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Sample records for replication-deficient adenovirus encoding

  1. Development of replication-deficient adenovirus malaria vaccines.

    Science.gov (United States)

    Hollingdale, Michael R; Sedegah, Martha; Limbach, Keith

    2017-03-01

    Malaria remains a major threat to endemic populations and travelers, including military personnel to these areas. A malaria vaccine is feasible, as radiation attenuated sporozoites induce nearly 100% efficacy. Areas covered: This review covers current malaria clinical trials using adenoviruses and pre-clinical research. Heterologous prime-boost regimens, including replication-deficient human adenovirus 5 (HuAd5) carrying malaria antigens, are efficacious. However, efficacy appears to be adversely affected by pre-existing anti-HuAd5 antibodies. Current strategies focus on replacing HuAd5 with rarer human adenoviruses or adenoviruses isolated from non-human primates (NHPs). The chimpanzee adenovirus ChAd63 is undergoing evaluation in clinical trials including infants in malaria-endemic areas. Key antigens have been identified and are being used alone, in combination, or with protein subunit vaccines. Gorilla adenoviruses carrying malaria antigens are also currently being evaluated in preclinical models. These replacement adenovirus vectors will be successfully used to develop vaccines against malaria, as well as other infectious diseases. Expert commentary: Simplified prime-boost single shot regimens, dry-coated live vector vaccines or silicon microneedle arrays could be developed for malaria or other vaccines. Replacement vectors with similar or superior immunogenicity have rapidly advanced, and several are now in extensive Phase 2 and beyond in malaria as well as other diseases, notably Ebola.

  2. Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice.

    Science.gov (United States)

    Bassi, Maria R; Larsen, Mads A B; Kongsgaard, Michael; Rasmussen, Michael; Buus, Søren; Stryhn, Anette; Thomsen, Allan R; Christensen, Jan P

    2016-02-01

    The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.

  3. Proteins encoded near the adenovirus late messenger RNA leader segments

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, J.B.; Anderson, C.W.

    1983-01-01

    Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the ''i''-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the i-leader segment.

  4. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    DEFF Research Database (Denmark)

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn

    2007-01-01

    Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein to that e......Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin......-cell-independent immunity from adenovirus vectors offers prospects for vaccination against opportunistic pathogens in AIDS patients and possibly immunotherapy in chronic virus infections....

  5. CEA启动子启动双自杀基因CD/TK重组AdEasy XL腺病毒构建%The construct of replication deficient AdEasy XL adenovirus containing CEA promoter, CD and TK gene

    Institute of Scientific and Technical Information of China (English)

    王天宝; 汪建平; 董文广; 钟女奇; 周军

    2005-01-01

    目的构建含有CEA启动子(CEA promoter,Cp)启动的双自杀基因CD与TK的复制缺陷型腺病毒.方法设计带有限制性内切酶酶切序列的PCR引物.在2μl T4 DNA ligase作用下,Cp插入pAdTrack(pAT)的MCS,形成pAT.Cp;CD插入pAT.Cp的MCS,形成pAT.Cp.C;TK插入pAT.Cp.C的MCS,形成pAT.Cp.C.T.5 μg pAT.Cp.C.T经4 μl Pme Ⅰ线化后,转化BJ5183-AD-1,1μl PacⅠ酶切鉴定.重组AdEasy质粒转化XL10-Gold细胞,大量扩增提取纯化,转染5 × 105 AD-293细胞,荧光显微镜观察,制备带有Cp.C.T重组腺病毒(recombinant adenovirus with Cp.C.T,RA-Cp.C.T),测定病毒滴度.结果pAT.Cp,pAT.Cp.C,pAT.Cp.C.T双酶切以及PCR均见长约500 bp的Cp,1 300 bp的CD,1 100 bp的TK.对照质粒DNA转化BJ5183-AD-1效率为7.36×106 cfu/μg.线化pAT.Cp.C.T转化BJ5183-AD-1,形成重组AdEasy质粒DNA,Pac Ⅰ酶切见典型长约3.0或4.5kb与30.0kbDNA片断.重组质粒转染AD-293出现绿色荧光.RA-Cp.C.T滴度为5.67×107 pfu/ml.结论RA-Cp.C.T构建正确;AdEasy XL腺病毒载体系统转导Cp.C.T具有高效性,易于观察转染效果.

  6. Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

    Science.gov (United States)

    Freimuth, Paul I.

    2010-04-06

    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  7. ADENOVIRUS-MEDIATED WILD-TYPE P53 EXPRESSION SUPPRESSES GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Xia Yongjing; Jiang Lei; Li Hongxia; Hu Yajun; Yi Lin; Hu Shixue; Xu Hongji

    1998-01-01

    Objective: To study the growth suppression of lung adenocarcinoma cell by the introduction of wild-type P53gene and explore a gene therapy approach for lung adenocarcinoma. Methods: A replication-deficient adenovirus vector encoding a wild-type P53 was constructed and transfected into the cultured human lung adenocarcinoma cell line GLC-82. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The cell growth rate and cell cycle were analysed by cell-counting and flow cytometry. Results: Wild-type P53 gene could be quickly and effectively transfected into the cells by adenovirus vector. Wild-type P53 expression could inhibit GLC-82 cell proliferation and induce apoptosis.Conclusion: The results indicated that recombinant adenovirus expressing wild-type P53 might be useful vector for gene therapy of human lung adenocarcinoma.

  8. Adenovirus-based vaccine against Listeria monocytogenes

    DEFF Research Database (Denmark)

    Jensen, Søren; Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech

    2013-01-01

    The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC...... class II-associated invariant chain (Ii) greatly enhances both the presentation of most target Ags, as well as overall protection against viral infection, such as lymphocytic choriomeningitis virus (LCMV). The present study extends this vaccination concept to include protection against intracellular...... bacteria, using Listeria monocytogenes as a model organism. Protection in C57BL/6 mice against recombinant L. monocytogenes expressing an immunodominant epitope of the LCMV glycoprotein (GP33) was greatly accelerated, augmented, and prolonged following vaccination with an adenoviral vaccine encoding GP...

  9. In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

    Institute of Scientific and Technical Information of China (English)

    Dennis Hoffmann; Wibke Bayer; Oliver Wildner

    2007-01-01

    AIM: To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and "F (MV-FMG), encoded by an adenovirus vector Ad.MV-H/ F, alone or in combination with local coexpression of cytokines (IL-2, IL-12, IL-18, IL-21 or GM-CSF), can serve as a platform for inducing tumor-specific immune responses in colon cancer.METHODS: We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector-treated tumor. We assessed the induction of a tumor-specific cytotoxic T lymphocyte (CTL) response with a lactate dehydrogenase (LDH) release assay.RESULTS: We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion. In addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and IL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response.CONCLUSION: Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

  10. Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

    Science.gov (United States)

    Murakami, Pete; Havenga, Menzo; Fawaz, Farah; Vogels, Ronald; Marzio, Giuseppe; Pungor, Erno; Files, Jim; Do, Linh; Goudsmit, Jaap; McCaman, Michael

    2004-01-01

    The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 × 1012 vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad. PMID:15163713

  11. Mucosal immunization with recombinant adenovirus encoding soluble globular head of hemagglutinin protects mice against lethal influenza virus infection.

    Science.gov (United States)

    Kim, Joo Young; Choi, Youngjoo; Nguyen, Huan H; Song, Man Ki; Chang, Jun

    2013-12-01

    Influenza virus is one of the major sources of respiratory tract infection. Due to antigenic drift in surface glycoproteins the virus causes annual epidemics with severe morbidity and mortality. Although hemagglutinin (HA) is one of the highly variable surface glycoproteins of the influenza virus, it remains the most attractive target for vaccine development against seasonal influenza infection because antibodies generated against HA provide virus neutralization and subsequent protection against the virus infection. Combination of recombinant adenovirus (rAd) vector-based vaccine and mucosal administration is a promising regimen for safe and effective vaccination against influenza. In this study, we constructed rAd encoding the globular head region of HA from A/Puerto Rico/8/34 virus as vaccine candidate. The rAd vaccine was engineered to express high level of the protein in secreted form. Intranasal or sublingual immunization of mice with the rAd-based vaccine candidates induced significant levels of sustained HA-specific mucosal IgA and IgG. When challenged with lethal dose of homologous virus, the vaccinated mice were completely protected from the infection. The results demonstrate that intranasal or sublingual vaccination with HA-encoding rAd elicits protective immunity against infection with homologous influenza virus. This finding underlines the potential of our recombinant adenovirus-based influenza vaccine candidate for both efficacy and rapid production.

  12. Anti-tumor Immunity Elicited by Adenovirus Encoding AdhTrp2 or AdmTrp2 without Vitiligo

    Institute of Scientific and Technical Information of China (English)

    Hongju LIU; Xianzhi XIONG; Zuoya LI; Jianbao XIN; Xiaonan TAO; Yu HU

    2008-01-01

    To compare the difference in tumor immunity and autoimmunity elicited by adenovirus (Ad) encoding human or murine tyrosinase-related protein 2 (AdhTRP2 or AdmTRP2), and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2, C57BL/6 mice were im-munized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 105, 106, 107 and 108 separately (10 mice for each dose). Two weeks after the immunization, in vivo CTL assay and in- tracellular staining (ICS) of IFN-γ were carried out to analyze the dose-effect relationship. Tumor growth and vitiligo (as an sign of autoimmunity) were observed until 3 months after challenge with 105 B I6F10 tumor cells. The results showed that Ad encoding AdmTrp2 induced weak tumor im- mune response. Similar immunization with AdhTrp-2 elicited stronger protective immunity. CTL activity and IFN-γ-produced CD8+T cells were directly proportional to dose of AdhTrp2 or AdmTrp2. Moreover, AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor. In the whole process of this experiment, no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2. It is concluded that anti-melanoma responses induced by genetic vaccina- tion expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong anti-gen-specific cytotoxic T cell response without vitiligo.

  13. Effectiveness of intracavernous delivery of adenovirus encoding Smad7 gene on erectile function in a mouse model of cavernous nerve injury.

    Science.gov (United States)

    Song, Kang Moon; Chung, Jae-Seung; Choi, Min Ji; Jin, Hai-Rong; Yin, Guo Nan; Kwon, Mi-Hye; Park, Jin-Mi; Kim, Woo Jean; Lee, Sang-Jin; Kim, Seong-Jin; Ryu, Ji-Kan; Suh, Jun-Kyu

    2014-01-01

    Men with erectile dysfunction (ED) respond poorly to oral phosphodiesterase-5 inhibitors following radical prostatectomy. Recent studies have reported that up-regulation of transforming growth factor-β1 (TGF-β1) and activation of the Smad signaling pathway play important roles in cavernous fibrosis and in the deterioration of erectile function in a mouse model of cavernous nerve injury (CNI) and in patients with spinal cord injury. The mothers against decapentaplegic homolog 7 (Smad7) is known to inhibit the phosphorylation of Smad2 and Smad3. To investigate the effectiveness of adenoviruses encoding Smad7 gene (Ad-Smad7) on erectile function in a mouse model of CNI. Twelve-week-old C57BL/6J mice were used and distributed into 7 groups: sham operation group, untreated CNI group, and CNI groups receiving a single intracavernous injection of adenovirus encoding LacZ (1 × 10(8) virus particles [vp]/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 10(7), 1 × 10(8), 2 × 10(8), or 1 × 10(9) vp/20 μL). Two weeks after bilateral cavernous nerve crushing and treatment, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. The highest erectile response was noted in CNI mice treated with Ad-Smad7 at a dose of 1 × 10(8)  vp, which reached up to 82-85% of sham control values. Local delivery of Ad-Smad7 significantly decreased endothelial cell apoptosis and the production of extracellular matrix proteins, including plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV, and induced endothelial nitric oxide synthase phosphorylation in the corpus cavernosum tissue of CNI mice. The adenovirus-mediated gene transfer of Smad7 successfully restored erectile function by enhancing endothelial cell function and through antifibrotic effects. These findings suggest that inhibition of the TGF-β signaling pathway by use of

  14. Induction of leukemia-specific CD8+ cytotoxic T cells with autologous myeloid leukemic cells maturated with a fiber-modified adenovirus encoding TNF-alpha.

    Science.gov (United States)

    Saudemont, Aurore; Corm, Selim; Wickham, Thomas; Hetuin, Dominique; Quesnel, Bruno

    2005-06-01

    Acute myeloid leukemia (AML) cells can be differentiated into dendritic cells (DCs) using appropriate combinations of cytokines but generation of autologous antileukemic cytotoxic T cells using leukemic DCs remains difficult. Transduction by adenoviral vectors has been reported to induce efficient maturation of monocyte-derived DCs but AML cells are generally resistant to adenoviral gene transfer. In this study we tested the effects of adenoviral TNF-alpha gene transfer on maturation of AML cells using the fiber-modified AdTNF.F(pK7) adenovirus. All samples expressed high and sustained levels of TNF-alpha following transduction. AdTNF.F(pK7) induced significantly greater maturation of AML cells into antigen-presenting cells (APC) than did recombinant TNF-alpha or control adenoviral vector. Maturation of leukemic cells into APCs was mediated at least partially via a PI3K/mTOR pathway, as the inhibitors LY294002, wortmannin, and rapamycin inhibited the maturation effect induced by the AdTNF.F(pK7) adenovirus. In addition, CD8+ T cells expanded with AdTNF.F(pK7)-transduced AML cells showed greater expansion and specific CD8+ CTL activity against autologous AML cells than T cells expanded by other means. Thus, fiber-modified adenoviral vectors encoding TNF-alpha are able to maturate AML cells into APCs with high efficacy and reproducibility, providing a useful tool to generate efficiently specific CD8+ CTLs against leukemic disease.

  15. Immune response is an important aspect of the antitumor effect produced by a CD40L-encoding oncolytic adenovirus.

    Science.gov (United States)

    Diaconu, Iulia; Cerullo, Vincenzo; Hirvinen, Mari L M; Escutenaire, Sophie; Ugolini, Matteo; Pesonen, Saila K; Bramante, Simona; Parviainen, Suvi; Kanerva, Anna; Loskog, Angelica S I; Eliopoulos, Aristides G; Pesonen, Sari; Hemminki, Akseli

    2012-05-01

    Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimulatory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40 ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immune mechanisms, including a T-helper type 1 (T(H)1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression. In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimeric Ad5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter for tumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibited tumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)-mediated oncolysis resulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immunogenicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated with induction of the T(H)1 cytokines IFN-γ, RANTES, and TNF-α. Tumors treated with Ad5/3-CMV-mCD40L also displayed an enhanced presence of macrophages and cytotoxic CD8(+) T cells but not B cells. Together, our findings show that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis, induction of T-cell responses, and upregulation of T(H)1 cytokines.

  16. Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

    Science.gov (United States)

    Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

    2008-12-01

    With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

  17. Striking similarities are exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII

    Energy Technology Data Exchange (ETDEWEB)

    Rosa, M.D.; Gottlieb, E.; Lerner, M.R.; Steitz, J.A.

    1981-09-01

    The nucleotide sequence of the region of the Epstein-Barr virus genome that specified two small ribonucleic acids (RNAs), EBER 1 and EBER 2, has been determined. Both of these RNAs are encoded by the right-hand 1,000 base pairs of the EcoRI J fragment of EBV deoxyribonucleic acid. EBER 1 is 166 (167) nucleotides long and EBER 2 is 172 +- 1 nucleotides long; the heterogeneity resides at the 3' termini. The EBER genes are separated by 161 base pairs and are transcribed from the same deoxyribonucleic acid strand. In vitro, both EBER genes can be transcribed by RNA polymerase III; sequences homologous to previously identified RNA polymerase III intragenic transcription control regions are present. Striking similarities are therefore apparent both between the EBERs and the two adenovirus-associated RNAs, VAI and VAII, and between the regions of the two viral genomes that specify these small RNAs. We have shown that VAII RNA as well as VAI RNA and the EBERs exist in ribonucleoprotein complexes which are precipitable by anti-La antibodies associated with systemic lupus erythematosus. Finally the authors have demonstrated that the binding of protein(s) from uninfected cells confers antigenicity on each of the four virus-encoded small RNAs.

  18. Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain

    Directory of Open Access Journals (Sweden)

    Shim Byoung-Shik

    2012-09-01

    Full Text Available Abstract Background Sublingual (s.l. administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S (rADV-S of severe acute respiratory syndrome-associated coronavirus (SARS-CoV induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV. Results Here, we show that s.l. administration of rADV-S induced serum SARS-CoV neutralizing and airway IgA antibodies in mice. These antibody responses are comparable to those induced by intranasal (i.n. administration. In addition, s.l. immunization induced antigen-specific CD8+ T cell responses in the lungs that are superior to those induced by intramuscular immunization. Importantly, unlike i.n. administration, s.l. immunization with rADV did not redirect the rADV vector to the olfactory bulb. Conclusion Our study indicates that s.l. immunization with rADV-S is safe and effective in induction of a broad spectrum of immune responses and presumably protection against infection with SARS-CoV.

  19. Effect of HIV-1 envelope cytoplasmic tail on adenovirus primed virus encoded virus-like particle immunizations

    DEFF Research Database (Denmark)

    Andersson, Anne Marie C; Ragonnaud, Emeline; Seaton, Kelly E.

    2016-01-01

    /c mice followed by sequential boosting with chimpanzee type 63, and chimpanzee type 3 adenoviral vectors encoding SIVmac239 Gag and full length consensus Env. Both vaccine regimens induced increasing titers of binding antibody responses after each immunization, and significant differences in immune......The low number of envelope (Env) spikes presented on native HIV-1 particles is a major impediment for HIV-1 prophylactic vaccine development. We designed virus-like particle encoding adenoviral vectors utilizing SIVmac239 Gag as an anchor for full length and truncated HIV-1 M consensus Env....... Truncated Env overexpressed VRC01 and 17b binding antigen on the surface of transduced cells while the full length Env vaccine presented more and similar amounts of antigen binding to the trimer conformation sensitive antibodies PGT151 and PGT145, respectively. The adenoviral vectors were used to prime Balb...

  20. DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity.

    Directory of Open Access Journals (Sweden)

    Ilin Chuang

    Full Text Available BACKGROUND: Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. METHODOLOGY/PRINCIPAL FINDINGS: The vaccine regimen was three monthly doses of two DNA plasmids (DNA followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad. The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1. The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea, possibly related to immunization, was severe (Grade 3, preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27% were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102 and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270 and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019. Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. SIGNIFICANCE: The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%. Protection

  1. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs.

    Science.gov (United States)

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard

    2013-01-01

    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.

  2. Breadth of T cell responses after immunization with adenovirus vectors encoding ancestral antigens or polyvalent papillomavirus antigens

    DEFF Research Database (Denmark)

    Ragonnaud, Emeline; Pedersen, Anders Gorm; Holst, Peter Johannes

    2017-01-01

    - either by inducing cross-reactive T cells or by administering a polyvalent vaccine. To test these strategies, we designed 3 ancestral and 2 circulating sequences based on the two domains of the E1 and E2 proteins of papillomaviruses (PVs) that exhibit the highest degree of conservation in comparison...... to the other PV proteins. The PV sequences were fused to a T cell adjuvant, the murine invariant chain and encoded in a recombinant adenoviral vector which was administered to naïve outbred mice. By measuring T cell responses induced by these different vaccines and towards peptide pools representing 3...... circulating strains and a putative ancestor of oncogenic HPVs, we showed that the ancestral vaccine antigen has to be approximately 90% identical to the circulating PVs before a marked drop of ~90% mean CD8+ T cell responses ensues. Interestingly, the combination of two or three type-specific PV vaccines did...

  3. Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

    Directory of Open Access Journals (Sweden)

    Lucile Tran

    Full Text Available Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT imaging of gene expression to determine whether local virus administration (direct injection in the kidney could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting.

  4. Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using direct homologous challenge

    Science.gov (United States)

    The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularl...

  5. Therapeutic induction of angiogenesis by direct myocardial administration of an adenovirus vector encoding human hepatocyte growth factor gene and its safety

    Institute of Scientific and Technical Information of China (English)

    WU Danli; ZHANG Yourong; LAO Miaofen; YUAN Lizhen; WANG Lan; HA Xiaoqin; WU Zuze(WU Cutse)

    2004-01-01

    After the study in vitro and in rats, we assessed further the effects and safety of local angiogen therapy using intramyocardial delivery of an adenovirus carrying hepatocyte growth factor gene (Ad-HGF) in a canine ischemia model. The angiogenic activity of Ad-HGF was evaluated from three aspects. First, the augmentation of collateral vessel development was assessed by angiography 30 d after surgery. The results showed that the density of collateral vessels in treated group was higher than that of control group. Secondly, infarct size was evaluated by TTC staining and image analysis. The results showed that the infarct size of treated group was smaller than that of control group. Thirdly, the myocardial regional blood flow was determined by the method of colored microspheres. The results showed that the blood flow recovered to the level before ligation in treated group, but that of the control group was lower than normal level. In addition, during the study of chronic toxicity, we tested the anti-adenovirus antibodies by neutralization method. The antibodies yielded after the fourth injection decreased slowly from peak level and disappeared 12 weeks after drug withdrawal. Overall, Ad-HGF can promote angiogenesis in ischemic myocardium and reduce infarct size.So this method may be considered as a therapeutic angiogenesis induction strategy for ischemic disease including myocardial infarction and peripheral artery disease. At the same time, Ad-HGF could induce the yield of anti-adenovirus antibodies to neutralize adenovirus, which may be the mechanism of adenovirus clearance.

  6. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

    Energy Technology Data Exchange (ETDEWEB)

    Papaneri, Amy B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Wirblich, Christoph [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Cann, Jennifer A.; Cooper, Kurt [Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Jahrling, Peter B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Schnell, Matthias J., E-mail: matthias.schnell@jefferson.edu [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Jefferson Vaccine Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Blaney, Joseph E., E-mail: jblaney@niaid.nih.gov [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States)

    2012-12-05

    We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RV{Delta}G-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RV{Delta}G-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RV{Delta}G-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RV{Delta}G-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

  7. Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Yuanhui [Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052 (China); College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China); He, Jinsheng, E-mail: jshhe@bjtu.edu.cn [College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China); Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Zheng, Xianxian [Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Wu, Qiang [Department of Pathology, Anhui Medical University, Hefei, Anhui, 230032 (China); Zhang, Mei; Wang, Xiaobo [Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Wang, Yan [Department of Pathology, Anhui Medical University, Hefei, Anhui, 230032 (China); Xie, Can; Tang, Qian; Wei, Wei [Department of Immunology, Anhui Medical University, Hefei, Anhui, 230032 (China); Wang, Min; Song, Jingdong; Qu, Jianguo [Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052 (China); Zhang, Ying; Wang, Xin [College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China); Hong, Tao [Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052 (China); College of Life Sciences and Bioengineering, Beijing Jiaotong University, 3 Shangyuan Residence, Haidian District, Beijing, 100044 (China)

    2009-04-17

    Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

  8. Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines

    Science.gov (United States)

    Celma, Cristina C.; Stewart, Meredith; Wernike, Kerstin; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; De Clercq, Kris; Zientara, Stephan; van Rijn, Piet A.; Beer, Martin

    2016-01-01

    ABSTRACT Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new

  9. Adenovirus structure.

    Science.gov (United States)

    Rux, John J; Burnett, Roger M

    2004-12-01

    Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.

  10. Construction and expression of recombinant adenovirus vector encoding BDNF%重组大鼠脑源性神经营养因子腺病毒的构建及体外表达分析

    Institute of Scientific and Technical Information of China (English)

    郎洪刚; 曹文斌; 牛道立; 何芬

    2011-01-01

    Objective To construct adenovirus vector encoding rat brain derived neurotrophic factor (BDNF) gene and identify the expression in vitro.Methods The specific BDNF sequence was cloned into the plasmid of pAdTrack-CMV to construct the BDNF expression plasmid pAd-BDNF.The recombinant plasmids were identified hy DNA sequencing and restriction digestion.The homologous recomhination between the recombinant vector and the adenovirus hone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector Ad-BDNF.The infecting recomhinant virus particles Ad-BDNF was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293 cells.The recombinant adenovirus vector infected the hela cell, the expression of BDNF was detected by RT-PCR, Western blot and immunocytochemistry.Results The sequence results of pAd-BDNF were consistent with expectancy.After homologous recombination and packaging with 293 cells , the recombinant Ad-BDNF adenovirus was obtained.RT-PCR, Western blot and immunocytochemistry suggested that the BDNF was expressed.Conclusions The Ad-BDNF was constructed successfully.It is confirmed that the interest proteins were expressed in the infected cells , which lays the basis for its application in the treatment of the neurodamaged diseases.%目的 构建含有大鼠脑源性神经营养因子(BDNF)基因的重组腺病毒(AD)载体,并分析其体外表达情况.方法 将采用RT-PCR技术获取的大鼠BDNF的cDNA基因定向克隆入穿梭质粒pAdTrack-CMV中.通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-BDNF,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Ad-BDNF).重组病毒感染体外培养的Hela细胞后,用RT-PCR、Western blot及免疫细胞化学检测细胞BDNF基因及蛋白表达情况.结果 pAd-BDNF测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-BDNF,重组病毒

  11. Structure of adenovirus bound to cellular receptor car

    Science.gov (United States)

    Freimuth, Paul I.

    2007-01-02

    Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

  12. Adenovirus-mediated Gene Transfer of MMP-2 into Cultured Porcine Trabecular Meshwork Cells

    OpenAIRE

    2012-01-01

    This study aimed to use adenoviral gene transfer to express matrix metalloproteinase (MMP)-2 in cultured porcine trabecular meshwork cells and to evaluate the duration of adenovirus-mediated MMP-2 expression and its enzymatic activity. MMP-2 cDNA was synthesized by ligating three segments of MMP-2 cDNA obtained by reverse transcription-polymerase chain reaction (RT-PCR) with mRNA extracted from mouse lungs. MMP-2 cDNA was inserted into replication-deficient adenoviral vectors. Western blottin...

  13. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses.

    Directory of Open Access Journals (Sweden)

    Maria Abildgaard Steffensen

    Full Text Available Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii. To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

  14. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses.

    Science.gov (United States)

    Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Holst, Peter Johannes; Bassi, Maria Rosaria; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup

    2012-01-01

    Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

  15. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Holst, Peter Johannes

    2012-01-01

    in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory...... directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated......8 T-cell memory even in individuals with pre-existing vector immunity....

  16. Early host cell reactivation of an oxidatively damaged adenovirus-encoded reporter gene requires the Cockayne syndrome proteins CSA and CSB.

    Science.gov (United States)

    Leach, Derrik M; Rainbow, Andrew J

    2011-03-01

    Reduced host cell reactivation (HCR) of a reporter gene containing 8-oxoguanine (8-oxoG) lesions in Cockayne syndrome (CS) fibroblasts has previously been attributed to increased 8-oxoG-mediated inhibition of transcription resulting from a deficiency in repair. This interpretation has been challenged by a report suggesting reduced expression from an 8-oxoG containing reporter gene occurs in all cells by a mechanism involving gene inactivation by 8-oxoG DNA glycosylase and this inactivation is strongly enhanced in the absence of the CS group B (CSB) protein. The observation of reduced gene expression in the absence of CSB protein led to speculation that decreased HCR in CS cells results from enhanced gene inactivation rather than reduced gene reactivation. Using an adenovirus-based β-galactosidase (β-gal) reporter gene assay, we have examined the effect of methylene blue plus visible light (MB + VL)-induced 8-oxoG lesions on the time course of gene expression in normal and CSA and CSB mutant human SV40-transformed fibroblasts, repair proficient and CSB mutant Chinese hamster ovary (CHO) cells and normal mouse embryo fibroblasts. We demonstrate that MB + VL treatment of the reporter leads to reduced expression of the damaged β-gal reporter relative to control at early time points following infection in all cells, consistent with in vivo inhibition of RNA polII-mediated transcription. In addition, we have demonstrated HCR of reporter gene expression occurs in all cell types examined. A significant reduction in the rate of gene reactivation in human SV40-transformed cells lacking functional CSA or CSB compared to normal cells was found. Similarly, a significant reduction in the rate of reactivation in CHO cells lacking functional CSB (CHO-UV61) was observed compared to the wild-type parental counterpart (CHO-AA8). The data presented demonstrate that expression of an oxidatively damaged reporter gene is reactivated over time and that CSA and CSB are required for

  17. Adenovirus DNA Replication

    OpenAIRE

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new deve...

  18. Purification of Vero cell derived live replication deficient influenza A and B virus by ion exchange monolith chromatography.

    Science.gov (United States)

    Banjac, Marko; Roethl, Elisabeth; Gelhart, Franz; Kramberger, Petra; Jarc, Barbara Lah; Jarc, Marko; Strancar, Aleš; Muster, Thomas; Peterka, Matjaž

    2014-05-01

    We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A ΔNS1-H1N1, ΔNS1-H3N2, ΔNS1-H5N1 and ΔNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while ΔNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ΔNS1-H1N1 virus were 1.9E+10 TCID50/ml, 1.0E+10 TCID50/ml and 8.9E+08 TCID50/ml, respectively. Purification of ΔNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8±32.3% and 87±30.8%. Total protein removal varied from 93.3±0.4% to 98.6±0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Chromatin structure of adenovirus DNA throughout infection

    OpenAIRE

    Giberson, Andrea N.; Davidson, Adam R.; Parks, Robin J.

    2011-01-01

    For more than half a century, researchers have studied the basic biology of Adenovirus (Ad), unraveling the subtle, yet profound, interactions between the virus and the host. These studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. In the infecting virion, the viral DNA is tightly condensed in a virally encoded protamine-like protein which must be remodeled within...

  20. CD40 ligand induced cytotoxicity in carcinoma cells is enhanced by inhibition of metalloproteinase cleavage and delivery via a conditionally-replicating adenovirus

    Directory of Open Access Journals (Sweden)

    Young Lawrence S

    2010-03-01

    Full Text Available Abstract Background CD40 and its ligand (CD40L play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death. The most profound induction of cell death in carcinoma cells is induced by membrane-bound rather than recombinant soluble CD40L, but like other TNF family ligands, it is cleaved from the membrane by matrix metalloproteinases. Results We have generated a replication-deficient adenovirus expressing a mutant CD40L that is resistant to metalloproteinase cleavage such that ligand expression is retained at the cell membrane. Here we show that the mutated, cleavage-resistant form of CD40L is a more potent inducer of apoptosis than wild-type ligand in CD40-positive carcinoma cell lines. Since transgene expression via replication-deficient adenovirus vectors in vivo is low, we have also engineered a conditionally replicating E1A-CR2 deleted adenovirus to express mutant CD40L, resulting in significant amplification of ligand expression and consequent enhancement of its therapeutic effect. Conclusions Combined with numerous studies demonstrating its immunotherapeutic potential, these data provide a strong rationale for the exploitation of the CD40-CD40L pathway for the treatment of solid tumours.

  1. Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice

    DEFF Research Database (Denmark)

    Bassi, Maria R; Larsen, Mads Andreas Bay; Kongsgaard, Michael

    2016-01-01

    The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should...

  2. Initiation of adenovirus DNA replication.

    OpenAIRE

    Reiter, T; Fütterer, J; Weingärtner, B; Winnacker, E L

    1980-01-01

    In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared ...

  3. Intramuscular delivery of adenovirus serotype 5 vector expressing humanized protective antigen induces rapid protection against anthrax that may bypass intranasally originated preexisting adenovirus immunity.

    Science.gov (United States)

    Wu, Shipo; Zhang, Zhe; Yu, Rui; Zhang, Jun; Liu, Ying; Song, Xiaohong; Yi, Shaoqiong; Liu, Ju; Chen, Jianqin; Yin, Ying; Xu, Junjie; Hou, Lihua; Chen, Wei

    2014-02-01

    Developing an effective anthrax vaccine that can induce a rapid and sustained immune response is a priority for the prevention of bioterrorism-associated anthrax infection. Here, we developed a recombinant replication-deficient adenovirus serotype 5-based vaccine expressing the humanized protective antigen (Ad5-PAopt). A single intramuscular injection of Ad5-PAopt resulted in rapid and robust humoral and cellular immune responses in Fisher 344 rats. Animals intramuscularly inoculated with a single dose of 10⁸ infectious units of Ad5-PAopt achieved 100% protection from challenge with 10 times the 50% lethal dose (LD₅₀) of anthrax lethal toxin 7 days after vaccination. Although preexisting intranasally induced immunity to Ad5 slightly weakened the humoral and cellular immune responses to Ad5-PAopt via intramuscular inoculation, 100% protection was achieved 15 days after vaccination in Fisher 344 rats. The protective efficacy conferred by intramuscular vaccination in the presence of preexisting intranasally induced immunity was significantly better than that of intranasal delivery of Ad5-PAopt and intramuscular injection with recombinant PA and aluminum adjuvant without preexisting immunity. As natural Ad5 infection often occurs via the mucosal route, the work here largely illuminates that intramuscular inoculation with Ad5-PAopt can overcome the negative effects of immunity induced by prior adenovirus infection and represents an efficient approach for protecting against emerging anthrax.

  4. Structure of Human Adenovirus

    OpenAIRE

    Nemerow, Glen R.; Phoebe L Stewart; Reddy, Vijay S.

    2012-01-01

    A detailed structural analysis of the entire human adenovirus capsid has been stymied by the complexity and size of this 150 MDa macromolecular complex. Over the past 10 years, the steady improvements in viral genome manipulation concomitant with advances in crystallographic techniques and data processing software has allowed structure determination of this virus by X-ray diffraction at 3.5 Å resolution. The virus structure revealed the location, folds, and interactions of major and minor (ce...

  5. Toxic activity of the CdtB component of Haemophilus ducreyi cytolethal distending toxin expressed from an adenovirus 5 vector.

    Science.gov (United States)

    Wising, Catharina; Magnusson, Maria; Ahlman, Karin; Lindholm, Leif; Lagergård, Teresa

    2010-02-01

    The Haemophilus ducreyi cytolethal distending toxin (HdCDT) catalytic subunit CdtB has DNase-like activity and mediates DNA damage after its delivery into target cells. We constructed a replication-deficient adenovirus type 5 (Ad5) vector expressing CdtB and investigated the toxic properties of this vector on HeLa cells. Ad5CdtB caused loss of cell viability, morphologic changes, and cell cycle arrest, findings similar to HdCDT intoxication. This confirmed that CdtB is responsible for the toxicity of the holotoxin when expressed in cells following transduction by an adenoviral vector, and indicated a possible potential of this novel strategy in studies of activity of intracellular products and in gene therapy of cancer.

  6. Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease.

    Science.gov (United States)

    Lu, Zengjun; Bao, Huifang; Cao, Yimei; Sun, Pu; Guo, Jianhun; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Li, Dong; Liu, Zaixin; Xie, Qingge

    2008-12-19

    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

  7. Adenovirus infection in immunocompromised patients

    Directory of Open Access Journals (Sweden)

    Sylwia Rynans

    2013-09-01

    Full Text Available Human adenoviruses belong to the Adenoviridae family and they are divided into seven species, including 56 types. Adenoviruses are common opportunistic pathogens that are rarely associated with clinical symptoms in immunocompetent patients. However, they are emerging pathogens causing morbidity and mortality in recipients of hematopoietic stem cell and solid organ transplants, HIV infected patients and patients with primary immune deficiencies. Clinical presentation ranges from asymptomatic viraemia to respiratory and gastrointestinal disease, haemorrhagic cystitis and severe disseminated illness. There is currently no formally approved therapy for the treatment of adenovirus infections.This article presents current knowledge about adenoviruses, their pathogenicity and information about available methods to diagnose and treat adenoviral infections.

  8. Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Puntel, Mariana [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Ghulam, Muhammad A.K.M. [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Farrokhi, Catherine [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Kroeger, Kurt M.; Salem, Alireza [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Lacayo, Liliana [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Pechnick, Robert N. [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Psychiatry and Behavioral Neurosciences, David Geffen School of Medicine, University of California, Los Angeles, CA (United States); Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Palmer, Donna; Ng, Philip [Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 (United States); and others

    2013-05-01

    Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at

  9. Targeting species D adenoviruses replication to counteract the epidemic keratoconjunctivitis.

    Science.gov (United States)

    Nikitenko, Natalia A; Speiseder, Thomas; Groitl, Peter; Spirin, Pavel V; Prokofjeva, Maria M; Lebedev, Timofey D; Rubtsov, Petr M; Lam, Elena; Riecken, Kristoffer; Fehse, Boris; Dobner, Thomas; Prassolov, Vladimir S

    2015-06-01

    Human adenoviruses are non-enveloped DNA viruses causing various infections; their pathogenicity varies dependent on virus species and type. Although acute infections can sometimes take severe courses, they are rarely fatal in immune-competent individuals. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are hyperacute and highly contagious infections of the eye caused by human adenovirus types within species D. Currently there is no causal treatment available to counteract these diseases effectively. The E2B region of the adenovirus genome encodes for the viral DNA polymerase, which is required for adenoviral DNA replication. Here we propose novel model systems to test this viral key factor, DNA polymerase, as a putative target for the development of efficient antiviral therapy based on RNA interference. Using our model cell lines we found that different small interfering RNAs mediate significant suppression (up to 90%) of expression levels of viral DNA polymerase upon transfection. Moreover, permanent expression of short hairpin RNA based on the most effective small interfering RNA led to a highly significant, more than tenfold reduction in replication for different human group D adenoviruses involved in ocular infections.

  10. Effect of adenovirus-mediated gene transfection of vascular endothelial growth factor on survival of random flaps in rats

    Institute of Scientific and Technical Information of China (English)

    崔磊; 李发成; 张群; 钱云良; 关文祥

    2003-01-01

    Objective: To evaluate the effect of local application of vascular endothelial growth factor (VEGF) via adenovirus-mediated gene transfer on survival of full thickness flaps selected randomly in rats.Methods: Thirty Sprague-Dawley rats weighing 480-520 g were used in this study. A dorsal flap (8 cm×2 cm) in full thickness with the pedicle located at the level of the iliac crest was designed. Then the rats received 1 012 pfu replication-deficient recombinant adenovirus carrying VEGF (AdCMV-VEGF group, n=10), 1 012 pfu recombinant β-galactosidase adenovirus (AdCMV-Gal group, n=10) and 1 ml saline (saline group, n=10), respectively, in the distal two thirds of the proposed flap by means of subdermal injection at 8 different locations. Three days after treatment, the flaps were elevated as originally designed and sutured back in situ. The survival rate of the flaps was evaluated on day 7 after operation. Results: The survival rate of the flaps in the AdCMV-VEGF group increased significantly as compared with those of the AdCMV-Gal group (P<0.01) and the saline group (P<0.01). Immunohistochemical staining showed that VEGF was expressed in the survival flaps injected with AdCMV-VEGF. Histological analysis showed that more granulation tissues and angiogenesis were observed in the AdCMV-VEGF group than those in the AdCMV-Gal and the saline groups.Conclusions: Local application of adenovirus-mediated VEGF165 cDNA 05- efficiently improve the survival of ischemic skin flaps.

  11. RNAi suppressor P19 can be broadly exploited for enhanced adenovirus replication and microRNA knockdown experiments.

    Science.gov (United States)

    Rauschhuber, Christina; Mueck-Haeusl, Martin; Zhang, Wenli; Nettelbeck, Dirk M; Ehrhardt, Anja

    2013-01-01

    RNA interference (RNAi) is a key regulator of various biological systems including viral infection. Within a virus life cycle gene products can be modulated by the RNA interference (RNAi) pathway which can crucially impact productive virus replication. Herein we explored the RNA interference suppressor protein P19 derived from a plant virus and we found that P19 enhanced adenovirus replication up to 100-fold. Critical factors responsible for this observation were overexpression of adenovirus encoded genes on mRNA and protein levels. To investigate the impact of this phenomenon on recombinant viruses, we exploited its feasibility for therapeutic and genomic applications. We found that P19 significantly increased recombinant adenovirus yields enabling up-scaling for preclinical and clinical studies. Moreover, adenoviruses possessed significantly higher oncolytic activity by expression of P19. Finally, we show that introducing a p19 expression cassette into high-capacity adenovirus provides a strategy to analyze RNAi knockdown in a tissue-specific manner.

