introduction * Why Most Models in Science Are Not Fictional * Typically Fictional Models in Science * Modeling the Unobservable * Fictional Models for the Unobservable? * References

  15. From Chaos to Order and Back? A Revisionist Reflection on the California Master Plan for Higher Education@50 and Thoughts about Its Future. Research & Occasional Paper Series: CSHE.7.10

    ERIC Educational Resources Information Center

    Douglass, John Aubrey

    2010-01-01

    In 1960, California developed a "master plan" for its already famed public higher education system. It was and continues to be arguably the single most influential effort to plan the future of a system of higher education in the annals of American higher education. Despite popular belief, however, the California Master Plan for Higher…

  16. An Examination of Revisionist Theories of the First Amendment Cited in Dinesh D'Souza's "Illiberal Education": Arguments for Abridgement of Freedom of Speech on Campus To Protect the Educational Opportunities of Women and Minorities.

    ERIC Educational Resources Information Center

    Nowell, W. Robert, III

    Recently, some critics have argued that the academy has become infected by a new brand of censorship which threatens the openness central to a university's purposes. Dinesh D'Souza has argued that this censorship is a manifestation of influence enjoyed by leftist radical professors and students. D'Souza is correct that the First Amendment is being…

  17. A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant

    PubMed Central

    Agnihothram, Sudhakar; Yount, Boyd L.; Donaldson, Eric F.; Huynh, Jeremy; Menachery, Vineet D.; Gralinski, Lisa E.; Graham, Rachel L.; Becker, Michelle M.; Tomar, Sakshi; Scobey, Trevor D.; Osswald, Heather L.; Whitmore, Alan; Gopal, Robin; Ghosh, Arun K.; Mesecar, Andrew; Zambon, Maria; Heise, Mark; Denison, Mark R.; Baric, Ralph S.

    2014-01-01

    ABSTRACT Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. PMID:24667706

  18. Mental Models, Conceptual Models, and Modelling.

    ERIC Educational Resources Information Center

    Greca, Ileana Maria; Moreira, Marco Antonio

    2000-01-01

    Reviews science education research into representations constructed by students in their interactions with the world, its phenomena, and artefacts. Features discussions of mental models, conceptual models, and the activity of modeling. (Contains 30 references.) (Author/WRM)

  19. Promoting Models

    NASA Astrophysics Data System (ADS)

    Li, Qin; Zhao, Yongxin; Wu, Xiaofeng; Liu, Si

    There can be multitudinous models specifying aspects of the same system. Each model has a bias towards one aspect. These models often override in specific aspects though they have different expressions. A specification written in one model can be refined by introducing additional information from other models. The paper proposes a concept of promoting models which is a methodology to obtain refinements with support from cooperating models. It refines a primary model by integrating the information from a secondary model. The promotion principle is not merely an academic point, but also a reliable and robust engineering technique which can be used to develop software and hardware systems. It can also check the consistency between two specifications from different models. A case of modeling a simple online shopping system with the cooperation of the guarded design model and CSP model illustrates the practicability of the promotion principle.

  20. EXPRESSION AND SELF-ASSEMBLY OF NORWALK VIRUS CAPSID PROTEIN FROM VENEZUELAN EQUINE ENCEPHALITIS VIRUS REPLICONS. (R826139)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  1. Development of a nanoparticle-based oral vaccine for Atlantic salmon against ISAV using an alphavirus replicon as adjuvant.

    PubMed

    Rivas-Aravena, Andrea; Fuentes, Yazmin; Cartagena, Julio; Brito, Tania; Poggio, Verónica; La Torre, José; Mendoza, Hegaly; Gonzalez-Nilo, Fernando; Sandino, Ana María; Spencer, Eugenio

    2015-07-01

    Adjuvants used in vaccine aquaculture are frequently harmful for the fish, causing melanosis, granulomas and kidney damage. Along with that, vaccines are mostly administered by injection, causing pain and stress to the fish. We used the DNA coding for the replicase of alphavirus as adjuvant (Ad) of a vaccine against ISAV. The Ad and an inactivated ISAV (V) were loaded in chitosan nanoparticles (NPs) to be administered orally to Atlantic salmon. NP-Ad was able to deliver the DNA ex vivo and in vivo. Oral administration of the NPs stimulated the expression of immune molecules, but did not stimulate the humoral response. Although the vaccination with NP-V results in a modest protection of fish against ISAV, NP-V administered together with NP-Ad caused a protection of 77%. Therefore, the DNA coding for the replicase of alphavirus could be administered orally and can potentiate the immuneprotection of a virine against infection.

  2. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis

    USDA-ARS?s Scientific Manuscript database

    Aim: Plasmid characterization has particular clinical importance because genes encoding significant traits including antimicrobial resistance are frequently carried on plasmids. The objective of this study was to examine the distribution of multidrug resistance (MDR) in Escherichia coli in relation ...

  3. Venezuelan Equine Encephalitis Virus replicon particles can induce rapid protection against Foot-and-Mouth Disease Virus

    USDA-ARS?s Scientific Manuscript database

    We have previously shown that swine pretreated with a replication-defective human adenovirus vector (Ad5) containing the porcine type I interferon gene (poIFN-alpha/Beta) are sterilely protected when challenged one day later with Foot-and-Mouth Disease Virus (FMDV), but the dose required is relativ...

  4. A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.

    PubMed

    Jailani, A Abdul Kader; Solanki, Vikas; Roy, Anirban; Sivasudha, T; Mandal, Bikash

    2017-03-02

    A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits.

  5. Molecular Virology of Hepatitis E Virus

    PubMed Central

    Ahmad, Imran; Holla, R. Prasida; Jameel, Shahid

    2011-01-01

    This review details the molecular virology of the hepatitis E virus (HEV). While replicons and in vitro infection systems have recently become available, a lot of information on HEV has been generated through comparisons with better-studied positive-strand RNA viruses and through subgenomic expression of viral open reading frames. These models are now being verified with replicon and infection systems. We provide here the current knowledge on the HEV genome and its constituent proteins - ORF1, ORF2 and ORF3. Based on the available information, we also modify the existing model of the HEV life cycle. PMID:21345356

  6. Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model.

    PubMed

    Sumpter, Rhea; Wang, Chunfu; Foy, Eileen; Loo, Yueh-Ming; Gale, Michael

    2004-11-01

    Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-beta production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-alpha treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.

  7. Modeling Methods

    USGS Publications Warehouse

    Healy, Richard W.; Scanlon, Bridget R.

    2010-01-01

    Simulation models are widely used in all types of hydrologic studies, and many of these models can be used to estimate recharge. Models can provide important insight into the functioning of hydrologic systems by identifying factors that influence recharge. The predictive capability of models can be used to evaluate how changes in climate, water use, land use, and other factors may affect recharge rates. Most hydrological simulation models, including watershed models and groundwater-flow models, are based on some form of water-budget equation, so the material in this chapter is closely linked to that in Chapter 2. Empirical models that are not based on a water-budget equation have also been used for estimating recharge; these models generally take the form of simple estimation equations that define annual recharge as a function of precipitation and possibly other climatic data or watershed characteristics.Model complexity varies greatly. Some models are simple accounting models; others attempt to accurately represent the physics of water movement through each compartment of the hydrologic system. Some models provide estimates of recharge explicitly; for example, a model based on the Richards equation can simulate water movement from the soil surface through the unsaturated zone to the water table. Recharge estimates can be obtained indirectly from other models. For example, recharge is a parameter in groundwater-flow models that solve for hydraulic head (i.e. groundwater level). Recharge estimates can be obtained through a model calibration process in which recharge and other model parameter values are adjusted so that simulated water levels agree with measured water levels. The simulation that provides the closest agreement is called the best fit, and the recharge value used in that simulation is the model-generated estimate of recharge.

  8. Supermatrix models

    SciTech Connect

    Yost, S.A.

    1991-05-01

    Radom matrix models based on an integral over supermatrices are proposed as a natural extension of bosonic matrix models. The subtle nature of superspace integration allows these models to have very different properties from the analogous bosonic models. Two choices of integration slice are investigated. One leads to a perturbative structure which is reminiscent of, and perhaps identical to, the usual Hermitian matrix models. Another leads to an eigenvalue reduction which can be described by a two component plasma in one dimension. A stationary point of the model is described.

  9. ENTRAINMENT MODELS

    EPA Science Inventory

    This presentation presented information on entrainment models. Entrainment models use entrainment hypotheses to express the continuity equation. The advantage is that plume boundaries are known. A major disadvantage is that the problems that can be solved are rather simple. The ...

  10. ENTRAINMENT MODELS

    EPA Science Inventory

    This presentation presented information on entrainment models. Entrainment models use entrainment hypotheses to express the continuity equation. The advantage is that plume boundaries are known. A major disadvantage is that the problems that can be solved are rather simple. The ...

  11. Radiation Models

    ERIC Educational Resources Information Center

    James, W. G. G.

    1970-01-01

    Discusses the historical development of both the wave and the corpuscular photon model of light. Suggests that students should be informed that the two models are complementary and that each model successfully describes a wide range of radiation phenomena. Cites 19 references which might be of interest to physics teachers and students. (LC)

  12. Radiation Models

    ERIC Educational Resources Information Center

    James, W. G. G.

    1970-01-01

    Discusses the historical development of both the wave and the corpuscular photon model of light. Suggests that students should be informed that the two models are complementary and that each model successfully describes a wide range of radiation phenomena. Cites 19 references which might be of interest to physics teachers and students. (LC)

  13. Physical observables of the Ising spin glass in 6 -ɛ dimensions: Asymptotical behavior around the critical fixed point

    NASA Astrophysics Data System (ADS)

    Temesvári, T.

    2017-07-01

    The asymptotical behavior of physical quantities, like the order parameter, the replicon, and longitudinal masses, is studied around the zero-field spin-glass transition point when a small external magnetic field is applied. An effective field theory to model this asymptotics contains a small perturbation in its Lagrangian which breaks the zero-field symmetry. A first-order renormalization group supplemented by perturbational results provides the scaling functions. The perturbative zero of the scaling function for the replicon mass defines a generic Almeida-Thouless surface stemming from the zero-field fixed point.

  14. Hydrological models are mediating models

    NASA Astrophysics Data System (ADS)

    Babel, L. V.; Karssenberg, D.

    2013-08-01

    Despite the increasing role of models in hydrological research and decision-making processes, only few accounts of the nature and function of models exist in hydrology. Earlier considerations have traditionally been conducted while making a clear distinction between physically-based and conceptual models. A new philosophical account, primarily based on the fields of physics and economics, transcends classes of models and scientific disciplines by considering models as "mediators" between theory and observations. The core of this approach lies in identifying models as (1) being only partially dependent on theory and observations, (2) integrating non-deductive elements in their construction, and (3) carrying the role of instruments of scientific enquiry about both theory and the world. The applicability of this approach to hydrology is evaluated in the present article. Three widely used hydrological models, each showing a different degree of apparent physicality, are confronted to the main characteristics of the "mediating models" concept. We argue that irrespective of their kind, hydrological models depend on both theory and observations, rather than merely on one of these two domains. Their construction is additionally involving a large number of miscellaneous, external ingredients, such as past experiences, model objectives, knowledge and preferences of the modeller, as well as hardware and software resources. We show that hydrological models convey the role of instruments in scientific practice by mediating between theory and the world. It results from these considerations that the traditional distinction between physically-based and conceptual models is necessarily too simplistic and refers at best to the stage at which theory and observations are steering model construction. The large variety of ingredients involved in model construction would deserve closer attention, for being rarely explicitly presented in peer-reviewed literature. We believe that devoting

  15. Model Experiments and Model Descriptions

    NASA Technical Reports Server (NTRS)

    Jackman, Charles H.; Ko, Malcolm K. W.; Weisenstein, Debra; Scott, Courtney J.; Shia, Run-Lie; Rodriguez, Jose; Sze, N. D.; Vohralik, Peter; Randeniya, Lakshman; Plumb, Ian

    1999-01-01

    The Second Workshop on Stratospheric Models and Measurements Workshop (M&M II) is the continuation of the effort previously started in the first Workshop (M&M I, Prather and Remsberg [1993]) held in 1992. As originally stated, the aim of M&M is to provide a foundation for establishing the credibility of stratospheric models used in environmental assessments of the ozone response to chlorofluorocarbons, aircraft emissions, and other climate-chemistry interactions. To accomplish this, a set of measurements of the present day atmosphere was selected. The intent was that successful simulations of the set of measurements should become the prerequisite for the acceptance of these models as having a reliable prediction for future ozone behavior. This section is divided into two: model experiment and model descriptions. In the model experiment, participant were given the charge to design a number of experiments that would use observations to test whether models are using the correct mechanisms to simulate the distributions of ozone and other trace gases in the atmosphere. The purpose is closely tied to the needs to reduce the uncertainties in the model predicted responses of stratospheric ozone to perturbations. The specifications for the experiments were sent out to the modeling community in June 1997. Twenty eight modeling groups responded to the requests for input. The first part of this section discusses the different modeling group, along with the experiments performed. Part two of this section, gives brief descriptions of each model as provided by the individual modeling groups.

  16. Model Experiments and Model Descriptions

    NASA Technical Reports Server (NTRS)

    Jackman, Charles H.; Ko, Malcolm K. W.; Weisenstein, Debra; Scott, Courtney J.; Shia, Run-Lie; Rodriguez, Jose; Sze, N. D.; Vohralik, Peter; Randeniya, Lakshman; Plumb, Ian

    1999-01-01

    The Second Workshop on Stratospheric Models and Measurements Workshop (M&M II) is the continuation of the effort previously started in the first Workshop (M&M I, Prather and Remsberg [1993]) held in 1992. As originally stated, the aim of M&M is to provide a foundation for establishing the credibility of stratospheric models used in environmental assessments of the ozone response to chlorofluorocarbons, aircraft emissions, and other climate-chemistry interactions. To accomplish this, a set of measurements of the present day atmosphere was selected. The intent was that successful simulations of the set of measurements should become the prerequisite for the acceptance of these models as having a reliable prediction for future ozone behavior. This section is divided into two: model experiment and model descriptions. In the model experiment, participant were given the charge to design a number of experiments that would use observations to test whether models are using the correct mechanisms to simulate the distributions of ozone and other trace gases in the atmosphere. The purpose is closely tied to the needs to reduce the uncertainties in the model predicted responses of stratospheric ozone to perturbations. The specifications for the experiments were sent out to the modeling community in June 1997. Twenty eight modeling groups responded to the requests for input. The first part of this section discusses the different modeling group, along with the experiments performed. Part two of this section, gives brief descriptions of each model as provided by the individual modeling groups.

  17. ICRF modelling

    SciTech Connect

    Phillips, C.K.

    1985-12-01

    This lecture provides a survey of the methods used to model fast magnetosonic wave coupling, propagation, and absorption in tokamaks. The validity and limitations of three distinct types of modelling codes, which will be contrasted, include discrete models which utilize ray tracing techniques, approximate continuous field models based on a parabolic approximation of the wave equation, and full field models derived using finite difference techniques. Inclusion of mode conversion effects in these models and modification of the minority distribution function will also be discussed. The lecture will conclude with a presentation of time-dependent global transport simulations of ICRF-heated tokamak discharges obtained in conjunction with the ICRF modelling codes. 52 refs., 15 figs.

  18. Climate Models

    NASA Technical Reports Server (NTRS)

    Druyan, Leonard M.

    2012-01-01

    Climate models is a very broad topic, so a single volume can only offer a small sampling of relevant research activities. This volume of 14 chapters includes descriptions of a variety of modeling studies for a variety of geographic regions by an international roster of authors. The climate research community generally uses the rubric climate models to refer to organized sets of computer instructions that produce simulations of climate evolution. The code is based on physical relationships that describe the shared variability of meteorological parameters such as temperature, humidity, precipitation rate, circulation, radiation fluxes, etc. Three-dimensional climate models are integrated over time in order to compute the temporal and spatial variations of these parameters. Model domains can be global or regional and the horizontal and vertical resolutions of the computational grid vary from model to model. Considering the entire climate system requires accounting for interactions between solar insolation, atmospheric, oceanic and continental processes, the latter including land hydrology and vegetation. Model simulations may concentrate on one or more of these components, but the most sophisticated models will estimate the mutual interactions of all of these environments. Advances in computer technology have prompted investments in more complex model configurations that consider more phenomena interactions than were possible with yesterday s computers. However, not every attempt to add to the computational layers is rewarded by better model performance. Extensive research is required to test and document any advantages gained by greater sophistication in model formulation. One purpose for publishing climate model research results is to present purported advances for evaluation by the scientific community.

  19. Phenomenological models

    SciTech Connect

    Braby, L.A.

    1990-09-01

    The biological effects of ionizing radiation exposure are the result of a complex sequence of physical, chemical, biochemical, and physiological interactions. One way to begin a search for an understanding of health effects of radiation is through the development of phenomenological models of the response. Many models have been presented and tested in the slowly evolving process of characterizing cellular response. A range of models covering different endpoints and phenomena has developed in parallel. Many of these models employ similar assumptions about some underlying processes while differing about the nature of others. An attempt is made to organize many of the models into groups with similar features and to compare the consequences of those features with the actual experimental observations. It is assumed that by showing that some assumptions are inconsistent with experimental observations, the job of devising and testing mechanistic models can be simplified. 43 refs., 13 figs.

  20. Cloud Modeling

    NASA Technical Reports Server (NTRS)

    Tao, Wei-Kuo; Moncrieff, Mitchell; Einaud, Franco (Technical Monitor)

    2001-01-01

    Numerical cloud models have been developed and applied extensively to study cloud-scale and mesoscale processes during the past four decades. The distinctive aspect of these cloud models is their ability to treat explicitly (or resolve) cloud-scale dynamics. This requires the cloud models to be formulated from the non-hydrostatic equations of motion that explicitly include the vertical acceleration terms since the vertical and horizontal scales of convection are similar. Such models are also necessary in order to allow gravity waves, such as those triggered by clouds, to be resolved explicitly. In contrast, the hydrostatic approximation, usually applied in global or regional models, does allow the presence of gravity waves. In addition, the availability of exponentially increasing computer capabilities has resulted in time integrations increasing from hours to days, domain grids boxes (points) increasing from less than 2000 to more than 2,500,000 grid points with 500 to 1000 m resolution, and 3-D models becoming increasingly prevalent. The cloud resolving model is now at a stage where it can provide reasonably accurate statistical information of the sub-grid, cloud-resolving processes poorly parameterized in climate models and numerical prediction models.

