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Sample records for vaccinia virus morphogenesis

  1. Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

    Science.gov (United States)

    Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

    2017-08-01

    The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into

  2. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    Science.gov (United States)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  3. Brazilian Vaccinia Viruses and Their Origins

    Centers for Disease Control (CDC) Podcasts

    Smallpox was eradicated more than 25 years ago, but live viruses used in vaccines may have survived to cause animal and human illness today. Dr. Inger Damon, Acting Branch Chief of the Poxvirus and Rabies Branch at CDC, discusses efforts to determine origins and spread of vaccinia viruses in Brazil.

  4. Brazilian Vaccinia Viruses and Their Origins

    Centers for Disease Control (CDC) Podcasts

    2007-07-30

    Smallpox was eradicated more than 25 years ago, but live viruses used in vaccines may have survived to cause animal and human illness today. Dr. Inger Damon, Acting Branch Chief of the Poxvirus and Rabies Branch at CDC, discusses efforts to determine origins and spread of vaccinia viruses in Brazil.  Created: 7/30/2007 by Emerging Infectious Diseases.   Date Released: 7/30/2007.

  5. Vaccinia Virus Infections in a Martial Arts Gym

    Centers for Disease Control (CDC) Podcasts

    This podcast discusses an outbreak of vaccinia virus in Maryland in 2008. Christine Hughes, a health scientist with the Poxvirus and Rabies Branch at CDC, and co-author of a paper in the April 2011 issue of CDC's journal, discusses vaccinia virus infections in a martial arts gym.

  6. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    Science.gov (United States)

    Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

    1984-04-01

    The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

  7. Vaccinia Virus Infections in a Martial Arts Gym

    Centers for Disease Control (CDC) Podcasts

    2011-04-04

    This podcast discusses an outbreak of vaccinia virus in Maryland in 2008. Christine Hughes, a health scientist with the Poxvirus and Rabies Branch at CDC, and co-author of a paper in the April 2011 issue of CDC's journal, discusses vaccinia virus infections in a martial arts gym.  Created: 4/4/2011 by National Center for Emerging Zoonotic and Infectious Diseases (NCEZID).   Date Released: 4/5/2011.

  8. Immunodomination during peripheral vaccinia virus infection.

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    Leon C W Lin

    Full Text Available Immunodominance is a fundamental property of CD8(+ T cell responses to viruses and vaccines. It had been observed that route of administration alters immunodominance after vaccinia virus (VACV infection, but only a few epitopes were examined and no mechanism was provided. We re-visited this issue, examining a panel of 15 VACV epitopes and four routes, namely intradermal (i.d., subcutaneous (s.c., intraperitoneal (i.p. and intravenous (i.v. injection. We found that immunodominance is sharpened following peripheral routes of infection (i.d. and s.c. compared with those that allow systemic virus dissemination (i.p. and i.v.. This increased immunodominance was demonstrated with native epitopes of VACV and with herpes simplex virus glycoprotein B when expressed from VACV. Responses to some subdominant epitopes were altered by as much as fourfold. Tracking of virus, examination of priming sites, and experiments restricting virus spread showed that priming of CD8(+ T cells in the spleen was necessary, but not sufficient to broaden responses. Further, we directly demonstrated that immunodomination occurs more readily when priming is mainly in lymph nodes. Finally, we were able to reduce immunodominance after i.d., but not i.p. infection, using a VACV expressing the costimulators CD80 (B7-1 and CD86 (B7-2, which is notable because VACV-based vaccines incorporating these molecules are in clinical trials. Taken together, our data indicate that resources for CD8(+ T cell priming are limiting in local draining lymph nodes, leading to greater immunodomination. Further, we provide evidence that costimulation can be a limiting factor that contributes to immunodomination. These results shed light on a possible mechanism of immunodomination and highlight the need to consider multiple epitopes across the spectrum of immunogenicities in studies aimed at understanding CD8(+ T cell immunity to viruses.

  9. Resistance to Two Heterologous Neurotropic Oncolytic Viruses, Semliki Forest Virus and Vaccinia Virus, in Experimental Glioma

    Science.gov (United States)

    Le Boeuf, Fabrice; Lemay, Chantal; De Silva, Naomi; Diallo, Jean-Simon; Cox, Julie; Becker, Michelle; Choi, Youngmin; Ananth, Abhirami; Sellers, Clara; Breton, Sophie; Roy, Dominic; Falls, Theresa; Brun, Jan; Hemminki, Akseli; Hinkkanen, Ari; Bell, John C.

    2013-01-01

    Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference. PMID:23221568

  10. Susceptibility of different leukocyte cell types to Vaccinia virus infection

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    Sánchez-Puig Juana M

    2004-11-01

    Full Text Available Abstract Background Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus. Results We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP, and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells. Conclusions When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines.

  11. Vaccinia virus vectors: new strategies for producing recombinant vaccines.

    Science.gov (United States)

    Hruby, D E

    1990-01-01

    The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector. PMID:2187593

  12. Vaccinia virus as a subhelper for AAV replication and packaging

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    Andrea R Moore

    Full Text Available Adeno-associated virus (AAV has been widely used as a gene therapy vector to treat a variety of disorders. While these vectors are increasingly popular and successful in the clinic, there is still much to learn about the viruses. Understanding the biology of these viruses is essential in engineering better vectors and generating vectors more efficiently for large-scale use. AAV requires a helper for production and replication making this aspect of the viral life cycle crucial. Vaccinia virus (VV has been widely cited as a helper virus for AAV. However, to date, there are no detailed analyses of its helper function. Here, the helper role of VV was studied in detail. In contrast to common belief, we demonstrated that VV was not a sufficient helper virus for AAV replication. Vaccinia failed to produce rAAV and activate AAV promoters. While this virus could not support rAAV production, Vaccinia could initiate AAV replication and packaging when AAV promoter activation is not necessary. This activity is due to the ability of Vaccinia-driven Rep78 to transcribe in the cytoplasm and subsequently translate in the nucleus and undergo typical functions in the AAV life cycle. As such, VV is subhelper for AAV compared to complete helper functions of adenovirus.

  13. Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus.

    NARCIS (Netherlands)

    K.J. Stittelaar (Koert); G. van Amerongen (Geert); I. Kondova (Ivanela); R.F. van Lavieren (Rob); F.H. Pistoor (Frank); H.G.M. Niesters (Bert); G.J.J. van Doornum (Gerard); B.A.M. van der Zeijst (Ben); L. Mateo (Luis); P.J. Chaplin (Paul); A.D.M.E. Osterhaus (Albert); T. Kuiken (Thijs)

    2005-01-01

    textabstractThe use of classical smallpox vaccines based on vaccinia virus (VV) is associated with severe complications in both naive and immune individuals. Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of VV, has been proven to be safe in humans and

  14. Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase

    International Nuclear Information System (INIS)

    Slabaugh, M.B.; Mathews, C.K.

    1986-01-01

    Repeated passage of vaccinia virus in increasing concentrations of hydroxyurea followed by plaque purification resulted in the isolation of variants capable of growth in 5 mM hydroxyurea, a drug concentration which inhibited the reproduction of wild-type vaccinia virus 1000-fold. Analyses of viral protein synthesis by using [ 35 S]methionine pulse-labeling at intervals throughout the infection cycle revealed that all isolates overproduced a 34,000-molecular-weight (MW) early polypeptide. Measurement of ribonucleoside-diphosphate reductase activity after infection indicated that 4- to 10-fold more activity was induced by hydroxyurea-resistant viruses than by the wild-type virus. A two-step partial purification resulted in a substantial enrichment for the 34,000-MW protein from extracts of wild-type and hydroxyurea-resistant-virus-infected, but not mock-infected, cells. In the presence of the drug, the isolates incorporated [ 3 H]thymidine into DNA earlier and a rate substantially greater than that of the wild type, although the onset of DNA synthesis was delayed in both cases. The drug resistance trait was markedly unstable in all isolates. In the absence of selective pressure, plaque-purified isolated readily segregated progeny that displayed a wide range of resistance phenotypes. The results of this study indicate that vaccinia virus encodes a subunit of ribonucleotide reductase which is 34,000-MW early protein whose overproduction confers hydroxyurea resistance on reproducing viruses

  15. Mapping the active site of vaccinia virus RNA triphosphatase

    International Nuclear Information System (INIS)

    Gong Chunling; Shuman, Stewart

    2003-01-01

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

  16. Early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization.

    Science.gov (United States)

    Vennema, H; de Groot, R J; Harbour, D A; Dalderup, M; Gruffydd-Jones, T; Horzinek, M C; Spaan, W J

    1990-01-01

    The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis was recombined into the genome of vaccinia virus. The recombinant induced spike-protein-specific, in vitro neutralizing antibodies in mice. When kittens were immunized with the recombinant, low titers of neutralizing antibodies were obtained. After challenge with feline infectious peritonitis virus, these animals succumbed earlier than did the control group immunized with wild-type vaccinia virus (early death syndrome). Images PMID:2154621

  17. Clinical signs, diagnosis, and case reports of Vaccinia virus infections

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    Daniela Carla Medeiros Silva

    Full Text Available Vaccinia virus is responsible for a zoonosis that usually affects cattle and human beings in Brazil. The initial clinical signs of the infection are focal red skin areas, fever, and general symptoms similar to those of a cold. Then, pustules and ulcerated lesions surrounded by edema and erythema follow, as well as local lymphadenopathy that can last for weeks. Cure and healing of the lesions occur over several weeks, leaving a typical scar in the skin of people and animals affected. The infection definitive diagnosis is made through morphological characterization of the virus by use of electron microscopy, followed by PCR for specific viral genes. Since 1963, circulating orthopoxviruses in infectious outbreaks in several regions of Brazil have been reported. Later, the etiological agent of those infections was characterized as samples of Vaccinia virus. In addition, the widespread use of those viruses in research laboratories and mass vaccination of militaries have contributed to increase the cases of those infections worldwide. Thus, several epidemiological and clinical studies are required, as well as studies of viral immunology, public health, and economic impact, because little is known about those Vaccinia virus outbreaks in Brazil.

  18. Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

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    Antonio Postigo

    2017-05-01

    Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

  19. Studies on the serological relationships between avian pox, sheep pox, goat pox and vaccinia viruses

    Science.gov (United States)

    Uppal, P. K.; Nilakantan, P. R.

    1970-01-01

    By using neutralization, complement fixation and immunogel-diffusion tests, it has been demonstrated that cross-reactions occur between various avian pox viruses and between sheep pox and goat pox viruses. No such reactions were demonstrated between avian pox viruses and vaccinia virus or between avian pox and sheep pox and goat pox viruses. Furthermore, no serological relationship was demonstrable between vaccinia virus and sheep pox and goat pox viruses. PMID:4989854

  20. Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

    International Nuclear Information System (INIS)

    Burles, Kristin; Irwin, Chad R.; Burton, Robyn-Lee; Schriewer, Jill; Evans, David H.; Buller, R. Mark; Barry, Michele

    2014-01-01

    Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. - Highlights: • B4R-null viruses show reduced plaque size, and decreased ability to spread. • B4R-null viruses formed mature and extracellular virions; and rearranged actin. • Virus devoid of EVM154, the B4R homolog, was attenuated during infection. • Initial characterization suggests that B4 may play a role in virus spread. • Unidentified mediators of actin tail formation may exist in vaccinia virus

  1. Initial characterization of Vaccinia Virus B4 suggests a role in virus spread

    Energy Technology Data Exchange (ETDEWEB)

    Burles, Kristin; Irwin, Chad R.; Burton, Robyn-Lee [Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 (Canada); Schriewer, Jill [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO (United States); Evans, David H. [Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 (Canada); Buller, R. Mark [Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO (United States); Barry, Michele, E-mail: michele.barry@ualberta.ca [Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2 (Canada)

    2014-05-15

    Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus. - Highlights: • B4R-null viruses show reduced plaque size, and decreased ability to spread. • B4R-null viruses formed mature and extracellular virions; and rearranged actin. • Virus devoid of EVM154, the B4R homolog, was attenuated during infection. • Initial characterization suggests that B4 may play a role in virus spread. • Unidentified mediators of actin tail formation may exist in vaccinia virus.

  2. Analysis of variola and vaccinia virus neutralization assays for smallpox vaccines.

    Science.gov (United States)

    Hughes, Christine M; Newman, Frances K; Davidson, Whitni B; Olson, Victoria A; Smith, Scott K; Holman, Robert C; Yan, Lihan; Frey, Sharon E; Belshe, Robert B; Karem, Kevin L; Damon, Inger K

    2012-07-01

    Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.

  3. Protection of Mice from Lethal Vaccinia Virus Infection by Vaccinia Virus Protein Subunits with a CpG Adjuvant

    Directory of Open Access Journals (Sweden)

    Sarah Reeman

    2017-12-01

    Full Text Available Smallpox vaccination carries a high risk of adverse events in recipients with a variety of contra-indications for live vaccines. Although alternative non-replicating vaccines have been described in the form of replication-deficient vaccine viruses, DNA vaccines, and subunit vaccines, these are less efficacious than replicating vaccines in animal models. DNA and subunit vaccines in particular have not been shown to give equivalent protection to the traditional replicating smallpox vaccine. We show here that combinations of the orthopoxvirus A27, A33, B5 and L1 proteins give differing levels of protection when administered in different combinations with different adjuvants. In particular, the combination of B5 and A27 proteins adjuvanted with CpG oligodeoxynucleotides (ODN gives a level of protection in mice that is equivalent to the Lister traditional vaccine in a lethal vaccinia virus challenge model.

  4. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    OpenAIRE

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

  5. Intrafamilial Transmission of Vaccinia virus during a Bovine Vaccinia Outbreak in Brazil: A New Insight in Viral Transmission Chain

    Science.gov (United States)

    Pereira Oliveira, Graziele; Tavares Silva Fernandes, André; Lopes de Assis, Felipe; Augusto Alves, Pedro; Moreira Franco Luiz, Ana Paula; Barcelos Figueiredo, Leandra; Costa de Almeida, Cláudia Maria; Pires Ferreira Travassos, Carlos Eurico; de Souza Trindade, Giliane; Santos Abrahão, Jônatas; Geessien Kroon, Erna

    2014-01-01

    Bovine vaccinia (BV) is an emerging zoonosis caused by the Vaccinia virus (VACV), genus Orthopoxvirus (OPV), Poxviridae family. In general, human cases are related to direct contact with sick cattle but there is a lack of information about human-to-human transmission of VACV during BV outbreaks. In this study, we epidemiologically and molecularly show a case of VACV transmission between humans in São Francisco de Itabapoana County, Rio de Janeiro state. Our group collected samples from the patients, a 49-year-old patient and his son. Our results showed that patients had developed anti-OPV IgG or IgM antibodies and presented neutralizing antibodies against OPV. The VACV isolates displayed high identity (99.9%) and were grouped in the same phylogenetic tree branch. Our data indicate that human-to-human VACV transmission occurred during a BV outbreak, raising new questions about the risk factors of the VACV transmission chain. PMID:24615135

  6. Non-coding RNAs and heme oxygenase-1 in vaccinia virus infection

    International Nuclear Information System (INIS)

    Meseda, Clement A.; Srinivasan, Kumar; Wise, Jasen; Catalano, Jennifer; Yamada, Kenneth M.; Dhawan, Subhash

    2014-01-01

    Highlights: • Heme oxygenase-1 (HO-1) induction inhibited vaccinia virus infection of macrophages. • Reduced infectivity inversely correlated with increased expression of non-coding RNAs. • The regulation of HO-1 and ncRNAs suggests a novel host defense response against vaccinia virus infection. - Abstract: Small nuclear RNAs (snRNAs) are <200 nucleotide non-coding uridylate-rich RNAs. Although the functions of many snRNAs remain undetermined, a population of snRNAs is produced during the early phase of infection of cells by vaccinia virus. In the present study, we demonstrate a direct correlation between expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), suppression of selective snRNA expression, and inhibition of vaccinia virus infection of macrophages. Hemin induced HO-1 expression, completely reversed virus-induced host snRNA expression, and suppressed vaccinia virus infection. This involvement of specific virus-induced snRNAs and associated gene clusters suggests a novel HO-1-dependent host-defense pathway in poxvirus infection

  7. De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.

    Directory of Open Access Journals (Sweden)

    Matthew D Greseth

    2014-03-01

    Full Text Available The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis. Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia

  8. De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.

    Science.gov (United States)

    Greseth, Matthew D; Traktman, Paula

    2014-03-01

    The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

  9. JST Thesaurus Headwords and Synonyms: vaccinia virus [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term vaccinia virus 名詞 一般 * * * * ワクシニ...アウイルス ワクシニアウイルス ワクシニアウイルス Thesaurus2015 200906001583798830 C LS07 UNKNOWN_2 vaccinia virus

  10. Can vaccinia virus be replaced by MVA virus for testing virucidal activity of chemical disinfectants?

    Directory of Open Access Journals (Sweden)

    Rapp Ingrid

    2010-06-01

    Full Text Available Abstract Background Vaccinia virus strain Lister Elstree (VACV is a test virus in the DVV/RKI guidelines as representative of the stable enveloped viruses. Since the potential risk of laboratory-acquired infections with VACV persists and since the adverse effects of vaccination with VACV are described, the replacement of VACV by the modified vaccinia Ankara strain (MVA was studied by testing the activity of different chemical biocides in three German laboratories. Methods The inactivating properties of different chemical biocides (peracetic acid, aldehydes and alcohols were tested in a quantitative suspension test according to the DVV/RKI guideline. All tests were performed with a protein load of 10% fetal calf serum with both viruses in parallel using different concentrations and contact times. Residual virus was determined by endpoint dilution method. Results The chemical biocides exhibited similar virucidal activity against VACV and MVA. In three cases intra-laboratory differences were determined between VACV and MVA - 40% (v/v ethanol and 30% (v/v isopropanol are more active against MVA, whereas MVA seems more stable than VACV when testing with 0.05% glutardialdehyde. Test accuracy across the three participating laboratories was high. Remarkably inter-laboratory differences in the reduction factor were only observed in two cases. Conclusions Our data provide valuable information for the replacement of VACV by MVA for testing chemical biocides and disinfectants. Because MVA does not replicate in humans this would eliminate the potential risk of inadvertent inoculation with vaccinia virus and disease in non-vaccinated laboratory workers.

  11. Use of a recombinant vaccinia virus expressing interferon gamma for post-exposure protection against vaccinia and ectromelia viruses.

    Directory of Open Access Journals (Sweden)

    Susan A Holechek

    Full Text Available Post-exposure vaccination with vaccinia virus (VACV has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV. Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.

  12. Relationship between RNA polymerase II and efficiency of vaccinia virus replication

    International Nuclear Information System (INIS)

    Wilton, S.; Dales, S.

    1989-01-01

    It is clear from previous studies that host transcriptase or RNA polymerase II (pol II) has a role in poxvirus replication. To elucidate the participation of this enzyme further, in this study the authors examined several parameters related to pol II during the cycle of vaccinia virus infection in L-strain fibroblasts, HeLa cells, and L 6 H 9 rat myoblasts. Nucleocytoplasmic transposition of pol II into virus factories and virions was assessed by immunofluorescence and immunoblotting by using anti-pol II immunoglobulin G. RNA polymerase activities were compared in nuclear extracts containing cured enzyme preparations. Rates of translation into cellular or viral polypeptides were ascertained by labeling with [ 35 S]methionine. In L and HeLa cells, which produced vaccinia virus more abundantly, the rate of RNA polymerase and translation in controls and following infection were higher than in myoblasts. The data on synthesis and virus formation could be correlated with observations on transmigration of pol II, which was more efficient and complete in L and HeLa cells. The stimulus for pol II to leave the nucleus required the expression of both early and late viral functions. On the basis of current and past information, the authors suggest that mobilization of pol II depends on the efficiency of vaccinia virus replication and furthermore that control over vaccinia virus production by the host is related to the content or availability (or both) of pol II in different cell types

  13. Mutations Conferring Resistance to Viral DNA Polymerase Inhibitors in Camelpox Virus Give Different Drug-Susceptibility Profiles in Vaccinia Virus

    Czech Academy of Sciences Publication Activity Database

    Duraffour, S.; Andrei, G.; Topalis, D.; Krečmerová, Marcela; Crance, J. M.; Garin, D.; Snoeck, R.

    2012-01-01

    Roč. 86, č. 13 (2012), s. 7310-7325 ISSN 0022-538X Institutional support: RVO:61388963 Keywords : camelpox virus * CMLV * vaccinia virus VACV * acyclic nucleoside phosphonates * HPMPDAP * cidofovir * drug resistance Subject RIV: CC - Organic Chemistry Impact factor: 5.076, year: 2012

  14. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens

    NARCIS (Netherlands)

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Pijlman, Gorben P.

    2016-01-01

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious

  15. Differential antigen requirements for protection against systemic and intranasal vaccinia virus challenges in mice

    NARCIS (Netherlands)

    Kaufman, David R.; Goudsmit, Jaap; Holterman, Lennart; Ewald, Bonnie A.; Denholtz, Matthew; Devoy, Colleen; Giri, Ayush; Grandpre, Lauren E.; Heraud, Jean-Michel; Franchini, Genoveffa; Seaman, Michael S.; Havenga, Menzo J. E.; Barouch, Dan H.

    2008-01-01

    The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding

  16. Attenuation of vaccinia virus by the expression of human Flt3 ligand

    Czech Academy of Sciences Publication Activity Database

    Žurková, K.; Hainz, P.; Kryštofová, J.; Kutinová, L.; Šanda, Miloslav; Němečková, Š.

    2010-01-01

    Roč. 7, č. 1 (2010), 109/1-109/15 ISSN 1743-422X Institutional research plan: CEZ:AV0Z40550506 Keywords : vaccinia virus * antibodies * virulence Subject RIV: CE - Biochemistry Impact factor: 2.546, year: 2010

  17. Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus.

    Science.gov (United States)

    Yasui, Fumihiko; Itoh, Yasushi; Ikejiri, Ai; Kitabatake, Masahiro; Sakaguchi, Nobuo; Munekata, Keisuke; Shichinohe, Shintaro; Hayashi, Yukiko; Ishigaki, Hirohito; Nakayama, Misako; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa; Kohara, Michinori

    2016-11-28

    H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.

  18. Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68

    International Nuclear Information System (INIS)

    Ascierto, Maria Libera; Bedognetti, Davide; Uccellini, Lorenzo; Rossano, Fabio; Ascierto, Paolo A; Stroncek, David F; Restifo, Nicholas P; Wang, Ena; Szalay, Aladar A; Marincola, Francesco M; Worschech, Andrea; Yu, Zhiya; Adams, Sharon; Reinboth, Jennifer; Chen, Nanhai G; Pos, Zoltan; Roychoudhuri, Rahul; Di Pasquale, Giovanni

    2011-01-01

    Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo. In the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection. We observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection. Our findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection

  19. Middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies

    NARCIS (Netherlands)

    F. Song (Fei); R. Fux (Robert); L.B.V. Provacia (Lisette); A. Volz (Asisa); M. Eickmann; S. Becker (Stephan); A.D.M.E. Osterhaus (Albert); B.L. Haagmans (Bart); G. Suttera (Gerd)

    2013-01-01

    textabstractMiddle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic

  20. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    International Nuclear Information System (INIS)

    Holzer, Georg W.; Mayrhofer, Josef; Gritschenberger, Werner; Falkner, Falko G.

    2005-01-01

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes

  1. A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions

    International Nuclear Information System (INIS)

    Szajner, Patricia; Jaffe, Howard; Weisberg, Andrea S.; Moss, Bernard

    2004-01-01

    Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood. Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm. In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase. To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis. In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides. Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3. The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10. Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants. Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature

  2. Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis

    International Nuclear Information System (INIS)

    Drillien, R.; Spehner, D.; Kirn, A.

    1978-01-01

    Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by uv irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective

  3. Vectores recombinantes basados en el virus Vaccinia modificado de Ankara (MVA) como vacunas contra la leishmaniasis

    OpenAIRE

    Pérez Jiménez, Eva; Larraga, Vicente; Esteban, Mariano

    2005-01-01

    Vectores recombinantes basados en el virus vaccinia modificado de Ankara (MVA) como vacunas contra la leishmaniasis. Los vectores de la invención contienen secuencias codificantes de la proteína LACK, preferentemente insertadas en el locus de hemaglutinina del virus y bajo el control de un promotor que permite su expresión a lo largo del ciclo de infección del virus. Son vectores seguros, estables, que dan lugar a una potente respuesta inmune que confiere protección frente a la leishmaniasis,...

  4. Modified vaccinia virus ankara recombinants are as potent as vaccinia recombinants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses.

    Science.gov (United States)

    Hodge, James W; Poole, Diane J; Aarts, Wilhelmina M; Gómez Yafal, Alicia; Gritz, Linda; Schlom, Jeffrey

    2003-11-15

    Cancer vaccine regimens use various strategies to enhance immune responses to specific tumor-associated antigens (TAAs), including the increasing use of recombinant poxviruses [vaccinia (rV) and fowlpox (rF)] for delivery of the TAA to the immune system. However, the use of replication competent vectors with the potential of adverse reactions have made attenuation a priority for next-generation vaccine strategies. Modified vaccinia Ankara (MVA) is a replication defective form of vaccinia virus. Here, we investigated the use of MVA encoding a tumor antigen gene, carcinoembryonic antigen (CEA), in addition to multiple costimulatory molecules (B7-1, intercellular adhesion molecule-1, and lymphocyte function-associated antigen-3 designated TRICOM). Vaccination of mice with MVA-CEA/TRICOM induced potent CD4+ and CD8+ T-cell responses specific for CEA. MVA-CEA/TRICOM could be administered twice in vaccinia naïve mice and only a single time in vaccinia-immune mice before being inhibited by antivector-immune responses. The use of MVA-CEA/TRICOM in a diversified prime and boost vaccine regimen with rF-CEA/TRICOM, however, induced significantly greater levels of both CD4+ and CD8+ T-cell responses specific for CEA than that seen with rV-CEA/TRICOM prime and rF-CEA/TRICOM boost. In a self-antigen tumor model, the diversified MVA-CEA/TRICOM/rF-CEA/ TRICOM vaccination regimen resulted in a significant therapeutic antitumor response as measured by increased survival, when compared with the diversified prime and boost regimen, rV-CEA/TRICOM/rF-CEA/TRICOM. The studies reported here demonstrate that MVA, when used as a prime in a diversified vaccination, is clearly comparable with the regimen using the recombinant vaccinia in both the induction of cellular immune responses specific for the "self"-TAA transgene and in antitumor activity.

  5. Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

    Directory of Open Access Journals (Sweden)

    Felipe L Assis

    Full Text Available Since 1999, several Vaccinia virus (VACV isolates, the etiological agents of bovine vaccinia (BV, have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005 molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.

  6. Modified Vaccinia Virus Ankara: History, Value in Basic Research, and Current Perspectives for Vaccine Development.

    Science.gov (United States)

    Volz, A; Sutter, G

    2017-01-01

    Safety tested Modified Vaccinia virus Ankara (MVA) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. Historically, MVA was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain Ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. Adapted to growth in avian cells MVA lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. As a biologically well-characterized mutant virus, MVA facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. As extremely safe viral vectors MVA vaccines have been found immunogenic and protective in various preclinical infection models. Multiple recombinant MVA currently undergo clinical testing for vaccination against human immunodeficiency viruses, Mycobacterium tuberculosis or Plasmodium falciparum. The versatility of the MVA vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the Middle East Respiratory Syndrome. Recent advances include promising results from the clinical testing of recombinant MVA-producing antigens of highly pathogenic avian influenza virus H5N1 or Ebola virus. This review summarizes our current knowledge about MVA as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology. © 2017 Elsevier Inc. All rights reserved.

  7. Host range, growth property, and virulence of the smallpox vaccine: Vaccinia virus Tian Tan strain

    International Nuclear Information System (INIS)

    Fang Qing; Yang Lin; Zhu Weijun; Liu Li; Wang Haibo; Yu Wenbo; Xiao Genfu; Tien Po; Zhang Linqi; Chen Zhiwei

    2005-01-01

    Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene. Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens

  8. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

    International Nuclear Information System (INIS)

    Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

    1986-01-01

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells

  9. Preclinical evaluation of oncolytic vaccinia virus for therapy of canine soft tissue sarcoma.

    Directory of Open Access Journals (Sweden)

    Ivaylo Gentschev

    Full Text Available Virotherapy using oncolytic vaccinia virus (VACV strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.

  10. Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

    Science.gov (United States)

    Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

    2015-01-01

    ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

  11. Cambios en virus vaccinia durante la síntesis de RNA in vitro

    Directory of Open Access Journals (Sweden)

    Julio Enrique Ospina

    1971-01-01

    Full Text Available Observaciones al microscopio electrónico de virus vaccinia previamente incubados en una mezcla para la reacción de RNA polimerasa in vitro, demuestran características alteraciones morfológicas en los virus. Estructuras similares a vesículas y ocasionalmente túbulos se formaron a partir de la membrana externa del virus. Uno de los sustituyentes de la reacción de RNA polimerasa in vitro, mercaptoetanol 0.007M, es el causante de esta alteración. El cambio morfológico se acompaña de pérdida de la infectividad viral. La presencia de grupos sulfhidrilo en la mezcla de la reacción enzimática es esencial para la ocurrencia de la síntesis de RNA de vaccinia in vitro. Esta condición no se pudo sustituir por choque térmico a 70C. ni por digestión parcial del virus por tripsina. Una gran variedad de compuestos con grupos sulfhidrilo pueden reemplazar el mercaptoetanol con efectividad variable. El más activo de ellos fué el ditiotreitol. Un período de latencia de 8 minutos ocurre entre la adición de vaccinia a la mezcla completa para la reacción de RNA polimerasa y la detección de síntesis de RNA. Los datos recolectados sugieren que cambios dependientes del mercaptoetanol ocurren durante este período.

  12. Improved protection conferred by vaccination with a recombinant vaccinia virus that incorporates a foreign antigen into the extracellular enveloped virion

    International Nuclear Information System (INIS)

    Kwak, Heesun; Mustafa, Waleed; Speirs, Kendra; Abdool, Asha J.; Paterson, Yvonne; Isaacs, Stuart N.

    2004-01-01

    Recombinant poxviruses have shown promise as vaccine vectors. We hypothesized that improved cellular immune responses could be developed to a foreign antigen by incorporating it as part of the extracellular enveloped virion (EEV). We therefore constructed a recombinant vaccinia virus that replaced the cytoplasmic domain of the B5R protein with a test antigen, HIV-1 Gag. Mice immunized with the virus expressing Gag fused to B5R had significantly better primary CD4 T-cell responses than recombinant virus expressing HIV-Gag from the TK-locus. The CD8 T-cell responses were less different between the two groups. Importantly, although we saw differences in the immune response to the test antigen, the vaccinia virus-specific immune responses were similar with both constructs. When groups of vaccinated mice were challenged 30 days later with a recombinant Listeria monocytogenes that expresses HIV-Gag, mice inoculated with the virus that expresses the B5R-Gag fusion protein had lower colony counts of Listeria in the liver and spleen than mice vaccinated with the standard recombinant. Thus, vaccinia virus expressing foreign antigen incorporated into EEV may be a better vaccine strategy than standard recombinant vaccinia virus

  13. Thy1+ NK [corrected] cells from vaccinia virus-primed mice confer protection against vaccinia virus challenge in the absence of adaptive lymphocytes.

    Directory of Open Access Journals (Sweden)

    Geoffrey O Gillard

    2011-08-01

    Full Text Available While immunological memory has long been considered the province of T- and B-lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1(+ subset of natural killer (NK cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1(+ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.

  14. Effect of Interferon, Polyacrylic Acid, and Polymethacrylic Acid on Tail Lesions in Mice Infected with Vaccinia Virus

    Science.gov (United States)

    De Clercq, E.; De Somer, P.

    1968-01-01

    Intravenous inoculation of mice with vaccinia virus produced characteristic lesions of the tail surface which were suppressed by intraperitoneal administration of interferon and polyacrylic acid (PAA). Polymethacrylic acid (PMAA) stimulated the formation of vaccinia virus lesions. For full activity, both interferon and PAA must be given prior to infection. PAA was still significantly effective at small dose levels (3 mg/kg) and achieved protection for at least 4 weeks. Protection increased with increasing molecular weight of the polymer. The mode of action of PAA is discussed. PMID:5676405

  15. Long-lasting stability of vaccinia virus (orthopoxvirus) in food and environmental samples.

    Science.gov (United States)

    Essbauer, S; Meyer, H; Porsch-Ozcürümez, M; Pfeffer, M

    2007-01-01

    Poxviruses are known to remain infectious in the scabs of patients for months to years. The aim of this study was to investigate viral stability in storm water, food or gauze spiked with vaccinia virus strain Munich 1 (VACV M1). Storm water, storm water supplemented with either fetal calf serum (FCS) or potting soil was stored at two different temperatures (refrigerator, room temperature; 4 degrees C/25 degrees C). In addition, we analysed the viability of VACV M1 on the surface of bread, salad, sausages and gauze bandages stored at 4 degrees C. Samples were titrated in MA 104 cells and the presence of viral DNA was demonstrated by orthopoxvirus-specific PCRs. After 2 weeks, reisolation of VACV M1 from all kinds of food, bandage and water samples except for storm water supplemented with potting soil was possible. Viral DNA was detected in almost all samples by PCR. Prolonged experiments with VACV M1-spiked storm water and storm water supplemented with FCS revealed that samples kept at 4.5 degrees C are infectious for up to 166 days. Our data demonstrate that VACV M1 has a longlasting stability in water and food. The results obtained during this study should be taken into account for risk assessment calculations for poxvirus transmission. Implying that variola virus and vaccinia virus behave in a similar way, our data call for sophisticated countermeasures in cases of a variola release in biological warfare.

  16. Review of Vaccinia Virus and Baculovirus Viability Versus Virucides

    Science.gov (United States)

    2008-03-01

    25 6.4 Lignin ......................................................................................... 25 6.5...a lower pH (4.83 - 5.22), the virus rapidly inactivated over a month (Tomas et al., 1973). 16 The effects of alkalis on baculoviruses are important...of antioxidant and oxidative enzymes on UV inactivation by inhibiting the generation of highly reactive free radicals within HzSNPV. Water suspensions

  17. Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide

    International Nuclear Information System (INIS)

    Altmann, S.E.; Jones, J.C.; Schultz-Cherry, S.; Brandt, C.R.

    2009-01-01

    Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC 50 of 15 μM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC 50 > 200 μM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of β-galactosidase expression from an early viral promoter with an EC 50 of 45 μM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p < 0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.

  18. Modulation of gene expression in a human cell line caused by poliovirus, vaccinia virus and interferon

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    Hoddevik Gunnar

    2007-03-01

    Full Text Available Abstract Background The project was initiated to describe the response of a human embryonic fibroblast cell line to the replication of two different viruses, and, more specifically, to look for candidate genes involved in viral defense. For this purpose, the cells were synchronously infected with poliovirus in the absence or presence of interferon-alpha, or with vaccinia virus, a virus that is not inhibited by interferon. By comparing the changes in transcriptosome due to these different challenges, it should be possible to suggest genes that might be involved in defense. Results The viral titers were sufficient to yield productive infection in a majority of the cells. The cells were harvested in triplicate at various time-points, and the transcriptosome compared with mock infected cells using oligo-based, global 35 k microarrays. While there was very limited similarities in the response to the different viruses, a large proportion of the genes up-regulated by interferon-alpha were also up-regulated by poliovirus. Interferon-alpha inhibited poliovirus replication, but there were no signs of any interferons being induced by poliovirus. The observations suggest that the cells do launch an antiviral response to poliovirus in the absence of interferon. Analyses of the data led to a list of candidate antiviral genes. Functional information was limited, or absent, for most of the candidate genes. Conclusion The data are relevant for our understanding of how the cells respond to poliovirus and vaccinia virus infection. More annotations, and more microarray studies with related viruses, are required in order to narrow the list of putative defence-related genes.

  19. Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication

    International Nuclear Information System (INIS)

    Evans, E.V.A.

    1989-01-01

    The poxvirus, vaccinia, is large DNA virus which replicates in the cytoplasma of the host cell. The virus is believed to encode most or all of the functions required for the temporally regulated transcription and replication of its 186 kilobase genome. Physical and genetic autonomy from the host make vaccinia a useful eukaryotic organism in which to study replication genes and proteins, using a combination of biochemical and genetic techniques. Essential viral functions for replication are identified by conditional lethal mutants that fail to synthesize DNA at the non-permissive temperatures. One such group contains the non-complementing alleles ts17, ts24, ts69 (WR strain). Studies were undertaken to define the phenotype of ts mutants, and to identify and characterize the affected gene and protein. Mutant infection was essentially normal at 32 degree C, but at 39 degree C the mutants did not incorporate 3 H-thymidine into nascent viral DNA or synthesize late viral proteins. If mutant cultures were shifted to non-permissive conditions at the height of replication, DNA synthesis was halted rapidly, implying that the mutants are defective in DNA elongation. The gene affected in the WR mutants and in ts6389, a DNA-minus mutant of the IHD strain, was mapped by marker rescue and corresponds to open reading frame 5 (orfD5) of the viral HindIII D fragment

  20. Microbiota is an essential element for mice to initiate a protective immunity against Vaccinia virus.

    Science.gov (United States)

    Lima, Maurício T; Andrade, Ana C S P; Oliveira, Graziele P; Calixto, Rafael S; Oliveira, Danilo B; Souza, Éricka L S; Trindade, Giliane S; Nicoli, Jacques R; Kroon, Erna G; Martins, Flaviano S; Abrahão, Jônatas S

    2016-02-01

    The gastrointestinal tract of vertebrates harbors one of the most complex ecosystems known in microbial ecology and this indigenous microbiota almost always has a profound influence on host-parasite relationships, which can enhance or reduce the pathology of the infection. In this context, the impact of the microbiota during the infection of several viral groups remains poorly studied, including the family Poxviridae. Vaccinia virus (VACV) is a member of this family and is the causative agent of bovine vaccinia, responsible for outbreaks that affect bovines and humans. To determine the influence of the microbiota in the development of the disease caused by VACV, a comparative study using a murine model was performed. Germ-free and conventional, 6- to 7-week-old Swiss NIH mice were infected by tail scarification and intranasally with VACV. Moreover, immunosuppression and microbiota reposition were performed, to establish the interactions among the host's immune system, microbiota and VACV. The data demonstrate that the microbiota is essential for the effective immune response of mice against VACV in intranasal inoculation and to control the virus at the primary site of infection. Furthermore, this study is the first to show that Swiss conventional mice are refractory to the intranasal infection of VACV. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Expression of DAI by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity

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    Mari Hirvinen

    2016-01-01

    Full Text Available In oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. Vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. However, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DNA-dependent activator of IFN-regulatory factors (DAI to boost the innate immune system and to activate adaptive immune cells in the tumor. We showed that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways. Treatment with DAI-armed VV resulted in significant reduction in the size of syngeneic melanoma tumors in mice. When the mice were rechallenged with the same tumor, DAI-VV-treated mice completely rejected growth of the new tumor, which indicates immunity established against the tumor. We also showed enhanced control of growth of human melanoma tumors and elevated levels of human T-cells in DAI-VV-treated mice humanized with human peripheral blood mononuclear cells. We conclude that expression of DAI by an oncolytic VV is a promising way to amplify the vaccine potency of an oncolytic vaccinia virus to trigger the innate—and eventually the long-lasting adaptive immunity against cancer.

  2. Functional characterization of the vaccinia virus I5 protein

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    Stanitsa Eleni S

    2008-12-01

    Full Text Available The I5L gene is one of ~90 genes that are conserved throughout the chordopoxvirus family, and hence are presumed to play vital roles in the poxvirus life cycle. Previous work had indicated that the VP13 protein, a component of the virion membrane, was encoded by the I5L gene, but no additional studies had been reported. Using a recombinant virus that encodes an I5 protein fused to a V5 epitope tag at the endogenous locus (vI5V5, we show here that the I5 protein is expressed as a post-replicative gene and that the ~9 kDa protein does not appear to be phosphorylated in vivo. I5 does not appear to traffic to any cellular organelle, but ultrastructural and biochemical analyses indicate that I5 is associated with the membranous components of assembling and mature virions. Intact virions can be labeled with anti-V5 antibody as assessed by immunoelectron microscopy, indicating that the C' terminus of the protein is exposed on the virion surface. Using a recombinant virus which encodes only a TET-regulated copy of the I5V5 gene (vΔindI5V5, or one in which the I5 locus has been deleted (vΔI5, we also show that I5 is dispensable for replication in tissue culture. Neither plaque size nor the viral yield produced in BSC40 cells or primary human fibroblasts are affected by the absence of I5 expression.

  3. Effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model.

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    Clement A Meseda

    Full Text Available Antibodies to both infectious forms of vaccinia virus, the mature virion (MV and the enveloped virion (EV, as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.

  4. Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

    International Nuclear Information System (INIS)

    Villa, Nancy Y.; Bartee, Eric; Mohamed, Mohamed R.; Rahman, Masmudur M.; Barrett, John W.; McFadden, Grant

    2010-01-01

    Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1 - low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2 - the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3 - knockdown of PAK1 revealed that it is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.

  5. Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.

    Directory of Open Access Journals (Sweden)

    Sjoerd H E van den Worm

    Full Text Available Severe acute respiratory syndrome (SARS is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV. Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs. In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E. Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.

  6. [Experiments on disinfection of vaccinia virus embedded in scabs and/or at the hand].

    Science.gov (United States)

    Schümann, K; Grossgebauer, K

    1977-01-01

    Vaccinia viruses embedded in rabbit dermal scabs were subjected to physical and chemical disinfection procedures. Scabs were suspended in vitro without saline or in physiological saline, and left for 1 hour at 70 to 90 degrees C. A complete inactivation was achived only in those scab samples which had been incubated at 90 degrees C for 1 hour and suspended in physiological saline. Scabs which had been placed in a disinfecting apparatus (Vacudes 4000) filled with mattrasses consistently proved to be free of infectious vaccinia viruses in each of the chosen programs. In addition scabs were subjected to disinfection by means of chemical disinfecting agents. The scabs had been placed in a chemical disinfecting suspension and left there for 90 minutes. Complete disinfection was obtained with glutaraldehyde 2%, formaldehyde 2%, Lysoformin 2% or 3%, phenol 5% and chloramine T 2%. Complete disinfection was likewise achieved after 3 hours treatment with some alchohols (ethylalcohol 80%, isopropylalcohol 7%, n-propylalcohol 60%), Amocid 5% and formaldehyde 1%.0.5% formaldehyde caused complete disinfection when applied for 6 hours. The only exception was a Quat which did not disinfect fully even after 18 hours application. Concerning the tests to disinfect the hands complete disinfection occurs when using chloramine T (1.5%) or isopropylalcohol (70%) in 2 to 5 minutes. Further tests were performed with scabs which were placed in sick rooms that were terminally disinfected with formaline vapor. It could be confirmed that the usual terminal disinfection with formaldehyde vapor was unable to completely disinfect the scabs. It is necessary to double the amount of formaldehyde (10 g formaldehyde per cubic metre of space) and prolong the period of treatment to 24 hours to achieve a greater degree of disinfection rate.

  7. Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles

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    Karoliina Autio

    2014-01-01

    Full Text Available We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification.

  8. Isolation and identification of compounds from Kalanchoe pinnata having human alphaherpesvirus and vaccinia virus antiviral activity.

    Science.gov (United States)

    Cryer, Matthew; Lane, Kyle; Greer, Mary; Cates, Rex; Burt, Scott; Andrus, Merritt; Zou, Jiping; Rogers, Paul; Hansen, Marc D H; Burgado, Jillybeth; Panayampalli, Subbian Satheshkumar; Day, Craig W; Smee, Donald F; Johnson, Brent F

    2017-12-01

    Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage. This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV). Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1 WT. KPB-100 and KPB-200 inhibited HHV-2 at IC 50 values of 2.5 and 2.9 μg/mL, respectively, and VACV at IC 50 values of 3.1 and 7.4 μg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC 50 values of 1.63 and 13.2 μg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20 μg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC 50 value of 4.5 μg/mL in CPE inhibition assay and HHV-1 at an IC 90 of 3.0 μg/mL in VYR assay. Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.

  9. Increased ATP generation in the host cell is required for efficient vaccinia virus production

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    Hsu Che-Fang

    2009-09-01

    Full Text Available Abstract To search for cellular genes up-regulated by vaccinia virus (VV infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

  10. Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus.

    Science.gov (United States)

    Price, Daniel L; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G; Yu, Yong A; Szalay, Aladar A; Cappello, Joseph; Fong, Yuman; Wong, Richard J

    2016-02-01

    Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. © 2014 Wiley Periodicals, Inc.

  11. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    Energy Technology Data Exchange (ETDEWEB)

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

    2015-01-15

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  12. Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

    Science.gov (United States)

    Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

    2013-09-17

    Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

  13. Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus

    International Nuclear Information System (INIS)

    D'Costa, Susan M.; Antczak, James B.; Pickup, David J.; Condit, Richard C.

    2004-01-01

    Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor

  14. Extracts from rabbit skin inflamed by the vaccinia virus attenuate bupivacaine-induced spinal neurotoxicity in pregnant rats

    Institute of Scientific and Technical Information of China (English)

    Rui Cui; Shiyuan Xu; Liang Wang; Hongyi Lei; Qingxiang Cai; Hongfei Zhang; Dongmei Wang

    2013-01-01

    Extracts from rabbit skin inflamed by the vaccinia virus can relieve pain and promote repair of nerve injury. The present study intraperitoneally injected extracts from rabbit skin inflamed by the vaccinia virus for 3 and 4 days prior to and following intrathecal injection of bupivacaine into pregnant rats. The pain threshold test after bupivacaine injection showed that the maximum possible effect of tail-flick latency peaked 1 day after intrathecal injection of bupivacaine in the extract-pretreatment group, and gradually decreased, while the maximum possible effect in the bupivacaine group continued to increase after intrathecal injection of bupivacaine. Histological observation showed that after 4 days of intrathecal injection of bupivacaine, the number of shrunken, vacuolated, apoptotic and caspase-9-positive cells in the dorsal root ganglion in the extract-pretreatment group was significantly reduced compared with the bupivacaine group. These findings indicate that extracts from rabbit skin inflamed by the vaccinia virus can attenuate neurotoxicity induced by intrathecal injection of bupivacaine in pregnant rats, possibly by inhibiting caspase-9 protein expression and suppressing nerve cell apoptosis.

  15. Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus.

    Science.gov (United States)

    Xuan, X; Kojima, A; Murata, T; Mikami, T; Otsuka, H

    1997-01-01

    The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp 145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection.

  16. Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus

    Science.gov (United States)

    Rubins, Kathleen H.; Hensley, Lisa E.; Relman, David A.; Brown, Patrick O.

    2011-01-01

    Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection. PMID:21267444

  17. Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

    Directory of Open Access Journals (Sweden)

    Kathleen H Rubins

    2011-01-01

    Full Text Available Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV, an emerging human pathogen, and Vaccinia virus (VAC, a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA, or poly (I-C was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

  18. Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid, Recombinant Vaccinia Virus, and Replication-Defective Adenovirus Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene

    OpenAIRE

    Casimiro, Danilo R.; Chen, Ling; Fu, Tong-Ming; Evans, Robert K.; Caulfield, Michael J.; Davies, Mary-Ellen; Tang, Aimin; Chen, Minchun; Huang, Lingyi; Harris, Virginia; Freed, Daniel C.; Wilson, Keith A.; Dubey, Sheri; Zhu, De-Min; Nawrocki, Denise

    2003-01-01

    Cellular immune responses, particularly those associated with CD3+ CD8+ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defecti...

  19. Adverse Events Post Smallpox-Vaccination: Insights from Tail Scarification Infection in Mice with Vaccinia virus

    Science.gov (United States)

    Mota, Bruno E. F.; Gallardo-Romero, Nadia; Trindade, Giliane; Keckler, M. Shannon; Karem, Kevin; Carroll, Darin; Campos, Marco A.; Vieira, Leda Q.; da Fonseca, Flávio G.; Ferreira, Paulo C. P.; Bonjardim, Cláudio A.; Damon, Inger K.; Kroon, Erna G.

    2011-01-01

    Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1 −/−) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1 −/− with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1 −/−, and passive transfer of WT T cells to Rag1 −/− animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify

  20. A loss of function analysis of host factors influencing Vaccinia virus replication by RNA interference.

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    Philippa M Beard

    Full Text Available Vaccinia virus (VACV is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.