  12. A Plasmodium vivax plasmid DNA- and adenovirus-vectored malaria vaccine encoding blood stage antigens AMA1 and MSP142 in a prime/boost heterologous immunization regimen partially protects Aotus monkeys against blood stage challenge.

    Science.gov (United States)

    Obaldia, Nicanor; Stockelman, Michael G; Otero, William; Cockrill, Jennifer A; Ganeshan, Harini; Abot, Esteban N; Zhang, Jianfeng; Limbach, Keith; Charoenvit, Yupin; Doolan, Denise L; Tang, De-Chu C; Richie, Thomas L

    2017-02-08

    Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of P. falciparum it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside of Africa, stressing the importance of developing a vaccine against malaria. In this study we assess the immunogenicity and protective efficacy of two P. vivax antigens, AMA1 and MSP142 in a recombinant DNA plasmid prime/adenoviral vector (Ad) boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with DNA alone, Ad alone, prime/boost regimens of each antigen, prime/boost with both antigens, and empty vector controls, and then subjected to blood stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, based on their ability to induced the longest pre-patent period and time to peak parasitemia; the lowest peak and mean parasitemia; the smallest area under the parasitemia curve and the highest self-cured rate. Overall, pre-challenge MSP1 antibody titers strongly correlated with decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, P. vivax plasmid DNA/Ad5 vaccine encoding blood stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen, provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and regimen for further development.

  13. Adenovirus Type 11 Uses CD46 as a Cellular Receptor

    OpenAIRE

    Segerman, Anna; Atkinson, John P.; Marttila, Marko; Dennerquist, Veronica; Wadell, Göran; Arnberg, Niklas

    2003-01-01

    The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type...

  14. An Adenovirus-Vectored Influenza Vaccine Induces Durable Cross-Protective Hemagglutinin Stalk Antibody Responses in Mice

    Directory of Open Access Journals (Sweden)

    Eun Hye Kim

    2017-08-01

    Full Text Available Currently licensed vaccines against the influenza A virus (IAV need to be updated annually to match the constantly evolving antigenicity of the influenza virus glycoproteins, hemagglutinin (HA, and neuramidiase (NA. Attempts to develop universal vaccines that provide broad protection have resulted in some success. Herein, we have shown that a replication-deficient adenovirus expressing H5/M2e induced significant humoral immunity against the conserved HA stalk. Compared to the humoral responses induced by an inactivated influenza vaccine, the humoral responses induced by the adenovirus-vectored vaccine against the conserved stalk domain mediated cross-protection against heterosubtypic influenza viruses. Importantly, virus inactivation by formaldehyde significantly reduced the binding of monoclonal antibodies (mAbs to the conserved nucleoprotein (NP, M2e, and HA stalk. These results suggest that inactivation by formaldehyde significantly alters the antigenicity of the HA stalk, and suggest that the conformation of the intact HA stalk provided by vector-based vaccines is important for induction of HA stalk-binding Abs. Our study provides insight into the mechanism by which a vector-based vaccine induces broad protection by stimulation of cross-protective Abs targeting conserved domains of viral proteins. The findings support further strategies to develop a vectored vaccine as a universal influenza vaccine for the control of influenza epidemics and unpredicted pandemics.

  15. An Adenovirus-Vectored Influenza Vaccine Induces Durable Cross-Protective Hemagglutinin Stalk Antibody Responses in Mice.

    Science.gov (United States)

    Kim, Eun Hye; Han, Gye-Yeong; Nguyen, Huan

    2017-08-21

    Currently licensed vaccines against the influenza A virus (IAV) need to be updated annually to match the constantly evolving antigenicity of the influenza virus glycoproteins, hemagglutinin (HA), and neuramidiase (NA). Attempts to develop universal vaccines that provide broad protection have resulted in some success. Herein, we have shown that a replication-deficient adenovirus expressing H5/M2e induced significant humoral immunity against the conserved HA stalk. Compared to the humoral responses induced by an inactivated influenza vaccine, the humoral responses induced by the adenovirus-vectored vaccine against the conserved stalk domain mediated cross-protection against heterosubtypic influenza viruses. Importantly, virus inactivation by formaldehyde significantly reduced the binding of monoclonal antibodies (mAbs) to the conserved nucleoprotein (NP), M2e, and HA stalk. These results suggest that inactivation by formaldehyde significantly alters the antigenicity of the HA stalk, and suggest that the conformation of the intact HA stalk provided by vector-based vaccines is important for induction of HA stalk-binding Abs. Our study provides insight into the mechanism by which a vector-based vaccine induces broad protection by stimulation of cross-protective Abs targeting conserved domains of viral proteins. The findings support further strategies to develop a vectored vaccine as a universal influenza vaccine for the control of influenza epidemics and unpredicted pandemics.

  16. Oncolytic Adenoviruses in Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Ramon Alemany

    2014-02-01

    Full Text Available The therapeutic use of viruses against cancer has been revived during the last two decades. Oncolytic viruses replicate and spread inside tumors, amplifying their cytotoxicity and simultaneously reversing the tumor immune suppression. Among different viruses, recombinant adenoviruses designed to replicate selectively in tumor cells have been clinically tested by intratumoral or systemic administration. Limited efficacy has been associated to poor tumor targeting, intratumoral spread, and virocentric immune responses. A deeper understanding of these three barriers will be required to design more effective oncolytic adenoviruses that, alone or combined with chemotherapy or immunotherapy, may become tools for oncologists.

  17. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    Science.gov (United States)

    Li, Wenyan; Shen, Jun

    2016-01-01

    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

  18. Biobased monoliths for adenovirus purification.

    Science.gov (United States)

    Fernandes, Cláudia S M; Gonçalves, Bianca; Sousa, Margarida; Martins, Duarte L; Barroso, Telma; Pina, Ana Sofia; Peixoto, Cristina; Aguiar-Ricardo, Ana; Roque, A Cecília A

    2015-04-01

    Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

  19. Intravenous delivery of adenovirus-mediated soluble FLT-1 results in liver toxicity

    NARCIS (Netherlands)

    Mahasreshti, P.J.; Kataram, M.; Wang, Miao; Stockard, C.R.; Grizzle, W.E.; Carey, D.; Siegal, G.P.; Haisma, H.J.; Alvarez, R.D.; Curiel, D.T.

    2003-01-01

    Purpose: Vascular endothelial growth factor (VEGF) is a potent angiogenic agent and plays a major role in tumor growth and metastases. We have previously reported the locoregional (i.p.) delivery of adenovirus-mediated antiangiogenic soluble FLT-1 (sFLT-1; a naturally encoded potent VEGF antagonist)

  20. Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection.

    Directory of Open Access Journals (Sweden)

    Brian B Gowen

    Full Text Available Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

  1. Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal challenge with pneumonia virus of mice.

    Science.gov (United States)

    Maunder, Helen E; Taylor, Geraldine; Leppard, Keith N; Easton, Andrew J

    2015-11-27

    Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replication-deficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response in such animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8(+) T cell response and, to a lesser extent, a CD4(+) T cell response. These findings suggest that T-cell responses may be more important than serum IgG in the observed protection induced by rAd5N.

  2. A novel tetracycline-controlled transactivator-transrepressor system enables external control of oncolytic adenovirus replication.

    Science.gov (United States)

    Fechner, H; Wang, X; Srour, M; Siemetzki, U; Seltmann, H; Sutter, A P; Scherübl, H; Zouboulis, C C; Schwaab, R; Hillen, W; Schultheiss, H-P; Poller, W

    2003-09-01

    The use of restricted replication-competent adenoviruses (RRCAs) inducing tumor cell-specific lysis is a promising approach in cancer gene therapy. However, the use of RRCAs in humans carries considerable risk, since after injection into the patient, further regulation or inhibition of virus replication from the outside is impossible. Therefore, we have developed a novel system allowing external pharmacological control of RRCA replication. We show here that a tumor-selective E1B-deleted RRCA can be tightly regulated by use of doxycycline (dox)-controlled adenoviral E1A gene expression, which in turn determines vector replication. RRCA replication is switched on by addition and switched off by withdrawal of dox. The system results in efficient tumor cell killing after induction by dox, whereas cells are unaffected by the uninduced system. It was also employed for efficient external control of transgene expression from cotransfected replication-deficient adenovectors. Furthermore, the use of a liver cell-specific human alpha1-antitrypsin (hAAT)-promoter driving a tetracycline-controlled transcriptional silencer allowed specific protection of cells with hAAT-promoter activity in the absence of dox in vitro and in vivo, delineating a new principle of 'tissue protective' gene therapy. The concept of external control of RRCAs may help to improve the safety of cancer gene therapy.

  3. Heat shock and heat shock protein 70i enhance the oncolytic effect of replicative adenovirus.

    Science.gov (United States)

    Haviv, Y S; Blackwell, J L; Li, H; Wang, M; Lei, X; Curiel, D T

    2001-12-01

    Replication-competent viruses are currently being evaluated for their cancer cell-killing properties. These vectors are designed to induce tumor regression after selective viral propagation within the tumor. However, replication-competent viruses have not resulted heretofore in complete tumor eradication in the clinical setting. Recently, heat shock has been reported to partially alleviate replication restriction on an avian adenovirus (Ad) in a human lung cancer cell line. Therefore, we hypothesized that heat shock and overexpression of heat shock protein (hsp) would support the oncolytic effect of a replication-competent human Ad. To this end, we tested the oncolytic and burst kinetics of a replication-competent Ad after exposure to heat shock or to inducible hsp 70 overexpression by a replication-deficient Ad (Adhsp 70i). Heat-shock resulted in augmentation of Ad burst and oncolysis while decreasing total intracellular Ad DNA. Overexpression of hsp 70i also enhanced Ad-mediated oncolysis but did not decrease intracellular Ad DNA levels. We conclude that heat shock and Adhsp 70i enhance the Ad cell-killing potential via distinct mechanisms. A potential therapeutic implication would be the use of local hyperthermia to augment oncolysis by increasing the burst of replication-competent Ad. The role of hsp in Ad-mediated oncolysis should be additionally explored.

  4. Progress on adenovirus-vectored universal influenza vaccines.

    Science.gov (United States)

    Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8(+) T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches.

  5. Effect of CD4 gene expression on adenovirus replication.

    Science.gov (United States)

    Hotta, J; Shi, L; Ginsberg, H S

    1994-11-01

    The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.

  6. Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis.

    Science.gov (United States)

    Sheinin, R; Fabbro, J; Dubsky, M

    1985-01-01

    Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

  7. Adenovirus sequences required for replication in vivo.

    OpenAIRE

    Wang, K.; Pearson, G D

    1985-01-01

    We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

  8. Oncolytic Adenoviruses for Gynecologic Cancer

    OpenAIRE

    Bauerschmitz, Gerd Johannes

    2007-01-01

    Gene therapy is a promising novel approach for treating cancers resistant to or escaping currently available modalities. Treatment approaches are based on taking advantage of molecular differences between normal and tumor cells. Various strategies are currently in clinical development with adenoviruses as the most popular vehicle. Recent developments include improving targeting strategies for gene delivery to tumor cells with tumor specific promoters or infectivity enhancement. A rapidly deve...

  9. An Adenovirus-Vectored Nasal Vaccine Confers Rapid and Sustained Protection against Anthrax in a Single-Dose Regimen

    Science.gov (United States)

    Jex, Edward; Feng, Tsungwei; Sivko, Gloria S.; Baillie, Leslie W.; Goldman, Stanley; Van Kampen, Kent R.; Tang, De-chu C.

    2013-01-01

    Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine. PMID:23100479

  10. Replication-Defective Vector Based on a Chimpanzee Adenovirus

    OpenAIRE

    Farina, Steven F.; Gao, Guang-Ping; Xiang, Z. Q.; Rux, John J.; Burnett, Roger M.; Alvira, Mauricio R.; Marsh, Jonathan; Ertl, Hildegund C.J.; Wilson, James M.

    2001-01-01

    An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including ...

  11. Anti-Viral Drugs for Human Adenoviruses

    Directory of Open Access Journals (Sweden)

    Chor Wing Sing

    2010-10-01

    Full Text Available There are many stages in the development of a new drug for viral infection and such processes are even further complicated for adenovirus by the fact that there are at least 51 serotypes, forming six distinct groups (A–F, with different degree of infectivity. This review attempts to address the importance of developing pharmaceuticals for adenovirus and also review recent development in drug discovery for adenovirus, including newer strategies such as microRNA approaches. Different drug screening strategies will also be discussed.

  12. Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4.

    Science.gov (United States)

    Dias, J D; Hemminki, O; Diaconu, I; Hirvinen, M; Bonetti, A; Guse, K; Escutenaire, S; Kanerva, A; Pesonen, S; Löskog, A; Cerullo, V; Hemminki, A

    2012-10-01

    Promising clinical results have been achieved with monoclonal antibodies (mAbs) such as ipilimumab and tremelimumab that block cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152). However, systemic administration of these agents also has the potential for severe immune-related adverse events. Thus, local production might allow higher concentrations at the target while reducing systemic side effects. We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24aCTLA4 expressing complete human mAb specific for CTLA-4 and tested it in vitro, in vivo and in peripheral blood mononuclear cells (PBMCs) of normal donors and patients with advanced solid tumors. mAb expression was confirmed by western blotting and immunohistochemistry. Biological functionality was determined in a T-cell line and in PBMCs from cancer patients. T cells of patients, but not those of healthy donors, were activated by an anti-CTLA4mAb produced by Ad5/3-Δ24aCTLA4. In addition to immunological effects, a direct anti-CTLA-4-mediated pro-apoptotic effect was observed in vitro and in vivo. Local production resulted in 43-fold higher (P<0.05) tumor versus plasma anti-CTLA4mAb concentration. Plasma levels in mice remained below what has been reported safe in humans. Replication-competent Ad5/3-Δ24aCTLA4 resulted in 81-fold higher (P<0.05) tumor mAb levels as compared with a replication-deficient control. This is the first report of an oncolytic adenovirus producing a full-length human mAb. High mAb concentrations were seen at tumors with lower systemic levels. Stimulation of T cells of cancer patients by Ad5/3-Δ24aCTLA4 suggests feasibility of testing the approach in clinical trials.

  13. Induction of mucosal immunity by intranasal immunization with recombinant adenovirus expressing major epitopes of Porcine circovirus-2 capsid protein.

    Science.gov (United States)

    Liu, Yu-feng; Guo, Quan-hai; Chen, Lu; Zhao, Jun; Chang, Hong-tao; Wang, Xin-wei; Yang, Xia; Wang, Chuan-qing

    2013-07-15

    Porcine circovirus-2 (PCV-2) is primarily transmitted through mucosa, thus the mucosal immunity may constitute an essential feature of vaccination strategies against PCV-2 infection. Mucosal immunity elicited by recombinant replication-deficient adenovirus expressing the major epitopes of PCV-2 capsid protein (rAd/Cap/518) via intranasal (i.n.), intramuscular (i.m.) or oral routes in mice were evaluated. Immunization with rAd/Cap/518 via i.n. route induced higher titers of IgA in saliva, bronchoalveolar and intestinal lavage fluid compared with those immunized via i.m. route. The proportions of CD3+, CD3+CD4+ and CD3+CD8+ T cells were significantly increased in mice immunized with rAd/Cap/518 via i.n. route compared with the control group. Higher levels of IFN-γ were detected in the spleen and mesenteric lymph nodes of mice immunized with rAd/Cap/518 via i.n. route compared with other groups, yet IL-4 was not detected in any group. Real-time PCR analysis confirmed viral DNA loads in the i.m. or i.n. immunization group was lower than that seen in the rAd immunization. These results indicate that i.n. administration of rAd/Cap/518 can elicit humoral and Th1-type cellular protective immunity in both systemic and mucosal immune compartments in mice, representing a promising mucosal vaccine candidate against PCV-2.

  14. Co-application of ricin A chain and a recombinant adenovirus expressing ricin B chain as a novel approach for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Hong-bin WANG; Fei XIA; Jing GE; Juan YIN; Li-song TAN; Pei-de ZHANG; Jiang ZHONG

    2007-01-01

    Aim: To develop a novel ricin-based approach for the safe and effective therapy of cancer. Methods: The ricin A chain (RTA) was expressed in Escherichia coli in the form of a 6xHis-tagged fusion protein and purified with Ni2*-NTA affinity resin. A replication-deficient ricin B chain (RTB)-expression adenovirus green fluorescence protein (AdGFP-RTB) was constructed. RTA and AdGFP-RTB were tested for cytotoxicity either individually or in combination in human cell lines HEK293, HeLa, SMMC7721, and HL7702. Cell viability was determined with trypan blue staining or MTT assay. Results: The expression and release of RTB, as well as the entry of RTA into AdGFP-RTB-infected cells were confirmed. When RTA and AdGFP-RTB was used individually, neither was toxic to the cells. When they were applied together, significant cell death was observed in all of the cell lines tested. The cell-killing effect correlated with the amount of RTA protein used, with cell mortality at about 60% at 4.8 lag RTA in combination with AdGFP-RTB at 100 pfu/ceU. No major cell killing was seen when RTA was used in combination with a control adenovirus AdGFP. The treatment of healthy HeLa cells with the virus-flee supernatant from AdGFP-RTB/RTA-treated HeLa cells resulted in cell death,suggesting the formation of RTA/RTB complex, and a potential by-stander effect.Conclusion: The new approach was successful in vitro. Further modifications of the adenovirus vector, as well as an in vivo study are needed to confirm its poten-tial in cancer therapy.

  15. Enfermedad neurologica por adenovirus Neurologic disease due to adenovirus infection

    Directory of Open Access Journals (Sweden)

    Cristina L. Lema

    2005-06-01

    Full Text Available El objetivo de este trabajo fue determinar la prevalencia de adenovirus (ADV en las infecciones del sistema nervioso central (SNC. Se analizaron 108 muestras de líquido cefalorraquídeo (LCR provenientes de 79 casos de encefalitis, 7 meningitis y 22 de otras patologías neurológicas, recibidas en el período 2000-2002. Cuarenta y nueve (47.35% se obtuvieron de pacientes inmunocomprometidos. La presencia de ADV se investigó mediante reacción en cadena de la polimerasa en formato anidado (Nested-PCR. La identificación del genogrupo se realizó mediante análisis filogenético de la secuencia nucleotídica parcial de la región que codifica para la proteína del hexón. Se detectó la presencia de ADV en 6 de 108 (5.5% muestras de LCR analizadas. Todos los casos positivos pertenecieron a pacientes con encefalitis que fueron 79, (6/79, 7.6%. No se observó diferencia estadísticamente significativa entre los casos de infección por ADV en pacientes inmunocomprometidos e inmunocompetentes (p>0.05. Las cepas de ADV detectadas se agruparon en los genogrupos B1 y C. En conclusión, nuestros resultados describen el rol de los ADV en las infecciones neurológicas en Argentina. La información presentada contribuye al conocimiento de su epidemiología, en particular en casos de encefalitis.The aim of this study was to assess the prevalence of adenovirusm (ADV infections in neurological disorders. A total of 108 cerebrospinal fluid (CSF samples from 79 encephalitis cases, 7 meningitis and 22 other neurological diseases analysed in our laboratory between 2000 and 2002 were studied. Forty nine (47.4% belonged to immunocompromised patients. Viral genome was detected using nested polymerase chain reaction (Nested-PCR and ADV genotypes were identified using partial gene sequence analysis of hexon gene. Adenovirus were detected in 6 of 108 (5.5% CSF samples tested. All of these were from encephalitis cases, 6/79, representing 7.6% of them. No statistically

  16. Role of preterminal protein processing in adenovirus replication.

    Science.gov (United States)

    Webster, A; Leith, I R; Nicholson, J; Hounsell, J; Hay, R T

    1997-09-01

    Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5' end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.

  17. Regulation of human adenovirus replication by RNA interference

    OpenAIRE

    Nikitenko, N. A.; SPEISEDER T.; Lam, E; Rubtsov, P. M.; TONAEVA KH. D.; S. A. Borzenok; Dobner, T; Prassolov, V.S.

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to...

  18. Components of Adenovirus Genome Packaging

    Science.gov (United States)

    Ahi, Yadvinder S.; Mittal, Suresh K.

    2016-01-01

    Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

  19. An adenoviral cancer vaccine co-encoding a tumor associated antigen together with secreted 4-1BBL leads to delayed tumor progression

    DEFF Research Database (Denmark)

    Ragonnaud, Emeline; Andersson, Anne-Marie C; Pedersen, Anders Elm

    2016-01-01

    with the previous reports of increased anti-cancer efficacy using systemically delivered 4-1BB agonists, we incorporated a secreted version of 4-1BBL (Fc-4-1BBL) in our vaccine and co-expressed it with the Ii linked to TAA. In tumor bearing mice, this vaccine initially delayed tumor growth and slightly increased......Previous studies have shown promising results when using an agonistic anti-4-1BB antibody treatment against established tumors. While this is promising, this type of treatment can induce severe side effects. Therefore, we decided to incorporate the membrane form of 4-1BB ligand (4-1BBL......) in a replicative deficient adenovirus vaccine expressing the invariant chain (Ii) adjuvant fused to a tumor associated antigen (TAA). The Ii adjuvant increases and prolongs TAA specific CD8+ T cells as previously shown and local expression of 4-1BBL was chosen to avoid the toxicity associated with systemic...

  20. 表达人JNK基因重组腺病毒的构建和鉴定%Construction and identification of expressing human c-Jun N-terminal kinase(JNK)recombinant adenovirus

    Institute of Scientific and Technical Information of China (English)

    陈金虎; 刘慧霞; 张佳妮; 郭敏; 全养雅; 谭莺

    2008-01-01

    Objective To construct replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase by homologous recombination.Methods The linearized recombinant shuttle vector pAdTrack-CMV-WT-JNK was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells to construct replication deficient recombinant adenovirus,and then the recombinant adenovirus was detected by PCR and DNA sequencing.Results JNK recombinant adenoviral vector was effectively transfected into HEK 293 cells and was successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)was observed on the 5th day after transfection.The fragment of JNK gene was amplified by PCR and identified by sequencing.The animal experiment confirmed that Ad-WT-JNK was effectivety expressed in liver tissue. Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.And the achievement laid a foundation for further investigation of the function and application of JNK.%目的 制备表达人c-jun氨基末端激酶(JNK)复制缺陷型重组腺病毒.方法 将重组穿梭载体pAdTrack-CMV-WT-JNK线性化后,与pAdEasy-1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体.将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定.结果 JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5 d后可以观察到绿色荧光蛋白(GFP)明显表达,搜集的病毒经过PCR扩增得到特定JNK基因片段并测序鉴定.动物实验证实构建的Ad-WT-JNK能有效在肝组织表达.结论 该研究成功构建了JNK重组腺病毒载体及相应重组腺病毒颗粒,为进一步研究JNK的作用及应用JNK进行相关疾病的基因治疗奠定了基础.

  1. Structure, Function and Dynamics in Adenovirus Maturation

    OpenAIRE

    Mangel, Walter F.; Carmen San Martín

    2014-01-01

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkabl...

  2. Predicted structure of two adenovirus tumor antigens.

    OpenAIRE

    Perricaudet, M; Le Moullec, J M; Pettersson, U

    1980-01-01

    Early adenovirus type 2(Ad2) mRNA sequences have been cloned by using the pBR322 plasmid as a vector. Two clones that include sequences from region E1B were identified and their DNAs were characterized by hybridization, restriction enzyme cleavage, and DNA sequence analysis. The results showed that the clones were derived from two different spliced mRNAs. By combining our results with the established DNA sequence for region E1B of the closely related adenovirus type 5[Maat, J., van Beveren, C...

  3. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

    OpenAIRE

    Sun X; Zhang Z; Gong T; Zhao D.; Han J; Zeng Q

    2012-01-01

    Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb) in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5) vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, w...

  4. Construction and expression of recombinant adenovirus encoding MDA-7/IL-24 and its inhibitory effect on proliferation in human hepatoceHular carcinoma cells%MDA-7/IL-24真核表达载体的构建和重组腺病毒的制备及其对肝癌细胞生长抑制作用的研究

    Institute of Scientific and Technical Information of China (English)

    张翔; 孟艳玲; 王磊; 赵晶; 闫博; 陈锐; 张瑞; 杨安钢

    2011-01-01

    AIM: To construct recombinant plasmid and adenovirus harboring MDA-7 gene, and to investigate its biological function on the proliferation of human hepatocellular carcinoma cells. METHODS: The MDA-7 fragments from the T vectors were inserted into pCDNA-3 vector to construct expression plasmids named pCDNA3-MDA-7. To determine its effects on the proliferation of HCC cells, trans-fected the expression vector into cells and tested the ability of colony formation in cancer cells. Simultaneously, constructed recombinant adenovirus expressing MDA-7, and detected its effect on the proliferation of HCC cells by using MTT assay. RESULTS; Successfully constructed plasmid- and adenovirus-based system to express MDA-7. The data of colony formation assay and MTT test showed that MDA-7 can obviously suppress cell growth in HCC cells. CONCLUSION; MDA-7 may function as tumor suppressor in HCC cells, and the ade-novirus-mediated MDA-7 can be a novel strategy for the anti-HCC therapy. Our study established the foundation for future research on the effect of MDA-7 in HCC.%目的:构建含有MDA-7基因的的真核表达载体并制备重组腺病毒,转染/感染肝癌细胞后,观察其对细胞增殖的影响.方法:构建MDA-7的真核表达载体,将表达质粒转染肝癌细胞,利用平板克隆形成实验观察MDA-7分子对肿瘤细胞系克隆形成能力的影响.利用Adeasy-1腺病毒重组系统构建、包装并扩增含有MDA-7基因的重组腺病毒.利用MTT实验检测Ad-MDA-7对肿瘤细胞增殖的影响.结果:成功构建了MDA-7真核表达载体,利用平板克隆形成实验证实其可抑制肝癌细胞的克隆形成能力.成功地构建并包装了含有MDA-7基因的重组腺病毒,利用MTT实验证明其可抑制肝癌细胞的增殖.结论:MDA-7对肝癌细胞的增殖具有显著的抑制作用,为进一步研究其在肝癌细胞中的功能提供了重要的实验依据.

  5. Deaths from Adenovirus in the US Military

    Centers for Disease Control (CDC) Podcasts

    2012-03-26

    Dr. Joel Gaydos, science advisor for the Armed Forces Health Surveillance Center, and Dr. Robert Potter, a research associate for the Armed Forces Medical Examiner System, discuss deaths from adenovirus in the US military.  Created: 3/26/2012 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 3/29/2012.

  6. Structure and Uncoating of Immature Adenovirus

    Energy Technology Data Exchange (ETDEWEB)

    Perez-Berna, A.J.; Mangel, W.; Marabini, R.; Scheres, S. H. W., Menendez-Conejero, R.; Dmitriev, I. P.; Curiel, D. T.; Flint, S. J.; San Martin, C.

    2009-09-18

    Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particles as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.

  7. Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Jim Silver

    Full Text Available Replication-competent adenovirus type 5 (Ad5 vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells.

  8. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    Science.gov (United States)

    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha; Cochrane, Alan

    2017-02-01

    The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

  9. Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

    OpenAIRE

    Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Joseph T Bruder; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.

    2004-01-01

    Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetica...

  10. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

    Directory of Open Access Journals (Sweden)

    Sun X

    2012-02-01

    Full Text Available Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5 vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, we ionically complexed Ad5 with a newly synthesized copolymer, which we called APC, making an adenovirus shielded from NAb.Methods: APC, a cationic polyethylene glycol derivative, was synthesized via two steps of ring-opening copolymerization of ethylene oxide and allyl glycidyl ether, followed by the addition of 2-mercaptoethylamine. The copolymer or the control PEI-2k was ionically complexed to anionic Ad5 in 5% glucose, and in vitro transduction assays were carried out in coxsackievirus and adenovirus receptor-positive cells (A549 and coxsackievirus and adenovirus receptor-negative cells (B16 and SKOV3. The physical properties and morphology of adenovirus alone or the complexes were investigated respectively by zeta potential, size distribution, and transmission electron microscopy image. Then cytotoxicity of APC was examined using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Finally, the ability of APC to protect adenovirus from NAb was evaluated by transfection assays after a neutralizing effect.Results: APC was successfully synthesized and showed a low cytotoxicity. Positively charged Ad5/APC exhibited slightly increased diameter (130.2 ± 0.60 nm than naked Ad5 (115.6 ± 5.46 nm while Ad5/PEI-2k showed severe aggregation (1382 ± 79.9 nm. Ad5/APC achieved a gene transfection level as high as Ad5/PEI-2k in A549 or B16 cells, and significantly higher than Ad5/PEI-2k in SKOV3 cells. Most importantly, after the exposure to the neutralizing

  11. Vaccination with an adenoviral vector encoding the tumor antigen directly linked to invariant chain induces potent CD4(+) T-cell-independent CD8(+) T-cell-mediated tumor control

    DEFF Research Database (Denmark)

    Sorensen, Maria R; Holst, Peter J; Pircher, Hanspeter

    2009-01-01

    Antigen-specific immunotherapy is an attractive strategy for cancer control. In the context of antiviral vaccines, adenoviral vectors have emerged as a favorable means for immunization. Therefore, we chose a strategy combining use of these vectors with another successful approach, namely linkage...... of the vaccine antigen to invariant chain (Ii). To evaluate this strategy we used a mouse model, in which an immunodominant epitope (GP33) of the LCMV glycoprotein (GP) represents the tumor-associated neoantigen. Prophylactic vaccination of C57BL/6 mice with a replication-deficient human adenovirus 5 vector...... the tumor degradation. Finally, Ad-Ii-GP but not Ad-GP vaccination can break the immunological non-reactivity in GP transgenic mice indicating that our vaccine strategy will prove efficient also against endogenous tumor antigens....

  12. Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.

    Science.gov (United States)

    Abraham, N G; da Silva, J L; Lavrovsky, Y; Stoltz, R A; Kappas, A; Dunn, M W; Schwartzman, M L

    1995-10-01

    Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO). Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of

  13. Comparison of polystyrene nanoparticles and UV-inactivated antigen-displaying adenovirus for vaccine delivery in mice

    Science.gov (United States)

    2013-01-01

    Background Inert nanoparticles are attracting attention as carriers for protein-based vaccines. Here we evaluate the immunogenicity of the model antigen ovalbumin delivered on polystyrene particles and directly compare particulate delivery with adenovirus-based immunization. Findings Mice were vaccinated with soluble ovalbumin, ovalbumin-coated polystyrene particles of different sizes, or an adenovirus-based expression-display vector that encodes and displays a pIX-ovalbumin fusion protein. Antibody responses were clearly higher when ovalbumin was administered on polystyrene particles compared to soluble protein administration, regardless of the particle size. Compared to adenovirus-based immunization, antibody levels were lower if an equivalent amount of protein was delivered, and no cellular immune response was detectable. Conclusions We demonstrate in a side-by-side comparison that inert nanoparticles allow for the reduction of the administered antigen amount compared to immunization with soluble protein and induce strongly enhanced antibody responses, but responses are lower compared to adenovirus-based immunization. PMID:23560981

  14. Latest Insights on Adenovirus Structure and Assembly

    OpenAIRE

    Carmen San Martín

    2012-01-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat p...

  15. Enhanced structural stability of adenovirus nanocapsule

    Institute of Scientific and Technical Information of China (English)

    Ding Weng; Ziyue Karen Jiang; Jing Jin; Lily Wu; Yunfeng Lu

    2014-01-01

    Application of viral vector in gene therapy and vaccination is still limited by their structural stability, which significantly increased avoidable cost in storage and transportation. Herein a non-covalent conjugated low-pH degradable nanocapsule has been adopted to stabilize viral vectors. By utilizing a luciferase expressing adenovirus, AdCMVLuc, we succeeded in a raise of over 11 folds in AdCMVLuc's structural stability after 12 days storage at 4 1C.

  16. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    Energy Technology Data Exchange (ETDEWEB)

    HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

    2001-08-01

    The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5 (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).

  17. Coacervate microspheres as carriers of recombinant adenoviruses.

    Science.gov (United States)

    Kalyanasundaram, S; Feinstein, S; Nicholson, J P; Leong, K W; Garver, R I

    1999-01-01

    The therapeutic utility of recombinant adenoviruses (rAds) is limited in part by difficulties in directing the viruses to specific sites and by the requirement for bolus administration, both of which limit the efficiency of target tissue infection. As a first step toward overcoming these limitations, rAds were encapsulated in coacervate microspheres comprised of gelatin and alginate followed by stabilization with calcium ions. Ultrastructural evaluation showed that the microspheres formed in this manner were 0.8-10 microM in diameter, with viruses evenly distributed. The microspheres achieved a sustained release of adenovirus with a nominal loss of bioactivity. The pattern of release and the total amount of virus released was modified by changes in microsphere formulation. Administration of the adenovirus-containing microspheres to human tumor nodules engrafted in mice showed that the viral transgene was transferred to the tumor cells. It is concluded that coacervate microspheres can be used to encapsulate bioactive rAd and release it in a time-dependent manner.

  18. Non-classical export of an adenovirus structural protein.

    Science.gov (United States)

    Trotman, Lloyd C; Achermann, Dominik P; Keller, Stephan; Straub, Monika; Greber, Urs F

    2003-06-01

    The icosahedral capsids of Adenoviruses (Ads) consist of the hexon and stabilizing proteins building the facettes, and of the vertex protein penton base (Pb) anchoring the protruding fibers. The fibers bind to the Coxsackie virus B Ad cell surface receptor (CAR) and Pb to integrins. Here we describe a novel property of the Ad2 Pb. Pb was found to leave the infected cell and, upon exit, it attached to the surrounding noninfected cells forming a radial gradient with highest Pb levels on cells adjacent to the infected cell. The producer cells remained intact until at least 30 h post infection. At this point, Pb was not recovered from the extracellular medium, suggesting that its cell-cell spread might not involve free Pb. When viral particles were released at late stages of infection, soluble Pb was found in the extracellular medium and it randomly bound to noninfected cells. Nonlytic export of Pb occurred upon transient transfection with plasmid DNA, but plasmid-encoded fiber was not exported, indicating that cell-cell spread of Pb is autonomous of infection. Pb export was not affected by Brefeldin A-induced disruption of the Golgi apparatus, suggesting that it occurred via a nonclassical mechanism. Interestingly, the coexpression of Pb and fiber leads to both Pb and fiber export, termed 'protein abduction'. We suggest that fiber abduction might support viral dissemination in infected tissues by interfering with tissue integrity.

  19. Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

    Science.gov (United States)

    Guse, K; Dias, J D; Bauerschmitz, G J; Hakkarainen, T; Aavik, E; Ranki, T; Pisto, T; Särkioja, M; Desmond, R A; Kanerva, A; Hemminki, A

    2007-06-01

    Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

  20. A novel adenovirus in Chinstrap penguins (Pygoscelis antarctica) in Antarctica.

    Science.gov (United States)

    Lee, Sook-Young; Kim, Jeong-Hoon; Park, Yon Mi; Shin, Ok Sarah; Kim, Hankyeom; Choi, Han-Gu; Song, Jin-Won

    2014-05-07

    Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

  1. Molecular architecture and function of adenovirus DNA polymerase

    NARCIS (Netherlands)

    Brenkman, A.B. (Arjan Bernard)

    2003-01-01

    Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and

  2. A Novel Adenovirus in Chinstrap Penguins (Pygoscelis antarctica in Antarctica

    Directory of Open Access Journals (Sweden)

    Sook-Young Lee

    2014-05-01

    Full Text Available Adenoviruses (family Adenoviridae infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica, collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1, showed nucleotide (amino acid sequence identity of 71.8% (65.5% with South Polar skua 1 (SPSAdV-1, 71% (70% with raptor adenovirus 1 (RAdV-1, 71.4% (67.6% with turkey adenovirus 3 (TAdV-3 and 61% (61.6% with frog adenovirus 1 (FrAdV-1. Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™ cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

  3. Molecular confirmation of an adenovirus in brushtail possums (Trichosurus vulpecula).

    Science.gov (United States)

    Thomson, Darelle; Meers, Joanne; Harrach, Balázs

    2002-02-26

    Partial genome characterisation of a non-cultivable marsupial adenovirus is described. Adenovirus-like particles were found by electron microscopy (EM) in the intestinal contents of brushtail possums (Trichosurus vulpecula) in New Zealand. Using degenerate PCR primers complementary to the most conserved genome regions of adenoviruses, the complete nucleotide sequence of the penton base gene, and partial nucleotide sequences of the DNA polymerase, hexon, and pVII genes were obtained. Phylogenetic analysis of the penton base gene strongly suggested that the brushtail possum adenovirus (candidate PoAdV-1) belongs to the recently proposed genus Atadenovirus. Sequence analysis of the PCR products amplified from the intestinal contents of brushtail possums originating from different geographical regions of New Zealand identified a single genotype. This is the first report of molecular confirmation of an adenovirus in a marsupial.

  4. Pulsed UV-light inactivation of poliovirus and adenovirus.

    Science.gov (United States)

    Lamont, Y; Rzezutka, A; Anderson, J G; MacGregor, S J; Given, M J; Deppe, C; Cook, N

    2007-11-01

    To study the pulsed ultraviolet (UV) inactivation of poliovirus and adenovirus. Viral suspensions of 2 ml volume were exposed to varying numbers of polychromatic light pulses emitted from a xenon flashlamp. Ten pulses produced an approximately 4 log(10) reduction in poliovirus titre, and no infectious poliovirus remained after 25 pulses. With adenovirus, 10 pulses resulted in an approximately 1 log(10) reduction in infectivity. Adenovirus required 100 pulses to produce an approximately 3 log(10) reduction in infectivity, and 200 pulses to produce a greater than 4 log(10) reduction. Adenovirus was more resistant to pulsed UV treatment than poliovirus although both viruses showed susceptibility to the treatment. Pulsed UV-light treatment proved successful in the inactivation of poliovirus and adenovirus, and represents an alternative to continuous-wave UV treatment.

  5. Construction of a recombinant adenovirus Vector of human papillomavirus type 16 L1_E7c

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 L1 proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7c. Methods: Human papillomavirus type 16 L1 open reading frame without terminator codon (TAA) (5559- 7152) and E7c (682- 855) were amplified using PCR. The L1 and E7c fragments were inserted into pGEM-T easy vectors by T- A strategy, named pTAL1 and pTAE7c. pTAL1 was cut with Hind III and BglII, the pTAE7c with BamHI and ClaI. The L1 DNA fragment, E7c and pBluesscript SK were ligated together using T4 DNA ligase. pBSL1-E7c and pBSL1-E7c was digested with Hind III and Xhol. The L1-E7c fragment was inserted into adenovirus shuttle plasmids pCAl4, named pCAl4L1-E7c. DNA sequence results indicated that The L1-E7c DNA fragment can encode the HPV16L1-E7 fusion protein correctly. Results: The L1 and E7c DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7c, pBSL1-E7c and pCA14L1-E7c were constructed correctly. The pCAl4L1-E7c can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10. Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCALl-E7c for the further research.

  6. MHC class II-associated invariant chain linkage of antigen dramatically improves cell-mediated immunity induced by adenovirus vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Mandrup Jensen, Camilla Maria; Orskov, Cathrine

    2008-01-01

    The ideal vaccine induces a potent protective immune response, which should be rapidly induced, long-standing, and of broad specificity. Recombinant adenoviral vectors induce potent Ab and CD8+ T cell responses against transgenic Ags within weeks of administration, and they are among the most...... potent and versatile Ag delivery vehicles available. However, the impact of chronic infections like HIV and hepatitis C virus underscore the need for further improvements. In this study, we show that the protective immune response to an adenovirus-encoded vaccine Ag can be accelerated, enhanced......, broadened, and prolonged by tethering of the rAg to the MHC class II-associated invariant chain (Ii). Thus, adenovirus-vectored vaccines expressing lymphocytic choriomeningitis virus (LCMV)-derived glycoprotein linked to Ii increased the CD4+ and CD8+ T cell stimulatory capacity in vitro and in vivo...