  1. Ventilation Model

    SciTech Connect

    V. Chipman

    2002-10-05

    The purpose of the Ventilation Model is to simulate the heat transfer processes in and around waste emplacement drifts during periods of forced ventilation. The model evaluates the effects of emplacement drift ventilation on the thermal conditions in the emplacement drifts and surrounding rock mass, and calculates the heat removal by ventilation as a measure of the viability of ventilation to delay the onset of peak repository temperature and reduce its magnitude. The heat removal by ventilation is temporally and spatially dependent, and is expressed as the fraction of heat carried away by the ventilation air compared to the fraction of heat produced by radionuclide decay. One minus the heat removal is called the wall heat fraction, or the remaining amount of heat that is transferred via conduction to the surrounding rock mass. Downstream models, such as the ''Multiscale Thermohydrologic Model'' (BSC 2001), use the wall heat fractions as outputted from the Ventilation Model to initialize their post-closure analyses. The Ventilation Model report was initially developed to analyze the effects of preclosure continuous ventilation in the Engineered Barrier System (EBS) emplacement drifts, and to provide heat removal data to support EBS design. Revision 00 of the Ventilation Model included documentation of the modeling results from the ANSYS-based heat transfer model. The purposes of Revision 01 of the Ventilation Model are: (1) To validate the conceptual model for preclosure ventilation of emplacement drifts and verify its numerical application in accordance with new procedural requirements as outlined in AP-SIII-10Q, Models (Section 7.0). (2) To satisfy technical issues posed in KTI agreement RDTME 3.14 (Reamer and Williams 2001a). Specifically to demonstrate, with respect to the ANSYS ventilation model, the adequacy of the discretization (Section 6.2.3.1), and the downstream applicability of the model results (i.e. wall heat fractions) to initialize post

  2. Molecular modeling and residue interaction network studies on the mechanism of binding and resistance of the HCV NS5B polymerase mutants to VX-222 and ANA598.

    PubMed

    Xue, Weiwei; Jiao, Pingzu; Liu, Huanxiang; Yao, Xiaojun

    2014-04-01

    Hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) with essential functions in viral genome replication and represents a promising therapeutic target to develop direct-acting antivirals (DAAs). Multiple nonnucleoside inhibitors (NNIs) binding sites have been identified within the polymerase. VX-222 and ANA598 are two NNIs targeting thumb II site and palm I site of HCV NS5B polymerase, respectively. These two molecules have been shown to be very effective in phase II clinical trials. However, the emergence of resistant HCV replicon variants (L419M, M423T, I482L mutants to VX-222 and M414T, M414L, G554D mutants to ANA598) has significantly decreased their efficacy. To elucidate the molecular mechanism about how these mutations influenced the drug binding mode and decreased drug efficacy, we studied the binding modes of VX-222 and ANA598 to wild-type and mutant polymerase by molecular modeling approach. Molecular dynamics (MD) simulations results combined with binding free energy calculations indicated that the mutations significantly altered the binding free energy and the interaction for the drugs to polymerase. The further per-residue binding free energy decomposition analysis revealed that the mutations decreased the interactions with several key residues, such as L419, M423, L474, S476, I482, L497, for VX-222 and L384, N411, M414, Y415, Q446, S556, G557 for ANA598. These were the major origins for the resistance to these two drugs. In addition, by analyzing the residue interaction network (RIN) of the complexes between the drugs with wild-type and the mutant polymerase, we found that the mutation residues in the networks involved in the drug resistance possessed a relatively lower size of topology centralities. The shift of betweenness and closeness values of binding site residues in the mutant polymerase is relevant to the mechanism of drug resistance of VX-222 and ANA598. These results can provide an atomic-level understanding about

  3. Model Selection for Geostatistical Models

    SciTech Connect

    Hoeting, Jennifer A.; Davis, Richard A.; Merton, Andrew A.; Thompson, Sandra E.

    2006-02-01

    We consider the problem of model selection for geospatial data. Spatial correlation is typically ignored in the selection of explanatory variables and this can influence model selection results. For example, the inclusion or exclusion of particular explanatory variables may not be apparent when spatial correlation is ignored. To address this problem, we consider the Akaike Information Criterion (AIC) as applied to a geostatistical model. We offer a heuristic derivation of the AIC in this context and provide simulation results that show that using AIC for a geostatistical model is superior to the often used approach of ignoring spatial correlation in the selection of explanatory variables. These ideas are further demonstrated via a model for lizard abundance. We also employ the principle of minimum description length (MDL) to variable selection for the geostatistical model. The effect of sampling design on the selection of explanatory covariates is also explored.

  4. Modeling Sunspots

    ERIC Educational Resources Information Center

    Oh, Phil Seok; Oh, Sung Jin

    2013-01-01

    Modeling in science has been studied by education researchers for decades and is now being applied broadly in school. It is among the scientific practices featured in the "Next Generation Science Standards" ("NGSS") (Achieve Inc. 2013). This article describes modeling activities in an extracurricular science club in a high…

  5. Turbulence modeling

    NASA Technical Reports Server (NTRS)

    Bardina, Jorge E.

    1995-01-01

    The objective of this work is to develop, verify, and incorporate the baseline two-equation turbulence models which account for the effects of compressibility into the three-dimensional Reynolds averaged Navier-Stokes (RANS) code and to provide documented descriptions of the models and their numerical procedures so that they can be implemented into 3-D CFD codes for engineering applications.

  6. Dispersion Modeling.

    ERIC Educational Resources Information Center

    Budiansky, Stephen

    1980-01-01

    This article discusses the need for more accurate and complete input data and field verification of the various models of air pollutant dispension. Consideration should be given to changing the form of air quality standards based on enhanced dispersion modeling techniques. (Author/RE)

  7. Qualitative modeling.

    PubMed

    Forbus, Kenneth D

    2011-07-01

    Qualitative modeling concerns the representations and reasoning that people use to understand continuous aspects of the world. Qualitative models formalize everyday notions of causality and provide accounts of how to ground symbolic, relational representations in perceptual processes. This article surveys the basic ideas of qualitative modeling and their applications from a cognitive science perspective. It describes the basic principles of qualitative modeling, and a variety of qualitative representations that have been developed for quantities and for relationships between them, providing a kind of qualitative mathematics. Three ontological frameworks for organizing modeling knowledge (processes, components, and field) are summarized, along with research on automatically assembling models for particular tasks from such knowledge. Qualitative simulation and how it carves up time into meaningful units is discussed. We discuss several accounts of causal reasoning about dynamical systems, based on different choices of qualitative mathematics and ontology. Qualitative spatial reasoning is explored, both in terms of relational systems and visual reasoning. Applications of qualitative models of particular interest to cognitive scientists are described, including how they have been used to capture the expertise of scientists and engineers and how they have been used in education. Open questions and frontiers are also discussed, focusing on relationships between ideas developed in the qualitative modeling community and other areas of cognitive science. WIREs Cogni Sci 2011 2 374-391 DOI: 10.1002/wcs.115 For further resources related to this article, please visit the WIREs website.

  8. Modeling Sunspots

    ERIC Educational Resources Information Center

    Oh, Phil Seok; Oh, Sung Jin

    2013-01-01

    Modeling in science has been studied by education researchers for decades and is now being applied broadly in school. It is among the scientific practices featured in the "Next Generation Science Standards" ("NGSS") (Achieve Inc. 2013). This article describes modeling activities in an extracurricular science club in a high…

  9. Dispersion Modeling.

    ERIC Educational Resources Information Center

    Budiansky, Stephen

    1980-01-01

    This article discusses the need for more accurate and complete input data and field verification of the various models of air pollutant dispension. Consideration should be given to changing the form of air quality standards based on enhanced dispersion modeling techniques. (Author/RE)

  10. Climate models and model evaluation

    SciTech Connect

    Gates, W.L.

    1994-12-31

    This brief overview addresses aspects of the nature, uses, evaluation and limitations of climate models. A comprehensive global modeling capability has been achieved only for the physical climate system, which is characterized by processes that serve to transport and exchange momentum, heat and moisture within and between the atmosphere, ocean and land surface. The fundamental aim of climate modeling, and the justification for the use of climate models, is the need to achieve a quantitative understanding of the operation of the climate system and to exploit any potential predictability that may exist.

  11. OSPREY Model

    SciTech Connect

    Veronica J. Rutledge

    2013-01-01

    The absence of industrial scale nuclear fuel reprocessing in the U.S. has precluded the necessary driver for developing the advanced simulation capability now prevalent in so many other countries. Thus, it is essential to model complex series of unit operations to simulate, understand, and predict inherent transient behavior and feedback loops. A capability of accurately simulating the dynamic behavior of advanced fuel cycle separation processes will provide substantial cost savings and many technical benefits. The specific fuel cycle separation process discussed in this report is the off-gas treatment system. The off-gas separation consists of a series of scrubbers and adsorption beds to capture constituents of interest. Dynamic models are being developed to simulate each unit operation involved so each unit operation can be used as a stand-alone model and in series with multiple others. Currently, an adsorption model has been developed within Multi-physics Object Oriented Simulation Environment (MOOSE) developed at the Idaho National Laboratory (INL). Off-gas Separation and REcoverY (OSPREY) models the adsorption of off-gas constituents for dispersed plug flow in a packed bed under non-isothermal and non-isobaric conditions. Inputs to the model include gas, sorbent, and column properties, equilibrium and kinetic data, and inlet conditions. The simulation outputs component concentrations along the column length as a function of time from which breakthrough data is obtained. The breakthrough data can be used to determine bed capacity, which in turn can be used to size columns. It also outputs temperature along the column length as a function of time and pressure drop along the column length. Experimental data and parameters were input into the adsorption model to develop models specific for krypton adsorption. The same can be done for iodine, xenon, and tritium. The model will be validated with experimental breakthrough curves. Customers will be given access to

  12. Model hydrographs

    USGS Publications Warehouse

    Mitchell, W.D.

    1972-01-01

    Model hydrographs are composed of pairs of dimensionless ratios, arrayed in tabular form, which, when modified by the appropriate values of rainfall exceed and by the time and areal characteristics of the drainage basin, satisfactorily represent the flood hydrograph for the basin. Model bydrographs are developed from a dimensionless translation hydrograph, having a time base of T hours and appropriately modified for storm duration by routing through reservoir storage, S=kOx. Models fall into two distinct classes: (1) those for which the value of x is unity and which have all the characteristics of true unit hydrographs and (2) those for which the value of x is other than unity and to which the unit-hydrograph principles of proportionality and superposition do not apply. Twenty-six families of linear models and eight families of nonlinear models in tabular form from the principal subject of this report. Supplemental discussions describe the development of the models and illustrate their application. Other sections of the report, supplemental to the tables, describe methods of determining the hydrograph characteristics, T, k, and x, both from observed hydrograph and from the physical characteristics of the drainage basin. Five illustrative examples of use show that the models, when properly converted to incorporate actual rainfall excess and the time and areal characteristics of the drainage basins, do indeed satisfactorily represent the observed flood hydrographs for the basins.

  13. Stereometric Modelling

    NASA Astrophysics Data System (ADS)

    Grimaldi, P.

    2012-07-01

    These mandatory guidelines are provided for preparation of papers accepted for publication in the series of Volumes of The The stereometric modelling means modelling achieved with : - the use of a pair of virtual cameras, with parallel axes and positioned at a mutual distance average of 1/10 of the distance camera-object (in practice the realization and use of a stereometric camera in the modeling program); - the shot visualization in two distinct windows - the stereoscopic viewing of the shot while modelling. Since the definition of "3D vision" is inaccurately referred to as the simple perspective of an object, it is required to add the word stereo so that "3D stereo vision " shall stand for "three-dimensional view" and ,therefore, measure the width, height and depth of the surveyed image. Thanks to the development of a stereo metric model , either real or virtual, through the "materialization", either real or virtual, of the optical-stereo metric model made visible with a stereoscope. It is feasible a continuous on line updating of the cultural heritage with the help of photogrammetry and stereometric modelling. The catalogue of the Architectonic Photogrammetry Laboratory of Politecnico di Bari is available on line at: http://rappresentazione.stereofot.it:591/StereoFot/FMPro?-db=StereoFot.fp5&-lay=Scheda&-format=cerca.htm&-view

  14. A Model for Math Modeling

    ERIC Educational Resources Information Center

    Lin, Tony; Erfan, Sasan

    2016-01-01

    Mathematical modeling is an open-ended research subject where no definite answers exist for any problem. Math modeling enables thinking outside the box to connect different fields of studies together including statistics, algebra, calculus, matrices, programming and scientific writing. As an integral part of society, it is the foundation for many…

  15. A Model for Math Modeling

    ERIC Educational Resources Information Center

    Lin, Tony; Erfan, Sasan

    2016-01-01

    Mathematical modeling is an open-ended research subject where no definite answers exist for any problem. Math modeling enables thinking outside the box to connect different fields of studies together including statistics, algebra, calculus, matrices, programming and scientific writing. As an integral part of society, it is the foundation for many…

  16. Modeling complexes of modeled proteins.

    PubMed

    Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A

    2017-03-01

    Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C(α) RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Whole-genome Sequencing of the Saprophyte L. biflexa and Comparative Genomics of Pathogoenic and Saprophytic Leptospira Species

    USDA-ARS?s Scientific Manuscript database

    We present the complete genome sequences of Leptospira biflexa, a saprophyte that is used as a model for gene functional analysis in Leptospira spp. The L. biflexa genome has 3,730 protein-coding genes distributed across three circular replicons: two of which are chromosomes (3,600- and 278-kb in si...

  18. Genome Sequence of the Saprophyte Leptospira Biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis

    USDA-ARS?s Scientific Manuscript database

    Leptospira biflexa is a free-living spirochete found broadly distributed in aquatic environments, and is the primary model for studying gene function in Leptospira spp. The L. biflexa genome has 3,590 protein-coding genes (excluding transposases) distributed across three circular replicons: two of w...

  19. Environmental Modeling

    EPA Pesticide Factsheets

    EPA's modeling community is working to gain insights into certain parts of a physical, biological, economic, or social system by conducting environmental assessments for Agency decision making to complex environmental issues.

  20. Anchor Modeling

    NASA Astrophysics Data System (ADS)

    Regardt, Olle; Rönnbäck, Lars; Bergholtz, Maria; Johannesson, Paul; Wohed, Petia

    Maintaining and evolving data warehouses is a complex, error prone, and time consuming activity. The main reason for this state of affairs is that the environment of a data warehouse is in constant change, while the warehouse itself needs to provide a stable and consistent interface to information spanning extended periods of time. In this paper, we propose a modeling technique for data warehousing, called anchor modeling, that offers non-destructive extensibility mechanisms, thereby enabling robust and flexible management of changes in source systems. A key benefit of anchor modeling is that changes in a data warehouse environment only require extensions, not modifications, to the data warehouse. This ensures that existing data warehouse applications will remain unaffected by the evolution of the data warehouse, i.e. existing views and functions will not have to be modified as a result of changes in the warehouse model.

  1. Model Lungs.

    ERIC Educational Resources Information Center

    Taylor, Emma

    1991-01-01

    A cheap and simple model that can be made and used by pupils to study the human breathing mechanism is presented. A list of needed materials, procedures for construction, possible refinements, and method of use are included. (KR)

  2. Micromolecular modeling

    NASA Technical Reports Server (NTRS)

    Guillet, J. E.

    1984-01-01

    A reaction kinetics based model of the photodegradation process, which measures all important rate constants, and a computerized model capable of predicting the photodegradation rate and failure modes of a 30 year period, were developed. It is shown that the computerized photodegradation model for polyethylene correctly predicts failure of ELVAX 15 and cross linked ELVAX 150 on outdoor exposure. It is indicated that cross linking ethylene vinyl acetate (EVA) does not significantly change its degradation rate. It is shown that the effect of the stabilizer package is approximately equivalent on both polymers. The computerized model indicates that peroxide decomposers and UV absorbers are the most effective stabilizers. It is found that a combination of UV absorbers and a hindered amine light stabilizer (HALS) is the most effective stabilizer system.

  3. Programming models

    SciTech Connect

    Daniel, David J; Mc Pherson, Allen; Thorp, John R; Barrett, Richard; Clay, Robert; De Supinski, Bronis; Dube, Evi; Heroux, Mike; Janssen, Curtis; Langer, Steve; Laros, Jim

    2011-01-14

    A programming model is a set of software technologies that support the expression of algorithms and provide applications with an abstract representation of the capabilities of the underlying hardware architecture. The primary goals are productivity, portability and performance.

  4. Energy Models

    EPA Science Inventory

    Energy models characterize the energy system, its evolution, and its interactions with the broader economy. The energy system consists of primary resources, including both fossil fuels and renewables; power plants, refineries, and other technologies to process and convert these r...

  5. Energy Models

    EPA Science Inventory

    Energy models characterize the energy system, its evolution, and its interactions with the broader economy. The energy system consists of primary resources, including both fossil fuels and renewables; power plants, refineries, and other technologies to process and convert these r...

  6. PREDICTIVE MODELS

    SciTech Connect

    Ray, R.M. )

    1986-12-01

    PREDICTIVE MODELS is a collection of five models - CFPM, CO2PM, ICPM, PFPM, and SFPM - used in the 1982-1984 National Petroleum Council study of enhanced oil recovery (EOR) potential. Each pertains to a specific EOR process designed to squeeze additional oil from aging or spent oil fields. The processes are: 1) chemical flooding, where soap-like surfactants are injected into the reservoir to wash out the oil; 2) carbon dioxide miscible flooding, where carbon dioxide mixes with the lighter hydrocarbons making the oil easier to displace; 3) in-situ combustion, which uses the heat from burning some of the underground oil to thin the product; 4) polymer flooding, where thick, cohesive material is pumped into a reservoir to push the oil through the underground rock; and 5) steamflood, where pressurized steam is injected underground to thin the oil. CFPM, the Chemical Flood Predictive Model, models micellar (surfactant)-polymer floods in reservoirs, which have been previously waterflooded to residual oil saturation. Thus, only true tertiary floods are considered. An option allows a rough estimate of oil recovery by caustic or caustic-polymer processes. CO2PM, the Carbon Dioxide miscible flooding Predictive Model, is applicable to both secondary (mobile oil) and tertiary (residual oil) floods, and to either continuous CO2 injection or water-alternating gas processes. ICPM, the In-situ Combustion Predictive Model, computes the recovery and profitability of an in-situ combustion project from generalized performance predictive algorithms. PFPM, the Polymer Flood Predictive Model, is switch-selectable for either polymer or waterflooding, and an option allows the calculation of the incremental oil recovery and economics of polymer relative to waterflooding. SFPM, the Steamflood Predictive Model, is applicable to the steam drive process, but not to cyclic steam injection (steam soak) processes.