  1. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

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    Qicheng Zhang

    Full Text Available Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1 viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1 and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs. ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

  2. Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

    Science.gov (United States)

    Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

    2013-01-01

    Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

  3. Attenuation and immunogenicity of host-range extended modified vaccinia virus Ankara recombinants.

    Science.gov (United States)

    Melamed, Sharon; Wyatt, Linda S; Kastenmayer, Robin J; Moss, Bernard

    2013-09-23

    Modified vaccinia virus Ankara (MVA) is being widely investigated as a safe smallpox vaccine and as an expression vector to produce vaccines against other infectious diseases and cancer. MVA was isolated following more than 500 passages in chick embryo fibroblasts and suffered several major deletions and numerous small mutations resulting in replication defects in human and most other mammalian cells as well as severe attenuation of pathogenicity. Due to the host range restriction, primary chick embryo fibroblasts are routinely used for production of MVA-based vaccines. While a replication defect undoubtedly contributes to safety of MVA, it is worth considering whether host range and attenuation are partially separable properties. Marker rescue transfection experiments resulted in the creation of recombinant MVAs with extended mammalian cell host range. Here, we characterize two host-range extended rMVAs and show that they (i) have acquired the ability to stably replicate in Vero cells, which are frequently used as a cell substrate for vaccine manufacture, (ii) are severely attenuated in immunocompetent and immunodeficient mouse strains following intranasal infection, (iii) are more pathogenic than MVA but less pathogenic than the ACAM2000 vaccine strain at high intracranial doses, (iv) do not form lesions upon tail scratch in mice in contrast to ACAM2000 and (v) induce protective humoral and cell-mediated immune responses similar to MVA. The extended host range of rMVAs may be useful for vaccine production. Published by Elsevier Ltd.

  4. Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

    Science.gov (United States)

    Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

    2015-09-01

    Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

  5. Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

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    Ana Paula Carneiro Salgado

    2013-08-01

    Full Text Available Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV and Cowpox (CPXV and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

  6. ISG15 governs mitochondrial function in macrophages following vaccinia virus infection.

    Directory of Open Access Journals (Sweden)

    Sara Baldanta

    2017-10-01

    Full Text Available The interferon (IFN-stimulated gene 15 (ISG15 encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15-/- and ISG15+/+ bone marrow derived macrophages (BMDM after vaccinia virus (VACV infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS were pathways altered in ISG15-/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP and reactive oxygen species (ROS production was higher in ISG15+/+ BMDM than in ISG15-/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15-/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15+/+ macrophages than in ISG15-/- macrophages concomitant with a decrease in viral titer. Thus, ISG15-/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent.

  7. Hazard Characterization of Modified Vaccinia Virus Ankara Vector: What Are the Knowledge Gaps?

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    Malachy I. Okeke

    2017-10-01

    Full Text Available Modified vaccinia virus Ankara (MVA is the vector of choice for human and veterinary applications due to its strong safety profile and immunogenicity in vivo. The use of MVA and MVA-vectored vaccines against human and animal diseases must comply with regulatory requirements as they pertain to environmental risk assessment, particularly the characterization of potential adverse effects to humans, animals and the environment. MVA and recombinant MVA are widely believed to pose low or negligible risk to ecosystem health. However, key aspects of MVA biology require further research in order to provide data needed to evaluate the potential risks that may occur due to the use of MVA and MVA-vectored vaccines. The purpose of this paper is to identify knowledge gaps in the biology of MVA and recombinant MVA that are of relevance to its hazard characterization and discuss ongoing and future experiments aimed at providing data necessary to fill in the knowledge gaps. In addition, we presented arguments for the inclusion of uncertainty analysis and experimental investigation of verifiable worst-case scenarios in the environmental risk assessment of MVA and recombinant MVA. These will contribute to improved risk assessment of MVA and recombinant MVA vaccines.

  8. Role of the vaccinia virus O3 protein in cell entry can be fulfilled by its Sequence flexible transmembrane domain

    Energy Technology Data Exchange (ETDEWEB)

    Satheshkumar, P.S.; Chavre, James; Moss, Bernard, E-mail: bmoss@nih.gov

    2013-09-15

    The vaccinia virus O3 protein, a component of the entry–fusion complex, is encoded by all chordopoxviruses. We constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry–fusion complex and function in cell entry. Nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. Moreover, replication-competent mutant viruses were generated by randomization of 10 amino acids of the transmembrane domain. Of eight unique viruses, two contained only two amino acids in common with wild type and the remainder contained one or none within the randomized sequence. Although these mutant viruses formed normal size plaques, the entry–fusion complex did not co-purify with the mutant O3 proteins suggesting a less stable interaction. Thus, despite low specific sequence requirements, the transmembrane domain is sufficient for function in entry. - Highlights: • The 35 amino acid O3 protein is required for efficient vaccinia virus entry. • The transmembrane domain of O3 is necessary and sufficient for entry. • Mutagenesis demonstrated extreme sequence flexibility compatible with function.

  9. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human leucoc...

  10. Use of Bioclimatic Factors to Determine Potential Niche of Vaccinia Virus, an Emerging and Zoonotic Pathogen

    Science.gov (United States)

    Quiner, C. A.; Nakazawa, Y.

    2017-12-01

    Emerging and understudied pathogens often lack information that most commonly used analytical tools require, such as negative controls or baseline data making public health control of emerging pathogens challenging. In lieu of opportunities to collect more data from larger outbreaks or formal epidemiological studies, new analytical strategies, merging case data with publically available datasets, can be used to understand transmission patterns and drivers of disease emergence. Zoonotic infections with Vaccinia virus (VACV) were first reported in Brazil in 1999, VACV is an emerging zoonotic Orthopoxvirus, which primarily infects dairy cattle and farmers in close contact with infected cows. Prospective studies of emerging pathogens could provide critical data that would inform public health planning and response to outbreaks. By using the location of 87-recorded outbreaks and publicly available bioclimatic data we demonstrate one such approach. Using an Ecological Niche Model (ENM), we identify the environmental conditions under which VACV outbreaks have occurred, and determine additional locations in two affected South American countries that may be susceptible to transmission. Further, we show how suitability for the virus responds to different levels of various environmental factors and highlight the most important climatic factors in determining its transmission. The final ENM predicted all areas where Brazilian outbreaks occurred, two out of five Colombian outbreaks and identified new regions within Brazil that are suitable for transmission based on bioclimatic factors. Further, the most important factors in determining transmission suitability are precipitation of the wettest quarter, annual precipitation, mean temperature of the coldest quarter and mean diurnal range. The analyses here provide a means by which to study patterns of an emerging infectious disease, and regions that are potentially at risk for it, in spite of the paucity of critical data. Policy

  11. Protective Effect of Surfactant Protein D in Pulmonary Vaccinia Virus Infection: Implication of A27 Viral Protein

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    Julien Perino

    2013-03-01

    Full Text Available Vaccinia virus (VACV was used as a surrogate of variola virus (VARV (genus Orthopoxvirus, the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D, constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/- resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27.

  12. Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene: an oncolytic virus superior to dl1520 (ONYX-015) for human head and neck cancer.

    Science.gov (United States)

    Tysome, James R; Wang, Pengju; Alusi, Ghassan; Briat, Arnaud; Gangeswaran, Rathi; Wang, Jiwei; Bhakta, Vipul; Fodor, Istvan; Lemoine, Nick R; Wang, Yaohe

    2011-09-01

    Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

  13. In vitro susceptibility to ST-246 and Cidofovir corroborates the phylogenetic separation of Brazilian Vaccinia virus into two clades.

    Science.gov (United States)

    Pires, Mariana A; Rodrigues, Nathália F S; de Oliveira, Danilo B; de Assis, Felipe L; Costa, Galileu B; Kroon, Erna G; Mota, Bruno E F

    2018-04-01

    The Orthopoxvirus (OPV) genus of the Poxviridae family contains several human pathogens, including Vaccinia virus (VACV), which have been implicating in outbreaks of a zoonotic disease called Bovine Vaccinia in Brazil. So far, no approved treatment exists for OPV infections, but ST-246 and Cidofovir (CDV) are now in clinical development. Therefore, the objective of this work was to evaluate the susceptibility of five strains of Brazilian VACV (Br-VACV) to ST-246 and Cidofovir. The susceptibility of these strains to both drugs was evaluated by plaque reduction assay, extracellular virus's quantification in the presence of ST-246 and one-step growth curve in cells treated with CDV. Besides that, the ORFs F13L and E9L were sequenced for searching of polymorphisms associated with drug resistance. The effective concentration of 50% (EC 50 ) from both drugs varies significantly for different strains (from 0.0054 to 0.051 μM for ST-246 and from 27.14 to 61.23 μM for CDV). ST-246 strongly inhibits the production of extracellular virus for all isolates in concentrations as low as 0.1 μM and it was observed a relevant decrease of progeny production for all Br-VACV after CDV treatment. Sequencing of the F13L and E9L ORFs showed that Br-VACV do not present the polymorphism(s) associated with resistance to ST-246 and CDV. Taken together, our results showed that ST-246 and CDV are effective against diverse, wild VACV strains and that the susceptibility of Br-VACV to these drugs mirrored the phylogenetic split of these isolates into two groups. Thus, both ST-246 and CDV are of great interest as compounds to treat individuals during Bovine Vaccinia outbreaks in Brazil. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Interaction between the G3 and L5 proteins of the vaccinia virus entry–fusion complex

    OpenAIRE

    Wolfe, Cindy L.; Moss, Bernard

    2011-01-01

    The vaccinia virus entry-fusion complex (EFC) consists of 10 to 12 proteins that are embedded in the viral membrane and individually required for fusion with the cell and entry of the core into the cytoplasm. The architecture of the EFC is unknown except for information regarding two pair-wise interactions: A28 with H2 and A16 with G9. Here we used a technique to destabilize the EFC by repressing the expression of individual components and identified a third pair-wise interaction: G3 with L5....

  15. Structure and function of A41, a vaccinia virus chemokine binding protein.

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    Mohammad W Bahar

    2008-01-01

    Full Text Available The vaccinia virus (VACV A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI, and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM chemokine-chemokine receptor interactions.

  16. Systemically administered DNA and fowlpox recombinants expressing four vaccinia virus genes although immunogenic do not protect mice against the highly pathogenic IHD-J vaccinia strain.

    Science.gov (United States)

    Bissa, Massimiliano; Pacchioni, Sole Maria; Zanotto, Carlo; De Giuli Morghen, Carlo; Illiano, Elena; Granucci, Francesca; Zanoni, Ivan; Broggi, Achille; Radaelli, Antonia

    2013-12-26

    The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Safety and Immunogenicity of Modified Vaccinia Ankara-Bavarian Nordic Smallpox Vaccine in Vaccinia-Naive and Experienced Human Immunodeficiency Virus-Infected Individuals: An Open-Label, Controlled Clinical Phase II Trial

    Science.gov (United States)

    Overton, Edgar Turner; Stapleton, Jack; Frank, Ian; Hassler, Shawn; Goepfert, Paul A.; Barker, David; Wagner, Eva; von Krempelhuber, Alfred; Virgin, Garth; Meyer, Thomas Peter; Müller, Jutta; Bädeker, Nicole; Grünert, Robert; Young, Philip; Rösch, Siegfried; Maclennan, Jane; Arndtz-Wiedemann, Nathaly; Chaplin, Paul

    2015-01-01

    Background. First- and second-generation smallpox vaccines are contraindicated in individuals infected with human immunodeficiency virus (HIV). A new smallpox vaccine is needed to protect this population in the context of biodefense preparedness. The focus of this study was to compare the safety and immunogenicity of a replication-deficient, highly attenuated smallpox vaccine modified vaccinia Ankara (MVA) in HIV-infected and healthy subjects. Methods. An open-label, controlled Phase II trial was conducted at 36 centers in the United States and Puerto Rico for HIV-infected and healthy subjects. Subjects received 2 doses of MVA administered 4 weeks apart. Safety was evaluated by assessment of adverse events, focused physical exams, electrocardiogram recordings, and safety laboratories. Immune responses were assessed using enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT). Results. Five hundred seventy-nine subjects were vaccinated at least once and had data available for analysis. Rates of ELISA seropositivity were comparably high in vaccinia-naive healthy and HIV-infected subjects, whereas PRNT seropositivity rates were higher in healthy compared with HIV-infected subjects. Modified vaccinia Ankara was safe and well tolerated with no adverse impact on viral load or CD4 counts. There were no cases of myo-/pericarditis reported. Conclusions. Modified vaccinia Ankara was safe and immunogenic in subjects infected with HIV and represents a promising smallpox vaccine candidate for use in immunocompromised populations. PMID:26380340

  18. Viral exploitation of the MEK/ERK pathway - A tale of vaccinia virus and other viruses.

    Science.gov (United States)

    Bonjardim, Cláudio A

    2017-07-01

    The VACV replication cycle is remarkable in the sense that it is performed entirely in the cytoplasmic compartment of vertebrate cells, due to its capability to encode enzymes required either for regulating the macromolecular precursor pool or the biosynthetic processes. Although remarkable, this gene repertoire is not sufficient to confer the status of a free-living microorganism to the virus, and, consequently, the virus relies heavily on the host to successfully generate its progeny. During the complex virus-host interaction, viruses must deal not only with the host pathways to accomplish their temporal demands but also with pathways that counteract viral infection, including the inflammatory, innate and acquired immune responses. This review focuses on VACV and other DNA or RNA viruses that stimulate the MEK (MAPK - Mitogen Activated Protein Kinase)/ERK- Extracellular signal-Regulated Kinase) pathway as part of their replication cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Unpolarized release of vaccinia virus and HIV antigen by colchicine treatment enhances intranasal HIV antigen expression and mucosal humoral responses.

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    Full Text Available The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol in mucosal epithelial cells (specifically Caco-2 cell layers and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies.

  20. Biophysical analysis of bacterial and viral systems. A shock tube study of bio-aerosols and a correlated AFM/nanosims investigation of vaccinia virus

    Energy Technology Data Exchange (ETDEWEB)

    Gates, Sean Damien [Stanford Univ., CA (United States)

    2013-05-01

    The work presented herein is concerned with the development of biophysical methodology designed to address pertinent questions regarding the behavior and structure of select pathogenic agents. Two distinct studies are documented: a shock tube analysis of endospore-laden bio-aerosols and a correlated AFM/NanoSIMS study of the structure of vaccinia virus.

  1. Evaluation of radiation effects against C6 glioma in combination with vaccinia virus-p53 gene therapy

    Science.gov (United States)

    Gridley, D. S.; Andres, M. L.; Li, J.; Timiryasova, T.; Chen, B.; Fodor, I.; Nelson, G. A. (Principal Investigator)

    1998-01-01

    The primary objective of this study was to evaluate the antitumor effects of recombinant vaccinia virus-p53 (rVV-p53) in combination with radiation therapy against the C6 rat glioma, a p53 deficient tumor that is relatively radioresistant. VV-LIVP, the parental virus (Lister strain), was used as a control. Localized treatment of subcutaneous C6 tumors in athymic mice with either rVV-p53 or VV-LIVP together with tumor irradiation resulted in low tumor incidence and significantly slower tumor progression compared to the agents given as single modalities. Assays of blood and spleen indicated that immune system activation may account, at least partly, for the enhance tumor inhibition seen with combined treatment. No overt signs of treatment-related toxicity were noted.

  2. Absence of vaccinia virus detection in a remote region of the Northern Amazon forests, 2005-2015.

    Science.gov (United States)

    Costa, Galileu Barbosa; Lavergne, Anne; Darcissac, Edith; Lacoste, Vincent; Drumond, Betânia Paiva; Abrahão, Jônatas Santos; Kroon, Erna Geessien; de Thoisy, Benoît; de Souza Trindade, Giliane

    2017-08-01

    Vaccinia virus (VACV) circulates in Brazil and other South America countries and is responsible for a zoonotic disease that usually affects dairy cattle and humans, causing economic losses and impacting animal and human health. Furthermore, it has been detected in wild areas in the Brazilian Amazon. To better understand the natural history of VACV, we investigated its circulation in wildlife from French Guiana, a remote region in the Northern Amazon forest. ELISA and plaque reduction neutralization tests were performed to detect anti-orthopoxvirus antibodies. Real-time and standard PCR targeting C11R, A56R and A26L were applied to detect VACV DNA in serum, saliva and tissue samples. No evidence of VACV infection was found in any of the samples tested. These findings provide additional information on the VACV epidemiological puzzle. The virus could nevertheless be circulating at low levels that were not detected in areas where no humans or cattle are present.

  3. Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

    Science.gov (United States)

    Jancovich, James K; Chapman, Dave; Hansen, Debra T; Robida, Mark D; Loskutov, Andrey; Craciunescu, Felicia; Borovkov, Alex; Kibler, Karen; Goatley, Lynnette; King, Katherine; Netherton, Christopher L; Taylor, Geraldine; Jacobs, Bertram; Sykes, Kathryn; Dixon, Linda K

    2018-04-15

    African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia

  4. A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Meador, Lydia R. [Ira A. Fulton School of Engineering, Arizona State University, Tempe, AZ (United States); Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ (United States); Kessans, Sarah A. [Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ (United States); School of Life Sciences, Arizona State University, Tempe, AZ (United States); Kilbourne, Jacquelyn; Kibler, Karen V. [Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ (United States); Pantaleo, Giuseppe [Division of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne (Switzerland); Swiss Vaccine Research Institute, Lausanne (Switzerland); Roderiguez, Mariano Esteban [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologia – CSIC, Madrid (Spain); Blattman, Joseph N. [Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ (United States); School of Life Sciences, Arizona State University, Tempe, AZ (United States); Jacobs, Bertram L., E-mail: bjacobs@asu.edu [Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ (United States); School of Life Sciences, Arizona State University, Tempe, AZ (United States); Mor, Tsafrir S., E-mail: tsafrir.mor@asu.edu [Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, AZ (United States); School of Life Sciences, Arizona State University, Tempe, AZ (United States)

    2017-07-15

    Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. - Highlights: • We devised a prime/boost anti HIV-1 vaccination strategy modeled after RV144. • We used plant-derived virus-like particles (VLPs) consisting of Gag and dgp41. • We used attenuated, replicating vaccinia virus vectors expressing the same antigens. • The immunogens elicited strong cellular and humoral immune responses.

  5. Live vaccinia-rabies virus recombinants, but not an inactivated rabies virus cell culture vaccine, protect B-lymphocyte-deficient A/WySnJ mice against rabies: considerations of recombinant defective poxviruses for rabies immunization of immunocompromised individuals.

    Science.gov (United States)

    Lodmell, Donald L; Esposito, Joseph J; Ewalt, Larry C

    2004-09-03

    Presently, commercially available cell culture rabies vaccines for humans and animals consist of the five inactivated rabies virus proteins. The vaccines elicit a CD4+ helper T-cell response and a humoral B-cell response against the viral glycoprotein (G) resulting in the production of virus neutralizing antibody. Antibody against the viral nucleoprotein (N) is also present, but the mechanism(s) of its protection is unclear. HIV-infected individuals with low CD4+ T-lymphocyte counts and individuals undergoing treatment with immunosuppressive drugs have an impaired neutralizing antibody response after pre- and post-exposure immunization with rabies cell culture vaccines. Here we show the efficacy of live vaccinia-rabies virus recombinants, but not a cell culture vaccine consisting of inactivated rabies virus, to elicit elevated levels of neutralizing antibody in B-lymphocyte deficient A/WySnJ mice. The cell culture vaccine also failed to protect the mice, whereas a single immunization of a vaccinia recombinant expressing the rabies virus G or co-expressing G and N equally protected the mice up to 18 months after vaccination. The data suggest that recombinant poxviruses expressing the rabies virus G, in particular replication defective poxviruses such as canarypox or MVA vaccinia virus that undergo abortive replication in non-avian cells, or the attenuated vaccinia virus NYVAC, should be evaluated as rabies vaccines in immunocompromised individuals.

  6. The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

    Science.gov (United States)

    Gammon, Don B; Evans, David H

    2009-05-01

    Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

  7. Expression of the A56 and K2 proteins is sufficient to inhibit vaccinia virus entry and cell fusion.

    Science.gov (United States)

    Wagenaar, Timothy R; Moss, Bernard

    2009-02-01

    Many animal viruses induce cells to fuse and form syncytia. For vaccinia virus, this phenomenon is associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositol-anchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.

  8. Effect of Vaccinia virus infection on poly(ADP-ribose)synthesis and DNA metabolism in different cells

    Energy Technology Data Exchange (ETDEWEB)

    Topaloglou, A.; Ott, E.; Altmann, H. (Oesterreichisches Forschungszentrum Seibersdorf G.m.b.H. Inst. fuer Biologie); Zashukhina, G.D.; Sinelschikova, T.A. (AN SSSR, Moscow. Inst. Obshchej Genetiki)

    1983-07-14

    In Chang liver cells and rat spleen cells infected with Vaccinia virus, DNA synthesis, repair replication after UV irradiation and poly(ADP-ribose)(PAR) synthesis were determined. In the time post infection semiconservative DNA synthesis showed only a slight reduction. DNA repair replication was not very different from controls 4 hours p.i. but was enhanced 24 hours after infection compared to noninfected cells. PAR synthesis was also not changed very much 4 hours p.i. but was decreased significantly after 24 hours. The determination of radioactivity resulting from /sup 3/H-NAD, showed a marked reduction of PAR in the spacer region of chromatin 24 hours p.i., but in addition, PAR located in the core region, was reduced, too.

  9. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA.

    Directory of Open Access Journals (Sweden)

    Matthew G Cottingham

    2008-02-01

    Full Text Available The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20. The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA, now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+ T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+ T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins, in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006. In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+ T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant

  10. Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins

    Science.gov (United States)

    Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C.; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

    2017-01-01

    Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned. PMID:28877647

  11. Prime/boost immunotherapy of HPV16-induced tumors with E7 protein delivered by Bordetella adenylate cyclase and modified vaccinia virus Ankara

    Czech Academy of Sciences Publication Activity Database

    Macková, J.; Stasíková, J.; Kutinová, L.; Mašín, Jiří; Hainz, P.; Šimšová, Marcela; Gabriel, P.; Šebo, Peter; Němečková, P.

    2006-01-01

    Roč. 55, - (2006), s. 39-46 ISSN 0340-7004 R&D Projects: GA AV ČR IBS5020311; GA ČR GA310/04/0004; GA MZd NR8004 Grant - others:GA MZd NC6570 Institutional research plan: CEZ:AV0Z50200510 Keywords : vaccine * hpv-e7 * vaccinia virus Subject RIV: EE - Microbiology, Virology Impact factor: 4.313, year: 2006

  12. Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification Surtos de vírus Vaccinia-like nos Estados de São Paulo e Goiás, Brasil: detecção, isolamento e identificação viral

    Directory of Open Access Journals (Sweden)

    Teresa Keico Nagasse-Sugahara

    2004-04-01

    Full Text Available Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM in samples of 49 (66.21% patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.A partir de outubro de 2001, o Instituto Adolfo Lutz tem recebido amostras de líquido vesicular e crostas de lesões de pele de pacientes das regiões do Vale do Paraíba, Estado de São Paulo e do Vale do São Patricio, Estado de Goiás. Os dados clínicos e epidemiológicos sugeriam que os surtos poderiam ser causados por Cowpox virus ou Vaccinia virus. A maioria dos pacientes era ordenhadores que tinham lesões vesicopustulares nas mãos, braços, antebraços e alguns na face. A análise por microscopia eletrônica direta (MED detectou partículas com morfologia de vírus do gênero Orthopoxvirus em amostras de 49 (66,21% pacientes dos 74

  13. RNA-Seq Based Transcriptome Analysis of the Type I Interferon Host Response upon Vaccinia Virus Infection of Mouse Cells

    Directory of Open Access Journals (Sweden)

    Bruno Hernáez

    2017-01-01

    Full Text Available Vaccinia virus (VACV encodes the soluble type I interferon (IFN binding protein B18 that is secreted from infected cells and also attaches to the cell surface, as an immunomodulatory strategy to inhibit the host IFN response. By using next generation sequencing technologies, we performed a detailed RNA-seq study to dissect at the transcriptional level the modulation of the IFN based host response by VACV and B18. Transcriptome profiling of L929 cells after incubation with purified recombinant B18 protein showed that attachment of B18 to the cell surface does not trigger cell signalling leading to transcriptional activation. Consistent with its ability to bind type I IFN, B18 completely inhibited the IFN-mediated modulation of host gene expression. Addition of UV-inactivated virus particles to cell cultures altered the expression of a set of 53 cellular genes, including genes involved in innate immunity. Differential gene expression analyses of cells infected with replication competent VACV identified the activation of a broad range of host genes involved in multiple cellular pathways. Interestingly, we did not detect an IFN-mediated response among the transcriptional changes induced by VACV, even after the addition of IFN to cells infected with a mutant VACV lacking B18. This is consistent with additional viral mechanisms acting at different levels to block IFN responses during VACV infection.

  14. Daily ingestion of the probiotic Lactobacillus paracasei ST11 decreases Vaccinia virus dissemination and lethality in a mouse model.

    Science.gov (United States)

    Dos Santos Pereira Andrade, A C; Lima, M Teixeira; Oliveira, G Pereira; Calixto, R Silva; de Sales E Souza, É Lorenna; da Glória de Souza, D; de Almeida Leite, C M; Ferreira, J M Siqueira; Kroon, E G; de Oliveira, D Bretas; Dos Santos Martins, F; Abrahão, J S

    2017-02-07

    Vaccinia virus (VACV) is an important pathogen. Although studies have shown relationships between probiotics and viruses, the effect of probiotics on VACV infection is unknown. Therefore, this work aims to investigate the probiotics effects on VACV infection. Mice were divided into four groups, two non-infected groups, one receiving the probiotic, the other one not receiving it, and two groups infected intranasally with VACV Western Reserve (VACV-WR) receiving or not receiving the probiotic. Viral titres in organs and cytokine production in the lungs were analysed. Lung samples were also subjected to histological analysis. The intake of probiotic results in reduction in viral spread with a significant decrease of VACV titer on lung, liver and brain of treated group. In addition,treatment with the probiotic results in attenuated mice lung inflammation showing fewer lesions on histological findings and decreased lethality in mice infected with VACV. The ingestion of Lactobacillus paracasei ST11 (LPST11) after VACV infection resulted in 2/9 animal lethality compared with 4/9 in the VACV group. This is the first study on probiotics and VACV interactions, providing not only information about this interaction, but also proposing a model for future studies involving probiotics and other poxvirus.

  15. Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

    Science.gov (United States)

    Mirzakhanyan, Yeva; Gershon, Paul D

    2017-09-01

    The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size. Copyright © 2017 American Society for Microbiology.

  16. E3L and F1L Gene Functions Modulate the Protective Capacity of Modified Vaccinia Virus Ankara Immunization in Murine Model of Human Smallpox

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    Asisa Volz

    2018-01-01

    Full Text Available The highly attenuated Modified Vaccinia virus Ankara (MVA lacks most of the known vaccinia virus (VACV virulence and immune evasion genes. Today MVA can serve as a safety-tested next-generation smallpox vaccine. Yet, we still need to learn about regulatory gene functions preserved in the MVA genome, such as the apoptosis inhibitor genes F1L and E3L. Here, we tested MVA vaccine preparations on the basis of the deletion mutant viruses MVA-ΔF1L and MVA-ΔE3L for efficacy against ectromelia virus (ECTV challenge infections in mice. In non-permissive human tissue culture the MVA deletion mutant viruses produced reduced levels of the VACV envelope antigen B5. Upon mousepox challenge at three weeks after vaccination, MVA-ΔF1L and MVA-ΔE3L exhibited reduced protective capacity in comparison to wildtype MVA. Surprisingly, however, all vaccines proved equally protective against a lethal ECTV infection at two days after vaccination. Accordingly, the deletion mutant MVA vaccines induced high levels of virus-specific CD8+ T cells previously shown to be essential for rapidly protective MVA vaccination. These results suggest that inactivation of the anti-apoptotic genes F1L or E3L modulates the protective capacity of MVA vaccination most likely through the induction of distinct orthopoxvirus specific immunity in the absence of these viral regulatory proteins.

  17. Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity.

    Science.gov (United States)

    Ami, Yasushi; Izumi, Yasuyuki; Matsuo, Kazuhiro; Someya, Kenji; Kanekiyo, Masaru; Horibata, Shigeo; Yoshino, Naoto; Sakai, Koji; Shinohara, Katsuaki; Matsumoto, Sohkichi; Yamada, Takeshi; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

    2005-10-01

    Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

  18. Imaging characteristics, tissue distribution, and spread of a novel oncolytic vaccinia virus carrying the human sodium iodide symporter.

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    Dana Haddad

    Full Text Available INTRODUCTION: Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS. METHODS: GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide (131I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via (124I-positron emission tomography (PET. Detection of systemic administration of virus was investigated with both (124I-PET and 99m-technecium gamma-scintigraphy. RESULTS: GLV-1h153 successfully facilitated time-dependent intracellular uptake of (131I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05. In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 10(9 plaque-forming unit (PFU/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82 ± 0.46 (P<0.05 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via (124I-PET and 99m-technecium-scintigraphy. CONCLUSION: GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic

  19. Quantitative Analysis of MicroRNAs in Vaccinia virus Infection Reveals Diversity in Their Susceptibility to Modification and Suppression.

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    Amy H Buck

    Full Text Available Vaccinia virus (VACV is a large cytoplasmic DNA virus that causes dramatic alterations to many cellular pathways including microRNA biogenesis. The virus encodes a poly(A polymerase which was previously shown to add poly(A tails to the 3' end of cellular miRNAs, resulting in their degradation by 24 hours post infection (hpi. Here we used small RNA sequencing to quantify the impact of VACV infection on cellular miRNAs in human cells at both early (6 h and late (24 h times post infection. A detailed quantitative analysis of individual miRNAs revealed marked diversity in the extent of their modification and relative change in abundance during infection. Some miRNAs became highly modified (e.g. miR-29a-3p, miR-27b-3p whereas others appeared resistant (e.g. miR-16-5p. Furthermore, miRNAs that were highly tailed at 6 hpi were not necessarily among the most reduced at 24 hpi. These results suggest that intrinsic features of human cellular miRNAs cause them to be differentially polyadenylated and altered in abundance during VACV infection. We also demonstrate that intermediate and late VACV gene expression are required for optimal repression of some miRNAs including miR-27-3p. Overall this work reveals complex and varied consequences of VACV infection on host miRNAs and identifies miRNAs which are largely resistant to VACV-induced polyadenylation and are therefore present at functional levels during the initial stages of infection and replication.

  20. Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.

    Science.gov (United States)

    Joachim, Agricola; Munseri, Patricia J; Nilsson, Charlotta; Bakari, Muhammad; Aboud, Said; Lyamuya, Eligius F; Tecleab, Teghesti; Liakina, Valentina; Scarlatti, Gabriella; Robb, Merlin L; Earl, Patricia L; Moss, Bernard; Wahren, Britta; Mhalu, Fred; Ferrari, Guido; Sandstrom, Eric; Biberfeld, Gunnel

    2017-08-01

    We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.

  1. Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

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    Tkachenko, Anastasiya; Richter, Vladimir

    2017-01-01

    Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. PMID:28951871

  2. Interaction between the G3 and L5 proteins of the vaccinia virus entry-fusion complex

    International Nuclear Information System (INIS)

    Wolfe, Cindy L.; Moss, Bernard

    2011-01-01

    The vaccinia virus entry-fusion complex (EFC) consists of 10 to 12 proteins that are embedded in the viral membrane and individually required for fusion with the cell and entry of the core into the cytoplasm. The architecture of the EFC is unknown except for information regarding two pair-wise interactions: A28 with H2 and A16 with G9. Here we used a technique to destabilize the EFC by repressing the expression of individual components and identified a third pair-wise interaction: G3 with L5. These two proteins remained associated under several different EFC destabilization conditions and in each case were immunopurified together as demonstrated by Western blotting. Further evidence for the specific interaction of G3 and L5 was obtained by mass spectrometry. This interaction also occurred when G3 and L5 were expressed in uninfected cells, indicating that no other viral proteins were required. Thus, the present study extends our knowledge of the protein interactions important for EFC assembly and stability.

  3. Oral vaccination of wildlife using a vaccinia-rabies-glycoprotein recombinant virus vaccine (RABORAL V-RG®): a global review.

    Science.gov (United States)

    Maki, Joanne; Guiot, Anne-Laure; Aubert, Michel; Brochier, Bernard; Cliquet, Florence; Hanlon, Cathleen A; King, Roni; Oertli, Ernest H; Rupprecht, Charles E; Schumacher, Caroline; Slate, Dennis; Yakobson, Boris; Wohlers, Anne; Lankau, Emily W

    2017-09-22

    RABORAL V-RG ® is an oral rabies vaccine bait that contains an attenuated ("modified-live") recombinant vaccinia virus vector vaccine expressing the rabies virus glycoprotein gene (V-RG). Approximately 250 million doses have been distributed globally since 1987 without any reports of adverse reactions in wildlife or domestic animals since the first licensed recombinant oral rabies vaccine (ORV) was released into the environment to immunize wildlife populations against rabies. V-RG is genetically stable, is not detected in the oral cavity beyond 48 h after ingestion, is not shed by vaccinates into the environment, and has been tested for thermostability under a range of laboratory and field conditions. Safety of V-RG has been evaluated in over 50 vertebrate species, including non-human primates, with no adverse effects observed regardless of route or dose. Immunogenicity and efficacy have been demonstrated under laboratory and field conditions in multiple target species (including fox, raccoon, coyote, skunk, raccoon dog, and jackal). The liquid vaccine is packaged inside edible baits (i.e., RABORAL V-RG, the vaccine-bait product) which are distributed into wildlife habitats for consumption by target species. Field application of RABORAL V-RG has contributed to the elimination of wildlife rabies from three European countries (Belgium, France and Luxembourg) and of the dog/coyote rabies virus variant from the United States of America (USA). An oral rabies vaccination program in west-central Texas has essentially eliminated the gray fox rabies virus variant from Texas with the last case reported in a cow during 2009. A long-term ORV barrier program in the USA using RABORAL V-RG is preventing substantial geographic expansion of the raccoon rabies virus variant. RABORAL V-RG has also been used to control wildlife rabies in Israel for more than a decade. This paper: (1) reviews the development and historical use of RABORAL V-RG; (2) highlights wildlife rabies control

  4. Antibodies to the A27 protein of vaccinia virus neutralize and protect against infection but represent a minor component of Dryvax vaccine--induced immunity.

    Science.gov (United States)

    He, Yong; Manischewitz, Jody; Meseda, Clement A; Merchlinsky, Michael; Vassell, Russell A; Sirota, Lev; Berkower, Ira; Golding, Hana; Weiss, Carol D

    2007-10-01

    The smallpox vaccine Dryvax, which consists of replication-competent vaccinia virus, elicits antibodies that play a major role in protection. Several vaccinia proteins generate neutralizing antibodies, but their importance for protection is unknown. We investigated the potency of antibodies to the A27 protein of the mature virion in neutralization and protection experiments and the contributions of A27 antibodies to Dryvax-induced immunity. Using a recombinant A27 protein (rA27), we confirmed that A27 contains neutralizing determinants and that vaccinia immune globulin (VIG) derived from Dryvax recipients contains reactivity to A27. However, VIG neutralization was not significantly reduced when A27 antibodies were removed, and antibodies elicited by an rA27 enhanced the protection conferred by VIG in passive transfer experiments. These findings demonstrate that A27 antibodies do not represent the major fraction of neutralizing activity in VIG and suggest that immunity may be augmented by vaccines and immune globulins that include strong antibody responses to A27.

  5. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins.

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    Sofiya Fedosyuk

    2016-12-01

    Full Text Available Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1-83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1-83 structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1-240, we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88.

  6. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    Science.gov (United States)

    1990-02-01

    procaryotic systems (12. 45). Certain eucaryotic ically cleaved by a trypsin-like proteas: ito produce a recep- viruses are currently being explored as...19847. Proteolytic activation of anthrax toxin bound to cellular recep- ACKN()WEIX;NMNTS tor%.. p. 111-112. In F. Fehrenbach et al. ifed.). Bacterial

  7. The Orf virus E3L homologue is able to complement deletion of the vaccinia virus E3L gene in vitro but not in vivo

    International Nuclear Information System (INIS)

    Vijaysri, Sangeetha; Talasela, Latha; Mercer, Andrew A.; Mcinnes, Colin J.; Jacobs, Bertram L.; Langland, Jeffrey O.

    2003-01-01

    Orf virus (OV), the prototypic parapoxvirus, is resistant to the effects of interferon (IFN) and this function of OV has been mapped to the OV20.0L gene. The protein product of this gene shares 31% amino acid identity to the E3L-encoded protein of vaccinia virus (VV) that is required for the broad host range and IFN-resistant phenotype of VV in cells in culture and for virulence of the virus in vivo. In this study we investigated whether the distantly related OV E3L homologue could complement the deletion of E3L in VV. The recombinant VV (VV/ORF-E3L) expressing the OV E3L homologue in place of VV E3L was indistinguishable from wt VV in its cell-culture phenotype. But VV/ORF-E3L was over a 1000-fold less pathogenic than wt VV (LD 50 > 5 x 10 6 PFU, compared to LD 50 of wtVV = 4 x 10 3 PFU) following intranasal infection of mice. While wt VV spread to the lungs and brain and replicated to high titers in the brain of infected mice, VV/ORF-E3L could not be detected in the lungs or brain following intranasal infection. VV/ORF-E3L was at least 100,000-fold less pathogenic than wt VV on intracranial injection. Domain swap experiments demonstrate that the difference in pathogenesis maps to the C-terminal domain of these proteins. This domain has been shown to be required for the dsRNA binding function of the VV E3L

  8. Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

    Science.gov (United States)

    Soto-Acosta, Rubén; Bautista-Carbajal, Patricia; Syed, Gulam H; Siddiqui, Aleem; Del Angel, Rosa M

    2014-09-01

    Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

    Science.gov (United States)

    Castrucci, Maria R; Facchini, Marzia; Di Mario, Giuseppina; Garulli, Bruno; Sciaraffia, Ester; Meola, Monica; Fabiani, Concetta; De Marco, Maria A; Cordioli, Paolo; Siccardi, Antonio; Kawaoka, Yoshihiro; Donatelli, Isabella

    2014-05-01

    To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans. © 2013 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

  10. Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

    Science.gov (United States)

    Luteijn, Rutger David; Drexler, Ingo; Smith, Geoffrey L; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

    2018-04-20

    Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

  11. L1R, A27L, A33R and B5R vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines.

    Science.gov (United States)

    Pacchioni, Sole Maria; Bissa, Massimiliano; Zanotto, Carlo; Morghen, Carlo De Giuli; Illiano, Elena; Radaelli, Antonia

    2013-04-11

    The traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most effective control and new vectors have been developed to generate recombinant vaccines against smallpox that induce the same immunogenicity as the traditional one. As protective antibodies are mainly directed against the surface proteins of the two infectious forms of vaccinia, the intracellular mature virions and the extracellular virions, combined proteins from these viral forms can be used to better elicit a complete and protective immunity. Four novel viral recombinants were constructed based on the fowlpox genetic background, which independently express the vaccinia virus L1 and A27 proteins present on the mature virions, and the A33 and B5 proteins present on the extracellular virions. The correct expression of the transgenes was determined by RT-PCR, Western blotting, and immunofluorescence. Using immunoprecipitation and Western blotting, the ability of the proteins expressed by the four novel FPL1R, FPA27L, FPA33R and FPB5R recombinants to be recognized by VV-specific hyperimmune mouse sera was demonstrated. By neutralisation assays, recombinant virus particles released by infected chick embryo fibroblasts were shown not be recognised by hyperimmune sera. This thus demonstrates that the L1R, A27L, A33R and B5R gene products are not inserted into the new viral progeny. Fowlpox virus replicates only in avian species, but it is permissive for entry and transgene expression in mammalian cells, while being immunologically non-cross-reactive with vaccinia virus. These recombinants might

  12. Effects of nasal or pulmonary delivered treatments with an adenovirus vectored interferon (mDEF201 on respiratory and systemic infections in mice caused by cowpox and vaccinia viruses.

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    Donald F Smee

    Full Text Available An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201 was evaluated for efficacy against lethal cowpox (Brighton strain and vaccinia (WR strain virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl and pulmonary (50-µl, to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI, upper respiratory infections (URI, and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term or 56d (long-term prior to virus challenge. Single nasal sinus treatments of 10(6 and 10(7 PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201. Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 10(7 mDEF201 treatments, but a 10(6 dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 10(7 PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 10(5 to 10(7 PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

  13. Combination of intratumoral injections of vaccinia virus MVA expressing GM-CSF and immunization with DNA vaccine prolongs the survival of mice bearing HPV16 induced tumors with downregulated expression of MHC class I molecules

    Czech Academy of Sciences Publication Activity Database

    Němečková, Š.; Šmahel, M.; Hainz, P.; Macková, J.; Zurková, K.; Gabriel, P.; Indrová, Marie; Kutinová, L.

    2007-01-01

    Roč. 54, č. 4 (2007), s. 326-333 ISSN 0028-2685 R&D Projects: GA MZd NR8004 Institutional research plan: CEZ:AV0Z50520514 Keywords : vaccinia virus MVA expressing GM- CSF * DNA vaccine * HPV16 induced tumors Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.208, year: 2007

  14. Deletion of C7L and K1L genes leads to significantly decreased virulence of recombinant vaccinia virus TianTan.

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    Zheng Liu

    Full Text Available The vaccinia virus TianTan (VTT has been modified as an HIV vaccine vector in China and has shown excellent performance in immunogenicity and safety. However, its adverse effects in immunosuppressed individuals warrant the search for a safer vector in the following clinic trails. In this study, we deleted the C7L and K1L genes of VTT and constructed six recombinant vaccinia strains VTT△C7L, VTT△K1L, VTT△C7LK1L, VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag. The pathogenicity and immunogenicity of these recombinants were evaluated in mouse and rabbit models. Comparing to parental VTT, VTT△C7L and VTT△K1L showed significantly decreased replication capability in CEF, Vero, BHK-21 and HeLa cell lines. In particular, replication of VTT△C7LK1L decreased more than 10-fold in all four cell lines. The virulence of all these mutants were decreased in BALB/c mouse and rabbit models; VTT△C7LK1L once again showed the greatest attenuation, having resulted in no evident damage in mice and erythema of only 0.4 cm diameter in rabbits, compared to 1.48 cm for VTT. VTKgpe△C7L, VTKgpe△K1L and VTT△C7LK1L-gag elicited as strong cellular and humoral responses against HIV genes as did VTKgpe, while humoral immune response against the vaccinia itself was reduced by 4-8-fold. These data show that deletion of C7L and K1L genes leads to significantly decreased virulence without compromising animal host immunogenicity, and may thus be key to creating a more safe and effective HIV vaccine vector.

  15. Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

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    Mittra Arjun

    2011-03-01

    Full Text Available Abstract Introduction Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET utilizing carrier-free 124I radiotracer. Results GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P 124I-PET. Conclusion Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.

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

    Directory of Open Access Journals (Sweden)

    Juana M Sánchez-Puig

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  18. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Science.gov (United States)

    Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

    2012-01-01

    Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  19. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    Directory of Open Access Journals (Sweden)

    Satoshi Sekiguchi

    Full Text Available Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV, is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis, liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25, which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-/MxCre((+/- mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor TNF-α and (interleukin IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  20. Rapid Generation of Multiple Loci-Engineered Marker-free Poxvirus and Characterization of a Clinical-Grade Oncolytic Vaccinia Virus

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    Zong Sheng Guo

    2017-12-01

    Full Text Available Recombinant poxviruses, utilized as vaccine vectors and oncolytic viruses, often require manipulation at multiple genetic loci in the viral genome. It is essential for viral vectors to possess no adventitious mutations and no (antibiotic selection marker in the final product for human patients in order to comply with the guidance from the regulatory agencies. Rintoul et al. have previously developed a selectable and excisable marker (SEM system for the rapid generation of recombinant vaccinia virus. In the current study, we describe an improved methodology for rapid creation and selection of recombinant poxviruses with multiple genetic manipulations solely based on expression of a fluorescent protein and with no requirement for drug selection that can lead to cellular stress and the risk of adventitious mutations throughout the viral genome. Using this improved procedure combined with the SEM system, we have constructed multiple marker-free oncolytic poxviruses expressing different cytokines and other therapeutic genes. The high fidelity of inserted DNA sequences validates the utility of this improved procedure for generation of therapeutic viruses for human patients. We have created an oncolytic poxvirus expressing human chemokine CCL5, designated as vvDD-A34R-hCCL5, with manipulations at two genetic loci in a single virus. Finally, we have produced and purified this virus in clinical grade for its use in a phase I clinical trial and presented data on initial in vitro characterization of the virus.

  1. Transmission of vaccinia virus, possibly through sexual contact, to a woman at high risk for adverse complications.

    Science.gov (United States)

    Said, Maria A; Haile, Charles; Palabindala, Venkataraman; Barker, Naomi; Myers, Robert; Thompson, Ruth; Wilson, Lucy; Allan-Martinez, Frances; Montgomery, Jay; Monroe, Benjamin; Tack, Danielle; Reynolds, Mary; Damon, Inger; Blythe, David

    2013-12-01

    Severe adverse events, including eczema vaccinatum (EV), can result after smallpox vaccination. Persons at risk for EV include those with underlying dermatologic conditions, such as atopic dermatitis. We investigated a case of vaccinia infection, possibly acquired during sexual contact with a recently vaccinated military service member, in a female Maryland resident with atopic dermatitis. The U.S. Department of Defense's Vaccine Healthcare Centers Network (VHCN) and the Centers for Disease Control and Prevention (CDC) worked in conjunction with the patient's physician and the Maryland Department of Health and Mental Hygiene (DHMH) to confirm the diagnosis, ensure treatment, and prevent further transmission. Specimens collected from the patient were tested at the DHMH laboratories and were positive by real-time polymerase chain reaction for nonvariola orthopoxvirus. Testing at the CDC verified the presence of vaccinia-specific DNA signatures. Continuing spread of the patient's lesions led to the administration of vaccinia immune globulin and strict infection control measures to prevent tertiary transmission to vulnerable family members, also with atopic dermatitis. VHCN contacted the service member to reinforce vaccination site care and hygiene. This case underscores the importance of prevaccination education for those receiving the smallpox vaccine to protect contacts at risk for developing severe adverse reactions. Reprint & Copyright © 2013 Association of Military Surgeons of the U.S.

  2. ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense.

    Science.gov (United States)

    Monath, Thomas P; Caldwell, Joseph R; Mundt, Wolfgang; Fusco, Joan; Johnson, Casey S; Buller, Mark; Liu, Jian; Gardner, Bridget; Downing, Greg; Blum, Paul S; Kemp, Tracy; Nichols, Richard; Weltzin, Richard

    2004-10-01

    The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax.

  3. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway.

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    Peihong Dai

    2014-04-01

    Full Text Available Modified vaccinia virus Ankara (MVA is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs, which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs, but not in plasmacytoid dendritic cells (pDCs. Transcription factors IRF3 (IFN regulatory factor 3 and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1, are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase. MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1 and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.

  4. Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis

    International Nuclear Information System (INIS)

    Carrasco, L.; Bravo, R.

    1986-01-01

    The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with [ 3 H]glucosamine were detected in vaccinia-infected HeLa cells

  5. Mucosal immunization with PLGA-microencapsulated DNA primes a SIV-specific CTL response revealed by boosting with cognate recombinant modified vaccinia virus Ankara

    International Nuclear Information System (INIS)

    Sharpe, Sally; Hanke, Tomas; Tinsley-Bown, Anne; Dennis, Mike; Dowall, Stuart; McMichael, Andrew; Cranage, Martin

    2003-01-01

    Systemically administered DNA encoding a recombinant human immunodeficiency virus (HIV) derived immunogen effectively primes a cytotoxic T lymphocyte (CTL) response in macaques. In this further pilot study we have evaluated mucosal delivery of DNA as an alternative priming strategy. Plasmid DNA, pTH.HW, encoding a multi-CTL epitope gene, was incorporated into poly(D,L-lactic-co-glycolic acid) microparticles of less than 10 μm in diameter. Five intrarectal immunizations failed to stimulate a circulating vaccine-specific CTL response in 2 Mamu-A*01 + rhesus macaques. However, 1 week after intradermal immunization with a cognate modified vaccinia virus Ankara vaccine MVA.HW, CTL responses were detected in both animals that persisted until analysis postmortem, 12 weeks after the final boost. In contrast, a weaker and less durable response was seen in an animal vaccinated with the MVA construct alone. Analysis of lymphoid tissues revealed a disseminated CTL response in peripheral and regional lymph nodes but not the spleen of both mucosally primed animals

  6. Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

    Science.gov (United States)

    Liu, Liang; Cooper, Tamara; Eldi, Preethi; Garcia-Valtanen, Pablo; Diener, Kerrilyn R; Howley, Paul M; Hayball, John D

    2017-04-01

    Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

  7. Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

    Directory of Open Access Journals (Sweden)

    Jennifer H Stuart

    2016-12-01

    Full Text Available The type I interferon (IFN response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3 complex to the interferon stimulated response element (ISRE. Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

  8. Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

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    Leonie Unterholzner

    2011-09-01

    Full Text Available Recognition of viruses by pattern recognition receptors (PRRs causes interferon-β (IFN-β induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1 and IκB kinase-ε (IKKε, which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

  9. Modified Vaccinia Virus Ankara Vector Induces Specific Cellular and Humoral Responses in the Female Reproductive Tract, the Main HIV Portal of Entry.