  7. Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

    Science.gov (United States)

    Lewis, J B; Atkins, J F; Anderson, C W; Baum, P R; Gesteland, R F

    1975-04-01

    Cytoplasmic RNA, isolated from cells late after infection by adenovirus type 2 and fractionated by hybridization to specific fragments of adenovirus DNA produced by cleavage with the endonuclease R-EcoRI, was used as template for protein synthesis in cell-free mammalian extracts. Each of the R-EcoRI fragments of DNA selects RNA that encodes specific subsets of the viral polypeptides. From the known order of the R-EcoRI fragments, the following partial map is deduced: (III, IIIa, IVa2, V, P-VII, IX), (II, P-VI), 100K, IV-where the relative order of the components enclosed in parentheses has not yet been determined.

  8. Regulation of Human Adenovirus Replication by RNA Interference.

    Science.gov (United States)

    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  9. Investigation of gene therapy of adenovirus in immune suppression

    Institute of Scientific and Technical Information of China (English)

    Xi XIA; Beibei WANG; Li CAO; Gang CHEN; Peng WU; Yunping LU; Jianfeng ZHOU; Ding MA

    2008-01-01

    The aim of this paper is to investigate the safety of reconstructed adenovirus in immunosuppressive ther-apeutics and to explore the role of ciclosporin A in ant-agonizing the elimination of the vector. Several rats were given retroperitoneal injection of purified ADV-TK in order to obtain models. After 14 days' treatment of ciclos-porin A, samples of different periods were obtained, then stained with hematoxylin-eosin (HE) to detect inflam-mation reactions. Immunohistochemistry was used to examine the expression of adenovirus in organs. The results are as follows: (1) In HE stained sections of the organs, some transitory and reversible inflammation was detected. (2) In immunohistochemistry assay, recon-structed adenovirus decreased gradually as time went by in the control group, while it did not happen in the experi-mental group in which the adenovirus showed a relative increase compared with their counterparts (P<0.05). (3) The distributions of adenovirus in the liver, spleen and lung were higher than those in the other organs detected. Reconstructed adenovirus as a vector is definitely safe in immunosuppressive therapeutics, and ciclosporin A, to some extent, is able to consequently inhibit the immune response of the rats and prolong the existing period of adenovirus.

  10. Replication of adenovirus DNA-protein complex with purified proteins.

    OpenAIRE

    Ikeda, J E; Enomoto, T.; Hurwitz, J

    1981-01-01

    A protein fraction isolated from the cytosol of adenovirus-infected HeLa cells, which contained DNA polymerase alpha, catalyzed adenoviral DNA replication in the presence of adenovirus DNA binding protein, eukaryotic DNA polymerase beta, ATP, all four dNTPs, and MgCl2. DNA replication started at either end of exogenously added adenoviral DNA and was totally dependent on the presence of terminal 55,000-dalton proteins on the DNA template. The replicaton of adenovirus DNA in the system was sens...

  11. Construction and characterization of replication-deficient adenoviral vector containing the cDNA for human VEGF165 in an antisense orientation%反义VEGF165腺病毒重组体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王家宁; 黄永章; 王俊峰; 王卫民; 李瑞明; 张群林

    2001-01-01

    objective To construct the recombinant adenovirus vectorcontaining the cDNA for hVEGF165 in an antisense orientation for future study of tumor treatment by antisense hVEGF165 RNA strategy.Methods The VEGF165 cDNA has been extracted from pUCCAGGS/hVEGF165 with EcoRI and then inserted in an antisense orientation into the E1-deleted expression plasmid pHCMVsp1A shuttle vector, called pAd-ahVEGF165. pAd-ahVEGF165 was cotransfected with the plasmid pJM17 into the transformed human embryonic kidney cell line 293 cells by liposome-mediated method. Homologous recombination of the pAd-ahVEGF165 and pJM17 in 293 cells replaced the E1 region with the expression cassette from pAd-ahVEGF165. Ad-ahVEGF165 was confirmed by PCR.Ad-ahVEGF165 was propagated in 293 cells and then underwent CsCl density purification. subsequently, the preparations were didalyzed in dialysis buffer. The titer of each viral stock was determined by measuring the absorbance at 260nm. Results VEGF165 cDNA was successfully inserted into the shuttle vector pHCMVSPIA. pAd-ahVEGF165 was confirmed by NcoI and XhoI digestion. Ad-ahVEGF165 was characterized by PCR coamplification. The virus titer was 5.6×1011pfu/ml. Conclusions Ad-ahVEGF165 containing the antisense VEGF165 sequence was successfully constructd.This investigation provides the basis for future study of tumor treatment based on antisense VEGF RNA strategy.%目的:构建含人反义血管内皮生长因子165(VEGF165)基因的重组腺病毒载体,为采用反义VEGF165RNA防治肿瘤的研究奠定基础。方法:将人VEGF165 cDNA反向插入到穿梭质粒pHCMVSP1A的CMV启动子之下,即pAd-ahVEGF165。后者与pJM17通过脂质体共转染293细胞,经同源重组获得含人反义VEGF165基因的重组腺病毒Ad-ahVEGF165。通过PCR共扩增法鉴别Ad-ahVEGF165的正确与否。根据260nm的紫外光吸收值计算病毒滴度。结果:VEGF165 cDNA成功地反向插入了pHCMVSP1A载体,以重组病毒基因组DNA

  12. Pulsed UV‐light inactivation of poliovirus and adenovirus

    National Research Council Canada - National Science Library

    Lamont, Y; Rzeżutka, A; Anderson, J.G; MacGregor, S.J; Given, M.J; Deppe, C; Cook, N

    2007-01-01

    .... Significance and Impact of the Study:  Pulsed UV‐light treatment proved successful in the inactivation of poliovirus and adenovirus, and represents an alternative to continuous‐wave UV treatment.

  13. Proteome analysis of adenovirus using mass spectrometry.

    Science.gov (United States)

    Lind, Sara Bergström; Artemenko, Konstantin A; Pettersson, Ulf

    2014-01-01

    Analysis of proteins and their posttranslational modifications is important for understanding different biological events. For analysis of viral proteomes, an optimal protocol includes production of a highly purified virus that can be investigated with a high-resolving analytical method. In this Methods in Molecular Biology paper we describe a working strategy for how structural proteins in the Adenovirus particle can be studied using liquid chromatography-high-resolving mass spectrometry. This method provides information on the chemical composition of the virus particle. Further, knowledge about amino acids carrying modifications that could be essential for any part of the virus life cycle is collected. We describe in detail alternatives available for preparation of virus for proteome analysis as well as choice of mass spectrometric instrumentation suitable for this kind of analysis.

  14. Acute Hepatitis and Pancytopenia in Healthy Infant with Adenovirus

    Directory of Open Access Journals (Sweden)

    Amr Matoq

    2016-01-01

    Full Text Available Adenoviruses are a common cause of respiratory infection, pharyngitis, and conjunctivitis in infants and young children. They are known to cause hepatitis and liver failure in immunocompromised patients; they are a rare cause of hepatitis in immunocompetent patients and have been known to cause fulminant hepatic failure. We present a 23-month-old immunocompetent infant who presented with acute noncholestatic hepatitis, hypoalbuminemia, generalized anasarca, and pancytopenia secondary to adenovirus infection.

  15. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

    Science.gov (United States)

    Inesi, G; Lewis, D; Sumbilla, C; Nandi, A; Strock, C; Huff, K W; Rogers, T B; Johns, D C; Kessler, P D; Ordahl, C P

    1998-03-01

    Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

  16. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

    Directory of Open Access Journals (Sweden)

    Julius W Kim

    Full Text Available BACKGROUND: Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. CONCLUSIONS/SIGNIFICANCE: These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

  17. Identification of FAM111A as an SV40 host range restriction and adenovirus helper factor.

    Directory of Open Access Journals (Sweden)

    Debrah A Fine

    Full Text Available The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

  18. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

    Directory of Open Access Journals (Sweden)

    Briana Jill Williams

    Full Text Available Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs with prostate specific membrane antigen (PSMA have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells. To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ. Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

  19. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

    Science.gov (United States)

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K; Boling, Susan; Carroll, Jennifer L; Li, Xiao-Lin; Rogers, Donna L; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V; Curiel, David T; Mathis, J Michael

    2012-01-01

    Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

  20. Marine Lectins DlFBL and HddSBL Fused with Soluble Coxsackie-Adenovirus Receptor Facilitate Adenovirus Infection in Cancer Cells BUT Have Different Effects on Cell Survival

    Science.gov (United States)

    Wu, Bingbing; Mei, Shengsheng; Cui, Lianzhen; Zhao, Zhenzhen; Chen, Jianhong; Wu, Tao; Li, Gongchu

    2017-01-01

    Cancer development and progression are usually associated with glycosylation change, providing prognostic and diagnostic biomarkers, as well as therapeutic targets, for various cancers. In this work, Dicentrarchus labrax fucose binding lectin (DlFBL) and Haliotis discus discus sialic acid binding lectin (HddSBL) were genetically fused with soluble coxsackie-adenovirus receptor (sCAR), and produced through a bacterial expression system. Results showed that recombinant sCAR-DlFBL not only facilitated adenovirus Ad-EGFP infection in K562/ADR and U87MG cells, but also enhanced the cytotoxicity of adenovirus harboring gene encoding Pinellia pedatisecta agglutinin (PPA) or DlFBL (Ad-PPA or Ad-DlFBL) on U87MG cells through inducing apoptosis. Recombinant sCAR-HddSBL facilitated Ad-EGFP infection, but dramatically counteracted the cytotoxicity of both Ad-PPA and Ad-DlFBL in U87MG cells. Further analysis revealed that sCAR-HddSBL, but not sCAR-DlFBL, significantly upregulated transcription factor E2F1 levels in U87MG cells, which might be responsible for the adverse effect of sCAR-HddSBL on Ad-PPA and Ad-DlFBL. Taken together, our data suggested that sCAR-DlFBL could be further developed to redirect therapeutic adenoviruses to infect cancer cells such as U87MG, and the sCAR-lectin fusion proteins for adenoviral retargeting should be carefully examined for possible survival signaling induced by lectins, such as HddSBL. PMID:28335432

  1. Amino-terminal sequence of adenovirus type 2 proteins: hexon, fiber, component IX, and early protein 1B-15K

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Lewis, J.B.

    1980-07-15

    The partial amino-terminal amino acid sequence was determined for four adenovirus 2 proteins: hexon, fiber, component IX, and early protein E1B-15K. A comparison of these sequences with the nucleotide sequences of the region of the genome encoding each of these proteins has identified the initiation sites for protein synthesis. Each protein is initiated at the AUG codon nearest the 5' end of its mRNA. The initiating methionine is retained by fiber and component IX while it is removed from hexon and protein E1B-15K.

  2. Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigen bispecific adapter.

    Science.gov (United States)

    Li, Hua-Jung; Everts, Maaike; Pereboeva, Larisa; Komarova, Svetlana; Idan, Anat; Curiel, David T; Herschman, Harvey R

    2007-06-01

    Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their preference for liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. Many epithelial tumors (e.g., colon, lung, and breast) express carcinoembryonic antigen (CEA). To block the natural hepatic tropism of adenovirus and to "retarget" the virus to CEA-expressing tumors, we used a bispecific adapter protein (sCAR-MFE), which fuses the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23). sCAR-MFE untargets adenovirus-directed luciferase transgene expression in the liver by >90% following systemic vector administration. Moreover, sCAR-MFE can "retarget" adenovirus to CEA-positive epithelial tumor cells in cell culture, in s.c. tumor grafts, and in hepatic tumor grafts. The sCAR-MFE bispecific adapter should, therefore, be a powerful agent to retarget adenovirus vectors to epithelial tumor metastases.

  3. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Directory of Open Access Journals (Sweden)

    Annasara Lenman

    2015-02-01

    Full Text Available Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR protein. Human adenovirus type 52 (HAdV-52 is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK binds to CAR, and the knob domain of the short fiber (52SFK binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

  4. Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid.

    Science.gov (United States)

    Worgall, Stefan; Krause, Anja; Rivara, Michael; Hee, Kyung-Kim; Vintayen, Enrico V; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G

    2005-05-01

    Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

  5. Exploiting features of adenovirus replication to support mammalian kinase production.

    Science.gov (United States)

    Cotten, Matt; Stegmueller, Kerstin; Eickhoff, Jan; Hanke, Miriam; Herzberger, Katrin; Herget, Thomas; Choidas, Axel; Daub, Henrik; Godl, Klaus

    2003-11-01

    Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.

  6. Adenovirus 36 and Obesity: An Overview

    Directory of Open Access Journals (Sweden)

    Eleonora Ponterio

    2015-07-01

    Full Text Available There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36. Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed.

  7. Adenovirus 36 and Obesity: An Overview

    Science.gov (United States)

    Ponterio, Eleonora; Gnessi, Lucio

    2015-01-01

    There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36). Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv) mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed. PMID:26184280

  8. Latest insights on adenovirus structure and assembly.

    Science.gov (United States)

    San Martín, Carmen

    2012-05-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

  9. Increasing the Efficacy of Oncolytic Adenovirus Vectors

    Directory of Open Access Journals (Sweden)

    William S. M. Wold

    2010-08-01

    Full Text Available Oncolytic adenovirus (Ad vectors present a new modality to treat cancer. These vectors attack tumors via replicating in and killing cancer cells. Upon completion of the vector replication cycle, the infected tumor cell lyses and releases progeny virions that are capable of infecting neighboring tumor cells. Repeated cycles of vector replication and cell lysis can destroy the tumor. Numerous Ad vectors have been generated and tested, some of them reaching human clinical trials. In 2005, the first oncolytic Ad was approved for the treatment of head-and-neck cancer by the Chinese FDA. Oncolytic Ads have been proven to be safe, with no serious adverse effects reported even when high doses of the vector were injected intravenously. The vectors demonstrated modest anti-tumor effect when applied as a single agent; their efficacy improved when they were combined with another modality. The efficacy of oncolytic Ads can be improved using various approaches, including vector design, delivery techniques, and ancillary treatment, which will be discussed in this review.

  10. Latest Insights on Adenovirus Structure and Assembly

    Directory of Open Access Journals (Sweden)

    Carmen San Martín

    2012-05-01

    Full Text Available Adenovirus (AdV capsid organization is considerably complex, not only because of its large size (~950 Å and triangulation number (pseudo T = 25, but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

  11. Silencing E1A mRNA by RNA interference inhibits adenovirus replication.

    Science.gov (United States)

    Chung, Y-S; Kim, M-K; Lee, W-J; Kang, C

    2007-01-01

    The adenovirus family contains 51 human serotypes, and most human adenoviruses cause widespread respiratory tract infections. Adenovirus infections can result in severe complications in some cases, such as in adenovirus type 11 infection in immunocompromised patients. However, effective treatment methods for adenovirus infections are currently unavailable. This prompted the search for antiviral agents effective against adenovirus infections. In the present study, adenovirus E1A was targeted by RNA interference (RNAi) using synthetic small interfering RNAs (siRNAs) in an attempt to inhibit viral replication, since adenovirus E1A proteins are known to be involved in the transcriptional activation of the viral and cellular genes necessary for controlling the cell cycle and viral replication. The results indicated that the siRNAs effectively reduced the amount of adenovirus E1A mRNA and the levels of replicative intermediates. Additionally, siRNA-mediated gene silencing inhibited adenovirus replication by suppressing the E1A mRNA. These results suggest that the RNAi-mediated targeting of adenovirus E1A may have a potentially therapeutic effect in controlling adenovirus infections.

  12. Novel adenovirus encoded virus-like particles displaying the placental malaria associated VAR2CSA antigen

    DEFF Research Database (Denmark)

    Andersson, Anne-Marie C; dos Santos Marques Resende, Mafalda; Salanti, Ali

    2017-01-01

    The malaria parasite Plasmodium falciparum presents antigens on the infected erythrocyte surface that bind human receptors expressed on the vascular endothelium. The VAR2CSA mediated binding to a distinct chondroitin sulphate A (CSA) is a crucial step in the pathophysiology of placental malaria a...

  13. MART-1 adenovirus-transduced dendritic cell immunization in a murine model of metastatic central nervous system tumor.

    Science.gov (United States)

    Broder, Howard; Anderson, Andrea; Kremen, Thomas J; Odesa, Sylvia K; Liau, Linda M

    2003-01-01

    Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially. Genetic modifications of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector. Sixty-two C57BL/6 mice were immunized subcutaneously with AdVMART-1-transduced DCs (n = 23), untransduced DCs (n = 17), or sterile saline (n = 22). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes from representative animals in each group were harvested for standard cytotoxicity (CTL) and enzyme-linked immunospot (ELISPOT) assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with an adenoviral vector encoding the MART-1 antigen elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared to control-immunized mice with intracranial B16 tumors, AdMART-1-DC vaccination was able to elicit partial protection against central nervous system tumor challenge in vivo.

  14. Full genome sequence analysis of a novel adenovirus of rhesus macaque origin indicates a new simian adenovirus type and species

    Directory of Open Access Journals (Sweden)

    Daniel Malouli

    2014-09-01

    Full Text Available Multiple novel simian adenoviruses have been isolated over the past years and their potential to cross the species barrier and infect the human population is an ever present threat. Here we describe the isolation and full genome sequencing of a novel simian adenovirus (SAdV isolated from the urine of two independent, never co-housed, late stage simian immunodeficiency virus (SIV-infected rhesus macaques. The viral genome sequences revealed a novel type with a unique genome length, GC content, E3 region and DNA polymerase amino acid sequence that is sufficiently distinct from all currently known human- or simian adenovirus species to warrant classifying these isolates as a novel species of simian adenovirus. This new species, termed Simian mastadenovirus D (SAdV-D, displays the standard genome organization for the genus Mastadenovirus containing only one copy of the fiber gene which sets it apart from the old world monkey adenovirus species HAdV-G, SAdV-B and SAdV-C.

  15. Inhibition of adenovirus replication in vitro by trifluridine.

    Science.gov (United States)

    Lennette, D A; Eiferman, R A

    1978-09-01

    At present, there is no effective chemotherapeutic agent available for the treatment of adenoviral keratoconjunctivitis. Recent evidence suggests that trifluridine (3FT) may effectively inhibit the replication of some adenovirus serotypes known to cause keratoconjunctivitis. The ability of 3FT to inhibit two reference strains of adenoviruses, type 8 and type 19, was examined using cell cultures. Two second-passage isolates of adenoviruses, identified as serotype 13, were also tested. Compared with untreated, virusinfected cell cultures, drug-treated cell cultures developed a lesser degree of cytopathic effect following infection with all three serotypes. Virus production was reduced in the drug-treated cell cultures: approximately tenfold for type 8, more than 1,000-fold for type 19, and 5,000-fold for the type 13 isolates.

  16. Construction, production, and purification of recombinant adenovirus vectors.

    Science.gov (United States)

    Miravet, Susana; Ontiveros, Maria; Piedra, Jose; Penalva, Cristina; Monfar, Mercè; Chillón, Miguel

    2014-01-01

    Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.

  17. Solution structure of the coxsackievirus and adenovirus receptor domain 2

    OpenAIRE

    Jiang, Shaokai; Caffrey, Michael

    2007-01-01

    The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR–D1 and CAR–D2. In the present work, the solution structure of CAR–D2, consisting of residues 142–235 of human CAR, has been determined by NMR spectroscopy. CAR–D2 is shown to be a β-sandwich motif comprised of two β-sheets, which are stabilized by two disulfide bonds. The first β-sheet is comprised of β...

  18. Transport of human adenoviruses in porous media

    Science.gov (United States)

    Kokkinos, Petros; Syngouna, Vasiliki I.; Tselepi, Maria A.; Bellou, Maria; Chrysikopoulos, Constantinos V.; Vantarakis, Apostolos

    2015-04-01

    Groundwater may be contaminated with infective human enteric viruses from various wastewater discharges, sanitary landfills, septic tanks, agricultural practices, and artificial groundwater recharge. Coliphages have been widely used as surrogates of enteric viruses, because they share many fundamental properties and features. Although a large number of studies focusing on various factors (i.e. pore water solution chemistry, fluid velocity, moisture content, temperature, and grain size) that affect biocolloid (bacteria, viruses) transport have been published over the past two decades, little attention has been given toward human adenoviruses (hAdVs). The main objective of this study was to evaluate the effect of pore water velocity on hAdV transport in water saturated laboratory-scale columns packed with glass beads. The effects of pore water velocity on virus transport and retention in porous media was examined at three pore water velocities (0.39, 0.75, and 1.22 cm/min). The results indicated that all estimated average mass recovery values for hAdV were lower than those of coliphages, which were previously reported in the literature by others for experiments conducted under similar experimental conditions. However, no obvious relationship between hAdV mass recovery and water velocity could be established from the experimental results. The collision efficiencies were quantified using the classical colloid filtration theory. Average collision efficiency, α, values decreased with decreasing flow rate, Q, and pore water velocity, U, but no significant effect of U on α was observed. Furthermore, the surface properties of viruses and glass beads were used to construct classical DLVO potential energy profiles. The results revealed that the experimental conditions of this study were unfavorable to deposition and that no aggregation between virus particles is expected to occur. A thorough understanding of the key processes governing virus transport is pivotal for public

  19. Adenovirus respiratory tract infections in Peru.

    Directory of Open Access Journals (Sweden)

    Julia S Ampuero

    Full Text Available BACKGROUND: Currently, there is a paucity of data regarding human adenovirus (HAdv circulation in Andean regions of South America. To address this shortcoming, we report the clinical, phylogenetic, and epidemiologic characteristics of HAdv respiratory tract infection from a large sentinel surveillance study conducted among adults and children in Peru. METHODS/PRINCIPAL FINDINGS: Oropharyngeal swabs were collected from participants visiting any of 38 participating health centers, and viral pathogens were identified by immunofluorescence assay in cell culture. In addition, molecular characterization was performed on 226 randomly selected HAdv samples. Between 2000 and 2010, a total of 26,375 participants with influenza-like illness (ILI or severe acute respiratory infection (SARI were enrolled in the study. HAdv infection was identified in 2.5% of cases and represented 6.2% of all viral pathogens. Co-infection with a heterologous virus was found in 15.5% of HAdv cases. HAdv infection was largely confined to children under the age of 15, representing 88.6% of HAdv cases identified. No clinical characteristics were found to significantly distinguish HAdv infection from other respiratory viruses. Geographically, HAdv infections were more common in sites from the arid coastal regions than in the jungle or highland regions. Co-circulation of subgroups B and C was observed each year between 2006 and 2010, but no clear seasonal patterns of transmission were detected. CONCLUSIONS/SIGNIFICANCE: HAdv accounted for a significant fraction of those presenting with ILI and SARI in Peru and tended to affect the younger population disproportionately. Longitudinal studies will help better characterize the clinical course of patients with HAdv in Peru, as well as determine the role of co-infections in the evolution of illness.

  20. Identification of genes differentially expressed as result of adenovirus type 5- and adenovirus type 12-transformation

    Directory of Open Access Journals (Sweden)

    Kellam Paul

    2009-02-01

    Full Text Available Abstract Background Cells transformed by human adenoviruses (Ad exhibit differential capacities to induce tumours in immunocompetent rodents; for example, Ad12-transformed rodent cells are oncogenic whereas Ad5-transformed cells are not. The E1A gene determines oncogenic phenotype, is a transcriptional regulator and dysregulates host cell gene expression, a key factor in both cellular transformation and oncogenesis. To reveal differences in gene expression between cells transformed with oncogenic and non-oncogenic adenoviruses we have performed comparative analysis of transcript profiles with the aim of identifying candidate genes involved in the process of neoplastic transformation. Results Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK cells. Gene information was available for 193 transcripts and using gene ontology (GO classifications and literature searches it was possible to assign known or suggested functions to 166 of these identified genes. A subset of differentially-expressed genes from the microarray was further examined by real-time PCR and Western blotting using BRK cells immortalised by Ad12 E1A or Ad5 E1A in addition to Ad12 E1- or Ad5 E1-transformed BRK cells. Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells. Conclusion These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation. Further work will focus on investigating which splice variant of Ad E1A is responsible for the observed dysregulation at the pathway level, and the mechanisms of E1A-mediated transcriptional regulation.

  1. Adenovirus Infection in Children with Diarrhea Disease in ...

    African Journals Online (AJOL)

    ANNALS

    41(Ad41), can cause acute and severe diarrhea in young children worldwide. This study was ... specimen in adenovirus infected patients showed watery diarrhea in 87% (55/63), diarrhea with mucus in ..... However, the impact of these viruses ...

  2. Low seroprevalent species D adenovirus vectors as influenza vaccines.

    Directory of Open Access Journals (Sweden)

    Eric A Weaver

    Full Text Available Seasonal and pandemic influenza remains a constant threat. While standard influenza vaccines have great utility, the need for improved vaccine technologies have been brought to light by the 2009 swine flu pandemic, highly pathogenic avian influenza infections, and the most recent early and widespread influenza activity. Species C adenoviruses based on serotype 5 (AD5 are potent vehicles for gene-based vaccination. While potent, most humans are already immune to this virus. In this study, low seroprevalent species D adenoviruses Ad26, 28, and 48 were cloned and modified to express the influenza virus A/PR/8/34 hemagglutinin gene for vaccine studies. When studied in vivo, these species D Ad vectors performed quite differently as compared to species C Ad vectors depending on the route of immunization. By intramuscular injection, species D vaccines were markedly weaker than species C vaccines. In contrast, the species D vaccines were equally efficient as species C when delivered mucosally by the intranasal route. Intranasal adenovirus vaccine doses as low as 10(8 virus particles per mouse induced complete protection against a stringent lethal challenge dose of influenza. These data support translation of species D adenoviruses as mucosal vaccines and highlight the fundamental effects of differences in virus tropism on vaccine applications.

  3. Pharmacological Interventions for Improving Adenovirus Usage in Gene Therapy

    NARCIS (Netherlands)

    Haisma, Hidde J.; Bellu, Anna Rita

    2011-01-01

    Gene therapy may be an innovative and promising new treatment strategy for cancer but is limited due to a low efficiency and specificity of gene delivery to the target cells. Adenovirus is the preferred gene therapy vector for systemic delivery because of its unparalleled in vivo transduction effici

  4. Bioaccumulation of animal adenoviruses in the pink shrimp

    Directory of Open Access Journals (Sweden)

    Roger B. Luz

    2015-09-01

    Full Text Available Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100 Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

  5. Adenovirus infection reverses the antiviral state induced by human interferon.

    Science.gov (United States)

    Feduchi, E; Carrasco, L

    1987-04-06

    HeLa cells treated with human lymphoblastoid interferon do not synthesize poliovirus proteins. The antiviral state against poliovirus is reversed if cells are previously infected with adenovirus type 5. A late gene product seems to be involved in this reversion, since no effect is observed at early stages of infection or in the presence of aphidicolin.

  6. Pharmacological Interventions for Improving Adenovirus Usage in Gene Therapy

    NARCIS (Netherlands)

    Haisma, Hidde J.; Bellu, Anna Rita

    2011-01-01

    Gene therapy may be an innovative and promising new treatment strategy for cancer but is limited due to a low efficiency and specificity of gene delivery to the target cells. Adenovirus is the preferred gene therapy vector for systemic delivery because of its unparalleled in vivo transduction

  7. Bioaccumulation of animal adenoviruses in the pink shrimp.

    Science.gov (United States)

    Luz, Roger B; Staggemeier, Rodrigo; Fabres, Rafael B; Soliman, Mayra C; Souza, Fernanda G; Gonçalves, Raoni; Fausto, Ivone V; Rigotto, Caroline; Heinzelmann, Larissa S; Henzel, Andréia; Fleck, Juliane D; Spilki, Fernando R

    2015-01-01

    Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

  8. Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

    NARCIS (Netherlands)

    van Beusechem, VW; van Rijswijk, ALCT; van Es, HHG; Haisma, HJ; Pinedo, HM; Gerritsen, WR

    2000-01-01

    Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfil

  9. Effect of adenovirus gene transfer vectors on the immunologic functions of mouse dendritic cells.

    Science.gov (United States)

    Korst, Robert J; Mahtabifard, Ali; Yamada, Reiko; Crystal, Ronald G

    2002-03-01

    To address the effect of adenovirus (Ad) gene transfer vector transduction on the diverse functions of dendritic cells, we used an Ad vector encoding no transgene (AdNull) to transduce mouse bone-marrow-derived dendritic cells (BMDC). Initial experiments using an Ad vector encoding a marker gene (AdGFP, jellyfish green fluorescent protein) showed that the optimal ratio of infectious Ad particles to each cell was 100, when both transgene expression and resultant BMDC viability were taken into account. Exposure to AdNull resulted in upregulation of both surface activation markers (CD40, MHC class II, B7.1, B7.2, ICAM-1) and IL-12 expression by BMDC. AdNull activation of BMDC was observed in multiple strains of mice. Despite this, AdNull-transduced BMDC displayed only modestly impaired antigen uptake ability, as demonstrated in macropinocytosis and phagocytosis assays, in vitro. However, Ad-modified BMDC migrated to regional lymph nodes five times more efficiently than sham-transduced BMDC in vivo. In addition, Ad transduction significantly enhanced the ability of BMDC to present a model peptide antigen to T-lymphocyte hybridoma cells at low BMDC:T cell ratios. We conclude that Ad modification, in and of itself, induces a state of activation in mouse BMDC. This activation, albeit mild compared with that induced by other stimuli, produces measurable effects of the specific immunological functions of these antigen-presenting cells.

  10. Assessment of the Na/I symporter as a reporter gene to visualize oncolytic adenovirus propagation in peritoneal tumours

    Energy Technology Data Exchange (ETDEWEB)

    Merron, Andrew; McNeish, Iain A. [Queen Mary' s School of Medicine and Dentistry, Centre for Molecular Oncology, Institute of Cancer, London (United Kingdom); Baril, Patrick; Tran, Lucile; Vassaux, Georges [CHU Hotel Dieu, INSERM, Nantes (France); CHU de Nantes, Institut des Maladies de l' Appareil Digestif, Nantes (France); Martin-Duque, Pilar [Instituto Aragones de Ciencias de la Salud, Zaragoza (Spain); Vieja, Antonio de la [Instituto de Investigaciones Biomedicas, Madrid (Spain); Briat, Arnaud [INSERM U877, Grenoble (France); Harrington, Kevin J. [Chester Beatty Laboratories, Institute of Cancer Research, London (United Kingdom)

    2010-07-15

    In vivo imaging of the spread of oncolytic viruses using the Na/I symporter (NIS) has been proposed. Here, we assessed whether the presence of NIS in the viral genome affects the therapeutic efficacy of the oncolytic adenovirus dl922-947 following intraperitoneal administration, in a mouse model of peritoneal ovarian carcinoma. We generated AdAM7, a dl922-947 oncolytic adenovirus encoding the NIS coding sequence. Iodide uptake, NIS expression, infectivity and cell-killing activity of AdAM7, as well as that of relevant controls, were determined in vitro. In vivo, the propagation of this virus in the peritoneal cavity of tumour-bearing mice was determined using SPECT/CT imaging and its therapeutic efficacy was evaluated. In vitro infection of ovarian carcinoma IGROV-1 cells with ADAM7 led to functional expression of NIS. However, the insertion of NIS into the viral genome resulted in a loss of efficacy of the virus in terms of replication and cytotoxicity. In vivo, on SPECT/CT imaging AdAM7 was only detectable in the peritoneal cavity of animals bearing peritoneal ovarian tumours for up to 5 days after intraperitoneal administration. Therapeutic experiments in vivo demonstrated that AdAM7 is as potent as its NIS-negative counterpart. This study demonstrated that despite the detrimental effect observed in vitro, insertion of the reporter gene NIS in an oncolytic adenovirus did not affect its therapeutic efficacy in vivo. We conclude that NIS is a highly relevant reporter gene to monitor the fate of oncolytic adenovectors in live subjects. (orig.)

  11. Infection by Cx43 adenovirus increased chemotherapy sensitivity in human gastric cancer BGC-823 cells: not involving in induction of cell apoptosis.

    Science.gov (United States)

    Liu, Dan; Zhou, Hongfeng; Wu, Jin; Liu, Wentao; Li, Yongqing; Shi, Guangyue; Yue, Xiaolong; Sun, Xiwen; Zhao, Yanbin; Hu, Xiaowei; Wang, Tianjiao; Zhang, Xufeng

    2015-12-15

    There is a lower basal expression of Connexin43 (Cx43) in human gastric cancer BGC-823 cells. In the present study, BGC-823 cells were transfected with recombinant Cx43 adenovirus plasmid vector, and we explored the influences of Cx43 expression on cell proliferation, chemo-sensitivity, colony forming ability, invasion ability and apoptosis. Moreover, we also determined the expression of Pgp, Cx43, as well as apoptosis-related proteins (bcl-2, bax, caspase3 and caspase 9). MTT assay was performed to determine the proliferation of BGC-823 cells before and after Cx43 transfection. The influences of Cx43 infection on sensitivity of chemotherapy (including Doxorubicin, fluorouracil, oxaliplatin) were detected by MTT assay. Expression levels of Pgp, Cx43, as well as apoptosis-related proteins (bcl-2, bax, caspase-3 and caspase-9) in BGC-823 cells were determined by Western blotting analysis before and after the infection with Cx43 adenovirus. MDR expression was determined by RT-PCR before and after Cx43 infection. Invasive ability was detected by invasion chamber. Influence of Cx43 adenovirus infection on apoptosis of BGC-823 cells was determined by flow cytometry. After infection by Cx43 adenovirus, colony forming rate and invasive ability of BGC-823 cells were decreased. Flow cytometry results revealed that cell apoptosis were insignificantly increased. The data of MTT assay revealed that infection with Cx43 adenovirus, cell proliferation ability decreased and sensitivity to chemotherapy drugs (including doxorubicin, fluorouracil, oxaliplatin) increased. Results of Western blotting analysis revealed that increasing expression levels of Cx43, decreasing expression levels of Pgp, and insignificant changes of bcl-2, bax, caspase3 and caspase 9 were detected. RT-PCR revealed the expression of MDR1 gene, the gene encoding Pgp, decreased significantly (pinfected with Cx43-IRES2-EGFP recombinant adenovirus vector. Colony formation, invasive ability and cell proliferation all

  12. Adenovirus-based p53 gene therapy in ovarian cancer.

    Science.gov (United States)

    Santoso, J T; Tang, D C; Lane, S B; Hung, J; Reed, D J; Muller, C Y; Carbone, D P; Lucci, J A; Miller, D S; Mathis, J M

    1995-11-01

    Mutations of the p53 tumor suppressor gene are the most common molecular genetic abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53 as a molecular target for gene therapy in ovarian cancer, we constructed an adenovirus vector containing the wild-type p53 gene. The ability of this adenovirus construct (Ad-CMV-p53) to express p53 protein was examined by Western blot analysis in the H358 lung cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus containing the beta-galactosidase reporter gene under the control of the CMV promoter (Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53 mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the effect of these agents on the growth of 2774 cells was determined using an in vitro growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad-CMV-p53 showed expression of wild-type p53 protein. When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.) of 10 PFU/cell, > 90% of cells showed beta-galactosidase activity, demonstrating that these cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The ability of the adenovirus vector to mediate high-level expression of infected genes and the inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could be further developed into a therapeutic agent for ovarian cancer.

  13. Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.

    Science.gov (United States)

    Balagué, C; Noya, F; Alemany, R; Chow, L T; Curiel, D T

    2001-08-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

  14. Human genes for U2 small nuclear RNA map to a major adenovirus 12 modification site on chromosome 17.

    Science.gov (United States)

    Lindgren, V; Ares, M; Weiner, A M; Francke, U

    U2 RNA is one of the abundant, highly conserved species of small nuclear RNA (snRNA) molecules implicated in RNA processing. As is typical of mammalian snRNAs, human U1 and U2 are each encoded by a multigene family. In the human genome, defective copies of the genes (pseudogenes) far outnumber the authentic genes. The majority or all of the 35 to 100 bona fide U1 genes have at least 20 kilobases (kb) of nearly perfect 5' and 3' flanking homology in common with each other; these U1 genes are clustered loosely in chromosome band 1p36 (refs 5, 7) with intergenic distances exceeding 44 kb. In contrast, the 10 to 20 U2 genes are clustered tightly in a virtually perfect tandem array which has a strict 6-kb repeating unit. We report here the assignment, by in situ hybridization, of the U2 gene cluster to chromosome 17, bands q21-q22. Surprisingly, this region is one of three major adenovirus 12 modification sites which undergo chromosome decondensation ('uncoiling') in permissive human cells infected by highly oncogenic strains of adenovirus. The two other major modification sites, 1p36 and 1q21, coincide with the locations of U1 genes and class I U1 pseudogenes, respectively. We suggest that snRNA genes are the major targets of viral chromosome modification.

  15. Definition of a novel promoter for the major adenovirus-associated virus mRNA.

    Science.gov (United States)

    Green, M R; Roeder, R G

    1980-11-01

    A 660 nucleotide adenovirus-associated virus type 2 (AAV2) DNA fragment which encodes the 5' terminal leader and the entire intervening sequence of the major viral mRNA has been cloned into pBR322, and its primary sequence has been determined. The 5' terminal viral mRNA sequence was deduced by sequencing the reverse-transcriptase cDNA extension product of a 5' end-labeled DNA primer complementary to the RNA 5' terminal region. From combined DNA and RNA sequence analyses (which confirm our previous mapping data) we conclude that the major AAV2 transcript contains a 5' terminal leader sequence about 55 nucleotides in length encoded from a continuous region of DNA (near position 39 on the viral genome) 320 bases from the RNA body. The DNA sequences of the splice junctions are similar to those found for other class II genes. No other nucleotide sequence, indicative of promotion at another (upstream) site, is present at the 5' terminus. The DNA region encoding and flanking the leader sequence displays structural features expected for a class II gene promoter, including the canonical ATATAA sequence 23-25 bases upstream from the presumed initiation site. When the cloned viral DNA fragment is transcribed in vitro by RNA polymerase II in a cell-free system, a transcript is produced with a 5' end that is similar or identical to that found on the in vivo mRNA. Taken together these data strongly suggest that the major polysomal RNA may be generated from a transcription unit with a promoter at position 39, even though this transcription unit is part of a larger transcription unit with an upstream promoter near position 6. This indication of overlapping transcription units with independent promoters provides a major new insight into parvovirus gene expression.

  16. Dramatic Decline of Respiratory Illness Among US Military Recruits After the Renewed Use of Adenovirus Vaccines

    Science.gov (United States)

    2014-10-01

    Naval Health Research Center Dramatic Decline of Respiratory Illness Among US Military Recruits After the Renewed Use of Adenovirus Vaccines ...Renewed Use of Adenovirus Vaccines Jennifer M. Radin,1,2 Anthony W. Hawksworth,1 Patrick J. Blair,1 Dennis J. Faix,3 Rema Raman,4 Kevin L. Russell,5...hiatus, oral vaccines against adenovirus types 4 (Ad4) and 7 (Ad7) were again produced and administered to US military recruits. This study examined the

  17. A Replicating Adenovirus Capsid Display Recombinant Elicits Antibodies against Plasmodium falciparum Sporozoites in Aotus nancymaae Monkeys

    OpenAIRE

    Karen, Kasey A.; Deal, Cailin; Adams, Robert J; Nielsen, Carolyn; Ward, Cameron; Espinosa, Diego A.; Xie, Jane; Zavala,Fidel; Ketner, Gary

    2014-01-01

    Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodie...

  18. Dielectrophoresis and dielectrophoretic impedance detection of adenovirus and rotavirus

    Science.gov (United States)

    Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya

    2016-01-01

    The aim of this study is the electrical detection of pathogenic viruses, namely, adenovirus and rotavirus, using dielectrophoretic impedance measurement (DEPIM). DEPIM consists of two simultaneous processes: dielectrophoretic trapping of the target and measurement of the impedance change and increase in conductance with the number of trapped targets. This is the first study of applying DEPIM, which was originally developed to detect bacteria suspended in aqueous solutions, to virus detection. The dielectric properties of the viruses were also investigated in terms of their dielectrophoretic behavior. Although their estimated dielectric properties were different from those of bacteria, the trapped viruses increased the conductance of the microelectrode in a manner similar to that in bacteria detection. We demonstrated the electrical detection of viruses within 60 s at concentrations as low as 70 ng/ml for adenovirus and 50 ng/ml for rotavirus.