  7. Model selection for geostatistical models.

    PubMed

    Hoeting, Jennifer A; Davis, Richard A; Merton, Andrew A; Thompson, Sandra E

    2006-02-01

    We consider the problem of model selection for geospatial data. Spatial correlation is often ignored in the selection of explanatory variables, and this can influence model selection results. For example, the importance of particular explanatory variables may not be apparent when spatial correlation is ignored. To address this problem, we consider the Akaike Information Criterion (AIC) as applied to a geostatistical model. We offer a heuristic derivation of the AIC in this context and provide simulation results that show that using AIC for a geostatistical model is superior to the often-used traditional approach of ignoring spatial correlation in the selection of explanatory variables. These ideas are further demonstrated via a model for lizard abundance. We also apply the principle of minimum description length (MDL) to variable selection for the geostatistical model. The effect of sampling design on the selection of explanatory covariates is also explored. R software to implement the geostatistical model selection methods described in this paper is available in the Supplement.

  8. The French Revolution and "Revisionism."

    ERIC Educational Resources Information Center

    Langlois, Claude

    1990-01-01

    Outlines revisionist interpretations of the French Revolution that challenged the dominant historiographical tradition during the 1950s and 1960s. Distinguishes four central characteristics of revisionist works. Identifies a key split in current French Revolution historiography between reflection on nineteenth-century…

  9. The French Revolution and "Revisionism."

    ERIC Educational Resources Information Center

    Langlois, Claude

    1990-01-01

    Outlines revisionist interpretations of the French Revolution that challenged the dominant historiographical tradition during the 1950s and 1960s. Distinguishes four central characteristics of revisionist works. Identifies a key split in current French Revolution historiography between reflection on nineteenth-century…

  10. Scalable Models Using Model Transformation

    DTIC Science & Technology

    2008-07-13

    huge number of web documents. We have created a simplified demo using 5 worker machines in the Ptolemy II modeling and simulation environment [3], as...the pattern of the transformation rule matches any subgraph of the input model. When the TransformationRule actor is opened in the Ptolemy II GUI...tool developed in the Ptolemy II frame- work, existing tools include AGG [14], PROGRES [15], AToM3 [16], FUJABA [17], VIATRA2 [18], and GReAT [19

  11. Bioactive cembrane derivatives from the Indian Ocean soft coral, Sinularia kavarattiensis.

    PubMed

    Lillsunde, Katja-Emilia; Festa, Carmen; Adel, Harshada; De Marino, Simona; Lombardi, Valter; Tilvi, Supriya; Nawrot, Dorota A; Zampella, Angela; D'Souza, Lisette; D'Auria, Maria Valeria; Tammela, Päivi

    2014-07-03

    Marine organisms and their metabolites represent a unique source of potential pharmaceutical substances. In this study, we examined marine-derived substances for their bioactive properties in a cell-based Chikungunya virus (CHIKV) replicon model and for in vitro anti-inflammatory activity. In the screening of a marine sample library, crude extracts from the Indian soft coral, Sinularia kavarattiensis, showed promising activity against the CHIKV replicon. Bioassay-guided chemical fractionation of S. kavarattiensis resulted in the isolation of six known norcembranoids (1-6) and one new compound, named kavaranolide (7). The structures were elucidated on the basis of NMR and MS spectroscopic data. Compounds 1-3 and 5-7 were evaluated for their replicon-inhibiting potential in the CHIKV model by using a luminescence-based detection technique and live cell imaging. Compounds 1 and 2 showed moderate inhibition of the CHIKV replicon, but imaging studies also revealed cytotoxic properties. Moreover, the effects of the isolated compounds on primary microglial cells, an experimental model for neuroinflammation, were evaluated. Compound 2 was shown to modulate the immune response in microglial cells and to possess potential anti-inflammatory properties by dose-dependently reducing the release of pro- and anti-inflammatory cytokines.

  12. Bioactive Cembrane Derivatives from the Indian Ocean Soft Coral, Sinularia kavarattiensis

    PubMed Central

    Lillsunde, Katja-Emilia; Festa, Carmen; Adel, Harshada; De Marino, Simona; Lombardi, Valter; Tilvi, Supriya; Nawrot, Dorota A.; Zampella, Angela; D’Souza, Lisette; D’Auria, Maria Valeria; Tammela, Päivi

    2014-01-01

    Marine organisms and their metabolites represent a unique source of potential pharmaceutical substances. In this study, we examined marine-derived substances for their bioactive properties in a cell-based Chikungunya virus (CHIKV) replicon model and for in vitro anti-inflammatory activity. In the screening of a marine sample library, crude extracts from the Indian soft coral, Sinularia kavarattiensis, showed promising activity against the CHIKV replicon. Bioassay-guided chemical fractionation of S. kavarattiensis resulted in the isolation of six known norcembranoids (1–6) and one new compound, named kavaranolide (7). The structures were elucidated on the basis of NMR and MS spectroscopic data. Compounds 1–3 and 5–7 were evaluated for their replicon-inhibiting potential in the CHIKV model by using a luminescence-based detection technique and live cell imaging. Compounds 1 and 2 showed moderate inhibition of the CHIKV replicon, but imaging studies also revealed cytotoxic properties. Moreover, the effects of the isolated compounds on primary microglial cells, an experimental model for neuroinflammation, were evaluated. Compound 2 was shown to modulate the immune response in microglial cells and to possess potential anti-inflammatory properties by dose-dependently reducing the release of pro- and anti-inflammatory cytokines. PMID:25056629

  13. Mechanistic models

    SciTech Connect

    Curtis, S.B.

    1990-09-01

    Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) interaction'' of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

  14. Mechanistic models

    SciTech Connect

    Curtis, S.B.

    1990-09-01

    Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) ``interaction`` of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

  15. Do stroke models model stroke?

    PubMed Central

    Mergenthaler, Philipp; Meisel, Andreas

    2012-01-01

    Stroke is one of the leading causes of death worldwide and the biggest reason for long-term disability. Basic research has formed the modern understanding of stroke pathophysiology, and has revealed important molecular, cellular and systemic mechanisms. However, despite decades of research, most translational stroke trials that aim to introduce basic research findings into clinical treatment strategies – most notably in the field of neuroprotection – have failed. Among other obstacles, poor methodological and statistical standards, negative publication bias, and incomplete preclinical testing have been proposed as ‘translational roadblocks’. In this article, we introduce the models commonly used in preclinical stroke research, discuss some of the causes of failed translational success and review potential remedies. We further introduce the concept of modeling ‘care’ of stroke patients, because current preclinical research models the disorder but does not model care or state-of-the-art clinical testing. Stringent statistical methods and controlled preclinical trials have been suggested to counteract weaknesses in preclinical research. We conclude that preclinical stroke research requires (1) appropriate modeling of the disorder, (2) appropriate modeling of the care of stroke patients and (3) an approach to preclinical testing that is similar to clinical testing, including Phase 3 randomized controlled preclinical trials as necessary additional steps before new therapies enter clinical testing. PMID:23115201

  16. Modeling reality

    NASA Technical Reports Server (NTRS)

    Denning, Peter J.

    1990-01-01

    Although powerful computers have allowed complex physical and manmade hardware systems to be modeled successfully, we have encountered persistent problems with the reliability of computer models for systems involving human learning, human action, and human organizations. This is not a misfortune; unlike physical and manmade systems, human systems do not operate under a fixed set of laws. The rules governing the actions allowable in the system can be changed without warning at any moment, and can evolve over time. That the governing laws are inherently unpredictable raises serious questions about the reliability of models when applied to human situations. In these domains, computers are better used, not for prediction and planning, but for aiding humans. Examples are systems that help humans speculate about possible futures, offer advice about possible actions in a domain, systems that gather information from the networks, and systems that track and support work flows in organizations.

  17. Cosmological models

    NASA Astrophysics Data System (ADS)

    Harvey, Alex

    1993-10-01

    Two of the most common terms employed in discussing cosmological models are open and closed. They are occasionally misused either by not recognizing or by not making it clear that each term may be used to characterize, independently and simultaneously, both the dynamic behavior and spatial geometric structure of the model under discussion. In addition, the spatial geometric structure implied by the terms open and closed is itself often either misunderstood or misused. Lastly, the role played by the cosmological constant is often improperly slighted. This paper is intended to give several examples of the abuse of terminology and clarify the distinction by means of a brief, elementary overview of Friedmann-Robertson-Walker cosmological models.

  18. Aerothermal modeling

    NASA Technical Reports Server (NTRS)

    Sturgess, G. J.

    1982-01-01

    The objectives, approach, and status of a program to develop the computational fluid dynamics tools needed to improve combustor design and analysis are outlined. The calculation procedure selected consists of a finite difference solution of the time averaged, steady state, primitive variable, elliptic form of the Reynolds equations. Standard TEACH type numerics are used to solve the resulting equations. These include hybrid differencing, SIMPLE algorithm for the pressure field, line by line iterative solution using the ADI method and the tridiagonal matrix algorithm (TDMA). Convergence is facilitated by using under relaxation. The physical processes are modeled by a two equation eddy viscosity model for turbulence; combustion is represented by a simple, irreversible, one step chemical reaction whose rate is influenced only by the time scale of the turbulence. The model evaluation procedure is also described.

  19. Supernova models

    SciTech Connect

    Woosley, S.E.; Weaver, T.A.

    1980-01-01

    Recent progress in understanding the observed properties of Type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the /sup 56/Ni produced therein is reviewed. Within the context of this model for Type I explosions and the 1978 model for Type II explosions, the expected nucleosynthesis and gamma-line spectra from both kinds of supernovae are presented. Finally, a qualitatively new approach to the problem of massive star death and Type II supernovae based upon a combination of rotation and thermonuclear burning is discussed.

  20. Reflectance Modeling

    NASA Technical Reports Server (NTRS)

    Smith, J. A. (Principal Investigator)

    1985-01-01

    The overall goal of this work has been to develop a set of computational tools and media abstractions for the terrain bidirectional reflectance problem. The modeling of soil and vegetation surfaces has been emphasized with a gradual increase in the complexity of the media geometries treated. Pragmatic problems involved in the combined modeling of soil, vegetation, and atmospheric effects have been of interest and one of the objectives has been to describe the canopy reflectance problem in a classical radiative transfer sense permitting easier inclusion of our work by other workers in the radiative transfer field.

  1. Atmospheric Modeling

    EPA Science Inventory

    Although air quality models have been applied historically to address issues specific to ambient air quality standards (i.e., one criteria pollutant at a time) or welfare (e.g.. acid deposition or visibility impairment). they are inherently multipollutant based. Therefore. in pri...

  2. Painting models

    NASA Astrophysics Data System (ADS)

    Baart, F.; Donchyts, G.; van Dam, A.; Plieger, M.

    2015-12-01

    The emergence of interactive art has blurred the line between electronic, computer graphics and art. Here we apply this art form to numerical models. Here we show how the transformation of a numerical model into an interactive painting can both provide insights and solve real world problems. The cases that are used as an example include forensic reconstructions, dredging optimization, barrier design. The system can be fed using any source of time varying vector fields, such as hydrodynamic models. The cases used here, the Indian Ocean (HYCOM), the Wadden Sea (Delft3D Curvilinear), San Francisco Bay (3Di subgrid and Delft3D Flexible Mesh), show that the method used is suitable for different time and spatial scales. High resolution numerical models become interactive paintings by exchanging their velocity fields with a high resolution (>=1M cells) image based flow visualization that runs in a html5 compatible web browser. The image based flow visualization combines three images into a new image: the current image, a drawing, and a uv + mask field. The advection scheme that computes the resultant image is executed in the graphics card using WebGL, allowing for 1M grid cells at 60Hz performance on mediocre graphic cards. The software is provided as open source software. By using different sources for a drawing one can gain insight into several aspects of the velocity fields. These aspects include not only the commonly represented magnitude and direction, but also divergence, topology and turbulence .

  3. Entrepreneurship Models.

    ERIC Educational Resources Information Center

    Finger Lakes Regional Education Center for Economic Development, Mount Morris, NY.

    This guide describes seven model programs that were developed by the Finger Lakes Regional Center for Economic Development (New York) to meet the training needs of female and minority entrepreneurs to help their businesses survive and grow and to assist disabled and dislocated workers and youth in beginning small businesses. The first three models…

  4. Why model?

    PubMed

    Wolkenhauer, Olaf

    2014-01-01

    Next generation sequencing technologies are bringing about a renaissance of mining approaches. A comprehensive picture of the genetic landscape of an individual patient will be useful, for example, to identify groups of patients that do or do not respond to certain therapies. The high expectations may however not be satisfied if the number of patient groups with similar characteristics is going to be very large. I therefore doubt that mining sequence data will give us an understanding of why and when therapies work. For understanding the mechanisms underlying diseases, an alternative approach is to model small networks in quantitative mechanistic detail, to elucidate the role of gene and proteins in dynamically changing the functioning of cells. Here an obvious critique is that these models consider too few components, compared to what might be relevant for any particular cell function. I show here that mining approaches and dynamical systems theory are two ends of a spectrum of methodologies to choose from. Drawing upon personal experience in numerous interdisciplinary collaborations, I provide guidance on how to model by discussing the question "Why model?"

  5. Modeling Muscles

    ERIC Educational Resources Information Center

    Goodwyn, Lauren; Salm, Sarah

    2007-01-01

    Teaching the anatomy of the muscle system to high school students can be challenging. Students often learn about muscle anatomy by memorizing information from textbooks or by observing plastic, inflexible models. Although these mediums help students learn about muscle placement, the mediums do not facilitate understanding regarding integration of…

  6. Entrepreneurship Models.

    ERIC Educational Resources Information Center

    Finger Lakes Regional Education Center for Economic Development, Mount Morris, NY.

    This guide describes seven model programs that were developed by the Finger Lakes Regional Center for Economic Development (New York) to meet the training needs of female and minority entrepreneurs to help their businesses survive and grow and to assist disabled and dislocated workers and youth in beginning small businesses. The first three models…

  7. Modeling Convection

    ERIC Educational Resources Information Center

    Ebert, James R.; Elliott, Nancy A.; Hurteau, Laura; Schulz, Amanda

    2004-01-01

    Students must understand the fundamental process of convection before they can grasp a wide variety of Earth processes, many of which may seem abstract because of the scales on which they operate. Presentation of a very visual, concrete model prior to instruction on these topics may facilitate students' understanding of processes that are largely…

  8. Ensemble Models

    EPA Science Inventory

    Ensemble forecasting has been used for operational numerical weather prediction in the United States and Europe since the early 1990s. An ensemble of weather or climate forecasts is used to characterize the two main sources of uncertainty in computer models of physical systems: ...

  9. Modeling Muscles

    ERIC Educational Resources Information Center

    Goodwyn, Lauren; Salm, Sarah

    2007-01-01

    Teaching the anatomy of the muscle system to high school students can be challenging. Students often learn about muscle anatomy by memorizing information from textbooks or by observing plastic, inflexible models. Although these mediums help students learn about muscle placement, the mediums do not facilitate understanding regarding integration of…

  10. Atmospheric Modeling

    EPA Science Inventory

    Although air quality models have been applied historically to address issues specific to ambient air quality standards (i.e., one criteria pollutant at a time) or welfare (e.g.. acid deposition or visibility impairment). they are inherently multipollutant based. Therefore. in pri...

  11. Modeling Convection

    ERIC Educational Resources Information Center

    Ebert, James R.; Elliott, Nancy A.; Hurteau, Laura; Schulz, Amanda

    2004-01-01

    Students must understand the fundamental process of convection before they can grasp a wide variety of Earth processes, many of which may seem abstract because of the scales on which they operate. Presentation of a very visual, concrete model prior to instruction on these topics may facilitate students' understanding of processes that are largely…

  12. Ensemble Models

    EPA Science Inventory

    Ensemble forecasting has been used for operational numerical weather prediction in the United States and Europe since the early 1990s. An ensemble of weather or climate forecasts is used to characterize the two main sources of uncertainty in computer models of physical systems: ...

  13. Criticality Model

    SciTech Connect

    A. Alsaed

    2004-09-14

    The ''Disposal Criticality Analysis Methodology Topical Report'' (YMP 2003) presents the methodology for evaluating potential criticality situations in the monitored geologic repository. As stated in the referenced Topical Report, the detailed methodology for performing the disposal criticality analyses will be documented in model reports. Many of the models developed in support of the Topical Report differ from the definition of models as given in the Office of Civilian Radioactive Waste Management procedure AP-SIII.10Q, ''Models'', in that they are procedural, rather than mathematical. These model reports document the detailed methodology necessary to implement the approach presented in the Disposal Criticality Analysis Methodology Topical Report and provide calculations utilizing the methodology. Thus, the governing procedure for this type of report is AP-3.12Q, ''Design Calculations and Analyses''. The ''Criticality Model'' is of this latter type, providing a process evaluating the criticality potential of in-package and external configurations. The purpose of this analysis is to layout the process for calculating the criticality potential for various in-package and external configurations and to calculate lower-bound tolerance limit (LBTL) values and determine range of applicability (ROA) parameters. The LBTL calculations and the ROA determinations are performed using selected benchmark experiments that are applicable to various waste forms and various in-package and external configurations. The waste forms considered in this calculation are pressurized water reactor (PWR), boiling water reactor (BWR), Fast Flux Test Facility (FFTF), Training Research Isotope General Atomic (TRIGA), Enrico Fermi, Shippingport pressurized water reactor, Shippingport light water breeder reactor (LWBR), N-Reactor, Melt and Dilute, and Fort Saint Vrain Reactor spent nuclear fuel (SNF). The scope of this analysis is to document the criticality computational method. The criticality

  14. A self-perpetuating repressive state of a viral replication protein blocks superinfection by the same virus

    PubMed Central

    Zhang, Xiao-Feng; Sun, Rong; Guo, Qin; Zhang, Shaoyan; Li, Dawei

    2017-01-01

    Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy. PMID:28267773

  15. Models, Part V: Composition Models.

    ERIC Educational Resources Information Center

    Callison, Daniel

    2003-01-01

    Describes four models: The Authoring Cycle, a whole language approach that reflects the inquiry process; I-Search, an approach to research that uses the power of student interests; Cultural Celebration, using local heritage topics; and Science Lab Report, for the composition of a lab report. (LRW)

  16. Fibre Models

    NASA Astrophysics Data System (ADS)

    Herrmann, H. J.; Kun, F.