    Science.gov (United States)

    Marlin, Romain; Nugeyre, Marie-Thérèse; Tchitchek, Nicolas; Parenti, Matteo; Hocini, Hakim; Benjelloun, Fahd; Cannou, Claude; Dereuddre-Bosquet, Nathalie; Levy, Yves; Barré-Sinoussi, Françoise; Scarlatti, Gabriella; Le Grand, Roger; Menu, Elisabeth

    2017-09-01

    The female reproductive tract (FRT) is one of the major mucosal invasion sites for HIV-1. This site has been neglected in previous HIV-1 vaccine studies. Immune responses in the FRT after systemic vaccination remain to be characterized. Using a modified vaccinia virus Ankara (MVA) as a vaccine model, we characterized specific immune responses in all compartments of the FRT of nonhuman primates after systemic vaccination. Memory T cells were preferentially found in the lower tract (vagina and cervix), whereas APCs and innate lymphoid cells were mainly located in the upper tract (uterus and fallopian tubes). This compartmentalization of immune cells in the FRT was supported by transcriptomic analyses and a correlation network. Polyfunctional MVA-specific CD8 + T cells were detected in the blood, lymph nodes, vagina, cervix, uterus, and fallopian tubes. Anti-MVA IgG and IgA were detected in cervicovaginal fluid after a second vaccine dose. Thus, systemic vaccination with an MVA vector elicits cellular and Ab responses in the FRT. Copyright © 2017 by The American Association of Immunologists, Inc.

  10. Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

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    Dolores Rodríguez

    Full Text Available With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs carrying the CD8(+ T cell epitope (SYVPSAEQI of the circumsporozoite (CS protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS, and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV vectors from the Western Reserve (WR and modified virus Ankara (MVA strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.

  11. Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

    Science.gov (United States)

    Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J Ignacio; Esteban, Mariano

    2012-01-01

    With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.

  12. A recombinant modified vaccinia ankara vaccine encoding Epstein-Barr Virus (EBV) target antigens: a phase I trial in UK patients with EBV-positive cancer.

    Science.gov (United States)

    Taylor, Graham S; Jia, Hui; Harrington, Kevin; Lee, Lip Wai; Turner, James; Ladell, Kristin; Price, David A; Tanday, Manjit; Matthews, Jen; Roberts, Claudia; Edwards, Ceri; McGuigan, Lesley; Hartley, Andrew; Wilson, Steve; Hui, Edwin P; Chan, Anthony T C; Rickinson, Alan B; Steven, Neil M

    2014-10-01

    Epstein-Barr virus (EBV) is associated with several cancers in which the tumor cells express EBV antigens EBNA1 and LMP2. A therapeutic vaccine comprising a recombinant vaccinia virus, MVA-EL, was designed to boost immunity to these tumor antigens. A phase I trial was conducted to demonstrate the safety and immunogenicity of MVA-EL across a range of doses. Sixteen patients in the United Kingdom (UK) with EBV-positive nasopharyngeal carcinoma (NPC) received three intradermal vaccinations of MVA-EL at 3-weekly intervals at dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming units (pfu). Blood samples were taken at screening, after each vaccine cycle, and during the post-vaccination period. T-cell responses were measured using IFNγ ELISpot assays with overlapping EBNA1/LMP2 peptide mixes or HLA-matched epitope peptides. Polychromatic flow cytometry was used to characterize functionally responsive T-cell populations. Vaccination was generally well tolerated. Immunity increased after vaccination to at least one antigen in 8 of 14 patients (7/14, EBNA1; 6/14, LMP2), including recognition of epitopes that vary between EBV strains associated with different ethnic groups. Immunophenotypic analysis revealed that vaccination induced differentiation and functional diversification of responsive T-cell populations specific for EBNA1 and LMP2 within the CD4 and CD8 compartments, respectively. MVA-EL is safe and immunogenic across diverse ethnicities and thus suitable for use in trials against different EBV-positive cancers globally as well as in South-East Asia where NPC is most common. The highest dose (5 × 10(8) pfu) is recommended for investigation in current phase IB and II trials. ©2014 American Association for Cancer Research.

  13. Structure, morphogenesis and function of tubular structures induced by cowpea mosaic virus

    NARCIS (Netherlands)

    Kasteel, D.T.J.

    1999-01-01

    During systemic plant infection, viruses move from the initially infected cells through plasmodesmata to neighbouring cells. Different mechanisms have been proposed for this cell-to-cell movement. Cowpea mosaic virus (CPMV) employs one of the major movement mechanisms, i.e. tubule-guided

  14. Outbreaks of vesicular disease caused by Vaccinia virus in dairy cattle from Goiás State, Brazil (2010-2012

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    Fabiano J.F. de Sant'Ana

    2013-07-01

    Full Text Available Cases of vesicular and exanthematic disease by Vaccinia virus (VACV have been reported in dairy herds of several Brazilian regions, occasionally also affecting humans. The present article describes eight outbreaks of vesicular disease caused by VACV in dairy herds of six counties of Goiás state, Midwestern Brazil (2010-2012, involving a total of 122 cows, 12 calves and 11 people. Dairy cows (3 to 9 years old were affected in all cases and calves (2 to 9 months old were affected in five outbreaks, presenting oral lesions. The morbidity ranged between 8 and 100% in cows, and 1.5 to 31% in calves. In the cows, the clinical signs started with vesicles (2-7mm, painful and coalescent papules (3-8 mm, which resulted in ulcers (5-25mm and scabs in teats, and, occasionally, in the muzzle. The clinical course lasted from 16 to 26 days. The histopathology of bovine skin samples revealed superficial perivascular inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, macrophages and multifocal areas of acanthosis, spongiosis, hipergranulosis and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers. Eosinophilic inclusion bodies were noted in the cytoplasm of the keratinocytes. PCR to vgf gene of Orthopoxvirus was positive in samples collected from all outbreaks, and in some cases, genomic VACV sequences were identified by nucleotide sequencing of the PCR amplicons. Infectious virus was isolated in cell culture from scabs from one outbreak. Antibodies to Orthopoxvirus were detected in at least 3 or 4 animals in most outbreaks, by ELISA (outbreaks 1, 2, 3, 4, 5 and 7 or virus-neutralization (outbreak 6. Neutralizing titers ranging from 8 to 64 in outbreak 6. In all outbreaks, VACV infection was suspected based on the clinical and pathological findings and it was confirmed by laboratory tests. Upon the etiological confirmation, other agents associated with vesicular disease were discarded. In all outbreaks, at least

  15. Anti-tumoral effect of recombinant vaccinia virus through US guided injection in a rabbit model of hepatic VX2 carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Jong Young; Park, Byeong Ho; Kang, Myong Jin; Cho, Jin Han; Choi, Jong Cheol; Choi, Sun Seob; Nam, Kyung Jin; Hwang, Tae Ho; Jeong, Jin Sook [College of Medicine, Dong-A University, Busan (Korea, Republic of)

    2006-02-15

    The purpose of this study was to evaluate the anti-tumoral effect of recombinant vaccinia virus (rVV) (Thymidine kinase (-)/GM-CSF (+)) that was administered as a US guided intratumoral injection in a rabbit model of hepatic VX2 carcinoma. VX2 carcinoma was implanted in the livers of 12 rabbits. US was performed at every week interval to detect hepatic mass after the implantation of VX2 carcinoma. The accurate tumor size and volume was evaluated with CT when the tumor was detected on US. US guided injection of rVV (10{sup 9} pfu/ml) was preformed in three rabbits, intravenous injection of the same dose of rVV was done in two rabbits and another seven rabbits that were without any treatment were selected as a control group. We evaluated the change of the hepatic tumor size and extrahepatic metastasis on serial CT. Tumor specimens were harvested from rabbits that were killed at 8 weeks after VX2 implantation. These tissues were histoimmuopathologically compared to each other (the virus injection group and the control group). The differences between these groups were statistically assessed with student t-tests. Tumor growth was significantly suppressed in the US guided injection group compared with the intravenous injection group or the control group ({rho} < 0.01). The intravenous injection group showed statistically significant tumor suppression compared to the control group ({rho} < 0.01) until 2 weeks after virus injection. Quantification of the pulmonary metastatic nodules was performed in view of both the number and volume. The average number or volume of the pulmonary metastatic nodules in the US injection group was much smaller than these in the control group. Histopathologically, the tumors of the US guided injection group showed less extensive necrosis than those of the control group. Immunohistochemically, the tumor of the US guided injection group showed more prominent infiltration of CD4 (+) and CD8 (+) lymphocytes than did the tumors of the other group

  16. Improved survival in rhesus macaques immunized with modified vaccinia virus Ankara recombinants expressing simian immunodeficiency virus envelope correlates with reduction in memory CD4+ T-cell loss and higher titers of neutralizing antibody.

    Science.gov (United States)

    Ourmanov, Ilnour; Kuwata, Takeo; Goeken, Robert; Goldstein, Simoy; Iyengar, Ranjani; Buckler-White, Alicia; Lafont, Bernard; Hirsch, Vanessa M

    2009-06-01

    Previous studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high viremia and AIDS following challenge with a pathogenic strain of SIV. Although all animals became infected, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines compared with animals that received nonrecombinant MVA. Most importantly, the reduction in viremia resulted in a significant increase in median and cumulative survival. Continued analysis of these animals over the subsequent 9 years has shown that they maintain a survival advantage, although all but two of the macaques have progressed to AIDS. Importantly, improved survival correlated with preservation of memory CD4(+) T cells in the peripheral blood. The greatest survival advantage was observed in macaques immunized with regimens containing SIV Env, and the titer of neutralizing antibodies to the challenge virus prior to or shortly following challenge correlated with preservation of CD4(+) T cells. These data are consistent with a role for neutralizing antibodies in nonsterilizing protection from high viremia and associated memory CD4(+) T-cell loss.

  17. Phase II trial of Modified Vaccinia Ankara (MVA virus expressing 5T4 and high dose Interleukin-2 (IL-2 in patients with metastatic renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Mitcham Josephine

    2009-01-01

    Full Text Available Abstract Background Interleukin-2 (IL-2 induces durable objective responses in a small cohort of patients with metastatic renal cell carcinoma (RCC but the antigen(s responsible for tumor rejection are not known. 5T4 is a non-secreted membrane glycoprotein expressed on clear cell and papillary RCCs. A modified vaccinia virus Ankara (MVA encoding 5T4 was tested in combination with high-dose IL-2 to determine the safety, objective response rate and effect on humoral and cell-mediated immunity. Methods 25 patients with metastatic RCC who qualified for IL-2 were eligible and received three immunizations every three weeks followed by IL-2 (600,000 IU/kg after the second and third vaccinations. Blood was collected for analysis of humoral, effector and regulatory T cell responses. Results There were no serious vaccine-related adverse events. While no objective responses were observed, three patients (12% were rendered disease-free after nephrectomy or resection of residual metastatic disease. Twelve patients (48% had stable disease which was associated with improved median overall survival compared to patients with progressive disease (not reached vs. 28 months, p = 0.0261. All patients developed 5T4-specific antibody responses and 13 patients had an increase in 5T4-specific T cell responses. Although the baseline frequency of Tregs was elevated in all patients, those with stable disease showed a trend toward increased effector CD8+ T cells and a decrease in Tregs. Conclusion Vaccination with MVA-5T4 did not improve objective response rates of IL-2 therapy but did result in stable disease associated with an increase in the ratio of 5T4-specific effector to regulatory T cells in selected patients. Trial registration number ISRCTN83977250

  18. Drosophila S2 cells are non-permissive for vaccinia virus DNA replication following entry via low pH-dependent endocytosis and early transcription.

    Directory of Open Access Journals (Sweden)

    Zain Bengali

    Full Text Available Vaccinia virus (VACV, a member of the chordopox subfamily of the Poxviridae, abortively infects insect cells. We have investigated VACV infection of Drosophila S2 cells, which are useful for protein expression and genome-wide RNAi screening. Biochemical and electron microscopic analyses indicated that VACV entry into Drosophila S2 cells depended on the VACV multiprotein entry-fusion complex but appeared to occur exclusively by a low pH-dependent endocytic mechanism, in contrast to both neutral and low pH entry pathways used in mammalian cells. Deep RNA sequencing revealed that the entire VACV early transcriptome, comprising 118 open reading frames, was robustly expressed but neither intermediate nor late mRNAs were made. Nor was viral late protein synthesis or inhibition of host protein synthesis detected by pulse-labeling with radioactive amino acids. Some reduction in viral early proteins was noted by Western blotting. Nevertheless, synthesis of the multitude of early proteins needed for intermediate gene expression was demonstrated by transfection of a plasmid containing a reporter gene regulated by an intermediate promoter. In addition, expression of a reporter gene with a late promoter was achieved by cotransfection of intermediate genes encoding the late transcription factors. The requirement for transfection of DNA templates for intermediate and late gene expression indicated a defect in viral genome replication in VACV-infected S2 cells, which was confirmed by direct analysis. Furthermore, VACV-infected S2 cells did not support the replication of a transfected plasmid, which occurs in mammalian cells and is dependent on all known viral replication proteins, indicating a primary restriction of DNA synthesis.

  19. Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

    Science.gov (United States)

    2016-08-01

    RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI 1. INTRODUCTION 1.1 Background Vaccinia virus (VACV) is the active component of the...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in...PURIFICATION AND CHARACTERIZATION OF RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI ECBC-TR-1370

  20. Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

    Science.gov (United States)

    Müllers, Erik; Uhlig, Tobias; Stirnnagel, Kristin; Fiebig, Uwe; Zentgraf, Hanswalter; Lindemann, Dirk

    2011-02-01

    Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

  1. The Central Role of the Matrix Protein in Nipah Virus Assembly and Morphogenesis

    Science.gov (United States)

    2007-03-23

    prepared and isolated by sucrose gradient floatation . VLPs from the resulting fractions were adsorbed to copper grids and immunolabeled using antibodies...Grosjean, M. C. Courbot, V. Deubel, R. Buckland, and T. F. Wild. 2006. Antibody prophylaxis and therapy against Nipah virus infection in hamsters. J Virol

  2. Immunogenicity of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma in vitro model: studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic T lymphocytes

    Directory of Open Access Journals (Sweden)

    Heinrich B

    2017-05-01

    Full Text Available B Heinrich,1 J Klein,1 M Delic,1 K Goepfert,1 V Engel,1 L Geberzahn,1 M Lusky,2 P Erbs,2 X Preville,3 M Moehler1 1First Department of Internal Medicine, University Medical Center Mainz, Mainz, Germany; 2Transgene SA, Illkirch-Graffenstaden, 3Amoneta Diagnostics, Huningue, France Abstract: Oncolytic virotherapy is an emerging immunotherapeutic modality for cancer treatment. Oncolytic viruses with genetic modifications can further enhance the oncolytic effects on tumor cells and stimulate antitumor immunity. The oncolytic vaccinia viruses JX-594-GFP+/hGM-CSF (JX-GFP and TG6002 are genetically modified by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF or transforming 5-fluorocytosine (5-FC into 5-fluorouracil (5-FU. We compared their properties to kill tumor cells and induce an immunogenic type of cell death in a human melanoma cell model using SK29-MEL melanoma cells. Their influence on human immune cells, specifically regarding the activation of dendritic cells (DCs and the interaction with the autologous cytotoxic T lymphocyte (CTL clone, was investigated. Melanoma cells were infected with either JX-GFP or TG6002 alone or in combination with 5-FC and 5-FU. The influence of viral infection on cell viability followed a time- and multiplicity of infection dependent manner. Combination of virus treatment with 5-FU resulted in stronger reduction of cell viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction of cell viability in this setting. Expression of calreticulin and high mobility group 1 protein (HMGB1, markers of immunogenic cell death (ICD, could be detected after viral infection. Accordingly, DC maturation was noted after viral oncolysis. DCs presented stronger expression of activation and maturation markers. The autologous CTL clone IVSB expressed the activation marker CD69, but viral treatment failed to enhance cytotoxicity marker. In summary, vaccinia viruses JX-GFP and TG6002 lyse

  3. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia

    International Nuclear Information System (INIS)

    Jumat, Muhammad Raihan; Yan, Yan; Ravi, Laxmi Iyer; Wong, Puisan; Huong, Tra Nguyen; Li, Chunwei; Tan, Boon Huan; Wang, De Yun; Sugrue, Richard J.

    2015-01-01

    The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function

  4. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia

    Energy Technology Data Exchange (ETDEWEB)

    Jumat, Muhammad Raihan [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Yan, Yan [Department of Otolaryngology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore 119228 (Singapore); Ravi, Laxmi Iyer [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Wong, Puisan [Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore 117510 (Singapore); Huong, Tra Nguyen [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Li, Chunwei [Department of Otolaryngology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore 119228 (Singapore); Tan, Boon Huan [Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore 117510 (Singapore); Wang, De Yun [Department of Otolaryngology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore 119228 (Singapore); Sugrue, Richard J., E-mail: rjsugrue@ntu.edu.sg [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2015-10-15

    The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function.

  5. A novel system for constructing a recombinant highly-attenuated vaccinia virus strain (LC16m8) expressing foreign genes and its application for the generation of LC16m8-based vaccines against herpes simplex virus 2.

    Science.gov (United States)

    Omura, Natsumi; Yoshikawa, Tomoki; Fujii, Hikaru; Shibamura, Miho; Inagaki, Takuya; Kato, Hirofumi; Egawa, Kazutaka; Harada, Shizuko; Yamada, Souichi; Takeyama, Haruko; Saijo, Masayuki

    2018-04-27

    A novel system was developed for generating a highly-attenuated vaccinia virus LC16m8 (m8, third generation smallpox vaccine) that expresses foreign genes. The innovations in this system are its excisable selection marker, specificity of the integration site of a gene of interest, and easy identification of clones with the fluorescent signal. Using this system, recombinant m8s, which expressed either herpes simplex virus 2 (HSV-2) glycoprotein B (gB)-, gD-, or both gB and gD (gB+gD) were developed, and their efficacy was evaluated. First, the induction of a specific IgG against these HSV-2 glycoproteins in mice infected with each of these recombinant m8s was confirmed with an immunofluorescence assay. Next, mice pre-infected with each of the recombinant m8s were infected with HSV-2 at the lethal dose to examine the vaccine efficacy. The fatality rate in mice pre-infected with either of the recombinant gB+gD- or gD-expressing m8s significantly decreased in comparison with that of the control. The survival rate in both male and female mice pre-infected with either of the recombinant gB+gD- and gD-expressing m8s increased to 100 % and 60 %, respectively, while most of the control mice died. In summary, this new system might be applicable for generating a novel m8-based vaccine.

  6. Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition.

    Science.gov (United States)

    Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

    2016-01-01

    We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro . Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8 + and CD4 + ). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

  7. In silico-accelerated identification of conserved and immunogenic variola/vaccinia T-cell epitopes

    DEFF Research Database (Denmark)

    Moise, Leonard; McMurry, Julie A; Buus, Søren

    2009-01-01

    Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted...

  8. Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus

    Directory of Open Access Journals (Sweden)

    Barik Sailen

    2003-05-01

    Full Text Available Abstract Background Actin is required for the gene expression and morphogenesis of respiratory syncytial virus (RSV, a clinically important Pneumovirus of the Paramyxoviridae family. In HEp-2 cells, RSV infection also induces actin stress fibers, which may be important in the immunopathology of the RSV disease. Profilin, a major regulator of actin polymerization, stimulates viral transcription in vitro. Thus, we tested the role of profilin in RSV growth and RSV-actin interactions in cultured cells (ex vivo. Results We tested three cell lines: HEp-2 (human, A549 (human, and L2 (rat. In all three, RSV grew well and produced fused cells (syncytium, and two RSV proteins, namely, the phosphoprotein P and the nucleocapsid protein N, associated with profilin. In contrast, induction of actin stress fibers by RSV occurred in HEp-2 and L2 cells, but not in A549. Knockdown of profilin by RNA interference had a small effect on viral macromolecule synthesis but strongly inhibited maturation of progeny virions, cell fusion, and induction of stress fibers. Conclusions Profilin plays a cardinal role in RSV-mediated cell fusion and viral maturation. In contrast, interaction of profilin with the viral transcriptional proteins P and N may only nominally activate viral RNA-dependent RNA polymerase. Stress fiber formation is a cell-specific response to infection, requiring profilin and perhaps other signaling molecules that are absent in certain cell lines. Stress fibers per se play no role in RSV replication in cell culture. Clearly, the cellular architecture controls multiple steps of host-RSV interaction, some of which are regulated by profilin.

  9. The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

    Science.gov (United States)

    Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

    2016-10-17

    Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  10. Diseño y construcción de vectores de transferencia para la obtención de virus vaccinia Ankara modificado (MVA recombinantes Design and construction of transfer vectors in order to obtain recombinant modified vaccinia virus Ankara (MVA

    Directory of Open Access Journals (Sweden)

    M. F. Ferrer

    2007-09-01

    Full Text Available El virus vaccinia Ankara modificado (MVA constituye un buen candidato para el desarrollo de vectores virales de expresión no replicativos porque no replica en la mayoría de las células de mamíferos. Para la producción de MVA recombinantes es fundamental disponer de vectores de transferencia que, por recombinación homóloga con el genoma viral, permitan introducir los genes de interés en regiones no esenciales para la replicación in vitro. En este trabajo se diseñaron y obtuvieron los vectores de transferencia denominados VT-MHA y VT-MTK que portan las regiones correspondientes a las posiciones 1-303 y 608-948 del gen MVA165R y 1-244 y 325-534 del gen MVA086R, respectivamente, las que flanquean un sitio de clonado múltiple para la inserción de los genes foráneos. En dichos vectores se clonaron los casetes para la expresión de los genes lac Z o uid A, y la actividad de las enzimas marcadoras b-galactosidasa y b-glucuronidasa se confirmó in situ. Además, utilizando el vector denominado VT-MTK-GUS, se obtuvieron y aislaron MVA recombinantes puros que portan y expresan el gen uid A. Los resultados obtenidos constituyen las herramientas básicas para establecer la metodología de obtención de MVA recombinantes, con el propósito de desarrollar localmente vectores virales no replicativos candidatos a vacunas.Modified Vaccinia virus Ankara (MVA constitutes a good candidate for the development of non-replicative expression viral vectors because it does not replicate in most of mammalian cells. It is essential, for the production of recombinant MVA, the availability of transfer vectors which allow the introduction of desired genes into non-essential regions for in vitro viral replication, by homologous recombination with the viral genome. In the present work, the transfer vectors named VT-MHA and VT-MTK were designed and obtained. They carried genomic regions corresponding to 1- 303 and 608-948 positions of the MVA165R gene and 1-244 and

  11. 42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

    Science.gov (United States)

    2010-10-01

    ... inflammatory cells in the dermis of the skin at the vaccination or inoculation site. The diagnosis of PV may be... the mother that results from the placental transmission of the vaccinia virus during any time in the... membrane lesion containing an accumulation of white blood cells. (8) Recipient means a person to whom the...

  12. Oncolytic vaccinia therapy of squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Yu Yong A

    2009-07-01

    Full Text Available Abstract Background Novel therapies are necessary to improve outcomes for patients with squamous cell carcinomas (SCC of the head and neck. Historically, vaccinia virus was administered widely to humans as a vaccine and led to the eradication of smallpox. We examined the therapeutic effects of an attenuated, replication-competent vaccinia virus (GLV-1h68 as an oncolytic agent against a panel of six human head and neck SCC cell lines. Results All six cell lines supported viral transgene expression (β-galactosidase, green fluorescent protein, and luciferase as early as 6 hours after viral exposure. Efficient transgene expression and viral replication (>150-fold titer increase over 72 hrs were observed in four of the cell lines. At a multiplicity of infection (MOI of 1, GLV-1h68 was highly cytotoxic to the four cell lines, resulting in ≥ 90% cytotoxicity over 6 days, and the remaining two cell lines exhibited >45% cytotoxicity. Even at a very low MOI of 0.01, three cell lines still demonstrated >60% cell death over 6 days. A single injection of GLV-1h68 (5 × 106 pfu intratumorally into MSKQLL2 xenografts in mice exhibited localized intratumoral luciferase activity peaking at days 2–4, with gradual resolution over 10 days and no evidence of spread to normal organs. Treated animals exhibited near-complete tumor regression over a 24-day period without any observed toxicity, while control animals demonstrated rapid tumor progression. Conclusion These results demonstrate significant oncolytic efficacy by an attenuated vaccinia virus for infecting and lysing head and neck SCC both in vitro and in vivo, and support its continued investigation in future clinical trials.

  13. Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

    Science.gov (United States)

    Liu, Shuchang; Ruban, Ludmila; Wang, Yaohe; Zhou, Yuhong; Nesbeth, Darren N

    2017-02-01

    Vaccinia virus (VACV) is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h -1 and 0.044 h -1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h -1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

  14. Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells

    Directory of Open Access Journals (Sweden)

    Shuchang Liu

    2017-02-01

    Full Text Available Vaccinia virus (VACV is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS Dulbecco’s Modified Eagle Medium (DMEM were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h−1 and 0.044 h−1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h−1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

  15. Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters

    Directory of Open Access Journals (Sweden)

    Laura Fernández

    2018-04-01

    Full Text Available Leishmania-activated C-kinase antigen (LACK is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65 expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL. Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to Leishmania infantum (L. infantum or induce protection in L. infantum-infected hamsters [visceral leishmaniasis (VL model], has not yet been analyzed. The present work examines the ex vivo immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an L. infantum area. It also evaluates the vaccine potential of LACK against L. infantum infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response. Further, 78% of PBMC samples that responded to soluble Leishmania antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of Leishmania DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with L. infantum, and the parasite protective effect of prime/boost vaccination in hamsters with DNA

  16. The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells

    DEFF Research Database (Denmark)

    Jensen, Helle Lone; Norrild, Bodil

    2003-01-01

    Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus...

  17. Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase I trial.

    Directory of Open Access Journals (Sweden)

    Emma-Jo Hayton

    Full Text Available HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported.Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination.Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1 and predominantly transient (<48 hours. Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range of 633 (231-1533 post-vaccination, which is of no safety concern.These data demonstrate safety and good tolerability of the pSG2

  18. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

    Science.gov (United States)

    Dubrau, Danilo; Tortorici, M Alejandra; Rey, Félix A; Tautz, Norbert

    2017-02-01

    The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

  19. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

    Science.gov (United States)

    Rey, Félix A.

    2017-01-01

    The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. PMID:28151973

  20. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

    Directory of Open Access Journals (Sweden)

    Danilo Dubrau

    2017-02-01

    Full Text Available The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132, which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

  1. Pre-Clinical Efficacy and Safety of Experimental Vaccines Based on Non-Replicating Vaccinia Vectors against Yellow Fever

    Science.gov (United States)

    Schäfer, Birgit; Holzer, Georg W.; Joachimsthaler, Alexandra; Coulibaly, Sogue; Schwendinger, Michael; Crowe, Brian A.; Kreil, Thomas R.; Barrett, P. Noel; Falkner, Falko G.

    2011-01-01

    Background Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. Methodology/Principal Findings A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 105 TCID50. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. Conclusions/Significance The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice. PMID:21931732

  2. Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever.

    Directory of Open Access Journals (Sweden)

    Birgit Schäfer

    Full Text Available BACKGROUND: Currently existing yellow fever (YF vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D. Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. METHODOLOGY/PRINCIPAL FINDINGS: A gene encoding the precursor of the membrane and envelope (prME protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10(5 TCID(50. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. CONCLUSIONS/SIGNIFICANCE: The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.

  3. Development of a novel, guinea pig-specific IFN-γ ELISPOT assay and characterization of guinea pig cytomegalovirus GP83-specific cellular immune responses following immunization with a modified vaccinia virus Ankara (MVA)-vectored GP83 vaccine.

    Science.gov (United States)

    Gillis, Peter A; Hernandez-Alvarado, Nelmary; Gnanandarajah, Josephine S; Wussow, Felix; Diamond, Don J; Schleiss, Mark R

    2014-06-30

    The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Clustered epitopes within the Gag-Pol fusion protein DNA vaccine enhance immune responses and protection against challenge with recombinant vaccinia viruses expressing HIV-1 Gag and Pol antigens

    International Nuclear Information System (INIS)

    Bolesta, Elizabeth; Gzyl, Jaroslaw; Wierzbicki, Andrzej; Kmieciak, Dariusz; Kowalczyk, Aleksandra; Kaneko, Yutaro; Srinivasan, Alagarsamy; Kozbor, Danuta

    2005-01-01

    We have generated a codon-optimized hGagp17p24-Polp51 plasmid DNA expressing the human immunodeficiency virus type 1 (HIV-1) Gag-Pol fusion protein that consists of clusters of highly conserved cytotoxic T lymphocyte (CTL) epitopes presented by multiple MHC class I alleles. In the hGagp17p24-Polp51 construct, the ribosomal frameshift site had been deleted together with the potentially immunosuppressive Gag nucleocapsid (p15) as well as Pol protease (p10) and integrase (p31). Analyses of the magnitude and breadth of cellular responses demonstrated that immunization of HLA-A2/K b transgenic mice with the hGagp17p24-Polp51 construct induced 2- to 5-fold higher CD8 + T-cell responses to Gag p17-, p24-, and Pol reverse transcriptase (RT)-specific CTL epitopes than the full-length hGag-PolΔFsΔPr counterpart. The increases were correlated with higher protection against challenge with recombinant vaccinia viruses (rVVs) expressing gag and pol gene products. Consistent with the profile of Gag- and Pol-specific CD8 + T cell responses, an elevated level of type 1 cytokine production was noted in p24- and RT-stimulated splenocyte cultures established from hGagp17p24-Polp51-immunized mice compared to responses induced with the hGag-PolΔFsΔPr vaccine. Sera of mice immunized with the hGagp17p24-Polp51 vaccine also exhibited an increased titer of p24- and RT-specific IgG2 antibody responses. The results from our studies provide insights into approaches for boosting the breadth of Gag- and Pol-specific immune responses

  5. Combined Cytolytic Effects of a Vaccinia Virus Encoding a Single Chain Trimer of MHC-I with a Tax-Epitope and Tax-Specific CTLs on HTLV-I-Infected Cells in a Rat Model

    Directory of Open Access Journals (Sweden)

    Takashi Ohashi

    2014-01-01

    Full Text Available Adult T cell leukemia (ATL is a malignant lymphoproliferative disease caused by human T cell leukemia virus type I (HTLV-I. To develop an effective therapy against the disease, we have examined the oncolytic ability of an attenuated vaccinia virus (VV, LC16m8Δ (m8Δ, and an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL line, 4O1/C8, against an HTLV-I-infected rat T cell line, FPM1. Our results demonstrated that m8Δ was able to replicate in and lyse tumorigenic FPM1 cells but was incompetent to injure 4O1/C8 cells, suggesting the preferential cytolytic activity toward tumor cells. To further enhance the cytolysis of HTLV-I-infected cells, we modified m8Δ and obtained m8Δ/RT1AlSCTax180L, which can express a single chain trimer (SCT of rat major histocompatibility complex class I with a Tax-epitope. Combined treatment with m8Δ/RT1AlSCTax180L and 4O1/C8 increased the cytolysis of FPM1V.EFGFP/8R cells, a CTL-resistant subclone of FPM1, compared with that using 4O1/C8 and m8Δ presenting an unrelated peptide, suggesting that the activation of 4O1/C8 by m8Δ/RT1AlSCTax180L further enhanced the killing of the tumorigenic HTLV-I-infected cells. Our results indicate that combined therapy of oncolytic VVs with SCTs and HTLV-I-specific CTLs may be effective for eradication of HTLV-I-infected cells, which evade from CTL lysis and potentially develop ATL.

  6. Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

    International Nuclear Information System (INIS)

    Moore, Michael D.; Fu, William; Soheilian, Ferri; Nagashima, Kunio; Ptak, Roger G.; Pathak, Vinay K.; Hu, Wei-Shau

    2008-01-01

    Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC 90 ), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation

  7. L1R, A27L, A33R and B5R vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines

    OpenAIRE

    Pacchioni, Sole Maria; Bissa, Massimiliano; Zanotto, Carlo; Morghen, Carlo De Giuli; Illiano, Elena; Radaelli, Antonia

    2013-01-01

    Background The traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. However, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. Preventive vaccination still remains the most e...

  8. Frequency of adverse events after vaccination with different vaccinia strains.

    Directory of Open Access Journals (Sweden)

    Mirjam Kretzschmar

    2006-08-01

    Full Text Available BACKGROUND: Large quantities of smallpox vaccine have been stockpiled to protect entire nations against a possible reintroduction of smallpox. Planning for an appropriate use of these stockpiled vaccines in response to a smallpox outbreak requires a rational assessment of the risks of vaccination-related adverse events, compared to the risk of contracting an infection. Although considerable effort has been made to understand the dynamics of smallpox transmission in modern societies, little attention has been paid to estimating the frequency of adverse events due to smallpox vaccination. Studies exploring the consequences of smallpox vaccination strategies have commonly used a frequency of approximately one death per million vaccinations, which is based on a study of vaccination with the New York City Board of Health (NYCBH strain of vaccinia virus. However, a multitude of historical studies of smallpox vaccination with other vaccinia strains suggest that there are strain-related differences in the frequency of adverse events after vaccination. Because many countries have stockpiled vaccine based on the Lister strain of vaccinia virus, a quantitative evaluation of the adverse effects of such vaccines is essential for emergency response planning. We conducted a systematic review and statistical analysis of historical data concerning vaccination against smallpox with different strains of vaccinia virus. METHODS AND FINDINGS: We analyzed historical vaccination data extracted from the literature. We extracted data on the frequency of postvaccinal encephalitis and death with respect to vaccinia strain and age of vaccinees. Using a hierarchical Bayesian approach for meta-analysis, we estimated the expected frequencies of postvaccinal encephalitis and death with respect to age at vaccination for smallpox vaccines based on the NYCBH and Lister vaccinia strains. We found large heterogeneity between findings from different studies and a time-period effect

  9. Safety and immunogenicity of a modified-vaccinia-virus-Ankara-based influenza A H5N1 vaccine: a randomised, double-blind phase 1/2a clinical trial.

    Science.gov (United States)

    Kreijtz, Joost H C M; Goeijenbier, Marco; Moesker, Fleur M; van den Dries, Lennert; Goeijenbier, Simone; De Gruyter, Heidi L M; Lehmann, Michael H; Mutsert, Gerrie de; van de Vijver, David A M C; Volz, Asisa; Fouchier, Ron A M; van Gorp, Eric C M; Rimmelzwaan, Guus F; Sutter, Gerd; Osterhaus, Albert D M E

    2014-12-01

    Modified vaccinia virus Ankara (MVA) is a promising viral vector platform for the development of an H5N1 influenza vaccine. Preclinical assessment of MVA-based H5N1 vaccines showed their immunogenicity and safety in different animal models. We aimed to assess the safety and immunogenicity of the MVA-haemagglutinin-based H5N1 vaccine MVA-H5-sfMR in healthy individuals. In a single-centre, double-blind phase 1/2a study, young volunteers (aged 18-28 years) were randomly assigned with a computer-generated list in equal numbers to one of eight groups and were given one injection or two injections intramuscularly at an interval of 4 weeks of a standard dose (10(8) plaque forming units [pfu]) or a ten times lower dose (10(7) pfu) of the MVA-H5-sfMR (vector encoding the haemagglutinin gene of influenza A/Vietnam/1194/2004 virus [H5N1 subtype]) or MVA-F6-sfMR (empty vector) vaccine. Volunteers and physicians who examined and administered the vaccine were masked to vaccine assignment. Individuals who received the MVA-H5-sfMR vaccine were eligible for a booster immunisation 1 year after the first immunisation. Primary endpoint was safety. Secondary outcome was immunogenicity. The trial is registered with the Dutch Trial Register, number NTR3401. 79 of 80 individuals who were enrolled completed the study. No serious adverse events were identified. 11 individuals reported severe headache and lightheadedness, erythema nodosum, respiratory illness (accompanied by influenza-like symptoms), sore throat, or injection-site reaction. Most of the volunteers had one or more local (itch, pain, redness, and swelling) and systemic reactions (rise in body temperature, headache, myalgia, arthralgia, chills, malaise, and fatigue) after the first, second, and booster immunisations. Individuals who received the 10(7) dose had fewer systemic reactions. The MVA-H5-sfMR vaccine at 10(8) pfu induced significantly higher antibody responses after one and two immunisations than did 10(7) pfu when

  10. Features of the Env leader protein and the N-terminal Gag domain of feline foamy virus important for virus morphogenesis

    International Nuclear Information System (INIS)

    Geiselhart, Verena; Schwantes, Astrid; Bastone, Patrizia; Frech, Matthias; Loechelt, Martin

    2003-01-01

    Previous studies have shown that foamy virus (FV) particle budding, especially the involvement of the viral Env glycoprotein, is different from that of other (ortho) retroviruses: the N-terminal Env leader protein Elp is a constituent of released FV particles. A defined sequence in Elp required for particle budding binds to the MA domain of Gag. To extend these findings, we show that feline FV Elp is a membrane-anchored protein with the N-terminus located inside the particle. Thus, the internal/cytoplasmic domain of Elp has the correct topology for interacting with Gag during budding. In addition to Elp, an Elp-related protein of about 9 kDa was shown to be virion associated and is probably generated by cellular signal peptidases. Besides the function of Elp binding, the N-terminal domain of Gag was shown to be required for proper localization of feline FV Gag to the cytoplasm and the perinuclear/nuclear region

  11. Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.

    Directory of Open Access Journals (Sweden)

    Hua Cao

    Full Text Available Plasmacytoid dendritic cells (pDCs play important roles in antiviral innate immunity by producing type I interferon (IFN. In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i vaccinia virus, but not myxoma virus, expresses inhibitor(s of the poxvirus sensing pathway(s in pDCs; and (ii Heat-VAC infection fails to produce inhibitor(s but rather produces novel activator(s, likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029 lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating

  12. Innate Immune Response of Human Plasmacytoid Dendritic Cells to Poxvirus Infection Is Subverted by Vaccinia E3 via Its Z-DNA/RNA Binding Domain

    Science.gov (United States)

    Dai, Peihong; Wang, Weiyi; Li, Hao; Yuan, Jianda; Wang, Fangjin; Fang, Chee-Mun; Pitha, Paula M; Liu, Jia; Condit, Richard C; McFadden, Grant; Merghoub, Taha; Houghton, Alan N; Young, James W; Shuman, Stewart; Deng, Liang

    2012-01-01

    Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of

  13. Guinea pigs experimentally infected with vaccinia virus replicate and shed, but do not transmit the virus Cobaias infectadas experimentalmente com vírus vaccínia replicam e excretam, porém não transmitem o vírus

    Directory of Open Access Journals (Sweden)

    Juliana Felipetto Cargnelutti

    2012-06-01

    Full Text Available The origin of vaccinia viruses (VACV associated with vesicular disease in cattle and humans in Southeast Brazil remains uncertain, yet the role of wild species in virus transmission has been suggested. This study investigated the susceptibility and transmission potential by guinea pigs (Cavia porcellus - phylogenetically close to an abundant Brazilian rodent (Cavia aperea - to two VACV strains (P1V and P2V isolated from an outbreak of cutaneous disease in horses in Southern Brazil. Eight guinea pigs inoculated intranasally with P1V and P2V (10(6 TCID50.ml-1 did not develop clinical signs, but six animals shed virus in nasal secretions (day 1 to 9 post-inoculation - pi, developed viremia (between days 1 and 10 pi and seroconverted to VACV. In spite of virus replication and shedding, the virus was not transmitted to sentinel animals by direct or indirect contact (aerosols or through food and water contaminated with virus. These results demonstrate that, in spite of replicating and shedding the virus, guinea pigs do not transmit the virus upon experimental inoculation. This finding makes unlikely a possible participation of related species in VACV maintenance and transmission in nature.A origem dos vírus vaccínia (VACV, envolvidos em surtos de doença vesicular em bovinos e humanos no Sudeste do Brasil, permanece desconhecida, e a participação de espécies silvestres na manutenção e transmissão do vírus tem sido sugerida. O objetivo deste trabalho foi investigar a susceptibilidade e o potencial de transmissão por cobaias (Cavia porcellus - filogeneticamente relacionada a uma espécie de roedor, conhecido por preá (Cavia aperea, bastante abundante no país - a duas cepas de VACV (P1V e P2V isoladas de um surto de doença cutânea em equinos no Rio Grande do Sul. Oito cobaias inoculadas pela via intranasal com uma mistura das amostras P1V e P2V (10(6 DICC50.ml-1 não apresentaram sinais clínicos, porém seis animais excretaram o vírus nas

  14. Development and evaluation of single domain antibodies for vaccinia and the L1 antigen.

    Directory of Open Access Journals (Sweden)

    Scott A Walper

    Full Text Available There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10(-9 M to 7.0×10(-10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×10(5 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.

  15. Humoral Immunity to Primary Smallpox Vaccination: Impact of Childhood versus Adult Immunization on Vaccinia Vector Vaccine Development in Military Populations.

    Directory of Open Access Journals (Sweden)

    Bonnie M Slike

    Full Text Available Modified Vaccinia virus has been shown to be a safe and immunogenic vector platform for delivery of HIV vaccines. Use of this vector is of particular importance to the military, with the implementation of a large scale smallpox vaccination campaign in 2002 in active duty and key civilian personnel in response to potential bioterrorist activities. Humoral immunity to smallpox vaccination was previously shown to be long lasting (up to 75 years and protective. However, using vaccinia-vectored vaccine delivery for other diseases on a background of anti-vector antibodies (i.e. pre-existing immunity may limit their use as a vaccine platform, especially in the military. In this pilot study, we examined the durability of vaccinia antibody responses in adult primary vaccinees in a healthy military population using a standard ELISA assay and a novel dendritic cell neutralization assay. We found binding and neutralizing antibody (NAb responses to vaccinia waned after 5-10 years in a group of 475 active duty military, born after 1972, who were vaccinated as adults with Dryvax®. These responses decreased from a geometric mean titer (GMT of 250 to baseline (30 years with a GMT of 210 (range 112-3234. This data suggests limited durability of antibody responses in adult vaccinees compared to those vaccinated in childhood and further that adult vaccinia recipients may benefit similarly from receipt of a vaccinia based vaccine as those who are vaccinia naïve. Our findings may have implications for the smallpox vaccination schedule and support the ongoing development of this promising viral vector in a military vaccination program.

  16. Ascidian notochord morphogenesis

    OpenAIRE

    Jiang, Di; Smith, William C.

    2007-01-01

    The development of the notochord involves a complex set of cellular behaviors. While these morphogenic behaviors are common to all chordates, the ascidian provides a particularly attractive experimental model because of its relative simplicity. In particular, all notochord morphogenesis in ascidians takes place with only 40 cells, as opposed to the hundreds of cells in vertebrate models systems. Initial steps in ascidian notochord development convert a monolayer of epithelial-like cells in th...

  17. Sea Urchin Morphogenesis.

    Science.gov (United States)

    McClay, David R

    2016-01-01

    In the sea urchin morphogenesis follows extensive molecular specification. The specification controls the many morphogenetic events and these, in turn, precede patterning steps that establish the larval body plan. To understand how the embryo is built it was necessary to understand those series of molecular steps. Here an example of the historical sequence of those discoveries is presented as it unfolded over the last 50 years, the years during which major progress in understanding development of many animals and plants was documented by CTDB. In sea urchin development a rich series of experimental studies first established many of the phenomenological components of skeletal morphogenesis and patterning without knowledge of the molecular components. The many discoveries of transcription factors, signals, and structural proteins that contribute to the shape of the endoskeleton of the sea urchin larva then followed as molecular tools became available. A number of transcription factors and signals were discovered that were necessary for specification, morphogenesis, and patterning. Perturbation of the transcription factors and signals provided the means for assembling models of the gene regulatory networks used for specification and controlled the subsequent morphogenetic events. The earlier experimental information informed perturbation experiments that asked how patterning worked. As a consequence it was learned that ectoderm provides a series of patterning signals to the skeletogenic cells and as a consequence the skeletogenic cells secrete a highly patterned skeleton based on their ability to genotypically decode the localized reception of several signals. We still do not understand the complexity of the signals received by the skeletogenic cells, nor do we understand in detail how the genotypic information shapes the secreted skeletal biomineral, but the current knowledge at least outlines the sequence of events and provides a useful template for future

  18. Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

    Energy Technology Data Exchange (ETDEWEB)

    Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.; Moore, Ronald J.; Clauss, Therese RW; Monroe, Matthew E.; Du, Xiuxia; Adkins, Joshua N.; Wong, Scott; Smith, Richard D.

    2008-03-07

    Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® and X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.

  19. Molecular network, pathway, and functional analysis of time-dependent gene changes associated with pancreatic cancer susceptibility to oncolytic vaccinia virotherapy

    Directory of Open Access Journals (Sweden)

    Dana Haddad

    2016-01-01

    Conclusions: Our study reveals the ability to assess time-dependent changes in gene expression patterns in pancreatic cancer cells associated with infection and susceptibility to vaccinia viruses. This suggests that molecular assays may be useful to develop safer and more efficacious oncolyticvirotherapies and support the idea that these treatments may target pathways implicated in pancreatic cancer resistance to conventional therapies.

  20. The morphogenesis of feathers.

    Science.gov (United States)

    Yu, Mingke; Wu, Ping; Widelitz, Randall B; Chuong, Cheng-Ming

    2002-11-21

    Feathers are highly ordered, hierarchical branched structures that confer birds with the ability of flight. Discoveries of fossilized dinosaurs in China bearing 'feather-like' structures have prompted interest in the origin and evolution of feathers. However, there is uncertainty about whether the irregularly branched integumentary fibres on dinosaurs such as Sinornithosaurus are truly feathers, and whether an integumentary appendage with a major central shaft and notched edges is a non-avian feather or a proto-feather. Here, we use a developmental approach to analyse molecular mechanisms in feather-branching morphogenesis. We have used the replication-competent avian sarcoma retrovirus to deliver exogenous genes to regenerating flight feather follicles of chickens. We show that the antagonistic balance between noggin and bone morphogenetic protein 4 (BMP4) has a critical role in feather branching, with BMP4 promoting rachis formation and barb fusion, and noggin enhancing rachis and barb branching. Furthermore, we show that sonic hedgehog (Shh) is essential for inducing apoptosis of the marginal plate epithelia, which results in spaces between barbs. Our analyses identify the molecular pathways underlying the topological transformation of feathers from cylindrical epithelia to the hierarchical branched structures, and provide insights on the possible developmental mechanisms in the evolution of feather forms.

  1. Perithecium morphogenesis in Sordaria macrospora.

    Science.gov (United States)

    Lord, Kathryn M; Read, Nick D

    2011-04-01

    The perithecium of the self-fertile ascomycete Sordaria macrospora provides an excellent model in which to analyse fungal multicellular development. This study provides a detailed analysis of perithecium morphogenesis in the wild type and eight developmental mutants of S. macrospora, using a range of correlative microscopical techniques. Fundamentally, perithecia and other complex multicellular structures produced by fungi arise by hyphal aggregation and adhesion, and these processes are followed by specialization and septation of hyphal compartments within the aggregates. Perithecial morphogenesis can be divided into the ascogonial, protoperithecial, and perithecial stages of development. At least 13 specialized, morphologically distinct cell-types are involved in perithecium morphogenesis, and these fall into three basic classes: hyphae, conglutinate cells and spores. Conglutinate cells arise from hyphal adhesion and certain perithecial hyphae develop from conglutinate cells. Various hypha-conglutinate cell transitions play important roles during the development of the perithecial wall and neck. Copyright © 2010. Published by Elsevier Inc.

  2. Evaluating anti-Orthopoxvirus antibodies in individuals from Brazilian rural areas prior to the bovine vaccinia era

    Directory of Open Access Journals (Sweden)

    Poliana de Oliveira Figueiredo

    2015-09-01

    Full Text Available Vaccinia virus naturally circulates in Brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (BV. We retrospectively evaluated two populations from the Amazon and Southeast Regions. BV outbreaks had not been reported in these regions before sample collection. Neutralising antibodies were found in 13 individuals (n = 132 with titres ranging from 100 ≥ 6,400 neutralising units/mL. Univariate analysis identified age and vaccination as statistically significant risk factors in individuals from the Southeast Region. The absence of detectable antibodies in vaccinated individuals raises questions about the protection of smallpox vaccine years after vaccination and reinforces the need for surveillance of Orthopoxvirus in Brazilian populations without evidence of previous outbreaks.

  3. Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization

    International Nuclear Information System (INIS)

    Moussatche, Nissin; Condit, Richard C.