  19. Adenovirus-derived vectors for prostate cancer gene therapy.

    Science.gov (United States)

    de Vrij, Jeroen; Willemsen, Ralph A; Lindholm, Leif; Hoeben, Rob C; Bangma, Chris H; Barber, Chris; Behr, Jean-Paul; Briggs, Simon; Carlisle, Robert; Cheng, Wing-Shing; Dautzenberg, Iris J C; de Ridder, Corrina; Dzojic, Helena; Erbacher, Patrick; Essand, Magnus; Fisher, Kerry; Frazier, April; Georgopoulos, Lindsay J; Jennings, Ian; Kochanek, Stefan; Koppers-Lalic, Daniela; Kraaij, Robert; Kreppel, Florian; Magnusson, Maria; Maitland, Norman; Neuberg, Patrick; Nugent, Regina; Ogris, Manfred; Remy, Jean-Serge; Scaife, Michelle; Schenk-Braat, Ellen; Schooten, Erik; Seymour, Len; Slade, Michael; Szyjanowicz, Pio; Totterman, Thomas; Uil, Taco G; Ulbrich, Karel; van der Weel, Laura; van Weerden, Wytske; Wagner, Ernst; Zuber, Guy

    2010-07-01

    Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.

  20. Inhibitory effect of interferon-gamma on adenovirus replication and late transcription.

    Science.gov (United States)

    Mistchenko, A S; Diez, R A; Falcoff, R

    1989-06-15

    We have previously shown that human interferon-gamma inhibited adenovirus multiplication in vitro in a dose-dependent fashion. This action was previous to capsid proteins synthesis and did not involve virus adsorption nor penetration. In this report we have analysed viral mRNA levels at early (7 hr post infection (p.i.)) or late (20 hr p.i.) times, as well as DNA replication in Wish cells pretreated with interferon-gamma and infected with adenovirus 5. Controls included untreated cells as well as cells treated with interferon-alpha, to which adenovirus are reported to be resistant. Transcription of adenovirus regions E1, E4, L1 and L2 has been analysed by Northern blot. Adenovirus DNA replication was determined by DNA-DNA hybridization with total adenovirus 2 DNA. We have also searched for adenovirus E1A proteins by immunoblot with a specific monoclonal antibody. Although pretreatment of cells with either interferon-alpha or interferon-gamma resulted in reduced amounts of E1 and E4 mRNA in the early phase of infection (7 hr p.i.), the near complete inhibition of viral DNA and late transcription was only achieved by interferon-gamma. Immunoblot has shown the absence of the 48-kD E1A protein in cells pretreated with interferon-gamma. The lack of this regulatory adenovirus protein may be involved in the inhibitory mechanism of interferon-gamma on adenovirus.

  1. Addition of polyadenylate sequences to virus-specific RNA during adenovirus replication.

    Science.gov (United States)

    Philipson, L; Wall, R; Glickman, G; Darnell, J E

    1971-11-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA.

  2. ENTERIC ADENOVIRUS INFECTION IN INFANTS AND YOUNG CHILDREN WITH ACUTE GASTROENTERITIS IN TEHRAN

    Directory of Open Access Journals (Sweden)

    F. Jam-Afzon S. Modarres

    2006-09-01

    Full Text Available Adenoviruses are one of the most important etiological agents of serious gastroenteritis among infants and young children. Fecal specimens from patients with an acute gastroenteritis were evaluated for the presence of adenovirus (Ad40, 41 from April 2002 to February 2004. During the study, 1052 samples were collected from children under the age of 5 years in six educational and therapeutic pediatric centers. The specimens were tested for adenovirus (Ad40, 41 by EIA technique in the Virology Department of Pasteur Institute of Iran. Adenoviruses (Ad40, 41 were detected from 27(2.6% samples, but were not detected in 150 samples of healthy control group. In this study the highest rate of adenovirus was found in children aged 6 to 12 months (40.7%, but the male to female ratio inpatients was approximately equal. Adenovirus (Ad40, 41 infections peaked in the winter as 48.1% was detected from December to March. There were a statistically significant difference between age and infection (P < 0.001, also between season with adenovirus (Ad40, 41 infection (P = 0.005. Breast-feeding had a protective action against adenovirus (Ad40, 41 infection. This study revealed that enteric adenovirus (Ad40, 41 is an etiological agent of acute gastroenteritis among children in Tehran.

  3. Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

    Science.gov (United States)

    Zaiss, Anne K; Vilaysane, Akosua; Cotter, Matthew J; Clark, Sharon A; Meijndert, H Christopher; Colarusso, Pina; Yates, Robin M; Petrilli, Virginie; Tschopp, Jurg; Muruve, Daniel A

    2009-06-01

    Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

  4. E1A genes of adenovirus type 2 and type 5 are expressed at different levels

    DEFF Research Database (Denmark)

    Moritz, Constanze; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses are an extensively studied system for modeling oncogenesis and for experimental cancer therapy. The most commonly analyzed virus types are 2 and 5, and little distinction has been made between them in past studies. Adenoviruses used for therapeutic purposes are frequently hybrids...... region. We found that the hybrid viruses replicated with considerably lower efficiency than their type 5 counterparts in H1299 cells (dl309:WtD = 3-4, dl338:dl1520 > 10). Moreover, adenovirus type 2 E1A expression from the hybrid viruses was strongly reduced in comparison to adenovirus type 5 E1A...

  5. Optimization and evaluation of a method to detect adenoviruses in river water

    Data.gov (United States)

    U.S. Environmental Protection Agency — This dataset includes the recoveries of spiked adenovirus through various stages of experimental optimization procedures. This dataset is associated with the...

  6. Adenovirus-Mediated Gene Therapy Against Viral Biothreat Agents

    Science.gov (United States)

    2016-04-12

    34--- I lr_ Transworld Research Network 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India Recent Development in Gene Therapy , 2007: 77-94...ISBN: 81-7895-262-9 Editor: Jim Xiang Adenovirus-mediated gene therapy against viral biothreat agents Josh Q.H. Wu Chemical Biological Defence... therapy , which introduces therapeutic genes into mammalian cells to achieve therapeutic effective, hds a great potential for use as a defensive

  7. Replication-Uncoupled Histone Deposition during Adenovirus DNA Replication

    OpenAIRE

    Komatsu, Tetsuro; Nagata, Kyosuke

    2012-01-01

    In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of his...

  8. The Dual Nature of Nek9 in Adenovirus Replication

    OpenAIRE

    Jung, Richard; Radko, Sandi; Pelka, Peter

    2016-01-01

    To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to s...

  9. Molecular architecture of the preinitiation complex in adenovirus DNA replication

    OpenAIRE

    Mysiak, Monika Elzbieta

    2004-01-01

    After infection of a host cell, adenovirus (Ad) aims for generation of progeny viruses, and thus it rapidly replicates its genomic DNA. The replication process starts with the assembly of the preinitiation complex (PIC) on the origin DNA. The PIC consists of three viral proteins, DNA polymerase (pol), precursor terminal protein (pTP), DNA binding protein (DBP) and two transcription factors of the host cell, Nuclear Factor I (NFI) and Octamer binding protein (Oct-1). Both transcription factors...

  10. Oncolytic adenovirus-mediated therapy for prostate cancer

    Directory of Open Access Journals (Sweden)

    Sweeney K

    2016-07-01

    Full Text Available Katrina Sweeney, Gunnel Halldén Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Abstract: Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen–androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting localized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support

  11. Reassessing culture media and critical metabolites that affect adenovirus production.

    Science.gov (United States)

    Shen, Chun Fang; Voyer, Robert; Tom, Roseanne; Kamen, Amine

    2010-01-01

    Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 x 10(6) cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 x 10(6) cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 x 10(6) cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions.

  12. Oncolytic adenovirus-mediated therapy for prostate cancer

    Science.gov (United States)

    Sweeney, Katrina; Halldén, Gunnel

    2016-01-01

    Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen–androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses. PMID:27579296

  13. An Update on Canine Adenovirus Type 2 and Its Vectors

    Directory of Open Access Journals (Sweden)

    Eric J. Kremer

    2010-09-01

    Full Text Available Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2 biology and gives an overview of the generation of early region 1 (E1-deleted to helper-dependent (HD CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors.

  14. Rotavirus and adenovirus in Rondônia

    Directory of Open Access Journals (Sweden)

    Gleiciene Félix Magalhães

    2007-08-01

    Full Text Available Acute gastroenteritis is one of the most common diseases in humans worldwide. Viral gastroenteritis is a global problem in infants and young children. In this study the incidence of diarrhea was assessed in 877 hospitalized children under five years old, over a period of 24 months and distributed in 470 cases of diarrhea and 407 age-matched group with other pathologies, as control group. Two antigen detection techniques based on enzyme immunoassay (EIA and latex particles were used for detection of rotavirus and adenovirus. Rotavirus A was a major cause of gastroenteritis with 23.6% of cases, being 90% of these cases in young children. Adenovirus infections was detected by EIA with frequency of 6.4%. Rotavirus and adenovirus were detected in 10.1 and 1.7% of stools from control group, respectively. Interestingly, the frequency of the youngest children in the control group excreting Rotavirus A was comparable to that detected in stools from diarrheic children. We cannot rule out the existence of other enteric viruses because the etiology of 171 cases of diarrhea was not determined and active search for astrovirus and calicivirus was not done. This is the first study that shows the presence of enteric viruses in the infantile population from Western Brazilian Amazonia and it was important to help physicians in the treatment of viral gastroenteritis.

  15. Brain tumors induced in rats by human adenovirus type 12

    Directory of Open Access Journals (Sweden)

    Murao,Tsuyoshi

    1974-02-01

    Full Text Available Oncogenesis of human adenovirus type 12 in the brain of rats was examined. Newborn rats of Sprague-Dawley and Donryu strains were injected intracranially with human adenovirus type 12. The incidence of intracranial tumors was 91% (30/33 in SpragueDawley and 56% (14/25 in Donryu rats. Except for one tumor nodule located in the parietal cortex of a Sprague.Dawley rat, all tumors developed in the paraventricular areas or in the meninges. Tumors were quite similar histologically to those induced in hamsters and mice resembling the undifferentiated human brain tumors such as medulloblastoma, ependymoblastoma and embryonic gliomas. From the histological features and primary sites of tumor development, it is suggested that the tumors in the brain of rats induced by adenovirus type 12 originate from the embryonic cells in the paraventricular area and also from the undifferentiated supporting cells of the peripheral nerves in the leptomeninges.

  16. An outbreak of lethal adenovirus infection among different otariid species.

    Science.gov (United States)

    Inoshima, Yasuo; Murakami, Tomoaki; Ishiguro, Naotaka; Hasegawa, Kazuhiro; Kasamatsu, Masahiko

    2013-08-30

    An outbreak of fatal fulminant hepatitis at a Japanese aquarium involved 3 otariids: a California sea lion (Zalophus californianus), a South African fur seal (Arctocephalus pusillus) and a South American sea lion (Otaria flavescens). In a span of about a week in February 2012, 3 otariids showed diarrhea and were acutely low-spirited; subsequently, all three animals died within a period of 3 days. Markedly increased aspartate amino transferase and alanine amino transferase activities were observed. Necrotic hepatitis and eosinophilic intranuclear inclusion bodies in liver hepatocytes and intestinal epithelial cells were observed in the South American sea lion on histological examination. Otarine adenovirus DNA was detected from the livers of all three animals by polymerase chain reaction and determination of the sequences showed that all were identical. These results suggest that a single otarine adenovirus strain may have been the etiological agent of this outbreak of fatal fulminant hepatitis among the different otariid species, and it may be a lethal threat to wild and captive otariids. This is the first evidence of an outbreak of lethal adenovirus infection among different otariid species.

  17. A novel and simple method for construction of recombinant adenoviruses.

    Science.gov (United States)

    Tan, Rong; Li, Chunhua; Jiang, Sijing; Ma, Lixin

    2006-07-19

    Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.

  18. Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation.

    Science.gov (United States)

    Sharma, Gaurav; Moria, Nithesh; Williams, Martin; Krishnarjuna, Bankala; Pouton, Colin W

    2017-07-01

    Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

  19. The Golden Ratio Encoder

    CERN Document Server

    Daubechies, I; Wang, Y; Yilmaz, Ö

    2008-01-01

    This paper proposes a novel Nyquist-rate analog-to-digital (A/D) conversion algorithm which achieves exponential accuracy in the bit-rate despite using imperfect components. The proposed algorithm is based on a robust implementation of a beta-encoder where the value of the base beta is equal to golden mean. It was previously shown that beta-encoders can be implemented in such a way that their exponential accuracy is robust against threshold offsets in the quantizer element. This paper extends this result by allowing for imperfect analog multipliers with imprecise gain values as well. A formal computational model for algorithmic encoders and a general test bed for evaluating their robustness is also proposed.

  20. Adenovirus-based vaccines against avian-origin H5N1 influenza viruses.

    Science.gov (United States)

    He, Biao; Zheng, Bo-jian; Wang, Qian; Du, Lanying; Jiang, Shibo; Lu, Lu

    2015-02-01

    Since 1997, human infection with avian H5N1, having about 60% mortality, has posed a threat to public health. In this review, we describe the epidemiology of H5N1 transmission, advantages and disadvantages of different influenza vaccine types, and characteristics of adenovirus, finally summarizing advances in adenovirus-based H5N1 systemic and mucosal vaccines.

  1. Presence of protein at the termini of intracellular adenovirus type 5 DNA

    NARCIS (Netherlands)

    Wielink, P.S. van; Naaktgeboren, N.; Sussenbach, J.S.

    1979-01-01

    Adenovirus type 5 contains linear double-stranded DNA with protein covalently attached to the ends of the molecules. The presence of protein at the termini of intracellular viral DNA in adenovirus type 5-infected cells was investigated at different stages during the replication process. The intracel

  2. 101 an epidemic of adenovirus type·' bronchopneumonia in bantu ...

    African Journals Online (AJOL)

    1971-01-30

    Jan 30, 1971 ... in 10 (11°.{,). A diagnosis of cardiac failure was made in all but 3 of those ... like that of the 17 in whom adenovirus type-7 infection was proved by ... by antiserum prepared in horses to adenovirus type-7a, strain 5-1058 ... MATERlALS AND METHODS .... chromic anaemia was present in 32 cases. The mean ...

  3. Anatomical differences determine distribution of adenovirus after convection-enhanced delivery to the rat brain

    NARCIS (Netherlands)

    S. Idema (Sander); V. Caretti (Viola); M.L.M. Lamfers (Martine); V.W. Beusechem (Victor); D.P. Noske (David); W.P. Vandertop (Peter); C.M.F. Dirven (Clemens)

    2011-01-01

    textabstractBackground: Convection-enhanced delivery (CED) of adenoviruses offers the potential of widespread virus distribution in the brain. In CED, the volume of distribution (Vd) should be related to the volume of infusion (Vi) and not to dose, but when using adenoviruses contrasting results hav

  4. Presence of adenovirus species C in infiltrating lymphocytes of human sarcoma.

    Directory of Open Access Journals (Sweden)

    Karin Kosulin

    Full Text Available Human adenoviruses are known to persist in T-lymphocytes of tonsils, adenoids and intestinal tract. The oncogenic potential of different adenovirus types has been widely studied in rodents, in which adenovirus inoculation can induce multiple tumors such as undifferentiated sarcomas, adenocarcinomas and neuroectodermal tumors. However, the oncogenic potential of this virus has never been proven in human subjects. Using a highly sensitive broad-spectrum qRT-PCR, we have screened a set of different human sarcomas including leiomyosarcoma, liposarcoma and gastro intestinal stroma tumors. Primers binding the viral oncogene E1A and the capsid-coding gene Hexon were used to detect the presence of adenovirus DNA in tumor samples. We found that 18% of the tested leiomyosarcomas and 35% of the liposarcomas were positive for the presence of adenovirus DNA, being species C types the most frequently detected adenoviruses. However, only in one sample of the gastro intestinal stroma tumors the virus DNA could be detected. The occurrence of adenovirus in the tumor sections was confirmed by subsequent fluorescence in-situ-hybridization analysis and co-staining with the transcription factor Bcl11b gives evidence for the presence of the virus in infiltrating T-lymphocytes within the tumors. Together these data underline, for the first time, the persistence of adenovirus in T-lymphocytes infiltrated in muscular and fatty tissue tumor samples. If an impaired immune system leads to the viral persistence and reactivation of the virus is involved in additional diseases needs further investigation.

  5. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    Science.gov (United States)

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-03-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus.

  6. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Science.gov (United States)

    2010-01-01

    ... Type 2 Vaccine. 113.305 Section 113.305 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  7. Adenovirus Type 7 Pneumonia in Children Who Died from Measles-Associated Pneumonia, Hanoi, Vietnam, 2014.

    Science.gov (United States)

    Hai, Le Thanh; Thach, Hoang Ngoc; Tuan, Ta Anh; Nam, Dao Huu; Dien, Tran Minh; Sato, Yuko; Kumasaka, Toshio; Suzuki, Tadaki; Hanaoka, Nozomu; Fujimoto, Tsuguto; Katano, Harutaka; Hasegawa, Hideki; Kawachi, Shoji; Nakajima, Noriko

    2016-04-01

    During a 2014 measles outbreak in Vietnam, postmortem pathologic examination of hospitalized children who died showed that adenovirus type 7 pneumonia was a contributory cause of death in children with measles-associated immune suppression. Adenovirus type 7 pneumonia should be recognized as a major cause of secondary infection after measles.

  8. Interspecies differences in virus uptake versus cardiac function of the coxsackievirus and adenovirus receptor.

    NARCIS (Netherlands)

    Freiberg, F.; Sauter, M.; Pinkert, S.; Govindarajan, T.; Kaldrack, J.; Thakkar, M.; Fechner, H.; Klingel, K.; Gotthardt, M.

    2014-01-01

    The coxsackievirus and adenovirus receptor (CAR) is a cell contact protein with an important role in virus uptake. Its extracellular immunoglobulin domains mediate the binding to coxsackievirus and adenovirus as well as homophilic and heterophilic interactions between cells. The cytoplasmic tail

  9. A novel technology to target adenovirus vectors : application in cells involved in atherosclerosis

    NARCIS (Netherlands)

    Gras, Jan Cornelis Emile

    2007-01-01

    In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR re

  10. Negative Beta Encoder

    CERN Document Server

    Kohda, Tohru; Aihara, Kazuyuki

    2008-01-01

    A new class of analog-digital (A/D), digital-analog (D/A) converters as an alternative to conventional ones, called $\\beta$-encoder, has been shown to have exponential accuracy in the bit rates while possessing self-correction property for fluctuations of amplifier factor $\\beta$ and quantizer threshold $\

  11. DNA sequences encoding erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, F.K.

    1987-10-27

    A purified and isolated DNA sequence is described consisting essentially of a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of that of erythropoietin to allow possession of the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells, and to increase hemoglobin synthesis or iron uptake.

  12. Time-Encoded Imagers.

    Energy Technology Data Exchange (ETDEWEB)

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  13. HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a cancer targeting gene-viro-therapy strategy

    Directory of Open Access Journals (Sweden)

    Xu Hai-Neng

    2011-11-01

    Full Text Available Abstract Background In previously published studies, oncolytic adenovirus-mediated gene therapy has produced good results in targeting cancer cells. However, safety and efficacy, the two most important aspects in cancer therapy, remain serious challenges. The specific expression or deletion of replication related genes in an adenovirus has been frequently utilized to regulate the cancer cell specificity of a virus. Accordingly, in this study, we deleted 24 bp in E1A (bp924-bp947 and the entirety of E1B, including those genes encoding E1B 55kDa and E1B19kDa. We used the survivin promoter (SP to control E1A in order to construct a new adenovirus vector named Ad.SP.E1A(Δ24.ΔE1B (briefly Ad.SPDD. HCCS1 (hepatocellular carcinoma suppressor 1 is a novel tumor suppressor gene that is able to specifically induce apoptosis in cancer cells. The expression cassette AFP-HCCS1-WPRE-SV40 was inserted into Ad.SPDD to form Ad.SPDD-HCCS1, enabling us to improve the safety and efficacy of oncolytic-mediated gene therapy for liver cancer. Results Ad.SPDD showed a decreased viral yield and less toxicity in normal cells but enhanced toxicity in liver cancer cells, compared with the cancer-specific adenovirus ZD55 (E1B55K deletion. Ad.SPDD-HCCS1 exhibited a potent anti-liver-cancer ability and decreased toxicity in vitro. Ad.SPDD-HCCS1 also showed a measurable capacity to inhibit Huh-7 xenograft tumor growth on nude mice. The underlying mechanism of Ad.SPDD-HCCS1-induced liver cancer cell death was found to be via the mitochondrial apoptosis pathway. Conclusions These results demonstrate that Ad.SPDD-HCCS1 was able to elicit reduced toxicity and enhanced efficacy both in vitro and in vivo compared to a previously constructed oncolytic adenovirus. Ad.SPDD-HCCS1 could be a promising candidate for liver cancer therapy.

  14. Novel adenovirus detected in kowari (Dasyuroides byrnei) with pneumonia.

    Science.gov (United States)

    Gál, János; Mándoki, Míra; Sós, Endre; Kertész, Péter; Koroknai, Viktória; Bányai, Krisztián; Farkas, Szilvia L

    2017-02-15

    A male kowari (Dasyuroides byrnei) originating from a zoo facility was delivered for post mortem evaluation in Hungary. Acute lobar pneumonia with histopathologic changes resembling an adenovirus (AdV) infection was detected by light microscopic examination. The presence of an AdV was confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Although the exact taxonomic position of this novel marsupial origin virus could not be determined, pairwise identity analyses and phylogenetic calculations revealed that it is distantly related to other members in the family Adenoviridae.

  15. Estramustine phosphate reversibly inhibits an early stage during adenovirus replication.

    Science.gov (United States)

    Everitt, E; Ekstrand, H; Boberg, B; Hartley-Asp, B

    1990-01-01

    Estramustine phosphate, an estradiol-mustard conjugate, was shown to reversibly inhibit a stage during the first hour of productive adenovirus 2 infection of HeLa cells. This drug, employed in the therapy of advanced prostatic cancer, specifically interacts with microtubule-associated proteins (MAPs) of the cytoskeleton. The results obtained under physiological conditions in vivo suggest a MAPs-interference with the microtubule-mediated vectorial migration of the virus inoculum to the nucleus. Virus attachment, uncoating kinetics and the appearance of established uncoating intermediates were not affected.

  16. Three-Dimensional Structure of Canine Adenovirus Serotype 2 Capsid▿

    OpenAIRE

    Schoehn, Guy; El Bakkouri, Majida; Fabry, Céline M. S.; Billet, Oliver; Leandro F. Estrozi; Le, Van Long; Curiel, David T.; Kajava, Andrey V; Ruigrok, Rob W. H.; Eric J Kremer

    2008-01-01

    There are more than 100 known adenovirus (AdV) serotypes, including 50 human serotypes. Because AdV-induced disease is relatively species specific, vectors derived from nonhuman serotypes may have wider clinical potential based, in part, on the lack of ubiquitous memory immunity. Whereas a few of the human serotype capsids have been studied at the structural level, none of the nonhuman serotypes has been analyzed. The basis laid by the analysis of human AdV (hAdV) has allowed us to determine ...

  17. Suppression of experimental osteoarthritis by adenovirus-mediated double gene transfer

    Institute of Scientific and Technical Information of China (English)

    WANG Hai-jun; YU Chang-long; Kishi Hiroyuki; Motoki Kazumi; MAO Ze-bin; Muraguchi Atsushi

    2006-01-01

    Background Osteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor- α (TNF- α ) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-α receptor type Ⅰ (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis. Methods Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligment plus medial meniscectomy. Five days after surgery, approximately 1×108 plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1×108 pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1×108 pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue

  18. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, Eric A. [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Camacho, Zenaido T. [Department of Cell Biology, Department of Natural Sciences, Western New Mexico University, Silver City, NM 88062 (United States); Hillestad, Matthew L. [Nephrology Training Program, Mayo Clinic, Rochester, MN 55902 (United States); Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J. [Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN 55902 (United States); Mercier, George T. [Department of Physics, University of Houston, Houston, TX 77004 (United States); Barry, Michael A., E-mail: mab@mayo.edu [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Department of Immunology and Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55902 (United States)

    2015-08-15

    We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5.

  19. A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla).

    Science.gov (United States)

    Wevers, Diana; Leendertz, Fabian H; Scuda, Nelly; Boesch, Christophe; Robbins, Martha M; Head, Josephine; Ludwig, Carsten; Kühn, Joachim; Ehlers, Bernhard

    2010-11-05

    Adenoviruses (AdV) broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL), preterminal protein (pTP) and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2-10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B). Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

  20. A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla

    Directory of Open Access Journals (Sweden)

    Ludwig Carsten

    2010-11-01

    Full Text Available Abstract Adenoviruses (AdV broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL, preterminal protein (pTP and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2 - 10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B. Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

  1. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    Science.gov (United States)

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam

    2016-01-01

    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  2. Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

    Directory of Open Access Journals (Sweden)

    Anton V Borovjagin

    Full Text Available To explore gene therapy strategies for amelogenesis imperfecta (AI, a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5 vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3 fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(vβ3/α(vβ5 integrins and heparan sulfate proteoglycans (HSPGs highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

  3. Fluctuating expression of microRNAs in adenovirus infected cells.

    Science.gov (United States)

    Zhao, Hongxing; Chen, Maoshan; Tellgren-Roth, Christian; Pettersson, Ulf

    2015-04-01

    The changes in cellular microRNA (miRNA) expression during the course of an adenovirus type 2 infection in human lung fibroblast were studied by deep RNA sequencing. Expressions of 175 miRNAs with over 100 transcripts per million nucleotides were changed more than 1.5-fold. The expression patterns of these miRNAs changed dramatically during the course of the infection, from upregulation of the miRNAs known as tumor suppressors (such as miR-22, miR-320, let-7, miR-181b, and miR-155) and down-regulation of oncogenic miRNAs (such as miR-21 and miR-31) early to downregulation of tumor suppressor miRNAs (such as let-7 family, mir-30 family, 23/27 cluster) and upregulation of oncogenic miRNAs (include miR-125, miR-27, miR-191) late after infection. The switch in miRNA expression pattern occurred when adenovirus DNA replication started. Furthermore, deregulation of cellular miRNA expression was a step-wise and special sets of miRNAs were deregulated in different phases of infection.

  4. Genetic Characterization of Fowl Adenovirus Strains Isolated from Poultry in China.

    Science.gov (United States)

    Zhang, Hewei; Jin, Wenjie; Ding, Ke; Cheng, Xiangchao; Sun, Yaru; Wang, Jianke; Cheng, Shipeng; Wu, Hua; Zhang, Chunjie

    2017-09-01

    Fowl adenoviruses (FAdVs) infect chickens worldwide, resulting in global economic losses in the poultry industry. We examined the strains present in chickens in regions of China where infections are particularly prevalent. Fifteen FAdV strains were successfully isolated in the field. The L1 loop region of the hexon gene was sequenced to genetically identify the FAdV isolates. By comparing these sequences to adenovirus reference strain sequences using phylogenetics, 15 adenovirus strains were found to cluster into two distinct species. One cluster containing 12 strains belonged to the fowl adenoviruses C species and serotyped as FAdV-4. The other cluster containing three strains belonged to the fowl adenoviruses E species and serotyped as FAdV-10. To our knowledge, this is the first report of the existence of fowl adenoviruses E in China. Furthermore, at least two types of fowl adenovirus strains are predominant among poultry in China. Cumulatively, this study helps lays the groundwork for future research on the pathogenicity and potential treatment measures for FAdV infections in chickens.

  5. Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region.

    Science.gov (United States)

    Ono, Hidetaka A; Le, Long P; Davydova, Julia G; Gavrikova, Tatyana; Yamamoto, Masato

    2005-11-15

    To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.

  6. IMPROVEMENT OF HUMAN ISLET FUNCTION BY ADENOVIRUS MEDIATED HO-1 GENE TRANSFER

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.

  7. Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells.

    Science.gov (United States)

    Maeda, Mitsuko; Kida, Shinya; Hojo, Keiko; Eto, Yusuke; Gaob, Jian-Qing; Kurachi, Shinnosuke; Sekiguchi, Fumiko; Mizuguchi, Hiroyuki; Hayakawa, Takao; Mayumi, Tadanori; Nakagawa, Shinsaku; Kawasaki, Koichi

    2005-02-01

    The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.

  8. Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

    Directory of Open Access Journals (Sweden)

    Gastaldelli Michele

    2009-10-01

    Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

  9. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    Science.gov (United States)

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

  10. Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells.

    Science.gov (United States)

    Wang, Haili; Kroeber, Markus; Hanke, Michael; Ries, Rainer; Schmid, Carsten; Poller, Wolfgang; Richter, Wiltrud

    2004-02-01

    To develop new therapeutic options for the treatment of disc degeneration we tested the possibility of overexpression of active growth and differentiation factor (GDF) 5 and of transforming growth factor (TGF) beta(1) by adenoviral gene transfer and characterized its effect on cell proliferation and matrix synthesis of cultured rabbit and human intervertebral disc cells. Recombinant adenovirus encoding for GDF-5 or TGF-beta(1) was developed and transgene expression characterized by RT-PCR, western blot and ELISA. Growth and matrix synthesis of transduced cells was measured by [(3)H]thymidine or [(35)S]sulfate incorporation. Disc cells expressed the receptors BMPR1A, BMPR1B, and BMPR2, which are relevant for GDF-5 action. Adenovirus efficiently transferred the GDF-5 gene or the TGF-beta(1) gene to rabbit and human intervertebral disc cells. About 50 ng GDF-5 protein/10(6 )cells per 24 h or 7 ng TGF-beta(1) protein/10(6 )cells per 24 h was produced. According to western blotting, two GDF-5 forms, with molecular weights consistent with the activated GDF-5 dimer and the proform, were secreted over the 3 weeks following gene transfer. Overexpressed GDF-5 and TGF-beta(1) were bioactive and promoted growth of rabbit disc cells in monolayer culture. Our results suggest that ex vivo gene delivery of GDF-5 and TGF-beta(1) is an attractive approach for the release of mature and pre-GDF-5 in surrounding tissue. This leads us to hope that it will prove possible to improve the treatment of degenerative disc disease by means of ex vivo gene transfer of single or multiple growth factors.

  11. Molecular characterization of adenovirus circulating in Central and South America during the 2006–2008 period

    Science.gov (United States)

    García, Josefina; Sovero, Merly; Laguna‐Torres, Victor Alberto; Gomez, Jorge; Chicaiza, Wilson; Barrantes, Melvin; Sanchez, Felix; Jimenez, Mirna; Comach, Guillermo; De Rivera, Ivette L.; Agudo, Roberto; Arango, Ana E.; Barboza, Alma; Aguayo, Nicolas; Kochel, Tadeusz J.

    2009-01-01

    Background  Human Adenoviruses are recognized pathogens, causing a broad spectrum of diseases. Serotype identification is critical for epidemiological surveillance, detection of new strains and understanding of HAdvs pathogenesis. Little data is available about HAdvs subtypes in Latin America. Methods  In this study, we have molecularly characterized 213 adenoviruses collected from ILI presenting patients, during 2006‐08, in Central and South America. Results  Our results indicate that 161(76%) adenoviruses belong to subgroup C, 45 (21%) to subgroup B and 7 (3%) to subtype E4. PMID:19903214

  12. Spectrally encoded confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tearney, G.J.; Webb, R.H.; Bouma, B.E. [Wellman Laboratories of Photomedicine, Massachusetts General Hospital, 50 Blossom Street, BAR 703, Boston, Massachusetts 02114 (United States)

    1998-08-01

    An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique. {copyright} {ital 1998} {ital Optical Society of America}

  13. Immune responses against hepatitis C virus genotype 3a virus-like particles in mice: A novel VLP prime-adenovirus boost strategy.

    Science.gov (United States)

    Kumar, Anuj; Das, Soma; Mullick, Ranajoy; Lahiri, Priyanka; Tatineni, Ranjitha; Goswami, Debashree; Bhat, Prasanna; Torresi, Joseph; Gowans, Eric James; Karande, Anjali Anoop; Das, Saumitra

    2016-02-17

    Chronic hepatitis C virus (HCV) infection represents a major health threat to global population. In India, approximately 15-20% of cases of chronic liver diseases are caused by HCV infection. Although, new drug treatments hold great promise for HCV eradication in infected individuals, the treatments are highly expensive. A vaccine for preventing or treating HCV infection would be of great value, particularly in developing countries. Several preclinical trials of virus-like particle (VLP) based vaccine strategies are in progress throughout the world. Previously, using baculovirus based system, we have reported the production of hepatitis C virus-like particles (HCV-LPs) encoding structural proteins for genotype 3a, which is prevalent in India. In the present study, we have generated HCV-LPs using adenovirus based system and tried different immunization strategies by using combinations of both kinds of HCV-LPs with other genotype 3a-based immunogens. HCV-LPs and peptides based ELISAs were used to evaluate antibody responses generated by these combinations. Cell-mediated immune responses were measured by using T-cell proliferation assay and intracellular cytokine staining. We observed that administration of recombinant adenoviruses expressing HCV structural proteins as final booster enhances both antibody as well as T-cell responses. Additionally, reduction of binding of VLP and JFH1 virus to human hepatocellular carcinoma cells demonstrated the presence of neutralizing antibodies in immunized sera. Taken together, our results suggest that the combined regimen of VLP followed by recombinant adenovirus could more effectively inhibit HCV infection, endorsing the novel vaccine strategy.

  14. Detection of enteric Adenoviruses in South-African waters using gene probes

    CSIR Research Space (South Africa)

    Genthe, Bettina

    1995-01-01

    Full Text Available Gene probes developed locally for both enteric Adenoviruses 40 and 41 were used to determine whether these viruses were present in both raw and treated waters. Approximately sixty water samples were concentrated by ultra filtration and analysed...

  15. Coliphage and adenovirus concentrations at various points along the net-zero system

    Data.gov (United States)

    U.S. Environmental Protection Agency — Coliphage and adenovirus concentrations per liter. This dataset is associated with the following publication: Gassie, L., J. Englehardt, J. Wang, N. Brinkman, J....

  16. Liposomal enhancement of the immunogenicity of adenovirus type 5 hexon and fiber vaccines.

    Science.gov (United States)

    Kramp, W J; Six, H R; Drake, S; Kasel, J A

    1979-01-01

    Immunogenicity of adenovirus capsid proteins carried in liposomes was comparable to that with equivalent doses administered in Freund adjuvant, and both forms were more potent than aqueous vaccines. PMID:489132

  17. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

    Science.gov (United States)

    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S

    2016-01-01

    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  18. Role of coxsackievirus and adenovirus receptor in the pathogenesis of dilated cardiomyopathy and its influencing factor

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shuo; JIA Hai-bo; GONG Bin-sheng; ZHANG Shao-jun; LI Xia; YU Bo

    2008-01-01

    Background Although clinical treatment for heart failure and sudden death has been improved over the last few decades, the morbidity and mortality of dilated cardiomyopathy (DCM) have increased. So a better understanding of the underlying molecular events leading to DCM is urgent. Persistent viral infection (especially coxsackievirus group B) of the myocardium in viral myocarditis and DCM has never been neglected by experts. Recent data indicate that the up-regulation of coxsackievirus and adenovirus receptor (CAR) in viral cardiomyopathy contributes to viral infection as a key factor in the pathogenesis of this disease. This study aimed to investigate the role and regulatory mechanism of CAR in DCM by the bioinformatic method.Methods We identified the clusters of genes co-expressed with CAR by clustering algorithm based on the public available microarray dataset of DCM (Kittleson, et al. 2005), and mapped these genes into the protein-protein interaction networks to investigate the interaction relationship to each other at the protein level after confirming that the samples are characterized by the cluster of genes in correctly partitioning.Results The gene cluster GENESET 11 containing 33 genes including CAR with similar expression pattem was identified by cluster algorithm, of which 19 genes were found to have interaction information of the protein encoded by them in the current human protein interaction database. Especially, 12 genes present as critical nodes (called HUB node) at the protein level are involved in energy metabolism, signal transduction, viral infection, immuno-response, cell apoptosis, cell proliferation, tissue repair, etc.Conclusions The genes in GENESET 11 together with CAR may play a pathogenic role in the development of DCM, mainly involved in the mechanism of energy metabolism, signal transduction, viral infection, immuno-response, cell apoptosis and tissue repair.

  19. Adenovirus-mediated shRNA interference against HSV-1 replication in vitro.

    Science.gov (United States)

    Song, Bo; Liu, Xinjing; Wang, Qingzhi; Zhang, Rui; Yang, Ting; Han, Zhiqiang; Xu, Yuming

    2016-12-01

    The UL29 and UL28 proteins encoded by herpes simplex virus type 1 (HSV-1) are critical for its replication and packaging, respectively. Research has demonstrated that synthesized siRNA molecules targeting the UL29 gene are able to suppress HSV-2 replication and the UL28-null HSV-1 gene cannot form infectious viruses in vitro. Silencing the UL28 and UL29 genes by RNAi might lead to the development of novel antiviral agents for the treatment of HSV-1 infections. Two kinds of short hairpin RNAs (shRNAs) targeting the UL29 and UL28 genes were chemically synthesized and then delivered into cells by a replication-defective human adenovirus type 5 (Adv5) vector. (-) shRNAs targeting none of the genome of HSV-1 were used as the control. Vero cells were inoculated with Ad-UL28shRNA or Ad-UL29shRNA at a multiplicity of infection (MOI) of 100 and challenged 24 h later with HSV-1 at an MOI of 0.01 to inhibit HSV-1 replication, as measured by the level of the corresponding RNA and proteins. In addition, the amount of progeny virus was assessed at daily intervals. The antiviral effects of Ad-shRNAs at ongoing HSV-1 infection were explored at 12 h after inoculation of the HSV-1. The results showed that the shRNAs delivered by Adv5 significantly suppressed HSV-1 replication in vitro, as determined by the levels of viral RNA transcription, viral protein synthesis, and viral production. The Ad-UL28shRNA and Ad-UL29shRNA suppressed the replication of HSV-1, respectively, compared with the control group (P HSV-1 infection (P HSV-1 infection.

  20. Random sampling of the Central European bat fauna reveals the existence of numerous hitherto unknown adenoviruses.

    Science.gov (United States)

    Vidovszky, Márton; Kohl, Claudia; Boldogh, Sándor; Görföl, Tamás; Wibbelt, Gudrun; Kurth, Andreas; Harrach, Balázs

    2015-12-01

    From over 1250 extant species of the order Chiroptera, 25 and 28 are known to occur in Germany and Hungary, respectively. Close to 350 samples originating from 28 bat species (17 from Germany, 27 from Hungary) were screened for the presence of adenoviruses (AdVs) using a nested PCR that targets the DNA polymerase gene of AdVs. An additional PCR was designed and applied to amplify a fragment from the gene encoding the IVa2 protein of mastadenoviruses. All German samples originated from organs of bats found moribund or dead. The Hungarian samples were excrements collected from colonies of known bat species, throat or rectal swab samples, taken from live individuals that had been captured for faunistic surveys and migration studies, as well as internal organs of dead specimens. Overall, 51 samples (14.73%) were found positive. We detected 28 seemingly novel and six previously described bat AdVs by sequencing the PCR products. The positivity rate was the highest among the guano samples of bat colonies. In phylogeny reconstructions, the AdVs detected in bats clustered roughly, but not perfectly, according to the hosts' families (Vespertilionidae, Rhinolophidae, Hipposideridae, Phyllostomidae and Pteropodidae). In a few cases, identical sequences were derived from animals of closely related species. On the other hand, some bat species proved to harbour more than one type of AdV. The high prevalence of infection and the large number of chiropteran species worldwide make us hypothesise that hundreds of different yet unknown AdV types might circulate in bats.