    2007-12-01

    Fibre models have been introduced as simple models to describe failure. They are based on the probability distribution of broken fibres. The load redistribution after a fibre yields can be global or local and the first case can often be solved analytically. We will present an interpolation between these the local and the global case and apply it to experimental situations like the compression of granular packings. Introducing viscoelastic fibres allows to describe the creep of wood. It is even possible to deal analytically with a gradual degradation of fibres and consider damage as well as healing. In this way Basquin's law of fatigue can be reproduced and new universalities concerning the histograms of bursts and waiting times can be uncovered.

  17. Modeling Molecules

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The molecule modeling method known as Multibody Order (N) Dynamics, or MBO(N)D, was developed by Moldyn, Inc. at Goddard Space Flight Center through funding provided by the SBIR program. The software can model the dynamics of molecules through technology which stimulates low-frequency molecular motions and properties, such as movements among a molecule's constituent parts. With MBO(N)D, a molecule is substructured into a set of interconnected rigid and flexible bodies. These bodies replace the computation burden of mapping individual atoms. Moldyn's technology cuts computation time while increasing accuracy. The MBO(N)D technology is available as Insight II 97.0 from Molecular Simulations, Inc. Currently the technology is used to account for forces on spacecraft parts and to perform molecular analyses for pharmaceutical purposes. It permits the solution of molecular dynamics problems on a moderate workstation, as opposed to on a supercomputer.

  18. Nuclear Models

    NASA Astrophysics Data System (ADS)

    Fossión, Rubén

    2010-09-01

    The atomic nucleus is a typical example of a many-body problem. On the one hand, the number of nucleons (protons and neutrons) that constitute the nucleus is too large to allow for exact calculations. On the other hand, the number of constituent particles is too small for the individual nuclear excitation states to be explained by statistical methods. Another problem, particular for the atomic nucleus, is that the nucleon-nucleon (n-n) interaction is not one of the fundamental forces of Nature, and is hard to put in a single closed equation. The nucleon-nucleon interaction also behaves differently between two free nucleons (bare interaction) and between two nucleons in the nuclear medium (dressed interaction). Because of the above reasons, specific nuclear many-body models have been devised of which each one sheds light on some selected aspects of nuclear structure. Only combining the viewpoints of different models, a global insight of the atomic nucleus can be gained. In this chapter, we revise the the Nuclear Shell Model as an example of the microscopic approach, and the Collective Model as an example of the geometric approach. Finally, we study the statistical properties of nuclear spectra, basing on symmetry principles, to find out whether there is quantum chaos in the atomic nucleus. All three major approaches have been rewarded with the Nobel Prize of Physics. In the text, we will stress how each approach introduces its own series of approximations to reduce the prohibitingly large number of degrees of freedom of the full many-body problem to a smaller manageable number of effective degrees of freedom.

  19. Nuclear Models

    SciTech Connect

    Fossion, Ruben

    2010-09-10

    The atomic nucleus is a typical example of a many-body problem. On the one hand, the number of nucleons (protons and neutrons) that constitute the nucleus is too large to allow for exact calculations. On the other hand, the number of constituent particles is too small for the individual nuclear excitation states to be explained by statistical methods. Another problem, particular for the atomic nucleus, is that the nucleon-nucleon (n-n) interaction is not one of the fundamental forces of Nature, and is hard to put in a single closed equation. The nucleon-nucleon interaction also behaves differently between two free nucleons (bare interaction) and between two nucleons in the nuclear medium (dressed interaction).Because of the above reasons, specific nuclear many-body models have been devised of which each one sheds light on some selected aspects of nuclear structure. Only combining the viewpoints of different models, a global insight of the atomic nucleus can be gained. In this chapter, we revise the the Nuclear Shell Model as an example of the microscopic approach, and the Collective Model as an example of the geometric approach. Finally, we study the statistical properties of nuclear spectra, basing on symmetry principles, to find out whether there is quantum chaos in the atomic nucleus. All three major approaches have been rewarded with the Nobel Prize of Physics. In the text, we will stress how each approach introduces its own series of approximations to reduce the prohibitingly large number of degrees of freedom of the full many-body problem to a smaller manageable number of effective degrees of freedom.

  20. Modeling biomembranes.

    SciTech Connect

    Plimpton, Steven James; Heffernan, Julieanne; Sasaki, Darryl Yoshio; Frischknecht, Amalie Lucile; Stevens, Mark Jackson; Frink, Laura J. Douglas

    2005-11-01

    Understanding the properties and behavior of biomembranes is fundamental to many biological processes and technologies. Microdomains in biomembranes or ''lipid rafts'' are now known to be an integral part of cell signaling, vesicle formation, fusion processes, protein trafficking, and viral and toxin infection processes. Understanding how microdomains form, how they depend on membrane constituents, and how they act not only has biological implications, but also will impact Sandia's effort in development of membranes that structurally adapt to their environment in a controlled manner. To provide such understanding, we created physically-based models of biomembranes. Molecular dynamics (MD) simulations and classical density functional theory (DFT) calculations using these models were applied to phenomena such as microdomain formation, membrane fusion, pattern formation, and protein insertion. Because lipid dynamics and self-organization in membranes occur on length and time scales beyond atomistic MD, we used coarse-grained models of double tail lipid molecules that spontaneously self-assemble into bilayers. DFT provided equilibrium information on membrane structure. Experimental work was performed to further help elucidate the fundamental membrane organization principles.

  1. Model checking

    NASA Technical Reports Server (NTRS)

    Dill, David L.

    1995-01-01

    Automatic formal verification methods for finite-state systems, also known as model-checking, successfully reduce labor costs since they are mostly automatic. Model checkers explicitly or implicitly enumerate the reachable state space of a system, whose behavior is described implicitly, perhaps by a program or a collection of finite automata. Simple properties, such as mutual exclusion or absence of deadlock, can be checked by inspecting individual states. More complex properties, such as lack of starvation, require search for cycles in the state graph with particular properties. Specifications to be checked may consist of built-in properties, such as deadlock or 'unspecified receptions' of messages, another program or implicit description, to be compared with a simulation, bisimulation, or language inclusion relation, or an assertion in one of several temporal logics. Finite-state verification tools are beginning to have a significant impact in commercial designs. There are many success stories of verification tools finding bugs in protocols or hardware controllers. In some cases, these tools have been incorporated into design methodology. Research in finite-state verification has been advancing rapidly, and is showing no signs of slowing down. Recent results include probabilistic algorithms for verification, exploitation of symmetry and independent events, and the use symbolic representations for Boolean functions and systems of linear inequalities. One of the most exciting areas for further research is the combination of model-checking with theorem-proving methods.

  2. Molecular Modeling

    NASA Astrophysics Data System (ADS)

    Holmes, Jon L.

    1999-06-01

    Molecular modeling has trickled down from the realm of pharmaceutical and research laboratories into the realm of undergraduate chemistry instruction. It has opened avenues for the visualization of chemical concepts that previously were difficult or impossible to convey. I am sure that many of you have developed exercises using the various molecular modeling tools. It is the desire of this Journal to become an avenue for you to share these exercises among your colleagues. It is to this end that Ron Starkey has agreed to edit such a column and to publish not only the description of such exercises, but also the software documents they use. The WWW is the obvious medium to distribute this combination and so accepted submissions will appear online as a feature of JCE Internet. Typical molecular modeling exercise: finding conformation energies. Molecular Modeling Exercises and Experiments is the latest feature column of JCE Internet, joining Conceptual Questions and Challenge Problems, Hal's Picks, and Mathcad in the Chemistry Curriculum. JCE Internet continues to seek submissions in these areas of interest and submissions of general interest. If you have developed materials and would like to submit them, please see our Guide to Submissions for more information. The Chemical Education Resource Shelf, Equipment Buyers Guide, and WWW Site Review would also like to hear about chemistry textbooks and software, equipment, and WWW sites, respectively. Please consult JCE Internet Features to learn more about these resources at JCE Online. Email Announcements Would you like to be informed by email when the latest issue of the Journal is available online? when a new JCE Software title is shipping? when a new JCE Internet article has been published or is available for Open Review? when your subscription is about to expire? A new feature of JCE Online makes this possible. Visit our Guestbook to learn how. When

  3. Students' Models of Curve Fitting: A Models and Modeling Perspective

    ERIC Educational Resources Information Center

    Gupta, Shweta

    2010-01-01

    The Models and Modeling Perspectives (MMP) has evolved out of research that began 26 years ago. MMP researchers use Model Eliciting Activities (MEAs) to elicit students' mental models. In this study MMP was used as the conceptual framework to investigate the nature of students' models of curve fitting in a problem-solving environment consisting of…

  4. 10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: 1' = 400' HORIZONTAL, 1' = 100' VERTICAL), AND GREENVILLE BRIDGE MODEL (MODEL SCALE: 1' = 360' HORIZONTAL, 1' = 100' VERTICAL). - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

  5. Biomimetic modelling.

    PubMed Central

    Vincent, Julian F V

    2003-01-01

    Biomimetics is seen as a path from biology to engineering. The only path from engineering to biology in current use is the application of engineering concepts and models to biological systems. However, there is another pathway: the verification of biological mechanisms by manufacture, leading to an iterative process between biology and engineering in which the new understanding that the engineering implementation of a biological system can bring is fed back into biology, allowing a more complete and certain understanding and the possibility of further revelations for application in engineering. This is a pathway as yet unformalized, and one that offers the possibility that engineers can also be scientists. PMID:14561351

  6. Existing and Future Drugs for the Treatment of the Dark Side of Addiction.

    PubMed

    Koob, George F; Mason, Barbara J

    2016-01-01

    The identification of a heuristic framework for the stages of the addiction cycle that are linked to neurocircuitry changes in pathophysiology includes the binge/intoxication stage, the withdrawal/negative affect stage, and the preoccupation/anticipation (craving) stage, which represent neuroadaptations in three neurocircuits (basal ganglia, extended amygdala, and frontal cortex, respectively). The identification of excellent and validated animal models, the development of human laboratory models, and an enormous surge in our understanding of neurocircuitry and neuropharmacological mechanisms have provided a revisionist view of addiction that emphasizes the loss of brain reward function and gain of stress function that drive negative reinforcement (the dark side of addiction) as a key to compulsive drug seeking. Reversing the dark side of addiction not only explains much of the existing successful pharmacotherapies for addiction but also points to vast new opportunities for future medications to alleviate this major source of human suffering.

  7. Comparative Protein Structure Modeling Using MODELLER

    PubMed Central

    Webb, Benjamin; Sali, Andrej

    2016-01-01

    Comparative protein structure modeling predicts the three-dimensional structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and how to use the ModBase database of such models, and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:27322406

  8. Pre-Modeling Ensures Accurate Solid Models

    ERIC Educational Resources Information Center

    Gow, George

    2010-01-01

    Successful solid modeling requires a well-organized design tree. The design tree is a list of all the object's features and the sequential order in which they are modeled. The solid-modeling process is faster and less prone to modeling errors when the design tree is a simple and geometrically logical definition of the modeled object. Few high…

  9. Pre-Modeling Ensures Accurate Solid Models

    ERIC Educational Resources Information Center

    Gow, George

    2010-01-01

    Successful solid modeling requires a well-organized design tree. The design tree is a list of all the object's features and the sequential order in which they are modeled. The solid-modeling process is faster and less prone to modeling errors when the design tree is a simple and geometrically logical definition of the modeled object. Few high…

  10. Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

    PubMed Central

    Villarreal, L P

    1991-01-01

    The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and

  11. Modeling metrology for calibration of OPC models

    NASA Astrophysics Data System (ADS)

    Mack, Chris A.; Raghunathan, Ananthan; Sturtevant, John; Deng, Yunfei; Zuniga, Christian; Adam, Kostas

    2016-03-01

    Optical Proximity Correction (OPC) has continually improved in accuracy over the years by adding more physically based models. Here, we further extend OPC modeling by adding the Analytical Linescan Model (ALM) to account for systematic biases in CD-SEM metrology. The ALM was added to a conventional OPC model calibration flow and the accuracy of the calibrated model with the ALM was compared to the standard model without the ALM using validation data. Without using any adjustable parameters in the ALM, OPC validation accuracy was improved by 5%. While very preliminary, these results give hope that modeling metrology could be an important next step in OPC model improvement.

  12. Vector models and generalized SYK models

    NASA Astrophysics Data System (ADS)

    Peng, Cheng

    2017-05-01

    We consider the relation between SYK-like models and vector models by studying a toy model where a tensor field is coupled with a vector field. By integrating out the tensor field, the toy model reduces to the Gross-Neveu model in 1 dimension. On the other hand, a certain perturbation can be turned on and the toy model flows to an SYK-like model at low energy. A chaotic-nonchaotic phase transition occurs as the sign of the perturbation is altered. We further study similar models that possess chaos and enhanced reparameterization symmetries.

  13. Vector models and generalized SYK models

    DOE PAGES

    Peng, Cheng

    2017-05-23

    Here, we consider the relation between SYK-like models and vector models by studying a toy model where a tensor field is coupled with a vector field. By integrating out the tensor field, the toy model reduces to the Gross-Neveu model in 1 dimension. On the other hand, a certain perturbation can be turned on and the toy model flows to an SYK-like model at low energy. Furthermore, a chaotic-nonchaotic phase transition occurs as the sign of the perturbation is altered. We further study similar models that possess chaos and enhanced reparameterization symmetries.

  14. Commentary: "I hope i'll continue to grow": rubrics and reflective writing in medical education.

    PubMed

    Coulehan, Jack; Granek, Iris A

    2012-01-01

    One respected tradition in medical education holds that physicians should struggle to maintain sensibility, openness, and compassion in the face of strong contravening tendencies. However, today's medical education is structured around a more recent tradition, which maintains that physicians should struggle to develop emotional detachment as a prerequisite for objectivity. In this model, sensibility and reflective capacity are potentially subversive. Reflective writing is one component of a revisionist approach to medical education that explicitly addresses reflective "habits of the mind" as core competencies and builds on existential concerns voiced by medical students. In response to Wald and colleagues' study, the authors reflect on the role of repeated formative feedback in developing reflective capacity. Formative feedback is as critical in this process as it is in traditional clinical learning. The authors emphasize that well-designed rubrics can assist learners in delineating desired outcomes and teachers in providing appropriate guidance.

  15. Stability of Attachment Style in Adolescence: An Empirical Test of Alternative Developmental Processes.

    PubMed

    Jones, Jason D; Fraley, R Chris; Ehrlich, Katherine B; Stern, Jessica A; Lejuez, C W; Shaver, Phillip R; Cassidy, Jude

    2017-03-16

    Few studies have examined stability and change in attachment during adolescence. This 5-year longitudinal study (a) examined whether prototype or revisionist developmental dynamics better characterized patterns of stability and change in adolescent attachment (at T1, N = 176; Mage  = 14.0 years, SD = 0.9), (b) tested potential moderators of prototype-like attachment stability, and (c) compared attachment stability in adolescence to stability in adulthood. The results supported the prototype model, which assumes that there is a stable, enduring factor underlying stability and change in attachment. Exploratory moderation analyses revealed that family conflict, parental separation or divorce, minority status, and male sex might undermine the prototype-like stability of adolescent attachment. Stability of attachment was lower in adolescence relative to adulthood.

  16. Building Mental Models by Dissecting Physical Models

    ERIC Educational Resources Information Center

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to…

  17. Building Mental Models by Dissecting Physical Models

    ERIC Educational Resources Information Center

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to…

  18. I&C Modeling in SPAR Models

    SciTech Connect

    John A. Schroeder

    2012-06-01

    The Standardized Plant Analysis Risk (SPAR) models for the U.S. commercial nuclear power plants currently have very limited instrumentation and control (I&C) modeling [1]. Most of the I&C components in the operating plant SPAR models are related to the reactor protection system. This was identified as a finding during the industry peer review of SPAR models. While the Emergency Safeguard Features (ESF) actuation and control system was incorporated into the Peach Bottom Unit 2 SPAR model in a recent effort [2], various approaches to expend resources for detailed I&C modeling in other SPAR models are investigated.

  19. Forward model nonlinearity versus inverse model nonlinearity

    USGS Publications Warehouse

    Mehl, S.

    2007-01-01

    The issue of concern is the impact of forward model nonlinearity on the nonlinearity of the inverse model. The question posed is, "Does increased nonlinearity in the head solution (forward model) always result in increased nonlinearity in the inverse solution (estimation of hydraulic conductivity)?" It is shown that the two nonlinearities are separate, and it is not universally true that increased forward model nonlinearity increases inverse model nonlinearity. ?? 2007 National Ground Water Association.

  20. Modeling natural gas reservoirs: A simple model

    USGS Publications Warehouse

    Collier, Richard S.; Monash, E.A.

    1981-01-01

    A mathematical model is developed and tested for the production of natural gas with water encroachment and gas entrapment. The model is built on the material and volumetric balance relations, the Schilthuis water drive model, and a gas entrapment mechanism which assumes that the rate of gas entrapment is proportional to the volumetric rate of water influx. This model represents an alternative to the large grid models because of its low computer, maintenance, and manpower costs.

  1. SYSTEMIC, MUCOSAL AND HETEROTYPIC IMMUNE INDUCTION IN MICE INOCULATED WITH VENEZUELAN EQUINE ENCEPHALITIS REPLICONS EXPRESSING NORWALK VIRUS-LIKE PARTICLES. (R826139)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  2. Only one catalase, katG, is detectable in Rhizobium etli, and is encoded along with the regulator OxyR on a plasmid replicon.