    2015-01-01

    The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion

  4. ATP-independent DNA synthesis in Vaccinia-infected L cells

    International Nuclear Information System (INIS)

    Berger, N.A.; Kauff, R.A.; Sikorski, G.W.

    1978-01-01

    Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl 2 . DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes. (Auth.)

  5. Transforming growth factor alpha, Shope fibroma growth factor, and vaccinia growth factor can replace myxoma growth factor in the induction of myxomatosis in rabbits.

    Science.gov (United States)

    Opgenorth, A; Nation, N; Graham, K; McFadden, G

    1993-02-01

    The epidermal growth factor (EGF) homologues encoded by vaccinia virus, myxoma virus, and malignant rabbit fibroma virus have been shown to contribute to the pathogenicity of virus infection upon inoculation of susceptible hosts. However, since the primary structures of these growth factors and the disease profiles induced by different poxvirus genera vary substantially, the degree to which the various EGF homologues perform similar roles in viral pathogenesis remains unclear. In order to determine whether different EGF-like growth factors can perform qualitatively similar functions in the induction of myxomatosis in rabbits, we created recombinant myxoma virus variants in which the native growth factor, myxoma growth factor (MGF), was disrupted and replaced with either vaccinia virus growth factor, Shope fibroma growth factor, or rat transforming growth factor alpha. Unlike the control virus containing an inactivated MGF gene, which caused marked attenuation of the disease syndrome and substantially less proliferation of the epithelial cell layers in the conjunctiva and respiratory tract, the recombinant myxoma virus strains expressing heterologous growth factors produced infections which were both clinically and histopathologically indistinguishable from wild-type myxomatosis. We conclude that these poxviral and cellular EGF-like growth factors, which are diverse with respect to primary structure and origin, have similar biological functions in the context of myxoma virus pathogenesis and are mitogenic for the same target cells.

  6. Binding of glutathione to enterovirus capsids is essential for virion morphogenesis.

    NARCIS (Netherlands)

    Thibaut, H.J.; Linden, L. van der; Jiang, P.; Thys, B.; Canela, M.D.; Aguado, L.; Rombaut, B.; Wimmer, E.; Paul, A.; Perez-Perez, M.J.; Kuppeveld, F.J.M. van; Neyts, J.

    2014-01-01

    Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We

  7. Binding of glutathione to enterovirus capsids is essential for virion morphogenesis

    NARCIS (Netherlands)

    Thibaut, Hendrik Jan; van der Linden, Lonneke; Jiang, Ping; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Rombaut, Bart; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J M; Neyts, Johan

    Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We

  8. Surveillance guidelines for smallpox vaccine (vaccinia) adverse reactions.

    Science.gov (United States)

    Casey, Christine; Vellozzi, Claudia; Mootrey, Gina T; Chapman, Louisa E; McCauley, Mary; Roper, Martha H; Damon, Inger; Swerdlow, David L

    2006-02-03

    CDC and the U.S. Food and Drug Administration rely on state and local health departments, health-care providers, and the public to report the occurrence of adverse events after vaccination to the Vaccine Adverse Event Reporting System. With such data, trends can be accurately monitored, unusual occurrences of adverse events can be detected, and the safety of vaccination intervention activities can be evaluated. On January 24, 2003, the U.S. Department of Health and Human Services (DHHS) implemented a preparedness program in which smallpox (vaccinia) vaccine was administered to federal, state, and local volunteers who might be first responders during a biologic terrorism event. As part of the DHHS Smallpox Preparedness and Response Program, CDC in consultation with experts, established surveillance case definitions for adverse events after smallpox vaccination. Adverse reactions after smallpox vaccination identified during the 1960s surveillance activities were classified on the basis of clinical description and included eczema vaccinatum; fetal vaccinia; generalized vaccinia; accidental autoinoculation, nonocular; ocular vaccinia; progressive vaccinia; erythema multiforme major; postvaccinial encephalitis or encephalomyelitis; and pyogenic infection of the vaccination site. This report provides uniform criteria used for the surveillance case definition and classification for these previously recognized adverse reactions used during the DHHS Smallpox Preparedness and Response Program. Inadvertent inoculation was changed to more precisely describe this event as inadvertent autoinoculation and contact transmission, nonocular and ocular vaccinia. Pyogenic infection also was renamed superinfection of the vaccination site or regional lymph nodes. Finally, case definitions were developed for a new cardiac adverse reaction (myo/pericarditis) and for a cardiac adverse event (dilated cardiomyopathy) and are included in this report. The smallpox vaccine surveillance case

  9. Factors influencing the vaccinia-specific cytotoxic response of thymocytes from normal and chimeric mice

    International Nuclear Information System (INIS)

    Doherty, P.C.; Schwartz, D.H.; Bennink, J.R.; Korngold, R.

    1981-01-01

    Following adoptive transfer into irradiated recipients, thymocytes can be induced to respond strongly to vaccinia virus. High levels of cytotoxic T-lymphocyte (CTL) activity may be generated from thymus, but not from spleen, of 3-day-old mice. The capacity of thymocytes to differentiate into effector CTL tends to be lost with age. Some of this loss may reflect positive suppression: a single, low dose of cyclophosphamide allows the reemergence of responsiveness in at least one mouse strain. Thymocytes from [A leads to (A x B)F1] and [(A x B)F1 leads to A] chimeras show the response patterns that would by predicted from previous studies of lymph node and spleen cells. However, thymic function seems to be rapidly lost in the [A leads to (A x B)F1] Chimeras

  10. Smallpox virus plaque phenotypes: genetic, geographical and case fatality relationships.

    Science.gov (United States)

    Olson, Victoria A; Karem, Kevin L; Smith, Scott K; Hughes, Christine M; Damon, Inger K

    2009-04-01

    Smallpox (infection with Orthopoxvirus variola) remains a feared illness more than 25 years after its eradication. Historically, case-fatality rates (CFRs) varied between outbreaks (<1 to approximately 40 %), the reasons for which are incompletely understood. The extracellular enveloped virus (EEV) form of orthopoxvirus progeny is hypothesized to disseminate infection. Investigations with the closely related Orthopoxvirus vaccinia have associated increased comet formation (EEV production) with increased mouse mortality (pathogenicity). Other vaccinia virus genetic manipulations which affect EEV production inconsistently support this association. However, antisera against vaccinia virus envelope protect mice from lethal challenge, further supporting a critical role for EEV in pathogenicity. Here, we show that the increased comet formation phenotypes of a diverse collection of variola viruses associate with strain phylogeny and geographical origin, but not with increased outbreak-related CFRs; within clades, there may be an association of plaque size with CFR. The mechanisms for variola virus pathogenicity probably involves multiple host and pathogen factors.

  11. Subclinical bovine vaccinia: An important risk factor in the epidemiology of this zoonosis in cattle.

    Science.gov (United States)

    Rehfeld, Izabelle Silva; Matos, Ana Carolina Diniz; Guedes, Maria Isabel Maldonado Coelho; Costa, Aristóteles Gomes; Fraiha, Ana Luiza Soares; Lobato, Zélia Inês Portela

    2017-10-01

    Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that mainly affects lactating cows and dairy farm milkers. The epidemiological role(s) of other cattle categories such as dry cows, bulls, and heifers in BV remains unclear. This study was performed to investigate VACV in affected dairy cattle herds and perifocal farms during an outbreak in Brazil. Crusts from lesions of cows' teats were collected from all farms with BV outbreaks. Milk, feces, blood, and serum were collected from symptomatic and asymptomatic lactating cows. Blood and serum were also sampled from other cattle categories (calves, heifers, dry cows, and bulls). The samples were tested for VACV by PCR, and to confirm VACV viability, VACV-positive samples were inoculated in BSC-40 cells and stained using immunoperoxidase. Neutralizing antibodies were investigated using plaque reduction neutralization test. Viral DNA was detected in milk, blood, and feces samples of symptomatic and asymptomatic dairy cows and in blood samples from other cattle categories on farms with and without confirmed BV outbreak. In affected farms, viable virus was identified in feces and milk samples from lactating cows and in blood samples from asymptomatic dry cows. Viable VACV was also identified in feces from lactating cows and one bull's blood sample from perifocal farms. Neutralizing antibodies were detected in 81.6% of the herds affected by BV and in 53.8% of the herds on perifocal farms. The presented data indicate a potential source of viral dissemination, which contributes to the persistence and spread of VACV in the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Genital Autoinoculation with Vaccinia: A Look at Two Cases.

    Science.gov (United States)

    Whittington, Julie R; Rollene, Nanette L; Gist, Richard S

    2018-05-01

    Smallpox, or vaccinia, has been eradicated worldwide as a disease; however, it may be weaponized and is thus a required immunization when military members deploy to certain parts of the world. We report two unusual cases of genital autoinoculation following smallpox vaccination. Both patients' lesions resolved without sequelae within 20 d. We advocate for thorough education on this potential vaccination adverse event. These cases highlight the importance of a broad differential diagnosis when dealing with vulvar lesions, particularly in our military population.

  13. On the Morphogenesis of Feathers

    Science.gov (United States)

    Yu, Mingke; Wu, Ping; Widelitz, Randall B.; Chuong, Cheng-Ming

    2015-01-01

    The most unique character of the feather is its highly ordered hierarchical branched structure1, 2. This evolutionary novelty confers flight function to birds3–5. Recent discoveries of fossils in China have prompted keen interest in the origin and evolution of feathers6–14. However, controversy arises whether the irregularly branched integumentary fibers on dinosaurs such as Sinornithosaurus are truly feathers6, 11, and whether an integumentary appendage with a major central shaft and notched edges is a non-avian feather or a proto-feather8–10. Here we take a developmental approach to analyze molecular mechanisms in feather branching morphogenesis. We have used the replication competent avian sarcoma (RCAS) retrovirus15 to efficiently deliver exogenous genes to regenerating chicken flight feather follicles. We show that the antagonistic balance between noggin and bone morphogenetic protein 4 (BMP4) plays a critical role in feather branching, with BMP4 promoting rachis formation and barb fusion, and noggin enhancing rachis and barb branching. Furthermore we show that sonic hedgehog (SHH) is essential for apoptosis of the marginal plate epithelia to become spaces between barbs. Our analyses show the molecular pathways underlying the topological transformation of feathers from cylindrical epithelia to the hierarchical branched structures, and provide first clues on the possible developmental mechanisms in the evolution of feather forms. PMID:12442169

  14. Applications of pox virus vectors to vaccination: an update.

    OpenAIRE

    Paoletti, E

    1996-01-01

    Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease...

  15. Vaccinia scars associated with improved survival among adults in rural Guinea-Bissau.

    Directory of Open Access Journals (Sweden)

    Mette Lundsby Jensen

    Full Text Available BACKGROUND: In urban Guinea-Bissau, adults with a vaccinia scar had better survival but also a higher prevalence of HIV-2 infection. We therefore investigated the association between vaccinia scar and survival and HIV infection in a rural area of Guinea-Bissau. METHODOLOGY/PRINCIPAL FINDINGS: In connection with a study of HIV in rural Guinea-Bissau, we assessed vaccinia and BCG scars in 193 HIV-1 or HIV-2 infected and 174 uninfected participants. Mortality was assessed after 2(1/2-3 years of follow-up. The analyses were adjusted for age, sex, village, and HIV status. The prevalence of vaccinia scar was associated with age, village, and HIV-2 status but not with sex and schooling. Compared with individuals without any scar, individuals with a vaccinia scar had better survival (mortality rate ratio (MR = 0.22 (95% CI 0.08-0.61, the MR being 0.19 (95% CI 0.06-0.57 for women and 0.40 (95% CI 0.04-3.74 for men. Estimates were similar for HIV-2 infected and HIV-1 and HIV-2 uninfected individuals. The HIV-2 prevalence was higher among individuals with a vaccinia scar compared to individuals without a vaccinia scar (RR = 1.57 (95% CI 1.02-2.36. CONCLUSION: The present study supports the hypothesis that vaccinia vaccination may have a non-specific beneficial effect on adult survival.

  16. A global sensitivity analysis approach for morphogenesis models

    KAUST Repository

    Boas, Sonja E. M.; Navarro, Marí a; Merks, Roeland M. H.; Blom, Joke G.

    2015-01-01

    Morphogenesis is a developmental process in which cells organize into shapes and patterns. Complex, non-linear and multi-factorial models with images as output are commonly used to study morphogenesis. It is difficult to understand

  17. Bovine Vaccinia in dairy cattle and suspicion of vesicular disease on milkers in Brazil

    Directory of Open Access Journals (Sweden)

    Thaís Garcia da Silva

    2018-05-01

    Full Text Available ABSTRACT: Bovine vaccinia (BV is a vesicular disease induced by the Vaccinia virus (VACV that affects milk production and is an occupational zoonosis. This research had the following objectives: (i detection of VACV by qPCR in cattle with clinical suspicion of vesicular disease; (ii symptoms characterization in animals and milkers with clinical suspicion of the disease and virus detection in humans; and (iii identification of risk factors for infections of VACV in herds from several Brazilian states. A total of 471 bovine epithelial samples from dairy farms, in 15 Brazilian states, were evaluated between 2007 and 2012. The samples were tested by quantitative PCR (qPCR using SYBR Green® reagents, validated with a lower limit of detection of 100 TCID50/50µL (1.7x100 viral particles, and 45.1% of VACV positive samples were detected. Using official forms for epidemiological investigation (FORM-IN, the risk factors for VACV infections in cattle were determined to be farms with a lack of technological facilities (P=0.029 and the presence of rodents (P=0.001. There was an effect of seasonality in cattle with a higher occurrence of BV during the dry season. A total of 420 epidemiological questionnaires were applied at public health care centers, where 100% of the milkers had vesicular lesions on their hands (98.1% and on their arms (6.9%. The most frequent clinical symptoms in humans were: local swelling (74.2%, headache (20.7%, fever (10.4% and inguinal lymphadenopathy (74.2%. Only 19.98% of milkers aged between 39 and 58 years were seroreactive to VACV and were immunized with the human anti-smallpox vaccine. There was an increase in the frequency of BV in older individuals due to their natural decrease in specific immunity. It has been shown that the implementation of zootechnical management techniques and health planning are important for the prevention of BV in animals and humans.

  18. Programming Morphogenesis through Systems and Synthetic Biology.

    Science.gov (United States)

    Velazquez, Jeremy J; Su, Emily; Cahan, Patrick; Ebrahimkhani, Mo R

    2018-04-01

    Mammalian tissue development is an intricate, spatiotemporal process of self-organization that emerges from gene regulatory networks of differentiating stem cells. A major goal in stem cell biology is to gain a sufficient understanding of gene regulatory networks and cell-cell interactions to enable the reliable and robust engineering of morphogenesis. Here, we review advances in synthetic biology, single cell genomics, and multiscale modeling, which, when synthesized, provide a framework to achieve the ambitious goal of programming morphogenesis in complex tissues and organoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Binding of glutathione to enterovirus capsids is essential for virion morphogenesis.

    Directory of Open Access Journals (Sweden)

    Hendrik Jan Thibaut

    2014-04-01

    Full Text Available Enteroviruses (family of the Picornaviridae cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH, thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.

  20. Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

    Science.gov (United States)

    Thibaut, Hendrik Jan; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J. M.; Neyts, Johan

    2014-01-01

    Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis. PMID:24722756

  1. Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

    Science.gov (United States)

    Mast, Jan; Thiel, Heinz-Jürgen; König, Matthias

    2014-01-01

    Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis. PMID:24352462

  2. Optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains.

    Directory of Open Access Journals (Sweden)

    Huiqiang Wang

    Full Text Available Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV. In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.

  3. Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees); E.J. Tijhaar (Edwin); R.C. Huisman (Robin); W. Huisman (Willem); A. de Ronde; I.H. Darby; M.J. Francis; G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Albert)

    1995-01-01

    textabstractCats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein

  4. Vaccinia scars associated with better survival for adults. An observational study from Guinea-Bissau

    DEFF Research Database (Denmark)

    Aaby, Peter; Gustafson, Per; Roth, Adam Anders Edvin

    2006-01-01

    Live vaccines including BCG and measles may have non-targeted beneficial effects on childhood survival in areas with high mortality. The authors therefore undertook a survey of vaccinia scars to evaluate subsequent mortality....

  5. One more piece in the VACV ecological puzzle: could peridomestic rodents be the link between wildlife and bovine vaccinia outbreaks in Brazil?

    Science.gov (United States)

    Abrahão, Jônatas S; Guedes, Maria Isabel M; Trindade, Giliane S; Fonseca, Flávio G; Campos, Rafael K; Mota, Bruno F; Lobato, Zélia I P; Silva-Fernandes, André T; Rodrigues, Gisele O L; Lima, Larissa S; Ferreira, Paulo C P; Bonjardim, Cláudio A; Kroon, Erna G

    2009-10-19

    Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks - BV), affecting dairy cattle and milkers. Little is known about VACV's natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated. In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks. These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?

  6. Computational models of airway branching morphogenesis.

    Science.gov (United States)

    Varner, Victor D; Nelson, Celeste M

    2017-07-01

    The bronchial network of the mammalian lung consists of millions of dichotomous branches arranged in a highly complex, space-filling tree. Recent computational models of branching morphogenesis in the lung have helped uncover the biological mechanisms that construct this ramified architecture. In this review, we focus on three different theoretical approaches - geometric modeling, reaction-diffusion modeling, and continuum mechanical modeling - and discuss how, taken together, these models have identified the geometric principles necessary to build an efficient bronchial network, as well as the patterning mechanisms that specify airway geometry in the developing embryo. We emphasize models that are integrated with biological experiments and suggest how recent progress in computational modeling has advanced our understanding of airway branching morphogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Feedback, Lineages and Self-Organizing Morphogenesis.

    Directory of Open Access Journals (Sweden)

    Sameeran Kunche

    2016-03-01

    Full Text Available Feedback regulation of cell lineage progression plays an important role in tissue size homeostasis, but whether such feedback also plays an important role in tissue morphogenesis has yet to be explored. Here we use mathematical modeling to show that a particular feedback architecture in which both positive and negative diffusible signals act on stem and/or progenitor cells leads to the appearance of bistable or bi-modal growth behaviors, ultrasensitivity to external growth cues, local growth-driven budding, self-sustaining elongation, and the triggering of self-organization in the form of lamellar fingers. Such behaviors arise not through regulation of cell cycle speeds, but through the control of stem or progenitor self-renewal. Even though the spatial patterns that arise in this setting are the result of interactions between diffusible factors with antagonistic effects, morphogenesis is not the consequence of Turing-type instabilities.

  8. Imaging morphogenesis: technological advances and biological insights.

    Science.gov (United States)

    Keller, Philipp J

    2013-06-07

    Morphogenesis, the development of the shape of an organism, is a dynamic process on a multitude of scales, from fast subcellular rearrangements and cell movements to slow structural changes at the whole-organism level. Live-imaging approaches based on light microscopy reveal the intricate dynamics of this process and are thus indispensable for investigating the underlying mechanisms. This Review discusses emerging imaging techniques that can record morphogenesis at temporal scales from seconds to days and at spatial scales from hundreds of nanometers to several millimeters. To unlock their full potential, these methods need to be matched with new computational approaches and physical models that help convert highly complex image data sets into biological insights.

  9. Feedback, Lineages and Self-Organizing Morphogenesis

    Science.gov (United States)

    Calof, Anne L.; Lowengrub, John S.; Lander, Arthur D.

    2016-01-01

    Feedback regulation of cell lineage progression plays an important role in tissue size homeostasis, but whether such feedback also plays an important role in tissue morphogenesis has yet to be explored. Here we use mathematical modeling to show that a particular feedback architecture in which both positive and negative diffusible signals act on stem and/or progenitor cells leads to the appearance of bistable or bi-modal growth behaviors, ultrasensitivity to external growth cues, local growth-driven budding, self-sustaining elongation, and the triggering of self-organization in the form of lamellar fingers. Such behaviors arise not through regulation of cell cycle speeds, but through the control of stem or progenitor self-renewal. Even though the spatial patterns that arise in this setting are the result of interactions between diffusible factors with antagonistic effects, morphogenesis is not the consequence of Turing-type instabilities. PMID:26989903

  10. Apoptosis during budding morphogenesis of dentition

    Czech Academy of Sciences Publication Activity Database

    Peterková, Renata; Peterka, Miroslav; Viriot, L.; Lesot, H.

    2002-01-01

    Roč. 70, č. 7 (2002), s. 353 ISSN 0301-4681. [International Conference of the International Society of Differentiation /12./. Lyon, France, 14.09.2002-17.09.2002] R&D Projects: GA ČR GA304/02/0448 Institutional research plan: CEZ:AV0Z5039906 Keywords : morphogenesis of dentition Subject RIV: FF - HEENT, Dentistry Impact factor: 2.078, year: 2002

  11. Airway branching morphogenesis in three dimensional culture

    Directory of Open Access Journals (Sweden)

    Gudjonsson Thorarinn

    2010-11-01

    Full Text Available Abstract Background Lungs develop from the fetal digestive tract where epithelium invades the vascular rich stroma in a process called branching morphogenesis. In organogenesis, endothelial cells have been shown to be important for morphogenesis and the maintenance of organ structure. The aim of this study was to recapitulate human lung morphogenesis in vitro by establishing a three dimensional (3D co-culture model where lung epithelial cells were cultured in endothelial-rich stroma. Methods We used a human bronchial epithelial cell line (VA10 recently developed in our laboratory. This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human umbilical vein endothelial cells (HUVECs, to mimic the close interaction between these cell types during lung development. Morphogenesis and differentiation was monitored by phase contrast microscopy, immunostainings and confocal imaging. Results We found that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like structures, suggesting that lung epithelial branching is facilitated by the presence of endothelial cells. The VA10 derived epithelial structures display various complex patterns of branching and show partial alveolar type-II differentiation with pro-Surfactant-C expression. The epithelial origin of the branching VA10 colonies was confirmed by immunostaining. These bronchioalveolar-like structures were polarized with respect to integrin expression at the cell-matrix interface. The endothelial-induced branching was mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2 and sprouty-2 were expressed at the growing tips of the branching structures and the branching was inhibited by the FGFR-small molecule inhibitor SU5402. Discussion In this study we show that a human lung epithelial cell line can be induced by endothelial cells to

  12. Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis.

    Science.gov (United States)

    Andrade, Ana Cláudia Dos Santos Pereira; Rodrigues, Rodrigo Araújo Lima; Oliveira, Graziele Pereira; Andrade, Kétyllen Reis; Bonjardim, Cláudio Antônio; La Scola, Bernard; Kroon, Erna Geessien; Abrahão, Jônatas Santos

    2017-11-15

    Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii , causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus. IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a

  13. Normal morphogenesis of epithelial tissues and progression of epithelial tumors

    Science.gov (United States)

    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A.

    2011-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. PMID:21898857

  14. Smallpox virus resequencing GeneChips can also rapidly ascertain species status for some zoonotic non-variola orthopoxviruses.

    Science.gov (United States)

    Sulaiman, Irshad M; Sammons, Scott A; Wohlhueter, Robert M

    2008-04-01

    We recently developed a set of seven resequencing GeneChips for the rapid sequencing of Variola virus strains in the WHO Repository of the Centers for Disease Control and Prevention. In this study, we attempted to hybridize these GeneChips with some known non-Variola orthopoxvirus isolates, including monkeypox, cowpox, and vaccinia viruses, for rapid detection.

  15. A global sensitivity analysis approach for morphogenesis models

    NARCIS (Netherlands)

    S.E.M. Boas (Sonja); M.I. Navarro Jimenez (Maria); R.M.H. Merks (Roeland); J.G. Blom (Joke)

    2015-01-01

    textabstract{\\bf Background} %if any Morphogenesis is a developmental process in which cells organize into shapes and patterns. Complex, non-linear and multi-factorial models with images as output are commonly used to study morphogenesis. It is difficult to understand the relation between the

  16. [Behavior of Orf virus in permissive and nonpermissive systems].

    Science.gov (United States)

    Büttner, M; Czerny, C P; Schumm, M

    1995-04-01

    Dogs were immunized i.m. with attenuated poxvirus vaccines (vaccinia virus, Orf-virus) and a bovine herpesvirus-1 (BHV-1) vaccine. After intradermal (i.d.) application of the vaccine viruses a specific delayed type hypersensitivity (DTH) reaction of the skin occurred only with vaccinia virus. The i.d. application of Orf-virus caused a short-term, non-specific inflammatory reaction of the skin, even in dogs not immunized with Orf-virus. Out of 30 sera from Orf-virus immunized beagles (n = 4) only eight were found reactive to Orf-virus in a competition ELISA. Three sera from dogs not Orf-virus immunized but skin-tested with the virus contained low antibody titers. Using indirect immunofluorescence (IIF) in flow cytometry, the existence of Orf-virus antigens was examined on the surface and in the cytoplasm of permissive (BFK and Vero)- and questionable permissive MDCK cells. The canine kidney MDCK cell line was found to be non-permissive for Orf-virus replication; the occurrence of an Orf-(ecthyma contagiosum) like disease in dogs is unlikely.

  17. New frontiers in oncolytic viruses: optimizing and selecting for virus strains with improved efficacy

    Directory of Open Access Journals (Sweden)

    Lundstrom K

    2018-02-01

    Full Text Available Kenneth Lundstrom PanTherapeutics, Lutry, Switzerland Abstract: Oncolytic viruses have demonstrated selective replication and killing of tumor cells. Different types of oncolytic viruses – adenoviruses, alphaviruses, herpes simplex viruses, Newcastle disease viruses, rhabdoviruses, Coxsackie viruses, and vaccinia viruses – have been applied as either naturally occurring or engineered vectors. Numerous studies in animal-tumor models have demonstrated substantial tumor regression and prolonged survival rates. Moreover, clinical trials have confirmed good safety profiles and therapeutic efficacy for oncolytic viruses. Most encouragingly, the first cancer gene-therapy drug – Gendicine, based on oncolytic adenovirus type 5 – was approved in China. Likewise, a second-generation oncolytic herpes simplex virus-based drug for the treatment of melanoma has been registered in the US and Europe as talimogene laherparepvec. Keywords: immunotherapy, viral vectors, clinical trials, drug approval

  18. Phospholipid Homeostasis Regulates Dendrite Morphogenesis in Drosophila Sensory Neurons

    Directory of Open Access Journals (Sweden)

    Shan Meltzer

    2017-10-01

    Full Text Available Disruptions in lipid homeostasis have been observed in many neurodevelopmental disorders that are associated with dendrite morphogenesis defects. However, the molecular mechanisms of how lipid homeostasis affects dendrite morphogenesis are unclear. We find that easily shocked (eas, which encodes a kinase with a critical role in phospholipid phosphatidylethanolamine (PE synthesis, and two other enzymes in this synthesis pathway are required cell autonomously in sensory neurons for dendrite growth and stability. Furthermore, we show that the level of Sterol Regulatory Element-Binding Protein (SREBP activity is important for dendrite development. SREBP activity increases in eas mutants, and decreasing the level of SREBP and its transcriptional targets in eas mutants largely suppresses the dendrite growth defects. Furthermore, reducing Ca2+ influx in neurons of eas mutants ameliorates the dendrite morphogenesis defects. Our study uncovers a role for EAS kinase and reveals the in vivo function of phospholipid homeostasis in dendrite morphogenesis.

  19. Immunogenicity of recombinant feline infectious peritonitis virus spike protein in mice and kittens

    NARCIS (Netherlands)

    Horzinek, M.C.; Vennema, H.; Groot, R. de; Harbour, D.A.; Dalderup, M.; Gruffydd-Jones, T.; Spaan, W.J.M.

    1990-01-01

    The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis (FIVP) was recombined into the genome of vaccinia virus, strain WR. The recombinant induced spike protein specific, in vitro neutralizing antibodies in mkice. When kittens were immunized with the

  20. Morphogenesis of the infectious HIV-1 virion

    Directory of Open Access Journals (Sweden)

    Jun-Ichi eSakuragi

    2011-12-01

    Full Text Available The virion of HIV-1 is spherical and viral glycoprotein spikes (gp120, gp41 protrude from its envelope. The characteristic cone-shaped core exists within the virion, caging the ribonucleoprotein (RNP complex, which is comprised of viral RNA, nucleocapsid (NC and viral enzymes. The HIV-1 virion is budded and released from the infected cell as an immature donut-shaped particle. During or immediately after release, viral protease (PR is activated and subsequently processes the viral structural protein Gag. Through this maturation process, virions acquire infectivity, but its mechanism and transition of morphology largely remain unclear. Recent technological advances in experimental devices and techniques have made it possible to closely dissect the viral production site on the cell, the exterior – or even the interior – of an individual virion, and many new aspects on virion morphology and maturation. In this manuscript, I review the morphogenesis of HIV-1 virions. I focus on several studies, including some of our recent findings, which examined virion formation and/or maturation processes. The story of novel compound, which inhibits virion maturation, and the importance of maturation research are also discussed.

  1. Mechanical growth and morphogenesis of seashells

    KAUST Repository

    Moulton, D.E.

    2012-10-01

    Seashells grow through the local deposition of mass along the aperture. Many mathematical descriptions of the shapes of shells have been provided over the years, and the basic logarithmic coiling seen in mollusks can be simulated with few parameters. However, the developmental mechanisms underlying shell coiling are largely not understood and the ubiquitous presence of ornamentation such as ribs, tubercles, or spines presents yet another level of difficulty. Here we develop a general model for shell growth based entirely on the local geometry and mechanics of the aperture and mantle. This local description enables us to efficiently describe both arbitrary growth velocities and the evolution of the shell aperture itself. We demonstrate how most shells can be simulated within this framework. We then turn to the mechanics underlying the shell morphogenesis, and develop models for the evolution of the aperture. We demonstrate that the elastic response of the mantle during shell deposition provides a natural mechanism for the formation of three-dimensional ornamentation in shells. © 2012 Elsevier Ltd.

  2. Multiscale information modelling for heart morphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Abdulla, T; Imms, R; Summers, R [Department of Electronic and Electrical Engineering, Loughborough University, Loughborough (United Kingdom); Schleich, J M, E-mail: T.Abdulla@lboro.ac.u [LTSI Signal and Image Processing Laboratory, University of Rennes 1, Rennes (France)

    2010-07-01

    Science is made feasible by the adoption of common systems of units. As research has become more data intensive, especially in the biomedical domain, it requires the adoption of a common system of information models, to make explicit the relationship between one set of data and another, regardless of format. This is being realised through the OBO Foundry to develop a suite of reference ontologies, and NCBO Bioportal to provide services to integrate biomedical resources and functionality to visualise and create mappings between ontology terms. Biomedical experts tend to be focused at one level of spatial scale, be it biochemistry, cell biology, or anatomy. Likewise, the ontologies they use tend to be focused at a particular level of scale. There is increasing interest in a multiscale systems approach, which attempts to integrate between different levels of scale to gain understanding of emergent effects. This is a return to physiological medicine with a computational emphasis, exemplified by the worldwide Physiome initiative, and the European Union funded Network of Excellence in the Virtual Physiological Human. However, little work has been done on how information modelling itself may be tailored to a multiscale systems approach. We demonstrate how this can be done for the complex process of heart morphogenesis, which requires multiscale understanding in both time and spatial domains. Such an effort enables the integration of multiscale metrology.

  3. Multiscale information modelling for heart morphogenesis

    International Nuclear Information System (INIS)

    Abdulla, T; Imms, R; Summers, R; Schleich, J M

    2010-01-01

    Science is made feasible by the adoption of common systems of units. As research has become more data intensive, especially in the biomedical domain, it requires the adoption of a common system of information models, to make explicit the relationship between one set of data and another, regardless of format. This is being realised through the OBO Foundry to develop a suite of reference ontologies, and NCBO Bioportal to provide services to integrate biomedical resources and functionality to visualise and create mappings between ontology terms. Biomedical experts tend to be focused at one level of spatial scale, be it biochemistry, cell biology, or anatomy. Likewise, the ontologies they use tend to be focused at a particular level of scale. There is increasing interest in a multiscale systems approach, which attempts to integrate between different levels of scale to gain understanding of emergent effects. This is a return to physiological medicine with a computational emphasis, exemplified by the worldwide Physiome initiative, and the European Union funded Network of Excellence in the Virtual Physiological Human. However, little work has been done on how information modelling itself may be tailored to a multiscale systems approach. We demonstrate how this can be done for the complex process of heart morphogenesis, which requires multiscale understanding in both time and spatial domains. Such an effort enables the integration of multiscale metrology.

  4. Psoriasis herpeticum due to Varicella zoster virus: A Kaposi′s varicelliform eruption in erythrodermic psoriasis

    Directory of Open Access Journals (Sweden)

    Geeta Garg

    2012-01-01

    Full Text Available Kaposi′s varicelliform eruption (KVE or eczema herpeticum is characterized by disseminated papulovesicular eruption caused by a number of viruses like Herpes simplex virus I and II, Coxsackie virus, and Vaccinia and Small pox viruses in patients with pre-existing skin disease. The occurrence of KVE with psoriasis has been reported recently as a new entity psoriasis herpeticum. The rare causation of psoriasis herpeticum due to Varicella zoster virus in a patient with underlying psoriasis is being reported for the first time.

  5. Morphogenesis of mimivirus and its viral factories: an atomic force microscopy study of infected cells.

    Science.gov (United States)

    Kuznetsov, Yuri G; Klose, Thomas; Rossmann, Michael; McPherson, Alexander

    2013-10-01

    Amoebas infected with mimivirus were disrupted at sequential stages of virus production and were visualized by atomic force microscopy. The development of virus factories proceeded over 3 to 4 h postinfection and resulted from the coalescence of 0.5- to 2-μm vesicles, possibly bearing nucleic acid, derived from either the nuclear membrane or the closely associated rough endoplasmic reticulum. Virus factories actively producing virus capsids on their surfaces were imaged, and this allowed the morphogenesis of the capsids to be delineated. The first feature to appear on a virus factory surface when a new capsid is born is the center of a stargate, which is a pentameric protein oligomer. As the arms of the stargate grow from the pentamer, a rough disk the diameter of a capsid thickens around it. This marks the initial emergence of a protein-coated membrane vesicle. The capsid self-assembles on the vesicle. Hillocks capped by different pentameric proteins spontaneously appear on the emerging vesicle at positions that are ultimately occupied by 5-fold icosahedral vertices. A lattice of coat protein nucleates at each of the 5-fold vertices, but not at the stargate, and then spreads outward from the vertices over the surface, merging seamlessly to complete the icosahedral capsid. Filling with DNA and associated proteins occurs by the transfer of nucleic acid from the interior of the virus factory into the nearly completed capsids. The portal, through which the DNA enters, is sealed by a plug of protein having a diameter of about 40 nm. A layer of integument protein that anchors the surface fibers is acquired by the passage of capsids through a membrane enriched in the protein. The coating of surface fibers is similarly acquired when the integument protein-coated capsids pass through a second membrane that has a forest of surface fibers embedded on one side.

  6. The Fog signaling pathway: Insights into signaling in morphogenesis

    Science.gov (United States)

    Manning, Alyssa J.; Rogers, Stephen L.

    2014-01-01

    Epithelia form the building blocks of many tissue and organ types. Epithelial cells often form a contiguous 2-dimensional sheet that is held together by strong adhesions. The mechanical properties conferred by these adhesions allow the cells to undergo dramatic three-dimensional morphogenetic movements while maintaining cell–cell contacts during embryogenesis and post-embryonic development. The Drosophila Folded gastrulation pathway triggers epithelial cell shape changes that drive gastrulation and tissue folding and is one of the most extensively studied examples of epithelial morphogenesis. This pathway has yielded key insights into the signaling mechanisms and cellular machinery involved in epithelial remodeling. In this review, we discuss principles of morphogenesis and signaling that have been discovered through genetic and cell biological examination of this pathway. We also consider various regulatory mechanisms and the system's relevance to mammalian development. We propose future directions that will continue to broaden our knowledge of morphogenesis across taxa. PMID:25127992

  7. Morphogenesis and pattern formation in biological systems experiments and models

    CERN Document Server

    Noji, Sumihare; Ueno, Naoto; Maini, Philip

    2003-01-01

    A central goal of current biology is to decode the mechanisms that underlie the processes of morphogenesis and pattern formation. Concerned with the analysis of those phenomena, this book covers a broad range of research fields, including developmental biology, molecular biology, plant morphogenesis, ecology, epidemiology, medicine, paleontology, evolutionary biology, mathematical biology, and computational biology. In Morphogenesis and Pattern Formation in Biological Systems: Experiments and Models, experimental and theoretical aspects of biology are integrated for the construction and investigation of models of complex processes. This collection of articles on the latest advances by leading researchers not only brings together work from a wide spectrum of disciplines, but also provides a stepping-stone to the creation of new areas of discovery.

  8. A global sensitivity analysis approach for morphogenesis models

    KAUST Repository

    Boas, Sonja E. M.

    2015-11-21

    Background Morphogenesis is a developmental process in which cells organize into shapes and patterns. Complex, non-linear and multi-factorial models with images as output are commonly used to study morphogenesis. It is difficult to understand the relation between the uncertainty in the input and the output of such ‘black-box’ models, giving rise to the need for sensitivity analysis tools. In this paper, we introduce a workflow for a global sensitivity analysis approach to study the impact of single parameters and the interactions between them on the output of morphogenesis models. Results To demonstrate the workflow, we used a published, well-studied model of vascular morphogenesis. The parameters of this cellular Potts model (CPM) represent cell properties and behaviors that drive the mechanisms of angiogenic sprouting. The global sensitivity analysis correctly identified the dominant parameters in the model, consistent with previous studies. Additionally, the analysis provided information on the relative impact of single parameters and of interactions between them. This is very relevant because interactions of parameters impede the experimental verification of the predicted effect of single parameters. The parameter interactions, although of low impact, provided also new insights in the mechanisms of in silico sprouting. Finally, the analysis indicated that the model could be reduced by one parameter. Conclusions We propose global sensitivity analysis as an alternative approach to study the mechanisms of morphogenesis. Comparison of the ranking of the impact of the model parameters to knowledge derived from experimental data and from manipulation experiments can help to falsify models and to find the operand mechanisms in morphogenesis. The workflow is applicable to all ‘black-box’ models, including high-throughput in vitro models in which output measures are affected by a set of experimental perturbations.

  9. A global sensitivity analysis approach for morphogenesis models.

    Science.gov (United States)

    Boas, Sonja E M; Navarro Jimenez, Maria I; Merks, Roeland M H; Blom, Joke G

    2015-11-21

    Morphogenesis is a developmental process in which cells organize into shapes and patterns. Complex, non-linear and multi-factorial models with images as output are commonly used to study morphogenesis. It is difficult to understand the relation between the uncertainty in the input and the output of such 'black-box' models, giving rise to the need for sensitivity analysis tools. In this paper, we introduce a workflow for a global sensitivity analysis approach to study the impact of single parameters and the interactions between them on the output of morphogenesis models. To demonstrate the workflow, we used a published, well-studied model of vascular morphogenesis. The parameters of this cellular Potts model (CPM) represent cell properties and behaviors that drive the mechanisms of angiogenic sprouting. The global sensitivity analysis correctly identified the dominant parameters in the model, consistent with previous studies. Additionally, the analysis provided information on the relative impact of single parameters and of interactions between them. This is very relevant because interactions of parameters impede the experimental verification of the predicted effect of single parameters. The parameter interactions, although of low impact, provided also new insights in the mechanisms of in silico sprouting. Finally, the analysis indicated that the model could be reduced by one parameter. We propose global sensitivity analysis as an alternative approach to study the mechanisms of morphogenesis. Comparison of the ranking of the impact of the model parameters to knowledge derived from experimental data and from manipulation experiments can help to falsify models and to find the operand mechanisms in morphogenesis. The workflow is applicable to all 'black-box' models, including high-throughput in vitro models in which output measures are affected by a set of experimental perturbations.

  10. Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

    OpenAIRE

    Stephensen, C B; Welter, J; Thaker, S R; Taylor, J; Tartaglia, J; Paoletti, E

    1997-01-01

    Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice ...

  11. Vaccinia-based influenza vaccine overcomes previously induced immunodominance hierarchy for heterosubtypic protection.

    Science.gov (United States)

    Kwon, Ji-Sun; Yoon, Jungsoon; Kim, Yeon-Jung; Kang, Kyuho; Woo, Sunje; Jung, Dea-Im; Song, Man Ki; Kim, Eun-Ha; Kwon, Hyeok-Il; Choi, Young Ki; Kim, Jihye; Lee, Jeewon; Yoon, Yeup; Shin, Eui-Cheol; Youn, Jin-Won

    2014-08-01

    Growing concerns about unpredictable influenza pandemics require a broadly protective vaccine against diverse influenza strains. One of the promising approaches was a T cell-based vaccine, but the narrow breadth of T-cell immunity due to the immunodominance hierarchy established by previous influenza infection and efficacy against only mild challenge condition are important hurdles to overcome. To model T-cell immunodominance hierarchy in humans in an experimental setting, influenza-primed C57BL/6 mice were chosen and boosted with a mixture of vaccinia recombinants, individually expressing consensus sequences from avian, swine, and human isolates of influenza internal proteins. As determined by IFN-γ ELISPOT and polyfunctional cytokine secretion, the vaccinia recombinants of influenza expanded the breadth of T-cell responses to include subdominant and even minor epitopes. Vaccine groups were successfully protected against 100 LD50 challenges with PR/8/34 and highly pathogenic avian influenza H5N1, which contained the identical dominant NP366 epitope. Interestingly, in challenge with pandemic A/Cal/04/2009 containing mutations in the dominant epitope, only the group vaccinated with rVV-NP + PA showed improved protection. Taken together, a vaccinia-based influenza vaccine expressing conserved internal proteins improved the breadth of influenza-specific T-cell immunity and provided heterosubtypic protection against immunologically close as well as distant influenza strains. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Purification, crystallization and preliminary diffraction studies of an ectromelia virus glutaredoxin

    International Nuclear Information System (INIS)

    Bacik, John-Paul; Brigley, Angela M.; Channon, Lisa D.; Audette, Gerald F.; Hazes, Bart

    2005-01-01

    Ectromelia virus glutaredoxin has been crystallized in the presence of the reducing agent DTT. A diffraction data set has been collected and processed to 1.8 Å resolution. Ectromelia, vaccinia, smallpox and other closely related viruses of the orthopoxvirus genus encode a glutaredoxin gene that is not present in poxviruses outside of this genus. The vaccinia glutaredoxin O2L has been implicated as the reducing agent for ribonucleotide reductase and may thus play an important role in viral deoxyribonucleotide synthesis. As part of an effort to understand nucleotide metabolism by poxviruses, EVM053, the O2L ortholog of the ectromelia virus, has been crystallized. EVM053 crystallizes in space group C222 1 , with unit-cell parameters a = 61.98, b = 67.57, c = 108.55 Å. Diffraction data have been processed to 1.8 Å resolution and a self-rotation function indicates that there are two molecules per asymmetric unit

  13. Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

    Directory of Open Access Journals (Sweden)

    Gareth W Morgan

    2010-02-01

    Full Text Available Vaccinia virus (VACV uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC, a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs. Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD motif, which is conserved in the kinesin-1-binding sequence (KBS of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

  14. Isomyosin expression patterns during rat heart morphogenesis: an immunohistochemical study

    NARCIS (Netherlands)

    de Groot, I. J.; Lamers, W. H.; Moorman, A. F.

    1989-01-01

    An immunohistochemical study of cardiac alpha and beta myosin heavy chain (MHC) expression during rat heart morphogenesis was performed. In tubular hearts (embryonic days, ED10-11) coexpression of both cardiac alpha and beta MHC was found throughout the heart, except for the left free wall of the

  15. Collective cell migration in morphogenesis, regeneration and cancer.

    NARCIS (Netherlands)

    Friedl, P.H.A.; Gilmour, D.

    2009-01-01

    The collective migration of cells as a cohesive group is a hallmark of the tissue remodelling events that underlie embryonic morphogenesis, wound repair and cancer invasion. In such migration, cells move as sheets, strands, clusters or ducts rather than individually, and use similar actin- and

  16. Epithelial morphogenesis: the mouse eye as a model system.

    Science.gov (United States)

    Chauhan, Bharesh; Plageman, Timothy; Lou, Ming; Lang, Richard

    2015-01-01

    Morphogenesis is the developmental process by which tissues and organs acquire the shape that is critical to their function. Here, we review recent advances in our understanding of the mechanisms that drive morphogenesis in the developing eye. These investigations have shown that regulation of the actin cytoskeleton is central to shaping the presumptive lens and retinal epithelia that are the major components of the eye. Regulation of the actin cytoskeleton is mediated by Rho family GTPases, by signaling pathways and indirectly, by transcription factors that govern the expression of critical genes. Changes in the actin cytoskeleton can shape cells through the generation of filopodia (that, in the eye, connect adjacent epithelia) or through apical constriction, a process that produces a wedge-shaped cell. We have also learned that one tissue can influence the shape of an adjacent one, probably by direct force transmission, in a process we term inductive morphogenesis. Though these mechanisms of morphogenesis have been identified using the eye as a model system, they are likely to apply broadly where epithelia influence the shape of organs during development. © 2015 Elsevier Inc. All rights reserved.

  17. Expression and functional role of sprouty-2 in breast morphogenesis.

    Science.gov (United States)

    Sigurdsson, Valgardur; Ingthorsson, Saevar; Hilmarsdottir, Bylgja; Gustafsdottir, Sigrun M; Franzdottir, Sigridur Rut; Arason, Ari Jon; Steingrimsson, Eirikur; Magnusson, Magnus K; Gudjonsson, Thorarinn

    2013-01-01

    Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of

  18. Expression and functional role of sprouty-2 in breast morphogenesis.

    Directory of Open Access Journals (Sweden)

    Valgardur Sigurdsson

    Full Text Available Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs, including epidermal growth factor receptor (EGFR and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2 in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an

  19. Expression of Brucella Antigens in Vaccinia Virus to Prevent Brucellosis in Humans: Protection Studies in Mice

    National Research Council Canada - National Science Library

    Schurig, Gerhardt

    2000-01-01

    .... Based on our present studies and the finding that Brucella Cu/ZN SOD and L7/Ll2 proteins are protective antigens and that the presence of IL-12 is necessary at the moment of immunization, we conclude...

  20. The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding

    Czech Academy of Sciences Publication Activity Database

    Tarbouriech, N.; Ducournau, C.; Hutin, S.; Mas, P.J.; Man, Petr; Forest, E.; Hart, D.J.; Peyrefitte, Ch.N.; Burmeister, W.P.; Iseni, F.

    2017-01-01

    Roč. 8, NOV 13 (2017), s. 1-12, č. článku 1455. ISSN 2041-1723 R&D Projects: GA MŠk(CZ) LQ1604 Institutional support: RVO:61388971 Keywords : PROTEIN SECONDARY STRUCTURE * CRYSTAL-STRUCTURE * GENETIC-CHARACTERIZATION Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 12.124, year: 2016

  1. Investigation of radiation enhanced reactivation of cytoplasmic replicating human virus

    International Nuclear Information System (INIS)

    Bockstahler, L.E.; Haynes, K.F.; Stafford, J.E.

    1976-01-01

    When monolayers of CV-1 monkey kidney cells were exposed to ultraviolet (uv) radiation (0 to 200 erg/nm 2 ) or x rays (0 to 10 krads) before infection with uv-irradiated herpes simplex virus, an increase in the infectivity of this nuclear replicating virus occurred as measured by plaque formation. These phenomena are known as uv (Weigle) reactivation (WR) and x-ray reactivation (x-ray R). In this study the presence of WR and x-ray R was examined in CV-1 cells infected with uv-irradiated vaccinia virus or poliovirus, both cytoplasmic replicating viruses. Little or no WR or x-ray R was observed for either of these viruses. These results suggest that WR and x-ray R in mammalian cells may be restricted to viruses which are synthesized in the cell nucleus

  2. A pandemic influenza H1N1 live vaccine based on modified vaccinia Ankara is highly immunogenic and protects mice in active and passive immunizations.

    Directory of Open Access Journals (Sweden)

    Annett Hessel

    Full Text Available BACKGROUND: The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model. METHODOLOGY/PRINCIPAL FINDINGS: For this purpose, the hemagglutinin (HA and neuraminidase (NA genes of the influenza A/California/07/2009 (H1N1 strain (CA/07 were inserted into the replication-deficient modified vaccinia Ankara (MVA virus--a safe poxviral live vector--resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-gamma-secreting (IFN-gamma CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus. CONCLUSIONS/SIGNIFICANCE: The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for

  3. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus infected cells

    DEFF Research Database (Denmark)

    Kristensen, Thea; Normann, Preben; Gullberg, Maria

    2017-01-01

    . Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results...

  4. Polarized protein transport and lumen formation during epithelial tissue morphogenesis.

    Science.gov (United States)

    Blasky, Alex J; Mangan, Anthony; Prekeris, Rytis

    2015-01-01

    One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo.