  1. Adenovirus Vector-Derived VA-RNA-Mediated Innate Immune Responses

    Directory of Open Access Journals (Sweden)

    Hiroyuki Mizuguchi

    2011-07-01

    Full Text Available The major limitation of the clinical use of replication-incompetent adenovirus (Ad vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN, following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs. In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88 and toll-like receptor 9 (TLR9 play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs, which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs.

  2. [Is there a risk of zoonotic disease due to adenoviruses?].

    Science.gov (United States)

    Loustalot, Fabien; Creyssels, Sophie; Salinas, Sara; Benkõ, Mária; Harrach, Balázs; Mennechet, Franck J D; Kremer, Eric J

    2015-12-01

    Every year brings another round of zoonotic viral infections. Usually they fall under the radar, but the occasional lethal epidemic brings another scare to the public and new urgency to the medical community. The types of these viruses (DNA vs. RNA genomes, enveloped vs. proteinaceous) as well as the preceding host(s) vary. Over the last 20 years, bats have been identified as an enigmatic carrier for several pathogens that have jumped the species barrier and infected humans. Factors that favour the emergence of zoonotic pathogens include the increasing overlap of the human and animal habitats, cultural activities, and the host reservoir. In this context, we asked whether bat and/or nonhuman primate adenoviruses are a risk for human health.

  3. Going viral: a review of replication-selective oncolytic adenoviruses.

    Science.gov (United States)

    Larson, Christopher; Oronsky, Bryan; Scicinski, Jan; Fanger, Gary R; Stirn, Meaghan; Oronsky, Arnold; Reid, Tony R

    2015-08-21

    Oncolytic viruses have had a tumultuous course, from the initial anecdotal reports of patients having antineoplastic effects after natural viral infections a century ago to the development of current cutting-edge therapies in clinical trials. Adenoviruses have long been the workhorse of virotherapy, and we review both the scientific and the not-so-scientific forces that have shaped the development of these therapeutics from wild-type viral pathogens, turning an old foe into a new friend. After a brief review of the mechanics of viral replication and how it has been modified to engineer tumor selectivity, we give particular attention to ONYX-015, the forerunner of virotherapy with extensive clinical testing that pioneered the field. The findings from those as well as other oncolytic trials have shaped how we now view these viruses, which our immune system has evolved to vigorously attack, as promising immunotherapy agents.

  4. Adenovirus-mediated gene transfer to tumor cells.

    Science.gov (United States)

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting.

  5. Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

    Institute of Scientific and Technical Information of China (English)

    CHEN Qingyong; WANG Chunyou; CHEN Daoda; CHEN Jianying; JIANG Chunfang; ZHENG Hai

    2006-01-01

    The recombinant defective adenovirus vector carrying human PTEN tumor suppres sor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector AdPTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×1010 pfu/mL, and about 70 % breast cancer cells were infected with Ad PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

  6. Inclusion body hepatitis (IBH) outbreak associated with fowl adenovirus type 8b in broilers

    OpenAIRE

    2013-01-01

    The causative agent of inclusion body hepatitis (IBH) was identified as fowl adenovirus (FAdV) type 8b, a member of the Fowl adenovirus E species, based on PCR results of adenoviral polymerase and the hexon gene in an outbreak of acute mortality that affected a broiler flock of 12,000 animals. In two waves of elevated mortality rate, a total of 264 chickens were found dead. Affected birds showed ruffled feathers, depression, watery droppings and limping. Th...

  7. Frequency of Adenoviruses, Rotaviruses and Noroviruses Among Diarrhea Samples Collected From Infants of Zabol, Southeastern Iran

    OpenAIRE

    Sharifi-Rad, Javad; Hoseini Alfatemi, Seyedeh Mahsan; Sharifi-Rad, Mehdi; Miri, Abdolhossein

    2015-01-01

    Background: Viruses are one of the major reasons of gastrointestinal disease worldwide, and commonly infect children less than five years of age in developing countries. Objectives: The current study aimed to determine the frequency of adenoviruses, rotaviruses and noroviruses among diarrhea samples collected from infants of Zabol, south-east of Iran. This study is the first investigation of adenoviruses, rotaviruses and noroviruses among diarrhea samples in Zabol. Patients and Methods: In th...

  8. Repression of insulin gene expression by adenovirus type 5 E1a proteins.

    OpenAIRE

    1987-01-01

    Insulin gene transcription relies on enhancer and promoter elements which are active in pancreatic beta cells. We showed that adenovirus type 5 infection of HIT T-15 cells, a transformed hamster beta cell line, represses insulin gene transcription and mRNA levels. Using expression plasmids transiently introduced into HIT T-15 cells, we showed that adenovirus type 5 E1a transcription regulatory proteins repress insulin enhancer-promoter element activity as assayed with a surrogate xanthine-gua...

  9. Ganciclovir inhibits human adenovirus replication and pathogenicity in permissive immunosuppressed Syrian hamsters.

    Science.gov (United States)

    Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R Mark L; Toth, Karoly; Wold, William S M

    2014-12-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

  10. Crystal structure of human adenovirus at 3.5 Å resolution*

    OpenAIRE

    Reddy, Vijay S.; Natchiar, S. Kundhavai; Phoebe L Stewart; Nemerow, Glen R.

    2010-01-01

    Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here we report the X-ray structure at 3.5 Å resolution of the 150 megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber ...

  11. Comparison of adenovirus viruria in bone marrow transplant patients before and after transplantation

    OpenAIRE

    Saderi, H; Owlia, P.; K.A. Moghadam; B Bahar; S Faghih Zadeh

    2005-01-01

    Baekgrouund & purpose: In recent years, the role of adenoviruses in infection and disease in recipients of bone marrow transplantation (BMT) has been studied. It suppose that adenoviral infections are prevalant in these patients Due to using medicines for preventing transplant rejection. This study was performed to compare the incidence of adenoviruses in urine samples taken before and after BMT from individuals undergoing BMT. In addition, The correlation between age, sex, etiology and kind...

  12. Quantitative detection of human adenoviruses in wastewater and combined sewer overflows influencing a Michigan river.

    Science.gov (United States)

    Fong, Theng-Theng; Phanikumar, Mantha S; Xagoraraki, Irene; Rose, Joan B

    2010-02-01

    Enteric viruses are important pathogens found in contaminated surface waters and have previously been detected in waters of the Great Lakes. Human adenoviruses were monitored because of their high prevalence and persistence in aquatic environments. In this study, we quantified adenoviruses in wastewater, surface water, and combined sewer overflows (CSOs) by real-time PCR. Between August 2005 and August 2006, adenovirus concentrations in raw sewage, primary-treated effluent, secondary-treated effluent, and chlorinated effluent from a wastewater treatment plant in Michigan were examined. CSO samples (n = 6) were collected from a CSO retention basin in Grand Rapids, MI. Adenoviruses were detected in 100% of wastewater and CSO discharge samples. Average adenovirus DNA concentrations in sewage and CSOs were 1.15 x 10(6) viruses/liter and 5.35 x 10(5) viruses/liter, respectively. Adenovirus removal was <2 log(10) (99%) at the wastewater treatment plant. Adenovirus type 41 (60% of clones), type 12 (29%), type 40 (3%), type 2 (3%), and type 3 (3%) were isolated from raw sewage and primary effluents (n = 28). Six of 20 surface water samples from recreational parks at the lower Grand River showed virus concentrations above the real-time PCR detection limit (average, 7.8 x 10(3) viruses/liter). This research demonstrates that wastewater effluents and wastewater-impacted surface waters in the lower Grand River in Michigan contain high levels of viruses and may not be suitable for full-body recreational activities. High concentrations of adenovirus in these waters may be due to inefficient removal during wastewater treatment and to the high persistence of these viruses in the environment.

  13. Prevention of Adenovirus Infection with Recombinant Interferon 2b Medication in Preschool Institutions

    Directory of Open Access Journals (Sweden)

    L. V. Osidak

    2016-01-01

    Full Text Available This article presents the results of experimental (on cell cultures and clinical (in children’s groups studies of recombinant interferon alpha-2b medication (Grippferon. Our aim was to examine the virus-inhibitory activity of this medication (against adenovirus and its preventive effect (on causative agents of ARVI, which allows us to use this medication as a preventive measure against adenovirus (as well as any other infection in children’s groups.

  14. Use of cidofovir in pediatric patients with adenovirus infection

    Science.gov (United States)

    Ganapathi, Lakshmi; Arnold, Alana; Jones, Sarah; Patterson, Al; Graham, Dionne; Harper, Marvin; Levy, Ofer

    2016-01-01

    Background: Adenoviruses contribute to morbidity and mortality among immunocompromised pediatric patients including stem cell and solid organ transplant recipients. Cidofovir (CDV), an antiviral compound approved by the FDA in 1996, is used for treatment of adenoviral (ADV) infections in immunocompromised patients despite concern of potential nephrotoxicity.   Methods: We conducted a retrospective 5-year review at Boston Children’s Hospital of 16 patients (mean age = 6.5 years) receiving 19 courses of CDV. During therapy all pertinent data elements were reviewed to characterize potential response to therapy and incidence of renal dysfunction.   Results: Of the 19 CDV courses prescribed, 16 courses (84%) were in patients who had a positive blood ADV Polymerase chain reaction (PCR) alone or in combination with positive ADV PCR/ Direct Immunofluorescence Assay (DFA) at another site. Respiratory symptoms with or without pneumonia were the most common presentation (10/19, 53%). In the majority of blood positive courses (10/16, 63%), viral clearance was also accompanied by clinical response. This was not the case in four courses where patients expired despite viral clearance, including one in which death was directly attributable to adenovirus. There was reversible renal dysfunction observed during the use of CDV. Conclusions:  CDV appeared safe and reasonably tolerated for treatment of ADV in this pediatric population and was associated with viral response and clinical improvement in the majority of patients but reversible renal dysfunction was a side effect. Further studies of the efficacy of CDV for immunocompromised children with ADV infection are warranted. PMID:27239277

  15. Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component.

    Directory of Open Access Journals (Sweden)

    Cindy Tamminga

    Full Text Available BACKGROUND: A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge. METHODOLOGY/PRINCIPAL FINDINGS: NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP. It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1 that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al. Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at 1 x 1010 particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m] that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m. CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected. SIGNIFICANCE: The NMRC-MV-Ad-PfC vaccine expressing CSP was

  16. Intratracheal administration of recombinant adenovirus containing IL-18 gene in treatment of experimental metastatic lung carcinoma

    Institute of Scientific and Technical Information of China (English)

    CHEN Ji-quan; GAO Xue-tao; XIU Qing-yu; YU Yi-zhi; LUO Wen-tong

    2001-01-01

    To study the treatment of experimental metastatic lung carcinoma by intratracheal injection of IL-18 gene recombinant adenovirus. Methods: (1)The mouse IL-18 mRNA was detected by RT-PCR, and the concentration of IL-18 and associated cytokines in lung lavages and blood were determined by ELISA at different time points after intratracheal injection of IL-18 recombinant adenovirus. (2)The lung metastasis nodes, mouse survival periods and survival rates were evaluated. NK activity and CTL activity were determined by 51Cr 4 h release method. Results: (1)IL-18 mRNA was detectable in lung tissue 6 h after intratracheal use of IL-18 recombinant adenovirus, and the concentration of IL-18 in lung lavage was higher than that in pelipheral blood. Neither IL-18 mRNA nor IL-18 was detectable in control group. (2) Intratracheal use of IL-18 recombinant adenovirus resulted in increased CTL and NK activity, longer survival time and higher survival rates compared with the control group, showing significant therapeutic effect on experimental lung metastasis. Conclusion: Intratracheal use of adenovirus vector containing IL-18 gene has therapeutic effect on the lung metastasis, denoting that gene therapy of lung diseases could be applied through airway directly with recombinant adenovirus.

  17. Replication-competent human adenovirus 11p vectors can propagate in Vero cells

    Energy Technology Data Exchange (ETDEWEB)

    Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

    2016-08-15

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

  18. A molecular epidemiology survey of respiratory adenoviruses circulating in children residing in Southern Palestine.

    Directory of Open Access Journals (Sweden)

    Lina Qurei

    Full Text Available A molecular epidemiology survey was performed in order to establish and document the respiratory adenovirus pathogen profiles among children in Southern Palestine. Three hundred and thirty-eight hospitalized pediatric cases with adenovirus-associated respiratory tract infections were analyzed. Forty four cases out of the 338 were evaluated in more detail for the adenoviruses types present. All of the children resided in Southern Palestine, that is, in city, village and refugee camp environments within the districts of Hebron and Bethlehem. Human adenoviruses circulated throughout 2005-2010, with major outbreaks occurring in the spring months. A larger percent of the children diagnosed with adenoviral infections were male infants. DNA sequence analysis of the hexon genes from 44 samples revealed that several distinct adenovirus types circulated in the region; these were HAdV-C1, HAdV-C2, HAdV-B3 and HAdV-C5. However, not all of these types were detected within each year. This is the first study ever conducted in Palestine of the genetic epidemiology of respiratory adenovirus infections.

  19. Long-term gene therapy in the CNS: reversal of hypothalamic diabetes insipidus in the Brattleboro rat by using an adenovirus expressing arginine vasopressin.

    Science.gov (United States)

    Geddes, B J; Harding, T C; Lightman, S L; Uney, J B

    1997-12-01

    The ability of adenovirus (Ad) to transfect most cell types efficiently has already resulted in human gene therapy trials involving the systemic administration of adenoviral constructs. However, because of the complexity of brain function and the difficulty in noninvasively monitoring alterations in neuronal gene expression, the potential of Ad gene therapy strategies for treating disorders of the CNS has been difficult to assess. In the present study, we have used an Ad encoding the arginine vasopressin cDNA (AdAVP) in an AVP-deficient animal model of diabetes insipidus (the Brattleboro rat), which allowed us to monitor chronically the success of the gene therapy treatment by noninvasive assays. Injection of AdAVP into the supraoptic nuclei (SON) of the hypothalamus resulted in expression of AVP in magnocellular neurons. This was accompanied by reduced daily water intake and urine volume, as well as increased urine osmolality lasting 4 months. These data show that a single gene defect leading to a neurological disorder can be corrected with an adenovirus-based strategy. This study highlights the potential of using Ad gene therapy for the long-term treatment of disorders of the CNS.

  20. The Coxsackievirus-Adenovirus Receptor Protein Can Function as a Cellular Attachment Protein for Adenovirus Serotypes from Subgroups A, C, D, E, and F

    OpenAIRE

    Roelvink, Peter W.; Lizonova, Alena; Lee, Jennifer G. M.; Li, Yuan; Bergelson, Jeffrey M.; Finberg, Robert W.; Douglas E Brough; Kovesdi, Imre; Wickham, Thomas J.

    1998-01-01

    Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein. To date, only the cellular fiber receptor for subgroup C serotypes 2 and 5, the so-called coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. Previous data suggested that the fiber of the subgroup D serotype Ad9 also recognizes CAR, since Ad9 and Ad2 fiber knobs cross-blocked each other’s cellular binding. Recombinant fiber knobs and 3H-labeled Ad virions from serotypes representi...

  1. Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase.

    Science.gov (United States)

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek; Dewhurst, Stephen

    2012-10-01

    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.

  2. Mucosal Immunization with Recombinant Adenovirus Encoding Soluble Globular Head of Hemagglutinin Protects Mice Against Lethal Influenza Virus Infection

    OpenAIRE

    Kim, Joo Young; Choi, Youngjoo; Nguyen, Huan H.; Song, Man Ki; Chang, Jun

    2013-01-01

    Influenza virus is one of the major sources of respiratory tract infection. Due to antigenic drift in surface glycoproteins the virus causes annual epidemics with severe morbidity and mortality. Although hemagglutinin (HA) is one of the highly variable surface glycoproteins of the influenza virus, it remains the most attractive target for vaccine development against seasonal influenza infection because antibodies generated against HA provide virus neutralization and subsequent protection agai...

  3. Immune Protection of Nonhuman Primates Against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

    Science.gov (United States)

    2006-06-01

    then fusing to TGA. The resulting plasmid also contained four extra amino acids (aa) at the C terminus ( MAAS ). HEK293 cells and the derivative T-Ag...Phil Gomez, Charla Andrews, Judy Stein, J. P. Todd, Vi Dang, Srinivas Rao, Sarah Norris, and Martha Nason for helpful discussions. Disclaimer: The

  4. Adenovirus Recruits Dynein by an Evolutionary Novel Mechanism Involving Direct Binding to pH-Primed Hexon

    Directory of Open Access Journals (Sweden)

    Julian Scherer

    2011-08-01

    Full Text Available Following receptor-mediated uptake into endocytic vesicles and escape from the endosome, adenovirus is transported by cytoplasmic dynein along microtubules to the perinuclear region of the cell. How motor proteins are recruited to viruses for their own use has begun to be investigated only recently. We review here the evidence for a role for dynein and other motor proteins in adenovirus infectivity. We also discuss the implications of recent studies on the mechanism of dynein recruitment to adenovirus for understanding the relationship between pathogenic and physiological cargo recruitment and for the evolutionary origins of dynein-mediated adenovirus transport.

  5. A myocarditis outbreak with fatal cases associated with adenovirus subgenera C among children from Havana City in 2005.

    Science.gov (United States)

    Savón, Clara; Acosta, Belsy; Valdés, Odalys; Goyenechea, Angel; Gonzalez, Grehete; Piñón, Alexander; Más, Pedro; Rosario, Delfina; Capó, Virginia; Kourí, Vivian; Martínez, Pedro A; Marchena, Juan J; González, Guelsys; Rodriguez, Hermis; Guzmán, María G

    2008-10-01

    Among multiple causes of acute myocarditis, viral infection, especially that due to enteroviruses and adenoviruses, is the leading cause. In the summer 2005 an outbreak of a febrile syndrome accompanied by acute cardiac decompensation occurred in infants and young children in Havana City. Eleven patients had a rapid evolution of disease and there were 8 fatalities from cardiac failure secondary to myocarditis. The aim of the present study was to determine the etiological agent responsible for this outbreak. Children admitted to the pediatric hospitals of Havana City from July 3 to August 2 with this clinical presentation were studied. Forty samples of necropsy tissue, cerebrospinal fluid, stools and serum were tested by molecular methods for 14 respiratory viruses, 6 herpesviruses and generic enteroviruses and flavirus and alfaviruses. Viral isolation was performed in A-549 cells. Isolated viruses were typed by sequence analysis. Adenovirus genome was detected in 6 of the 8 fatal cases-the lungs in 5 (63%) and the myocardium in 3 (37%). In two fatal cases, viral genome was detected in both lung and myocardium. Adenovirus was isolated in five fatal cases. In all three non-fatal cases, adenovirus genome was detected and adenovirus was isolated into two. Sequence analysis showed that adenovirus type 5 was the only isolate from fatal cases and adenovirus 1 the only isolate in non-fatal cases. No other viruses were found by PCR or isolation techniques. Adenovirus was the etiologic agent implicated in this myocarditis outbreak and adenovirus type 5 was associated with fatal outcome.

  6. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    Science.gov (United States)

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

  7. Selecting Operations for Assembler Encoding

    Directory of Open Access Journals (Sweden)

    Tomasz Praczyk

    2010-04-01

    Full Text Available Assembler Encoding is a neuro-evolutionary method in which a neural network is represented in the form of a simple program called Assembler Encoding Program. The task of the program is to create the so-called Network Definition Matrix which maintains all the information necessary to construct the network. To generate Assembler Encoding Programs and the subsequent neural networks evolutionary techniques are used.
    The performance of Assembler Encoding strongly depends on operations used in Assembler Encoding Programs. To select the most effective operations, experiments in the optimization and the predator-prey problem were carried out. In the experiments, Assembler Encoding Programs equipped with different types of operations were tested. The results of the tests are presented at the end of the paper.

  8. Construction and expression of SET gene and siRNA recombinant adenovirus vectors

    Institute of Scientific and Technical Information of China (English)

    Xu Bo-qun; Lu Pin-hong; Li Ying; Xue Kai; Li Mei; Ma Xiang; Diao Fei-yan; Cui Yu-gui; Liu Jia-yin

    2010-01-01

    Objective: To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA (SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels.Methods: The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction (RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET. The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells. The expression of SET in AD293 cells was detected by Western blot. In addition, we constructed SET gene SiRNA recombinant adenovirus vector (Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results: The recombinant adenovirus vectors, both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET, were proven to be constructed successfully by the evidence of endonulease digestion and sequencing. AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP. The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector. On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion: The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells. It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

  9. The Human Cytomegalovirus MHC Class I Homolog UL18 Inhibits LIR-1+ but Activates LIR-1− NK Cells1

    OpenAIRE

    Prod’Homme, Virginie; Griffin, Cora; Rebecca J. Aicheler; Wang, Eddie C.Y.; McSharry, Brian P.; Rickards, Carole R.; Stanton, Richard J; Borysiewicz, Leszek K.; López-Botet, Miguel; Wilkinson, Gavin W. G.; Tomasec, Peter

    2007-01-01

    The inhibitory leukocyte Ig-like receptor 1 (LIR-1, also known as ILT2, CD85j, or LILRB1) was identified by its high affinity for the human CMV (HCMV) MHC class I homolog gpUL18. The role of this LIR-1-gpUL18 interaction in modulating NK recognition during HCMV infection has previously not been clearly defined. In this study, LIR-1+ NKL cell-mediated cytotoxicity was shown to be inhibited by transduction of targets with a replication-deficient adenovirus vector encoding UL18 (RAd-UL18). Fibro...

  10. Molecular epidemiology and surveillance of circulating rotavirus and adenovirus in Congolese children with gastroenteritis.

    Science.gov (United States)

    Mayindou, Gontran; Ngokana, Berge; Sidibé, Anissa; Moundélé, Victoire; Koukouikila-Koussounda, Felix; Christevy Vouvoungui, Jeannhey; Kwedi Nolna, Sylvie; Velavan, Thirumalaisamy P; Ntoumi, Francine

    2016-04-01

    Infectious Diarrhea caused by rotavirus and adenovirus, is a leading cause of death in children in sub-Sahara Africa but there is limited published data on the diverse rotavirus genotypes and adenovirus serotypes circulating in the Republic of Congo. In this study, we investigated the prevalence of severe diarrhea caused by rotavirus A (RVA) and Adenovirus serotype 40 and 41 in Congolese children hospitalized with severe gastroenteritis. Stool samples were collected from 655 Congolese children less than 60 months of age hospitalized with acute gastroenteritis between June 2012 and June 2013. Rotavirus and adenovirus antigens were tested using commercially available ELISA kits and the RVA G- and P- genotypes were identified by seminested multiplex RT-PCR. Three hundred and four (46.4%) children were tested positive for RVA. Adenovirus infection was found in 5.5% of the 564 tested children. Rotavirus infection was frequently observed in children between 6-12 months (55.9%). The dry season months recorded increased RVA infection while no seasonality of adenovirus infection was demonstrated. The most common RVA genotypes were G1 (57.5%), G2 (6.4%), G1G2 mixture (15.5%), P[8] (58%), P[6] (13.2%), and P[8]P[6] mixture (26%). Additionally, the genotype G12P[6] was significantly associated with increased vomiting. This first study on Congolese children demonstrates a high prevalence and clinical significance of existing rotavirus genotypes. Adenovirus prevalence is similar to that of other Central African countries. This baseline epidemiology and molecular characterization study will contribute significantly to the RVA surveillance after vaccine implementation in the country.

  11. Transduction and apoptosis induction in the rat prostate, using adenovirus vectors.

    Science.gov (United States)

    Kirkman, W; Chen, P; Schroeder, R; Feneley, M R; Rodriguez, R; Wickham, T J; King, C R; Bruder, J T

    2001-08-10

    Proapoptotic adenovirus vectors offer great promise for the treatment of cancer and nonmalignant conditions. Benign prostate hyperplasia (BPH) is a common nonmalignant enlargement of the prostate that involves epithelial, stromal, and smooth muscle components of the gland. We tested the hypothesis that an adenovirus vector expressing Fas ligand can be used to induce apoptosis in the prostate. We analyzed the efficiency of transduction and apoptosis induction in primary cultures of human prostate cells after adenovirus-mediated gene transfer. Efficient transduction was observed in primary prostate epithelial cells. Stromal and smooth muscle cells were more difficult to transduce, as no coxsackie-adenovirus receptor (CAR) expression was detectable on these cells. However, transduction was achieved in these cells when the multiplicity of infection was increased to 100 focal-forming units per cell, or when the vectors were delivered as calcium phosphate precipitates. Infection of all three primary prostate cell types with an adenovirus vector that expresses Fas ligand (AdFasL/G) resulted in rapid apoptosis. Direct injection of the rat prostate with an adenovirus vector carrying luciferase resulted in substantial luciferase expression. TUNEL analysis demonstrated that AdFasL/G administration induced low-level apoptosis in prostatic epithelial cells throughout the gland. As a first step toward enhancing the efficiency of prostate transduction in vivo, we tested an adenovirus vector that was engineered to have an expanded tropism. This vector, AdZ.F2K(pK7), was 10- to 500-fold more efficient than unmodified vectors in transducing prostate epithelial, smooth muscle, and stromal cells in culture. Moreover, AdZ.F2K(pK7) was more efficient than an unmodified vector at transducing the rat prostate in vivo, although the effect was dose dependent.

  12. A replicating adenovirus capsid display recombinant elicits antibodies against Plasmodium falciparum sporozoites in Aotus nancymaae monkeys.

    Science.gov (United States)

    Karen, Kasey A; Deal, Cailin; Adams, Robert J; Nielsen, Carolyn; Ward, Cameron; Espinosa, Diego A; Xie, Jane; Zavala, Fidel; Ketner, Gary

    2015-01-01

    Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Fiber mediated receptor masking in non-infected bystander cells restricts adenovirus cell killing effect but promotes adenovirus host co-existence.

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    Johan Rebetz

    Full Text Available The basic concept of conditionally replicating adenoviruses (CRAD as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI, and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses.

  14. Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

    Institute of Scientific and Technical Information of China (English)

    哈小琴; 苑宾; 李元敏; 劳妙芬; 吴祖泽

    2003-01-01

    A complementary DNA (cDNA) encoding human hepatocyte growth factor wasintroduced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homologous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities tohuman hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was detected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF,the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that ofthe normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereasno therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were observed to different extent on healed wound treated with Ad-HGF. Histopathologic examination revealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed

  15. Transient detection of E1-containing adenovirus in saliva after the delivery of a first-generation adenoviral vector to human parotid gland†

    Science.gov (United States)

    Zheng, Changyu; Nikolov, Nikolay P.; Alevizos, Ilias; Cotrim, Ana P.; Liu, Shuying; McCullagh, Linda; Chiorini, John A.; Illei, Gabor G.; Baum, Bruce J.

    2017-01-01

    Background Radiation-induced salivary hypofunction is a common side-effect of treatment for head and neck cancers. Patients suffer significant morbidity and there is no suitable conventional therapy. We are conducting a Phase I clinical trial, using a first-generation serotype 5 adenoviral (Ad5) vector encoding human aquaporin-1 (AdhAQP1) to treat such patients. One week after the administration of AdhAQP1 to an enrolled, generally healthy patient, E1-containing adenovirus was detected in parotid saliva. Methods The real-time quantitative polymerase chain reactuion (PCR) was used to measure the Ad5 E1 gene and AdhAQP1 in saliva and serum. PCR and sequencing were used to characterize viral/vector DNA extracted from saliva. The presence of infectious adenovirus was assessed by the inoculation of A549 cells with aliquots of saliva. Serum Ad5 neutralizing antibodies were measured by the inhibition of 293-cell transduction with an Ad5 vector encoding luciferase. Multiple clinical evaluations were performed. Results On day 7 after AdhAQP1 delivery, low levels of the Ad5 E1 gene were detected in parotid saliva (82 copies/μl). In addition, significant levels of AdhAQP1 were also detected (1.5 × 103 copies/μl). The patient was asymptomatic and subsequent analysis of parotid saliva samples prior to day 7 and after day 7 until day 42 was negative for both virus and vector. No virus or vector was detected in serum at any time. Detailed PCR analyses of DNA extracted from the day 7 parotid saliva sample suggested the absence of a recombination event, and no infectious virus was found. Conclusions The patient most likely had a latent Ad5 infection in the targeted parotid gland that was activated after gene transfer and was without clinical consequence. Published in 2009 by John Wiley & Sons, Ltd. PMID:19941317

  16. Genetic and Molecular Epidemiological Characterization of a Novel Adenovirus in Antarctic Penguins Collected between 2008 and 2013.

    Science.gov (United States)

    Lee, Sook-Young; Kim, Jeong-Hoon; Seo, Tae-Kun; No, Jin Sun; Kim, Hankyeom; Kim, Won-Keun; Choi, Han-Gu; Kang, Sung-Ho; Song, Jin-Won

    2016-01-01

    Antarctica is considered a relatively uncontaminated region with regard to the infectious diseases because of its extreme environment, and isolated geography. For the genetic characterization and molecular epidemiology of the newly found penguin adenovirus in Antarctica, entire genome sequencing and annual survey of penguin adenovirus were conducted. The entire genome sequences of penguin adenoviruses were completed for two Chinstrap penguins (Pygoscelis antarctica) and two Gentoo penguins (Pygoscelis papua). The whole genome lengths and G+C content of penguin adenoviruses were found to be 24,630-24,662 bp and 35.5-35.6%, respectively. Notably, the presence of putative sialidase gene was not identified in penguin adenoviruses by Rapid Amplification of cDNA Ends (RACE-PCR) as well as consensus specific PCR. The penguin adenoviruses were demonstrated to be a new species within the genus Siadenovirus, with a distance of 29.9-39.3% (amino acid, 32.1-47.9%) in DNA polymerase gene, and showed the closest relationship with turkey adenovirus 3 (TAdV-3) in phylogenetic analysis. During the 2008-2013 study period, the penguin adenoviruses were annually detected in 22 of 78 penguins (28.2%), and the molecular epidemiological study of the penguin adenovirus indicates a predominant infection in Chinstrap penguin population (12/30, 40%). Interestingly, the genome of penguin adenovirus could be detected in several internal samples, except the lymph node and brain. In conclusion, an analysis of the entire adenoviral genomes from Antarctic penguins was conducted, and the penguin adenoviruses, containing unique genetic character, were identified as a new species within the genus Siadenovirus. Moreover, it was annually detected in Antarctic penguins, suggesting its circulation within the penguin population.

  17. Genetic and Molecular Epidemiological Characterization of a Novel Adenovirus in Antarctic Penguins Collected between 2008 and 2013

    Science.gov (United States)

    Lee, Sook-Young; Kim, Jeong-Hoon; Seo, Tae-Kun; No, Jin Sun; Kim, Hankyeom; Kim, Won-keun; Choi, Han-Gu; Kang, Sung-Ho; Song, Jin-Won

    2016-01-01

    Antarctica is considered a relatively uncontaminated region with regard to the infectious diseases because of its extreme environment, and isolated geography. For the genetic characterization and molecular epidemiology of the newly found penguin adenovirus in Antarctica, entire genome sequencing and annual survey of penguin adenovirus were conducted. The entire genome sequences of penguin adenoviruses were completed for two Chinstrap penguins (Pygoscelis antarctica) and two Gentoo penguins (Pygoscelis papua). The whole genome lengths and G+C content of penguin adenoviruses were found to be 24,630–24,662 bp and 35.5–35.6%, respectively. Notably, the presence of putative sialidase gene was not identified in penguin adenoviruses by Rapid Amplification of cDNA Ends (RACE-PCR) as well as consensus specific PCR. The penguin adenoviruses were demonstrated to be a new species within the genus Siadenovirus, with a distance of 29.9–39.3% (amino acid, 32.1–47.9%) in DNA polymerase gene, and showed the closest relationship with turkey adenovirus 3 (TAdV-3) in phylogenetic analysis. During the 2008–2013 study period, the penguin adenoviruses were annually detected in 22 of 78 penguins (28.2%), and the molecular epidemiological study of the penguin adenovirus indicates a predominant infection in Chinstrap penguin population (12/30, 40%). Interestingly, the genome of penguin adenovirus could be detected in several internal samples, except the lymph node and brain. In conclusion, an analysis of the entire adenoviral genomes from Antarctic penguins was conducted, and the penguin adenoviruses, containing unique genetic character, were identified as a new species within the genus Siadenovirus. Moreover, it was annually detected in Antarctic penguins, suggesting its circulation within the penguin population. PMID:27309961

  18. Genetic and Molecular Epidemiological Characterization of a Novel Adenovirus in Antarctic Penguins Collected between 2008 and 2013.

    Directory of Open Access Journals (Sweden)

    Sook-Young Lee

    Full Text Available Antarctica is considered a relatively uncontaminated region with regard to the infectious diseases because of its extreme environment, and isolated geography. For the genetic characterization and molecular epidemiology of the newly found penguin adenovirus in Antarctica, entire genome sequencing and annual survey of penguin adenovirus were conducted. The entire genome sequences of penguin adenoviruses were completed for two Chinstrap penguins (Pygoscelis antarctica and two Gentoo penguins (Pygoscelis papua. The whole genome lengths and G+C content of penguin adenoviruses were found to be 24,630-24,662 bp and 35.5-35.6%, respectively. Notably, the presence of putative sialidase gene was not identified in penguin adenoviruses by Rapid Amplification of cDNA Ends (RACE-PCR as well as consensus specific PCR. The penguin adenoviruses were demonstrated to be a new species within the genus Siadenovirus, with a distance of 29.9-39.3% (amino acid, 32.1-47.9% in DNA polymerase gene, and showed the closest relationship with turkey adenovirus 3 (TAdV-3 in phylogenetic analysis. During the 2008-2013 study period, the penguin adenoviruses were annually detected in 22 of 78 penguins (28.2%, and the molecular epidemiological study of the penguin adenovirus indicates a predominant infection in Chinstrap penguin population (12/30, 40%. Interestingly, the genome of penguin adenovirus could be detected in several internal samples, except the lymph node and brain. In conclusion, an analysis of the entire adenoviral genomes from Antarctic penguins was conducted, and the penguin adenoviruses, containing unique genetic character, were identified as a new species within the genus Siadenovirus. Moreover, it was annually detected in Antarctic penguins, suggesting its circulation within the penguin population.

  19. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines.

    Directory of Open Access Journals (Sweden)

    Shakti Singh

    Full Text Available Adenoviruses (Ad are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV.

  20. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines

    Science.gov (United States)

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2016-01-01

    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV. PMID:26751211

  1. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  2. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  3. Isoform-specific regulation and localization of the coxsackie and adenovirus receptor in human airway epithelia.

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    Katherine J D A Excoffon

    Full Text Available Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR(Ex7 and CAR(Ex8. We found low-level expression of the CAR(Ex8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR(Ex7, CAR(Ex8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR(Ex7, CAR(Ex8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR(Ex8 providing a potential mechanism for the apical localization of CAR(Ex8 in airway epithelial. In summary, apical localization of CAR(Ex8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.

  4. Inactivation of human adenovirus by sequential disinfection with an alternative UV technology and free chlorine.

    Science.gov (United States)

    Lee, Jung-Keun; Shin, Gwy-Am

    2011-03-01

    There has been growing concern over human exposure to adenoviruses through drinking water due to the extreme resistance of human adenoviruses to the traditional UV technology (low-pressure (LP) UV). As an effort to develop an effective treatment strategy against human adenoviruses in drinking water, we determined the effectiveness of sequential disinfection with an alternative UV technology (medium-pressure (MP) UV) and free chlorine. Human adenovirus 2 (Ad2) was irradiated with a low dose of MP UV irradiation (10 mJ/cm(2)) through UV collimated apparatus and then exposed to a low dose of free chlorine (0.17 mg/L) at pH 8 and 5°C using a bench-scale chemical disinfection system. A significant inactivation (e.g. 4 log(10)) of Ad2 was achieved with the low doses of MP UV and free chlorine within a very short contact time (∼1.5 min) although there was no apparent synergistic effect on Ad2 between MP UV and free chlorine. Overall, it is likely that the sequential disinfection with UV irradiation and free chlorine should control the contamination of drinking water by human adenoviruses within practical doses of UV and free chlorine typically used in drinking water treatment processes.

  5. Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

    Science.gov (United States)

    Thorne, Barbara A; Quigley, Paulene; Nichols, Gina; Moore, Christine; Pastor, Eric; Price, David; Ament, Jon W; Takeya, Ryan K; Peluso, Richard W

    2008-01-01

    Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

  6. Innate functions of immunoglobulin M lessen liver gene transfer with helper-dependent adenovirus.

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    Carmen Unzu

    Full Text Available The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.

  7. Adenovirus-mediated nitric oxide synthase gene transfer.

    Science.gov (United States)

    Raman, Kathleen G; Shapiro, Richard A; Tzeng, Edith; Kibbe, Melina R

    2004-01-01

    The varied biological effects of nitric oxide (NO) have led to intense research into its diverse physiologic and pathophysiologic roles in multiple disease processes. It has been implicated in the development of altered vasomotor tone, intimal hyperplasia, atherosclerosis, impotence, host defense, and wound healing. Using the modern technologies of recombinant DNA and gene transfer using adenoviral vectors, the effects of NO derived from various NO synthase (NOS) enzymes can be studied in a variety of tissues and the therapeutic applications of NOS is possible. Such uses of NOS gene transfer have been investigated extensively in the vasculature where NO is critical to regulating vascular homeostasis. NOS gene therapy has the theoretical advantage of allowing NO delivery to be localized, thereby limiting potential adverse effects of NO. The benefits of adenoviral vectors in gene transfer include relatively high transduction efficiencies, both replicating and nonreplicating cells may be infected, and the high titers of adenovirus that can be produced. The methods described in this chapter include the cloning of the iNOS cDNA into a recombinant adenoviral vector, large-scale production of that vector AdiNOS preparation, and the use of the vector to transduce tissue in vitro and in vivo.

  8. Oncolytic Replication of E1b-Deleted Adenoviruses

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    Pei-Hsin Cheng

    2015-11-01

    Full Text Available Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.

  9. Prevalence of neutralising antibodies against adenoviruses in lizards and snakes.

    Science.gov (United States)

    Ball, Inna; Ofner, Sabine; Funk, Richard S; Griffin, Chris; Riedel, Ulf; Möhring, Jens; Marschang, Rachel E

    2014-10-01

    Adenoviruses (AdVs) are relatively common in lizards and snakes, and several genetically distinct AdVs have been isolated in cell culture. The aims of this study were to examine serological relationships among lizard and snake AdVs and to determine the frequency of AdV infections in these species. Isolates from a boa constrictor (Boa constrictor), a corn snake (Pantherophis gutattus) and a central bearded dragon (Pogona vitticeps), and two isolates from helodermatid lizards (Heloderma horridum and H. suspectum) were used in neutralisation tests for the detection of antibodies in plasma from 263 lizards from seven families (including 12 species) and from 141 snakes from four families (including 28 species) from the USA and Europe. Most lizard and snake samples had antibodies against a range of AdV isolates, indicating that AdV infection is common among these squamates. Neutralisation tests with polyclonal antibodies raised in rabbits demonstrated serological cross-reactivity between both helodermatid lizard isolates. However, squamate plasma showed different reactions to each of these lizard isolates in neutralisation tests.

  10. Severe Necrotizing Adenovirus Tubulointerstitial Nephritis in a Kidney Transplant Recipient

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    Ravi Parasuraman

    2013-01-01

    Full Text Available Adenoviruses (AdV are emerging pathogens with a prevalence of 11% viruria and 6.5% viremia in kidney transplant recipients. Although AdV infection is common, interstitial nephritis (ADVIN is rare with only 13 biopsy proven cases reported in the literature. We report a case of severe ADVIN with characteristic histological features that includes severe necrotizing granulomatous lesion with widespread tubular basement membrane rupture and hyperchromatic smudgy intranuclear inclusions in the tubular epithelial cells. The patient was asymptomatic at presentation, and the high AdV viral load (quantitative PCR>2,000,000 copies/mL in the urine and 646,642 copies/mL in the serum confirmed the diagnosis. The patient showed excellent response to a combination of immunosuppression reduction, intravenous cidofovir, and immunoglobulin therapy resulting in complete resolution of infection and recovery of allograft function. Awareness of characteristic biopsy findings may help to clinch the diagnosis early which is essential since the disseminated infection is associated with high mortality of 18% in kidney transplant recipients. Cidofovir is considered the agent of choice for AdV infection in immunocompromised despite lack of randomized trials, and the addition of intravenous immunoglobulin may aid in resolution of infection while help prevention of rejection.