    PubMed

    Vargas, María del Carmen; Encarnación, Sergio; Dávalos, Araceli; Reyes-Pérez, Agustín; Mora, Yolanda; García-de los Santos, Alejandro; Brom, Susana; Mora, Jaime

    2003-05-01

    The plasmid-borne Rhizobium etli katG gene encodes a dual-function catalase-peroxidase (KatG) (EC 1.11.1.7) that is inducible and heat-labile. In contrast to other rhizobia, katG was shown to be solely responsible for catalase and peroxidase activity in R. etli. An R. etli mutant that did not express catalase activity exhibited increased sensitivity to hydrogen peroxide (H(2)O(2)). Pre-exposure to a sublethal concentration of H(2)O(2) allowed R. etli to adapt and survive subsequent exposure to higher concentrations of H(2)O(2). Based on a multiple sequence alignment with other catalase-peroxidases, it was found that the catalytic domains of the R. etli KatG protein had three large insertions, two of which were typical of KatG proteins. Like the katG gene of Escherichia coli, the R. etli katG gene was induced by H(2)O(2) and was important in sustaining the exponential growth rate. In R. etli, KatG catalase-peroxidase activity is induced eightfold in minimal medium during stationary phase. It was shown that KatG catalase-peroxidase is not essential for nodulation and nitrogen fixation in symbiosis with Phaseolus vulgaris, although bacteroid proteome analysis indicated an alternative compensatory mechanism for the oxidative protection of R. etli in symbiosis. Next to, and divergently transcribed from the catalase promoter, an ORF encoding the regulator OxyR was found; this is the first plasmid-encoded oxyR gene described so far. Additionally, the katG promoter region contained sequence motifs characteristic of OxyR binding sites, suggesting a possible regulatory mechanism for katG expression.

  3. Diversity of plasmid replicons encoding the bla(CMY-2) gene in broad-spectrum cephalosporin-resistant Escherichia coli from livestock animals in Japan.

    PubMed

    Hiki, Mototaka; Usui, Masaru; Kojima, Akemi; Ozawa, Manao; Ishii, Yoshikazu; Asai, Tetsuo

    2013-03-01

    Broad-spectrum cephalosporin (BSC) resistance has increased in Escherichia coli isolates from broiler chickens in Japan since 2004. The purpose of this study was to understand the epidemiology of BSC-resistant E. coli in livestock animals. Among 3274 E. coli isolates from 1767 feces of apparently healthy animals on 1767 farms between 2004 and 2009, 118 ceftiofur (CTF)-resistant isolates (CTF MIC ≥4 μg/mL) were identified on 74 farms. After elimination of apparently clonal isolates from a single animal, 75 selected CTF-resistant isolates (62 isolates from 61 broiler chickens, 10 isolates from 10 layer chickens, two isolates from two cows, and one isolate from a pig) were characterized. The bla(CMY-2) gene was most frequently detected in 50 isolates, followed by bla(CTX-M) (CTX-M-2: six isolates; CTX-M-14: four isolates; CTX-M-25: two isolates; CTX-M-1: one isolate) and bla(SHV) (SHV-12: seven isolates; SHV-2, SHV-2a, SHV-5: one isolate each). In particular, 42 of 62 broiler chicken isolates harbored bla(CMY-2). Pulsed-field gel electrophoresis analyses using XbaI revealed divergent profiles among the BSC-resistant isolates. The incompatibility groups of bla(CMY-2) plasmids from 34 of the 42 broiler chicken isolates belonged to IncIγ (10 isolates), IncA/C (nine isolates), IncB/O (seven isolates) and IncI1 (six isolates), or were nontypeable (two isolates). Co-transmission of resistance to non-β-lactam antibiotics was observed in transconjugants with IncA/C plasmids, but not with IncI1, IncIγ, and IncB/O plasmids except for one isolate with IncB/O. Our findings suggest that the bla(CMY-2) gene is a key player in BSC-resistant E. coli isolates and that coselection is unlikely to be associated with the abundance of bla(CMY-2) plasmids, except for IncA/C plasmids.

  4. Ralstonia solanacearum fatty acid composition is determined by interaction of two 3-ketoacyl-acyl carrier protein reductases encoded on separate replicons.

    PubMed

    Feng, Sai-Xiang; Ma, Jin-Cheng; Yang, Ji; Hu, Zhe; Zhu, Lei; Bi, Hong-Kai; Sun, Yi-Rong; Wang, Hai-Hong

    2015-10-22

    FabG is the only known enzyme that catalyzes reduction of the 3-ketoacyl-ACP intermediates of bacterial fatty acid synthetic pathways. However, there are two Ralstonia solanacearum genes, RSc1052 (fabG1) and RSp0359 (fabG2), annotated as encoding putative 3-ketoacyl-ACP reductases. Both FabG homologues possess the conserved catalytic triad and the N-terminal cofactor binding sequence of the short chain dehydrogenase/reductase (SDR) family. Thus, it seems reasonable to hypothesize that RsfabG1 and RsfabG2 both encode functional 3-ketoacyl-ACP reductases and play important roles in R. solanacearum fatty acid synthesis and growth. Complementation of Escherichia coli fabG temperature-sensitive mutant with R. solanacearum fabGs encoded plasmids was carried out to test the function of RsfabGs in fatty acid biosynthesis. RsFabGs proteins were purified by nickel chelate chromatography and fatty acid biosynthetic reaction was reconstituted to investigate the 3-ketoacyl-ACP reductase activity of RsFabGs in vitro. Disruption of both RsfabG genes was done via DNA homologous recombination to test the function of both RsfabG in vivo. And more we also carried out pathogenicity tests on tomato plants using RsfabG mutant strains.  We report that expression of either of the two proteins (RsFabG1 and RsFabG2) restores growth of the E. coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrate that both proteins restore fatty acid synthetic ability to extracts of the E. coli strain. The RsfabG1 gene carried on the R. solanacearum chromosome is essential for growth of the bacterium, as is the case for fabG in E. coli. In contrast, the null mutant strain with the megaplasmid-encoded RsfabG2 gene is viable but has a fatty acid composition that differs significantly from that of the wild type strain. Our study also shows that RsFabG2 plays a role in adaptation to high salt concentration and low pH, and in pathogenesis of disease in tomato plants. R. solanacearum encodes two 3-ketoacyl-ACP reductases that both have functions in fatty acid synthesis. We supply the first evidence that, like other enzymes in the bacterial fatty acid biosynthetic pathway, one bacterium may simultaneously possess two or more 3-oxoacyl-ACP reductase isozymes.

  5. Pulsed-field gel electrophoresis analysis of the genome of Rhodococcus fascians: genome size and linear and circular replicon composition in virulent and avirulent strains.

    PubMed

    Pisabarro, A; Correia, A; Martín, J F

    1998-05-01

    Total DNA of virulent and avirulent strains of Rhodococcus fascians was resolved by pulsed-field gel electrophoresis (PFGE) into a discrete number of fragments by digestion with the endonucleases AseI and DraI. Restriction endonucleases PacI, PmeI, and SwaI yielded no fragments upon digestion of R. fascians genome, and all the other tested endonucleases recognizing 6 bp released too many fragments. The genome size was 5.6 megabases for the type strain R. fascians DSM 20669, and 5.8 megabases for the virulent R. fascians D188 strain. However the genome size of R. fascians CECT 3001 (NRRL B15096) was 8.0 megabases. No linear chromosome in the megabase range was observed under pulse conditions in which Saccharomyces cerevisiae and Schizosaccharomyces pombe chromosomes were perfectly resolved, suggesting that the R. fascians chromosome is circular. A new linear plasmid pIRN640 of 640 kb was found in the avirulent R. fascians CECT 3001 that did not hybridize with a probe internal to the fas region of pFiD188 known to be involved in plant pathogenicity in the virulent strain R. fascians D188. Virulence was correlated in all strains tested with the presence of the fas region. The AseI and DraI bands corresponding to the extrachromosomal elements were identified providing the basis for a physical map of this organism.

  6. SYSTEMIC, MUCOSAL AND HETEROTYPIC IMMUNE INDUCTION IN MICE INOCULATED WITH VENEZUELAN EQUINE ENCEPHALITIS REPLICONS EXPRESSING NORWALK VIRUS-LIKE PARTICLES. (R826139)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  7. Engineering Structurally Configurable Models with Model Transformation

    DTIC Science & Technology

    2008-12-15

    model in the case of Simulink, and a dataflow model in the case of LabVIEW). Research modeling tools such as Ptolemy II [14], ForSyDe [21], SPEX [30...functionality of our model transformation tool built in the Ptolemy II framework, and its application to large models of distributed and parallel embedded...in Ptolemy II, the same idea can be applied to other modeling tools such as Simulink, LabVIEW, ForSyDe, SPEX and ModHel’X. Moreover, the recent OMG

  8. Modeling cholera outbreaks.

    PubMed

    Chao, Dennis L; Longini, Ira M; Morris, J Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating models and the need to tailor models for different outbreak scenarios.

  9. Modeling cholera outbreaks

    PubMed Central

    Longini, Ira M.; Morris, J. Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating models and the need to tailor models for different outbreak scenarios. PMID:23412687

  10. Uncertainty Modeling Via Frequency Domain Model Validation

    NASA Technical Reports Server (NTRS)

    Waszak, Martin R.; Andrisani, Dominick, II

    1999-01-01

    Abstract The majority of literature on robust control assumes that a design model is available and that the uncertainty model bounds the actual variations about the nominal model. However, methods for generating accurate design models have not received as much attention in the literature. The influence of the level of accuracy of the uncertainty model on closed loop performance has received even less attention. The research reported herein is an initial step in applying and extending the concept of model validation to the problem of obtaining practical uncertainty models for robust control analysis and design applications. An extension of model validation called 'sequential validation' is presented and applied to a simple spring-mass-damper system to establish the feasibility of the approach and demonstrate the benefits of the new developments.

  11. Air Quality Dispersion Modeling - Alternative Models

    EPA Pesticide Factsheets

    Models, not listed in Appendix W, that can be used in regulatory applications with case-by-case justification to the Reviewing Authority as noted in Section 3.2, Use of Alternative Models, in Appendix W.

  12. Model selection for logistic regression models

    NASA Astrophysics Data System (ADS)

    Duller, Christine

    2012-09-01

    Model selection for logistic regression models decides which of some given potential regressors have an effect and hence should be included in the final model. The second interesting question is whether a certain factor is heterogeneous among some subsets, i.e. whether the model should include a random intercept or not. In this paper these questions will be answered with classical as well as with Bayesian methods. The application show some results of recent research projects in medicine and business administration.

  13. China model: Energy modeling the modern dynasty

    SciTech Connect

    Shaw, Jason

    1996-05-01

    In this paper a node-based microeconomic analysis is used to model the Chinese energy system. This model is run across multiple periods employing Lagrangian Relaxation techniques to achieve general equilibrium. Later, carbon dioxide emissions are added and the model is run to answer the question, {open_quotes}How can greenhouse gas emissions be reduced{close_quotes}?

  14. "Bohr's Atomic Model."

    ERIC Educational Resources Information Center

    Willden, Jeff

    2001-01-01

    "Bohr's Atomic Model" is a small interactive multimedia program that introduces the viewer to a simplified model of the atom. This interactive simulation lets students build an atom using an atomic construction set. The underlying design methodology for "Bohr's Atomic Model" is model-centered instruction, which means the central model of the…

  15. "Bohr's Atomic Model."

    ERIC Educational Resources Information Center

    Willden, Jeff

    2001-01-01

    "Bohr's Atomic Model" is a small interactive multimedia program that introduces the viewer to a simplified model of the atom. This interactive simulation lets students build an atom using an atomic construction set. The underlying design methodology for "Bohr's Atomic Model" is model-centered instruction, which means the central model of the…

  16. Forest-fire models

    Treesearch

    Haiganoush Preisler; Alan Ager

    2013-01-01

    For applied mathematicians forest fire models refer mainly to a non-linear dynamic system often used to simulate spread of fire. For forest managers forest fire models may pertain to any of the three phases of fire management: prefire planning (fire risk models), fire suppression (fire behavior models), and postfire evaluation (fire effects and economic models). In...

  17. Cloud Scene Simulation Modeling the Enhanced Model

    DTIC Science & Technology

    1992-04-01

    AD-A265 958 PL-TR-92-2106 CLOUD SCENE SIMULATION MODELING THE ENHANCED MODEL Maureen E. Cianciolo R. Gary Rasmussen TASC 55 Walkers Brook Drive...SUBTITLE 5. FUNDING NUMBERS Cloud Scene Simulation Modeling PF 62101,F The Enhanced Model PR 6670 TA 09 WU BE 6,AUTHOR(S) Contracl Fl1 9628-90-C-0022 7...the cloud field. 37 REFERENCES 1. Cianciolo, M.E., Hersh, J.S., and M.P. Ramos-Johnson, Cloud scene simulation modeling interim technical report, TASC

  18. Bohr model as an algebraic collective model

    SciTech Connect

    Rowe, D. J.; Welsh, T. A.; Caprio, M. A.

    2009-05-15

    Developments and applications are presented of an algebraic version of Bohr's collective model. Illustrative examples show that fully converged calculations can be performed quickly and easily for a large range of Hamiltonians. As a result, the Bohr model becomes an effective tool in the analysis of experimental data. The examples are chosen both to confirm the reliability of the algebraic collective model and to show the diversity of results that can be obtained by its use. The focus of the paper is to facilitate identification of the limitations of the Bohr model with a view to developing more realistic, computationally tractable models.

  19. Building mental models by dissecting physical models.

    PubMed

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to ensure focused learning; models that are too constrained require less supervision, but can be constructed mechanically, with little to no conceptual engagement. We propose "model-dissection" as an alternative to "model-building," whereby instructors could make efficient use of supervisory resources, while simultaneously promoting focused learning. We report empirical results from a study conducted with biology undergraduate students, where we demonstrate that asking them to "dissect" out specific conceptual structures from an already built 3D physical model leads to a significant improvement in performance than asking them to build the 3D model from simpler components. Using questionnaires to measure understanding both before and after model-based interventions for two cohorts of students, we find that both the "builders" and the "dissectors" improve in the post-test, but it is the latter group who show statistically significant improvement. These results, in addition to the intrinsic time-efficiency of "model dissection," suggest that it could be a valuable pedagogical tool. © 2015 The International Union of Biochemistry and Molecular Biology.

  20. Modeling regional wind erosion using different model

    NASA Astrophysics Data System (ADS)

    Guo, Zhongling; Chang, Chunping; Wang, Rende; Li, Jifeng; Li, Qing

    2017-04-01

    Wind erosion is an important factor causing soil degradation in arid and semi-arid regions. The need to quantitatively evaluate wind induced soil erosion yields many wind erosion models. These models include Wind Erosion Equation (WEQ), Revised Wind Erosion Equation (RWEQ),Wind Erosion Predicted System (WEPS) etc. at a field scale and Wind Erosion Assessment Model (WEAM), Integrated Wind Erosion Modeling System (IWEMS), AUStralian Land Erodibility Model (AUSLEM) etc. at a regional scale. The challenge of precisely estimating wind erosion at a regional scale still remain to date. To assess regional wind erosion, WEQ, RWEQ and WEPS have been scaled up to regional versions. However, no attempt is performed to compare these models for regional wind erosion modeling. In this study, the regional versions of WEQ, RWEQ, WEPS and WEAM, IWEMS, AUSLEM will be selected to model regional wind erosion of farmlands in the Kangbao County of northern China with annual soil loss by wind erosion based on 137 Cs analysis. Remote sensing image is used to determine the size and shape of local farmlands. Weather data of 2000-2010, China Soil Survey and published soil data, crops rotations etc. are compiled to generate raster layers of inputs for selected models using ArcGIS 10.2. These models were rebuilt based on ArcGIS Model-builder Module. Spatial distribution of annual soil loss by wind erosion determined from different model will be tested using annual soil loss data by 137 Cs analysis. Performances of these models will be investigated, and restrictions of these models will be further ascertained.

  1. Geologic Framework Model Analysis Model Report

    SciTech Connect

    R. Clayton

    2000-12-19

    The purpose of this report is to document the Geologic Framework Model (GFM), Version 3.1 (GFM3.1) with regard to data input, modeling methods, assumptions, uncertainties, limitations, and validation of the model results, qualification status of the model, and the differences between Version 3.1 and previous versions. The GFM represents a three-dimensional interpretation of the stratigraphy and structural features of the location of the potential Yucca Mountain radioactive waste repository. The GFM encompasses an area of 65 square miles (170 square kilometers) and a volume of 185 cubic miles (771 cubic kilometers). The boundaries of the GFM were chosen to encompass the most widely distributed set of exploratory boreholes (the Water Table or WT series) and to provide a geologic framework over the area of interest for hydrologic flow and radionuclide transport modeling through the unsaturated zone (UZ). The depth of the model is constrained by the inferred depth of the Tertiary-Paleozoic unconformity. The GFM was constructed from geologic map and borehole data. Additional information from measured stratigraphy sections, gravity profiles, and seismic profiles was also considered. This interim change notice (ICN) was prepared in accordance with the Technical Work Plan for the Integrated Site Model Process Model Report Revision 01 (CRWMS M&O 2000). The constraints, caveats, and limitations associated with this model are discussed in the appropriate text sections that follow. The GFM is one component of the Integrated Site Model (ISM) (Figure l), which has been developed to provide a consistent volumetric portrayal of the rock layers, rock properties, and mineralogy of the Yucca Mountain site. The ISM consists of three components: (1) Geologic Framework Model (GFM); (2) Rock Properties Model (RPM); and (3) Mineralogic Model (MM). The ISM merges the detailed project stratigraphy into model stratigraphic units that are most useful for the primary downstream models and the

  2. Educating with Aircraft Models

    ERIC Educational Resources Information Center

    Steele, Hobie

    1976-01-01

    Described is utilization of aircraft models, model aircraft clubs, and model aircraft magazines to promote student interest in aerospace education. The addresses for clubs and magazines are included. (SL)

  3. Modelling hot air balloons

    NASA Astrophysics Data System (ADS)

    Brimicombe, N. W.

    1991-07-01

    Hot air balloons can be modelled in a number of different ways. The most satisfactory, but least useful model is at a microscopic level. Macroscopic models are easier to use but can be very misleading.

  4. Photochemical Modeling Applications

    EPA Pesticide Factsheets

    Provides access to modeling applications involving photochemical models, including modeling of ozone, particulate matter (PM), and mercury for national and regional EPA regulations such as the Clean Air Interstate Rule (CAIR) and the Clean Air Mercury Rule

  5. Educating with Aircraft Models

    ERIC Educational Resources Information Center

    Steele, Hobie

    1976-01-01

    Described is utilization of aircraft models, model aircraft clubs, and model aircraft magazines to promote student interest in aerospace education. The addresses for clubs and magazines are included. (SL)

  6. Orbital Debris Modeling

    NASA Technical Reports Server (NTRS)

    Liou, J. C.