  5. EphB/syndecan-2 signaling in dendritic spine morphogenesis

    DEFF Research Database (Denmark)

    Ethell, I M; Irie, F; Kalo, M S

    2001-01-01

    We previously reported that the cell surface proteoglycan syndecan-2 can induce dendritic spine formation in hippocampal neurons. We demonstrate here that the EphB2 receptor tyrosine kinase phosphorylates syndecan-2 and that this phosphorylation event is crucial for syndecan-2 clustering and spine...... formation. Syndecan-2 is tyrosine phosphorylated and forms a complex with EphB2 in mouse brain. Dominant-negative inhibition of endogenous EphB receptor activities blocks clustering of endogenous syndecan-2 and normal spine formation in cultured hippocampal neurons. This is the first evidence that Eph...... receptors play a physiological role in dendritic spine morphogenesis. Our observations suggest that spine morphogenesis is triggered by the activation of Eph receptors, which causes tyrosine phosphorylation of target molecules, such as syndecan-2, in presumptive spines....

  6. Extending Graphic Statics for User-Controlled Structural Morphogenesis

    OpenAIRE

    Fivet, Corentin; Zastavni, Denis; Cap, Jean-François; Structural Morphology Group International Seminar 2011

    2011-01-01

    The first geometrical definitions of any structure are of primary importance when considering pertinence and efficiency in structural design processes. Engineering history has taught us how graphic statics can be a very powerful tool since it allows the designer to take shapes and forces into account simultaneously. However, current and past graphic statics methods are more suitable for analysis than structural morphogenesis. This contribution introduces new graphical methods that can supp...

  7. Inhibition of enveloped viruses infectivity by curcumin.

    Directory of Open Access Journals (Sweden)

    Tzu-Yen Chen

    Full Text Available Curcumin, a natural compound and ingredient in curry, has antiinflammatory, antioxidant, and anticarcinogenic properties. Previously, we reported that curcumin abrogated influenza virus infectivity by inhibiting hemagglutination (HA activity. This study demonstrates a novel mechanism by which curcumin inhibits the infectivity of enveloped viruses. In all analyzed enveloped viruses, including the influenza virus, curcumin inhibited plaque formation. In contrast, the nonenveloped enterovirus 71 remained unaffected by curcumin treatment. We evaluated the effects of curcumin on the membrane structure using fluorescent dye (sulforhodamine B; SRB-containing liposomes that mimic the viral envelope. Curcumin treatment induced the leakage of SRB from these liposomes and the addition of the influenza virus reduced the leakage, indicating that curcumin disrupts the integrity of the membranes of viral envelopes and of liposomes. When testing liposomes of various diameters, we detected higher levels of SRB leakage from the smaller-sized liposomes than from the larger liposomes. Interestingly, the curcumin concentration required to reduce plaque formation was lower for the influenza virus (approximately 100 nm in diameter than for the pseudorabies virus (approximately 180 nm and the vaccinia virus (roughly 335 × 200 × 200 nm. These data provide insights on the molecular antiviral mechanisms of curcumin and its potential use as an antiviral agent for enveloped viruses.

  8. Inhibition of Enveloped Viruses Infectivity by Curcumin

    Science.gov (United States)

    Wen, Hsiao-Wei; Ou, Jun-Lin; Chiou, Shyan-Song; Chen, Jo-Mei; Wong, Min-Liang; Hsu, Wei-Li

    2013-01-01

    Curcumin, a natural compound and ingredient in curry, has antiinflammatory, antioxidant, and anticarcinogenic properties. Previously, we reported that curcumin abrogated influenza virus infectivity by inhibiting hemagglutination (HA) activity. This study demonstrates a novel mechanism by which curcumin inhibits the infectivity of enveloped viruses. In all analyzed enveloped viruses, including the influenza virus, curcumin inhibited plaque formation. In contrast, the nonenveloped enterovirus 71 remained unaffected by curcumin treatment. We evaluated the effects of curcumin on the membrane structure using fluorescent dye (sulforhodamine B; SRB)-containing liposomes that mimic the viral envelope. Curcumin treatment induced the leakage of SRB from these liposomes and the addition of the influenza virus reduced the leakage, indicating that curcumin disrupts the integrity of the membranes of viral envelopes and of liposomes. When testing liposomes of various diameters, we detected higher levels of SRB leakage from the smaller-sized liposomes than from the larger liposomes. Interestingly, the curcumin concentration required to reduce plaque formation was lower for the influenza virus (approximately 100 nm in diameter) than for the pseudorabies virus (approximately 180 nm) and the vaccinia virus (roughly 335 × 200 × 200 nm). These data provide insights on the molecular antiviral mechanisms of curcumin and its potential use as an antiviral agent for enveloped viruses. PMID:23658730

  9. Embryo mechanics: balancing force production with elastic resistance during morphogenesis.

    Science.gov (United States)

    Davidson, Lance A

    2011-01-01

    Morphogenesis requires the spatial and temporal control of embryo mechanics, including force production and mechanical resistance to those forces, to coordinate tissue deformation and large-scale movements. Thus, biomechanical processes play a key role in directly shaping the embryo. Additional roles for embryo mechanics during development may include the patterning of positional information and to provide feedback to ensure the success of morphogenetic movements in shaping the larval body and organs. To understand the multiple roles of mechanics during development requires familiarity with engineering principles of the mechanics of structures, the viscoelastic properties of biomaterials, and the integration of force and stress within embryonic structures as morphogenesis progresses. In this chapter, we review the basic engineering principles of biomechanics as they relate to morphogenesis, introduce methods for quantifying embryo mechanics and the limitations of these methods, and outline a formalism for investigating the role of embryo mechanics in birth defects. We encourage the nascent field of embryo mechanics to adopt standard engineering terms and test methods so that studies of diverse organisms can be compared and universal biomechanical principles can be revealed. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Morphogenesis in bat wings: linking development, evolution and ecology.

    Science.gov (United States)

    Adams, Rick A

    2008-01-01

    The evolution of powered flight in mammals required specific developmental shifts from an ancestral limb morphology to one adapted for flight. Through studies of comparative morphogenesis, investigators have quantified points and rates of divergence providing important insights into how wings evolved in mammals. Herein I compare growth,development and skeletogenesis of forelimbs between bats and the more ancestral state provided by the rat (Rattus norvegicus)and quantify growth trajectories that illustrate morphological divergence both developmentally and evolutionarily. In addition, I discuss how wing shape is controlled during morphogenesis by applying multivariate analyses of wing bones and wing membranes and discuss how flight dynamics are stabilized during flight ontogeny. Further, I discuss the development of flight in bats in relation to the ontogenetic niche and how juveniles effect populational foraging patterns. In addition, I provide a hypothetical ontogenetic landscape model that predicts how and when selection is most intense during juvenile morphogenesis and test this model with data from a population of the little brown bat, Myotis lucifugus. (c) 2007 S. Karger AG, Basel

  11. Control of mucosal virus infection by influenza nucleoprotein-specific CD8+ cytotoxic T lymphocytes

    Directory of Open Access Journals (Sweden)

    Couch Robert B

    2007-06-01

    Full Text Available Abstract Background MHC class I-restricted CD8+ cytotoxic T lymphocytes (CTL are thought to play a major role in clearing virus and promoting recovery from influenza infection and disease. This has been demonstrated for clearance of influenza virus from the lungs of infected mice. However, human influenza infection is primarily a respiratory mucosal infection involving the nasopharynx and tracheobronchial tree. The role of CD8+ CTL directed toward the influenza nucleoprotein (NP in defense against influenza virus infection at the respiratory mucosa was evaluated in two separate adoptive transfer experiments. Methods Influenza nucleoprotein (NP-specific CD8+ CTL were generated from splenocytes obtained from Balb/c mice previously primed with influenza A/Taiwan/1/86 (H1N1 infection or with influenza A/PR/8/34 (H1N1-derived NP plasmid DNA vaccine followed by infection with A/Hong Kong/68 (H3N2 virus. After in vitro expansion by exposure to an influenza NP-vaccinia recombinant, highly purified CD8+ T cells exhibited significant lysis in vitro of P815 target cells infected with A/Hong Kong/68 (H3N2 virus while the CD8- fraction (CD4+ T cells, B cells and macrophages had no CTL activity. Purified CD8+ and CD8- T cells (1 × 107 were injected intravenously or interperitoneally into naive mice four hours prior to intranasal challenge with A/HK/68 (H3N2 virus. Results The adoptively transferred NP-vaccinia-induced CD8+ T cells caused significant reduction of virus titers in both the lungs and nasal passages when compared to CD8- cells. Neither CD8+ nor CD8- T cells from cultures stimulated with HIV gp120-vaccinia recombinant reduced virus titers. Conclusion The present data demonstrate that influenza NP-specific CD8+ CTL can play a direct role in clearance of influenza virus from the upper respiratory mucosal surfaces.

  12. Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins.

    Science.gov (United States)

    Zaitseva, Marina; Thomas, Antonia; Meseda, Clement A; Cheung, Charles Y K; Diaz, Claudia G; Xiang, Yan; Crotty, Shane; Golding, Hana

    2017-08-01

    Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 10 5  pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2-3 days and before primary lesions are visible at the site of scarification. Extension of survival in nude mice treated with a combination of anti-B5 and anti-L1 MAbs 24 h post challenge correlated with a significant reduction in viral load at the site of scarification and delayed systemic dissemination. Nude mice reconstituted with 10 4  T cells prior to challenge with IHD-J-Luc, and treated with MAbs post-challenge, survived infection, cleared the virus from all organs and scarification site, and developed anti-VACV IgG and VACV-specific polyfunctional CD8 + T cells that co-expressed the degranulation marker CD107a, and IFNγ and TNFα cytokines. All T cell reconstituted mice survived intranasal re-challenge with IHD-J-Luc (10 4  pfu) two months after the primary infection. Thus, using BLI to monitor VACV replication in a PV model, we showed that anti-VACV MAbs administered post challenge extended survival of nude mice and protected T cell reconstituted nude mice from lethality by reducing replication at the site of scarification and systemic dissemination of VACV. Published by Elsevier B.V.

  13. Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain

    DEFF Research Database (Denmark)

    Mikkelsen, Marianne; Holst, Peter Johannes; Bukh, Jens

    2011-01-01

    memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice...

  14. Molecular and Cellular Dynamics in the Skin, the Lymph Nodes, and the Blood of the Immune Response to Intradermal Injection of Modified Vaccinia Ankara Vaccine

    Directory of Open Access Journals (Sweden)

    Pierre Rosenbaum

    2018-04-01

    Full Text Available New vaccine design approaches would be greatly facilitated by a better understanding of the early systemic changes, and those that occur at the site of injection, responsible for the installation of a durable and oriented protective response. We performed a detailed characterization of very early infection and host response events following the intradermal administration of the modified vaccinia virus Ankara as a live attenuated vaccine model in non-human primates. Integrated analysis of the data obtained from in vivo imaging, histology, flow cytometry, multiplex cytokine, and transcriptomic analysis using tools derived from systems biology, such as co-expression networks, showed a strong early local and systemic inflammatory response that peaked at 24 h, which was then progressively replaced by an adaptive response during the installation of the host response to the vaccine. Granulocytes, macrophages, and monocytoid cells were massively recruited during the local innate response in association with local productions of GM-CSF, IL-1β, MIP1α, MIP1β, and TNFα. We also observed a rapid and transient granulocyte recruitment and the release of IL-6 and IL-1RA, followed by a persistent phase involving inflammatory monocytes. This systemic inflammation was confirmed by molecular signatures, such as upregulations of IL-6 and TNF pathways and acute phase response signaling. Such comprehensive approaches improve our understanding of the spatiotemporal orchestration of vaccine-elicited immune response, in a live-attenuated vaccine model, and thus contribute to rational vaccine development.

  15. Development and evaluation of recombinant MVA viruses expressing bohv-1 glycoprotein D

    OpenAIRE

    Ferrer, María Florencia

    2010-01-01

    El virus vaccinia Ankara modificado (MVA) es un virus altamente atenuado que se utiliza eficientemente como vector viral no replicativo para el desarrollo de nuevas vacunas. En este trabajo de Tesis se desarrolló un nuevo inmunógeno basado en MVA que expresa como antígeno de interés la glicoproteína D (versión secretada, gDs) del virus herpes bovino tipo I (BoHV-1), un agente infeccioso ampliamente distribuido en Argentina. Primeramente, se diseñó y construyó el vector de transferencia para o...

  16. Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex

    Directory of Open Access Journals (Sweden)

    Fuchs Pinhas

    2009-09-01

    Full Text Available Abstract Background Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes. This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. Results We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within" or by infection with a high amount of virus particles per cell (fusion "from without". Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Conclusion Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

  17. Genome-wide comparison of cowpox viruses reveals a new clade related to Variola virus.

    Directory of Open Access Journals (Sweden)

    Piotr Wojtek Dabrowski

    Full Text Available Zoonotic infections caused by several orthopoxviruses (OPV like monkeypox virus or vaccinia virus have a significant impact on human health. In Europe, the number of diagnosed infections with cowpox viruses (CPXV is increasing in animals as well as in humans. CPXV used to be enzootic in cattle; however, such infections were not being diagnosed over the last decades. Instead, individual cases of cowpox are being found in cats or exotic zoo animals that transmit the infection to humans. Both animals and humans reveal local exanthema on arms and legs or on the face. Although cowpox is generally regarded as a self-limiting disease, immunosuppressed patients can develop a lethal systemic disease resembling smallpox. To date, only limited information on the complex and, compared to other OPV, sparsely conserved CPXV genomes is available. Since CPXV displays the widest host range of all OPV known, it seems important to comprehend the genetic repertoire of CPXV which in turn may help elucidate specific mechanisms of CPXV pathogenesis and origin. Therefore, 22 genomes of independent CPXV strains from clinical cases, involving ten humans, four rats, two cats, two jaguarundis, one beaver, one elephant, one marah and one mongoose, were sequenced by using massive parallel pyrosequencing. The extensive phylogenetic analysis showed that the CPXV strains sequenced clearly cluster into several distinct clades, some of which are closely related to Vaccinia viruses while others represent different clades in a CPXV cluster. Particularly one CPXV clade is more closely related to Camelpox virus, Taterapox virus and Variola virus than to any other known OPV. These results support and extend recent data from other groups who postulate that CPXV does not form a monophyletic clade and should be divided into multiple lineages.

  18. Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

    Science.gov (United States)

    Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

    2018-04-03

    High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

  19. Phase 1 safety and immunogenicity evaluation of ADMVA, a multigenic, modified vaccinia Ankara-HIV-1 B'/C candidate vaccine.

    Directory of Open Access Journals (Sweden)

    Sandhya Vasan

    Full Text Available BACKGROUND: We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1x10(7 (low, 5x10(7 (mid, or 2.5x10(8 pfu (high] volunteers were randomized in a 3:1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA. ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNgamma ELISpot response rate to any HIV antigen was 0/12 (0% in the placebo group, 3/12 (25% in the low dosage group, 6/12 (50% in the mid dosage group, and 8/13 (62% in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%, 8/13 (62%, 6/12 (50% and 10/13 (77% in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement

  20. A Randomized, Double-Blind, Placebo-Controlled Phase II Trial Investigating the Safety and Immunogenicity of Modified Vaccinia Ankara Smallpox Vaccine (MVA-BN®) in 56-80-Year-Old Subjects.

    Science.gov (United States)

    Greenberg, Richard N; Hay, Christine M; Stapleton, Jack T; Marbury, Thomas C; Wagner, Eva; Kreitmeir, Eva; Röesch, Siegfried; von Krempelhuber, Alfred; Young, Philip; Nichols, Richard; Meyer, Thomas P; Schmidt, Darja; Weigl, Josef; Virgin, Garth; Arndtz-Wiedemann, Nathaly; Chaplin, Paul

    2016-01-01

    Modified Vaccinia Ankara MVA-BN® is a live, highly attenuated, viral vaccine under advanced development as a non-replicating smallpox vaccine. In this Phase II trial, the safety and immunogenicity of Modified Vaccinia Ankara MVA-BN® (MVA) was assessed in a 56-80 years old population. MVA with a virus titer of 1 x 108 TCID50/dose was administered via subcutaneous injection to 56-80 year old vaccinia-experienced subjects (N = 120). Subjects received either two injections of MVA (MM group) or one injection of Placebo and one injection of MVA (PM group) four weeks apart. Safety was evaluated by assessment of adverse events (AE), focused physical exams, electrocardiogram recordings and safety laboratories. Solicited AEs consisted of a set of pre-defined expected local reactions (erythema, swelling, pain, pruritus, and induration) and systemic symptoms (body temperature, headache, myalgia, nausea and fatigue) and were recorded on a memory aid for an 8-day period following each injection. The immunogenicity of the vaccine was evaluated in terms of humoral immune responses measured with a vaccinia-specific enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT) before and at different time points after vaccination. Vaccinations were well tolerated by all subjects. No serious adverse event related to MVA and no case of myopericarditis was reported. The overall incidence of unsolicited AEs was similar in both groups. For both groups immunogenicity responses two weeks after the final vaccination (i.e. Visit 4) were as follows: Seroconversion (SC) rates (doubling of titers from baseline) in vaccine specific antibody titers measured by ELISA were 83.3% in Group MM and 82.8% in Group PM (difference 0.6% with 95% exact CI [-13.8%, 15.0%]), and 90.0% for Group MM and 77.6% for Group PM measured by PRNT (difference 12.4% with 95% CI of [-1.1%, 27.0%]). Geometric mean titers (GMT) measured by ELISA two weeks after the final vaccination for Group

  1. Infection cycles of large DNA viruses: Emerging themes and underlying questions

    International Nuclear Information System (INIS)

    Mutsafi, Yael; Fridmann-Sirkis, Yael; Milrot, Elad; Hevroni, Liron; Minsky, Abraham

    2014-01-01

    The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these ‘nuclear-like’ organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the ‘stargate’ portal that is used for genome release. Such a ‘division of labor’ is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. - Highlights: • The discovery of giant DNA viruses blurs the distinction between viruses and cells. • Mimivirus and Vaccinia replicate exclusively in their host cytoplasm. • Mimivirus genome is delivered through a unique portal coined the Stargate. • Generation of Mimivirus internal membrane proceeds through a novel pathway

  2. Infection cycles of large DNA viruses: Emerging themes and underlying questions

    Energy Technology Data Exchange (ETDEWEB)

    Mutsafi, Yael, E-mail: yael.mutsafi@weizmann.ac.il; Fridmann-Sirkis, Yael; Milrot, Elad; Hevroni, Liron; Minsky, Abraham, E-mail: avi.minsky@weizmann.ac.il

    2014-10-15

    The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these ‘nuclear-like’ organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the ‘stargate’ portal that is used for genome release. Such a ‘division of labor’ is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. - Highlights: • The discovery of giant DNA viruses blurs the distinction between viruses and cells. • Mimivirus and Vaccinia replicate exclusively in their host cytoplasm. • Mimivirus genome is delivered through a unique portal coined the Stargate. • Generation of Mimivirus internal membrane proceeds through a novel pathway.

  3. Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

    International Nuclear Information System (INIS)

    Domber, E.A.

    1986-01-01

    These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

  4. Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus

    International Nuclear Information System (INIS)

    Upton, C.; McFadden, G.

    1986-01-01

    The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK Gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 187 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively

  5. Activation of cross-reactive mucosal T and B cell responses in human nasopharynx-associated lymphoid tissue in vitro by Modified Vaccinia Ankara-vectored influenza vaccines.

    Science.gov (United States)

    Mullin, Jennifer; Ahmed, Muhammed S; Sharma, Ravi; Upile, Navdeep; Beer, Helen; Achar, Priya; Puksuriwong, Suttida; Ferrara, Francesca; Temperton, Nigel; McNamara, Paul; Lambe, Teresa; Gilbert, Sarah C; Zhang, Qibo

    2016-03-29

    Recent efforts have been focused on the development of vaccines that could induce broad immunity against influenza virus, either through T cell responses to conserved internal antigens or B cell response to cross-reactive haemagglutinin (HA). We studied the capacity of Modified Vaccinia Ankara (MVA)-vectored influenza vaccines to induce cross-reactive immunity to influenza virus in human nasopharynx-associated lymphoid tissue (NALT) in vitro. Adenotonsillar cells were isolated and stimulated with MVA vaccines expressing either conserved nucleoprotein (NP) and matrix protein 1 (M1) (MVA-NP-M1) or pandemic H1N1 HA (MVA-pdmH1HA). The MVA vaccine uptake and expression, and T and B cell responses were analyzed. MVA-vectored vaccines were highly efficient infecting NALT and vaccine antigens were highly expressed by B cells. MVA-NP-M1 elicited T cell response with greater numbers of IFNγ-producing CD4+ T cells and tissue-resident memory T cells than controls. MVA-pdmH1HA induced cross-reactive anti-HA antibodies to a number of influenza subtypes, in an age-dependent manner. The cross-reactive antibodies include anti-avian H5N1 and mainly target HA2 domain. MVA vaccines are efficient in infecting NALT and the vaccine antigen is highly expressed by B cells. MVA vaccines expressing conserved influenza antigens induce cross-reactive T and B cell responses in human NALT in vitro, suggesting the potential as mucosal vaccines for broader immunity against influenza. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Giga-voxel computational morphogenesis for structural design

    DEFF Research Database (Denmark)

    Aage, Niels; Andreassen, Erik; Lazarov, Boyan Stefanov

    2017-01-01

    In the design of industrial products ranging from hearing aidsto automobiles and aeroplanes, material is distributed so as to maximize the performance and minimize the cost. Historically, human intuition and insight have driven the evolution of mechanical design, recently assisted by computer...... aeroplane wing designs, which translates into are duction in fuel consumption of about 40–200 tonnes per year per aeroplane. Our morphogenesis process is generally applicable, not only to mechanical design, but also to flow systems3, antennas4,nano-optics5 and micro-systems6,7...

  7. Physics and the canalization of morphogenesis: a grand challenge in organismal biology

    International Nuclear Information System (INIS)

    Von Dassow, Michelangelo; Davidson, Lance A

    2011-01-01

    Morphogenesis takes place against a background of organism-to-organism and environmental variation. Therefore, fundamental questions in the study of morphogenesis include: How are the mechanical processes of tissue movement and deformation affected by that variability, and in turn, how do the mechanic of the system modulate phenotypic variation? We highlight a few key factors, including environmental temperature, embryo size and environmental chemistry that might perturb the mechanics of morphogenesis in natural populations. Then we discuss several ways in which mechanics—including feedback from mechanical cues—might influence intra-specific variation in morphogenesis. To understand morphogenesis it will be necessary to consider whole-organism, environment and evolutionary scales because these larger scales present the challenges that developmental mechanisms have evolved to cope with. Studying the variation organisms express and the variation organisms experience will aid in deciphering the causes of birth defects

  8. Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis.

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2010-08-01

    Full Text Available In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV, is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20 and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20 were blocked in encapsidation (no virus after blind passages but could be rescued if the capsid and 2C(ATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i genome replication is known to be stringently linked to translation, (ii morphogenesis is known to be stringently linked to genome replication, (iii newly synthesized 2C(ATPase is an essential component of the replication complex, and (iv 2C(ATPase has specific affinity to capsid protein(s. These conditions lead to morphogenesis at the site where newly

  9. Dynamics of Spore Coat Morphogenesis in Bacillus subtilis

    Science.gov (United States)

    McKenney, Peter T.; Eichenberger, Patrick

    2011-01-01

    SUMMARY Spores of Bacillus subtilis are encased in a protective coat made up of at least 70 proteins. The structure of the spore coat has been examined using a variety of genetic, imaging and biochemical techniques, however, the majority of these studies have focused on mature spores. In this study we use a library of 41 spore coat proteins fused to the Green Fluorescent Protein (GFP) to examine spore coat morphogenesis over the time-course of sporulation. We found considerable diversity in the localization dynamics of coat proteins and were able to establish 6 classes based on localization kinetics. Localization dynamics correlate well with the known transcriptional regulators of coat gene expression. Previously, we described the existence of multiple layers in the mature spore coat. Here, we find that the spore coat initially assembles a scaffold that is organized into multiple layers on one pole of the spore. The coat then encases the spore in multiple coordinated waves. Encasement is driven, at least partially, by transcription of coat genes and deletion of sporulation transcription factors arrests encasement. We also identify the trans-compartment SpoIIIAH-SpoIIQ channel as necessary for encasement. This is the first demonstration of a forespore contribution to spore coat morphogenesis. PMID:22171814

  10. Conjoined twins: morphogenesis of the heart and a review.

    Science.gov (United States)

    Gilbert-Barness, Enid; Debich-Spicer, Diane; Opitz, John M

    2003-08-01

    Five cases of conjoined twins have been studied. These included three thoracopagus twins, one monocephalus diprosopus (prosop = face), and one dicephalus dipus dibrachus. The thoracopagus twins were conjoined only from the upper thorax to the umbilicus with a normal foregut. These three cases shared a single complex multiventricular heart, one with a four chambered heart with one atrium and one ventricle belonging to each twin with complex venous and arterial connection; two had a seven chambered heart with four atria and three ventricles. The mono-cephalus diprosopus twins had a single heart with tetralogy of Fallot. The dicephalus twins had two separate axial skeletons to the sacrum, two separate hearts were connected between the right atria with a shared inferior vena cava. Thoracopagus twinning is associated with complex cardiac malformations. The cardiac anlagen in cephalopagus or diprosopus are diverted and divided along with the entire rostral end of the embryonic disc and result in two relatively normal shared hearts. However, in thoracopagus twins the single heart is multiventricular and suggests very early union with fusion of the cardiac anlagen before significant differentiation. Cardiac morphogenesis in conjoined twins therefore appears to depend on the site of the conjoined fusion and the temporal and spatial influence that determines morphogenesis as well as abnormally oriented embryonic axes. Copyright 2003 Wiley-Liss, Inc.

  11. Tissue Motion and Assembly During Early Cardiovascular Morphogenesis

    Science.gov (United States)

    Rongish, Brenda

    2010-03-01

    Conventional dogma in the field of cardiovascular developmental biology suggests that cardiac precursor cells migrate to the embryonic midline to form a tubular heart. These progenitors are believed to move relative to their extracellular matrix (ECM); responding to stimulatory and inhibitory cues in their environment. The tubular heart that is formed by 30 hours post fertilization is comprised of two concentric layers: the muscular myocardium and the endothelial-like endocardium, which are separated by a thick layer of ECM believed to be secreted predominantly by the myocardial cells. Here we describe the origin and motility of fluorescently tagged endocardial precursors in transgenic (Tie1-YFP) quail embryos (R. Lansford, Caltech) using epifluorescence time-lapse imaging. To visualize the environment of migrating endocardial progenitors, we labeled two ECM components, fibronectin and fibrillin-2, via in vivo microinjection of fluorochrome-conjugated monoclonal antibodies. Dynamic imaging was performed at stages encompassing tubular heart assembly and early looping. We established the motion of endocardial precursor cells and presumptive cardiac ECM fibrils using both object tracking and particle image velocimetry (image cross correlation). We determined the relative importance of directed cell autonomous motility versus passive tissue movements in endocardial morphogenesis. The data show presumptive endocardial cells and cardiac ECM fibrils are swept passively into the anterior and posterior poles of the elongating tubular heart. These quantitative data indicate the contribution of cell autonomous motility displayed by endocardial precursors is limited. Thus, tissue motion drives most of the cell displacements during endocardial morphogenesis.

  12. Notochord morphogenesis in mice: Current understanding & open questions.

    Science.gov (United States)

    Balmer, Sophie; Nowotschin, Sonja; Hadjantonakis, Anna-Katerina

    2016-05-01

    The notochord is a structure common to all chordates, and the feature that the phylum Chordata has been named after. It is a rod-like mesodermal structure that runs the anterior-posterior length of the embryo, adjacent to the ventral neural tube. The notochord plays a critical role in embryonic tissue patterning, for example the dorsal-ventral patterning of the neural tube. The cells that will come to form the notochord are specified at gastrulation. Axial mesodermal cells arising at the anterior primitive streak migrate anteriorly as the precursors of the notochord and populate the notochordal plate. Yet, even though a lot of interest has centered on investigating the functional and structural roles of the notochord, we still have a very rudimentary understanding of notochord morphogenesis. The events driving the formation of the notochord are rapid, taking place over the period of approximately a day in mice. In this commentary, we provide an overview of our current understanding of mouse notochord morphogenesis, from the initial specification of axial mesendodermal cells at the primitive streak, the emergence of these cells at the midline on the surface of the embryo, to their submergence and organization of the stereotypically positioned notochord. We will also discuss some key open questions. Developmental Dynamics 245:547-557, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. The unfolded protein response is required for dendrite morphogenesis

    Science.gov (United States)

    Wei, Xing; Howell, Audrey S; Dong, Xintong; Taylor, Caitlin A; Cooper, Roshni C; Zhang, Jianqi; Zou, Wei; Sherwood, David R; Shen, Kang

    2015-01-01

    Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/ grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors. DOI: http://dx.doi.org/10.7554/eLife.06963.001 PMID:26052671

  14. The Role of B Cells for in Vivo T Cell Responses to a Friend Virus-Induced Leukemia

    Science.gov (United States)

    Schultz, Kirk R.; Klarnet, Jay P.; Gieni, Randall S.; Hayglass, Kent T.; Greenberg, Philip D.

    1990-08-01

    B cells can function as antigen-presenting cells and accessory cells for T cell responses. This study evaluated the role of B cells in the induction of protective T cell immunity to a Friend murine leukemia virus (F-MuLV)-induced leukemia (FBL). B cell-deficient mice exhibited significantly reduced tumor-specific CD4^+ helper and CD8^+ cytotoxic T cell responses after priming with FBL or a recombinant vaccinia virus containing F-MuLV antigens. Moreover, these mice had diminished T cell responses to the vaccinia viral antigens. Tumor-primed T cells transferred into B cell-deficient mice effectively eradicated disseminated FBL. Thus, B cells appear necessary for efficient priming but not expression of tumor and viral T cell immunity.

  15. The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

    Science.gov (United States)

    1995-04-13

    vaccinia virus-T7 RNA polymerase s y stem for e xpression of target genes . Mol . Cell . BioI . 7 : 2538-2544 . Gagneten , S ., Gout , 0 ., Dubois-Dalcq...glycoprotein. These results showed f or the first time that two murine CEA- related genes can be co-expressed in some cell lines from inbred mice...49 Southern Hybridization ................ . ............ 50 Subcloning of PCR Products and Gene Cloning ........ 51 Growth

  16. Prospective surveillance for cardiac adverse events in healthy adults receiving modified vaccinia Ankara vaccines: a systematic review.

    Directory of Open Access Journals (Sweden)

    Marnie L Elizaga

    Full Text Available Vaccinia-associated myo/pericarditis was observed during the US smallpox vaccination (DryVax campaign initiated in 2002. A highly-attenuated vaccinia strain, modified vaccinia Ankara (MVA has been evaluated in clinical trials as a safer alternative to DryVax and as a vector for recombinant vaccines. Due to the lack of prospectively collected cardiac safety data, the US Food and Drug Administration required cardiac screening and surveillance in all clinical trials of MVA since 2004. Here, we report cardiac safety surveillance from 6 phase I trials of MVA vaccines.Four clinical research organizations contributed cardiac safety data using common surveillance methods in trials administering MVA or recombinant MVA vaccines to healthy participants. 'Routine cardiac investigations' (ECGs and cardiac enzymes obtained 2 weeks after injections of MVA or MVA-HIV recombinants, or placebo-controls, and 'Symptom-driven cardiac investigations' are reported. The outcome measure is the number of participants who met the CDC-case definition for vaccinia-related myo/pericarditis or who experienced cardiac adverse events from an MVA vaccine.Four hundred twenty-five study participants had post-vaccination safety data analyzed, 382 received at least one MVA-containing vaccine and 43 received placebo; 717 routine ECGs and 930 cardiac troponin assays were performed. Forty-five MVA recipients (12% had additional cardiac testing performed; 22 for cardiac symptoms, 19 for ECG/laboratory changes, and 4 for cardiac symptoms with an ECG/laboratory change. No participant had evidence of symptomatic or asymptomatic myo/pericarditis meeting the CDC-case definition and judged to be related to an MVA vaccine.Prospective surveillance of MVA recipients for myo/pericarditis did not detect cardiac adverse reactions in 382 study participants.ClinicalTrials.gov NCT00082446 NCT003766090 NCT00252148 NCT00083603 NCT00301184 NCT00428337.

  17. Prospective surveillance for cardiac adverse events in healthy adults receiving modified vaccinia Ankara vaccines: a systematic review.

    Science.gov (United States)

    Elizaga, Marnie L; Vasan, Sandhya; Marovich, Mary A; Sato, Alicia H; Lawrence, Dale N; Chaitman, Bernard R; Frey, Sharon E; Keefer, Michael C

    2013-01-01

    Vaccinia-associated myo/pericarditis was observed during the US smallpox vaccination (DryVax) campaign initiated in 2002. A highly-attenuated vaccinia strain, modified vaccinia Ankara (MVA) has been evaluated in clinical trials as a safer alternative to DryVax and as a vector for recombinant vaccines. Due to the lack of prospectively collected cardiac safety data, the US Food and Drug Administration required cardiac screening and surveillance in all clinical trials of MVA since 2004. Here, we report cardiac safety surveillance from 6 phase I trials of MVA vaccines. Four clinical research organizations contributed cardiac safety data using common surveillance methods in trials administering MVA or recombinant MVA vaccines to healthy participants. 'Routine cardiac investigations' (ECGs and cardiac enzymes obtained 2 weeks after injections of MVA or MVA-HIV recombinants, or placebo-controls), and 'Symptom-driven cardiac investigations' are reported. The outcome measure is the number of participants who met the CDC-case definition for vaccinia-related myo/pericarditis or who experienced cardiac adverse events from an MVA vaccine. Four hundred twenty-five study participants had post-vaccination safety data analyzed, 382 received at least one MVA-containing vaccine and 43 received placebo; 717 routine ECGs and 930 cardiac troponin assays were performed. Forty-five MVA recipients (12%) had additional cardiac testing performed; 22 for cardiac symptoms, 19 for ECG/laboratory changes, and 4 for cardiac symptoms with an ECG/laboratory change. No participant had evidence of symptomatic or asymptomatic myo/pericarditis meeting the CDC-case definition and judged to be related to an MVA vaccine. Prospective surveillance of MVA recipients for myo/pericarditis did not detect cardiac adverse reactions in 382 study participants. ClinicalTrials.gov NCT00082446 NCT003766090 NCT00252148 NCT00083603 NCT00301184 NCT00428337.

  18. Infection cycles of large DNA viruses: emerging themes and underlying questions.

    Science.gov (United States)

    Mutsafi, Yael; Fridmann-Sirkis, Yael; Milrot, Elad; Hevroni, Liron; Minsky, Abraham

    2014-10-01

    The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these 'nuclear-like' organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the 'stargate' portal that is used for genome release. Such a 'division of labor' is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Inactivation of viruses in bubbling processes utilized for personal bioaerosol monitoring.

    Science.gov (United States)

    Agranovski, I E; Safatov, A S; Borodulin, A I; Pyankov, O V; Petrishchenko, V A; Sergeev, A N; Agafonov, A P; Ignatiev, G M; Sergeev, A A; Agranovski, V

    2004-12-01

    A new personal bioaerosol sampler has recently been developed and evaluated for sampling of viable airborne bacteria and fungi under controlled laboratory conditions and in the field. The operational principle of the device is based on the passage of air through porous medium immersed in liquid. This process leads to the formation of bubbles within the filter as the carrier gas passes through and thus provides effective mechanisms for aerosol removal. As demonstrated in previous studies, the culturability of sampled bacterium and fungi remained high for the entire 8-h sampling period. The present study is the first step of the evaluation of the new sampler for monitoring of viable airborne viruses. It focuses on the investigation of the inactivation rate of viruses in the bubbling process during 4 h of continuous operation. Four microbes were used in this study, influenza, measles, mumps, and vaccinia viruses. It was found that the use of distilled water as the collection fluid was associated with a relatively high decay rate. A significant improvement was achieved by utilizing virus maintenance fluid prepared by using Hank's solution with appropriate additives. The survival rates of the influenza, measles, and mumps viruses were increased by 1.4 log, 0.83 log, and 0.82 log, respectively, after the first hour of operation compared to bubbling through the sterile water. The same trend was observed throughout the entire 4-h experiment. There was no significant difference observed only for the robust vaccinia virus.

  20. Variations in resistance of viruses from different groups to chemico-physical decontamination methods

    Energy Technology Data Exchange (ETDEWEB)

    Mahnel, H

    1979-01-01

    The resistance of a total of 13 different viruses to some important chemico-physical influences was studied under uniform experimental conditions. Stability in tape water, thermostability and sensitivity to anodic oxidation, gamma radiation, some virucidal substances and several commercial disinfectants were tested. In evaluating the results, an attempt is made to rank the viruses investigated according to their sensitivity. On average a bovine parvovirus, and also a reovirus and three enteroviruses, proved most stable. These were followed by infectious canine hepatitis (adenoviruses). Newcastle disease (paramyxoviruses) and vaccinia (poxviruses) demonstrating less resistance. In all the tests an orthomyxovirus (influenza A), a rhabdovirus (pseudorabies) and a togavirus (sindbis) proved to have relatively low resistance.

  1. Mechanical basis of morphogenesis and convergent evolution of spiny seashells

    KAUST Repository

    Chirat, R.; Moulton, D. E.; Goriely, A.

    2013-01-01

    Convergent evolution is a phenomenon whereby similar traits evolved independently in not closely related species, and is often interpreted in functional terms. Spines in mollusk seashells are classically interpreted as having repeatedly evolved as a defense in response to shell-crushing predators. Here we consider the morphogenetic process that shapes these structures and underlies their repeated emergence. We develop a mathematical model for spine morphogenesis based on the mechanical interaction between the secreting mantle edge and the calcified shell edge to which the mantle adheres during shell growth. It is demonstrated that a large diversity of spine structures can be accounted for through small variations in control parameters of this natural mechanical process. This physical mechanism suggests that convergent evolution of spines can be understood through a generic morphogenetic process, and provides unique perspectives in understanding the phenotypic evolution of this second largest phylum in the animal kingdom.

  2. Some peculiarities of inflorescences morphogenesis in Brexia (Celastraceae

    Directory of Open Access Journals (Sweden)

    Ivan A. Savinov

    2013-04-01

    Full Text Available Comparative analysis of inflorescences structure for 6 species of the Brexia(according to the last revision by Schatz & Lowry II (2004 is conducted. For one species, B. madagascariensis, the shoots growth and development, inflorescence morphogenesis details are studied. It is determined inflorescences of Brexiaspecies (have described in literature as cymes, pseudo-umbellate, corymbiform, sessile in fascicles; and including for some species cauliflory presents a different variations of ancestral form transformation – dichasial system (closed thyrse. Apparently, presence of a big bracts may be consider as ancestral, plesiomorphic character for the genus; and derivate ones – reduction of bracts and presence of minute bracteoles in pedicel basis only. Inflorescences of Brexiain typically may be considered as bracteous. Process of reduction the number of clusters and separate flowers is accompanied by different variations of their transformations.

  3. Complex dynamics and morphogenesis an introduction to nonlinear science

    CERN Document Server

    Misbah, Chaouqi

    2017-01-01

    This book offers an introduction to the physics of nonlinear phenomena through two complementary approaches: bifurcation theory and catastrophe theory. Readers will be gradually introduced to the language and formalisms of nonlinear sciences, which constitute the framework to describe complex systems. The difficulty with complex systems is that their evolution cannot be fully predicted because of the interdependence and interactions between their different components. Starting with simple examples and working toward an increasing level of universalization, the work explores diverse scenarios of bifurcations and elementary catastrophes which characterize the qualitative behavior of nonlinear systems. The study of temporal evolution is undertaken using the equations that characterize stationary or oscillatory solutions, while spatial analysis introduces the fascinating problem of morphogenesis. Accessible to undergraduate university students in any discipline concerned with nonlinear phenomena (physics, mathema...

  4. The green seaweed Ulva: a model system to study morphogenesis.

    Science.gov (United States)

    Wichard, Thomas; Charrier, Bénédicte; Mineur, Frédéric; Bothwell, John H; Clerck, Olivier De; Coates, Juliet C

    2015-01-01

    Green macroalgae, mostly represented by the Ulvophyceae, the main multicellular branch of the Chlorophyceae, constitute important primary producers of marine and brackish coastal ecosystems. Ulva or sea lettuce species are some of the most abundant representatives, being ubiquitous in coastal benthic communities around the world. Nonetheless the genus also remains largely understudied. This review highlights Ulva as an exciting novel model organism for studies of algal growth, development and morphogenesis as well as mutualistic interactions. The key reasons that Ulva is potentially such a good model system are: (i) patterns of Ulva development can drive ecologically important events, such as the increasing number of green tides observed worldwide as a result of eutrophication of coastal waters, (ii) Ulva growth is symbiotic, with proper development requiring close association with bacterial epiphytes, (iii) Ulva is extremely developmentally plastic, which can shed light on the transition from simple to complex multicellularity and (iv) Ulva will provide additional information about the evolution of the green lineage.

  5. Mechanical basis of morphogenesis and convergent evolution of spiny seashells

    KAUST Repository

    Chirat, R.

    2013-03-25

    Convergent evolution is a phenomenon whereby similar traits evolved independently in not closely related species, and is often interpreted in functional terms. Spines in mollusk seashells are classically interpreted as having repeatedly evolved as a defense in response to shell-crushing predators. Here we consider the morphogenetic process that shapes these structures and underlies their repeated emergence. We develop a mathematical model for spine morphogenesis based on the mechanical interaction between the secreting mantle edge and the calcified shell edge to which the mantle adheres during shell growth. It is demonstrated that a large diversity of spine structures can be accounted for through small variations in control parameters of this natural mechanical process. This physical mechanism suggests that convergent evolution of spines can be understood through a generic morphogenetic process, and provides unique perspectives in understanding the phenotypic evolution of this second largest phylum in the animal kingdom.

  6. Turing mechanism underlying a branching model for lung morphogenesis.

    Science.gov (United States)

    Xu, Hui; Sun, Mingzhu; Zhao, Xin

    2017-01-01

    The mammalian lung develops through branching morphogenesis. Two primary forms of branching, which occur in order, in the lung have been identified: tip bifurcation and side branching. However, the mechanisms of lung branching morphogenesis remain to be explored. In our previous study, a biological mechanism was presented for lung branching pattern formation through a branching model. Here, we provide a mathematical mechanism underlying the branching patterns. By decoupling the branching model, we demonstrated the existence of Turing instability. We performed Turing instability analysis to reveal the mathematical mechanism of the branching patterns. Our simulation results show that the Turing patterns underlying the branching patterns are spot patterns that exhibit high local morphogen concentration. The high local morphogen concentration induces the growth of branching. Furthermore, we found that the sparse spot patterns underlie the tip bifurcation patterns, while the dense spot patterns underlies the side branching patterns. The dispersion relation analysis shows that the Turing wavelength affects the branching structure. As the wavelength decreases, the spot patterns change from sparse to dense, the rate of tip bifurcation decreases and side branching eventually occurs instead. In the process of transformation, there may exists hybrid branching that mixes tip bifurcation and side branching. Since experimental studies have reported that branching mode switching from side branching to tip bifurcation in the lung is under genetic control, our simulation results suggest that genes control the switch of the branching mode by regulating the Turing wavelength. Our results provide a novel insight into and understanding of the formation of branching patterns in the lung and other biological systems.

  7. Collective cell migration drives morphogenesis of the kidney nephron.

    Directory of Open Access Journals (Sweden)

    Aleksandr Vasilyev

    2009-01-01

    Full Text Available Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase-positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow-dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.

  8. Giga-voxel computational morphogenesis for structural design

    Science.gov (United States)

    Aage, Niels; Andreassen, Erik; Lazarov, Boyan S.; Sigmund, Ole

    2017-10-01

    In the design of industrial products ranging from hearing aids to automobiles and aeroplanes, material is distributed so as to maximize the performance and minimize the cost. Historically, human intuition and insight have driven the evolution of mechanical design, recently assisted by computer-aided design approaches. The computer-aided approach known as topology optimization enables unrestricted design freedom and shows great promise with regard to weight savings, but its applicability has so far been limited to the design of single components or simple structures, owing to the resolution limits of current optimization methods. Here we report a computational morphogenesis tool, implemented on a supercomputer, that produces designs with giga-voxel resolution—more than two orders of magnitude higher than previously reported. Such resolution provides insights into the optimal distribution of material within a structure that were hitherto unachievable owing to the challenges of scaling up existing modelling and optimization frameworks. As an example, we apply the tool to the design of the internal structure of a full-scale aeroplane wing. The optimized full-wing design has unprecedented structural detail at length scales ranging from tens of metres to millimetres and, intriguingly, shows remarkable similarity to naturally occurring bone structures in, for example, bird beaks. We estimate that our optimized design corresponds to a reduction in mass of 2-5 per cent compared to currently used aeroplane wing designs, which translates into a reduction in fuel consumption of about 40-200 tonnes per year per aeroplane. Our morphogenesis process is generally applicable, not only to mechanical design, but also to flow systems, antennas, nano-optics and micro-systems.

  9. Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes*

    Science.gov (United States)

    Hindman, Ryan; Gollnick, Paul

    2016-01-01

    Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5′ to 3′ translocase on single-stranded DNA. PMID:27189950

  10. A C-terminal, cysteine-rich site in poliovirus 2C(ATPase) is required for morphogenesis.

    Science.gov (United States)

    Wang, Chunling; Ma, Hsin-Chieh; Wimmer, Eckard; Jiang, Ping; Paul, Aniko V

    2014-06-01

    The morphogenesis of viruses belonging to the genus Enterovirus in the family Picornaviridae is still poorly understood despite decades-long investigations. However, we recently provided evidence that 2C(ATPase) gives specificity to poliovirus encapsidation through an interaction with capsid protein VP3. The polypeptide 2C(ATPase) is a highly conserved non-structural protein of enteroviruses with important roles in RNA replication, encapsidation and uncoating. We have identified a site (K279/R280) near the C terminus of the polypeptide that is required for morphogenesis. The aim of the current project was to search for additional functional sites near the C terminus of the 2C(ATPase) polypeptide, with particular interest in those that are required for encapsidation. We selected for analysis a cysteine-rich site of the polypeptide and constructed four mutants in which cysteines or a histidine was changed to an alanine. The RNA transcripts were transfected into HeLa cells yielding two lethal, one temperature-sensitive and one quasi-infectious mutants. All four mutants exhibited normal protein translation in vitro and three of them possessed severe RNA replication defects. The quasi-infectious mutant (C286A) yielded variants with a pseudo-reversion at the original site (A286D), but some also contained one additional mutation: A138V or M293V. The temperature-sensitive mutant (C272A/H273A) exhibited an encapsidation and possibly also an uncoating defect at 37 °C. Variants of this mutant revealed suppressor mutations at three different sites in the 2C(ATPase) polypeptide: A138V, M293V and K295R. We concluded that the cysteine-rich site near the C terminus of 2C(ATPase) is involved in encapsidation, possibly through an interaction with an upstream segment located between boxes A and B of the nucleotide-binding domain. © 2014 The Authors.

  11. Investigating Viruses during the Transformation of Molecular Biology.

    Science.gov (United States)

    Moss, Bernard

    2017-03-10

    This Reflections article describes my early work on viral enzymes and the discovery of mRNA capping, how my training in medicine and biochemistry merged as I evolved into a virologist, the development of viruses as vaccine vectors, and how scientific and technological developments during the 1970s and beyond set the stage for the interrogation of nearly every step in the reproductive cycle of vaccinia virus (VACV), a large DNA virus with about 200 genes. The reader may view this article as a work in progress, because I remain actively engaged in research at the National Institutes of Health (NIH) notwithstanding 50 memorable years there. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Spatial organization of adhesion: force-dependent regulation and function in tissue morphogenesis

    OpenAIRE

    Papusheva, Ekaterina; Heisenberg, Carl-Philipp

    2010-01-01

    The Heisenberg laboratory reviews the spatial organization of signalling complexes at cell–matrix and cell–cell contact sites and its impact on cell integrity, cellular polarity and tissue morphogenesis.

  13. A replicating modified vaccinia tiantan strain expressing an avian-derived influenza H5N1 hemagglutinin induce broadly neutralizing antibodies and cross-clade protective immunity in mice.

    Directory of Open Access Journals (Sweden)

    Haixia Xiao

    Full Text Available To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT as a vaccine vector, we constructed MVTTHA-QH and MVTTHA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTTHA-QH induced a significant level of neutralizing antibodies (Nabs against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4. Neutralization tests (NT and Haemagglutination inhibition (HI antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTTHA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTTS vaccine. However, MVTTHA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.