  11. Adenovirus as a gene therapy vector for hematopoietic cells.

    Science.gov (United States)

    Marini, F C; Yu, Q; Wickham, T; Kovesdi, I; Andreeff, M

    2000-06-01

    Adenovirus (Adv)-mediated gene transfer has recently gained new attention as a means to deliver genes for hematopoietic stem cell (HSC) or progenitor cell gene therapy. In the past, HSCs have been regarded as poor Adv targets, mainly because they lack the specific Adv receptors required for efficient and productive Adv infection. In addition, the nonintegrating nature of Adv has prevented its application to HSC and bone marrow transduction protocols where long-term expression is required. There is even controversy as to whether Adv can infect hematopoietic cells at all. In fact, the ability of Adv to infect epithelium-based targets and its inability to effectively transfect HSCs have been used in the development of eradication schemes that use Adv to preferentially infect and "purge" tumor cell-contaminating HSC grafts. However, there are data supporting the existence of productive Adv infections into HSCs. Such protocols involve the application of cytokine mixtures, high multiplicities of infection, long incubation periods, and more recently, immunological and genetic modifications to Adv itself to enable it to efficiently transfer genes into HSCs. This is a rapidly growing field, both in terms of techniques and applications. This review examines the two sides of the Adv/CD34 controversy as well as the current developments in this field.

  12. Epigenetic mechanisms in human adenovirus type 12 oncogenesis.

    Science.gov (United States)

    Doerfler, Walter

    2009-06-01

    For the past 30 years, my laboratory has concentrated its work on demonstrating that the epigenetic consequences of foreign DNA insertion into established mammalian genomes -de novo DNA methylation of the integrate and alterations of methylation patterns across the recipient genome - are essential elements in setting the stage towards oncogenic transformation. We have primarily studied human adenovirus type 12 (Ad12) which induces undifferentiated tumors in Syrian hamsters (Mesocricetus auratus) either at the site of subcutaneous Ad12 injection or intraperitoneally upon intramuscular injection. Up to 90% of the hamsters injected with Ad12 develop tumors within 3-6 weeks. Integration of foreign DNA, its de novo methylation, and the consequences of insertion on the cellular methylation and transcription profiles have been studied in detail. While viral infections are a frequent source of foreign genomes entering mammalian and other hosts and often their genomes, we have also pursued the fate of food-ingested foreign DNA in the mouse organism. The persistence of this DNA in the animals is transient and there is no evidence for the expression or germ line fixation of foreign DNA. Nevertheless, the occasional cell that carries integrated genomes from that foreign source deserves the oncologist's sustained interest.

  13. Serotype Chimeric Human Adenoviruses for Cancer GeneTherapy

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    Akseli Hemminki

    2010-09-01

    Full Text Available Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus,enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications.

  14. Usage of adenovirus expressing thymidine kinase mediated hepatocellular damage for enabling mouse liver repopulation with allogenic or xenogenic hepatocytes.

    Directory of Open Access Journals (Sweden)

    Daniel Moreno

    Full Text Available It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/- γc(-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk and two consecutive doses of ganciclovir (GCV. We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

  15. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Carolyn P.; Ayalew, Lisanework E. [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); Tikoo, Suresh K., E-mail: suresh.tik@usask.ca [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada (Canada)

    2012-10-10

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  16. Structural and Dynamic Characterization of the Molecular Hub Early Region 1A (E1A) from Human Adenovirus.

    Science.gov (United States)

    Hošek, Tomáš; Calçada, Eduardo O; Nogueira, Marcela Oliveira; Salvi, Michele; Pagani, Talita Duarte; Felli, Isabella C; Pierattelli, Roberta

    2016-09-05

    The small-DNA human adenovirus encodes one of the most versatile molecular hubs, the E1A protein. This protein is essential for productive viral infection in human cells and a vast amount of biologically relevant data are available on its interactions with host proteins. Up to now, however, no high-resolution structural and dynamic information on E1A is available despite its important biological role. Among the different spliced variants of E1A, two are expressed at high level in the early stage of infection. These are 243 and 289 residues isoforms. Herein, we present their NMR characterization, showing that they are both highly disordered, but also demonstrate a certain heterogeneous behavior in terms of structural and dynamic properties. Furthermore, we present the characterization of the isolated domain of the longer variant, known as CR3. This study opens the way to understanding at the molecular level how E1A functions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Combination of vorinostat and adenovirus-TRAIL exhibits a synergistic antitumor effect by increasing transduction and transcription of TRAIL in lung cancer cells

    National Research Council Canada - National Science Library

    Kim, D R; Park, M-Y; Lee, C-S; Shim, S-H; Yoon, H-I; Lee, J H; Sung, M-W; Kim, Y-S; Lee, C-T

    2011-01-01

    ... clinical response. Coxackie adenoviral receptor (CAR) is a major cellular receptor for adenovirus and is known as a gate for adenovirus and infection by adenovirus of tumor cells by binding with CAR expressed on the cell membrane. (2) Most lung cancer cell lines used in cancer research express variable amounts of CAR on cell membranes; h...

  18. Fiber-chimeric adenoviruses expressing fibers from serotype 16 and 50 improve gene transfer to human pancreatic adenocarcinoma

    NARCIS (Netherlands)

    Kuhlmann, K.F.D.; Geer, M.A. van; Bakker, C.T.; Dekker, J.E.M.; Havenga, M.J.E.; Oude Elferink, R.P.J.; Gouma, D.J.; Bosma, P.J.; Wesseling, J.G.

    2009-01-01

    Survival of patients with pancreatic cancer is poor. Adenoviral (Ad) gene therapy employing the commonly used serotype 5 reveals limited transduction efficiency due to the low amount of coxsackie-adenovirus receptor on pancreatic cancer cells. To identify fiber-chimeric adenoviruses with improved ge

  19. BS69 : A novel adenovirus E1A-associated protein that inhibits E1A transactivation

    NARCIS (Netherlands)

    Hateboer, G.; Gennissen, A.M.C.; Ramos, Y.F.M.; Kerkhoven, R.; Sonntag-Buck, V.; Stunnenberg, H.G.; Bernards, R.A.

    1995-01-01

    The adenovirus ElA gene products are nuclear phosphoproteins that can transactivate the other adenovirus early genes as well as several cellular genes, and can transform primary rodent cells in culture. Transformation and transactivation by ElA proteins is most likely to be mediated through binding

  20. Characterization of fastidious adenovirus types 40 and 41 by DNA restriction enzyme analysis and by neutralizing monoclonal antibodies.

    NARCIS (Netherlands)

    H.G.A.M. van der Avoort (Harrie); A.G. Wermenbol; T.P.L. Zomerdijk (Timo); J.A.F.W. Kleijne; J.A.A.M. van Asten (Jack); P. Jensma; A.D.M.E. Osterhaus (Albert); A.H. Kidd; J.C. de Jong (Jan)

    1989-01-01

    textabstractThe DNA of 48 strains of adenovirus type 40 (Ad40) and of 128 strains of adenovirus type 41 (Ad41), isolated between 1971 and 1986 from various countries, was characterized by restriction enzyme analysis using nine and ten restriction endonucleases respectively. Five new DNA variants of

  1. Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media

    Science.gov (United States)

    Murrah, Kyle A.; Turner, Roberta L.; Pang, Bing; Perez, Antonia C.; Reimche, Jennifer L.; King, Lauren B.; Wren, John; Gandhi, Uma; Swords, W. Edward; Ornelles, David A.

    2015-01-01

    Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease. PMID:25251686

  2. Isolated limb perfusion for local gene delivery: efficient and targeted adenovirus-mediated gene transfer into soft tissue sarcomas

    NARCIS (Netherlands)

    W.K. de Roos; J.H.W. de Wilt (Johannes); M.E. van der Kaaden; E.R. Manusama (Eric); M.W. de Vries; A. Bout; T.L.M. ten Hagen (Timo); D. Valerio (Dinko); A.M.M. Eggermont (Alexander)

    2000-01-01

    textabstractOBJECTIVE: To evaluate the potential of isolated limb perfusion (ILP) for efficient and tumor-specific adenovirus-mediated gene transfer in sarcoma-bearing rats. SUMMARY BACKGROUND DATA: A major concern in adenovirus-mediated gene therapy in cancer is the transfer of ge

  3. Application of mesenchymal stem cells as a vehicle to deliver replication-competent adenovirus for treating malignant glioma

    Institute of Scientific and Technical Information of China (English)

    Cui Hai; Yong-Min Jin; Wen-Biao Jin; Zhe-Zhu Han; Mei-Nv Cui; Xue-Zhe Piao; Xiong-Hu Shen; Song-Nan Zhang; Hong-Hua Sun

    2012-01-01

    Although gene therapy was regarded as a promising approach for glioma treatment,its therapeutic efficacy was often disappointing because of the lack of efficient drug delivery systems.Mesenchymal stem cells (MSCs) have been reported to have a tropism for brain tumors and thus could be used as delivery vehicles for glioma therapy.Therefore,in this study,we attempted to treat glioma by using MSCs as a vehicle for delivering replication-competent adenovirus.We firstly compared the infectivity of type 3,type 5,and type 35 fiber-modified adenoviruses in MSCs.We also determined suitable adenovirus titer in vitro and then used this titer to analyze the ability of MSCs to deliver replication-competent adenovirus into glioma in vivo.Our results indicated that type 35 fiber-modified adenovirus showed higher infectivity than did naked type 3 or type 5 fiber-modified adenovirus.MSCs carrying replication-competent adenovirus significantly inhibited tumor growth in vivo compared with other control groups.In conclusion,MSCs are an effective vehicle that can successfully transport replication-competent adenovirus into glioma,making it a potential therapeutic strategy for treating malignant glioma.

  4. PNA-encoded chemical libraries.

    Science.gov (United States)

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

  5. Identification and Classification of Adenovirus Particles in Digital Microscopic Images using Active Contours

    Directory of Open Access Journals (Sweden)

    Manjunatha Hiremath

    2014-06-01

    Full Text Available Medical imaging is the technique and process used to create images of the human body or medical science. Digital image processing is the use of computer algorithms to perform image processing on digital images. Microscope image processing dates back a half century when it was realized that some of the techniques of image capture and manipulation, first developed for television, could also be applied to images captured through the microscope. This paper presents semi-automated segmentation and identification of adenovirus particles using active contour with multi grid segmentation model. The geometric features are employed to identify the adenovirus particles in digital microscopic image. The min-max, 3 rules are used for recognition of adenovirus particles. The results are compared with manual method obtained by microbiologist.

  6. Protection of non-human primates against rabies with an adenovirus recombinant vaccine

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Z.Q. [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Greenberg, L. [Centers for Disease Control and Prevention, Atlanta, GA (United States); Ertl, H.C., E-mail: ertl@wistar.upenn.edu [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Rupprecht, C.E. [The Global Alliance for Rabies Control, Manhattan, KS (United States); Ross University School of Veterinary Medicine, Basseterre (Saint Kitts and Nevis)

    2014-02-15

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.

  7. Adenovirus-mediated transfection with glucose transporter 3 suppresses PC12 cell apoptosis following ischemic injury

    Institute of Scientific and Technical Information of China (English)

    Junliang Li; Xinke Xu; Shanyi Zhang; Meiguang Zheng; Zhonghua Wu; Yinlun Weng; Leping Ouyang; Jian Yu; Fangcheng Li

    2012-01-01

    In this study, we investigated the effects of adenovirus-mediated transfection of PC12 cells with glucose transporter 3 after ischemic injury. The results of flow cytometry and TUNEL showed that exogenous glucose transporter 3 significantly suppressed PC12 cell apoptosis induced by ischemic injury. The results of isotopic scintiscan and western blot assays showed that, the glucose uptake rate was significantly increased and nuclear factor kappaB expression was significantly decreased after adenovirus-mediated transfection of ischemic PC12 cells with glucose transporter 3. These results suggest that adenovirus-mediated transfection of cells with glucose transporter 3 elevates the energy metabolism of PC12 cells with ischemic injury, and inhibits cell apoptosis.

  8. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA...... mutational status, upon infection with VA-less adenoviruses in the presence of interferon, but also upon addition of the PKR activator polyIC to cells. When comparing two isogenic cell lines that differ solely with regard to the presence or absence of mutant Ras, no difference was observed concerning...

  9. Safety evaluation of adenovirus type 4 and type 7 vaccine live, oral in military recruits.

    Science.gov (United States)

    Choudhry, Azhar; Mathena, Julie; Albano, Jessica D; Yacovone, Margaret; Collins, Limone

    2016-08-31

    Before the widespread adoption of vaccination, adenovirus type 4 and type 7 were long associated with respiratory illnesses among military recruits. When supplies were depleted and vaccination was suspended in 1999 for approximately a decade, respiratory illnesses due to adenovirus infections resurged. In March 2011, a new live, oral adenovirus vaccine was licensed by the US Food and Drug Administration and was first universally administered to military recruits in October 2011, leading to rapid, dramatic elimination of the disease within a few months. As part of licensure, a postmarketing study (Sentinel Surveillance Plan) was performed to detect potential safety signals within 42days after immunization of military recruits. This study retrospectively evaluated possible adverse events related to vaccination using data from the Armed Forces Health Surveillance Branch Defense Medical Surveillance System (DMSS) database. Among 100,000 recruits who received the adenovirus vaccine, no statistically significant greater risk of prespecified medical events was observed within 42days after vaccination when compared with a historical cohort of 100,000 unvaccinated recruits. In an initial statistical analysis of International Classification of Disease, 9th Revision, Clinical Modification codes, a statistically significant higher risk for 19 other (not prespecified) medical events occurring in 5 or more recruits was observed among vaccinated compared with unvaccinated groups. After case record data abstraction for attribution and validation, two events (psoriasis [21 vs 7 cases] and serum reactions [12 vs 4 cases]) occurred more frequently in the vaccinated cohort. A causal relation of these rare events with adenovirus vaccination could not be established given confounding factors in the DMSS, such as coadministration of other vaccines and incomplete or inaccurate medical information, for some recruits. Prospective surveillance assessing these uncommon, but potentially

  10. Parotid enlargement due to adenovirus infection in patient with human immunodeficiency virus infection

    Directory of Open Access Journals (Sweden)

    Maria Irma Seixas Duarte

    1996-10-01

    Full Text Available The authors report a case of adenovirus- induced enlargement of the parotid gland involving a patient infected with human immunodeficiency virus (HIV. Physical examination revealed good general condition, no fever and bilateral enlargement of the parotid region, which was of increased consistency and slightly tender to palpation. Histological examination of the parotid gland demonstrated a slight periductal lymphomononuclear inflammatory infiltrate with the presence of focal points of necrosis. Tests to determine the presence of fungi and alcohol-acid resistent bacilli were negative. Immunohistochemistry for cytomegalovirus, heipes simplex, HIV p24 antigen and adenovirus showed positivity only for adenovirus in the epithelial nuclei of numerous gland ducts. Tins is the third case of this type reported in the literature, indicating the importance of including adenovirus in the differential diagnosis of this condition.Os autores relatam um caso de aumento da glândula parótida ocasionado por adenovirus, em paciente infectado pelo vírus da imunodeficiência humana. Ao exame físico, este se apresentava em bom estado geral, afebril e com aumento bilateral de parõtidas, de consistência aumentada e discretamente dolorosas ã palpação. O exame histológico da parótida demonstrou discreto infiltrado inflamatório linfomononuclear periductal com presença de focos de necrose, as pesquisas para fungos e bacilos ãlcool ãcido resistentes foram negativas. A técnica de imuno-histoquímica para citomegalovírus, beipes simples, antígeno p24 do HIVe adenovirus, somente evidenciou posítividade para o último. Este é o terceiro caso descrito na literatura, destacando a importância de incluir o adenovíms, no diagnóstico diferencial, deste acometimento.

  11. Intraductal delivery of adenoviruses targets pancreatic tumors in transgenic Ela-myc mice and orthotopic xenografts.

    Science.gov (United States)

    José, Anabel; Sobrevals, Luciano; Miguel Camacho-Sánchez, Juan; Huch, Meritxell; Andreu, Núria; Ayuso, Eduard; Navarro, Pilar; Alemany, Ramon; Fillat, Cristina

    2013-01-01

    Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTKT plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TKT oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p less than 0.0001) when combined with GCV. Of notice, both AduPARTKT and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors.

  12. Imaging of human sodium-iodide symporter gene expression mediated by recombinant adenovirus in skeletal muscle of living rats

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hyun Suk; Park, Seong-Wook [Department of Internal Medicine (Cardiology), Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, 138-736, Seoul (Korea); Lee, Heuiran; Kim, Sung Jin [Department of Microbiology, University of Ulsan College of Medicine, Seoul (Korea); Lee, Won Woo [Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam (Korea); Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea); Yang, You-Jung; Moon, Dae Hyuk [Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea)

    2004-09-01

    We evaluated the feasibility of non-invasive imaging of recombinant adenovirus-mediated human sodium-iodide symporter (hNIS) gene expression by {sup 99m}TcO{sub 4}{sup -} scintigraphy in skeletal muscle of rats. Replication-defective recombinant adenovirus encoding hNIS gene [Rad-CMV-hNIS 5 x 10{sup 7}, 2 x 10{sup 8} or 1 x 10{sup 9} plaque forming units (pfu)] or {beta}-galactosidase gene (Rad-CMV-LacZ 1 x 10{sup 9} pfu) was injected into the right biceps femoris muscle of rats (n=5-6 for each group). Three days after gene transfer, scintigraphy was performed using a gamma camera 30 min after injection of {sup 99m}TcO{sub 4}{sup -} (1.85 MBq). An additional two rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS underwent {sup 99m}TcO{sub 4}{sup -} scintigraphy with sodium perchlorate. After the imaging studies, rats were sacrificed for assessment of the biodistribution of {sup 99m}TcO{sub 4}{sup -} and measurement of hNIS mRNA expression. In all the rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS, hNIS expression was successfully imaged by {sup 99m}TcO{sub 4}{sup -} scintigraphy, while rats injected with Rad-CMV-LacZ or lower doses of Rad-CMV-hNIS failed to show uptake. The biodistribution studies indicated that a significantly different amount of {sup 99m}TcO{sub 4}{sup -} was retained in the liver (p<0.001) and the right muscle (p<0.05), with the highest uptake in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS. The muscular hNIS mRNA level quantified by real-time reverse transcription-polymerase chain reaction was significantly higher in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS (p<0.05), with a positive correlation with the imaging counts (r=0.810, p<0.05) and the biodistribution (r=0.847, p<0.001). Hot spots in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS were specifically inhibited by sodium perchlorate. This study illustrated that {sup 99m}TcO{sub 4}{sup -} scintigraphy can monitor Rad-CMV-hNIS-mediated gene expression in

  13. Compressed Encoding for Rank Modulation

    CERN Document Server

    Gad, Eyal En; Jiang,; Bruck, Jehoshua

    2011-01-01

    Rank modulation has been recently proposed as a scheme for storing information in flash memories. While rank modulation has advantages in improving write speed and endurance, the current encoding approach is based on the "push to the top" operation that is not efficient in the general case. We propose a new encoding procedure where a cell level is raised to be higher than the minimal necessary subset - instead of all - of the other cell levels. This new procedure leads to a significantly more compressed (lower charge levels) encoding. We derive an upper bound for a family of codes that utilize the proposed encoding procedure, and consider code constructions that achieve that bound for several special cases.

  14. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  15. Self-Organising Stochastic Encoders

    CERN Document Server

    Luttrell, Stephen

    2010-01-01

    The processing of mega-dimensional data, such as images, scales linearly with image size only if fixed size processing windows are used. It would be very useful to be able to automate the process of sizing and interconnecting the processing windows. A stochastic encoder that is an extension of the standard Linde-Buzo-Gray vector quantiser, called a stochastic vector quantiser (SVQ), includes this required behaviour amongst its emergent properties, because it automatically splits the input space into statistically independent subspaces, which it then separately encodes. Various optimal SVQs have been obtained, both analytically and numerically. Analytic solutions which demonstrate how the input space is split into independent subspaces may be obtained when an SVQ is used to encode data that lives on a 2-torus (e.g. the superposition of a pair of uncorrelated sinusoids). Many numerical solutions have also been obtained, using both SVQs and chains of linked SVQs: (1) images of multiple independent targets (encod...

  16. Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange

    Directory of Open Access Journals (Sweden)

    Farley Daniel C

    2010-12-01

    Full Text Available Abstract Background Adenovirus serotype 5 (Ad5 has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results We have used LoxP/Cre recombination mediated cassette exchange (RMCE to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be

  17. Lifelong persistent infection of hamster brain by human adenovirus type 6.

    Directory of Open Access Journals (Sweden)

    Yabe,Yoshiro

    1988-02-01

    Full Text Available To establish an experimental persistent infection of the brain with human adenoviruses, adenovirus type 6 (ad 6 was inoculated intracerebrally into young adult hamsters. Hamsters appeared languid for a few days after inoculation, but recovered rapidly. By cocultivation of tissue fragments with HeLa cells, ad 6 was always recovered from the brains of hamsters throughout their lives, as long as 29 months, indicating the establishment of a lifelong persistent infection. Except for the first few days after inoculation, however, attempts to recover virus by inoculation of tissue extracts onto HeLa cells or by cultivation of tissue fragments alone were unsuccessful.

  18. Characterization of an upstream regulatory element of adenovirus L1 poly (A) site.

    Science.gov (United States)

    Liu, Li

    2005-06-20

    The transition from early to late stage infection by adenovirus involves a change in mRNA expression from the adenovirus major late transcription unit (AdMLTU). This early to late switch centers around alternative selection of one of five poly (A) sites (L1-L5) that code for the major structural proteins of Adenovirus. During the early stage of infection, steady state mRNA is primarily derived from the L1 poly (A) site. During the late stage of infection, each of the MLTU poly (A) sites is represented in the steady state mRNA pool (Falck-Pedersen, E., Logan, J., 1989. Regulation of poly(A) site selection in adenovirus. J. Virol. 63 (2), 532-541.). Using transient transfection of a plasmid expressing Chloramphenicol Acetyl Transferase with a tandem poly (A) minigene system (L13) (DeZazzo, J.D., Falck-Pedersen, E., Imperiale, M.J., 1991. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol. Cell. Biol. 11 (12), 5977-5984; Prescott, J., Falck-Pedersen, E., 1994. Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites. Mol. Cell. Biol. 14 (7), 4682-4693.), it has been demonstrated that the promoter-proximal L1 poly (A) site which is poorly recognized by the 3' end processing machinery, contains an upstream repressor element (URE) that influences steady state levels of mRNA (Prescott, J.C., Liu, L., Falck-Pedersen, E., 1997. Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation. Mol. Cell. Biol. 17 (4), 2207-2216.). In this study, we have further characterized the elements that mediate L1URE function. These studies indicate that the L1 upstream regulatory element (L1 URE) contains a complex RNA architecture that serves to repress gene expression through multiple sub-effectors. The L1URE functions when located upstream of a heterologous poly (A) site, and is able to strongly suppress steady state m

  19. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor.

    Science.gov (United States)

    Wigand, R

    1979-01-01

    The inhibition of adenovirus multiplication by adenine arabinoside was determined by yield reduction in one-step multiplication cycle. Inhibition was greatly enhanced by an adenosine deaminase inhibitor (2-deoxycoformycin) in concentrations down to 10 ng/ml. Adenovirus types from four subgroups showed similar results. However, the enhancing effect of adenosine deaminase inhibitor was great in HeLa cells, moderate in human fibroblasts, and negligible in Vero cells. This difference could be explained by different concentrations of adenosine deaminase found in cell homogenates.

  20. Modulation of breast cancer resistance protein mediated atypical multidrug resistance using RNA interference delivered by adenovirus

    Institute of Scientific and Technical Information of China (English)

    LI Wen-tong; ZHOU Geng-yin; WANG Chun-ling; GUO Cheng-hao; SONG Xian-rang; CHI Wei-ling

    2005-01-01

    @@ Clinical multidrug resistance (MDR) of malignancies to many antineoplastic agents is the major obstacle in the successful treatment of cancer. The emergence of breast cancer resistance protein (BCRP), a member of the adenosine triphosphate (ATP) binding cassette (ABC) transporter family, has necessitated the development of antagonists. To overcome the BCRP-mediated atypical MDR, RNA interference (RNAi) delivered by adenovirus targeting BCRP mRNA was used to inhibit the atypical MDR expression by infecting MCF-7/MX100 cell lines with constructed RNAi adenovirus.

  1. Community-acquired adenovirus pneumonia in a patient with chronic lymphatic leukaemia.

    Science.gov (United States)

    Larsen, I K; Nielsen, H

    2005-03-01

    Described here is a severe case of community-acquired adenovirus pneumonia that occurred in a previously healthy 54-year-old male who was later determined to have stage A chronic lymphatic leukemia. The clinical presentation was consistent with that of atypical pneumonia. Testing with PCR revealed adenovirus in a bronchoalveolar lavage sample, while all other tests to determine a bacterial or virological etiology were negative. Further examination of the patient revealed the previously undiagnosed chronic lymphatic leukemia. Following treatment with human immunoglobulin and oxygen therapy with continuous positive airway pressure support the patient recovered from the pneumonia completely.

  2. Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polym...

  3. Experimental study of Human Adenoviruses interactions with clays

    Science.gov (United States)

    Bellou, Maria; Syngouna, Vasiliki; Paparrodopoulos, Spyros; Vantarakis, Apostolos; Chrysikopoulos, Constantinos

    2014-05-01

    Clays are used to establish low permeability liners in landfills, sewage lagoons, water retention ponds, golf course ponds, and hazardous waste sites. Human adenoviruses (HAdVs) are waterborne viruses which have been used as viral indicators of fecal pollution. The objective of this study was to investigate the survival of HAdV in static and dynamic clay systems. The clays used as a model were crystalline aluminosilicates: kaolinite and bentonite. The adsorption and survival of HAdVs onto these clays were characterized at two different controlled temperatures (4 and 25o C) under static and dynamic batch conditions. Control tubes, in the absence of clay, were used to monitor virus inactivation due to factors other than adsorption to clays (e.g. inactivation or sorption onto the tubes walls). For both static and dynamic batch experiments, samples were collected for a maximum period of seven days. This seven day time - period was determined to be sufficient for the virus-clay systems to reach equilibrium. To infer the presence of infectious HAdV particles, all samples were treated with Dnase and the extraction of viral nucleid acid was performed using a commercial viral RNA kit. All samples were analyzed by Real - Time PCR which was used to quantify viral particles in clays. Samples were also tested for virus infectivity by A549 cell cultures. Exposure time intervals in the range of seven days (0.50-144 hours) resulted in a load reduction of 0.74 to 2.96 logs for kaolinite and a reduction of 0.89 to 2.92 for bentonite. Furthermore, virus survival was higher onto bentonite than kaolinite (p

  4. Improving Adenovirus Based Gene Transfer: Strategies to Accomplish Immune Evasion

    Directory of Open Access Journals (Sweden)

    Andrea Amalfitano

    2010-09-01

    Full Text Available Adenovirus (Ad based gene transfer vectors continue to be the platform of choice for an increasing number of clinical trials worldwide. In fact, within the last five years, the number of clinical trials that utilize Ad based vectors has doubled, indicating growing enthusiasm for the numerous positive characteristics of this gene transfer platform. For example, Ad vectors can be easily and relatively inexpensively produced to high titers in a cGMP compliant manner, can be stably stored and transported, and have a broad applicability for a wide range of clinical conditions, including both gene therapy and vaccine applications. Ad vector based gene transfer will become more useful as strategies to counteract innate and/or pre-existing adaptive immune responses to Ads are developed and confirmed to be efficacious. The approaches attempting to overcome these limitations can be divided into two broad categories: pre-emptive immune modulation of the host, and selective modification of the Ad vector itself. The first category of methods includes the use of immunosuppressive drugs or specific compounds to block important immune pathways, which are known to be induced by Ads. The second category comprises several innovative strategies inclusive of: (1 Ad-capsid-display of specific inhibitors or ligands; (2 covalent modifications of the entire Ad vector capsid moiety; (3 the use of tissue specific promoters and local administration routes; (4 the use of genome modified Ads; and (5 the development of chimeric or alternative serotype Ads. This review article will focus on both the promise and the limitations of each of these immune evasion strategies, and in the process delineate future directions in developing safer and more efficacious Ad-based gene transfer strategies.

  5. Genomic and phylogenetic analyses of murine adenovirus 2.

    Science.gov (United States)

    Hemmi, Silvio; Vidovszky, Márton Z; Ruminska, Justyna; Ramelli, Sandra; Decurtins, Willy; Greber, Urs F; Harrach, Balázs

    2011-09-01

    Murine adenoviruses (MAdV) are supposedly the oldest members of the genus Mastadenovirus. Currently, there are three distinct MAdV types known with rather different tropism and pathology. Here we report and annotate the DNA sequence of the full genome of MAdV-2. It was found to consist of 35,203 bp thus being considerably larger than the genomes of the other two MAdV types. The increased size of the MAdV-2 genome is generally due to larger genes and ORFs, although some differences in the number of ORFs were observed for the early regions E1, E3 and E4. The homologue of the 19K gene of E1B from MAdV-2 codes for 330 amino acids (aa) and is almost twice as large as from other mastadenoviruses. Accordingly, only the N-terminal half (155aa) has homology to the 19K protein. A homologue of the gene of the 12.5K protein was identified in the E3 region of MAdV-2, but not in MAdV-1 or MAdV-3. The other gene of yet unknown function in the E3 region of MAdV-2 seems to be unique. The E4 region of MAdV-2 contains three ORFs. One has similarity to the 34K gene of other AdVs. Two unique ORFs in the E4 region of MAdV-2 have no homology to any of the five and six ORFs in the E4 region of MAdV-1 or MAdV-3, respectively. Phylogenetic analyses showed that the three murine AdVs have a close common ancestor. They likely formed the first branching of the lineage of mastadenoviruses, and seem to be the most ancient representatives of this genus. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. [Molecular epideiological and clinical feature of human calicivirus and adenovirus among children with diarrhea less than 5 years old from 2010 to 2011 in Lanzhou, Gansu province].

    Science.gov (United States)

    Wang, Yong-Xia; Li, Dan-Di; Jin, Yu; Zhang, Qing; Wang, Hong; Kong, Xiang-Yu; Li, Yu-Ning; Duan, Zhao-Jun

    2012-02-01

    To investigate the clinical and molecular epidemiology characteristics of calicivirus and adenovirus in children for viral diarrhea in Lanzhou. Stool samples were collected from 295 children with diarrhea at the First Hospital of Lanzhou University, Gansu Province,China, between July 2010 and June 2011. Reverse transcription-polymerase chain reaction (RT-PCR) or PCR were used to detected calicivirus and adenovirus. The adenovirus positive samples were typed by nested PCR and multiple PCR. Of the 295 specimens, 13.2% (39/295) were positive for calicivirus, and 5.1% (15/295) were adenovirus. Typing and Phylogenetic analysis revealed that novirus GII-3 and adenovirus 41 were the dominant strains. Both calicivirus and adenovirus predominately affect children under the age of 2. In seasonal distribution, there was no obvious peak. Human calicivirus and adenovirus are important pathogens of viral diarrhea,it is important to develop long-term systematic surveillance.

  7. Differential effects of adenovirus-p16 on bladder cancer cell lines can be overcome by the addition of butyrate.

    Science.gov (United States)

    Lee, C T; Seol, J Y; Park, K H; Yoo, C G; Kim, Y W; Ahn, C; Song, Y W; Han, S K; Han, J S; Kim, S; Lee, J S; Shim, Y S

    2001-01-01

    High frequency of p16 alteration and high local recurrence rate of bladder cancer make this cancer an ideal target for p16 gene therapy. However, a low transduction rate of p16 via adenoviral vector causes an inconsistent result. In this study, we have tested adenovirus-p16 in several bladder cancer cell lines and investigated a way of improving the low transduction rate. Adenovirus-p16 showed a strong antitumor effect on bladder cancer cell lines (253J and T24) with strong Coxackie-adenoviral receptor (CAR) expression but little antitumor effect on bladder cancer cell lines (J82 and HT1376) with little CAR expression. In this study, we suggest a simple way of overcoming the differential effects of the adenovirus. The addition of butyrate to media was found to increase the transduction rate of adenovirus remarkably and increase the antitumor effect of adenovirus-p16 in bladder cancer cell lines with little CAR expression. Butyrate effects were related with increased CAR expression on the cell surface as well as increased transgene expression from adenoviral vector. From these observations, application of adenovirus-p16 gene therapy with butyrate can overcome the obstacle of low gene transfer and enhance the antitumor effect of adenovirus-p16 in bladder cancer.

  8. Multidimensionally encoded magnetic resonance imaging.

    Science.gov (United States)

    Lin, Fa-Hsuan

    2013-07-01

    Magnetic resonance imaging (MRI) typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here, we propose the multidimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel radiofrequency coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled.

  9. Downmodulation of El A Protein Expression as a Novel Strategy to Design Cancer-Selective Adenoviruses

    Directory of Open Access Journals (Sweden)

    Hong Jiang

    2005-08-01

    Full Text Available Oncolytic adenoviruses are being tested as potential therapies for human malignant tumors, including gliomas. Here we report for the first time that a mutation in the E1A gene results in low levels of ElA protein, conditioning the replication of mutant adenoviruses specifically to cancer cells. In this study, we compared the oncolytic potencies of three mutant adenoviruses encompassing deletions within the CRi (Delta-39, CR2 (Delta-24 regions, or both regions (Delta-24/39 of the ElA protein. Delta-39, Delta-24 induced a cytopathic effect with similar efficiency in glioma cells, a comparable capacity for replication. Importantly, the activity of Delta-39 was significantly attenuated compared to Delta-24 in proliferating normal human astrocytes. Direct analyses of the activation of E2F-1 promoter demonstrated the inability of Delta-39 to induce S-phase-related transcriptional activity in normal cells. Interestingly, ElA protein levels in cells infected with Delta-39 were remarkably downmodulated. Furthermore, protein stability studies revealed enhanced degradation of CRi mutant ElA proteins, inhibition of the proteasome activity resulted in the striking rescue of ElA levels. We conclude that the level of ElA protein is a critical determinant of oncolytic phenotype, we propose a completely novel strategy for the design, construction of conditionally replicative adenoviruses.

  10. The presence of enterovirus, adenovirus, and parvovirus B19 in myocardial tissue samples from autopsies

    DEFF Research Database (Denmark)

    Nielsen, Trine Skov; Hansen, Jakob; Nielsen, Lars Peter

    2014-01-01

    of adenovirus, enterovirus, and parvovirus B19 (PVB) in myocardial autopsy samples from myocarditis related deaths and in non-inflamed control hearts in an effort to clarify their significance as the causes of myocarditis in a forensic material. METHODS: We collected all autopsy cases diagnosed with myocarditis...

  11. Inhibitory effects of silver nanoparticles against adenovirus type 3 in vitro.

    Science.gov (United States)

    Chen, Nana; Zheng, Yang; Yin, Jianjian; Li, Xiujing; Zheng, Conglong

    2013-11-01

    Adenoviruses are associated with respiratory, ocular, or gastrointestinal disease. With various species and high morbidity, adenoviruses are increasingly recognized as significant viral pathogen among pediatric and immunocompromised patients. However, there is almost no specific drug for treatment. Silver nanoparticles are demonstrated to be virucidal against influenza A (H1N1) virus, human immunodeficiency virus and Hepatitis B virus. Currently, there is no data regarding whether the silver nanoparticles inhibit the adenovirus or not. The aim of this study is to investigate the effect of silver nanoparticles on adenovirus type 3 (Ad3). The results revealed that HeLa cells infected with silver nanoparticles treated Ad3 did not show obvious CPE. The viability of HeLa cells infected with silver nanoparticles treated Ad3 was significantly higher than that of cells infected with untreated Ad3. There was a significant difference of fluorescence intensity between the cells infected with silver nanoparticles treated and untreated Ad3. The transmission electron microscopy (TEM) showed that silver nanoparticles could directly damage the structure of Ad3 particle. The PCR amplification products of DNA isolated from silver nanoparticles treated Ad3 was decreased in a dose-dependent manner. The decreased DNA loads were also confirmed by real-time PCR experiment. The present study indicates silver nanoparticles exhibit remarkably inhibitory effects on Ad3 in vitro, which suggests silver nanoparticles could be a potential antiviral agent for inhibiting Ad3 infection. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Analysis of the expression of coxsackievirus and adenovirus receptor in five colon cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the expression of coxsackievirus and adenovirus receptor (CAR) and adenovirus-mediated reporter gene transfer in five human colon cancer cell lines.METHODS: Expression of CAR-specific mRNA and protein was analyzed by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Adenovirusbased gene delivery was evaluated by infection of cells with adenoviral vector carrying the green fluorescent protein (GFP) gene.RESULTS: All the colon cancer cell lines examined (HT29,LS180, SW480, SW948 and SW1116) expressed CAR full-length mRNA and an alternatively-spliced variant that lacks the transmembrane coding exon. All cell lines were detected as CAR-positive by Western blot analysis.Further, all cells we examined were efficiently infected with adenoviral vector-GFP.CONCLUSION: The data indicated that the five colon cancer cell lines tested expressed adenovirus primary receptor and could be efficiently infected by adenoviral vectors. Therefore, these cell lines will be useful for adenovirus-based gene transfer and research.

  13. Detection of adenovirus hexon sequence in a cat by polymerase chain reaction(short communication)

    NARCIS (Netherlands)

    Horzinek, M.C.; Lakatos, B.; Farkas, J.; Egberink, H.F.; Vennema, H.; Benko, M.

    1999-01-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal

  14. Molecular Epidemiology of Adenovirus Type 7 in the United States, 1966-2000

    Science.gov (United States)

    2002-03-01

    Med Trop Sao Paulo 1997;39:185-9. GF, et al. Extrapulmonary manifestations of adenovirus type 7 pneumonia 31. Golovina GI, Zolotaryov FN, Yurlova TI...residues. J Virol 1996;70:1836-44. 22. McNeill KM, Ridgely Benton F, Monteith SC, Tuchscherer MA, Gaydos 44. Kajon AE, Murtagh P, Garcia Franco S, Freire

  15. Adenovirus-mediated interteukin-13 gene therapy attenuates acute kidney allograft injury

    NARCIS (Netherlands)

    Sandovici, Maria; Deelmani, Leo E.; van Goor, Harry; Helfrich, Wijnand; de Zeeuw, Dick; Henning, Robert H.

    2007-01-01

    Background Kidney transplantation is possible by virtue of systemic immunosuppression, which is in turn accompanied by serious side effects. The search for novel therapeutic agents and strategies is ongoing. Here we investigate the effects of adenovirus-mediated gene therapy with interleukin (IL)-13

  16. High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Fei Zhong; Zhen-Yu Zhong; Shuang Liang; Xiu-Jin Li

    2006-01-01

    AIM: To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation.METHODS: The human adenovirus vector was used to express the soluble S-protein (corresponding to 1~1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells.RESULTS: Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 μg/106cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule.CONCLUSION: The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.

  17. The relevance of coagulation factor X protection of adenoviruses in human sera.

    Science.gov (United States)

    Duffy, M R; Doszpoly, A; Turner, G; Nicklin, S A; Baker, A H

    2016-07-01

    Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5-FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad-FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy.

  18. Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

    Directory of Open Access Journals (Sweden)

    Sarah R Klein

    Full Text Available Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

  19. An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

    NARCIS (Netherlands)

    Kim, Julius W.; Glasgow, Joel N.; Nakayama, Masaharu; Ak, Ferhat; Ugai, Hideyo; Curiel, David T.