    2012-01-01

    Presentation outlne: (1) The NASA Orbital Debris (OD) Engineering Model -- A mathematical model capable of predicting OD impact risks for the ISS and other critical space assets (2) The NASA OD Evolutionary Model -- A physical model capable of predicting future debris environment based on user-specified scenarios (3) The NASA Standard Satellite Breakup Model -- A model describing the outcome of a satellite breakup (explosion or collision)

  7. Modeling of geothermal systems

    SciTech Connect

    Bodvarsson, G.S.; Pruess, K.; Lippmann, M.J.

    1985-03-01

    During the last decade the use of numerical modeling for geothermal resource evaluation has grown significantly, and new modeling approaches have been developed. In this paper we present a summary of the present status in numerical modeling of geothermal systems, emphasizing recent developments. Different modeling approaches are described and their applicability discussed. The various modeling tasks, including natural-state, exploitation, injection, multi-component and subsidence modeling, are illustrated with geothermal field examples. 99 refs., 14 figs.

  8. Continuous system modeling

    NASA Technical Reports Server (NTRS)

    Cellier, Francois E.

    1991-01-01

    A comprehensive and systematic introduction is presented for the concepts associated with 'modeling', involving the transition from a physical system down to an abstract description of that system in the form of a set of differential and/or difference equations, and basing its treatment of modeling on the mathematics of dynamical systems. Attention is given to the principles of passive electrical circuit modeling, planar mechanical systems modeling, hierarchical modular modeling of continuous systems, and bond-graph modeling. Also discussed are modeling in equilibrium thermodynamics, population dynamics, and system dynamics, inductive reasoning, artificial neural networks, and automated model synthesis.

  9. Interfacing materials models with fire field models

    SciTech Connect

    Nicolette, V.F.; Tieszen, S.R.; Moya, J.L.

    1995-12-01

    For flame spread over solid materials, there has traditionally been a large technology gap between fundamental combustion research and the somewhat simplistic approaches used for practical, real-world applications. Recent advances in computational hardware and computational fluid dynamics (CFD)-based software have led to the development of fire field models. These models, when used in conjunction with material burning models, have the potential to bridge the gap between research and application by implementing physics-based engineering models in a transient, multi-dimensional tool. This paper discusses the coupling that is necessary between fire field models and burning material models for the simulation of solid material fires. Fire field models are capable of providing detailed information about the local fire environment. This information serves as an input to the solid material combustion submodel, which subsequently calculates the impact of the fire environment on the material. The response of the solid material (in terms of thermal response, decomposition, charring, and off-gassing) is then fed back into the field model as a source of mass, momentum and energy. The critical parameters which must be passed between the field model and the material burning model have been identified. Many computational issues must be addressed when developing such an interface. Some examples include the ability to track multiple fuels and species, local ignition criteria, and the need to use local grid refinement over the burning material of interest.

  10. Comparative protein structure modeling using MODELLER.

    PubMed

    Eswar, Narayanan; Webb, Ben; Marti-Renom, Marc A; Madhusudhan, M S; Eramian, David; Shen, Min-Yi; Pieper, Ursula; Sali, Andrej

    2007-11-01

    Functional characterization of a protein sequence is a common goal in biology, and is usually facilitated by having an accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. (c) 2007 by John Wiley & Sons, Inc.

  11. Regularized Structural Equation Modeling.

    PubMed

    Jacobucci, Ross; Grimm, Kevin J; McArdle, John J

    A new method is proposed that extends the use of regularization in both lasso and ridge regression to structural equation models. The method is termed regularized structural equation modeling (RegSEM). RegSEM penalizes specific parameters in structural equation models, with the goal of creating easier to understand and simpler models. Although regularization has gained wide adoption in regression, very little has transferred to models with latent variables. By adding penalties to specific parameters in a structural equation model, researchers have a high level of flexibility in reducing model complexity, overcoming poor fitting models, and the creation of models that are more likely to generalize to new samples. The proposed method was evaluated through a simulation study, two illustrative examples involving a measurement model, and one empirical example involving the structural part of the model to demonstrate RegSEM's utility.

  12. Model Reduction of Viscoelastic Finite Element Models

    NASA Astrophysics Data System (ADS)

    Park, C. H.; Inman, D. J.; Lam, M. J.

    1999-01-01

    This paper examines a method of adding viscoelastic properties to finite element models by using additional co-ordinates to account for the frequency dependence usually associated with such damping materials. Several such methods exist and all suffer from an increase in order of the final finite model which is undesirable in many applications. Here we propose to combine one of these methods, the GHM (Golla-Hughes-McTavish) method, with model reduction techniques to remove the objection of increased model order. The result of combining several methods is an ability to add the effects of visoelastic components to finite element or other analytical models without increasing the order of the system. The procedure is illustrated by a numerical example. The method proposed here results in a viscoelastic finite element of a structure without increasing the order of the original model.

  13. ROCK PROPERTIES MODEL ANALYSIS MODEL REPORT

    SciTech Connect

    Clinton Lum

    2002-02-04

    The purpose of this Analysis and Model Report (AMR) is to document Rock Properties Model (RPM) 3.1 with regard to input data, model methods, assumptions, uncertainties and limitations of model results, and qualification status of the model. The report also documents the differences between the current and previous versions and validation of the model. The rock properties models are intended principally for use as input to numerical physical-process modeling, such as of ground-water flow and/or radionuclide transport. The constraints, caveats, and limitations associated with this model are discussed in the appropriate text sections that follow. This work was conducted in accordance with the following planning documents: WA-0344, ''3-D Rock Properties Modeling for FY 1998'' (SNL 1997, WA-0358), ''3-D Rock Properties Modeling for FY 1999'' (SNL 1999), and the technical development plan, Rock Properties Model Version 3.1, (CRWMS M&O 1999c). The Interim Change Notice (ICNs), ICN 02 and ICN 03, of this AMR were prepared as part of activities being conducted under the Technical Work Plan, TWP-NBS-GS-000003, ''Technical Work Plan for the Integrated Site Model, Process Model Report, Revision 01'' (CRWMS M&O 2000b). The purpose of ICN 03 is to record changes in data input status due to data qualification and verification activities. These work plans describe the scope, objectives, tasks, methodology, and implementing procedures for model construction. The constraints, caveats, and limitations associated with this model are discussed in the appropriate text sections that follow. The work scope for this activity consists of the following: (1) Conversion of the input data (laboratory measured porosity data, x-ray diffraction mineralogy, petrophysical calculations of bound water, and petrophysical calculations of porosity) for each borehole into stratigraphic coordinates; (2) Re-sampling and merging of data sets; (3) Development of geostatistical simulations of porosity; (4

  14. Neurometric Modeling: Computational Modeling of Individual Brains

    DTIC Science & Technology

    2011-05-16

    Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS Neural networks, computational neuroscience, fMRI ...obtained using functional MRI. Algorithmic processing of these measurements can exploit a variety of statistical machine learning methods to... statistical machine learning methods to synthesize a new kind of neuro-cognitive model, which we call neurometric models. These executable models could be

  15. Better models are more effectively connected models

    NASA Astrophysics Data System (ADS)

    Nunes, João Pedro; Bielders, Charles; Darboux, Frederic; Fiener, Peter; Finger, David; Turnbull-Lloyd, Laura; Wainwright, John

    2016-04-01

    The concept of hydrologic and geomorphologic connectivity describes the processes and pathways which link sources (e.g. rainfall, snow and ice melt, springs, eroded areas and barren lands) to accumulation areas (e.g. foot slopes, streams, aquifers, reservoirs), and the spatial variations thereof. There are many examples of hydrological and sediment connectivity on a watershed scale; in consequence, a process-based understanding of connectivity is crucial to help managers understand their systems and adopt adequate measures for flood prevention, pollution mitigation and soil protection, among others. Modelling is often used as a tool to understand and predict fluxes within a catchment by complementing observations with model results. Catchment models should therefore be able to reproduce the linkages, and thus the connectivity of water and sediment fluxes within the systems under simulation. In modelling, a high level of spatial and temporal detail is desirable to ensure taking into account a maximum number of components, which then enables connectivity to emerge from the simulated structures and functions. However, computational constraints and, in many cases, lack of data prevent the representation of all relevant processes and spatial/temporal variability in most models. In most cases, therefore, the level of detail selected for modelling is too coarse to represent the system in a way in which connectivity can emerge; a problem which can be circumvented by representing fine-scale structures and processes within coarser scale models using a variety of approaches. This poster focuses on the results of ongoing discussions on modelling connectivity held during several workshops within COST Action Connecteur. It assesses the current state of the art of incorporating the concept of connectivity in hydrological and sediment models, as well as the attitudes of modellers towards this issue. The discussion will focus on the different approaches through which connectivity

  16. Integrity modelling of tropospheric delay models

    NASA Astrophysics Data System (ADS)

    Rózsa, Szabolcs; Bastiaan Ober, Pieter; Mile, Máté; Ambrus, Bence; Juni, Ildikó

    2017-04-01

    The effect of the neutral atmosphere on signal propagation is routinely estimated by various tropospheric delay models in satellite navigation. Although numerous studies can be found in the literature investigating the accuracy of these models, for safety-of-life applications it is crucial to study and model the worst case performance of these models using very low recurrence frequencies. The main objective of the INTegrity of TROpospheric models (INTRO) project funded by the ESA PECS programme is to establish a model (or models) of the residual error of existing tropospheric delay models for safety-of-life applications. Such models are required to overbound rare tropospheric delays and should thus include the tails of the error distributions. Their use should lead to safe error bounds on the user position and should allow computation of protection levels for the horizontal and vertical position errors. The current tropospheric model from the RTCA SBAS Minimal Operational Standards has an associated residual error that equals 0.12 meters in the vertical direction. This value is derived by simply extrapolating the observed distribution of the residuals into the tail (where no data is present) and then taking the point where the cumulative distribution has an exceedance level would be 10-7.While the resulting standard deviation is much higher than the estimated standard variance that best fits the data (0.05 meters), it surely is conservative for most applications. In the context of the INTRO project some widely used and newly developed tropospheric delay models (e.g. RTCA MOPS, ESA GALTROPO and GPT2W) were tested using 16 years of daily ERA-INTERIM Reanalysis numerical weather model data and the raytracing technique. The results showed that the performance of some of the widely applied models have a clear seasonal dependency and it is also affected by a geographical position. In order to provide a more realistic, but still conservative estimation of the residual

  17. Biosphere Model Report

    SciTech Connect

    M. A. Wasiolek

    2003-10-27

    The purpose of this report is to document the biosphere model, the Environmental Radiation Model for Yucca Mountain, Nevada (ERMYN), which describes radionuclide transport processes in the biosphere and associated human exposure that may arise as the result of radionuclide release from the geologic repository at Yucca Mountain. The biosphere model is one of the process models that support the Yucca Mountain Project (YMP) Total System Performance Assessment (TSPA) for the license application (LA), the TSPA-LA. The ERMYN model provides the capability of performing human radiation dose assessments. This report documents the biosphere model, which includes: (1) Describing the reference biosphere, human receptor, exposure scenarios, and primary radionuclides for each exposure scenario (Section 6.1); (2) Developing a biosphere conceptual model using site-specific features, events, and processes (FEPs), the reference biosphere, the human receptor, and assumptions (Section 6.2 and Section 6.3); (3) Building a mathematical model using the biosphere conceptual model and published biosphere models (Sections 6.4 and 6.5); (4) Summarizing input parameters for the mathematical model, including the uncertainty associated with input values (Section 6.6); (5) Identifying improvements in the ERMYN model compared with the model used in previous biosphere modeling (Section 6.7); (6) Constructing an ERMYN implementation tool (model) based on the biosphere mathematical model using GoldSim stochastic simulation software (Sections 6.8 and 6.9); (7) Verifying the ERMYN model by comparing output from the software with hand calculations to ensure that the GoldSim implementation is correct (Section 6.10); and (8) Validating the ERMYN model by corroborating it with published biosphere models; comparing conceptual models, mathematical models, and numerical results (Section 7).

  18. Biosphere Model Report

    SciTech Connect

    D. W. Wu

    2003-07-16

    The purpose of this report is to document the biosphere model, the Environmental Radiation Model for Yucca Mountain, Nevada (ERMYN), which describes radionuclide transport processes in the biosphere and associated human exposure that may arise as the result of radionuclide release from the geologic repository at Yucca Mountain. The biosphere model is one of the process models that support the Yucca Mountain Project (YMP) Total System Performance Assessment (TSPA) for the license application (LA), the TSPA-LA. The ERMYN model provides the capability of performing human radiation dose assessments. This report documents the biosphere model, which includes: (1) Describing the reference biosphere, human receptor, exposure scenarios, and primary radionuclides for each exposure scenario (Section 6.1); (2) Developing a biosphere conceptual model using site-specific features, events, and processes (FEPs), the reference biosphere, the human receptor, and assumptions (Section 6.2 and Section 6.3); (3) Building a mathematical model using the biosphere conceptual model and published biosphere models (Sections 6.4 and 6.5); (4) Summarizing input parameters for the mathematical model, including the uncertainty associated with input values (Section 6.6); (5) Identifying improvements in the ERMYN model compared with the model used in previous biosphere modeling (Section 6.7); (6) Constructing an ERMYN implementation tool (model) based on the biosphere mathematical model using GoldSim stochastic simulation software (Sections 6.8 and 6.9); (7) Verifying the ERMYN model by comparing output from the software with hand calculations to ensure that the GoldSim implementation is correct (Section 6.10); and (8) Validating the ERMYN model by corroborating it with published biosphere models; comparing conceptual models, mathematical models, and numerical results (Section 7).

  19. Updated seismic solar model

    NASA Astrophysics Data System (ADS)

    Dziembowski, W. A.; Goode, Philip R.; Pamyatnykh, A. A.; Sienkiewicz, R.

    1995-05-01

    Recently released low-l solar oscillation data from the BISON network are combined with BBSO data to obtain an updated solar seismic model of the Sun's interior. For the core, the solar seismic model from the new data is more consistent with the current standard solar models than our earlier seismic model. An astrophysical solution to the solar neutrino problem fades away.

  20. The Instrumental Model

    ERIC Educational Resources Information Center

    Yeates, Devin Rodney

    2011-01-01

    The goal of this dissertation is to enable better predictive models by engaging raw experimental data through the Instrumental Model. The Instrumental Model captures the protocols and procedures of experimental data analysis. The approach is formalized by encoding the Instrumental Model in an XML record. Decoupling the raw experimental data from…

  1. Biomass Scenario Model

    SciTech Connect

    2015-09-01

    The Biomass Scenario Model (BSM) is a unique, carefully validated, state-of-the-art dynamic model of the domestic biofuels supply chain which explicitly focuses on policy issues, their feasibility, and potential side effects. It integrates resource availability, physical/technological/economic constraints, behavior, and policy. The model uses a system dynamics simulation (not optimization) to model dynamic interactions across the supply chain.

  2. Models for Ammunition Management

    DTIC Science & Technology

    1977-08-01

    Analysis Operations Research Management Models Mobilization Planning Computer Programming Ammunition Management Economic Analysis Production Planning...ammunition managers on a unique set of nine modern computer models specifically developed to support the conventional ammunition management decision...DECISION MODELS DIRECTORATE ROCK ISLAND, ILLINOIS 61201 r ABSTRACT This special management report presents a unique set of nine computer models

  3. Qualitative Student Models.

    ERIC Educational Resources Information Center

    Clancey, William J.

    The concept of a qualitative model is used as the focus of this review of qualitative student models in order to compare alternative computational models and to contrast domain requirements. The report is divided into eight sections: (1) Origins and Goals (adaptive instruction, qualitative models of processes, components of an artificial…

  4. Generative Models of Disfluency

    ERIC Educational Resources Information Center

    Miller, Timothy A.

    2010-01-01

    This thesis describes a generative model for representing disfluent phenomena in human speech. This model makes use of observed syntactic structure present in disfluent speech, and uses a right-corner transform on syntax trees to model this structure in a very natural way. Specifically, the phenomenon of speech repair is modeled by explicitly…

  5. Models of Counselling Centres.

    ERIC Educational Resources Information Center

    Calgary Univ. (Alberta).

    University counseling centers usually follow one of a variety of themes or "models," although not in pure form. Perhaps the oldest is the vocational counseling model, which concentrates on helping students find suitable careers. In the psychotherapy model, most student concerns are seen for their personal content. Another model, student affairs…

  6. Multimodeling and Model Abstraction

    USDA-ARS?s Scientific Manuscript database

    The multiplicity of models of the same process or phenomenon is the commonplace in environmental modeling. Last 10 years brought marked interest to making use of the variety of conceptual approaches instead of attempting to find the best model or using a single preferred model. Two systematic approa...

  7. The Instrumental Model

    ERIC Educational Resources Information Center

    Yeates, Devin Rodney

    2011-01-01

    The goal of this dissertation is to enable better predictive models by engaging raw experimental data through the Instrumental Model. The Instrumental Model captures the protocols and procedures of experimental data analysis. The approach is formalized by encoding the Instrumental Model in an XML record. Decoupling the raw experimental data from…

  8. Qualitative Student Models.

    ERIC Educational Resources Information Center

    Clancey, William J.

    The concept of a qualitative model is used as the focus of this review of qualitative student models in order to compare alternative computational models and to contrast domain requirements. The report is divided into eight sections: (1) Origins and Goals (adaptive instruction, qualitative models of processes, components of an artificial…

  9. AIDS Epidemiological models

    NASA Astrophysics Data System (ADS)

    Rahmani, Fouad Lazhar

    2010-11-01

    The aim of this paper is to present mathematical modelling of the spread of infection in the context of the transmission of the human immunodeficiency virus (HIV) and the acquired immune deficiency syndrome (AIDS). These models are based in part on the models suggested in the field of th AIDS mathematical modelling as reported by ISHAM [6].

  10. HRM: HII Region Models

    NASA Astrophysics Data System (ADS)

    Wenger, Trey V.; Kepley, Amanda K.; Balser, Dana S.

    2017-07-01

    HII Region Models fits HII region models to observed radio recombination line and radio continuum data. The algorithm includes the calculations of departure coefficients to correct for non-LTE effects. HII Region Models has been used to model star formation in the nucleus of IC 342.