  14. The CRC orthologue from Pisum sativum shows conserved functions in carpel morphogenesis and vascular development.

    Science.gov (United States)

    Fourquin, Chloé; Primo, Amparo; Martínez-Fernández, Irene; Huet-Trujillo, Estefanía; Ferrándiz, Cristina

    2014-11-01

    CRABS CLAW (CRC) is a member of the YABBY family of transcription factors involved in carpel morphogenesis, floral determinacy and nectary specification in arabidopsis. CRC orthologues have been functionally characterized across angiosperms, revealing additional roles in leaf vascular development and carpel identity specification in Poaceae. These studies support an ancestral role of CRC orthologues in carpel development, while roles in vascular development and nectary specification appear to be derived. This study aimed to expand research on CRC functional conservation to the legume family in order to better understand the evolutionary history of CRC orthologues in angiosperms. CRC orthologues from Pisum sativum and Medicago truncatula were identified. RNA in situ hybridization experiments determined the corresponding expression patterns throughout flower development. The phenotypic effects of reduced CRC activity were investigated in P. sativum using virus-induced gene silencing. CRC orthologues from P. sativum and M. truncatula showed similar expression patterns, mainly restricted to carpels and nectaries. However, these expression patterns differed from those of other core eudicots, most importantly in a lack of abaxial expression in the carpel and in atypical expression associated with the medial vein of the ovary. CRC downregulation in pea caused defects in carpel fusion and style/stigma development, both typically associated with CRC function in eudicots, but also affected vascular development in the carpel. The data support the conserved roles of CRC orthologues in carpel fusion, style/stigma development and nectary development. In addition, an intriguing new aspect of CRC function in legumes was the unexpected role in vascular development, which could be shared by other species from widely diverged clades within the angiosperms, suggesting that this role could be ancestral rather than derived, as so far generally accepted. © The Author 2014. Published by

  15. Radiation enhanced reactivation of nuclear replicating mammalian viruses

    International Nuclear Information System (INIS)

    Bockstahler, L.E.; Lytle, C.D.

    1977-01-01

    When CV-1 monkey kidney cells were UV-irradiated (0 to 18 J/m 2 ) or X-irradiated (0 to 10 krads) before infection with UV-irradiated simian adenovirus 7 (SA7) or simian virus 40 (SV40), increases in the infectivity of these nuclear replicating viruses as measured by plaque formation were observed. These radiation enhanced reactivations, UV enhanced reactivation (UVER) and X-ray enhanced reactivation (X-ray ER), occurred both when virus infection immediately followed irradiation of the cells (except for X-ray ER with SA7) and when virus infection was delayed until 3 to 5 days after cell irradiation. While there was little difference in the levels of reactivation of UV-irradiated SV40 between immediate and delayed infection, delayed infection resulted in higher levels of reactivation of SA7. X-ray enhanced reactivation of UV-irradiated Herpes simplex virus persisted for several days but did not increase. Thus, X-ray enhanced and UV enhanced reactivations of these mammalian viruses were relatively long-lived effects. Essentially no UVER or X-ray ER was found in CV-1 cells for either immediate or delayed infection with UV-irradiated vaccinia virus or poliovirus, both of which replicate in the cell cytoplasm. These results suggest UVER and X-ray ER in mammalian cells may be restricted to viruses which are replicated in the cell nucleus. (author)

  16. In vitro inhibition of monkeypox virus production and spread by Interferon-β

    Directory of Open Access Journals (Sweden)

    Johnston Sara C

    2012-01-01

    Full Text Available Abstract Background The Orthopoxvirus genus contains numerous virus species that are capable of causing disease in humans, including variola virus (the etiological agent of smallpox, monkeypox virus, cowpox virus, and vaccinia virus (the prototypical member of the genus. Monkeypox is a zoonotic disease that is endemic in the Democratic Republic of the Congo and is characterized by systemic lesion development and prominent lymphadenopathy. Like variola virus, monkeypox virus is a high priority pathogen for therapeutic development due to its potential to cause serious disease with significant health impacts after zoonotic, accidental, or deliberate introduction into a naïve population. Results The purpose of this study was to investigate the prophylactic and therapeutic potential of interferon-β (IFN-β for use against monkeypox virus. We found that treatment with human IFN-β results in a significant decrease in monkeypox virus production and spread in vitro. IFN-β substantially inhibited monkeypox virus when introduced 6-8 h post infection, revealing its potential for use as a therapeutic. IFN-β induced the expression of the antiviral protein MxA in infected cells, and constitutive expression of MxA was shown to inhibit monkeypox virus infection. Conclusions Our results demonstrate the successful inhibition of monkeypox virus using human IFN-β and suggest that IFN-β could potentially serve as a novel safe therapeutic for human monkeypox disease.

  17. Ebola Virus and Marburg Virus

    Science.gov (United States)

    Ebola virus and Marburg virus Overview Ebola virus and Marburg virus are related viruses that cause hemorrhagic fevers — illnesses marked by severe bleeding (hemorrhage), organ failure and, in many ...

  18. Emergent properties during dorsal closure in Drosophila morphogenesis

    International Nuclear Information System (INIS)

    Peralta, X G; Toyama, Y; Edwards, G S; Kiehart, D P

    2008-01-01

    Dorsal closure is an essential stage of Drosophila development that is a model system for research in morphogenesis and biological physics. Dorsal closure involves an orchestrated interplay between gene expression and cell activities that produce shape changes, exert forces and mediate tissue dynamics. We investigate the dynamics of dorsal closure based on confocal microscopic measurements of cell shortening in living embryos. During the mid-stages of dorsal closure we find that there are fluctuations in the width of the leading edge cells but the time-averaged analysis of measurements indicate that there is essentially no net shortening of cells in the bulk of the leading edge, that contraction predominantly occurs at the canthi as part of the process for zipping together the two leading edges of epidermis and that the rate constant for zipping correlates with the rate of movement of the leading edges. We characterize emergent properties that regulate dorsal closure, i.e., a velocity governor and the coordination and synchronization of tissue dynamics

  19. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

    Full Text Available Embryonic heart morphogenesis (EHM is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling.

  20. The green seaweed Ulva: A model system to study morphogenesis

    Directory of Open Access Journals (Sweden)

    Thomas eWichard

    2015-02-01

    Full Text Available Green macroalgae, mostly represented by the Ulvophyceae, the main multicellular branch of the Chlorophyceae, constitute important primary producers of marine and brackish coastal ecosystems. Ulva or sea lettuce species are some of the most abundant representatives, being ubiquitous in coastal benthic communities around the world. Nonetheless the genus also remains largely understudied. This review highlights Ulva as an exciting novel model organism for studies of algal growth, development and morphogenesis as well as mutualistic interactions. The key reasons that Ulva is potentially such a good model system are: (i patterns of Ulva development can drive ecologically important events, such as the increasing number of green tides observed worldwide as a result of eutrophication of coastal waters, (ii Ulva growth is symbiotic, with proper development requiring close association with bacterial epiphytes, (iii Ulva is extremely developmentally plastic, which can shed light on the transition from simple to complex multicellularity and (iv Ulva will provide additional information about the evolution of the green lineage.

  1. Modeling the morphogenesis of brine channels in sea ice.

    Science.gov (United States)

    Kutschan, B; Morawetz, K; Gemming, S

    2010-03-01

    Brine channels are formed in sea ice under certain constraints and represent a habitat of different microorganisms. The complex system depends on a number of various quantities as salinity, density, pH value, or temperature. Each quantity governs the process of brine channel formation. There exists a strong link between bulk salinity and the presence of brine drainage channels in growing ice with respect to both the horizontal and vertical planes. We develop a suitable phenomenological model for the formation of brine channels both referring to the Ginzburg-Landau theory of phase transitions as well as to the chemical basis of morphogenesis according to Turing. It is possible to conclude from the critical wave number on the size of the structure and the critical parameters. The theoretically deduced transition rates have the same magnitude as the experimental values. The model creates channels of similar size as observed experimentally. An extension of the model toward channels with different sizes is possible. The microstructure of ice determines the albedo feedback and plays therefore an important role for large-scale global circulation models.

  2. The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis.

    Science.gov (United States)

    Huang, Yung-An; Kao, Chun-Wei; Liu, Kuang-Kai; Huang, Hou-Syun; Chiang, Ming-Han; Soo, Ching-Ren; Chang, Huan-Cheng; Chiu, Tzai-Wen; Chao, Jui-I; Hwang, Eric

    2014-11-05

    Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs in vitro and in vivo were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).

  3. The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis

    Science.gov (United States)

    Huang, Yung-An; Kao, Chun-Wei; Liu, Kuang-Kai; Huang, Hou-Syun; Chiang, Ming-Han; Soo, Ching-Ren; Chang, Huan-Cheng; Chiu, Tzai-Wen; Chao, Jui-I.; Hwang, Eric

    2014-11-01

    Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs in vitro and in vivo were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).

  4. NFIX Regulates Neural Progenitor Cell Differentiation During Hippocampal Morphogenesis

    Science.gov (United States)

    Heng, Yee Hsieh Evelyn; McLeay, Robert C.; Harvey, Tracey J.; Smith, Aaron G.; Barry, Guy; Cato, Kathleen; Plachez, Céline; Little, Erica; Mason, Sharon; Dixon, Chantelle; Gronostajski, Richard M.; Bailey, Timothy L.; Richards, Linda J.; Piper, Michael

    2014-01-01

    Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix−/− mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix−/− mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix−/− mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure. PMID:23042739

  5. Study on Seed Morphogenesis of Orobanchaceae in Taiwan

    Directory of Open Access Journals (Sweden)

    Jao-Shien Chen

    2011-11-01

    Full Text Available Seed morphogenesis of Orobanchaceae was not completely investigated previously. Here, we observed seed development of Orobanchaceous species in Taiwan using light and scanning electron microscopies. Results indicated that seeds of Aeginetia indica, Boschniakia himalaica, and Orobanche caerulescens all consisted of embryo, endosperm and testa. Ontogeny of the embryo in A. indica was Solanad type, while in both B. himalaica and O. caerulescens was Onagrad type. The mature embryos of the three species lacked embryonic organs, and their endosperm development was the cellular type and, at maturity, appeared as several cell layers of storage tissue. Ontogeny of the testa was all non-multiplicative, with the residues of the outermost cell layer and reticulately-thickened secondary walls of its cells at maturity. Mature seeds of A. indica and O. caerulescens were ovate whereas those of B. himalaica were oblate. As for Christisonia hookeri, due to lack of samples, only the cellular-typed endosperm was determined. The comparative development of Orobanchaceous seeds was discussed.

  6. Morphogenesis of Mammary Glands in Buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Amit Challana

    2014-01-01

    Full Text Available The present research was elucidated on the morphogenesis of mammary gland of buffalo during prenatal development. Total of 16 foetuses ranging from 1.2 cm (34 days to 108 cm CVRL (curved crown rump length (317 days were used for study. The study revealed that mammary line was first observed at 1.2 cm CVRL (34 days, mammary hillock at 1.7 cm (37 days, and mammary bud at 2.6 cm CVRL (41 days foetuses. Epidermal cone was found at 6.7 cm CVRL (58 days whereas primary and secondary ducts were observed at 7.4 cm CVRL (62 days and 15 cm CVRL (96 days, respectively. Connective tissue whorls were reported at 18.2 cm CVRL (110 days and internal elastic lamina and muscle layers at 24.1 cm CVRL (129 days. Lobules were observed at 29.3 cm CVRL (140 days, rosette of furstenberg at 39.5 cm CVRL (163 days, and keratin plug at 45.5 cm CVRL (176 days foetus. Primordia of sweat and sebaceous glands around hair follicle were seen at 21.2 cm CVRL (122 days of foetal life. Differentiation of all the skin layers along with cornification was observed at 69 cm (229 days in group III foetuses.

  7. Heart morphogenesis gene regulatory networks revealed by temporal expression analysis.

    Science.gov (United States)

    Hill, Jonathon T; Demarest, Bradley; Gorsi, Bushra; Smith, Megan; Yost, H Joseph

    2017-10-01

    During embryogenesis the heart forms as a linear tube that then undergoes multiple simultaneous morphogenetic events to obtain its mature shape. To understand the gene regulatory networks (GRNs) driving this phase of heart development, during which many congenital heart disease malformations likely arise, we conducted an RNA-seq timecourse in zebrafish from 30 hpf to 72 hpf and identified 5861 genes with altered expression. We clustered the genes by temporal expression pattern, identified transcription factor binding motifs enriched in each cluster, and generated a model GRN for the major gene batteries in heart morphogenesis. This approach predicted hundreds of regulatory interactions and found batteries enriched in specific cell and tissue types, indicating that the approach can be used to narrow the search for novel genetic markers and regulatory interactions. Subsequent analyses confirmed the GRN using two mutants, Tbx5 and nkx2-5 , and identified sets of duplicated zebrafish genes that do not show temporal subfunctionalization. This dataset provides an essential resource for future studies on the genetic/epigenetic pathways implicated in congenital heart defects and the mechanisms of cardiac transcriptional regulation. © 2017. Published by The Company of Biologists Ltd.

  8. Aquaporin 2 promotes cell migration and epithelial morphogenesis.

    Science.gov (United States)

    Chen, Ying; Rice, William; Gu, Zhizhan; Li, Jian; Huang, Jianmin; Brenner, Michael B; Van Hoek, Alfred; Xiong, Jianping; Gundersen, Gregg G; Norman, Jim C; Hsu, Victor W; Fenton, Robert A; Brown, Dennis; Lu, Hua A Jenny

    2012-09-01

    The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin β1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin β1 by mutating the RGD motif led to reduced endocytosis, retention of integrin β1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin β1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.

  9. Luteolin suppresses cancer cell proliferation by targeting vaccinia-related kinase 1.

    Directory of Open Access Journals (Sweden)

    Ye Seul Kim

    Full Text Available Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1 is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF, histone H3, and the cAMP response element (CRE-binding protein (CREB. In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.

  10. Virus-Like-Vaccines against HIV.

    Science.gov (United States)

    Andersson, Anne-Marie C; Schwerdtfeger, Melanie; Holst, Peter J

    2018-02-11

    Protection against chronic infections has necessitated the development of ever-more potent vaccination tools. HIV seems to be the most challenging foe, with a remarkable, poorly immunogenic and fragile surface glycoprotein and the ability to overpower the cell immune system. Virus-like-particle (VLP) vaccines have emerged as potent inducers of antibody and helper T cell responses, while replication-deficient viral vectors have yielded potent cytotoxic T cell responses. Here, we review the emerging concept of merging these two technologies into virus-like-vaccines (VLVs) for the targeting of HIV. Such vaccines are immunologically perceived as viruses, as they infect cells and produce VLPs in situ, but they only resemble viruses, as the replication defective vectors and VLPs cannot propagate an infection. The inherent safety of such a platform, despite robust particle production, is a distinct advantage over live-attenuated vaccines that must balance safety and immunogenicity. Previous studies have delivered VLVs encoded in modified Vaccinia Ankara vectors and we have developed the concept into a single-reading adenovirus-based technology capable of eliciting robust CD8⁺ and CD4⁺ T cells responses and trimer binding antibody responses. Such vaccines offer the potential to display the naturally produced immunogen directly and induce an integrated humoral and cellular immune response.

  11. Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

    Directory of Open Access Journals (Sweden)

    George G Daaboul

    Full Text Available Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm, while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm. Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

  12. Ebolavirus Vaccines: Progress in the Fight Against Ebola Virus Disease.

    Science.gov (United States)

    Wu, Xiao-Xin; Yao, Hang-Ping; Wu, Nan-Ping; Gao, Hai-Nv; Wu, Hai-Bo; Jin, Chang-Zhong; Lu, Xiang-Yun; Xie, Tian-Shen; Li, Lan-Juan

    2015-01-01

    Ebolaviruses are highly infectious pathogens that cause lethal Ebola virus disease (EVD) in humans and non-human primates (NHPs). Due to their high pathogenicity and transmissibility, as well as the potential to be misused as a bioterrorism agent, ebolaviruses would threaten the health of global populations if not controlled. In this review, we describe the origin and structure of ebolaviruses and the development of vaccines from the beginning of the 1980s, including conventional ebolavirus vaccines, DNA vaccines, Ebola virus-like particles (VLPs), vaccinia virus-based vaccines, Venezuelan equine encephalitis virus (VEEV)-like replicon particles, Kunjin virus-based vaccine, recombinant Zaire Ebolavirusx2206;VP30, recombinant cytomegalovirus (CMV)-based vaccines, recombinant rabies virus (RABV)-based vaccines, recombinant paramyxovirus-based vaccines, adenovirus-based vaccines and vesicular stomatitis virus (VSV)-based vaccines. No licensed vaccine or specific treatment is currently available to counteract ebolavirus infection, although DNA plasmids and several viral vector approaches have been evaluated as promising vaccine platforms. These vaccine candidates have been confirmed to be successful in protecting NHPs against lethal infection. Moreover, these vaccine candidates were successfully advanced to clinical trials. The present review provides an update of the current research on Ebola vaccines, with the aim of providing an overview on current prospects in the fight against EVD. © 2015 The Author(s) Published by S. Karger AG, Basel.

  13. Ebolavirus Vaccines: Progress in the Fight Against Ebola Virus Disease

    Directory of Open Access Journals (Sweden)

    Xiao-Xin Wu

    2015-11-01

    Full Text Available Ebolaviruses are highly infectious pathogens that cause lethal Ebola virus disease (EVD in humans and non-human primates (NHPs. Due to their high pathogenicity and transmissibility, as well as the potential to be misused as a bioterrorism agent, ebolaviruses would threaten the health of global populations if not controlled. In this review, we describe the origin and structure of ebolaviruses and the development of vaccines from the beginning of the 1980s, including conventional ebolavirus vaccines, DNA vaccines, Ebola virus-like particles (VLPs, vaccinia virus-based vaccines, Venezuelan equine encephalitis virus (VEEV-like replicon particles, Kunjin virus-based vaccine, recombinant Zaire Ebolavirus∆VP30, recombinant cytomegalovirus (CMV-based vaccines, recombinant rabies virus (RABV-based vaccines, recombinant paramyxovirus-based vaccines, adenovirus-based vaccines and vesicular stomatitis virus (VSV-based vaccines. No licensed vaccine or specific treatment is currently available to counteract ebolavirus infection, although DNA plasmids and several viral vector approaches have been evaluated as promising vaccine platforms. These vaccine candidates have been confirmed to be successful in protecting NHPs against lethal infection. Moreover, these vaccine candidates were successfully advanced to clinical trials. The present review provides an update of the current research on Ebola vaccines, with the aim of providing an overview on current prospects in the fight against EVD.

  14. Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

    Science.gov (United States)

    Miura, Jiro; Sakai, Manabu; Uchida, Hitoshi; Nakamura, Wataru; Nohara, Kanji; Maruyama, Yusuke; Hattori, Atsuhiko; Sakai, Takayoshi

    2015-01-01

    Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development. PMID:25876057

  15. Conserved RNA-Binding Proteins Required for Dendrite Morphogenesis in Caenorhabditis elegans Sensory Neurons

    Science.gov (United States)

    Antonacci, Simona; Forand, Daniel; Wolf, Margaret; Tyus, Courtney; Barney, Julia; Kellogg, Leah; Simon, Margo A.; Kerr, Genevieve; Wells, Kristen L.; Younes, Serena; Mortimer, Nathan T.; Olesnicky, Eugenia C.; Killian, Darrell J.

    2015-01-01

    The regulation of dendritic branching is critical for sensory reception, cell−cell communication within the nervous system, learning, memory, and behavior. Defects in dendrite morphology are associated with several neurologic disorders; thus, an understanding of the molecular mechanisms that govern dendrite morphogenesis is important. Recent investigations of dendrite morphogenesis have highlighted the importance of gene regulation at the posttranscriptional level. Because RNA-binding proteins mediate many posttranscriptional mechanisms, we decided to investigate the extent to which conserved RNA-binding proteins contribute to dendrite morphogenesis across phyla. Here we identify a core set of RNA-binding proteins that are important for dendrite morphogenesis in the PVD multidendritic sensory neuron in Caenorhabditis elegans. Homologs of each of these genes were previously identified as important in the Drosophila melanogaster dendritic arborization sensory neurons. Our results suggest that RNA processing, mRNA localization, mRNA stability, and translational control are all important mechanisms that contribute to dendrite morphogenesis, and we present a conserved set of RNA-binding proteins that regulate these processes in diverse animal species. Furthermore, homologs of these genes are expressed in the human brain, suggesting that these RNA-binding proteins are candidate regulators of dendrite development in humans. PMID:25673135

  16. Nanoscale morphogenesis of nylon-sputtered plasma polymer particles

    Science.gov (United States)

    Choukourov, Andrei; Shelemin, Artem; Pleskunov, Pavel; Nikitin, Daniil; Khalakhan, Ivan; Hanuš, Jan

    2018-05-01

    Sub-micron polymer particles are highly important in various fields including astrophysics, thermonuclear fusion and nanomedicine. Plasma polymerization offers the possibility to produce particles with tailor-made size, crosslink density and chemical composition to meet the requirements of a particular application. However, the mechanism of nucleation and growth of plasma polymer particles as well as diversity of their morphology remain far from being clear. Here, we prepared nitrogen-containing plasma polymer particles by rf magnetron sputtering of nylon in a gas aggregation cluster source with variable length. The method allowed the production of particles with roughly constant chemical composition and number density but with the mean size changing from 80 to 320 nm. Atomic Force Microscopy with super-sharp probes was applied to study the evolution of the particle surface topography as they grow in size. Height–height correlation and power spectral density functions were obtained to quantify the roughness exponent α  =  0.78, the growth exponent β  =  0.35, and the dynamic exponent 1/z  =  0.50. The set of critical exponents indicates that the particle surface evolves in a self-affine mode and the overall particle growth is caused by the accretion of polymer-forming species from the gas phase and not by coagulation. Redistribution of the incoming material over the surface coupled with the inhomogeneous distribution of inner stress is suggested as the main factor that determines the morphogenesis of the plasma polymer particles.

  17. Re-growth, morphogenesis and differentiation during starfish arm regeneration

    KAUST Repository

    Khadra, Yousra Ben

    2015-06-25

    The red starfish Echinaster sepositus is an excellent model for studying arm regeneration processes following traumatic amputation. The initial repair phase was described in a previous paper in terms of the early cicatrisation phenomena, and tissue and cell involvement. In this work we attempt to provide a further comprehensive description of the later regenerative stages in this species. Here we present the results of a detailed microscopic and submicroscopic investigation of the long regenerative phase, which can be subdivided into two sub-phases: early and advanced regenerative phases. The early regenerative phase (1-6 weeks p.a.) is characterized by tissue rearrangement, morphogenetic processes and initial differentiation events (mainly neurogenesis and skeletogenesis). The advanced regenerative phase (after 6 weeks p.a.) is characterized by further differentiation processes (early myogenesis), and obvious morphogenesis and re-growth of the regenerate. As in other starfish, the regenerative process in E. sepositus is relatively slow in comparison with that of crinoids and many ophiuroids, which is usually interpreted as resulting mainly from size-related aspects and of the more conspicuous involvement of morphallactic processes. Light and electron microscopy analyses suggest that some of the amputated structures, such as muscles, are not able to replace their missing parts by directly regrowing them from the remaining tissues, whereas others tissues, such as the skeleton and the radial nerve cord, appear to undergo direct re-growth. The overall process is in agreement with the distalization-intercalation model proposed by Agata and co-workers (1). Further experiments are needed to confirm this hypothesis. This article is protected by copyright. All rights reserved.

  18. A spatially-averaged mathematical model of kidney branching morphogenesis

    KAUST Repository

    Zubkov, V.S.

    2015-08-01

    © 2015 Published by Elsevier Ltd. Kidney development is initiated by the outgrowth of an epithelial ureteric bud into a population of mesenchymal cells. Reciprocal morphogenetic responses between these two populations generate a highly branched epithelial ureteric tree with the mesenchyme differentiating into nephrons, the functional units of the kidney. While we understand some of the mechanisms involved, current knowledge fails to explain the variability of organ sizes and nephron endowment in mice and humans. Here we present a spatially-averaged mathematical model of kidney morphogenesis in which the growth of the two key populations is described by a system of time-dependant ordinary differential equations. We assume that branching is symmetric and is invoked when the number of epithelial cells per tip reaches a threshold value. This process continues until the number of mesenchymal cells falls below a critical value that triggers cessation of branching. The mathematical model and its predictions are validated against experimentally quantified C57Bl6 mouse embryonic kidneys. Numerical simulations are performed to determine how the final number of branches changes as key system parameters are varied (such as the growth rate of tip cells, mesenchyme cells, or component cell population exit rate). Our results predict that the developing kidney responds differently to loss of cap and tip cells. They also indicate that the final number of kidney branches is less sensitive to changes in the growth rate of the ureteric tip cells than to changes in the growth rate of the mesenchymal cells. By inference, increasing the growth rate of mesenchymal cells should maximise branch number. Our model also provides a framework for predicting the branching outcome when ureteric tip or mesenchyme cells change behaviour in response to different genetic or environmental developmental stresses.

  19. Pancreatic morphogenesis and extracellular matrix organization during rat development.

    Science.gov (United States)

    Hisaoka, M; Haratake, J; Hashimoto, H

    1993-07-01

    pancreas, especially in the branching epithelial morphogenesis, and the major alterations appeared prior to distinct acinar cell differentiation.

  20. Shape self-regulation in early lung morphogenesis.

    Directory of Open Access Journals (Sweden)

    Raphaël Clément

    Full Text Available The arborescent architecture of mammalian conductive airways results from the repeated branching of lung endoderm into surrounding mesoderm. Subsequent lung's striking geometrical features have long raised the question of developmental mechanisms involved in morphogenesis. Many molecular actors have been identified, and several studies demonstrated the central role of Fgf10 and Shh in growth and branching. However, the actual branching mechanism and the way branching events are organized at the organ scale to achieve a self-avoiding tree remain to be understood through a model compatible with evidenced signaling. In this paper we show that the mere diffusion of FGF10 from distal mesenchyme involves differential epithelial proliferation that spontaneously leads to branching. Modeling FGF10 diffusion from sub-mesothelial mesenchyme where Fgf10 is known to be expressed and computing epithelial and mesenchymal growth in a coupled manner, we found that the resulting laplacian dynamics precisely accounts for the patterning of FGF10-induced genes, and that it spontaneously involves differential proliferation leading to a self-avoiding and space-filling tree, through mechanisms that we detail. The tree's fine morphological features depend on the epithelial growth response to FGF10, underlain by the lung's complex regulatory network. Notably, our results suggest that no branching information has to be encoded and that no master routine is required to organize branching events at the organ scale. Despite its simplicity, this model identifies key mechanisms of lung development, from branching to organ-scale organization, and could prove relevant to the development of other branched organs relying on similar pathways.

  1. A spatially-averaged mathematical model of kidney branching morphogenesis

    KAUST Repository

    Zubkov, V.S.; Combes, A.N.; Short, K.M.; Lefevre, J.; Hamilton, N.A.; Smyth, I.M.; Little, M.H.; Byrne, H.M.

    2015-01-01

    © 2015 Published by Elsevier Ltd. Kidney development is initiated by the outgrowth of an epithelial ureteric bud into a population of mesenchymal cells. Reciprocal morphogenetic responses between these two populations generate a highly branched epithelial ureteric tree with the mesenchyme differentiating into nephrons, the functional units of the kidney. While we understand some of the mechanisms involved, current knowledge fails to explain the variability of organ sizes and nephron endowment in mice and humans. Here we present a spatially-averaged mathematical model of kidney morphogenesis in which the growth of the two key populations is described by a system of time-dependant ordinary differential equations. We assume that branching is symmetric and is invoked when the number of epithelial cells per tip reaches a threshold value. This process continues until the number of mesenchymal cells falls below a critical value that triggers cessation of branching. The mathematical model and its predictions are validated against experimentally quantified C57Bl6 mouse embryonic kidneys. Numerical simulations are performed to determine how the final number of branches changes as key system parameters are varied (such as the growth rate of tip cells, mesenchyme cells, or component cell population exit rate). Our results predict that the developing kidney responds differently to loss of cap and tip cells. They also indicate that the final number of kidney branches is less sensitive to changes in the growth rate of the ureteric tip cells than to changes in the growth rate of the mesenchymal cells. By inference, increasing the growth rate of mesenchymal cells should maximise branch number. Our model also provides a framework for predicting the branching outcome when ureteric tip or mesenchyme cells change behaviour in response to different genetic or environmental developmental stresses.

  2. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA)

    DEFF Research Database (Denmark)

    Cottingham, Matthew G; Andersen, Rikke F; Spencer, Alexandra J

    2008-01-01

    -length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A...

  3. Progress toward a universal H5N1 vaccine: a recombinant modified vaccinia virus Ankara-expressing trivalent hemagglutinin vaccine.

    Directory of Open Access Journals (Sweden)

    Mookkan Prabakaran

    Full Text Available The rapid evolution of new sublineages of H5N1 influenza poses the greatest challenge in control of H5N1 infection by currently existing vaccines. To overcome this, an MVAtor vector expressing three H5HA antigens A/Vietnam/1203/04, A/Indonesia/669/06 and A/Anhui/01/05 (MVAtor-tri-HA vector was developed to elicit broad cross-protection against diverse clades by covering amino acid variations in the major neutralizing epitopes of HA among H5N1 subtypes.BALB/c mice and guinea pigs were immunized i.m. with 8×107 TCID50/animal of MVAtor-tri-HA vector. The immunogenicity and cross-protective immunity of the MVAtor-tri-HA vector was evaluated against diverse clades of H5N1 strains.The results showed that mice immunized with MVAtor-tri-HA vector induced robust cross-neutralizing immunity to diverse H5N1 clades. In addition, the MVAtor-tri-HA vector completely protected against 10 MLD50 of a divergent clade of H5N1 infection (clade 7. Importantly, the serological surveillance of post-vaccinated guinea pig sera demonstrated that MVAtor-tri-HA vector was able to elicit strong cross-clade neutralizing immunity against twenty different H5N1 strains from six clades that emerged between 1997 and 2012.The present findings revealed that incorporation of carefully selected HA genes from divergent H5N1 strains within a single vector could be an effective approach in developing a vaccine with broad coverage to prevent infection during a pandemic situation.

  4. Vectores recombinantes basados en el virus modificado de ankara (MVA), con deleción en el gen C6L, como vacunas contra el VIH/SIDA y otras enfermedades

    OpenAIRE

    García-Arriaza, J.; Gómez, Carmen E.; Esteban, Mariano

    2011-01-01

    [ES] La presente invención engloba dentro de los campos de la biología molecular y de la biotecnología. Especificamente se refiere a virus recombinantes basados en el virus modificado de Ankara (MVA) que expresan los antigenos gp120 y Gag-Pol-Nef del virus de la inmunodeficienciahumana (VIH-1) de subtipo B (MVA-B), sobre los que see ha delecionado el gen de vaccinia C6L, y que han sido diseñados para utilizarse como vacunas contra el VIH/SIDA y otras enfermedades.

  5. Vectores recombinantes basados en el virus modificado de Ankara (MVA), con deleción en el gen C6L, como vacunas contra el VIH/SIDA y otras enfermedades

    OpenAIRE

    García-Arriaza, J.; Gómez, Carmen E.; Esteban, Mariano

    2011-01-01

    La presente invención se engloba dentro de los campos de la biología molecular y de la biotecnología. Específicamente se refiere a virus recombinantes basados en el virus modificado de Ankara (MVA) que expresan los antígenos gp120 y Gag-Pol-Nef del virus de la inmunodeficiencia humana (VIH-1) de subtipo B (MVA-B), sobre los que se ha delecionado el gen de vaccinia C6L, y que han sido diseñados para utilizarse como vacunas contra el VIH/SIDA y otras enfermedades.

  6. The Morphogenesis and Biology of a Morbillivirus from MCF Cattle.

    Science.gov (United States)

    1979-01-01

    eye signs are minimal although fever, dairrhea and lymphadenopathy are common. The mild form is frequently self limiting and often observed in... Hayflick , 1965) and electron microscopic examination of infected cells proved them free of mycoplasma. Electron Microscopic Investigations of Virus...consistently isolated from leukocytes (62% of samples taken) than from any other source. Therefore, when time or space limitations precluded the examination

  7. The ureteric bud epithelium: morphogenesis and roles in metanephric kidney patterning.

    Science.gov (United States)

    Nagalakshmi, Vidya K; Yu, Jing

    2015-03-01

    The mammalian metanephric kidney is composed of two epithelial components, the collecting duct system and the nephron epithelium, that differentiate from two different tissues -the ureteric bud epithelium and the nephron progenitors, respectively-of intermediate mesoderm origin. The collecting duct system is generated through reiterative ureteric bud branching morphogenesis, whereas the nephron epithelium is formed in a process termed nephrogenesis, which is initiated with the mesenchymal-epithelial transition of the nephron progenitors. Ureteric bud branching morphogenesis is regulated by nephron progenitors, and in return, the ureteric bud epithelium regulates nephrogenesis. The metanephric kidney is physiologically divided along the corticomedullary axis into subcompartments that are enriched with specific segments of these two epithelial structures. Here, we provide an overview of the major molecular and cellular processes underlying the morphogenesis and patterning of the ureteric bud epithelium and its roles in the cortico-medullary patterning of the metanephric kidney. © 2015 Wiley Periodicals, Inc.

  8. Regulation of Epithelial Morphogenesis by the G-Protein Coupled Receptor Mist and its Ligand Fog*

    Science.gov (United States)

    Manning, Alyssa J.; Peters, Kimberly A.; Peifer, Mark; Rogers, Stephen L.

    2014-01-01

    Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells, and used RNAi-based screening to identify Mist, a Drosophila G protein-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes. PMID:24222713

  9. Supplementary Material for: A global sensitivity analysis approach for morphogenesis models

    KAUST Repository

    Boas, Sonja

    2015-01-01

    Abstract Background Morphogenesis is a developmental process in which cells organize into shapes and patterns. Complex, non-linear and multi-factorial models with images as output are commonly used to study morphogenesis. It is difficult to understand the relation between the uncertainty in the input and the output of such ‘black-box’ models, giving rise to the need for sensitivity analysis tools. In this paper, we introduce a workflow for a global sensitivity analysis approach to study the impact of single parameters and the interactions between them on the output of morphogenesis models. Results To demonstrate the workflow, we used a published, well-studied model of vascular morphogenesis. The parameters of this cellular Potts model (CPM) represent cell properties and behaviors that drive the mechanisms of angiogenic sprouting. The global sensitivity analysis correctly identified the dominant parameters in the model, consistent with previous studies. Additionally, the analysis provided information on the relative impact of single parameters and of interactions between them. This is very relevant because interactions of parameters impede the experimental verification of the predicted effect of single parameters. The parameter interactions, although of low impact, provided also new insights in the mechanisms of in silico sprouting. Finally, the analysis indicated that the model could be reduced by one parameter. Conclusions We propose global sensitivity analysis as an alternative approach to study the mechanisms of morphogenesis. Comparison of the ranking of the impact of the model parameters to knowledge derived from experimental data and from manipulation experiments can help to falsify models and to find the operand mechanisms in morphogenesis. The workflow is applicable to all ‘black-box’ models, including high-throughput in vitro models in which output measures are affected by a set of experimental perturbations.

  10. The case for applying tissue engineering methodologies to instruct human organoid morphogenesis.

    Science.gov (United States)

    Marti-Figueroa, Carlos R; Ashton, Randolph S

    2017-05-01

    Three-dimensional organoids derived from human pluripotent stem cell (hPSC) derivatives have become widely used in vitro models for studying development and disease. Their ability to recapitulate facets of normal human development during in vitro morphogenesis produces tissue structures with unprecedented biomimicry. Current organoid derivation protocols primarily rely on spontaneous morphogenesis processes to occur within 3-D spherical cell aggregates with minimal to no exogenous control. This yields organoids containing microscale regions of biomimetic tissues, but at the macroscale (i.e. 100's of microns to millimeters), the organoids' morphology, cytoarchitecture, and cellular composition are non-biomimetic and variable. The current lack of control over in vitro organoid morphogenesis at the microscale induces aberrations at the macroscale, which impedes realization of the technology's potential to reproducibly form anatomically correct human tissue units that could serve as optimal human in vitro models and even transplants. Here, we review tissue engineering methodologies that could be used to develop powerful approaches for instructing multiscale, 3-D human organoid morphogenesis. Such technological mergers are critically needed to harness organoid morphogenesis as a tool for engineering functional human tissues with biomimetic anatomy and physiology. Human PSC-derived 3-D organoids are revolutionizing the biomedical sciences. They enable the study of development and disease within patient-specific genetic backgrounds and unprecedented biomimetic tissue microenvironments. However, their uncontrolled, spontaneous morphogenesis at the microscale yields inconsistences in macroscale organoid morphology, cytoarchitecture, and cellular composition that limits their standardization and application. Integration of tissue engineering methods with organoid derivation protocols could allow us to harness their potential by instructing standardized in vitro morphogenesis

  11. MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

    Science.gov (United States)

    Wang, Chia-Hui; Lee, Daniel Y; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B

    2008-06-18

    Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

  12. Effects of light quality on flowering and morphogenesis in Hyoscyamus niger L.

    NARCIS (Netherlands)

    Hattab, El A.H.

    1968-01-01

    The present paper is concerned with bolting and morphogenesis of Hyoscyamus niger L. as reactions upon radiation in the visible spectrum.

    Experiments are described in which Hyoscyamus plants were exposed to light of various well defined spectral regions. The light of these

  13. Cardiac septation: a late contribution of the embryonic primary myocardium to heart morphogenesis

    NARCIS (Netherlands)

    Lamers, Wouter H.; Moorman, Antoon F. M.

    2002-01-01

    Heart morphogenesis comprises 2 major consecutive steps, viz. chamber formation followed by septation. Septation is the remodeling of the heart from a single-channel peristaltic pump to a dual-channel, synchronously contracting device with 1-way valves. In the human heart, septation occurs between 4

  14. Growth and morphogenesis of shoot initials of Douglas fir, Pseudotsuga menziesii (Mirb.) Franco, in vitro

    NARCIS (Netherlands)

    Evers, P.W.

    1984-01-01

    An optimalized method of micropropagation of Douglas fir is described. Seasonal changes were found in optima for nitrate and sucrose in the medium and in the optimum for the light intensity during the culture of shoot initials. Differences in morphogenesis were obtained from shoot initials that had

  15. S1P transporter SPNS2 regulates proper postnatal retinal morphogenesis.

    Science.gov (United States)

    Fang, Chao; Bian, Ganlan; Ren, Pan; Xiang, Jie; Song, Jun; Yu, Caiyong; Zhang, Qian; Liu, Ling; Chen, Kun; Liu, Fangfang; Zhang, Kun; Wu, Chunfeng; Sun, Ruixia; Hu, Dan; Ju, Gong; Wang, Jian

    2018-02-08

    Spinster homolog 2 (SPNS2) is the membrane transporter of sphingosine-1-phosphate (S1P), and it participates in several physiologic processes by activating different S1P receptors (S1PRs). However, its functions in the nervous system remain largely unclear. We explored the important role of SPNS2 in the process of retinal morphogenesis using a spns2-deficient rat model. In the absence of the functional SPNS2 transporter, we observed progressively aggravating laminar disorganization of the epithelium at the postnatal stage of retinal development. Disrupted cell polarity, delayed cell-cycle exit of retinal progenitor cells, and insufficient migration of newborn neurons were proposed in this study as potential mechanisms accounting for this structural disorder. In addition, we analyzed the expression profiles of spns2 and s1prs, and proposed that SPNS2 regulated retinal morphogenesis by establishing the S1P level in the eye and activating S1PR3 signaling. These data indicate that SPNS2 is indispensable for normal retinal morphogenesis and provide new insights on the role of S1P in the developing retina using an established in vivo model.-Fang, C., Bian, G., Ren, P., Xiang, J., Song, J., Yu, C., Zhang, Q., Liu, L., Chen, K., Liu, F., Zhang, K., Wu, C., Sun, R., Hu, D., Ju, G., Wang, J. S1P transporter SPNS2 regulates proper postnatal retinal morphogenesis.

  16. Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette

    2004-01-01

    elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion......A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene...... of CD8+ T cells reduced, but did not abolish, enhancement of disease. Mice vaccinated with a construct encoding a class I-restricted lymphocytic choriomeningitis virus epitope and beta2m suffered more severe weight loss after RSV infection than unvaccinated RSV-infected mice, although RSV-specific CD8...

  17. Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase?

    Science.gov (United States)

    Wildy, P; Gell, P G; Rhodes, J; Newton, A

    1982-07-01

    Proteose-peptone-activated mouse macrophages can prevent productive infection by herpes simplex virus in neighboring cells in vitro whether or not those cells belong to the same animal species. The effect does not require contact between the macrophages and the infected cells, may be prevented by adding extra arginine to the medium, and may be reversed when extra arginine is added 24 h after the macrophages. Arginase activity was found both intracellularly and released from the macrophages. The extracellular enzyme is quite stable; 64% activity was found after 48 h of incubation at 37 degrees C in tissue culture medium. No evidence was found that the inefficiency of virus replication in macrophages was due to self-starvation by arginase. As might be predicted macrophages can, by the same mechanism, limit productive infection by vaccinia virus.

  18. Slug controls stem/progenitor cell growth dynamics during mammary gland morphogenesis.

    Directory of Open Access Journals (Sweden)

    Mayssa Nassour

    Full Text Available Morphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT "master genes". EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question.Using a Slug-lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10-20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres.We conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism coordinating cell lineage dynamics and morphogenesis, and

  19. Estudo comparativo das inclusões do alastrim e da vacina no macaco (Macacus rhesus A comparison of the inclusion bodies of alastrim and vaccinia in the monkey (Macacus rhesus

    Directory of Open Access Journals (Sweden)

    C. Magarinos Torres

    1934-02-01

    Full Text Available Vesiculas e pustulas contendo numerosas inclusões citoplasmicas nas celulas epidermicas, foram regularmente produzidas no macaco (Macacus rhesus, quer com o virus do alastrim, quer com o da vacina, após inoculação endovenosa e sem previa escarificação. O virus do alastrim parece menos virulento para essa especie de macaco que o da vacina. Ao passo que 12 macacos rhesus injetados por via endovenosa com sete amostras diferentes de virus do alastrim, após apresentarem com regularidade um infecção experimental, sobreviveram e se conservaram em boa saúde, a injecção endovenosa do virus da vacina recentemente preparado (polpa bruta produziu a morte em 2, dentre 4 animais experimentados. 2. - Foram notadas diferenças pequenas, mas nitidas, na morfologia das inclusões do alastrim e da vacina, em material fixado no liquido de Helly, incluido em parafina e corado pela hematoxilina-eosina. Dizem elas respeito ao numero de inclusões encontradas em cada celula epidermica e às suas reações de coloração. 3. - As inclusões do alastrim, quando apresentam grandes dimensões, conservam-se unicas ou solitarias no citoplasma das celulas epidermicas do macaco rhesus, e coram-se em tonalidade que varia do azul escuro ao cinzento-azulado. Comtudo, em celulas que sofreram necrose, ou naquelas contendo 2 a 4 inclusões de pequenas dimensões, por vezes elas se mostram coradas em roseo. 4. - As inclusões da vacina, quando em faze adeantada de desenvolvimento, são multiplas nas celulas epidermicas do macaco rhesus e mostram, regularmente, uma policromatofilia caracteristica.1. - Vesicles and pustules containing numerous cytoplasmic inclusion bodies within the epidermal cells were regularly produced in monkeys (Macacus rhesus by intravenous inoculation either of alastrim virus or of recently prepared vaccine emulsion, no previous scarifications being required. Alastrim virus seems less virulent for this species of monkey than the virus of vaccinia is

  20. Coated microneedle arrays for transcutaneous delivery of live virus vaccines.

    Science.gov (United States)

    Vrdoljak, Anto; McGrath, Marie G; Carey, John B; Draper, Simon J; Hill, Adrian V S; O'Mahony, Conor; Crean, Abina M; Moore, Anne C

    2012-04-10

    Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8(+) T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Coated microneedle arrays for transcutaneous delivery of live virus vaccines

    Science.gov (United States)

    Vrdoljak, Anto; McGrath, Marie G.; Carey, John B.; Draper, Simon J.; Hill, Adrian V.S.; O’Mahony, Conor; Crean, Abina M.; Moore, Anne C.

    2016-01-01

    Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8+ T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices. PMID:22245683

  2. ECHO virus

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/001340.htm ECHO virus To use the sharing features on this page, please enable JavaScript. Enteric cytopathic human orphan (ECHO) viruses are a group of viruses that can lead ...

  3. Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

    Science.gov (United States)

    Milrot, Elad; Mutsafi, Yael; Fridmann-Sirkis, Yael; Shimoni, Eyal; Rechav, Katya; Gurnon, James R; Van Etten, James L; Minsky, Abraham

    2016-01-01

    The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway. © 2015 John Wiley & Sons Ltd.

  4. CRIM1 Complexes with ß-catenin and Cadherins, Stabilizes Cell-Cell Junctions and Is Critical for Neural Morphogenesis

    OpenAIRE

    Ponferrada, Virgilio G.; Fan, Jieqing; Vallance, Jefferson E.; Hu, Shengyong; Mamedova, Aygun; Rankin, Scott A.; Kofron, Matthew; Zorn, Aaron M.; Hegde, Rashmi S.; Lang, Richard A.

    2012-01-01

    In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using C...

  5. A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

    Directory of Open Access Journals (Sweden)

    Sanchita Das

    Full Text Available CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR. The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively. The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus.

  6. A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

    Science.gov (United States)

    Das, Sanchita; Rundell, Mark S; Mirza, Aashiq H; Pingle, Maneesh R; Shigyo, Kristi; Garrison, Aura R; Paragas, Jason; Smith, Scott K; Olson, Victoria A; Larone, Davise H; Spitzer, Eric D; Barany, Francis; Golightly, Linnie M

    2015-01-01

    CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

  7. Protective immunity provided by HLA-A2 epitopes for fusion and hemagglutinin proteins of measles virus

    International Nuclear Information System (INIS)

    Oh, Sang Kon; Stegman, Brian; Pendleton, C. David; Ota, Martin O.; Pan, C.-H.; Griffin, Diane E.; Burke, Donald S.; Berzofsky, Jay A.

    2006-01-01

    Natural infection and vaccination with a live-attenuated measles virus (MV) induce CD8 + T-cell-mediated immune responses that may play a central role in controlling MV infection. In this study, we show that newly identified human HLA-A2 epitopes from MV hemagglutinin (H) and fusion (F) proteins induced protective immunity in HLA-A2 transgenic mice challenged with recombinant vaccinia viruses expressing F or H protein. HLA-A2 epitopes were predicted and synthesized. Five and four peptides from H and F, respectively, bound to HLA-A2 molecules in a T2-binding assay, and four from H and two from F could induce peptide-specific CD8 + T cell responses in HLA-A2 transgenic mice. Further experiments proved that three peptides from H (H9-567, H10-250, and H10-516) and one from F protein (F9-57) were endogenously processed and presented on HLA-A2 molecules. All peptides tested in this study are common to 5 different strains of MV including Edmonston. In both A2K b and HHD-2 mice, the identified peptide epitopes induced protective immunity against recombinant vaccinia viruses expressing H or F. Because F and H proteins induce neutralizing antibodies, they are major components of new vaccine strategies, and therefore data from this study will contribute to the development of new vaccines against MV infection

  8. Dicer activity in neural crest cells is essential for craniofacial organogenesis and pharyngeal arch artery morphogenesis

    Science.gov (United States)

    Nie, Xuguang; Wang, Qin; Jiao, Kai

    2014-01-01

    MicroRNAs (miRNAs) play important roles in regulating gene expression during numerous biological/pathological processes. Dicer encodes an RNase III endonuclease that is essential for generating most, if not all, functional miRNAs. In this work, we applied a conditional gene inactivation approach to examine the function of Dicer during neural crest cell (NCC) development. Mice with NCC-specific inactivation of Dicer died perinatally. Cranial and cardiac NCC migration into target tissues was not affected by Dicer disruption, but their subsequent development was disturbed. NCC derivatives and their associated mesoderm-derived cells displayed massive apoptosis, leading to severe abnormalities during craniofacial morphogenesis and organogenesis. In addition, the 4th pharyngeal arch artery (PAA) remodeling was affected, resulting in interrupted aortic arch artery type B (IAA-B) in mutant animals. Taken together, our results show that Dicer activity in NCCs is essential for craniofacial development and pharyngeal arch artery morphogenesis. PMID:21256960

  9. An integrated miRNA functional screening and target validation method for organ morphogenesis.

    Science.gov (United States)

    Rebustini, Ivan T; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A; Maas, Richard L

    2016-03-16

    The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.

  10. Semaphorin-Plexin Signaling Controls Mitotic Spindle Orientation during Epithelial Morphogenesis and Repair

    DEFF Research Database (Denmark)

    Xia, Jingjing; Swiercz, Jakub M.; Bañón-Rodríguez, Inmaculada

    2015-01-01

    Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we...... show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading...... to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central...