    2013-01-01

    Background: Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene

  20. Analysis of enterovirus and adenovirus presence in swimming pools in Cyprus from 2007-2008.

    Science.gov (United States)

    Bashiardes, S; Koptides, D; Pavlidou, S; Richter, J; Stavrou, N; Kourtis, C; Papageorgiou, G T; Christodoulou, C G

    2011-01-01

    An analysis was carried out to determine the presence of enteroviruses and adenoviruses in public swimming pools in Cyprus. The effectiveness of the commonly implemented disinfection procedure of chlorination was confirmed by determination of bacteriological markers. Analysis of viral presence was carried out by sampling random swimming pools from the five major cities in Cyprus during a period of 21 months spanning from April 2007 to December 2008. A 10 I sample was taken from each swimming pool to be tested and was subsequently concentrated via membrane filtration using a new methodological approach for virus elution. Concentrated samples were analysed using of a Real Time Polymerase Chain Reaction (PCR) TaqMan probe based approach to detect the presence of enteroviruses and adenoviruses. Over the period of 21 months a total of 126 swimming pools were sampled and analysed. In four swimming pools enteroviruses were detected, in one pool echovirus 18 was identified, in two pools echovirus 30 was identified and in one other pool poliovirus Sabin 1 was identified. Similarly, in four swimming pools adenoviruses were detected, in all four adenovirus 41 was identified. Bacteriological marker analysis showed that 98% of pools complied with Cyprus regulations.

  1. Detection of adenovirus hexon sequence in a cat by polymerase chain reaction(short communication)

    NARCIS (Netherlands)

    Horzinek, M.C.; Lakatos, B.; Farkas, J.; Egberink, H.F.; Vennema, H.; Benko, M.

    1999-01-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal sam

  2. Valganciclovir Inhibits Human Adenovirus Replication and Pathology in Permissive Immunosuppressed Female and Male Syrian Hamsters

    Directory of Open Access Journals (Sweden)

    Karoly Toth

    2015-03-01

    Full Text Available Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5 infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients.

  3. (13) C-metabolic flux analysis of human adenovirus infection: Implications for viral vector production.

    Science.gov (United States)

    Carinhas, Nuno; Koshkin, Alexey; Pais, Daniel A M; Alves, Paula M; Teixeira, Ana P

    2017-01-01

    Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2-(13) C]glucose or [U-(13) C]glutamine to evaluate metabolic alterations in the amniocyte-derived, E1-transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5). Nonstationary (13) C-metabolic flux analysis revealed increased fluxes of glycolysis (17%) and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth-arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5-fold), but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80%). Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195-207. © 2016 Wiley Periodicals, Inc.

  4. Transcriptional activation by the E1A regions of adenovirus types 40 and 41

    NARCIS (Netherlands)

    Loon, A.E. van; Gilardi, P.; Perricaudet, M.; Rozijn, Th. H.; Sussenbach, J.S.

    1987-01-01

    In order to establish whether the poor growth of the two fastidious adenoviruses types 40 and 41 (Ad40 and Ad41) in HeLa cells is due to a reduced trans-activation by the early region to (E1A), we have determined the trans-activating effect of this region on the expression of the chloramphenicol ace

  5. Valganciclovir inhibits human adenovirus replication and pathology in permissive immunosuppressed female and male Syrian hamsters.

    Science.gov (United States)

    Toth, Karoly; Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Sagartz, John E; Buller, Robert Mark L; Wold, William S M

    2015-03-23

    Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate) is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5) infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients.

  6. The role of the adenovirus DNA binding protein in DNA replication and recombination

    NARCIS (Netherlands)

    Breukelen, B. van

    2003-01-01

    Replication of adenovirus DNA in infected cells is an efficient process that, compared to cellular replication, has the use of a protein primer as a hallmark. The mechanism of this DNA replication process and especially the role of one of the replication proteins, the DNA binding protein DBP, is the

  7. Adenovirus respiratory infection: significant increase in diagnosis using PCR comparing with antigen detection and culture methods

    Directory of Open Access Journals (Sweden)

    Elenice Stroparo

    2010-12-01

    Full Text Available Adenovirus (AdV respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF, and a specific nested polymerase chain reaction (PCR, to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%. In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.

  8. Detection of adenovirus hexon sequence in a cat by polymerase chain reaction(short communication)

    NARCIS (Netherlands)

    Horzinek, M.C.; Lakatos, B.; Farkas, J.; Egberink, H.F.; Vennema, H.; Benko, M.

    1999-01-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal sam

  9. Crystallographic Structure of Porcine Adenovirus Type 4 Fiber Head and Galectin Domains▿

    Science.gov (United States)

    Guardado-Calvo, Pablo; Muñoz, Eva M.; Llamas-Saiz, Antonio L.; Fox, Gavin C.; Kahn, Richard; Curiel, David T.; Glasgow, Joel N.; van Raaij, Mark J.

    2010-01-01

    Adenovirus isolate NADC-1, a strain of porcine adenovirus type 4, has a fiber containing an N-terminal virus attachment region, shaft and head domains, and a C-terminal galectin domain connected to the head by an RGD-containing sequence. The crystal structure of the head domain is similar to previously solved adenovirus fiber head domains, but specific residues for binding the coxsackievirus and adenovirus receptor (CAR), CD46, or sialic acid are not conserved. The structure of the galectin domain reveals an interaction interface between its two carbohydrate recognition domains, locating both sugar binding sites face to face. Sequence evidence suggests other tandem-repeat galectins have the same arrangement. We show that the galectin domain binds carbohydrates containing lactose and N-acetyl-lactosamine units, and we present structures of the galectin domain with lactose, N-acetyl-lactosamine, 3-aminopropyl-lacto-N-neotetraose, and 2-aminoethyl-tri(N-acetyl-lactosamine), confirming the domain as a bona fide galectin domain. PMID:20686025

  10. Adenovirus DNA replication in vitro is stimulated by RNA from uninfected HeLa cells

    NARCIS (Netherlands)

    Vliet, P.C. van der; Dam, D. van; Kwant, M.M.

    1984-01-01

    Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, r

  11. Effect of neutralizing sera on factor X-mediated adenovirus serotype 5 gene transfer

    NARCIS (Netherlands)

    Parker, A.L.; Waddington, S.N.; Buckley, S.M.K.; Custers, J.; Havenga, M.J.E.; Rooijen, N. van; Goudsmit, J.; McVey, J.H.; Nicklin, S.A.; Baker, A.H.

    2009-01-01

    The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated

  12. The cleaved N-terminus of pVI binds peripentonal hexons in mature adenovirus

    NARCIS (Netherlands)

    Snijder, Joost; Benevento, Marco; Moyer, Crystal L; Reddy, Vijay; Nemerow, Glen R; Heck, Albert J R

    2014-01-01

    Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle

  13. Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection

    DEFF Research Database (Denmark)

    Carson, Christian T; Orazio, Nicole I; Lee, Darwin V

    2009-01-01

    The protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild-type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral...

  14. Acute Respiratory Disease in US Army Trainees 3 Years after Reintroduction of Adenovirus Vaccine1

    Science.gov (United States)

    McCormic, Zachary D.; Gaydos, Joel C.; Hawksworth, Anthony W.; Jordan, Nikki N.

    2017-01-01

    The 1999 cessation of vaccination against adenovirus types 4 and 7 among US Army trainees resulted in reemergence of acute respiratory disease (ARD) outbreaks. The 2011 implementation of a replacement vaccine led to dramatic and sustained decreases in ARD cases, supporting continuation of vaccination in this population at high risk for ARD. PMID:27748651

  15. Fatal Fulminant Hepatic Failure from Adenovirus in Allogeneic Bone Marrow Transplant Patients

    Directory of Open Access Journals (Sweden)

    Jatin M. Vyas

    2012-01-01

    Full Text Available We report two cases of fatal hepatic failure in patients who received matched unrelated bone marrow transplantation. Both patients presented with high fevers, abnormal liver functions tests, and hypodense lesions in the liver by CT scan. Histologic examination of postmortem liver samples demonstrated extensive necrosis, and immunohistochemistry was positive for adenovirus.

  16. Effect of organic carbon on sorption of human adenovirus to soil particles and laboratory containers

    Science.gov (United States)

    A key factor controlling the relationship between virus release and human exposure is how virus particles interact with soils, sediments and other solid particles in the environment and in engineered treatment systems. Finding no previous investigations of human adenovirus (HAdV)...

  17. Delta-24-RGD oncolytic adenovirus elicits anti-glioma immunity in an immunocompetent mouse model

    NARCIS (Netherlands)

    H. Jiang (Hao); K. Clise-Dwyer (Karen); K.E. Ruisaard (Kathryn); X. Fan (Xuejun); W. Tian (Weihua); J. Gumin (Joy); M.L.M. Lamfers (Martine); A. Kleijn (Anne); F.F. Lang (Frederick); S. Yung (Sun); L.M. Vence (Luis); C. Gomez-Manzano (Candelaria); J. Fueyo (Juan)

    2014-01-01

    textabstractBackground: Emerging evidence suggests anti-cancer immunity is involved in the therapeutic effect induced by oncolytic viruses. Here we investigate the effect of Delta-24-RGD oncolytic adenovirus on innate and adaptive anti-glioma immunity. Design: Mouse GL261-glioma model was set up in

  18. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ..., shall be prepared from virus-bearing cell culture fluids. Only Master Seed Virus which has been... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...

  19. Life-Threatening Adenovirus Infections in the Setting of the Immunocompromised Allogeneic Stem Cell Transplant Patients

    Directory of Open Access Journals (Sweden)

    Cedar J. Fowler

    2010-01-01

    Full Text Available A single institution case series of adenovirus infections after allogeneic hematopoietic stem cell transplantation is presented to highlight the consideration for adenovirus infections as an etiology in patients with rapid hepatic or other sudden organ deterioration in the setting of apparent GVHD stabilization. The series also highlights that survival is limited with these infections often due in part to concomitant opportunistic infections. In addition, the pathophysiological events, such as GVHD and hepatic dysfunction, may complicate the clinical picture and delay therapy of an opportunistic infection. This is particularly true for adenoviral infections as they also have a distinct clinical picture in immunocompromised patients when compared to immune competent patients. Adenovirus infections also have the additional challenge that its treatment, cidofovir, has associated toxicities that can delay its administration. Recent developments has yielded an assay that can be used in the early detection and for serial determinations of adenovirus in patients with advanced GVHD, as well as a new therapeutic agent currently undergoing clinical trials.

  20. Virally encoded 7TM receptors

    DEFF Research Database (Denmark)

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

    2001-01-01

    A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion...... in various parts of the viral life cyclus. Most of the receptors encoded by human pathogenic virus are still orphan receptors, i.e. the endogenous ligand is unknown. In the few cases where it has been possible to characterize these receptors pharmacologically, they have been found to bind a broad spectrum...... expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets....

  1. Synaptic encoding of temporal contiguity

    Directory of Open Access Journals (Sweden)

    Srdjan eOstojic

    2013-04-01

    Full Text Available Often we need to perform tasks in an environment that changes stochastically. In these situations it is important to learn the statistics of sequences of events in order to predict the future and the outcome of our actions. The statistical description of many of these sequences can be reduced to the set of probabilities that a particular event follows another event (temporal contiguity. Under these conditions, it is important to encode and store in our memory these transition probabilities. Here we show that for a large class of synaptic plasticity models, the distribution of synaptic strengths encodes transitions probabilities. Specifically, when the synaptic dynamics depend on pairs of contiguous events and the synapses can remember multiple instances of the transitions, then the average synaptic weights are a monotonic function of the transition probabilities. The synaptic weights converge to the distribution encoding the probabilities also when the correlations between consecutive synaptic modifications are considered. We studied how this distribution depends on the number of synaptic states for a specific model of a multi-state synapse with hard bounds. In the case of bistable synapses, the average synaptic weights are a smooth function of the transition probabilities and the accuracy of the encoding depends on the learning rate. As the number of synaptic states increases, the average synaptic weights become a step function of the transition probabilities. We finally show that the information stored in the synaptic weights can be read out by a simple rate-based neural network. Our study shows that synapses encode transition probabilities under general assumptions and this indicates that temporal contiguity is likely to be encoded and harnessed in almost every neural circuit in the brain.

  2. Caveolin-1 associated adenovirus entry into human corneal cells.

    Directory of Open Access Journals (Sweden)

    Mohammad A Yousuf

    Full Text Available The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC, caused by viruses within human adenovirus species D (HAdV-D, is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with

  3. Comparison of adenovirus viruria in bone marrow transplant patients before and after transplantation

    Directory of Open Access Journals (Sweden)

    H. Saderi

    2005-01-01

    Full Text Available Baekgrouund & purpose: In recent years, the role of adenoviruses in infection and disease in recipients of bone marrow transplantation (BMT has been studied. It suppose that adenoviral infections are prevalant in these patients Due to using medicines for preventing transplant rejection. This study was performed to compare the incidence of adenoviruses in urine samples taken before and after BMT from individuals undergoing BMT. In addition, The correlation between age, sex, etiology and kind of transplantation and adenovirus infection was studied.Materials and Methods: From 11 November 2002 to 12 June 2003, 91 patients received BMT in Hematology, Oncology and Bone Marrow Transplantation center of Tehran University of Medical Sciences. From 72 patients, 2 urine samples were taken before and 4 weeks after transplantation The DNA samples were examined by PCR using primers that detect 134 bp sequence in Hexon gene shared in human adenovirus DNA was extracted using Phenol-Chloroform method and concentrated by sodium acetate and ethanol.In Faculty of Medicine, Shahed University.Results: Adenovirus DNA was found in 39 patients (54.2% before transplantation and in 37 patients (51.4% after that. Both before and after BMT samples were negative for adenoviruses in 21 patients and positive in 25 patients. In 14 patients only before BMT and in 12 patients only after BMT urine samples were positive. No statistical difference between before and after BMT viruria was shown by McNemar’s test. Also, no statistical difference was shown between mean age of infected and non-infected patients by t-test. In spite of higher frequency of males, malignant and allogeneic transplant among studied patients, there was no statistical difference between two mentioned patients (P>0.05.Conclusion: In the present study, increase of prevalence of viruria in patients after bone marrow transplantation was not seen. In adition, there was no correlation between patients with various

  4. Adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro

    Institute of Scientific and Technical Information of China (English)

    Sheng Qiang; Zhen-Fang Du; Min Huang

    2014-01-01

    Objective: To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro. Methods: NDRG2 was harvested by RT-PCR, confirmed by DNA sequencing, and then cloned into the eukaryotic expression vector pIRES2-EGFP, which encodes green fluorescent protein (GFP), to construct pIRES2-EGFP-NDRG2 plasmid. OS-RC-2 cells with NDRG2 negative expression were transfected with pIRES2-EGFP-NDRG2 plasmid. The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes. After colony-forming cell assays, cell proliferation detection and MTT assays, the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells. Cell cycle was determined by flow cytometry. Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle. Results: A eukaryotic expression vector pIRES2-EGFP-NDRG2 was successfully constructed. After NDRG2 transfection, the growth of OS-RC-2 cells was inhibited. Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present, and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion. Conclusions: NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.

  5. THE BIOLOGICAL CHARACTERISTICS OF ADENOVIRUS-MEDIATED IL-18 GENE-MODIFIED MURINE COLORECTAL ADENOCARCINOMA CELL IN VIVO AND IN VITRO

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: Interleukin 18 (IL-18) is a strong activator of NK cells and promotes the generation of IL-2, IFN-g, and GM-CSF. In the present study, we constructed adenovirus encoding IL-18 gene (AdIL-18), and observed the biological characteristics of IL-18 gene-modified murine colorectal adenocarcinoma cell (CT26) in vivo and in vitro. Methods: Gene modification was mediated by adenovirus. The proliferation of the cells was determined by MTT and IL-18 was assayed by ELISA. The cytotoxicity of NK and CTL was detected by four-hour 51Cr release assay. Results: IL-18 gene modification had no effect on the proliferation and morphology of CT-26 cells in vitro, but the growth of IL-18-modified CT26 cells was obviously inhibited in vivo. In addition, although IL-18-modified CT26 cells could form tumor nodules in vivo as well as LacZ-modified CT26 cells or wild-type CT26 cells, the mean survival time of the mice inoculated with IL-18-modified CT26 cells was significantly prolonged as compared with that of control groups. Thus, the anti-tumor immune responses were induced in the group of mice inoculated with IL-18-modified CT26 cells, which might be related to the activation of NK cells and CTL. However, all the three groups ultimately died of tumor.free facility for all experiments. CT26, Yac-1 and 293 cells were from the American Type Culture Collection (ATCC, Manassas, VA). All cell lines were cultured in RPMI1640 (GIBCO-BRL, Grandisland, NY) supple-mented with penicillin (100 units/ml), streptomycin (100 mg/ml), 2-mercaptoethanol (5′10-5 M), and 10% FCS (GIBCO-BRL) at 37℃ in a humidified atmosphere of 5% CO2 in air.

  6. Isolation and identification of Adenovirus in hospitalized children, under five years, with acute respiratory disease, in Havana, Cuba

    Directory of Open Access Journals (Sweden)

    Tania Pumariega

    2000-12-01

    Full Text Available Nine Adenovirus (Ad strains isolated in Cuba, from 128 nasopharingeal swab specimens of children below five years old, with acute respiratory diseases, during 1996 and 1997, were studied by restriction enzyme analysis of genomic DNA with two endonucleases BamH I and Sma I. All different fragment patterns were compared with the respective prototypes. The identified adenoviruses were Ad 1 (n=4, Ad 2 (n=1 and Ad 6 (n=4. Males were more frequently infected than females. The analysis of the occurrence of these Adenovirus strains of subgenus C revealed that Ad 1 and Ad 6 were the predominant serotypes in 1996 and in 1997, respectively.

  7. Adenovirus, calicivirus and astrovirus detection in fecal samples of hospitalized children with acute gastroenteritis from Campo Grande, MS, Brazil

    Directory of Open Access Journals (Sweden)

    Marcia Sueli Assis Andreasi

    2008-11-01

    Full Text Available We analyzed fecal samples from hospitalized children up to three years of age with acute gastroenteritis at Campo Grande, Mato Grosso do Sul, Brazil, from May 2000-January 2004. Astrovirus and calicivirus were detected by Reverse Transcription-Polymerase Chain Reaction and adenovirus was detected using the Rotavirus and Adenovirus combined immunoenzyme assay. Astrovirus, adenovirus and calicivirus were detected at rates of 3.1%, 3.6% and 7.6%, respectively. These results re-emphasize the need for the establishment of regional vigilance systems to evaluate the impact of enteric viruses on viral gastroenteritis.

  8. [Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases].

    Science.gov (United States)

    Kiseleva, E K; Khil'ko, S N; Grigor'ev, V G; Diachenko, N S; Vantsak, N P

    1986-08-01

    Hexon capsomers of human adenovirus type 1 (h1) labeled by iodine 125 were digested in a native state (trimers) by trypsin, chymotrypsin or papain, and the resulting hydrolysates were analyzed by SDS-PAGE. In each case, a discrete and temporally stable pattern of relatively large fragments was revealed. The degree of hexon polypeptide hydrolysis was maximal for papain, intermediate for chymotrypsin and minimal for trypsin, the largest fragments in the digest being 32, 40 and 80 kD, respectively. At room temperature, all the electrophoretically discernible hexon proteolytical fragments were held together in structures resembling intact hexon trimers and could be regarded as "hexon cores", of which papain hexon cores were the most stable during SDS-PAGE. Radioimmunoprecipitation analysis revealed a complete absence of native hexon antigenicity in thermodenaturated fragments of hexon protease digests, while native trypsin, chymotrypsin and papain hexon cores could be precipitated by hexon-specific antibodies. The immunoprecipitated material contained all of the hexon fragments found in appropriate hexon cores and retained the structure of the original cores. Trypsin, chymotrypsin and papain hexon cores were shown to possess at least part of native Ad h1 hexon antigenic determinants of each of the following specificities: species-specific (epsilon), cross-reactive with hexon of human adenoviruses (h3 and h6), simian adenovirus (sim 16), bovine adenoviruses (bos 3 and bos 7) and avian adenovirus (Aviadenovirus gal 1 or CELO). Thus, the full spectrum of known hexon antigenic determinants (species-specific to intergenus-crossreactive) is at least portly stable against protease attack of native hexon capsomers.

  9. Experimental cross-species infection of common marmosets by titi monkey adenovirus.

    Directory of Open Access Journals (Sweden)

    Guixia Yu

    Full Text Available Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV, as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus. Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.

  10. The Revolution in Viral Genomics as Exemplified by the Bioinformatic Analysis of Human Adenoviruses

    Directory of Open Access Journals (Sweden)

    Sarah Torres

    2010-06-01

    Full Text Available Over the past 30 years, genomic and bioinformatic analysis of human adenoviruses has been achieved using a variety of DNA sequencing methods; initially with the use of restriction enzymes and more currently with the use of the GS FLX pyrosequencing technology. Following the conception of DNA sequencing in the 1970s, analysis of adenoviruses has evolved from 100 base pair mRNA fragments to entire genomes. Comparative genomics of adenoviruses made its debut in 1984 when nucleotides and amino acids of coding sequences within the hexon genes of two human adenoviruses (HAdV, HAdV–C2 and HAdV–C5, were compared and analyzed. It was determined that there were three different zones (1-393, 394-1410, 1411-2910 within the hexon gene, of which HAdV–C2 and HAdV–C5 shared zones 1 and 3 with 95% and 89.5% nucleotide identity, respectively. In 1992, HAdV-C5 became the first adenovirus genome to be fully sequenced using the Sanger method. Over the next seven years, whole genome analysis and characterization was completed using bioinformatic tools such as blastn, tblastx, ClustalV and FASTA, in order to determine key proteins in species HAdV-A through HAdV-F. The bioinformatic revolution was initiated with the introduction of a novel species, HAdV-G, that was typed and named by the use of whole genome sequencing and phylogenetics as opposed to traditional serology. HAdV bioinformatics will continue to advance as the latest sequencing technology enables scientists to add to and expand the resource databases. As a result of these advancements, how novel HAdVs are typed has changed. Bioinformatic analysis has become the revolutionary tool that has significantly accelerated the in-depth study of HAdV microevolution through comparative genomics.

  11. High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

    Institute of Scientific and Technical Information of China (English)

    CONG Tie-chuan; LU Zhe-ming; LI Yong; ZHENG Li; QIN Yong

    2007-01-01

    Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses.Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E.coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pacl and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000.Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture.After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk.Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

  12. p53/E1b58kDa complex regulates adenovirus replication.

    Science.gov (United States)

    Ridgway, P J; Hall, A R; Myers, C J; Braithwaite, A W

    1997-10-27

    We have explored a role for the adenovirus (Ad5) E1b58kDa/p53 protein complex in adenovirus replication. This was done by using virus mutants containing different defects in the E1b58kDa gene and cell lines that express either a wild-type p53 protein or a mutant p53 protein. We find that infection of wild-type p53-containing cells with wild-type Ad5 causes a shutoff of p53 and alpha-actin protein synthesis by distinct mechanisms, but neither occurs in mutant p53 cells. Our data also indicate that the shutoff is dependent on formation of the p53/E1b complex and may also involve another virus protein, E4ORF6. Following from these observations we asked whether failure to form the complex resulted in impaired adenovirus replication. Our experiments showed that neither wild-type Ad5 nor the E1b mutant dl338 could replicate in cells expressing a mutant p53 protein, but that wild-type adenovirus replicated well in wild-type p53-expressing cells. Collectively, our data suggest that the interaction between p53 and the E1b58kDa protein is necessary for efficient adenovirus replication. This is the first time such a direct link between the complex and virus replication has been demonstrated. These data raise serious questions about the usefulness of E1b-defective viruses in tumor therapy.

  13. Coxsackie and adenovirus receptor is a modifier of cardiac conduction and arrhythmia vulnerability in the setting of myocardial ischemia. : Coxsackie and adenovirus receptor is a modifier of cardiac conduction and arrhythmia vulnerability in the setting of myocardial ischemia.

    NARCIS (Netherlands)

    Marsman, Roos F; Bezzina, Connie R; Freiberg, Fabian; Verkerk, Arie O; Adriaens, Michiel E; Podliesna, Svitlana; Chen, Chen; Purfürst, Bettina; Spallek, Bastian; Koopmann, Tamara T; Baczko, Istvan; Dos Remedios, Cristobal G; George, Alfred L; Bishopric, Nanette H; Lodder, Elisabeth M; de Bakker, Jacques M; Fischer, Robert; Coronel, Ruben; Wilde, Arthur A; Gotthardt, Michael; Remme, Carol A

    2014-01-01

    The aim of this study was to investigate the modulatory effect of the coxsackie and adenovirus receptor (CAR) on ventricular conduction and arrhythmia vulnerability in the setting of myocardial ischemia.

  14. Co-expression of Erns and E2 genes of classical swine fever virus by replication-defective recombinant adenovirus completely protects pigs against virulent challenge with classical swine fever virus.

    Science.gov (United States)

    Sun, Yongke; Yang, Yuai; Zheng, Huanli; Xi, Dongmei; Lin, Mingxing; Zhang, Xiaomin; Yang, Linfu; Yan, Yulin; Chu, Xiaohui; Bi, Baoliang

    2013-04-01

    The objective of this study was to construct a recombinant adenovirus for future CSFV vaccines used in the pig industry for the reduction of losses involved in CSF outbreaks. The Erns and E2 genes of classical swine fever virus (CSFV), which encode the two main protective glycoproteins from the "Shimen" strain of CSFV, were combined and inserted into the replication-defective human adenovirus type-5 and named the rAd-Erns-E2. Nine pigs were randomly assigned to three treatment groups (three pigs in each group) including the rAd-Erns-E2, hAd-CMV control and DMEM control. Intramuscular vaccination with 2×10(6) TCID(50) of the rAd-Erns-E2 was administered two times with an interval of 21 days. At 42 days post inoculation, pigs in all groups were challenged with a lethal dose of 1×10(3) TCID(50) CSFV "Shimen" strain. Observation of clinical signs was made and the existence of CSFV RNA was detected. Animals in the hAd-CMV and DMEM groups showed severe clinical CSF symptoms and were euthanized from 7 to 10 days after the challenge. However, no adverse clinical CSF signs were observed in vaccinated pigs after the administration of rAd-Erns-E2 and even after CSFV challenge. Neither CSFV RNA nor pathological changes were detected in the tissues of interest of the above vaccinated pigs. These results implied that the recombination adenovirus carrying the Erns-E2 genes could be used to prevent swine from classical swine fever.

  15. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    Science.gov (United States)

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  16. Interleukin-encoding adenoviral vectors as genetic adjuvant for vaccination against retroviral infection.

    Directory of Open Access Journals (Sweden)

    Inga Ohs

    Full Text Available Interleukins (IL are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4(+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV surface envelope protein gp70 (Ad.pIXgp70 in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4(+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4(+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity.

  17. Interleukin-encoding adenoviral vectors as genetic adjuvant for vaccination against retroviral infection.

    Science.gov (United States)

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4(+) T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4(+) T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4(+) T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity.

  18. DETECTION OF INFECTIOUS ADENOVIRUS IN TERTIARY TREATED AND UV DISINFECTED WASTEWATER DURING A UV DISINFECTION PILOT STUDY

    Science.gov (United States)

    An infectious enteric adenovirus was isolated from urban wastewater receiving tertiary treatment and ultraviolet (UV) disinfection. A pilot study was undertaken to investigate the efficacy of UV disinfection (low pressure, high intensity radiation) of total and fecal coliform bac...

  19. Use of adenovirus vector expressing the mouse full estrogen receptor alpha gene to infect mouse primary neurons

    Institute of Scientific and Technical Information of China (English)

    Xiao HU; Lei Lou; Jun Yuan; Xing Wan; Jianyi Wang; Xinyue Qin

    2010-01-01

    Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation.Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to clarify the undedying mechanisms,in the present study,an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells.Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection,followed by 100 multiplicity of infection.Results showed overexpression of estrogen receptor a mRNA and protein in the infected nerve cells.Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.

  20. The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices

    Data.gov (United States)

    U.S. Environmental Protection Agency — The data to support the evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water...

  1. Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle.

    Science.gov (United States)

    Medina, Gisselle N; Montiel, Nestor; Diaz-San Segundo, Fayna; Sturza, Diego; Ramirez-Medina, Elizabeth; Grubman, Marvin J; de los Santos, Teresa

    2015-11-25

    Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4(+) and CD8(+) gamma interferon (IFN-γ)(+) cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle.

  2. Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle

    Science.gov (United States)

    Medina, Gisselle N.; Montiel, Nestor; Diaz-San Segundo, Fayna; Sturza, Diego; Ramirez-Medina, Elizabeth; Grubman, Marvin J.

    2015-01-01

    Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4+ and CD8+ gamma interferon (IFN-γ)+ cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle. PMID:26607309

  3. Efficient induction of cross-presentating human B cell by transduction with human adenovirus type 7 vector.

    Science.gov (United States)

    Peng, Ying; Lai, Meimei; Lou, Yunyan; Liu, Yanqing; Wang, Huiyan; Zheng, Xiaoqun

    2016-01-01

    Although human autologous B cells represent a promising alternative to dendritic cells (DCs) for easy large-scale preparation, the naive human B cells are always poor at antigen presentation. The safe and effective usage record of human adenovirus type 7 (HAdV7) live vaccines makes it attractive as a promising vaccine vector candidate. To investigate whether HAdV7 vector could be used to induce the human B cells cross-presentation, in the present study, we constructed the E3-defective recombinant HAdV7 vector encoding green fluorescent protein (GFP) and carcinoembryonic antigen (CEA). We demonstrated that naive human B cells can efficiently be transduced, and that the MAPKs/NF-κB pathway can be activated by recombinant HAdV7. We proved that cytokine TNF-α, IL-6 and IL-10, surface molecule MHC class I and the CD86, antigen-processing machinery (APM) compounds ERp57, TAP-1, and TAP-2. were upregulated in HAdV7 transduced human B cells. We also found that CEA-specific IFNγ expression, degranulation, and in vitro and ex vivo cytotoxicities are induced in autologous CD8(+) T cells presensitized by HAd7CEA modified human B cells. Meanwhile, our evidences clearly show that Toll-like receptors 9 (TLR9) antagonist IRS 869 significantly eliminated most of the HAdV7 initiated B cell activation and CD8(+) T cells response, supporting the role and contribution of TLR9 signaling in HAdV7 induced human B cell cross-presentation. Besides a better understanding of the interactions between recombinant HAdV7 and human naive B cells, to our knowledge, the present study provides the first evidence to support the use of HAdV7-modified B cells as a vehicle for vaccines and immunotherapy.

  4. Regeneration of adenovirus specific T-cells after allogeneic, hematopietic stem cell transplantation in children and adolescents

    OpenAIRE

    2010-01-01

    Adenovirus is a significant cause of morbidity and mortality in pediatric allogeneic hematopoietic stem cell transplant recipients, and control of infection seems to require antigen-specific T cells. Aim of this Work was to estimate the Regeneration of Adenovirus-specific T-cells In 26 children (Age 8 months -25 years) over 6 months after HSCT and to investigate the effects of the transplantations parameter, Virus reactivation and the Transplantations complications on the adv-specific cell...

  5. Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein.

    Science.gov (United States)

    Gonzalez, R; Vereecque, R; Wickham, T J; Vanrumbeke, M; Kovesdi, I; Bauters, F; Fenaux, P; Quesnel, B

    1999-03-01

    Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML.

  6. Molecular epidemiological study of adenovirus infecting western lowland gorillas and humans in and around Moukalaba-Doudou National Park (Gabon).

    Science.gov (United States)

    Nkogue, Chimène Nze; Horie, Masayuki; Fujita, Shiho; Ogino, Michiko; Kobayashi, Yuki; Mizukami, Keijiro; Masatani, Tatsunori; Ezzikouri, Sayeh; Matsuu, Aya; Mizutani, Tetsuya; Ozawa, Makoto; Yamato, Osamu; Ngomanda, Alfred; Yamagiwa, Juichi; Tsukiyama-Kohara, Kyoko

    2016-10-01

    Adenoviruses are widespread in human population as well as in great apes, although the data about the naturally occurring adenovirus infections remain rare. We conducted the surveillance of adenovirus infection in wild western lowland gorillas in Moukalaba-Doudou National Park (Gabon), in order to investigate naturally occurring adenovirus in target gorillas and tested specifically a possible zoonotic transmission with local people inhabiting the vicinity of the park. Fecal samples were collected from western lowland gorillas and humans, and analyzed by PCR. We detected adenoviral genes in samples from both gorillas and the local people living around the national park, respectively: the overall prevalence rates of adenovirus were 24.1 and 35.0 % in gorillas and humans, respectively. Sequencing revealed that the adenoviruses detected in the gorillas were members of Human mastadenovirus B (HAdV-B), HAdV-C, or HAdV-E, and those in the humans belonged to HAdV-C or HAdV-D. Although HAdV-C members were detected in both gorillas and humans, phylogenetic analysis revealed that the virus detected in gorillas are genetically distinct from those detected in humans. The HAdV-C constitutes a single host lineage which is compatible with the host-pathogen divergence. However, HAdV-B and HAdV-E are constituted by multiple host lineages. Moreover, there is no evidence of zoonotic transmission thus far. Since the gorilla-to-human transmission of adenovirus has been shown before, the current monitoring should be continued in a broader scale for getting more insights in the natural history of naturally occurring adenoviruses and for the safe management of gorillas' populations.

  7. Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

  8. Identification of a nonstructural DNA-binding protein (DBP as an antigen with diagnostic potential for human adenovirus.

    Directory of Open Access Journals (Sweden)

    Li Guo

    Full Text Available BACKGROUND: Human adenoviruses (HAdVs have been implicated as important agents in a wide range of human illnesses. To date, 58 distinct HAdV serotypes have been identified and can be grouped into six species. For the immunological diagnosis of adenoviruses, the hexon protein, a structural protein, has been used. The potential of other HAdV proteins has not been fully addressed. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a nonstructural antigenic protein, the DNA binding protein (DBP of human adenovirus 5 and 35 (Ad5, Ad35 - was identified using immunoproteomic technology. The expression of Ad5 and Ad35 DBP in insect cells could be detected by rhesus monkey serum antibodies and healthy adult human serum positive for Ad5 and Ad35. Recombinant DBPs elicited high titer antibodies in mice. Their conserved domain displayed immunological cross-reactions with heterologous DBP antibodies in Western blot assays. DBP-IgM ELISA showed higher sensitivity adenovirus IgM detection than the commercial Adenovirus IgM Human ELISA Kit. A Western blot method developed based on Ad5 DBP was highly consistent with (χ(2 = 44.9, P<0.01 the Western blot assay for the hexon protein in the detection of IgG, but proved even more sensitive. CONCLUSIONS/SIGNIFICANCE: The HAdV nonstructural protein DBP is an antigenic protein that could serve as an alternative common antigen for adenovirus diagnosis.

  9. Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    Hongyu Yang; Hong Qi; Junjie Zou; Xiwei Zhang

    2008-01-01

    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

  10. Encoding information into precipitation structures

    Science.gov (United States)

    Martens, Kirsten; Bena, Ioana; Droz, Michel; Rácz, Zoltan

    2008-12-01

    Material design at submicron scales would be profoundly affected if the formation of precipitation patterns could be easily controlled. It would allow the direct building of bulk structures, in contrast to traditional techniques which consist of removing material in order to create patterns. Here, we discuss an extension of our recent proposal of using electrical currents to control precipitation bands which emerge in the wake of reaction fronts in A+ + B- → C reaction-diffusion processes. Our main result, based on simulating the reaction-diffusion-precipitation equations, is that the dynamics of the charged agents can be guided by an appropriately designed time-dependent electric current so that, in addition to the control of the band spacing, the width of the precipitation bands can also be tuned. This makes straightforward the encoding of information into precipitation patterns and, as an amusing example, we demonstrate the feasibility by showing how to encode a musical rhythm.

  11. Geometric Hyperplanes: Desargues Encodes Doily

    CERN Document Server

    Saniga, Metod

    2011-01-01

    It is shown that the structure of the generalized quadrangle of order two is fully encoded in the properties of the Desargues configuration. A point of the quadrangle is represented by a geometric hyperplane of the Desargues configuration and its line by a set of three hyperplanes such that one of them is the complement of the symmetric difference of the remaining two and they all share a pair of non-collinear points.

  12. Vector Encoding in Biochemical Networks

    Science.gov (United States)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  13. Inhibitory Effect of Pulmonary Carcinoma by Adenovirus-Mediated CD/UPRT Gene

    Institute of Scientific and Technical Information of China (English)

    HUANG Qi; CHEN Dayu; FU Xiangning; ZU Yukun

    2006-01-01

    The cell killing effects and bystander effects of double suicide gene on pulmonary carcinoma cells were explored. Lung adenocarcinoma cells (A549) were transfected with different titers of adenovirus vector and followed with different concentrations of 5-FC after a recombinant adenovirus vector carrying CD/UPRT gene (Ad-CD/UPRT) was constructed. The cell viability was measured by MTT assay 4 days later. The cell viability was dropped to 30.57 %-8.62 % after 10 MOI of Ad-CD/UPRT transfected and 5-FC (10-1000 μg/mL) administration. Furthermore, Ad-CD/UPRT-infected A549 cells showed a profound neighbor cell killing effect in the same methods. These results suggested that Ad-CD/UPRT/5-FC system can effectively suppress growth of lung adenocarcinoma cells, which may provide a novel and powerful candidate for lung cancer gene therapy strategies.

  14. Future prospects for the development of cost-effective Adenovirus vaccines

    DEFF Research Database (Denmark)

    Fougeroux, Cyrielle; Holst, Peter J

    2017-01-01

    Vaccination is one of the most efficient tools for disease prevention, and a continuously growing field of research. However, despite progress, we still need more efficient and cost-effective vaccines that would improve access to those in need. In this review, we will describe the status of virus......-vectored vaccine technology with a focus on adenoviral-based vaccines. Adenovirus (Ad) vaccines have proven to be efficient in military vaccinations against Ad4 and Ad7 and as highly efficient vectored vaccines against rabies. The question of how other adenovirus-based vaccines can become as efficient...... as the rabies vaccine is the underlying theme in this review. Here, we will first give an overview of the basic properties of vectored vaccines, followed by an introduction to the characteristics of adenoviral vectors and previously tested modifications of the vector backbone and expression cassettes...

  15. Inclusion body hepatitis (IBH outbreak associated with fowl adenovirus type 8b in broilers

    Directory of Open Access Journals (Sweden)

    Zadravec M.

    2013-01-01

    Full Text Available The causative agent of inclusion body hepatitis (IBH was identified as fowl adenovirus (FAdV type 8b, a member of the Fowl adenovirus E species, based on PCR results of adenoviral polymerase and the hexon gene in an outbreak of acute mortality that affected a broiler flock of 12,000 animals. In two waves of elevated mortality rate, a total of 264 chickens were found dead. Affected birds showed ruffled feathers, depression, watery droppings and limping. The most common pathological lesions seen on necropsy were pale, swollen and friable livers. On histological examination, acute hepatitis characterized by necrosis of hepatocytes, with large basophilic intranuclear inclusion bodies, were observed. In addition, infectious bursal disease virus and infectious bronchitis virus were detected in the same flock.

  16. Identification of a novel adenovirus in a cotton-top tamarin (Saguinus oedipus).

    Science.gov (United States)

    Hall, Natalie H; Archer, Linda L; Childress, April L; Wellehan, James F X

    2012-03-01

    A novel adenovirus was identified in a cotton-top tamarin (Saguinus oedipus) with diarrhea by negative-staining electron microscopy of feces, consensus polymerase chain reaction, and sequencing. Partial sequences were obtained from the DNA-dependent DNA polymerase, the p52k gene, and the hexon. Bayesian and maximum likelihood phylogenetic analyses indicated that the virus is a member of the genus Mastadenovirus, and is herein termed Saguinus siadenovirus 1. The phylogeny of the mastadenoviruses is similar to that of their hosts, supporting coevolution. Support for this was strongest in the analysis of the predicted hexon protein. The obtained sequences were GC-rich, which may suggest a lack of recent host jumps. The diversity and evolution of the adenoviruses of platyrrhine primates merits further investigation. Additional study of the association of this virus with diarrhea is indicated.