  11. Talk about toy models

    NASA Astrophysics Data System (ADS)

    Luczak, Joshua

    2017-02-01

    Scientific models are frequently discussed in philosophy of science. A great deal of the discussion is centred on approximation, idealisation, and on how these models achieve their representational function. Despite the importance, distinct nature, and high presence of toy models, they have received little attention from philosophers. This paper hopes to remedy this situation. It aims to elevate the status of toy models: by distinguishing them from approximations and idealisations, by highlighting and elaborating on several ways the Kac ring, a simple statistical mechanical model, is used as a toy model, and by explaining why toy models can be used to successfully carry out important work without performing a representational function.

  12. Calibrated Properties Model

    SciTech Connect

    C. Ahlers; H. Liu

    2000-03-12

    The purpose of this Analysis/Model Report (AMR) is to document the Calibrated Properties Model that provides calibrated parameter sets for unsaturated zone (UZ) flow and transport process models for the Yucca Mountain Site Characterization Project (YMP). This work was performed in accordance with the ''AMR Development Plan for U0035 Calibrated Properties Model REV00. These calibrated property sets include matrix and fracture parameters for the UZ Flow and Transport Model (UZ Model), drift seepage models, drift-scale and mountain-scale coupled-processes models, and Total System Performance Assessment (TSPA) models as well as Performance Assessment (PA) and other participating national laboratories and government agencies. These process models provide the necessary framework to test conceptual hypotheses of flow and transport at different scales and predict flow and transport behavior under a variety of climatic and thermal-loading conditions.

  13. Equivalent Dynamic Models.

    PubMed

    Molenaar, Peter C M

    2017-02-16

    Equivalences of two classes of dynamic models for weakly stationary multivariate time series are discussed: dynamic factor models and autoregressive models. It is shown that exploratory dynamic factor models can be rotated, yielding an infinite set of equivalent solutions for any observed series. It also is shown that dynamic factor models with lagged factor loadings are not equivalent to the currently popular state-space models, and that restriction of attention to the latter type of models may yield invalid results. The known equivalent vector autoregressive model types, standard and structural, are given a new interpretation in which they are conceived of as the extremes of an innovating type of hybrid vector autoregressive models. It is shown that consideration of hybrid models solves many problems, in particular with Granger causality testing.

  14. Approaching School Change: An Anthropologist's View

    ERIC Educational Resources Information Center

    Lopate, Carol

    1974-01-01

    A reexamination of the functionalist interpretation of education by revisionist historians that posits an innate, biological need for freedom and autonomy in order to explain and analyze possible sources for educational change. (EH)

  15. The Question of Modernism and Postmodernism.

    ERIC Educational Resources Information Center

    Smith, Ralph A.

    1995-01-01

    Argues that, far from being a dangerous and destructive break with the past, various factions of postmodernism actually parallel modernist beliefs. Both movements share groups whose creative revisionists approach seeks a constructive accommodation. Similar parallel destructive approaches also exist. (MJP)

  16. Feudalism and the French Revolution.

    ERIC Educational Resources Information Center

    Kaiser, Thomas E.

    1979-01-01

    Reviews and questions the traditional established interpretation that the French Revolution was about feudalism. Concludes that revisionist historians have cast doubt upon the orthodox theory but that they have not supplied an alternative explanation. (Author/DB)

  17. Feudalism and the French Revolution.

    ERIC Educational Resources Information Center

    Kaiser, Thomas E.

    1979-01-01

    Reviews and questions the traditional established interpretation that the French Revolution was about feudalism. Concludes that revisionist historians have cast doubt upon the orthodox theory but that they have not supplied an alternative explanation. (Author/DB)

  18. Knowledge and information modeling.

    PubMed

    Madsen, Maria

    2010-01-01

    This chapter gives an educational overview of: * commonly used modelling methods what they represent * the importance of selecting the tools and methods suited to the health information system being designed * how the quality of the information or knowledge model is determined by the quality of the system requirements specification * differentiating between the purpose of information models and knowledge models * the benefits of the openEHR approach for health care data modeling.

  19. Introduction to Adjoint Models

    NASA Technical Reports Server (NTRS)

    Errico, Ronald M.

    2015-01-01

    In this lecture, some fundamentals of adjoint models will be described. This includes a basic derivation of tangent linear and corresponding adjoint models from a parent nonlinear model, the interpretation of adjoint-derived sensitivity fields, a description of methods of automatic differentiation, and the use of adjoint models to solve various optimization problems, including singular vectors. Concluding remarks will attempt to correct common misconceptions about adjoint models and their utilization.

  20. Stable models of superacceleration

    SciTech Connect

    Kaplinghat, Manoj; Rajaraman, Arvind

    2007-05-15

    We discuss an instability in a large class of models where dark energy is coupled to matter. In these models the mass of the scalar field is much larger than the expansion rate of the Universe. We find models in which this instability is absent, and show that these models generically predict an apparent equation of state for dark energy smaller than -1, i.e., superacceleration. These models have no acausal behavior or ghosts.

  1. WASP TRANSPORT MODELING AND WASP ECOLOGICAL MODELING

    EPA Science Inventory

    A combination of lectures, demonstrations, and hands-on excercises will be used to introduce pollutant transport modeling with the U.S. EPA's general water quality model, WASP (Water Quality Analysis Simulation Program). WASP features include a user-friendly Windows-based interfa...

  2. Model Shrinkage for Discriminative Language Models

    NASA Astrophysics Data System (ADS)

    Oba, Takanobu; Hori, Takaaki; Nakamura, Atsushi; Ito, Akinori

    This paper describes a technique for overcoming the model shrinkage problem in automatic speech recognition (ASR), which allows application developers and users to control the model size with less degradation of accuracy. Recently, models for ASR systems tend to be large and this can constitute a bottleneck for developers and users without special knowledge of ASR with respect to introducing the ASR function. Specifically, discriminative language models (DLMs) are usually designed in a high-dimensional parameter space, although DLMs have gained increasing attention as an approach for improving recognition accuracy. Our proposed method can be applied to linear models including DLMs, in which the score of an input sample is given by the inner product of its features and the model parameters, but our proposed method can shrink models in an easy computation by obtaining simple statistics, which are square sums of feature values appearing in a data set. Our experimental results show that our proposed method can shrink a DLM with little degradation in accuracy and perform properly whether or not the data for obtaining the statistics are the same as the data for training the model.

  3. WASP TRANSPORT MODELING AND WASP ECOLOGICAL MODELING

    EPA Science Inventory

    A combination of lectures, demonstrations, and hands-on excercises will be used to introduce pollutant transport modeling with the U.S. EPA's general water quality model, WASP (Water Quality Analysis Simulation Program). WASP features include a user-friendly Windows-based interfa...

  4. Geochemistry Model Validation Report: External Accumulation Model

    SciTech Connect

    K. Zarrabi

    2001-09-27

    The purpose of this Analysis and Modeling Report (AMR) is to validate the External Accumulation Model that predicts accumulation of fissile materials in fractures and lithophysae in the rock beneath a degrading waste package (WP) in the potential monitored geologic repository at Yucca Mountain. (Lithophysae are voids in the rock having concentric shells of finely crystalline alkali feldspar, quartz, and other materials that were formed due to entrapped gas that later escaped, DOE 1998, p. A-25.) The intended use of this model is to estimate the quantities of external accumulation of fissile material for use in external criticality risk assessments for different types of degrading WPs: U.S. Department of Energy (DOE) Spent Nuclear Fuel (SNF) codisposed with High Level Waste (HLW) glass, commercial SNF, and Immobilized Plutonium Ceramic (Pu-ceramic) codisposed with HLW glass. The scope of the model validation is to (1) describe the model and the parameters used to develop the model, (2) provide rationale for selection of the parameters by comparisons with measured values, and (3) demonstrate that the parameters chosen are the most conservative selection for external criticality risk calculations. To demonstrate the applicability of the model, a Pu-ceramic WP is used as an example. The model begins with a source term from separately documented EQ6 calculations; where the source term is defined as the composition versus time of the water flowing out of a breached waste package (WP). Next, PHREEQC, is used to simulate the transport and interaction of the source term with the resident water and fractured tuff below the repository. In these simulations the primary mechanism for accumulation is mixing of the high pH, actinide-laden source term with resident water; thus lowering the pH values sufficiently for fissile minerals to become insoluble and precipitate. In the final section of the model, the outputs from PHREEQC, are processed to produce mass of accumulation

  5. Iteron Plasmids.

    PubMed

    Konieczny, Igor; Bury, Katarzyna; Wawrzycka, Aleksandra; Wegrzyn, Katarzyna

    2014-12-01

    Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge and understanding of the structure of iteron-containing replicons, the structure of the iteron plasmid encoded replication initiation proteins, and the molecular mechanisms for iteron plasmid DNA replication initiation. We also discuss the current understanding of control mechanisms affecting the plasmid copy number and how host chaperone proteins and proteases can affect plasmid maintenance in bacterial cells.

  6. Practical Marginalized Multilevel Models

    PubMed Central

    Griswold, Michael E.; Swihart, Bruce J.; Caffo, Brian S.; Zeger, Scott L.

    2013-01-01

    Clustered data analysis is characterized by the need to describe both systematic variation in a mean model and cluster-dependent random variation in an association model. Marginalized multilevel models embrace the robustness and interpretations of a marginal mean model, while retaining the likelihood inference capabilities and flexible dependence structures of a conditional association model. Although there has been increasing recognition of the attractiveness of marginalized multilevel models, there has been a gap in their practical application arising from a lack of readily available estimation procedures. We extend the marginalized multilevel model to allow for nonlinear functions in both the mean and association aspects. We then formulate marginal models through conditional specifications to facilitate estimation with mixed model computational solutions already in place. We illustrate the MMM and approximate MMM approaches on a cerebrovascular deficiency crossover trial using SAS and an epidemiological study on race and visual impairment using R. Datasets, SAS and R code are included as supplemental materials. PMID:24357884

  7. Model Validation Status Review

    SciTech Connect

    E.L. Hardin

    2001-11-28

    The primary objective for the Model Validation Status Review was to perform a one-time evaluation of model validation associated with the analysis/model reports (AMRs) containing model input to total-system performance assessment (TSPA) for the Yucca Mountain site recommendation (SR). This review was performed in response to Corrective Action Request BSC-01-C-01 (Clark 2001, Krisha 2001) pursuant to Quality Assurance review findings of an adverse trend in model validation deficiency. The review findings in this report provide the following information which defines the extent of model validation deficiency and the corrective action needed: (1) AMRs that contain or support models are identified, and conversely, for each model the supporting documentation is identified. (2) The use for each model is determined based on whether the output is used directly for TSPA-SR, or for screening (exclusion) of features, events, and processes (FEPs), and the nature of the model output. (3) Two approaches are used to evaluate the extent to which the validation for each model is compliant with AP-3.10Q (Analyses and Models). The approaches differ in regard to whether model validation is achieved within individual AMRs as originally intended, or whether model validation could be readily achieved by incorporating information from other sources. (4) Recommendations are presented for changes to the AMRs, and additional model development activities or data collection, that will remedy model validation review findings, in support of licensing activities. The Model Validation Status Review emphasized those AMRs that support TSPA-SR (CRWMS M&O 2000bl and 2000bm). A series of workshops and teleconferences was held to discuss and integrate the review findings. The review encompassed 125 AMRs (Table 1) plus certain other supporting documents and data needed to assess model validity. The AMRs were grouped in 21 model areas representing the modeling of processes affecting the natural and

  8. Trapped Radiation Model Uncertainties: Model-Data and Model-Model Comparisons

    NASA Technical Reports Server (NTRS)

    Armstrong, T. W.; Colborn, B. L.

    2000-01-01

    The standard AP8 and AE8 models for predicting trapped proton and electron environments have been compared with several sets of flight data to evaluate model uncertainties. Model comparisons are made with flux and dose measurements made on various U.S. low-Earth orbit satellites (APEX, CRRES, DMSP. LDEF, NOAA) and Space Shuttle flights, on Russian satellites (Photon-8, Cosmos-1887, Cosmos-2044), and on the Russian Mir space station. This report gives the details of the model-data comparisons -- summary results in terms of empirical model uncertainty factors that can be applied for spacecraft design applications are given in a companion report. The results of model-model comparisons are also presented from standard AP8 and AE8 model predictions compared with the European Space Agency versions of AP8 and AE8 and with Russian trapped radiation models.

  9. Trapped Radiation Model Uncertainties: Model-Data and Model-Model Comparisons

    NASA Technical Reports Server (NTRS)

    Armstrong, T. W.; Colborn, B. L.

    2000-01-01

    The standard AP8 and AE8 models for predicting trapped proton and electron environments have been compared with several sets of flight data to evaluate model uncertainties. Model comparisons are made with flux and dose measurements made on various U.S. low-Earth orbit satellites (APEX, CRRES, DMSP, LDEF, NOAA) and Space Shuttle flights, on Russian satellites (Photon-8, Cosmos-1887, Cosmos-2044), and on the Russian Mir Space Station. This report gives the details of the model-data comparisons-summary results in terms of empirical model uncertainty factors that can be applied for spacecraft design applications are given in a combination report. The results of model-model comparisons are also presented from standard AP8 and AE8 model predictions compared with the European Space Agency versions of AP8 and AE8 and with Russian-trapped radiation models.

  10. Multiple model inference.

    SciTech Connect

    Swiler, Laura Painton; Urbina, Angel

    2010-07-01

    This paper compares three approaches for model selection: classical least squares methods, information theoretic criteria, and Bayesian approaches. Least squares methods are not model selection methods although one can select the model that yields the smallest sum-of-squared error function. Information theoretic approaches balance overfitting with model accuracy by incorporating terms that penalize more parameters with a log-likelihood term to reflect goodness of fit. Bayesian model selection involves calculating the posterior probability that each model is correct, given experimental data and prior probabilities that each model is correct. As part of this calculation, one often calibrates the parameters of each model and this is included in the Bayesian calculations. Our approach is demonstrated on a structural dynamics example with models for energy dissipation and peak force across a bolted joint. The three approaches are compared and the influence of the log-likelihood term in all approaches is discussed.

  11. Modeling nonstationary longitudinal data.

    PubMed

    Núñez-Antón, V; Zimmerman, D L

    2000-09-01

    An important theme of longitudinal data analysis in the past two decades has been the development and use of explicit parametric models for the data's variance-covariance structure. A variety of these models have been proposed, of which most are second-order stationary. A few are flexible enough to accommodate nonstationarity, i.e., nonconstant variances and/or correlations that are not a function solely of elapsed time between measurements. We review five nonstationary models that we regard as most useful: (1) the unstructured covariance model, (2) unstructured antedependence models, (3) structured antedependence models, (4) autoregressive integrated moving average and similar models, and (5) random coefficients models. We evaluate the relative strengths and limitations of each model, emphasizing when it is inappropriate or unlikely to be useful. We present three examples to illustrate the fitting and comparison of the models and to demonstrate that nonstationary longitudinal data can be modeled effectively and, in some cases, quite parsimoniously. In these examples, the antedependence models generally prove to be superior and the random coefficients models prove to be inferior. We conclude that antedependence models should be given much greater consideration than they have historically received.

  12. Modeling volatility using state space models.

    PubMed

    Timmer, J; Weigend, A S

    1997-08-01

    In time series problems, noise can be divided into two categories: dynamic noise which drives the process, and observational noise which is added in the measurement process, but does not influence future values of the system. In this framework, we show that empirical volatilities (the squared relative returns of prices) exhibit a significant amount of observational noise. To model and predict their time evolution adequately, we estimate state space models that explicitly include observational noise. We obtain relaxation times for shocks in the logarithm of volatility ranging from three weeks (for foreign exchange) to three to five months (for stock indices). In most cases, a two-dimensional hidden state is required to yield residuals that are consistent with white noise. We compare these results with ordinary autoregressive models (without a hidden state) and find that autoregressive models underestimate the relaxation times by about two orders of magnitude since they do not distinguish between observational and dynamic noise. This new interpretation of the dynamics of volatility in terms of relaxators in a state space model carries over to stochastic volatility models and to GARCH models, and is useful for several problems in finance, including risk management and the pricing of derivative securities. Data sets used: Olsen & Associates high frequency DEM/USD foreign exchange rates (8 years). Nikkei 225 index (40 years). Dow Jones Industrial Average (25 years).

  13. Energy-consumption modelling

    SciTech Connect

    Reiter, E.R.

    1980-01-01

    A highly sophisticated and accurate approach is described to compute on an hourly or daily basis the energy consumption for space heating by individual buildings, urban sectors, and whole cities. The need for models and specifically weather-sensitive models, composite models, and space-heating models are discussed. Development of the Colorado State University Model, based on heat-transfer equations and on a heuristic, adaptive, self-organizing computation learning approach, is described. Results of modeling energy consumption by the city of Minneapolis and Cheyenne are given. Some data on energy consumption in individual buildings are included.

  14. Stochastic modeling of rainfall

    SciTech Connect

    Guttorp, P.

    1996-12-31

    We review several approaches in the literature for stochastic modeling of rainfall, and discuss some of their advantages and disadvantages. While stochastic precipitation models have been around at least since the 1850`s, the last two decades have seen an increased development of models based (more or less) on the physical processes involved in precipitation. There are interesting questions of scale and measurement that pertain to these modeling efforts. Recent modeling efforts aim at including meteorological variables, and may be useful for regional down-scaling of general circulation models.

  15. Reduced Vector Preisach Model

    NASA Technical Reports Server (NTRS)

    Patel, Umesh D.; Torre, Edward Della; Day, John H. (Technical Monitor)

    2002-01-01

    A new vector Preisach model, called the Reduced Vector Preisach model (RVPM), was developed for fast computations. This model, derived from the Simplified Vector Preisach model (SVPM), has individual components that like the SVPM are calculated independently using coupled selection rules for the state vector computation. However, the RVPM does not require the rotational correction. Therefore, it provides a practical alternative for computing the magnetic susceptibility using a differential approach. A vector version, using the framework of the DOK model, is implemented. Simulation results for the reduced vector Preisach model are also presented.

  16. Reliability model generator

    NASA Technical Reports Server (NTRS)

    McMann, Catherine M. (Inventor); Cohen, Gerald C. (Inventor)

    1991-01-01

    An improved method and system for automatically generating reliability models for use with a reliability evaluation tool is described. The reliability model generator of the present invention includes means for storing a plurality of low level reliability models which represent the reliability characteristics for low level system components. In addition, the present invention includes means for defining the interconnection of the low level reliability models via a system architecture description. In accordance with the principles of the present invention, a reliability model for the entire system is automatically generated by aggregating the low level reliability models based on the system architecture description.