  11. Reverse engineering the mechanical and molecular pathways in stem cell morphogenesis.

    Science.gov (United States)

    Lu, Kai; Gordon, Richard; Cao, Tong

    2015-03-01

    The formation of relevant biological structures poses a challenge for regenerative medicine. During embryogenesis, embryonic cells differentiate into somatic tissues and undergo morphogenesis to produce three-dimensional organs. Using stem cells, we can recapitulate this process and create biological constructs for therapeutic transplantation. However, imperfect imitation of nature sometimes results in in vitro artifacts that fail to recapitulate the function of native organs. It has been hypothesized that developing cells may self-organize into tissue-specific structures given a correct in vitro environment. This proposition is supported by the generation of neo-organoids from stem cells. We suggest that morphogenesis may be reverse engineered to uncover its interacting mechanical pathway and molecular circuitry. By harnessing the latent architecture of stem cells, novel tissue-engineering strategies may be conceptualized for generating self-organizing transplants. Copyright © 2013 John Wiley & Sons, Ltd.

  12. Essential role for fibrillin-2 in zebrafish notochord and vascular morphogenesis.

    Science.gov (United States)

    Gansner, John M; Madsen, Erik C; Mecham, Robert P; Gitlin, Jonathan D

    2008-10-01

    Recent studies demonstrate that lysyl oxidase cuproenzymes are critical for zebrafish notochord formation, but the molecular mechanisms of copper-dependent notochord morphogenesis are incompletely understood. We, therefore, conducted a forward genetic screen for zebrafish mutants that exhibit notochord sensitivity to lysyl oxidase inhibition, yielding a mutant with defects in notochord and vascular morphogenesis, puff daddygw1 (pfdgw1). Meiotic mapping and cloning reveal that the pfdgw1 phenotype results from disruption of the gene encoding the extracellular matrix protein fibrillin-2, and the spatiotemporal expression of fibrillin-2 is consistent with the pfdgw1 phenotype. Furthermore, each aspect of the pfdgw1 phenotype is recapitulated by morpholino knockdown of fibrillin-2. Taken together, the data reveal a genetic interaction between fibrillin-2 and the lysyl oxidases in notochord formation and demonstrate the importance of fibrillin-2 in specific early developmental processes in zebrafish. Copyright (c) 2008 Wiley-Liss, Inc.

  13. Gibberellin influence on the morphogenesis of the moss Bryum argenteum Hedw. in in vitro conditions

    Directory of Open Access Journals (Sweden)

    Sabovljević Aneta

    2010-01-01

    Full Text Available The moss Bryum argenteum Hedw. was treated with gibberellins as well as some inhibitors of gibberellin biosynthesis in order to investigate their influence on B. argenteum morphogenesis. Generally, gibberellins have not been chemically identified in bryophytes, while other groups of classical phytohormones (auxins, cytokinins, abscisic acid and ethylene have been chemically identified in these plants. The in vitro culture of the moss Bryum argenteum was established from sterilized spores. The apical shoots of untreated gametophytes grown in vitro were used to investigate the influence of different substances on secondary protonema and on the growth and multiplication of the gametophytes. B. argenteum reacts differently to the growth regulators applied. Both gibberellins applied in vitro (GA3 and GA7 have a positive effect on B. argenteum morphogenesis. Shoot multiplication was negatively affected by three tested growth retardants (ancymidol, BX-112 and chlorocholine chloride, while these substances did not have such strong effects on the moss protonema development.

  14. Estrogenic effect of soy isoflavones on mammary gland morphogenesis and gene expression profile

    DEFF Research Database (Denmark)

    Thomsen, Anni R.; Almstrup, Kristian; Nielsen, John E.

    2006-01-01

    We examined the effect of 17 beta-estradiol (E2) and soy isoflavones' exposure on morphogenesis and global gene expression in the murine mammary gland. Three exposure regimens were applied: isoflavones added to the diet throughout either the lactational period (via the dams) or the postweaning...... period and E2 administered orally during the lactational period. Whole mounts of mammary glands were evaluated both in juvenile and adult animals with respect to branching morphogenesis and terminal end bud (TEB) formation. At postnatal day (PND) 28, we observed a significant increase in branching...... isoflavone and E2 exposure was further substantiated by changes in gene expression, since the same groups of genes were up- and downregulated, particularly in the E2 and postweaning isoflavone regimen. All changes in gene expression correlated with changes in the cellular composition of the gland, i.e., more...

  15. Exocrine Gland Morphogenesis: Insights into the Role of Amphiregulin from Development to Disease.

    Science.gov (United States)

    Sisto, Margherita; Lorusso, Loredana; Ingravallo, Giuseppe; Lisi, Sabrina

    2017-12-01

    Amphiregulin (AREG) is a well-characterized member of the epidermal growth factor (EGF) family and is one of the ligands of the EGF receptor (EGFR). AREG plays a key role in mammalian development and in the control of branching morphogenesis in various organs. Furthermore, AREG participates in a wide range of physiological and pathological processes activating the major intracellular signalling cascades governing cell survival, proliferation and motility. In this article, we review current advances in exocrine glands morphogenesis, focusing on the salivary gland, and discuss the essential aspects of AREG structure, function and regulation, and its differential role within the EGFR family of ligands. Finally, we identify emerging aspects in AREG research applied to mammary gland development and the salivary gland autoimmune disease, Sjögren's syndrome.

  16. A novel ALS-associated variant in UBQLN4 regulates motor axon morphogenesis

    Science.gov (United States)

    Edens, Brittany M; Yan, Jianhua; Miller, Nimrod; Deng, Han-Xiang; Siddique, Teepu; Ma, Yongchao C

    2017-01-01

    The etiological underpinnings of amyotrophic lateral sclerosis (ALS) are complex and incompletely understood, although contributions to pathogenesis by regulators of proteolytic pathways have become increasingly apparent. Here, we present a novel variant in UBQLN4 that is associated with ALS and show that its expression compromises motor axon morphogenesis in mouse motor neurons and in zebrafish. We further demonstrate that the ALS-associated UBQLN4 variant impairs proteasomal function, and identify the Wnt signaling pathway effector beta-catenin as a UBQLN4 substrate. Inhibition of beta-catenin function rescues the UBQLN4 variant-induced motor axon phenotypes. These findings provide a strong link between the regulation of axonal morphogenesis and a new ALS-associated gene variant mediated by protein degradation pathways. DOI: http://dx.doi.org/10.7554/eLife.25453.001 PMID:28463112

  17. Should remaining stockpiles of smallpox virus (variola) be destroyed?

    Science.gov (United States)

    Weinstein, Raymond S

    2011-04-01

    In 2011, the World Health Organization will recommend the fate of existing smallpox stockpiles, but circumstances have changed since the complete destruction of these cultures was first proposed. Recent studies suggest that variola and its experimental surrogate, vaccinia, have a remarkable ability to modify the human immune response through complex mechanisms that scientists are only just beginning to unravel. Further study that might require intact virus is essential. Moreover, modern science now has the capability to recreate smallpox or a smallpox-like organism in the laboratory in addition to the risk of nature re-creating it as it did once before. These factors strongly suggest that relegating smallpox to the autoclave of extinction would be ill advised.

  18. Septins from the phytopathogenic fungus Ustilago maydis are required for proper morphogenesis but dispensable for virulence.

    Directory of Open Access Journals (Sweden)

    Isabel Alvarez-Tabarés

    Full Text Available BACKGROUND: Septins are a highly conserved family of GTP-binding proteins involved in multiple cellular functions, including cell division and morphogenesis. Studies of septins in fungal cells underpin a clear correlation between septin-based structures and fungal morphology, providing clues to understand the molecular frame behind the varied morphologies found in fungal world. METHODOLOGY/PRINCIPAL FINDINGS: Ustilago maydis genome has the ability to encode four septins. Here, using loss-of-function as well as GFP-tagged alleles of these septin genes, we investigated the roles of septins in the morphogenesis of this basidiomycete fungus. We described that septins in U. maydis could assemble into at least three different structures coexisting in the same cell: bud neck collars, band-like structures at the growing tip, and long septin fibers that run from pole to pole near the cell cortex. We also found that in the absence of septins, U. maydis cells lost their elongated shape, became wider at the central region and ended up losing their polarity, pointing to an important role of septins in the morphogenesis of this fungus. These morphological defects were alleviated in the presence of an osmotic stabilizer suggesting that absence of septins affected the proper formation of the cell wall, which was coherent with a higher sensitivity of septin defective cells to drugs that affect cell wall construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed the role of septins in virulence and found that in spite of the described morphological defects, septin mutants were virulent in corn plants. CONCLUSIONS/SIGNIFICANCE: Our results indicated a major role of septins in morphogenesis in U. maydis. However, in contrast to studies in other fungal pathogens, in which septins were reported to be necessary during the infection process, we found a minor role of septins during corn infection by U. maydis.

  19. FLI-1 Flightless-1 and LET-60 Ras control germ line morphogenesis in C. elegans

    Directory of Open Access Journals (Sweden)

    Dentler William L

    2008-05-01

    Full Text Available Abstract Background In the C. elegans germ line, syncytial germ line nuclei are arranged at the cortex of the germ line as they exit mitosis and enter meiosis, forming a nucleus-free core of germ line cytoplasm called the rachis. Molecular mechanisms of rachis formation and germ line organization are not well understood. Results Mutations in the fli-1 gene disrupt rachis organization without affecting meiotic differentiation, a phenotype in C. elegans referred to here as the germ line morphogenesis (Glm phenotype. In fli-1 mutants, chains of meiotic germ nuclei spanned the rachis and were partially enveloped by invaginations of germ line plasma membrane, similar to nuclei at the cortex. Extensions of the somatic sheath cells that surround the germ line protruded deep inside the rachis and were associated with displaced nuclei in fli-1 mutants. fli-1 encodes a molecule with leucine-rich repeats and gelsolin repeats similar to Drosophila flightless 1 and human Fliih, which have been shown to act as cytoplasmic actin regulators as well as nuclear transcriptional regulators. Mutations in let-60 Ras, previously implicated in germ line development, were found to cause the Glm phenotype. Constitutively-active LET-60 partially rescued the fli-1 Glm phenotype, suggesting that LET-60 Ras and FLI-1 might act together to control germ line morphogenesis. Conclusion FLI-1 controls germ line morphogenesis and rachis organization, a process about which little is known at the molecular level. The LET-60 Ras GTPase might act with FLI-1 to control germ line morphogenesis.

  20. Proliferation and apoptosis in early molar morphogenesis - voles as models in odontogenesis

    Czech Academy of Sciences Publication Activity Database

    Šetková, Jana; Lesot, H.; Matalová, Eva; Witter, K.; Matulová, Petra; Míšek, Ivan

    2006-01-01

    Roč. 50, 5 (2006), s. 481-489 ISSN 0214-6282 R&D Projects: GA ČR GA304/04/0101; GA MŠk OC B23.001 Grant - others:COST STSM B23-00981 Institutional research plan: CEZ:AV0Z50450515 Keywords : tooth development * morphogenesis * Microtus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.577, year: 2006

  1. Review of aragonite and calcite crystal morphogenesis in thermal spring systems

    Science.gov (United States)

    Jones, Brian

    2017-06-01

    Aragonite and calcite crystals are the fundamental building blocks of calcareous thermal spring deposits. The diverse array of crystal morphologies found in these deposits, which includes monocrystals, mesocrystals, skeletal crystals, dendrites, and spherulites, are commonly precipitated under far-from-equilibrium conditions. Such crystals form through both abiotic and biotic processes. Many crystals develop through non-classical crystal growth models that involve the arrangement of nanocrystals in a precisely controlled crystallographic register. Calcite crystal morphogenesis has commonly been linked to a ;driving force;, which is a conceptual measure of the distance of the growth conditions from equilibrium conditions. Essentially, this scheme indicates that increasing levels of supersaturation and various other parameters that produce a progressive change from monocrystals and mesocrystals to skeletal crystals to crystallographic and non-crystallographic dendrites, to dumbbells, to spherulites. Despite the vast amount of information available from laboratory experiments and natural spring systems, the precise factors that control the driving force are open to debate. The fact that calcite crystal morphogenesis is still poorly understood is largely a reflection of the complexity of the factors that influence aragonite and calcite precipitation. Available information indicates that variations in calcite crystal morphogenesis can be attributed to physical and chemical parameters of the parent water, the presence of impurities, the addition of organic or inorganic additives to the water, the rate of crystal growth, and/or the presence of microbes and their associated biofilms. The problems in trying to relate crystal morphogenesis to specific environmental parameters arise because it is generally impossible to disentangle the controlling factor(s) from the vast array of potential parameters that may act alone or in unison with each other.

  2. Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

    Science.gov (United States)

    Barriga, Elias H; Franze, Kristian; Charras, Guillaume; Mayor, Roberto

    2018-02-22

    Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis.

  3. Topological laser speckle analyzer of differentiation and proliferation activity during morphogenesis in cell cultures

    OpenAIRE

    Notchenko A.V.; Gradov O.V.

    2011-01-01

    An automated system for morpho-topological determination of cell division phases and structur al differentiation of tissues during morphogenesis was implemented on the basis of topological properties of cell cultures, considered within the framework of set and manifold theories. A simple robotic hardware and software system based on Zeiss microscope with a modified stage and a Velleman manipulator KSR-1 allow to control the laser module position, carrying out the angular irradiation of s...

  4. MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

    Directory of Open Access Journals (Sweden)

    Chia-Hui Wang

    Full Text Available Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

  5. Gibberellin influence on the morphogenesis of the moss Bryum argenteum Hedw. in in vitro conditions

    OpenAIRE

    Sabovljević Aneta; Sabovljević Marko; Grubišić D.

    2010-01-01

    The moss Bryum argenteum Hedw. was treated with gibberellins as well as some inhibitors of gibberellin biosynthesis in order to investigate their influence on B. argenteum morphogenesis. Generally, gibberellins have not been chemically identified in bryophytes, while other groups of classical phytohormones (auxins, cytokinins, abscisic acid and ethylene) have been chemically identified in these plants. The in vitro culture of the moss Bryum argenteum was established from sterilized spores. Th...

  6. SACE_0012, a TetR-Family Transcriptional Regulator, Affects the Morphogenesis of Saccharopolyspora erythraea

    OpenAIRE

    Yin, Xiaojuan; Xu, Xinqiang; Wu, Hang; Yuan, Li; Huang, Xunduan; Zhang, Buchang

    2013-01-01

    Saccharopolyspora erythraea, a mycelium-forming actinomycete, produces a clinically important antibiotic erythromycin. Extensive investigations have provided insights into erythromycin biosynthesis in S. erythraea, but knowledge of its morphogenesis remains limited. By gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE_0012 was identified to be a negative regulator of mycelium formation of S. erythraea A226. Detected by quantitative real-time PCR,...

  7. Regulation of cellulase expression, sporulation, and morphogenesis by velvet family proteins in Trichoderma reesei.

    Science.gov (United States)

    Liu, Kuimei; Dong, Yanmei; Wang, Fangzhong; Jiang, Baojie; Wang, Mingyu; Fang, Xu

    2016-01-01

    Homologs of the velvet protein family are encoded by the ve1, vel2, and vel3 genes in Trichoderma reesei. To test their regulatory functions, the velvet protein-coding genes were disrupted, generating Δve1, Δvel2, and Δvel3 strains. The phenotypic features of these strains were examined to identify their functions in morphogenesis, sporulation, and cellulase expression. The three velvet-deficient strains produced more hyphal branches, indicating that velvet family proteins participate in the morphogenesis in T. reesei. Deletion of ve1 and vel3 did not affect biomass accumulation, while deletion of vel2 led to a significantly hampered growth when cellulose was used as the sole carbon source in the medium. The deletion of either ve1 or vel2 led to the sharp decrease of sporulation as well as a global downregulation of cellulase-coding genes. In contrast, although the expression of cellulase-coding genes of the ∆vel3 strain was downregulated in the dark, their expression in light condition was unaffected. Sporulation was hampered in the ∆vel3 strain. These results suggest that Ve1 and Vel2 play major roles, whereas Vel3 plays a minor role in sporulation, morphogenesis, and cellulase expression.

  8. Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish

    Science.gov (United States)

    Swartz, Mary E.; McCarthy, Neil; Norrie, Jacqueline L.; Eberhart, Johann K.

    2016-01-01

    The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face. PMID:27122171

  9. Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition

    Directory of Open Access Journals (Sweden)

    Antônio Jesus Dorighetto Cogo

    2018-02-01

    Full Text Available Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

  10. The ERM protein Moesin is essential for neuronal morphogenesis and long-term memory in Drosophila.

    Science.gov (United States)

    Freymuth, Patrick S; Fitzsimons, Helen L

    2017-08-29

    Moesin is a cytoskeletal adaptor protein that plays an important role in modification of the actin cytoskeleton. Rearrangement of the actin cytoskeleton drives both neuronal morphogenesis and the structural changes in neurons that are required for long-term memory formation. Moesin has been identified as a candidate memory gene in Drosophila, however, whether it is required for memory formation has not been evaluated. Here, we investigate the role of Moesin in neuronal morphogenesis and in short- and long-term memory formation in the courtship suppression assay, a model of associative memory. We found that both knockdown and overexpression of Moesin led to defects in axon growth and guidance as well as dendritic arborization. Moreover, reduction of Moesin expression or expression of a constitutively active phosphomimetic in the adult Drosophila brain had no effect on short term memory, but prevented long-term memory formation, an effect that was independent of its role in development. These results indicate a critical role for Moesin in both neuronal morphogenesis and long-term memory formation.

  11. Differentiated roles for MreB-actin isologues and autolytic enzymes in Bacillus subtilis morphogenesis.

    Science.gov (United States)

    Domínguez-Cuevas, Patricia; Porcelli, Ida; Daniel, Richard A; Errington, Jeff

    2013-09-01

    Cell morphogenesis in most bacteria is governed by spatiotemporal growth regulation of the peptidoglycan cell wall layer. Much is known about peptidoglycan synthesis but regulation of its turnover by hydrolytic enzymes is much less well understood. Bacillus subtilis has a multitude of such enzymes. Two of the best characterized are CwlO and LytE: cells lacking both enzymes have a lethal block in cell elongation. Here we show that activity of CwlO is regulated by an ABC transporter, FtsEX, which is required for cell elongation, unlike cell division as in Escherichia coli. Actin-like MreB proteins are thought to play a key role in orchestrating cell wall morphogenesis. B. subtilis has three MreB isologues with partially differentiated functions. We now show that the three MreB isologues have differential roles in regulation of the CwlO and LytE systems and that autolysins control different aspects of cell morphogenesis. The results add major autolytic activities to the growing list of functions controlled by MreB isologues in bacteria and provide new insights into the different specialized functions of essential cell wall autolysins. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  12. Quantification of local matrix deformations and mechanical properties during capillary morphogenesis in 3D.

    Science.gov (United States)

    Kniazeva, Ekaterina; Weidling, John W; Singh, Rahul; Botvinick, Elliot L; Digman, Michelle A; Gratton, Enrico; Putnam, Andrew J

    2012-04-01

    Reciprocal mechanical interactions between cells and the extracellular matrix (ECM) are thought to play important instructive roles in branching morphogenesis. However, most studies to date have failed to characterize these interactions on a length scale relevant to cells, especially in three-dimensional (3D) matrices. Here we utilized two complementary methods, spatio-temporal image correlation spectroscopy (STICS) and laser optical tweezers-based active microrheology (AMR), to quantify endothelial cell (EC)-mediated deformations of individual ECM elements and the local ECM mechanical properties, respectively, during the process of capillary morphogenesis in a 3D cell culture model. In experiments in which the ECM density was systematically varied, STICS revealed that the rate at which ECs deformed individual ECM fibers on the microscale positively correlated with capillary sprouting on the macroscale. ECs expressing constitutively active V14-RhoA displaced individual matrix fibers at significantly faster rates and displayed enhanced capillary sprouting relative to wild-type cells, while those expressing dominant-negative N19-RhoA behaved in an opposite fashion. In parallel, AMR revealed a local stiffening of the ECM proximal to the tips of sprouting ECs. By quantifying the dynamic physical properties of the cell-ECM interface in both space and time, we identified a correlation linking ECM deformation rates and local ECM stiffening at the microscale with capillary morphogenesis at the macroscale. This journal is © The Royal Society of Chemistry 2012

  13. Quantification of local matrix deformations and mechanical properties during capillary morphogenesis in 3D†‡

    Science.gov (United States)

    Kniazeva, Ekaterina; Weidling, John W.; Singh, Rahul; Botvinick, Elliot L.; Digman, Michelle A.; Gratton, Enrico

    2013-01-01

    Reciprocal mechanical interactions between cells and the extracellular matrix (ECM) are thought to play important instructive roles in branching morphogenesis. However, most studies to date have failed to characterize these interactions on a length scale relevant to cells, especially in three-dimensional (3D) matrices. Here we utilized two complementary methods, spatio-temporal image correlation spectroscopy (STICS) and laser optical tweezers-based active microrheology (AMR), to quantify endothelial cell (EC)-mediated deformations of individual ECM elements and the local ECM mechanical properties, respectively, during the process of capillary morphogenesis in a 3D cell culture model. In experiments in which the ECM density was systematically varied, STICS revealed that the rate at which ECs deformed individual ECM fibers on the microscale positively correlated with capillary sprouting on the macroscale. ECs expressing constitutively active V14-RhoA displaced individual matrix fibers at significantly faster rates and displayed enhanced capillary sprouting relative to wild-type cells, while those expressing dominant-negative N19-RhoA behaved in an opposite fashion. In parallel, AMR revealed a local stiffening of the ECM proximal to the tips of sprouting ECs. By quantifying the dynamic physical properties of the cell-ECM interface in both space and time, we identified a correlation linking ECM deformation rates and local ECM stiffening at the microscale with capillary morphogenesis at the macroscale. PMID:22281872

  14. PAR-Complex and Crumbs Function During Photoreceptor Morphogenesis and Retinal Degeneration.

    Science.gov (United States)

    Pichaud, Franck

    2018-01-01

    The fly photoreceptor has long been used as a model to study sensory neuron morphogenesis and retinal degeneration. In particular, elucidating how these cells are built continues to help further our understanding of the mechanisms of polarized cell morphogenesis, intracellular trafficking and the causes of human retinal pathologies. The conserved PAR complex, which in flies consists of Cdc42-PAR6-aPKC-Bazooka, and the transmembrane protein Crumbs (Crb) are key players during photoreceptor morphogenesis. While the PAR complex regulates polarity in many cell types, Crb function in polarity is relatively specific to epithelial cells. Together Cdc42-PAR6-aPKC-Bazooka and Crb orchestrate the differentiation of the photoreceptor apical membrane (AM) and zonula adherens (ZA) , thus allowing these cells to assemble into a neuro-epithelial lattice. In addition to its function in epithelial polarity, Crb has also been shown to protect fly photoreceptors from light-induced degeneration, a process linked to Rhodopsin expression and trafficking. Remarkably, mutations in the human Crumbs1 (CRB1) gene lead to retinal degeneration, making the fly photoreceptor a powerful disease model system.

  15. PAR-Complex and Crumbs Function During Photoreceptor Morphogenesis and Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    Franck Pichaud

    2018-03-01

    Full Text Available The fly photoreceptor has long been used as a model to study sensory neuron morphogenesis and retinal degeneration. In particular, elucidating how these cells are built continues to help further our understanding of the mechanisms of polarized cell morphogenesis, intracellular trafficking and the causes of human retinal pathologies. The conserved PAR complex, which in flies consists of Cdc42-PAR6-aPKC-Bazooka, and the transmembrane protein Crumbs (Crb are key players during photoreceptor morphogenesis. While the PAR complex regulates polarity in many cell types, Crb function in polarity is relatively specific to epithelial cells. Together Cdc42-PAR6-aPKC-Bazooka and Crb orchestrate the differentiation of the photoreceptor apical membrane (AM and zonula adherens (ZA, thus allowing these cells to assemble into a neuro-epithelial lattice. In addition to its function in epithelial polarity, Crb has also been shown to protect fly photoreceptors from light-induced degeneration, a process linked to Rhodopsin expression and trafficking. Remarkably, mutations in the human Crumbs1 (CRB1 gene lead to retinal degeneration, making the fly photoreceptor a powerful disease model system.

  16. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta

    International Nuclear Information System (INIS)

    Hirai, Yohei; Radisky, Derek; Boudreau, Rosanne; Simian, Marina; Stevens, Mary E.; Oka, Yumiko; Takebe, Kyoko; Niwa, Shinichiro; Bissell, Mina J.

    2002-01-01

    We have previously shown that epimorphin, a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which epimorphin mediates luminal morphogenesis. Treatment of cells with epimorphin to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, as constitutive expression of LIP was sufficient to produce lumen formation, while constitutive expression of LAP blocked epimorphin-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which epimorphin expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for epimorphin in luminal morphogenesis through control of C/EBPbeta expression

  17. SACE_0012, a TetR-family transcriptional regulator, affects the morphogenesis of Saccharopolyspora erythraea.

    Science.gov (United States)

    Yin, Xiaojuan; Xu, Xinqiang; Wu, Hang; Yuan, Li; Huang, Xunduan; Zhang, Buchang

    2013-12-01

    Saccharopolyspora erythraea, a mycelium-forming actinomycete, produces a clinically important antibiotic erythromycin. Extensive investigations have provided insights into erythromycin biosynthesis in S. erythraea, but knowledge of its morphogenesis remains limited. By gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE_0012 was identified to be a negative regulator of mycelium formation of S. erythraea A226. Detected by quantitative real-time PCR, the relative transcription of SACE_7115, the amfC homolog for an aerial mycelium formation protein, was dramatically increased in SACE_0012 mutant, whereas erythromycin biosynthetic gene eryA, a pleiotropic regulatory gene bldD, and the genes SACE_2141, SACE_6464, SACE_6040, that are the homologs to the sporulation regulators WhiA, WhiB, WhiG, were not differentially expressed. SACE_0012 disruption could not restore its defect of aerial development in bldD mutant, and also did not further accelerate the mycelium formation in the mutant of SACE_7040 gene, that was previously identified to be a morphogenesis repressor. Furthermore, the transcriptional level of SACE_0012 had not markedly changed in bldD and SACE_7040 mutant over A226. Taken together, these results suggest that SACE_0012 is a negative regulator of S. erythraea morphogenesis by mainly increasing the transcription of amfC gene, independently of the BldD regulatory system.

  18. Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant

    Directory of Open Access Journals (Sweden)

    NELSON ACOSTA-RIVERO

    2009-01-01

    Full Text Available In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-γ producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.

  19. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

    Science.gov (United States)

    Chervyakova, Olga V; Zaitsev, Valentin L; Iskakov, Bulat K; Tailakova, Elmira T; Strochkov, Vitaliy M; Sultankulova, Kulyaisan T; Sandybayev, Nurlan T; Stanbekova, Gulshan E; Beisenov, Daniyar K; Abduraimov, Yergali O; Mambetaliyev, Muratbay; Sansyzbay, Abylay R; Kovalskaya, Natalia Y; Nemchinov, Lev G; Hammond, Rosemarie W

    2016-06-07

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

  20. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Olga V. Chervyakova

    2016-06-01

    Full Text Available The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122, orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

  1. Hendra and Nipah viruses: pathogenesis, animal models and recent breakthroughs in vaccination

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    Weingartl HM

    2015-09-01

    Full Text Available Hana M Weingartl National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Winnipeg, MB, Canada Abstract: Hendra and Nipah viruses are two highly pathogenic zoonotic members of the genus Henipavirus, family Paramyxoviridae, requiring work under biosafety level 4 conditions due to a lack of effective therapy and human vaccines. Several vaccine candidates were protective in animal models: recombinant vaccinia virus expressing Nipah virus (NiV F and G proteins in hamsters against NiV; recombinant ALVAC–NiV F and G in swine against NiV; recombinant Hendra virus (HeV soluble G protein (sGHeV against HeV and NiV in cats, ferrets, horses, and African green monkeys (AGM; recombinant vesicular stomatitis virus-based vectors expressing NiV F or G against NiV in hamsters and ferrets; measles virus-based NiV G vaccine candidate in hamsters and AGMs against NiV; and adenoassociated virus expressing NiG protein, which protected hamsters against NiV. The sGHeV was licensed for use in horses (Equivac HeV® in 2012. It is the first vaccine candidate licensed against a biosafety level 4 agent. With the development of suitable animal models (ferret, hamster and, importantly, AGM, progress can be made toward development of a human vaccine.Keywords: henipavirus, equine, swine, human infection, animal models, vaccine candidates

  2. The Ebola virus VP35 protein is a suppressor of RNA silencing.

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    Joost Haasnoot

    2007-06-01

    Full Text Available RNA silencing or interference (RNAi is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.

  3. Transmissibility of the monkeypox virus clades via respiratory transmission: investigation using the prairie dog-monkeypox virus challenge system.

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    Christina L Hutson

    Full Text Available Monkeypox virus (MPXV is endemic within Africa where it sporadically is reported to cause outbreaks of human disease. In 2003, an outbreak of human MPXV occurred in the US after the importation of infected African rodents. Since the eradication of smallpox (caused by an orthopoxvirus (OPXV related to MPXV and cessation of routine smallpox vaccination (with the live OPXV vaccinia, there is an increasing population of people susceptible to OPXV diseases. Previous studies have shown that the prairie dog MPXV model is a functional animal model for the study of systemic human OPXV illness. Studies with this model have demonstrated that infected animals are able to transmit the virus to naive animals through multiple routes of exposure causing subsequent infection, but were not able to prove that infected animals could transmit the virus exclusively via the respiratory route. Herein we used the model system to evaluate the hypothesis that the Congo Basin clade of MPXV is more easily transmitted, via respiratory route, than the West African clade. Using a small number of test animals, we show that transmission of viruses from each of the MPXV clade was minimal via respiratory transmission. However, transmissibility of the Congo Basin clade was slightly greater than West African MXPV clade (16.7% and 0% respectively. Based on these findings, respiratory transmission appears to be less efficient than those of previous studies assessing contact as a mechanism of transmission within the prairie dog MPXV animal model.

  4. Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

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    Zanotto Carlo

    2011-11-01

    Full Text Available Abstract Background Human papilloma virus (HPV-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1 have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species. Methods A new fowlpox virus recombinant encoding HPV-L1 (FPL1 was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays. Results The FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector. Conclusion This FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  6. Preventing the return of smallpox: molecular modeling studies on thymidylate kinase from Variola virus.

    Science.gov (United States)

    Guimarães, Ana Paula; Ramalho, Teodorico Castro; França, Tanos Celmar Costa

    2014-01-01

    Smallpox was one of the most devastating diseases in the human history and still represents a serious menace today due to its potential use by bioterrorists. Considering this threat and the non-existence of effective chemotherapy, we propose the enzyme thymidylate kinase from Variola virus (VarTMPK) as a potential target to the drug design against smallpox. We first built a homology model for VarTMPK and performed molecular docking studies on it in order to investigate the interactions with inhibitors of Vaccinia virus TMPK (VacTMPK). Subsequently, molecular dynamics (MD) simulations of these compounds inside VarTMPK and human TMPK (HssTMPK) were carried out in order to select the most promising and selective compounds as leads for the design of potential VarTMPK inhibitors. Results of the docking and MD simulations corroborated to each other, suggesting selectivity towards VarTMPK and, also, a good correlation with the experimental data.

  7. High-throughput screening to enhance oncolytic virus immunotherapy

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    Allan KJ

    2016-04-01

    Full Text Available KJ Allan,1,2 David F Stojdl,1–3 SL Swift1 1Children’s Hospital of Eastern Ontario (CHEO Research Institute, 2Department of Biology, Microbiology and Immunology, 3Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada Abstract: High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms – including those based on herpes simplex virus, reovirus, and vaccinia virus – have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. Keywords: oncolytic, virus, screen, high-throughput, cancer, chemical, genomic, immunotherapy

  8. Three-dimensional visualization of forming Hepatitis C virus-like particles by electron-tomography

    Energy Technology Data Exchange (ETDEWEB)

    Badia-Martinez, Daniel; Peralta, Bibiana [Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio (Spain); Andres, German; Guerra, Milagros [Electron Microscopy Unit, Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Campus Cantoblanco, 28049 Madrid (Spain); Gil-Carton, David [Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio (Spain); Abrescia, Nicola G.A., E-mail: nabrescia@cicbiogune.es [Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio (Spain); IKERBASQUE, Basque Foundation for Science, 48011 Bilbao (Spain)

    2012-09-01

    Hepatitis C virus infects almost 170 million people per year but its assembly pathway, architecture and the structures of its envelope proteins are poorly understood. Using electron tomography of plastic-embedded sections of insect cells, we have visualized the morphogenesis of recombinant Hepatitis C virus-like particles. Our data provide a three-dimensional sketch of viral assembly at the endoplasmic reticulum showing different budding stages and contiguity of buds. This latter phenomenon could play an important role during the assembly of wt-HCV and explain the size-heterogeneity of its particles.

  9. Three-dimensional visualization of forming Hepatitis C virus-like particles by electron-tomography

    International Nuclear Information System (INIS)

    Badia-Martinez, Daniel; Peralta, Bibiana; Andrés, German; Guerra, Milagros; Gil-Carton, David; Abrescia, Nicola G.A.

    2012-01-01

    Hepatitis C virus infects almost 170 million people per year but its assembly pathway, architecture and the structures of its envelope proteins are poorly understood. Using electron tomography of plastic-embedded sections of insect cells, we have visualized the morphogenesis of recombinant Hepatitis C virus-like particles. Our data provide a three-dimensional sketch of viral assembly at the endoplasmic reticulum showing different budding stages and contiguity of buds. This latter phenomenon could play an important role during the assembly of wt-HCV and explain the size-heterogeneity of its particles.

  10. Chikungunya virus

    Science.gov (United States)

    Chikungunya virus infection; Chikungunya ... Where Chikungunya is Found Before 2013, the virus was found in Africa, Asia, Europe, and the Indian and Pacific oceans. In late 2013, outbreaks occurred for the first time in the ...

  11. Zika Virus

    Science.gov (United States)

    ... through blood transfusions. There have been outbreaks of Zika virus in the United States, Africa, Southeast Asia, the ... not travel to areas where there is a Zika virus outbreak. If you do decide to travel, first ...

  12. Zika Virus

    Science.gov (United States)

    ... Funding CDC Activities For Healthcare Providers Clinical Evaluation & Disease Sexual Transmission HIV Infection & Zika Virus Testing for Zika Test Specimens – At Time of Birth Diagnostic Tests Understanding Zika Virus Test Results ...

  13. Targeting Poxvirus Decapping Enzymes and mRNA Decay to Generate an Effective Oncolytic Virus

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    Hannah Burgess

    2018-03-01

    Full Text Available Through the action of two virus-encoded decapping enzymes (D9 and D10 that remove protective caps from mRNA 5′-termini, Vaccinia virus (VACV accelerates mRNA decay and limits activation of host defenses. D9- or D10-deficient VACV are markedly attenuated in mice and fail to counter cellular double-stranded RNA-responsive innate immune effectors, including PKR. Here, we capitalize upon this phenotype and demonstrate that VACV deficient in either decapping enzyme are effective oncolytic viruses. Significantly, D9- or D10-deficient VACV displayed anti-tumor activity against syngeneic mouse tumors of different genetic backgrounds and human hepatocellular carcinoma xenografts. Furthermore, D9- and D10-deficient VACV hyperactivated the host anti-viral enzyme PKR in non-tumorigenic cells compared to wild-type virus. This establishes a new genetic platform for oncolytic VACV development that is deficient for a major pathogenesis determinant while retaining viral genes that support robust productive replication like those required for nucleotide metabolism. It further demonstrates how VACV mutants unable to execute a fundamental step in virus-induced mRNA decay can be unexpectedly translated into a powerful anti-tumor therapy. Keywords: oncolytic virus, mRNA decay, decapping

  14. Real-Time PCR Assay To Detect Smallpox Virus

    Science.gov (United States)

    Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.

    2003-01-01

    We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler

  15. Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center.

    Directory of Open Access Journals (Sweden)

    Gabriela N Condezo

    2017-04-01

    Full Text Available Adenovirus (AdV morphogenesis is a complex process, many aspects of which remain unclear. In particular, it is not settled where in the nucleus assembly and packaging occur, and whether these processes occur in a sequential or a concerted manner. Here we use immunofluorescence and immunoelectron microscopy (immunoEM to trace packaging factors and structural proteins at late times post infection by either wildtype virus or a delayed packaging mutant. We show that representatives of all assembly factors are present in the previously recognized peripheral replicative zone, which therefore is the AdV assembly factory. Assembly intermediates and abortive products observed in this region favor a concurrent assembly and packaging model comprising two pathways, one for capsid proteins and another one for core components. Only when both pathways are coupled by correct interaction between packaging proteins and the genome is the viral particle produced. Decoupling generates accumulation of empty capsids and unpackaged cores.

  16. Strategies to obtain multiple recombinant modified vaccinia Ankara vectors. Applications to influenza vaccines.

    Science.gov (United States)

    Barbieri, Andrea; Panigada, Maddalena; Soprana, Elisa; Di Mario, Giuseppina; Gubinelli, Francesco; Bernasconi, Valentina; Recagni, Marta; Donatelli, Isabella; Castrucci, Maria R; Siccardi, Antonio G

    2018-01-01

    As a vaccination vector, MVA has been widely investigated both in animal models and humans. The construction of recombinant MVA (rMVA) relies on homologous recombination between an acceptor virus and a donor plasmid in infected/transfected permissive cells. Our construction strategy "Red-to-Green gene swapping" - based on the exchange of two fluorescent markers within the flanking regions of MVA deletion ΔIII, coupled to fluorescence activated cell sorting - is here extended to a second insertion site, within the flanking regions of MVA deletion ΔVI. Exploiting this strategy, both double and triple rMVA were constructed, expressing as transgenes the influenza A proteins HA, NP, M1, and PB1. Upon validation of the harbored transgenes co-expression, double and triple recombinants rMVA(ΔIII)-NP-P2A-M1 and rMVA(ΔIII)-NP-P2A-M1-(ΔVI)-PB1 were assayed for in vivo immunogenicity and protection against lethal challenge. In vivo responses were identical to those obtained with the reported combinations of single recombinants, supporting the feasibility and reliability of the present improvement and the extension of Red-to-Green gene swapping to insertion sites other than ΔIII. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Exploring bacteria-induced growth and morphogenesis in the green macroalga order Ulvales (Chlorophyta

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    Thomas eWichard

    2015-03-01

    Full Text Available Green macroalgae, such as Ulvales, lose their typical morphology completely when grown under axenic conditions or in the absence of the appropriate microbiome. As a result, slow growing aberrant phenotypes or even callus-like morphotypes are observed in Ulvales. The cross-kingdom interactions between marine algae and microorganisms are hence not only restricted by the exchange of macronutrients, including vitamins and nutrients, but also by infochemicals such as bacterial morphogenetic compounds. The latter are a fundamental trait mediating the mutualism within the chemosphere where the organisms interact with each other via compounds in their surroundings.Approximately 60 years ago, pilot studies demonstrated that certain bacteria promote growth, whereas other bacteria induce morphogenesis; this is particularly true for the order of Ulvales. However, only slow progress was made towards the underlying mechanism due to the complexity of, for example, algal cultivation techniques, and the lack of standardized experiments in the laboratory.A breakthrough in this research was the discovery of the morphogenetic compound thallusin, which was isolated from an epiphytic bacterium and induces normal germination and restores the foliaceous morphotypes of Monostroma. Owing to the low concentration, the purification and structure elucidation of highly biologically active morphogenetic compounds is still challenging. Recently, it was found that only the combination of two specific bacteria from the Rhodobacteraceae and Flavobacteriaceae can completely recover the growth and morphogenesis of axenic Ulva mutabilis cultures forming a symbiotic tripartite community by chemical communication.This review combines literature detailing evidence of bacteria-induced morphogenesis in Ulvales. A set of standardized experimental approaches is further proposed for the preparation of axenic algal tissues, bacteria isolation, co-cultivation experiments, and the analysis of

  18. Time-lapse analysis and mathematical characterization elucidate novel mechanisms underlying muscle morphogenesis.

    Directory of Open Access Journals (Sweden)

    Chelsi J Snow

    2008-10-01

    Full Text Available Skeletal muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction (MTJ. In vertebrates, a great deal is known about muscle specification as well as how somitic cells, as a cohort, generate the early myotome. However, the cellular mechanisms that generate long muscle fibers from short cells and the molecular factors that limit elongation are unknown. We show that zebrafish fast muscle fiber morphogenesis consists of three discrete phases: short precursor cells, intercalation/elongation, and boundary capture/myotube formation. In the first phase, cells exhibit randomly directed protrusive activity. The second phase, intercalation/elongation, proceeds via a two-step process: protrusion extension and filling. This repetition of protrusion extension and filling continues until both the anterior and posterior ends of the muscle fiber reach the MTJ. Finally, both ends of the muscle fiber anchor to the MTJ (boundary capture and undergo further morphogenetic changes as they adopt the stereotypical, cylindrical shape of myotubes. We find that the basement membrane protein laminin is required for efficient elongation, proper fiber orientation, and boundary capture. These early muscle defects in the absence of either lamininbeta1 or laminingamma1 contrast with later dystrophic phenotypes in lamininalpha2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development. Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon. Taken together, our results define three phases of muscle fiber morphogenesis and show that the critical second phase of elongation proceeds by a repetitive process of protrusion extension and protrusion filling. Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.

  19. Rac1 modulates mammalian lung branching morphogenesis in part through canonical Wnt signaling.

    Science.gov (United States)

    Danopoulos, Soula; Krainock, Michael; Toubat, Omar; Thornton, Matthew; Grubbs, Brendan; Al Alam, Denise

    2016-12-01

    Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity, and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment, and polarization. Furthermore, Rac1 modulates both noncanonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation. Copyright © 2016 the American Physiological Society.

  20. Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

    Science.gov (United States)

    Song, Hongman; Bush, Ronald A.; Vijayasarathy, Camasamudram; Fariss, Robert N.; Kjellstrom, Sten; Sieving, Paul A.

    2014-01-01

    Purpose. Dominant-active RAC1 rescues photoreceptor structure in Drosophila rhodopsin-null mutants, indicating an important role in morphogenesis. This report assesses the morphogenetic effect of activated RAC1 during mammalian rod photoreceptor development using transgenic mice that express constitutively active (CA) RAC1. Methods. Transgenic mice were generated by expressing CA RAC1 under control of the Rhodopsin promoter, and morphological features of the photoreceptors were evaluated by histology, immunohistochemistry, and transmission electron microscopy. Function was evaluated by electroretinography. Potential protein partners of CA RAC1 were identified by co-immunoprecipitation of retinal extracts. Results. Constitutively active RAC1 expression in differentiating rods disrupted outer retinal lamination as early as postnatal day (P)6, and many photoreceptor cell nuclei were displaced apically into the presumptive subretinal space. These photoreceptors did not develop normal inner and outer segments and had abnormal placement of synaptic elements. Some photoreceptor nuclei were also mislocalized into the inner nuclear layer. Extensive photoreceptor degeneration was subsequently observed in the adult animal. Constitutively active RAC1 formed a complex with the polarity protein PAR6 and with microtubule motor dynein in mouse retina. The normal localization of the PAR6 complex was disrupted in CA RAC1-expressing rod photoreceptors. Conclusions. Constitutively active RAC1 had a profound negative effect on mouse rod cell viability and development. Rod photoreceptors in the CA RAC1 retina exhibited a defect in polarity and migration. Constitutively active RAC1 disrupted rod morphogenesis and gave a phenotype resembling that found in the Crumbs mutant. PAR6 and dynein are two potential downstream effectors that may be involved in CA RAC1-mediated defective mouse photoreceptor morphogenesis. PMID:24651551

  1. Modulating Wnt Signaling Rescues Palate Morphogenesis in Pax9 Mutant Mice.

    Science.gov (United States)

    Li, C; Lan, Y; Krumlauf, R; Jiang, R

    2017-10-01

    Cleft palate is a common birth defect caused by disruption of palatogenesis during embryonic development. Although mutations disrupting components of the Wnt signaling pathway have been associated with cleft lip and palate in humans and mice, the mechanisms involving canonical Wnt signaling and its regulation in secondary palate development are not well understood. Here, we report that canonical Wnt signaling plays an important role in Pax9-mediated regulation of secondary palate development. We found that cleft palate pathogenesis in Pax9-deficient embryos is accompanied by significantly reduced expression of Axin2, an endogenous target of canonical Wnt signaling, in the developing palatal mesenchyme, particularly in the posterior regions of the palatal shelves. We found that expression of Dkk2, encoding a secreted Wnt antagonist, is significantly increased whereas the levels of active β-catenin protein, the essential transcriptional coactivator of canonical Wnt signaling, is significantly decreased in the posterior regions of the palatal shelves in embryonic day 13.5 Pax9-deficent embryos in comparison with control littermates. We show that small molecule-mediated inhibition of Dickkopf (DKK) activity in utero during palatal shelf morphogenesis partly rescued secondary palate development in Pax9-deficient embryos. Moreover, we found that genetic inactivation of Wise, which is expressed in the developing palatal shelves and encodes another secreted antagonist of canonical Wnt signaling, also rescued palate morphogenesis in Pax9-deficient mice. Furthermore, whereas Pax9 del/del embryos exhibit defects in palatal shelf elevation/reorientation and significant reduction in accumulation of hyaluronic acid-a high molecular extracellular matrix glycosaminoglycan implicated in playing an important role in palatal shelf elevation-80% of Pax9 del/del ;Wise -/- double-mutant mouse embryos exhibit rescued palatal shelf elevation/reorientation, accompanied by restored

  2. CDKL5, a protein associated with rett syndrome, regulates neuronal morphogenesis via Rac1 signaling.

    Science.gov (United States)

    Chen, Qian; Zhu, Yong-Chuan; Yu, Jing; Miao, Sheng; Zheng, Jing; Xu, Li; Zhou, Yang; Li, Dan; Zhang, Chi; Tao, Jiong; Xiong, Zhi-Qi

    2010-09-22

    Mutations in cyclin-dependent kinase-like 5 (CDKL5), also known as serine/threonine kinase 9 (STK9), have been identified in patients with Rett syndrome (RTT) and X-linked infantile spasm. However, the function of CDKL5 in the brain remains unknown. Here, we report that CDKL5 is a critical regulator of neuronal morphogenesis. We identified a neuron-specific splicing variant of CDKL5 whose expression was markedly induced during postnatal development of the rat brain. Downregulating CDKL5 by RNA interference (RNAi) in cultured cortical neurons inhibited neurite growth and dendritic arborization, whereas overexpressing CDKL5 had opposite effects. Furthermore, knocking down CDKL5 in the rat brain by in utero electroporation resulted in delayed neuronal migration, and severely impaired dendritic arborization. In contrast to its proposed function in the nucleus, we found that CDKL5 regulated dendrite development through a cytoplasmic mechanism. In fibroblasts and in neurons, CDKL5 colocalized and formed a protein complex with Rac1, a critical regulator of actin remodeling and neuronal morphogenesis. Overexpression of Rac1 prevented the inhibition of dendrite growth caused by CDKL5 knockdown, and the growth-promoting effect of ectopically expressed CDKL5 on dendrites was abolished by coexpressing a dominant-negative form of Rac1. Moreover, CDKL5 was required for brain-derived neurotrophic factor (BDNF)-induced activation of Rac1. Together, these results demonstrate a critical role of CDKL5 in neuronal morphogenesis and identify a Rho GTPase signaling pathway which may contribute to CDKL5-related disorders.

  3. Cell-based multi-parametric model of cleft progression during submandibular salivary gland branching morphogenesis.

    Directory of Open Access Journals (Sweden)

    Shayoni Ray

    Full Text Available Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft

  4. Hypoxia-inducible factor 1α regulates branching morphogenesis during kidney development.

    Science.gov (United States)

    Tsuji, Kenji; Kitamura, Shinji; Makino, Hirofumi

    2014-04-25

    The kidneys are exposed to hypoxic conditions during development. Hypoxia-inducible factor (HIF), an important mediator of the response to hypoxia, is believed to have an important role in development. However, the relationship between HIF and branching morphogenesis has not been elucidated clearly. In this study, we examined whether HIF regulates kidney development. We harvested kidneys from day 13 rat embryos (E13Ks) and cultured the organs under normoxic (20% O2/5% CO2) or hypoxic (5% O2/5% CO2) conditions. We evaluated the kidneys based on morphology and gene expression. E13Ks cultured under hypoxic conditions had significantly more ureteric bud (UB) branching than the E13Ks cultured under normoxic conditions. In addition, the mRNA levels of GDNF and GDNF receptor (GFR-α1), increased under hypoxic conditions in E13Ks. When we cultured E13Ks with the HIF-1α inhibitor digoxin or with siRNA targeting HIF-1α under hypoxic conditions, we did not observe increased UB branching. In addition, the expression of GDNF and GFR-α1 was inhibited under hypoxic conditions when the kidneys were treated with siRNA targeting HIF-1α. We also elucidated that hypoxia inhibited UB cell apoptosis and promoted the expression of FGF7 mRNA levels in metanephric mesenchymal (MM) cells in vitro. These findings suggest that hypoxic condition has important roles in inducing branching morphogenesis during kidney development. Hypoxia might mediate branching morphogenesis via not only GDNF/Ret but also FGF signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Exploring bacteria-induced growth and morphogenesis in the green macroalga order Ulvales (Chlorophyta)

    Science.gov (United States)

    Wichard, Thomas

    2015-01-01

    Green macroalgae, such as Ulvales, lose their typical morphology completely when grown under axenic conditions or in the absence of the appropriate microbiome. As a result, slow growing aberrant phenotypes or even callus-like morphotypes are observed in Ulvales. The cross-kingdom interactions between marine algae and microorganisms are hence not only restricted by the exchange of macronutrients, including vitamins and nutrients, but also by infochemicals such as bacterial morphogenetic compounds. The latter are a fundamental trait mediating the mutualism within the chemosphere where the organisms interact with each other via compounds in their surroundings. Approximately 60 years ago, pilot studies demonstrated that certain bacteria promote growth, whereas other bacteria induce morphogenesis; this is particularly true for the order of Ulvales. However, only slow progress was made towards the underlying mechanism due to the complexity of, for example, algal cultivation techniques, and the lack of standardized experiments in the laboratory. A breakthrough in this research was the discovery of the morphogenetic compound thallusin, which was isolated from an epiphytic bacterium and induces normal germination restoring the foliaceous morphotypes of Monostroma. Owing to the low concentration, the purification and structure elucidation of highly biologically active morphogenetic compounds are still challenging. Recently, it was found that only the combination of two specific bacteria from the Rhodobacteraceae and Flavobacteriaceae can completely recover the growth and morphogenesis of axenic Ulva mutabilis cultures forming a symbiotic tripartite community by chemical communication. This review combines literature detailing evidences of bacteria-induced morphogenesis in Ulvales. A set of standardized experimental approaches is further proposed for the preparation of axenic algal tissues, bacteria isolation, co-cultivation experiments, and the analysis of the chemosphere

  6. hmmr mediates anterior neural tube closure and morphogenesis in the frog Xenopus.

    Science.gov (United States)

    Prager, Angela; Hagenlocher, Cathrin; Ott, Tim; Schambony, Alexandra; Feistel, Kerstin

    2017-10-01

    Development of the central nervous system requires orchestration of morphogenetic processes which drive elevation and apposition of the neural folds and their fusion into a neural tube. The newly formed tube gives rise to the brain in anterior regions and continues to develop into the spinal cord posteriorly. Conspicuous differences between the anterior and posterior neural tube become visible already during neural tube closure (NTC). Planar cell polarity (PCP)-mediated convergent extension (CE) movements are restricted to the posterior neural plate, i.e. hindbrain and spinal cord, where they propagate neural fold apposition. The lack of CE in the anterior neural plate correlates with a much slower mode of neural fold apposition anteriorly. The morphogenetic processes driving anterior NTC have not been addressed in detail. Here, we report a novel role for the breast cancer susceptibility gene and microtubule (MT) binding protein Hmmr (Hyaluronan-mediated motility receptor, RHAMM) in anterior neurulation and forebrain development in Xenopus laevis. Loss of hmmr function resulted in a lack of telencephalic hemisphere separation, arising from defective roof plate formation, which in turn was caused by impaired neural tissue narrowing. hmmr regulated polarization of neural cells, a function which was dependent on the MT binding domains. hmmr cooperated with the core PCP component vangl2 in regulating cell polarity and neural morphogenesis. Disrupted cell polarization and elongation in hmmr and vangl2 morphants prevented radial intercalation (RI), a cell behavior essential for neural morphogenesis. Our results pinpoint a novel role of hmmr in anterior neural development and support the notion that RI is a major driving force for anterior neurulation and forebrain morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Infecção em humanos por varíola bovina na microrregião de Itajubá, Estado de Minas Gerais: relato de caso Human infection by the cowpox virus in the microregion of Itajubá, state of Minas Gerais: case report

    Directory of Open Access Journals (Sweden)

    Afonso Carlos da Silva

    2008-10-01

    Full Text Available Os autores relatam três casos de varíola bovina em humanos, ordenhadores manuais em vacas infectadas, na microrregião de Itajubá, MG. As técnicas diagnósticas foram: isolamento de amostra semelhante ao vírus vaccinia de secreções das lesões cutâneas, reação em cadeia de polimerase, microscopia eletrônica e anticorpos para Orthopoxvirus no sangue dos pacientes.The authors report three human cases of cowpox infection, among farm workers who were manually milking infected cows, in the microregion of Itajubá, Minas Gerais. The diagnostic techniques used were: isolation of samples similar to the vaccinia virus, from skin lesion secretions; polymerase chain reaction; electronic microscopy; and antibodies for Orthopoxvirus in the patients' blood.

  8. Coupling gene expression and multicellular morphogenesis during fruiting body formation in Myxococcus xanthus

    DEFF Research Database (Denmark)

    Søgaard-Andersen, L.; Overgaard, M.; Lobedanz, S.

    2003-01-01

    xanthus illustrates this coupling in the construction of a multicellular structure. Fruiting body formation involves two stages: aggregation of cells into mounds and the position-specific sporulation of cells that have accumulated inside mounds. Developmental gene expression propels these two processes...... morphogenesis. Accumulation of the C-signal is tightly regulated and involves transcriptional activation of the csgA gene and proteolysis of the full-length CsgA protein to produce the shorter cell surface-associated 17 kDa C-signal protein. The C-signal induces aggregation, sporulation and developmental gene...

  9. Complex interactions between GSK3 and aPKC in Drosophila embryonic epithelial morphogenesis.

    Directory of Open Access Journals (Sweden)

    Nicole A Kaplan

    Full Text Available Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.

  10. Morphogenesis and calcification of the statoconia in the chick (Gallus domesticus) embryo - Implications for future studies

    Science.gov (United States)

    Fermin, C. D.; Igarashi, M.

    1985-01-01

    The morphogenesis of the statoconia in the chick, Gallus domesticus, injected with a carbon anhydrase inhibitor is studied. The preparation of the embryo specimens for analysis is described. The early, middle, and late stages of embryonic development are examined. The data reveal that acetozolamide inhibits statoconia formation in the middle stage of development and the calcification process follows statoconia formation. The spatial relationship between the development of type 1 and type 2 hair cells and the appearance and maturation of the statoconia is investigated.

  11. Bicaudal C1 promotes pancreatic NEUROG3+ endocrine progenitor differentiation and ductal morphogenesis

    DEFF Research Database (Denmark)

    Lemaire, Laurence A; Goulley, Joan; Kim, Yung Hae

    2015-01-01

    that line the ducts during development, and in the ducts after birth, but not in differentiated endocrine or acinar cells. Genetic inactivation of Bicc1 leads to ductal cell over-proliferation and cyst formation. Transcriptome comparison between WT and Bicc1 KO pancreata, before the phenotype onset, reveals......(+) endocrine progenitor production. Its deletion leads to a late but sustained endocrine progenitor decrease, resulting in a 50% reduction of endocrine cells. We show that BICC1 functions downstream of ONECUT1 in the pathway controlling both NEUROG3(+) endocrine cell production and ductal morphogenesis...

  12. The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

    OpenAIRE

    Keddie, J S; Carroll, B; Jones, J D; Gruissem, W

    1996-01-01

    The defective chloroplasts and leaves-mutable (dcl-m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild-type in appearance. Within dark green sectors of dcl-m l...

  13. Chikungunya Virus Vaccines: Viral Vector-Based Approaches.

    Science.gov (United States)

    Ramsauer, Katrin; Tangy, Frédéric

    2016-12-15

    In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  14. [The development of therapeutic vaccine for hepatitis C virus].

    Science.gov (United States)

    Kimura, Kiminori; Kohara, Michinori

    2012-10-01

    Chronic hepatitis C caused by infection with the hepatitis C virus(HCV)is a global health problem. HCV causes persistent infection that can lead to chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The therapeutic efficacy of antiviral drugs is not optimal in patients with chronic infection; furthermore, an effective vaccine has not yet been developed. To design an effective HCV vaccine, generation of a convenient animal model of HCV infection is necessary. Recently, we used the Cre/loxP switching system to generate an immunocompetent mouse model of HCV expression, thereby enabling the study of host immune responses against HCV proteins. At present vaccine has not yet been shown to be therapeutically effective against chronic HCV infection. We examined the therapeutic effects of a recombinant vaccinia virus(rVV)encoding HCV protein in a mouse model. we generated rVVs for 3 different HCV proteins and found that one of the recombinant viruses encoding a nonstructural protein(rVV-N25)resolved pathological chronic hepatitis C symptoms in the liver. We propose the possibility that rVV-N25 immunization has the potential for development of an effective therapeutic vaccine for HCV induced chronic hepatitis. The utilization of the therapeutic vaccine can protect progress to chronic hepatitis, and as a consequence, leads to eradication of hepatocellular carcinoma. In this paper, we summarized our current study for HCV therapeutic vaccine and review the vaccine development to date.

  15. Canonical TGF-β Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

    Science.gov (United States)

    Nakashima, Hideyuki; Tsujimura, Keita; Irie, Koichiro; Ishizu, Masataka; Pan, Miao; Kameda, Tomonori; Nakashima, Kinichi

    2018-05-16

    Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases. SIGNIFICANCE STATEMENT Canonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our

  16. Recent advances in the development of vaccines for Ebola virus disease.

    Science.gov (United States)

    Ohimain, Elijah Ige

    2016-01-04

    Ebola virus is one of the most dangerous microorganisms in the world causing hemorrhagic fevers in humans and non-human primates. Ebola virus (EBOV) is a zoonotic infection, which emerges and re-emerges in human populations. The 2014 outbreak was caused by the Zaire strain, which has a kill rate of up to 90%, though 40% was recorded in the current outbreak. The 2014 outbreak is larger than all 20 outbreaks that have occurred since 1976, when the virus was first discovered. It is the first time that the virus was sustained in urban centers and spread beyond Africa into Europe and USA. Thus far, over 22,000 cases have been reported with about 50% mortality in one year. There are currently no approved therapeutics and preventive vaccines against Ebola virus disease (EVD). Responding to the devastating effe1cts of the 2014 outbreak and the potential risk of global spread, has spurred research for the development of therapeutics and vaccines. This review is therefore aimed at presenting the progress of vaccine development. Results showed that conventional inactivated vaccines produced from EBOV by heat, formalin or gamma irradiation appear to be ineffective. However, novel vaccines production techniques have emerged leading to the production of candidate vaccines that have been demonstrated to be effective in preclinical trials using small animal and non-human primates (NHP) models. Some of the promising vaccines have undergone phase 1 clinical trials, which demonstrated their safety and immunogenicity. Many of the candidate vaccines are vector based such as Vesicular Stomatitis Virus (VSV), Rabies Virus (RABV), Adenovirus (Ad), Modified Vaccinia Ankara (MVA), Cytomegalovirus (CMV), human parainfluenza virus type 3 (HPIV3) and Venezuelan Equine Encephalitis Virus (VEEV). Other platforms include virus like particle (VLP), DNA and subunit vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections.

    Science.gov (United States)

    O'Bryan, Joel M; Woda, Marcia; Co, Mary; Mathew, Anuja; Rothman, Alan L

    2013-08-26

    Declining telomere length (TL) is associated with T cell senescence. While TL in naïve and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4+ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years. VACV- and CMV-specific T cells had longer average TL than IAV-specific CD4+ T cells. Although most virus-specific cells were CD45RA-, we observed a minor population of BrdU+ CD45RA+ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naïve T cell repertoire. TLs in memory CD4+ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory.

  18. Genetic Variability of Myxoma Virus Genomes

    Science.gov (United States)

    Braun, Christoph; Thürmer, Andrea; Daniel, Rolf; Schultz, Anne-Kathrin; Bulla, Ingo; Schirrmeier, Horst; Mayer, Dietmar; Neubert, Andreas

    2016-01-01

    ABSTRACT Myxomatosis is a recurrent problem on rabbit farms throughout Europe despite the success of vaccines. To identify gene variations of field and vaccine strains that may be responsible for changes in virulence, immunomodulation, and immunoprotection, the genomes of 6 myxoma virus (MYXV) strains were sequenced: German field isolates Munich-1, FLI-H, 2604, and 3207; vaccine strain MAV; and challenge strain ZA. The analyzed genomes ranged from 147.6 kb (strain MAV) to 161.8 kb (strain 3207). All sequences were affected by several mutations, covering 24 to 93 open reading frames (ORFs) and resulted in amino acid substitutions, insertions, or deletions. Only strains Munich-1 and MAV revealed the deletion of 10 ORFs (M007L to M015L) and 11 ORFs (M007L to M008.1L and M149R to M008.1R), respectively. Major differences were observed in the 27 immunomodulatory proteins encoded by MYXV. Compared to the reference strain Lausanne, strains FLI-H, 2604, 3207, and ZA showed the highest amino acid identity (>98.4%). In strains Munich-1 and MAV, deletion of 5 and 10 ORFs, respectively, was observed, encoding immunomodulatory proteins with ankyrin repeats or members of the family of serine protease inhibitors. Furthermore, putative immunodominant surface proteins with homology to vaccinia virus (VACV) were investigated in the sequenced strains. Only strain MAV revealed above-average frequencies of amino acid substitutions and frameshift mutations. Finally, we performed recombination analysis and found signs of recombination in vaccine strain MAV. Phylogenetic analysis showed a close relationship of strain MAV and the MSW strain of Californian MYXV. However, in a challenge model, strain MAV provided full protection against lethal challenges with strain ZA. IMPORTANCE Myxoma virus (MYXV) is pathogenic for European rabbits and two North American species. Due to sophisticated strategies in immune evasion and oncolysis, MYXV is an important model virus for immunological and

  19. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

    International Nuclear Information System (INIS)

    Li Jianwei; Faber, Milosz; Papaneri, Amy; Faber, Marie-Luise; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard

    2006-01-01

    Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus

  20. Spatiotemporal mechanical variation reveals critical role for rho kinase during primitive streak morphogenesis.

    Science.gov (United States)

    Henkels, Julia; Oh, Jaeho; Xu, Wenwei; Owen, Drew; Sulchek, Todd; Zamir, Evan

    2013-02-01

    Large-scale morphogenetic movements during early embryo development are driven by complex changes in biochemical and biophysical factors. Current models for amniote primitive streak morphogenesis and gastrulation take into account numerous genetic pathways but largely ignore the role of mechanical forces. Here, we used atomic force microscopy (AFM) to obtain for the first time precise biomechanical properties of the early avian embryo. Our data reveal that the primitive streak is significantly stiffer than neighboring regions of the epiblast, and that it is stiffer than the pre-primitive streak epiblast. To test our hypothesis that these changes in mechanical properties are due to a localized increase of actomyosin contractility, we inhibited actomyosin contractility via the Rho kinase (ROCK) pathway using the small-molecule inhibitor Y-27632. Our results using several different assays show the following: (1) primitive streak formation was blocked; (2) the time-dependent increase in primitive streak stiffness was abolished; and (3) convergence of epiblast cells to the midline was inhibited. Taken together, our data suggest that actomyosin contractility is necessary for primitive streak morphogenesis, and specifically, ROCK plays a critical role. To better understand the underlying mechanisms of this fundamental process, future models should account for the findings presented in this study.

  1. Micro/nano-computed tomography technology for quantitative dynamic, multi-scale imaging of morphogenesis.

    Science.gov (United States)

    Gregg, Chelsea L; Recknagel, Andrew K; Butcher, Jonathan T

    2015-01-01

    Tissue morphogenesis and embryonic development are dynamic events challenging to quantify, especially considering the intricate events that happen simultaneously in different locations and time. Micro- and more recently nano-computed tomography (micro/nanoCT) has been used for the past 15 years to characterize large 3D fields of tortuous geometries at high spatial resolution. We and others have advanced micro/nanoCT imaging strategies for quantifying tissue- and organ-level fate changes throughout morphogenesis. Exogenous soft tissue contrast media enables visualization of vascular lumens and tissues via extravasation. Furthermore, the emergence of antigen-specific tissue contrast enables direct quantitative visualization of protein and mRNA expression. Micro-CT X-ray doses appear to be non-embryotoxic, enabling longitudinal imaging studies in live embryos. In this chapter we present established soft tissue contrast protocols for obtaining high-quality micro/nanoCT images and the image processing techniques useful for quantifying anatomical and physiological information from the data sets.

  2. Molecular Biology of Feather Morphogenesis: A Testable Model for Evo-Devo Research

    Science.gov (United States)

    WIDELITZ, RANDALL B.; JIANG, TING XIN; YU, MINGKE; SHEN, TED; SHEN, JEN-YEE; WU, PING; YU, ZHICAO; CHUONG, CHENG-MING

    2015-01-01

    Darwin’s theory describes the principles that are responsible for evolutionary change of organisms and their attributes. The actual mechanisms, however, need to be studied for each species and each organ separately. Here we have investigated the mechanisms underlying these principles in the avian feather. Feathers comprise one of the most complex and diverse epidermal organs as demonstrated by their shape, size, patterned arrangement and pigmentation. Variations can occur at several steps along each level of organization, leading to highly diverse forms and functions. Feathers develop gradually during ontogeny through a series of steps that may correspond to the evolutionary steps that were taken during the phylogeny from a reptilian ancestor to birds. These developmental steps include 1) the formation of feather tract fields on the skin surfaces; 2) periodic patterning of the individual feather primordia within the feather tract fields; 3) feather bud morphogenesis establishing anterio - posterior (along the cranio - caudal axis) and proximo - distal axes; 4) branching morphogenesis to create the rachis, barbs and barbules within a feather bud; and 5) gradual modulations of these basic morphological parameters within a single feather or across a feather tract. Thus, possibilities for variation in form and function of feathers occur at every developmental step. In this paper, principles guiding feather tract formation, distributions of individual feathers within the tracts and variations in feather forms are discussed at a cellular and molecular level. PMID:12949772

  3. A protocadherin-cadherin-FLRT3 complex controls cell adhesion and morphogenesis.

    Directory of Open Access Journals (Sweden)

    Xuejun Chen

    2009-12-01

    Full Text Available Paraxial protocadherin (PAPC and fibronectin leucine-rich domain transmembrane protein-3 (FLRT3 are induced by TGFbeta signaling in Xenopus embryos and both regulate morphogenesis by inhibiting C-cadherin mediated cell adhesion.We have investigated the functional and physical relationships between PAPC, FLRT3, and C-cadherin. Although neither PAPC nor FLRT3 are required for each other to regulate C-cadherin adhesion, they do interact functionally and physically, and they form a complex with cadherins. By itself PAPC reduces cell adhesion physiologically to induce cell sorting, while FLRT3 disrupts adhesion excessively to cause cell dissociation. However, when expressed together PAPC limits the cell dissociating and tissue disrupting activity of FLRT3 to make it effective in physiological cell sorting. PAPC counteracts FLRT3 function by inhibiting the recruitment of the GTPase RND1 to the FLRT3 cytoplasmic domain.PAPC and FLRT3 form a functional complex with cadherins and PAPC functions as a molecular "governor" to maintain FLRT3 activity at the optimal level for physiological regulation of C-cadherin adhesion, cell sorting, and morphogenesis.

  4. TMEM45A Is Dispensable for Epidermal Morphogenesis, Keratinization and Barrier Formation.

    Directory of Open Access Journals (Sweden)

    Aurélie Hayez

    Full Text Available TMEM45A gene encodes an initially uncharacterized predicted transmembrane protein. We previously showed that this gene is highly expressed in keratinocytes where its expression correlates with keratinization, suggesting a role in normal epidermal physiology. To test this hypothesis, we generated TMEM45A knockout mice and found that these mice develop without any evident phenotype. The morphology of the epidermis assessed by histology and by labelling differentiation markers in immunofluorescence was not altered. Toluidine blue permeability assay showed that the epidermal barrier develops normally during embryonic development. We also showed that depletion of TMEM45A in human keratinocytes does not alter their potential to form in vitro 3D-reconstructed epidermis. Indeed, epidermis with normal morphogenesis were generated from TMEM45A-silenced keratinocytes. Their expression of differentiation markers quantified by RT-qPCR and evidenced by immunofluorescence labelling as well as their barrier function estimated by Lucifer yellow permeability were similar to the control epidermis. In summary, TMEM45A gene expression is dispensable for epidermal morphogenesis, keratinization and barrier formation. If this protein plays a role in the epidermis, its experimental depletion can possibly be compensated by other proteins in the two experimental models analyzed in this study.

  5. Influence of plasmogenes on the productivity of morphogenesis in strawberry (Fragaria x ananassa Duch.

    Directory of Open Access Journals (Sweden)

    Jadwiga Żebrowska

    2012-12-01

    Full Text Available Plasmogenes are largely located in mitochondria or plastids and they can influence the inheritance of many plant characteristics. This phenomenon is called cytoplasmic inheritance and can be detected on the basis of the expression of a trait in progeny F1 obtained from single and reciprocal crosses. The aim of this study was to examine the cytoplasmic inheritance of in vitro productivity of morphogenesis in three genotypes of Fragaria x ananassa Duch., i.e. the cultivars 'Dukat', 'Teresa' and the breeding clone no. 590. Single and reciprocal crosses were done according to Griffi ng's method 3. The value of general combining ability (GCA indicated cv. 'Teresa' as the best maternal component for crossing and 'Dukat' as the worst. The negative reciprocal cross effects (rij revealed the cytoplasmic inheritance for cv. 'Dukat' as maternal form and positive rij for the breeding clone no. 590 indicated the nuclear inheritance of morphogenetic ability. Cv. 'Teresa', as maternal component, showed nuclear inheritance of that trait in crossing with cv. 'Dukat' and with 590 cytoplasmic inheritance. The productivity of morphogenesis in strawberry depended on the parental combination and the direction of crossing.

  6. Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

    Directory of Open Access Journals (Sweden)

    Jennifer S. Liu

    2012-11-01

    Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

  7. p38α MAPK Is Required for Tooth Morphogenesis and Enamel Secretion*

    Science.gov (United States)

    Greenblatt, Matthew B.; Kim, Jung-Min; Oh, Hwanhee; Park, Kwang Hwan; Choo, Min-Kyung; Sano, Yasuyo; Tye, Coralee E.; Skobe, Ziedonis; Davis, Roger J.; Park, Jin Mo; Bei, Marianna; Glimcher, Laurie H.; Shim, Jae-Hyuck

    2015-01-01

    An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678–27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38αK14 mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and β4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel. PMID:25406311

  8. Morphology, morphogenesis, and phylogeny of an Anteholosticha intermedia (Ciliophora, Urostylida) population from the United States.

    Science.gov (United States)

    Chen, Lingyun; Wu, Weining; El-Serehy, Hamed A; Hu, Xiaozhong; Clamp, John C

    2018-04-30

    A distinct population of Anteholosticha intermedia was isolated from soil in the Great Smoky Mountains of North Carolina, USA, and its morphology, morphogenesis and molecular phylogeny investigated by microscopic observations of live and protargol-prepared specimens and analyses of the sequence of small subunit (SSU) rDNA. Our population closely resembles the populations from Austria and Korea. Members of the genus Anteholosticha have been regarded as ontogenetically diverse, which is confirmed by the present work. The most noteworthy ontogenetic feature of the American population of A. intermedia is that the oral primordium in the proter appears apokinetally at the posterior end of the undulating membranes anlage at the beginning of division and then dedifferentiates midway through morphogenesis. Molecular phylogenetic analyses demonstrate, with high support, that the American population of A. intermedia is clearly distinct from congeners and branches as part of a sister lineage to the Bakuella-Urostyla clade that belongs to the major clade comprising the order Urostylida. Copyright © 2018 Elsevier GmbH. All rights reserved.

  9. Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-Vibrio association.

    Science.gov (United States)

    Koropatnick, Tanya; Goodson, Michael S; Heath-Heckman, Elizabeth A C; McFall-Ngai, Margaret

    2014-02-01

    The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical, and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function, and biochemistry of the cells as part of the morphogenic program.

  10. A Transient Exposure to Symbiosis-Competent Bacteria Induces Light Organ Morphogenesis in the Host Squid.

    Science.gov (United States)

    Doino, J A; McFall-Ngai, M J

    1995-12-01

    Recent studies of the symbiotic association between the Hawaiian sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri have shown that colonization of juvenile squid with symbiosis-competent bacteria induces morphogenetic changes of the light organ. These changes occur over a 4-day period and include cell death and tissue regression of the external ciliated epithelium. In the absence of bacterial colonization, morphogenesis does not occur. To determine whether the bacteria must be present throughout the morphogenetic process, we used the antibiotic chloramphenicol to clear the light organ of bacteria at various times during the initial colonization. We provide evidence in this study that a transient, 12-hour exposure to symbiosis-competent bacteria is necessary and sufficient to induce tissue regression in the light organ over the next several days. Further, we show that successful entrance into the light organ is necessary to induce morphogenesis, suggesting that induction results from bacterial interaction with internal crypt cells and not with the external ciliated epithelium. Finally, no difference in development was observed when the light organ was colonized by a mutant strain of V. fischeri that did not produce autoinducer, a potential light organ morphogen.

  11. Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana.

    Science.gov (United States)

    Zhu, Chen; Wang, Li; Chen, Jinhua; Liu, Chenglan; Zeng, Huiming; Wang, Huafang

    2017-07-17

    Kalanchoe daigremontiana reproduces asexually by producing plantlets along the leaf margin. The aim of this study was to identify the function of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 gene in Kalanchoe daigremontiana (KdSOC1) during plantlet morphogenesis. In this study, KdSOC1 gene expression was detected at stem cell niche during in vitro somatic embryogenesis and plantlet morphogenesis. Disrupting endogenous auxin transportation suppressed the KdSOC1 gene response. Knockdown of the KdSOC1 gene caused a defect in cotyledon formation during the early heart stage of somatic embryogenesis. Over-expression (OE) of the KdSOC1 gene resulted in asymmetric plantlet distribution, a reduced number of plantlets, thicker leaves, and thicker vascular fibers. Higher KdPIN1 gene expression and auxin content were found in OE plant compared to those of wild-type plant leaves, which indicated possible KdSOC1 gene role in affecting auxin distribution and accumulation. KdSOC1 gene OE in DR5-GUS Arabidopsis reporting lines resulted in an abnormal auxin response pattern during different stages of somatic embryogenesis. In summary, the KdSOC1 gene OE might alter auxin distribution and accumulation along leaf margin to initiate plantlet formation and distribution, which is crucial for plasticity during plantlet formation under various environmental conditions.

  12. The APC tumor suppressor is required for epithelial cell polarization and three-dimensional morphogenesis

    Science.gov (United States)

    Lesko, Alyssa C.; Goss, Kathleen H.; Yang, Frank F.; Schwertner, Adam; Hulur, Imge; Onel, Kenan; Prosperi, Jenifer R.

    2015-01-01

    The Adenomatous Polyposis Coli (APC) tumor suppressor has been previously implicated in the control of apical-basal polarity; yet, the consequence of APC loss-of-function in epithelial polarization and morphogenesis has not been characterized. To test the hypothesis that APC is required for the establishment of normal epithelial polarity and morphogenesis programs, we generated APC-knockdown epithelial cell lines. APC depletion resulted in loss of polarity and multi-layering on permeable supports, and enlarged, filled spheroids with disrupted polarity in 3D culture. Importantly, these effects of APC knockdown were independent of Wnt/β-catenin signaling, but were rescued with either full-length or a carboxy (c)-terminal segment of APC. Moreover, we identified a gene expression signature associated with APC knockdown that points to several candidates known to regulate cell-cell and cell-matrix communication. Analysis of epithelial tissues from mice and humans carrying heterozygous APC mutations further support the importance of APC as a regulator of epithelial behavior and tissue architecture. These data also suggest that the initiation of epithelial-derived tumors as a result of APC mutation or gene silencing may be driven by loss of polarity and dysmorphogenesis. PMID:25578398

  13. Gonad morphogenesis defects drive hybrid male sterility in asymmetric hybrid breakdown of Caenorhabditis nematodes.

    Science.gov (United States)

    Dey, Alivia; Jin, Qi; Chen, Yen-Chu; Cutter, Asher D

    2014-01-01

    Determining the causes and evolution of reproductive barriers to gene flow between populations, speciation, is the key to understanding the origin of diversity in nature. Many species manifest hybrid breakdown when they intercross, characterized by increasingly exacerbated problems in later generations of hybrids. Recently, Caenorhabditis nematodes have emerged as a genetic model for studying speciation, and here we investigate the nature and causes of hybrid breakdown between Caenorhabditis remanei and C. latens. We quantify partial F1 hybrid inviability and extensive F2 hybrid inviability; the ~75% F2 embryonic arrest occurs primarily during gastrulation or embryonic elongation. Moreover, F1 hybrid males exhibit Haldane's rule asymmetrically for both sterility and inviability, being strongest when C. remanei serves as maternal parent. We show that the mechanism by which sterile hybrid males are incapable of transferring sperm or a copulatory plug involves defective gonad morphogenesis, which we hypothesize results from linker cell defects in migration and/or cell death during development. This first documented case of partial hybrid male sterility in Caenorhabditis follows expectations of Darwin's corollary to Haldane's rule for asymmetric male fitness, providing a powerful foundation for molecular dissection of intrinsic reproductive barriers and divergence of genetic pathways controlling organ morphogenesis. © 2014 Wiley Periodicals, Inc.

  14. A computational framework for 3D mechanical modeling of plant morphogenesis with cellular resolution.

    Directory of Open Access Journals (Sweden)

    Frédéric Boudon

    2015-01-01

    Full Text Available The link between genetic regulation and the definition of form and size during morphogenesis remains largely an open question in both plant and animal biology. This is partially due to the complexity of the process, involving extensive molecular networks, multiple feedbacks between different scales of organization and physical forces operating at multiple levels. Here we present a conceptual and modeling framework aimed at generating an integrated understanding of morphogenesis in plants. This framework is based on the biophysical properties of plant cells, which are under high internal turgor pressure, and are prevented from bursting because of the presence of a rigid cell wall. To control cell growth, the underlying molecular networks must interfere locally with the elastic and/or plastic extensibility of this cell wall. We present a model in the form of a three dimensional (3D virtual tissue, where growth depends on the local modulation of wall mechanical properties and turgor pressure. The model shows how forces generated by turgor-pressure can act both cell autonomously and non-cell autonomously to drive growth in different directions. We use simulations to explore lateral organ formation at the shoot apical meristem. Although different scenarios lead to similar shape changes, they are not equivalent and lead to different, testable predictions regarding the mechanical and geometrical properties of the growing lateral organs. Using flower development as an example, we further show how a limited number of gene activities can explain the complex shape changes that accompany organ outgrowth.

  15. Plant morphogenesis, auxin, and the signal-trafficking network incompleteness theorem

    Directory of Open Access Journals (Sweden)

    Karl J. Niklas

    2012-03-01

    Full Text Available Plant morphogenesis (the development of form and function requires signal-trafficking and cross-talking among all levels of organization to coordinate the operation of metabolic and genomic networked systems. Many if not all of these biological features can be rendered as logic circuits supervising the operation of one or more signal-activated metabolic or genome networks. This approach simplifies complex morphogenetic phenomena and allows for their aggregation into diagrams of larger, more "global" networked systems. This conceptualization is illustrated for morphogenesis in model plants such as maize (Zea mays and Thale cress (Arabidopsis thaliana from an evolutionary perspective. The phytohormone indole-acetic acid (IAA is used as an example for a well-known signaling chemical and discussed in terms of the logic circuits and signal-activated sub-systems for hormone-mediated wall loosening and cell expansion as well as polar/lateral intercellular IAA transport. For each of these phenomena, a circuit/sub-system diagram highlights missing components, either in the logic circuit or in the sub-system it supervises, that must be identified experimentally if each of these basic phenomena is to be fully understood within a phylogen

  16. ABA content in shoots and roots of pea mutants af and tl as related to their growth and morphogenesis

    Czech Academy of Sciences Publication Activity Database

    Kof, E.M.; Vinogradova, I.A.; Oorzhak, A.S.; Karyagin, V.V.; Kalibernaya, Z.V.; Macháčková, Ivana; Kondykov, I.V.; Chuvasheva, E.S.

    2006-01-01

    Roč. 53, č. 3 (2006), s. 359-365 ISSN 1021-4437 Institutional research plan: CEZ:AV0Z50380511 Keywords : Pisum sativum * af and tl leaf mutants * morphogenesis Subject RIV: EF - Botanics Impact factor: 0.321, year: 2006

  17. A novel cell binding site in the coiled‐coil domain of laminin involved in capillary morphogenesis

    DEFF Research Database (Denmark)

    Sanz, Laura; García-Bermejo, Laura; Blanco, Francisco J

    2003-01-01

    Recently, we reported the isolation and characterization of an anti‐laminin antibody that modulates the extracellular matrix‐dependent morphogenesis of endothelial cells. Here we use this antibody to precisely map the binding site responsible for mediating this biologically important interaction....

  18. Multidisciplinary Inquiry-Based Investigation Learning Using an Ex Ovo Chicken Culture Platform: Role of Vitamin A on Embryonic Morphogenesis

    Science.gov (United States)

    Buskohl, Philip R.; Gould, Russell A.; Curran, Susan; Archer, Shivaun D.; Butcher, Jonathan T.

    2012-01-01

    Embryonic development offers a unique perspective on the function of many biological processes because of embryos' heightened sensitivity to environmental factors. This hands-on lesson investigates the effects of elevated vitamin A on the morphogenesis of chicken embryos. The active form of vitamin A (retinoic acid) is applied to shell-less (ex…

  19. Full-length fibronectin drives fibroblast accumulation at the surface of collagen microtissues during cell-induced tissue morphogenesis

    NARCIS (Netherlands)

    Foolen, J.; Shiu, J.-Y.; Mitsi, M.; Zhang, Y.; Chen, C.; Vogel, Viola

    2016-01-01

    Generating and maintaining gradients of cell density and extracellular matrix (ECM) components is a prerequisite for the development of functionality of healthy tissue. Therefore, gaining insights into the drivers of spatial organization of cells and the role of ECM during tissue morphogenesis is

  20. A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

    NARCIS (Netherlands)

    Siegel, Gabriele; Obernosterer, Gregor; Fiore, Roberto; Oehmen, Martin; Bicker, Silvia; Christensen, Mette; Khudayberdiev, Sharof; Leuschner, Philipp F; Busch, Clara J L; Kane, Christina; Hübel, Katja; Dekker, Frank; Hedberg, Christian; Rengarajan, Balamurugan; Drepper, Carsten; Waldmann, Herbert; Kauppinen, Sakari; Greenberg, Michael E; Draguhn, Andreas; Rehmsmeier, Marc; Martinez, Javier; Schratt, Gerhard M; Dekker, Frank

    The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific

  1. Hepatitis E Virus Mutations: Functional and Clinical Relevance

    Directory of Open Access Journals (Sweden)

    Hoang van Tong

    2016-09-01

    Full Text Available Hepatitis E virus (HEV infection is a major cause of acute hepatitis and affects more than 20 million individuals, with three million symptomatic cases and 56,000 recognized HEV-related deaths worldwide. HEV is endemic in developing countries and is gaining importance in developed countries, due to increased number of autochthone cases. Although HEV replication is controlled by the host immune system, viral factors (especially specific viral genotypes and mutants can modulate HEV replication, infection and pathogenesis. Limited knowledge exists on the contribution of HEV genome variants towards pathogenesis, susceptibility and to therapeutic response. Nonsynonymous substitutions can modulate viral proteins structurally and thus dysregulate virus-host interactions. This review aims to compile knowledge and discuss recent advances on the casual role of HEV heterogeneity and its variants on viral morphogenesis, pathogenesis, clinical outcome and antiviral resistance.

  2. Use of a Vaccinia Construct Expressing the Circumsporozoite Protein in the Analysis of Protective Immunity to Plasmodium yoelii

    Science.gov (United States)

    1988-01-01

    William R. Majarian, 2 ,5 Frank A. Robey, 3 Walter Weiss, 1 and Stephen L. Hoffman 1 lInfectious Diseases Department, Naval Medical Research Institute...autoradiography. Recombinant viruses which were positive in this assay were subject to 3 rounds of plaque purification. Finally, plaque purified virus was...mechanisms in the protective immunity elicited by inmunization with irradiated sporozoites (3,7,8,9). In an attempt to induce a protective cellular immune

  3. Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a distinct set of mammalian proapoptotic proteins.

    Science.gov (United States)

    Okamoto, Toru; Campbell, Stephanie; Mehta, Ninad; Thibault, John; Colman, Peter M; Barry, Michele; Huang, David C S; Kvansakul, Marc

    2012-11-01

    Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.

  4. A Julia set model of field-directed morphogenesis: developmental biology and artificial life.

    Science.gov (United States)

    Levin, M

    1994-04-01

    One paradigm used in understanding the control of morphogenetic events is the concept of positional information, where sub-organismic components (such as cells) act in response to positional cues. It is important to determine what kinds of spatiotemporal patterns may be obtained by such a method, and what the characteristics of such a morphogenetic process might be. This paper presents a computer model of morphogenesis based on gene activity driven by interpreting a positional information field. In this model, the interactions of mutually regulating developmental genes are viewed as a map from R2 to R2, and are modeled by the complex number algebra. Functions in complex variables are used to simulate genetic interactions resulting in position-dependent differentiation. This is shown to be equivalent to computing modified Julia sets, and is seen to be sufficient to produce a very rich set of morphologies which are similar in appearance and several important characteristics to those of real organisms. The properties of this model can be used to study the potential role of fields and positional information as guiding factors in morphogenesis, as the model facilitates the study of static images, time-series (movies) and experimental alterations of the developmental process. It is thus shown that gene interactions can be modeled as a multi-dimensional algebra, and that only two interacting genes are sufficient for (i) complex pattern formation, (ii) chaotic differentiation behavior, and (iii) production of sharp edges from a continuous positional information field. This model is meant to elucidate the properties of the process of positional information-guided biomorphogenesis, not to serve as a simulation of any particular organism's development. Good quantitative data are not currently available on the interplay of gene products in morphogenesis. Thus, no attempt is made to link the images produced with actual pictures of any particular real organism. A brief

  5. Ret and Etv4 Promote Directed Movements of Progenitor Cells during Renal Branching Morphogenesis.

    Directory of Open Access Journals (Sweden)

    Paul Riccio

    2016-02-01

    Full Text Available Branching morphogenesis of the epithelial ureteric bud forms the renal collecting duct system and is critical for normal nephron number, while low nephron number is implicated in hypertension and renal disease. Ureteric bud growth and branching requires GDNF signaling from the surrounding mesenchyme to cells at the ureteric bud tips, via the Ret receptor tyrosine kinase and coreceptor Gfrα1; Ret signaling up-regulates transcription factors Etv4 and Etv5, which are also critical for branching. Despite extensive knowledge of the genetic control of these events, it is not understood, at the cellular level, how renal branching morphogenesis is achieved or how Ret signaling influences epithelial cell behaviors to promote this process. Analysis of chimeric embryos previously suggested a role for Ret signaling in promoting cell rearrangements in the nephric duct, but this method was unsuited to study individual cell behaviors during ureteric bud branching. Here, we use Mosaic Analysis with Double Markers (MADM, combined with organ culture and time-lapse imaging, to trace the movements and divisions of individual ureteric bud tip cells. We first examine wild-type clones and then Ret or Etv4 mutant/wild-type clones in which the mutant and wild-type sister cells are differentially and heritably marked by green and red fluorescent proteins. We find that, in normal kidneys, most individual tip cells behave as self-renewing progenitors, some of whose progeny remain at the tips while others populate the growing UB trunks. In Ret or Etv4 MADM clones, the wild-type cells generated at a UB tip are much more likely to remain at, or move to, the new tips during branching and elongation, while their Ret-/- or Etv4-/- sister cells tend to lag behind and contribute only to the trunks. By tracking successive mitoses in a cell lineage, we find that Ret signaling has little effect on proliferation, in contrast to its effects on cell movement. Our results show that Ret

  6. Phytophthora viruses.

    Science.gov (United States)

    Cai, Guohong; Hillman, Bradley I

    2013-01-01

    Phytophthora sp. is a genus in the oomycetes, which are similar to filamentous fungi in morphology and habitat, but phylogenetically more closely related to brown algae and diatoms and fall in the kingdom Stramenopila. In the past few years, several viruses have been characterized in Phytophthora species, including four viruses from Phytophthora infestans, the late blight pathogen, and an endornavirus from an unnamed Phytophthora species from Douglas fir. Studies on Phytophthora viruses have revealed several interesting systems. Phytophthora infestans RNA virus 1 (PiRV-1) and PiRV-2 are likely the first members of two new virus families; studies on PiRV-3 support the establishment of a new virus genus that is not affiliated with established virus families; PiRV-4 is a member of Narnaviridae, most likely in the genus Narnavirus; and Phytophthora endornavirus 1 (PEV1) was the first nonplant endornavirus at the time of reporting. Viral capsids have not been found in any of the above-mentioned viruses. PiRV-1 demonstrated a unique genome organization that requires further examination, and PiRV-2 may have played a role in late blight resurgence in 1980s-1990s. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Schmallenberg Virus

    Indian Academy of Sciences (India)

    IAS Admin

    explore the potential of this infection crossing the species barrier and thereby .... The virus targets mainly the brain of the unborn animal resulting in neurological ... The virus is located in the blood of the adult infected animal or in the central ...

  8. Zika Virus

    Science.gov (United States)

    ... with facebook share with twitter share with linkedin Zika Virus Credit: NIAID A female Aedes mosquito. This type of mosquito can transmit Zika, ... transmitted to humans through the bite of infected Aedes aegypti mosquitoes. Zika virus can be transmitted from an infected pregnant woman ...

  9. CHANDIPURA VIRUS

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. CHANDIPURA VIRUS. First isolated from a village called Chandipura near Nagpur in 1965 in India. Belongs to rhabdoviridae family. Used as a Model System to study RNA virus multiplication in the infected cell at molecular level. Notes:

  10. TDZ pulsing evaluation on the in vitro morphogenesis of peach palm.

    Science.gov (United States)

    Graner, Erika Mendes; Oberschelp, Gustavo Pedro Javier; Brondani, Gilvano Ebling; Batagin-Piotto, Katherine Derlene; de Almeida, Cristina Vieira; de Almeida, Marcílio

    2013-04-01

    Peach palm (Bactris gasipaes Kunth.) cropping is an excellent alternative to native species exploitation; nevertheless, the problems with seed germination and conventional propagation justify the use of in vitro culturing. Aiming to asses TDZ pulsing effect on B. gasipaes morphogenesis, explants obtained from unarmed microplants were maintained in two treatments, half of them in MS free medium (without growth regulator) and the other half in MS with TDZ (0.36 μM). Both groups were transferred to growth regulator-free MS medium following 14 days of culture. After 84 days of culture, TDZ pulsing increased the growth and development of the shoots, restricted the growth and development of the roots, with no influence on adventitious bud induction or somatic embryogenesis. Furthermore, development of prickles, thickening of roots and chlorotic leaves were noted under TDZ pulsing. Leaf sheath histological analysis showed an epidermal origin and no vascularization of these prickles.

  11. Uncovering the Number and Clonal Dynamics of Mesp1 Progenitors during Heart Morphogenesis

    Directory of Open Access Journals (Sweden)

    Samira Chabab

    2016-01-01

    Full Text Available The heart arises from distinct sources of cardiac progenitors that independently express Mesp1 during gastrulation. The precise number of Mesp1 progenitors that are specified during the early stage of gastrulation, and their clonal behavior during heart morphogenesis, is currently unknown. Here, we used clonal and mosaic tracing of Mesp1-expressing cells combined with quantitative biophysical analysis of the clonal data to define the number of cardiac progenitors and their mode of growth during heart development. Our data indicate that the myocardial layer of the heart derive from ∼250 Mesp1-expressing cardiac progenitors born during gastrulation. Despite arising at different time points and contributing to different heart regions, the temporally distinct cardiac progenitors present very similar clonal dynamics. These results provide insights into the number of cardiac progenitors and their mode of growth and open up avenues to decipher the clonal dynamics of progenitors in other organs and tissues.

  12. Effect of oxygen on morphogenesis and polypeptide expression by Mucor racemosus

    International Nuclear Information System (INIS)

    Phillips, G.J.; Borgia, P.T.

    1985-01-01

    The morphology of Mucor racemosus in cultures continuously sparged with nitrogen gas was investigated. When appropriate precautions were taken to prevent oxygen from entering the cultures, the morphology of the cells was uniformly yeastlike irrespective of the N 2 flow rate. When small amounts of oxygen entered the cultures the resulting microaerobic conditions evoked mycelial development. Polypeptides synthesized by aerobic mycelia, microaerobic mycelia, anaerobic yeasts, and yeasts grown in a CO 2 atmosphere were compared by two-dimensional gel electrophoresis. The results indicated that a large number of differences in polypeptide expression exist when microaerobic mycelia or anaerobic yeasts are compared with aerobic mycelia and that these alterations correlate with a change from an oxidative to a fermentative metabolic mode. The authors hypothesize that oxygen regulates the expression of polypeptides involved in both the metabolic mode and in morphogenesis

  13. Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.

    Science.gov (United States)

    Ellis, Kathryn; Bagwell, Jennifer; Bagnat, Michel

    2013-03-04

    The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H(+)-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

  14. Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis

    Science.gov (United States)

    Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas

    2016-01-01

    The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. PMID:27605377

  15. Evolution of experimental deciduomata in the rat: cellular filiation and morphogenesis

    International Nuclear Information System (INIS)

    Dupont, Henri; Sartor, Pierre; Dupont, M.-A.; Duluc, A.-J.; Mayer, Gaston

    1978-01-01

    The morphogenesis of experimental deciduomata and the genesis of polyploidy in this tissue were studied by radioautographic and ultrastructural techniques in the rat. The growth is effected by mitotic renewal of diploid cells which are in post-synthetic resting period. They themselves organize by forming cellular strings. The level of Polyploidy is determined by the modality of division. Mitotic divisions without cytodieresis lead to a substantial pool of binucleated cells (2n + 2n) in mesometrial and antimesometrial areas. Mitotic divisions for binucleated cells with a single spindle can create tetraploid mononucleated cells. This mitotic type of division limits the polyploidy to 4 n in mesometrial cells. In antimesometrial area the evolution continues above 4 n nuclei by means of endomitosis, without any filiation between mono-and binucleated cells. In some respects, this evolution agrees with results obtained in hepatic tissue in the rat [fr

  16. The role of nitric oxide and hemoglobin in plant development and morphogenesis

    DEFF Research Database (Denmark)

    Hebelstrup, Kim; Shah, Jay K; Igamberdiev, Abir U

    2013-01-01

    effects on control of plant growth and development, such as shoot and root architecture. All plants are able to express non-symbiotic hemoglobins at low concentration. Their function is generally not related to oxygen transport or storage; instead they effectively oxidize NO to NO3– and thereby control...... the local cellular NO concentration. In this review, we analyze available data on the role of NO and plant hemoglobins in morphogenetic processes in plants. The comparison of the data suggests that hemoglobin gene expression in plants modulates development and morphogenesis of organs, such as roots...... and shoots, through the localized control of NO, and that hemoglobin gene expression should always be considered a modulating factor in processes controlled directly or indirectly by NO in plants....

  17. Myoepithelial Cells: Their Origin and Function in Lacrimal Gland Morphogenesis, Homeostasis, and Repair.

    Science.gov (United States)

    Makarenkova, Helen P; Dartt, Darlene A

    2015-09-01

    Lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes the aqueous layer of the tear film. LG epithelium is composed of ductal, acinar, and myoepithelial cells (MECs) bordering the basal lamina and separating the epithelial layer from the extracellular matrix. Mature MECs have contractile ability and morphologically resemble smooth muscle cells; however, they exhibit features typical for epithelial cells, such as the presence of specific cytokeratin filaments. Increasing evidence supports the assertion that myoepithelial cells (MECs) play key roles in the lacrimal gland development, homeostasis, and stabilizing the normal structure and polarity of LG secretory acini. MECs take part in the formation of extracellular matrix gland and participate in signal exchange between epithelium and stroma. MECs have a high level of plasticity and are able to differentiate into several cell lineages. Here, we provide a review on some of the MEC characteristics and their role in LG morphogenesis, maintenance, and repair.

  18. Reassessing the Roles of PIN Proteins and Anticlinal Microtubules during Pavement Cell Morphogenesis1[OPEN

    Science.gov (United States)

    Sawchuk, Megan G.; Scarpella, Enrico

    2018-01-01

    The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that lon