  17. Active registration models

    NASA Astrophysics Data System (ADS)

    Marstal, Kasper; Klein, Stefan

    2017-02-01

    We present the Active Registration Model (ARM) that couples medical image registration with regularization using a statistical model of intensity. Inspired by Active Appearance Models (AAMs), the statistical model is embedded in the registration procedure as a regularization term that penalize differences between a target image and a synthesized model reconstruction of that image. We demonstrate that the method generalizes AAMs to 3D images, many different transformation models, and many different gradient descent optimization methods. The method is validated on magnetic resonance images of human brains.

  18. Program management model study

    NASA Technical Reports Server (NTRS)

    Connelly, J. J.; Russell, J. E.; Seline, J. R.; Sumner, N. R., Jr.

    1972-01-01

    Two models, a system performance model and a program assessment model, have been developed to assist NASA management in the evaluation of development alternatives for the Earth Observations Program. Two computer models were developed and demonstrated on the Goddard Space Flight Center Computer Facility. Procedures have been outlined to guide the user of the models through specific evaluation processes, and the preparation of inputs describing earth observation needs and earth observation technology. These models are intended to assist NASA in increasing the effectiveness of the overall Earth Observation Program by providing a broader view of system and program development alternatives.

  19. A future of the model organism model.

    PubMed

    Rine, Jasper

    2014-03-01

    Changes in technology are fundamentally reframing our concept of what constitutes a model organism. Nevertheless, research advances in the more traditional model organisms have enabled fresh and exciting opportunities for young scientists to establish new careers and offer the hope of comprehensive understanding of fundamental processes in life. New advances in translational research can be expected to heighten the importance of basic research in model organisms and expand opportunities. However, researchers must take special care and implement new resources to enable the newest members of the community to engage fully with the remarkable legacy of information in these fields.

  20. Modeling Guru: Knowledge Base for NASA Modelers

    NASA Astrophysics Data System (ADS)

    Seablom, M. S.; Wojcik, G. S.; van Aartsen, B. H.

    2009-05-01

    Modeling Guru is an on-line knowledge-sharing resource for anyone involved with or interested in NASA's scientific models or High End Computing (HEC) systems. Developed and maintained by the NASA's Software Integration and Visualization Office (SIVO) and the NASA Center for Computational Sciences (NCCS), Modeling Guru's combined forums and knowledge base for research and collaboration is becoming a repository for the accumulated expertise of NASA's scientific modeling and HEC communities. All NASA modelers and associates are encouraged to participate and provide knowledge about the models and systems so that other users may benefit from their experience. Modeling Guru is divided into a hierarchy of communities, each with its own set forums and knowledge base documents. Current modeling communities include those for space science, land and atmospheric dynamics, atmospheric chemistry, and oceanography. In addition, there are communities focused on NCCS systems, HEC tools and libraries, and programming and scripting languages. Anyone may view most of the content on Modeling Guru (available at http://modelingguru.nasa.gov/), but you must log in to post messages and subscribe to community postings. The site offers a full range of "Web 2.0" features, including discussion forums, "wiki" document generation, document uploading, RSS feeds, search tools, blogs, email notification, and "breadcrumb" links. A discussion (a.k.a. forum "thread") is used to post comments, solicit feedback, or ask questions. If marked as a question, SIVO will monitor the thread, and normally respond within a day. Discussions can include embedded images, tables, and formatting through the use of the Rich Text Editor. Also, the user can add "Tags" to their thread to facilitate later searches. The "knowledge base" is comprised of documents that are used to capture and share expertise with others. The default "wiki" document lets users edit within the browser so others can easily collaborate on the

  1. A future of the model organism model

    PubMed Central

    Rine, Jasper

    2014-01-01

    Changes in technology are fundamentally reframing our concept of what constitutes a model organism. Nevertheless, research advances in the more traditional model organisms have enabled fresh and exciting opportunities for young scientists to establish new careers and offer the hope of comprehensive understanding of fundamental processes in life. New advances in translational research can be expected to heighten the importance of basic research in model organisms and expand opportunities. However, researchers must take special care and implement new resources to enable the newest members of the community to engage fully with the remarkable legacy of information in these fields. PMID:24577733

  2. Biosphere Model Report

    SciTech Connect

    D.W. Wu; A.J. Smith

    2004-11-08

    The purpose of this report is to document the biosphere model, the Environmental Radiation Model for Yucca Mountain, Nevada (ERMYN), which describes radionuclide transport processes in the biosphere and associated human exposure that may arise as the result of radionuclide release from the geologic repository at Yucca Mountain. The biosphere model is one of the process models that support the Yucca Mountain Project (YMP) Total System Performance Assessment (TSPA) for the license application (LA), TSPA-LA. The ERMYN provides the capability of performing human radiation dose assessments. This report documents the biosphere model, which includes: (1) Describing the reference biosphere, human receptor, exposure scenarios, and primary radionuclides for each exposure scenario (Section 6.1); (2) Developing a biosphere conceptual model using site-specific features, events, and processes (FEPs) (Section 6.2), the reference biosphere (Section 6.1.1), the human receptor (Section 6.1.2), and approximations (Sections 6.3.1.4 and 6.3.2.4); (3) Building a mathematical model using the biosphere conceptual model (Section 6.3) and published biosphere models (Sections 6.4 and 6.5); (4) Summarizing input parameters for the mathematical model, including the uncertainty associated with input values (Section 6.6); (5) Identifying improvements in the ERMYN compared with the model used in previous biosphere modeling (Section 6.7); (6) Constructing an ERMYN implementation tool (model) based on the biosphere mathematical model using GoldSim stochastic simulation software (Sections 6.8 and 6.9); (7) Verifying the ERMYN by comparing output from the software with hand calculations to ensure that the GoldSim implementation is correct (Section 6.10); (8) Validating the ERMYN by corroborating it with published biosphere models; comparing conceptual models, mathematical models, and numerical results (Section 7).

  3. Develop a Model Component

    NASA Technical Reports Server (NTRS)

    Ensey, Tyler S.

    2013-01-01

    During my internship at NASA, I was a model developer for Ground Support Equipment (GSE). The purpose of a model developer is to develop and unit test model component libraries (fluid, electrical, gas, etc.). The models are designed to simulate software for GSE (Ground Special Power, Crew Access Arm, Cryo, Fire and Leak Detection System, Environmental Control System (ECS), etc. .) before they are implemented into hardware. These models support verifying local control and remote software for End-Item Software Under Test (SUT). The model simulates the physical behavior (function, state, limits and 110) of each end-item and it's dependencies as defined in the Subsystem Interface Table, Software Requirements & Design Specification (SRDS), Ground Integrated Schematic (GIS), and System Mechanical Schematic.(SMS). The software of each specific model component is simulated through MATLAB's Simulink program. The intensiv model development life cycle is a.s follows: Identify source documents; identify model scope; update schedule; preliminary design review; develop model requirements; update model.. scope; update schedule; detailed design review; create/modify library component; implement library components reference; implement subsystem components; develop a test script; run the test script; develop users guide; send model out for peer review; the model is sent out for verifictionlvalidation; if there is empirical data, a validation data package is generated; if there is not empirical data, a verification package is generated; the test results are then reviewed; and finally, the user. requests accreditation, and a statement of accreditation is prepared. Once each component model is reviewed and approved, they are intertwined together into one integrated model. This integrated model is then tested itself, through a test script and autotest, so that it can be concluded that all models work conjointly, for a single purpose. The component I was assigned, specifically, was a

  4. Lie Markov models.

    PubMed

    Sumner, J G; Fernández-Sánchez, J; Jarvis, P D

    2012-04-07

    Recent work has discussed the importance of multiplicative closure for the Markov models used in phylogenetics. For continuous-time Markov chains, a sufficient condition for multiplicative closure of a model class is ensured by demanding that the set of rate-matrices belonging to the model class form a Lie algebra. It is the case that some well-known Markov models do form Lie algebras and we refer to such models as "Lie Markov models". However it is also the case that some other well-known Markov models unequivocally do not form Lie algebras (GTR being the most conspicuous example). In this paper, we will discuss how to generate Lie Markov models by demanding that the models have certain symmetries under nucleotide permutations. We show that the Lie Markov models include, and hence provide a unifying concept for, "group-based" and "equivariant" models. For each of two and four character states, the full list of Lie Markov models with maximal symmetry is presented and shown to include interesting examples that are neither group-based nor equivariant. We also argue that our scheme is pleasing in the context of applied phylogenetics, as, for a given symmetry of nucleotide substitution, it provides a natural hierarchy of models with increasing number of parameters. We also note that our methods are applicable to any application of continuous-time Markov chains beyond the initial motivations we take from phylogenetics. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  5. Aggregation in ecosystem models and model stability

    NASA Astrophysics Data System (ADS)

    Giricheva, Evgeniya

    2015-05-01

    Using a multimodal approach to research ecosystems improves usage of available information on an object. This study presents several models of the Bering Sea ecosystem. The ecosystem is considered as a closed object, that is, the influence of the environment is not provided. We then add the links with the external medium in the models. The models differ in terms of the degree and method of grouping components. Our method is based on the differences in habitat and food source of groups, which allows us to determine the grouping of species with a greater effect on system dynamics. In particular, we determine whether benthic fish aggregation or pelagic fish aggregation can change the consumption structure of some groups of species, and consequently, the behavior of the entire model system.

  6. Nonlinear Modeling by Assembling Piecewise Linear Models

    NASA Technical Reports Server (NTRS)

    Yao, Weigang; Liou, Meng-Sing

    2013-01-01

    To preserve nonlinearity of a full order system over a parameters range of interest, we propose a simple modeling approach by assembling a set of piecewise local solutions, including the first-order Taylor series terms expanded about some sampling states. The work by Rewienski and White inspired our use of piecewise linear local solutions. The assembly of these local approximations is accomplished by assigning nonlinear weights, through radial basis functions in this study. The efficacy of the proposed procedure is validated for a two-dimensional airfoil moving at different Mach numbers and pitching motions, under which the flow exhibits prominent nonlinear behaviors. All results confirm that our nonlinear model is accurate and stable for predicting not only aerodynamic forces but also detailed flowfields. Moreover, the model is robustness-accurate for inputs considerably different from the base trajectory in form and magnitude. This modeling preserves nonlinearity of the problems considered in a rather simple and accurate manner.

  7. Aerosol Modeling for the Global Model Initiative

    NASA Technical Reports Server (NTRS)

    Weisenstein, Debra K.; Ko, Malcolm K. W.

    2001-01-01

    The goal of this project is to develop an aerosol module to be used within the framework of the Global Modeling Initiative (GMI). The model development work will be preformed jointly by the University of Michigan and AER, using existing aerosol models at the two institutions as starting points. The GMI aerosol model will be tested, evaluated against observations, and then applied to assessment of the effects of aircraft sulfur emissions as needed by the NASA Subsonic Assessment in 2001. The work includes the following tasks: 1. Implementation of the sulfur cycle within GMI, including sources, sinks, and aqueous conversion of sulfur. Aerosol modules will be added as they are developed and the GMI schedule permits. 2. Addition of aerosol types other than sulfate particles, including dust, soot, organic carbon, and black carbon. 3. Development of new and more efficient parameterizations for treating sulfate aerosol nucleation, condensation, and coagulation among different particle sizes and types.

  8. PREDICTIVE MODELS. Enhanced Oil Recovery Model

    SciTech Connect

    Ray, R.M.

    1992-02-26

    PREDICTIVE MODELS is a collection of five models - CFPM, CO2PM, ICPM, PFPM, and SFPM - used in the 1982-1984 National Petroleum Council study of enhanced oil recovery (EOR) potential. Each pertains to a specific EOR process designed to squeeze additional oil from aging or spent oil fields. The processes are: 1 chemical flooding, where soap-like surfactants are injected into the reservoir to wash out the oil; 2 carbon dioxide miscible flooding, where carbon dioxide mixes with the lighter hydrocarbons making the oil easier to displace; 3 in-situ combustion, which uses the heat from burning some of the underground oil to thin the product; 4 polymer flooding, where thick, cohesive material is pumped into a reservoir to push the oil through the underground rock; and 5 steamflood, where pressurized steam is injected underground to thin the oil. CFPM, the Chemical Flood Predictive Model, models micellar (surfactant)-polymer floods in reservoirs, which have been previously waterflooded to residual oil saturation. Thus, only true tertiary floods are considered. An option allows a rough estimate of oil recovery by caustic or caustic-polymer processes. CO2PM, the Carbon Dioxide miscible flooding Predictive Model, is applicable to both secondary (mobile oil) and tertiary (residual oil) floods, and to either continuous CO2 injection or water-alternating gas processes. ICPM, the In-situ Combustion Predictive Model, computes the recovery and profitability of an in-situ combustion project from generalized performance predictive algorithms. PFPM, the Polymer Flood Predictive Model, is switch-selectable for either polymer or waterflooding, and an option allows the calculation of the incremental oil recovery and economics of polymer relative to waterflooding. SFPM, the Steamflood Predictive Model, is applicable to the steam drive process, but not to cyclic steam injection (steam soak) processes.

  9. Solid Waste Projection Model: Model user's guide

    SciTech Connect

    Stiles, D.L.; Crow, V.L.

    1990-08-01

    The Solid Waste Projection Model (SWPM) system is an analytical tool developed by Pacific Northwest Laboratory (PNL) for Westinghouse Hanford company (WHC) specifically to address solid waste management issues at the Hanford Central Waste Complex (HCWC). This document, one of six documents supporting the SWPM system, contains a description of the system and instructions for preparing to use SWPM and operating Version 1 of the model. 4 figs., 1 tab.

  10. Bounding Species Distribution Models

    NASA Technical Reports Server (NTRS)

    Stohlgren, Thomas J.; Jarnevich, Cahterine S.; Morisette, Jeffrey T.; Esaias, Wayne E.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used [Current Zoology 57 (5): 642-647, 2011].

  11. Ginocchio model with isospin

    NASA Astrophysics Data System (ADS)

    Okai, Tadashi; Otsuka, Takaharu; Arima, Akito

    1992-02-01

    We study the sp(8) subgroup of the isospin-invariant Ginnocchio model. The allowed quantum numbers are determined in terms of Young's diagrams. Using this result, we discuss the excitation energy of a model hamiltonian.

  12. Bounding species distribution models

    USGS Publications Warehouse

    Stohlgren, T.J.; Jarnevich, C.S.; Esaias, W.E.; Morisette, J.T.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used. ?? 2011 Current Zoology.

  13. SEDIMENT GEOCHEMICAL MODEL

    EPA Science Inventory

    Until recently, sediment geochemical models (diagenetic models) have been only able to explain sedimentary flux and concentration profiles for a few simplified geochemical cycles (e.g., nitrogen, carbon and sulfur). However with advances in numerical methods, increased accuracy ...

  14. Consistent model driven architecture

    NASA Astrophysics Data System (ADS)

    Niepostyn, Stanisław J.

    2015-09-01

    The goal of the MDA is to produce software systems from abstract models in a way where human interaction is restricted to a minimum. These abstract models are based on the UML language. However, the semantics of UML models is defined in a natural language. Subsequently the verification of consistency of these diagrams is needed in order to identify errors in requirements at the early stage of the development process. The verification of consistency is difficult due to a semi-formal nature of UML diagrams. We propose automatic verification of consistency of the series of UML diagrams originating from abstract models implemented with our consistency rules. This Consistent Model Driven Architecture approach enables us to generate automatically complete workflow applications from consistent and complete models developed from abstract models (e.g. Business Context Diagram). Therefore, our method can be used to check practicability (feasibility) of software architecture models.

  15. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  16. Model comparison in ANOVA.

    PubMed

    Rouder, Jeffrey N; Engelhardt, Christopher R; McCabe, Simon; Morey, Richard D

    2016-12-01

    Analysis of variance (ANOVA), the workhorse analysis of experimental designs, consists of F-tests of main effects and interactions. Yet, testing, including traditional ANOVA, has been recently critiqued on a number of theoretical and practical grounds. In light of these critiques, model comparison and model selection serve as an attractive alternative. Model comparison differs from testing in that one can support a null or nested model vis-a-vis a more general alternative by penalizing more flexible models. We argue this ability to support more simple models allows for more nuanced theoretical conclusions than provided by traditional ANOVA F-tests. We provide a model comparison strategy and show how ANOVA models may be reparameterized to better address substantive questions in data analysis.

  17. Bounding Species Distribution Models

    NASA Technical Reports Server (NTRS)

    Stohlgren, Thomas J.; Jarnevich, Cahterine S.; Morisette, Jeffrey T.; Esaias, Wayne E.

    2011-01-01

    Species distribution models are increasing in popularity for mapping suitable habitat for species of management concern. Many investigators now recognize that extrapolations of these models with geographic information systems (GIS) might be sensitive to the environmental bounds of the data used in their development, yet there is no recommended best practice for "clamping" model extrapolations. We relied on two commonly used modeling approaches: classification and regression tree (CART) and maximum entropy (Maxent) models, and we tested a simple alteration of the model extrapolations, bounding extrapolations to the maximum and minimum values of primary environmental predictors, to provide a more realistic map of suitable habitat of hybridized Africanized honey bees in the southwestern United States. Findings suggest that multiple models of bounding, and the most conservative bounding of species distribution models, like those presented here, should probably replace the unbounded or loosely bounded techniques currently used [Current Zoology 57 (5): 642-647, 2011].

  18. Of Molecules and Models.

    ERIC Educational Resources Information Center

    Brinner, Bonnie

    1992-01-01

    Presents an activity in which models help students visualize both the DNA process and transcription. After constructing DNA, RNA messenger, and RNA transfer molecules; students model cells, protein synthesis, codons, and RNA movement. (MDH)

  19. Adequacy of kinetic models

    SciTech Connect

    Kiperman, S.L.

    1995-01-01

    The problems associated with the accuracy of kinetic models in heterogeneous catalysis and their adequacy to experimental data and reaction mechanisms are considered. The prospects for the further improvement and use of these models is also explored.

  20. Models (Part 1).

    ERIC Educational Resources Information Center

    Callison, Daniel

    2002-01-01

    Defines models and describes information search models that can be helpful to instructional media specialists in meeting users' abilities and information needs. Explains pathfinders and Kuhlthau's information search process, including the pre-writing information search process. (LRW)