  17. Adenovirus-mediated transfer of RA538 gene and its antitumor effect

    Institute of Scientific and Technical Information of China (English)

    程金科; 林晨; 隗玥; 张雪艳; 邢嵘; 牟巨伟; 王秀琴; 吴旻

    1999-01-01

    The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of cmyc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.

  18. Chronic Activation of Innate Immunity Correlates With Poor Prognosis in Cancer Patients Treated With Oncolytic Adenovirus.

    Science.gov (United States)

    Taipale, Kristian; Liikanen, Ilkka; Juhila, Juuso; Turkki, Riku; Tähtinen, Siri; Kankainen, Matti; Vassilev, Lotta; Ristimäki, Ari; Koski, Anniina; Kanerva, Anna; Diaconu, Iulia; Cerullo, Vincenzo; Vähä-Koskela, Markus; Oksanen, Minna; Linder, Nina; Joensuu, Timo; Lundin, Johan; Hemminki, Akseli

    2016-02-01

    Despite many clinical trials conducted with oncolytic viruses, the exact tumor-level mechanisms affecting therapeutic efficacy have not been established. Currently there are no biomarkers available that would predict the clinical outcome to any oncolytic virus. To assess the baseline immunological phenotype and find potential prognostic biomarkers, we monitored mRNA expression levels in 31 tumor biopsy or fluid samples from 27 patients treated with oncolytic adenovirus. Additionally, protein expression was studied from 19 biopsies using immunohistochemical staining. We found highly significant changes in several signaling pathways and genes associated with immune responses, such as B-cell receptor signaling (P immunity before treatment is associated with inferior survival in patients treated with oncolytic adenovirus. Conversely, lack of chronic innate inflammation at baseline may predict improved treatment outcome, as suggested by good overall prognosis.

  19. Establishment of an agamid cell line and isolation of adenoviruses from central bearded dragons (Pogona vitticeps).

    Science.gov (United States)

    Ball, Inna; Hoferer, Marc; Marschang, Rachel E

    2014-03-01

    A cell line was established from whole 6-8-week-old central bearded dragon (Pogona vitticeps) embryos. Cells were mid-sized and showed an elongated and polymorphic form. The cell line grew in a monolayer and has been serially passaged for 17 passages at time of publication. This cell line has been used with samples from adenovirus polymerase chain reaction (PCR)-positive bearded dragons, and 2 virus isolates have been obtained so far. The isolates show a clear cytopathic effect in inoculated cells. Both virus isolates have been serially passaged on this cell line, and have been identified by PCR amplification and sequencing of a portion of the DNA-dependent DNA polymerase gene and show 100% nucleotide identity to the corresponding region of an agamid adenovirus. Electron microscopic examination of supernatant from infected cells demonstrated the presence of nonenveloped particles, with a diameter of approximately 80 nm in both virus isolates.

  20. A retrospective investigation of canine adenovirus (CAV infection in adult dogs in Turkey : article

    Directory of Open Access Journals (Sweden)

    S. Gur

    2009-05-01

    Full Text Available Canine adenovirus (CAV type 1 and 2, respectively, cause infectious canine hepatitis and infectious canine laryngotracheitis in members of the families Canidae and Ursidae worldwide. Both of these infections are acute diseases, especially in young dogs. The aim of this study was to conduct a serological investigation of canine adenovirus infection. For this purpose, serumsamples were collected from native pure-bred Kangal (n = 11, and Akbash dogs (n = 17 and Turkish Greyhounds (n=15 in Eskisehir and Konya provinces. None ofthe dogs were previously vaccinated against CAV types. Indirect ELISA detected 88.2 %, 93.3 % and 100 % prevalences in Akbash, Greyhound and Kangal dogs, respectively. The remainder of the samples (n = 51 were collected at the Afyonkarahisar Municipality Shelter. Fourty-two of these dogs (82.3 % were detected as seropositive. In total, 82 of 94 dogs (87.2 % were found to be positive for CAV serum antibodies.

  1. Assessment of the Incidence of Enteric Adenovirus Species and Serotypes in Surface Waters in the Eastern Cape Province of South Africa: Tyume River as a Case Study

    Directory of Open Access Journals (Sweden)

    Timothy Sibanda

    2012-01-01

    Full Text Available TaqMan real-time PCR was used for the detection and quantitation of adenoviruses in Tyume River water samples over a 12-month period. A total of 72 samples were analysed, and 22 samples were positive for adenovirus. Of the positive samples, 18 were collected from downstream sampling points. Among the downstream sampling points, adenovirus detection rate increased with distance downstream, being 28%, 33%, and 39% for Alice, Drayini, and Manqulweni, respectively. The Alice sampling site had the highest concentrations of adenovirus ranging between 6.54×103 genome copies/L and 8.49×104 genome copies/L. The observed trend could have been expected considering the level of anthropogenic activities in areas along the lower stretch of Tyume River, with the major one being the effluent of treated and semi treated sewage from wastewater treatment facilities. Adenovirus detection was sporadic at most sampling sites. Multiplex conventional PCR was used for the detection of clinically important adenovirus species B, C, and F and their serotypes. Species C and F adenoviruses were detected in 77% and 18% of the samples, respectively. Most adenovirus positive samples were obtained from areas of increased population densities. The presence of adenoviruses may confirm the risk of its transmission to the human population.

  2. Trans activation of plasmid-borne promoters by adenovirus and several herpes group viruses.

    OpenAIRE

    Everett, R D; Dunlop, M

    1984-01-01

    This paper describes experiments to test the ability of a number of viruses of the Herpes group, and also Adenovirus-2 and SV40, to activate transcription from the Herpes simplex virus-1 glycoprotein D and the rabbit beta-globin promoters. Plasmids containing these genes were transfected into HeLa cells which were then infected with various viruses. Transcriptional activation in trans of the plasmid-borne promoters was monitored by quantitative S1 nuclease analysis of total cytoplasmic RNA is...

  3. Expression and function of DMT1 without IRE in C6 cells mediated by recombinant adenovirus

    Institute of Scientific and Technical Information of China (English)

    Xixun DU; Huamin XU; Hong JIANG; Jun WANG; Lei WANG; Junxia XIE

    2009-01-01

    Divalent metal transporter 1 (DMT1) is a ferrous iron import protein. The improper expression of DMT1 is involved in neurodegenerative diseases. In the present study, we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element (DMT1-IRE) and investigated its expression and function in the C6 glioma cell line. The DMTI-IRE gene, obtained by RT-PCR, was cloned into the shuttle plasmid-ing pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene. Linearized plasmid pAdTrack-CMV-DMTI-IRE was subsequently co-transformed into Escher-iehia coli (E. coli) BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with Pme I. Pac I-digested pAdEasy 1-DMT 1-IRE was then transfected into El-transformed human embryonic kidney cells (HEK293 cells), in which recombinant adenoviruses were generated within 7 to 10 days. The results demon-strated that we obtained the DMTI-IRE gene. pAdEasyl-DMT1-IRE yielded a large fragment, plus a smaller fragment of 4.5kb after digestion with Pac I. PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE, indicating the successful construction of recombi-nant adenovirus plasmid containing DMT1-IRE. GFP fluorescence further confirmed the generation of recombi-nant AdDMTI-IRE adenovirus. AdDMTI-IRE could efficiently infect C6 glioma cells. And cell viability decreased in AdDMT1-IRE infected cells after iron overload compared to the control. These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.

  4. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2

    OpenAIRE

    Zhang, Zheng; WANG, GUOXIAN; Li, Chen; Liu, Danping

    2013-01-01

    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2+ ...

  5. Comparative Studies on the Structure of Human Adenovirus Genomes 4, 7 and 21.

    Science.gov (United States)

    1980-02-01

    the DNA was tested by labeling the protein moiety with [ I ] using chloramine T a t e oxidizing agent (11). This procedure labels the tyrosine residue...Isolation and Characterization of Adenovirus Type 7 DNA-terminal protein complex 5 4) Physical Mapping of Ad7 Genome with several restriction enzymes 7 5...We purified the Ad 7 DNA- protein complex to establish conditions for DNA- transfection in Human Embryonic Kidney cells in vitro. We labeled the protein

  6. Differential Innate Immune Stimulation Elicited by Adenovirus and Poxvirus Vaccine Vectors

    OpenAIRE

    Teigler, Jeffrey Edward

    2014-01-01

    Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited b...

  7. Calcium gluconate in phosphate buffered saline increases gene delivery with adenovirus type 5.

    Directory of Open Access Journals (Sweden)

    Marko T Ahonen

    Full Text Available BACKGROUND: Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. METHODS/RESULTS: We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. CONCLUSION: In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.

  8. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    Science.gov (United States)

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad

    2010-01-01

    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  9. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Directory of Open Access Journals (Sweden)

    Ling Zhang

    Full Text Available Recombinant interferon-γ (IFNγ production in cultured lentivirus (LV was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5. The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041, and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032, which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  10. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Science.gov (United States)

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle

    2013-01-01

    MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  11. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Directory of Open Access Journals (Sweden)

    Erkko Ylösmäki

    Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  12. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Science.gov (United States)

    Zhang, Ling; Yin, Sen; Tan, Wanlong; Xiao, Dong; Weng, Yunceng; Wang, Wenjing; Li, Tingting; Shi, Junwen; Shuai, Lifang; Li, Hongwei; Zhou, Jianhua; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  13. Functional role of the Coxsackie and Adenovirus Receptor (CAR) in colorectal carcinomas

    OpenAIRE

    Küster, Katrin

    2009-01-01

    Der Coxsackie- und Adenovirus Rezeptor (CAR) ist als Bestandteil von Tight Junctions (TJ) an interzellulären Adhäsionsprozessen beteiligt und scheint eine wichtige Rolle in der Karzinogenese zu spielen. Diese ist jedoch insbesondere bei Entstehung von Darmkrebs weitgehend unklar. Ziel der Arbeit war es daher, die funktionelle Bedeutung, mögliche Interaktionspartner sowie die Expressionsregulation von CAR im kolorektalen Karzinom zu analysieren. In den Zelllinien CaCo2, Colo205, DLD1, HCT116, ...

  14. Interaction between mouse adenovirus type 1 and cell surface heparan sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Liesbeth Lenaerts

    Full Text Available Application of human adenovirus type 5 (Ad5 derived vectors for cancer gene therapy has been limited by the poor cell surface expression, on some tumor cell types, of the primary Ad5 receptor, the coxsackie-adenovirus-receptor (CAR, as well as the accumulation of Ad5 in the liver following interaction with blood coagulation factor X (FX and subsequent tethering of the FX-Ad5 complex to heparan sulfate proteoglycan (HSPG on liver cells. As an alternative vector, mouse adenovirus type 1 (MAV-1 is particularly attractive, since this non-human adenovirus displays pronounced endothelial cell tropism and does not use CAR as a cellular attachment receptor. We here demonstrate that MAV-1 uses cell surface heparan sulfate proteoglycans (HSPGs as primary cellular attachment receptor. Direct binding of MAV-1 to heparan sulfate-coated plates proved to be markedly more efficient compared to that of Ad5. Experiments with modified heparins revealed that the interaction of MAV-1 to HSPGs depends on their N-sulfation and, to a lesser extent, 6-O-sulfation rate. Whereas the interaction between Ad5 and HSPGs was enhanced by FX, this was not the case for MAV-1. A slot blot assay demonstrated the ability of MAV-1 to directly interact with FX, although the amount of FX complexed to MAV-1 was much lower than observed for Ad5. Analysis of the binding of MAV-1 and Ad5 to the NCI-60 panel of different human tumor cell lines revealed the preference of MAV-1 for ovarian carcinoma cells. Together, the data presented here enlarge our insight into the HSPG receptor usage of MAV-1 and support the development of an MAV-1-derived gene vector for human cancer therapy.

  15. Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

    Institute of Scientific and Technical Information of China (English)

    PAN Xin-ting; ZHU Qing-yun; LI De-chun; YANG Ji-cheng; ZHANG Zi-xiang; ZHU Xing-guo; ZHAO Hua

    2008-01-01

    Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhlL-24) on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n=10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0x1010 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P <0.05). The intratumoral MVD decreased significantly in the treated tumors (P <0.05).Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

  16. Adenovirus Vectors Expressing Hantavirus Proteins Protect Hamsters against Lethal Challenge with Andes Virus ▿

    OpenAIRE

    2009-01-01

    Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdGN and AdGC) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infe...

  17. Murine adenovirus infection of SCID mice induces hepatic lesions that resemble human Reye syndrome.

    OpenAIRE

    Pirofski, L.; Horwitz, M S; Scharff, M. D.; Factor, S. M.

    1991-01-01

    Murine adenovirus type 1 (MAV-1) infection of CB-17 SCID mice (which are homozygous for the severe combined immunodeficiency mutation) induces hepatic histopathologic and ultrastructural features that are strikingly similar to human Reye syndrome. Gross pathologic examination of MAV-1-infected mice revealed only pale yellow liver tissue. Histopathologic studies of tissue from MAV-1-infected mice revealed diffuse hepatic injury manifested by microvesicular fatty degenerative changes of hepatoc...

  18. Phylogenetic analysis of the genomes of two strains of human adenovirus type 3

    Institute of Scientific and Technical Information of China (English)

    RONG ZHOU; XIAO Bo SU; QI WEI ZIIANG; QI YI ZENG; BING ZHU; CHU Yu ZHANG; Hou Bo WU; ZAO HE WU; SI TANG GONG

    2007-01-01

    Human adenovirus type 3 (HAdV-3) is widely prevalent all over the world, especially in Asia. The objective of this study is to carry out complete genomic DNA sequencing and the phylogenetic analysis for two strains (Guangzhou01 and Guangzhou02) of HAdV-3 wild virus isolated from South China. Nasopharyngeal secretion aspirate specimens of sick children were inoculated into HEp-2 and HeLa culture tubes, and the cultures were identified by neutralization assay with type-specific reference rabbit antisermn. Type-specific primers were also utilized to confirm the serotype. The restriction fragments of HAdV genome DNA were cloned into pBlueScript SK ( + ) vectors and sequenced, and the 5' and 3'ends of the linear HAdV-3 genome were directly sequenced with double purified genomic DNA as templates. General features of the HAdV-3 genome sequences were explored by using several bio-software.Phylogenetic analysis was done with MEGA 3.0 software. The genomic sequences of Guangzhou01 and Guangzhou02 possess the same 4 early regions and 5 late regions and have 39 ceding sequences and two RNA coding sequences. Other non-ceding regions are conservative. Inverted repeats and palindromes were identified in the genome sequences. The genomes of group B human adenovirus as well as HAdV-3have close phylogenetic relationship with that of chimpanzee adenovirus type 21. The genomie lengths of these two isolated strains are 35 273 bp and 35 269 bp, respectively. The phylogenetie analysis showed that HAdV-B species has some relationship with eertain types of chimpanzee adenovirus.

  19. Acute Respiratory Disease in US Army Trainees 3 Years after Reintroduction of Adenovirus Vaccine

    Science.gov (United States)

    2017-01-15

    hospitalized (5). Routine use of oral adenovirus type 4 and 7 (AdV- 4 and -7) vaccine began in 1971 and eventually included year-round vaccination, resulting in...use of benzathine penicil- lin G prophylaxis for group A β-hemolytic streptococcus infections coupled with a surveillance artifact introduced when...1,100–2,700 hospitalizations , and 13,000 febrile infections among military recruits. Vaccination costs $150 per person, pro- viding a net savings of

  20. Antitumor bioactivity of adenovirus-mediated p27mt in colorectal cancer cell line SW480

    Institute of Scientific and Technical Information of China (English)

    Ze-Qun sun; Chang-Sheng Deng; Shao-Yong Xu; Yong Du

    2008-01-01

    AIM: To explore the antitumor bioactivity of adenovirus-mediated mutant type p27kip1 gene in a colorectal cancer cell line SW480.METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27kip1 gene (ad-p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber end SW480 tumor cell growth inhibition in vitro and in vivo.RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ed-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G1/S arrest in SW480 cells and inhibited celt migration.CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.

  1. Prevalence of Rotavirus, Adenovirus, Norovirus, and Astrovirus Infections and Coinfections among Hospitalized Children in Northern France▿

    Science.gov (United States)

    Tran, Adissa; Talmud, Déborah; Lejeune, Benoît; Jovenin, Nicolas; Renois, Fanny; Payan, Christopher; Leveque, Nicolas; Andreoletti, Laurent

    2010-01-01

    From January to December 2007, 973 stool specimens were prospectively collected from children hospitalized for gastroenteritis signs or from neonates and premature cases who were born in two French hospital settings in the north of France. They were tested by rapid enzyme immunoassay (EIA) analyses for rotavirus and adenovirus and by two commercially available ELISA tests for the detection of norovirus and astrovirus. The overall rates of prevalence for rotavirus, norovirus, adenovirus, and astrovirus were 21, 13, 5, and 1.8%, respectively, and they did not significantly differ between the two hospital settings (P = 0.12). Mixed virus infections were detected in 32 (3.3%) of the 973 study children and were associated with norovirus in 21 (66%) infants, including 5 premature cases. From fall to spring, norovirus infections accounted for 52% of documented gastroenteritidis viral infections at a time when rotavirus was epidemic, resulting in mixed norovirus and rotavirus gastrointestinal tract infections. Of the 367 documented viral gastroenteritis cases, 15 (4.1%) were identified as nosocomial infections, 5 of which occurred in premature cases. These findings highlight the need to implement norovirus and astrovirus ELISA detection assays in association with rapid EIA rotavirus and adenovirus detection assays for the clinical diagnosis and the nosocomial prevention of gastroenteritis viral infections in pediatric departments. PMID:20305010

  2. An armed oncolytic adenovirus system,ZD55-gene,demonstrating potent antitumoral efficacy

    Institute of Scientific and Technical Information of China (English)

    ZI LAI ZHANG; WEI GUO ZOU; CHUN XIA LUO; BING HUA LI; JIN HUI WANG; LAN YING SUN; QI JUN QIAN; XIN YUAN LIU

    2003-01-01

    ONYXONYX-015 is an attractive therapeutic adenovirus for cancer because it can selectively replicate in tumor cells and kill them.To date,clinicaltrials of this adenovirus have demonstrated marked safety but not potent enough when it was used alone.In this paper,we put forward a novel concept of Gene-Viro Therapy strategy and in this way,we constructed an armed therapeutic onco1ytic adenovirus system,ZD55-gene,whichis not only deleted of E1B 55-kD gene similar to ONYX-015,but also armed with foreign antitumor gene.ZD55-gene exhibited similar cytopathic effects and replication Kinetics to that of ONYX-015 in vitro.Importantly,the carried gene 1s expressed and the expression level can increase with the replication of virus.Consequently,a significant antitumoral efficacy was observed when ZD55-CD/5-FU was used as an example in nude mice with subcutaneous human SW620 colon cancer.Our data demonstratedthat ZD55-gene,which utilizingthe Gene-ViroTherapy strategy,is more efficacious than each individual component in vivo.

  3. Stimulation of innate immunity by in vivo cyclic di-GMP synthesis using adenovirus.

    Science.gov (United States)

    Koestler, Benjamin J; Seregin, Sergey S; Rastall, David P W; Aldhamen, Yasser A; Godbehere, Sarah; Amalfitano, Andrea; Waters, Christopher M

    2014-11-01

    The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

  4. Adenovirus KH901 promotes 5-FU antitumor efficacy and S phase in LoVo cells.

    Science.gov (United States)

    Peng, Wei; Li, Jin; Yin, X G; Xu, J F; Cheng, L Z

    2012-06-01

    A combination of oncolytic and chemotherapeutic agents has been used to kill cancer cells. However, the effect of oncolytic adenoviruses on the cell cycle remains to be determined. Cytotoxicity assays were performed to determine cell death in cells treated with 5-fluorouracil (5-FU) alone or in combination with the oncolytic adenovirus KH901. Dynamic changes in the cell cycle, cell proliferation, and apoptosis-related proteins including p-AKT, Bcl-2, Bax, and caspase 3 were investigated after treatment with 5-FU with or without KH901. A higher proportion of S-phase cells were observed after treatment with KH901 and 5-FU than with 5-FU alone. p-AKT, Bcl-2, and Bax expression was increased upon treatment with KH901, whereas the expression of caspase-3 was not induced upon treatment with KH901 with or without 5-FU. KH901 exhibited significant potential as an oncolytic adenovirus and increased cell death in combination with 5-FU in LoVo cells, as compared to 5-FU alone. In conclusion, KH901 stimulates LoVo cells to enter the S-phase by activation of p-AKT, which could partly explain its synergistic effect with 5-FU on LoVo cell cytotoxicity.

  5. A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein.

    Science.gov (United States)

    Fingerut, E; Gutter, B; Gallili, G; Michael, A; Pitcovski, J

    2003-06-20

    In this study, the effectiveness of antibodies against the hexon, fiber or a fiber fragment of an avian adenovirus egg-drop syndrome (EDS), in neutralizing the virus was tested. The fiber protein is responsible for binding the virus to the target cell. The fiber fragment knob-s comprises the carboxy-terminal knob domain and 34 amino acids of the immediately adjacent shaft domain of the adenovirus fiber protein. The hexon, fiber capsid protein and knob-s were produced in E. coli and injected into chickens. Antibodies that were produced against the whole fiber protein showed some hemagglutination inhibition (HI) activity. Antibodies produced against the knob-s protein showed HI activity and serum neutralization (SN) activity similar to the positive control-whole virus vaccine. We assume that production of only part of the fiber enables the protein produced in E. coli to fold correctly. Antibodies produced against the hexon protein showed no SN activity. In summary, knob-s induced SN and HI antibodies against EDS virus at a rate similar to the whole virus and were significantly more efficient than the full-length fiber. The recombinant knob-s protein may be used as a vaccine against pathogenic adenovirus infections.

  6. The Serological and Virological Investigation of Canine Adenovirus Infection on the Dogs

    Directory of Open Access Journals (Sweden)

    Oya Bulut

    2013-01-01

    Full Text Available Two types of Canine Adenovirus (CAVs, Canine Adenovirus type 1 (CAV-1, the virus which causes infectious canine hepatitis, and Canine Adenovirus type 2 (CAV-2, which causes canine infectious laryngotracheitis, have been found in dogs. In this study, blood samples taken from 111 dogs, which were admitted to the Internal Medicine Clinic of Selcuk University, Faculty of Veterinary Medicine, with clinical symptoms. Seventy-seven dogs were sampled from Isparta and Burdur dog shelters by random sampling, regardless of the clinical findings. Dogs showed a systemic disease, characterized by fever, diarrhea, vomiting, oculonasal discharge, conjunctivitis, severe moist cough, signs of pulmonary disease and dehydration. Two dogs had corneal opacity and photophobia. In serological studies, 188 serum samples were investigated on the presence of CAV antibodies by ELISA. Total 103 (103/188–54.7% blood samples were detected to be positive for CAV antibodies by ELISA. However, 85 (85/188–45.2% blood samples were negative. Blood leukocyte samples from dogs were processed and inoculated onto confluent monolayers of MDCK cells using standard virological techniques. After third passage, cells were examined by direct immunoflourescence test for virus isolation. But positive result was not detected. In conclusion, this study clearly demonstrates the high prevalence of CAV infection in dogs.

  7. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Directory of Open Access Journals (Sweden)

    Sandy Ibanes

    Full Text Available When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2 vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  8. Construction and in vitro Study of an E1B-Defective Adenovirus

    Institute of Scientific and Technical Information of China (English)

    Xue Feng; Joshua Mallam Nock; Zhu Hua-bin; Dong Chang-yuan; Qi Yi-peng

    2004-01-01

    An E1B-defective adenovirus named r1/Ad was constructed by homologous recombination. The construction, selection and propagation of recombinant virus was done in the human embryonic kidney 293 cells (HEK293). The in vitro study demonstrated that the recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as the human glioblastoma tumor cells (U251) and human bladder tumer cells (EJ) but not in the normal cells with functional p53 such as the human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated that the U251 cells were more sensitive to the infection of r1/Ad than that of EJ cells under identical conditions. In this paper, it was found that r1/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus. This may help to determine the safety of using any E1B-defective adenoviruses in cancer gene therapy.

  9. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Science.gov (United States)

    Ibanes, Sandy; Kremer, Eric J

    2013-01-01

    When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  10. Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41.

    Science.gov (United States)

    Lemiale, Franck; Haddada, Hedi; Nabel, Gary J; Brough, Douglas E; King, C Richter; Gall, Jason G D

    2007-03-01

    Replication-defective adenovirus vectors, primarily developed from serotype 5 (Ad5) viruses, have been widely used for gene transfer and vaccination approaches. Vectors based on other serotypes of adenovirus could be used in conjunction with, or in place of, Ad5 vectors. In this study, Ad41, an enteric adenovirus usually described as 'non-cultivable' or 'fastidious,' has been successfully cloned, rescued and propagated on 293-ORF6 cells. The complementation capabilities of this cell line allow generation of Ad41 vectors at titers comparable to those obtained for Ad5 vectors. Mice immunized with an Ad41 vector containing an HIV envelope (Env) gene mounted anti-Env cellular and humoral immune responses. Ad41-Env vectors appear to be particularly attractive when used in heterologous prime-boost regimens, where they induce significantly higher cellular immune responses to HIV-Env than Ad5-based regimens. Ad41-based constructs are attractive vaccine vectors alone or in combination with Ad5 adenovectors, since each vector type can provide circumvention of pre-existing immunity to the other.

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

    Directory of Open Access Journals (Sweden)

    Ashish V Chintakuntlawar

    2010-04-01

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

  12. Combination of adenovirus and cross-linked low molecular weight PEI improves efficiency of gene transduction

    Energy Technology Data Exchange (ETDEWEB)

    Han Jianfeng; Zhao Dong; Zhong Zhirong; Zhang Zhirong; Gong Tao; Sun Xun, E-mail: xunsun22@gmail.com [Key Laboratory of Drug Targeting and Novel Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, 610041 (China)

    2010-03-12

    Recombinant adenovirus (Ad)-mediated gene therapy is an exciting novel strategy in cancer treatment. However, poor infection efficiency with coxsackievirus and adenovirus receptor (CAR) down-regulated cancer cell lines is one of the major challenges for its practical and extensive application. As an alternative method of viral gene delivery, a non-viral carrier using cationic materials could compensate for the limitation of adenovirus. In our study, adenovectors were complexed with a new synthetic polymer PEI-DEG-bis-NPC (PDN) based on polyethylenimine (PEI), and then the properties of the vehicle were characterized by measurement of size distribution, zeta potential and transmission electron microscopy (TEM). Enhancement of gene transduction by Ad/PDN complexes was observed in both CAR-overexpressing cell lines (A549) and CAR-lacking cell lines (MDCK, CHO, LLC), as a result of facilitating binding and cell uptake of adenoviral particles by the cationic component. Ad/PDN complexes also promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. These data suggest that a combination of viral and non-viral gene delivery methods may offer a new approach to successful cancer gene therapy.

  13. EXPRESSION AND GLYCOSYLATION OF ROTAVIRUS STRAIN SAIl VP4 PROTEIN IN A RECOMBINANT ADENOVIRUS

    Institute of Scientific and Technical Information of China (English)

    孙茂盛; 昝云红; 马雁冰; 张光明; 杜秋江; 戴长柏

    2001-01-01

    Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal.The chimera gene Was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co-transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation. Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed productin recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence as-say. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosyla-tion of VP4 protein. Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicityand immunogenicity of expressed protein.

  14. Application of conditionally replicating adenoviruses in tumor early diagnosis technology, gene-radiation therapy and chemotherapy.

    Science.gov (United States)

    Li, Shun; Ou, Mengting; Wang, Guixue; Tang, Liling

    2016-10-01

    Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future.

  15. Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.

    Science.gov (United States)

    Doucas, V; Ishov, A M; Romo, A; Juguilon, H; Weitzman, M D; Evans, R M; Maul, G G

    1996-01-15

    Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins.

  16. Bak and Bax function to limit adenovirus replication through apoptosis induction.

    Science.gov (United States)

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-05-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.

  17. Adenovirus-mediated sphingomyelin synthase 2 increases atherosclerotic lesions in ApoE KO mice

    Directory of Open Access Journals (Sweden)

    Zhao Yarui

    2011-01-01

    Full Text Available Abstract Background Sphingomyelin synthase 2 (SMS2 contributes to de novo sphingomyelin (SM biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis. Methods The Adenovirus containing SMS2 gene was given into 10-week ApoE KO C57BL/6J mice by femoral intravenous injection. In the control group, the Adenovirus containing GFP was given. To confirm this model, we took both mRNA level examination (RT-PCR and protein level examination (SMS activity assay. Result We generated recombinant adenovirus vectors containing either human SMS2 cDNA (AdV-SMS2 or GFP cDNA (AdV-GFP. On day six after intravenous infusion of 2 × 1011 particle numbers into ten-week-old apoE KO mice, AdV-SMS2 treatment significantly increased liver SMS2 mRNA levels and SMS activity (by 2.7-fold, 2.3-fold, p Conclusions Our results present direct morphological evidence for the pro-atherogenic capabilities of SMS2. SMS2 could be a potential target for treating atherosclerosis.

  18. THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Jiang Lei; Xia Yongjing; Li Hongxia; Hu Yajun; Hu Shixue; Xu Hongji

    1998-01-01

    Objective:To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene,on the growth of lung adenocarcinoma cell line GLC-82 and explore a gene therapy approach for lung adenocarcinoma. Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method. Infection effects were detected by biochemical staining of β-gal and immunohistochemical analysis of Rb protein. The Rb cDNA of infected cells were determined by PCR. The cell growth rate and cell cycle were observed by cell-counting and flow cytometry. Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein. The transfection of wild-type Rb gene could suppress GLC-82 cell proliferation and decrease the cellular DNA synthesis. Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer.

  19. CAR chasing: canine adenovirus vectors-all bite and no bark?

    Science.gov (United States)

    Kremer, Eric J

    2004-02-01

    This review deals primarily with canine adenovirus serotype 2 (CAV-2) vectors and gives a simplified overview of how the various domains of virology, cellular and molecular biology, as well as immunology, come into play when trying to understand and ameliorate adenovirus (Ad)-mediated gene transfer. The generation of early region 1 (E1)-deleted (DeltaE1) CAV-2 vectors, the lack of pre-existing humoral immunity, trafficking, the use of the coxsackie B adenovirus receptor (CAR), the surprising neuronal tropism, and the ability to migrate via axons to afferent regions of the central and peripheral nervous system, are described. Due to these intrinsic properties, CAV-2 vectors may be powerful tools for the study of the pathophysiology and potential treatment of neurodegenerative diseases like lysosomal storage disorders, Parkinson's, Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and others. Other potential uses include anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, pain therapy, cancer therapy (e.g. K9 CRAds), and gene transfer to other somatic tissues.

  20. Serologic study on the outbreak of acute upper respiratory tract Infections caused by adenovirus 3

    Institute of Scientific and Technical Information of China (English)

    JIANG Lufang; JU Liwen; JIANG Renjie; LIN Yuzun; ZHOU Liandi; YU Shunzhang; JIANG Qingwu

    2007-01-01

    From April to June,2004,an outbreak of acute upper respiratory tract infections(AURTI)occurred in the north area of Jiangsu Province,China.Twenty throat swabs were collected with 13 of them presenting an adenovirus (Ad)-like cytopathogenic effect on HEp-2.These were verified as Ad by the electron microscope,direct immunofluorescence assay and Ad primer-mediated PCR.Moreover,they were identified as adenovirus type 3(Ad3)by type-specific PCR and sequencing of the amplification products.Subsequent serologic studies were carried out to finally diagnose and document the outbreak.The neutralization test of paired serum of six in nine cases show obviously increased antibodies titers.The positive rate of IgM,IgG and recovery phase neutralization antibodies of the cases were 3.7%,44.4%and 59.5%respectively while those of the controls were 0%,8.3%and 33.3%respectively.The Pvalues of Chi-Square were 0.510,0.018 and 0.226 respectively.The concordance between IgG detected by ELISA and neutralization antibodies detected by the neutralization test was 61.4%and the Pvalue of Kappa was 0.070.By the serologic study,we can definitively diagnose that this outbreak of acute respiratory infections was caused by Adenovirus 3.

  1. Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation.

    Science.gov (United States)

    Bounty, Sarah; Rodriguez, Roberto A; Linden, Karl G

    2012-12-01

    Adenovirus has consistently been observed to be the most resistant known pathogen to disinfection by ultraviolet light. This has had an impact on regulations set by the United States Environmental Protection Agency regarding the use of UV disinfection for virus inactivation in groundwater and surface water. In this study, enhancement of UV inactivation of adenovirus was evaluated when hydrogen peroxide was added to create an advanced oxidation process (AOP). While 4 log reduction of adenovirus was determined to require a UV dose (UV fluence) of about 200 mJ/cm(2) from a low pressure (LP) UV source (emitting at 253.7 nm), addition of 10 mg/L H(2)O(2) achieved 4 log inactivation at a dose of 120 mJ/cm(2). DNA damage was assessed using a novel nested PCR approach, and similar levels of DNA damage between the two different treatments were noted, suggesting the AOP enhancement in inactivation was not due to additional DNA damage. Hydroxyl radicals produced in the advanced oxidation process are likely able to damage parts of the virus not targeted by LPUV, such as attachment proteins, enhancing the UV-induced inactivation. The AOP-enhanced inactivation potential was modeled in three natural waters. This research sheds light on the inactivation mechanisms of viruses with ultraviolet light and in the presence of hydroxyl radicals and provides a practical means to enhance inactivation of this UV-resistant virus.

  2. E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

    Energy Technology Data Exchange (ETDEWEB)

    Turner, Roberta L. [Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Wilkinson, John C., E-mail: john.wilkinson@ndsu.edu [Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Ornelles, David A., E-mail: ornelles@wakehealth.edu [Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States)

    2014-05-15

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.

  3. The cleaved N-terminus of pVI binds peripentonal hexons in mature adenovirus.

    Science.gov (United States)

    Snijder, Joost; Benevento, Marco; Moyer, Crystal L; Reddy, Vijay; Nemerow, Glen R; Heck, Albert J R

    2014-05-01

    Mature human adenovirus particles contain four minor capsid proteins, in addition to the three major capsid proteins (penton base, hexon and fiber) and several proteins associated with the genomic core of the virion. Of the minor capsid proteins, VI plays several crucial roles in the infection cycle of the virus, including hexon nuclear targeting during assembly, activation of the adenovirus proteinase (AVP) during maturation and endosome escape following cell entry. VI is translated as a precursor (pVI) that is cleaved at both N- and C-termini by AVP. Whereas the role of the C-terminal fragment of pVI, pVIc, is well established as an important co-factor of AVP, the role of the N-terminal fragment, pVIn, is currently elusive. In fact, the fate of pVIn following proteolytic cleavage is completely unknown. Here, we use a combination of proteomics-based peptide identification, native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to show that pVIn is associated with mature human adenovirus, where it binds at the base of peripentonal hexons in a pH-dependent manner. Our findings suggest a possible role for pVIn in targeting pVI to hexons for proper assembly of the virion and timely release of the membrane lytic mature VI molecule. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Clinical data analysis of 19 cases of community-acquired adenovirus pneumonia in immunocompetent adults.

    Science.gov (United States)

    Yu, Hong-Xia; Zhao, Mao-Mao; Pu, Zeng-Hui; Wang, Yun-Qiang; Liu, Yan

    2015-01-01

    The aim of this study was to investigate the characteristics of clinical manifestations, laboratory tests and imaging changes of community-acquired adenovirus pneumonia in immunocompetent adults. A retrospective study was performed on 19 adult community-acquired adenovirus pneumonia cases in Yantai, whereby the clinical data were collected and analyzed. Of 19 cases, 14 (73.68%) had fever and 17 (89.47%) had cough symptoms. Moreover, 14 cases (73.68%) had normal white blood cell counts, while 11 cases (57.89%) exhibited a reduction in lymphocyte proportion. Among the 19 cases, 17 cases exhibited lesions in a single lung, while 2 cases involved bilateral lungs. The lesions predominantly exhibited ground glass-like changes. The clinical manifestations of adult community-acquired adenovirus pneumonia patients with normal immune functions were mild, with such presenting symptoms as fever, cough, and sputum; most patients did not exhibit high levels of white blood cells or low lymphocyte counts, and the imaging features (ground glass-like effusion) were indicative of single-lung involvement.

  5. Adenovirus-mediated eNOS expression augments liver injury after ischemia/reperfusion in mice.

    Directory of Open Access Journals (Sweden)

    Arun P Palanisamy

    Full Text Available Hepatic ischemia/reperfusion (l/R injury continues to be a critical problem. The role of nitric oxide in liver I/R injury is still controversial. This study examines the effect of endothelial nitric oxide synthase (eNOS over-expression on hepatic function following I/R. Adenovirus expressing human eNOS (Ad-eNOS was administered by tail vein injection into C57BL/6 mice. Control mice received either adenovirus expressing LacZ or vehicle only. Sixty minutes of total hepatic ischemia was performed 3 days after adenovirus treatment, and mice were sacrificed after 6 or 24 hrs of reperfusion to assess hepatic injury. eNOS over expression caused increased liver injury as evidenced by elevated AST and ALT levels and decreased hepatic ATP content. While necrosis was not pervasive in any group, TUNEL demonstrated significantly increased apoptosis in Ad-eNOS infected livers. Western blotting demonstrated increased levels of protein nitration and upregulation of the pro-apoptotic proteins bax and p53. Our data suggest that over-expression of eNOS is detrimental in the setting of hepatic I/R.

  6. EXPRESSION AND GLYCOSYLATION OF ROTAVIRUS STRAIN SA11 VP4 PROTEIN IN A RECOMBINANT ADENOVIRUS

    Institute of Scientific and Technical Information of China (English)

    孙茂盛; 昝云红; 马雁冰; 张光明; 杜秋江; 戴长柏

    2001-01-01

    Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain.``Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terninal.``The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co-transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation.``Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein.``Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.``

  7. Hall effect encoding of brushless dc motors

    Science.gov (United States)

    Berard, C. A.; Furia, T. J.; Goldberg, E. A.; Greene, R. C.

    1970-01-01

    Encoding mechanism integral to the motor and using the permanent magnets embedded in the rotor eliminates the need for external devices to encode information relating the position and velocity of the rotating member.

  8. Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell.

    Science.gov (United States)

    Fang, Lin; Cheng, Qian; Liu, Wenshun; Zhang, Jie; Ge, Yan; Zhang, Qi; Li, Liantao; Liu, Junjie; Zheng, Junnian

    2016-06-02

    ASBTARCT Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy.

  9. Development of a Minor Histocompatibility Antigen Vaccine Regimen in the Canine Model of Hematopoietic Cell Transplantation.

    Science.gov (United States)

    Rosinski, Steven Lawrence; Stone, Brad; Graves, Scott S; Fuller, Deborah H; De Rosa, Stephen C; Spies, Gregory A; Mize, Gregory J; Fuller, James T; Storb, Rainer

    2015-10-01

    Minor histocompatibility antigen (miHA) vaccines have the potential to augment graft-versus-tumor effects without graft-versus-host disease (GVHD). We used mixed hematopoietic chimerism in the canine model of major histocompatibility complex-matched allogeneic hematopoietic cell transplantation as a platform to develop a miHA vaccination regimen. We engineered DNA plasmids and replication-deficient human adenovirus type 5 constructs encoding large sections of canine SMCY and the entire canine SRY gene. Priming with replication-deficient human adenovirus type 5 constructs and boosting with ex vivo plasmid-transfected dendritic cells and cutaneous delivery of plasmids with a particle-mediated epidermal delivery device (PMED) in 2 female dogs induced antigen-specific T-cell responses. Similar responses were observed after a prime-boost vaccine regimen in three female hematopoietic cell transplantation donors. Subsequent donor lymphocyte infusion resulted in a significant change of chimerism in 1 of 3 male recipients without any signs of graft-versus-host disease. The change in chimerism in the recipient occurred in association with the development of CD4+ and CD8+ T-cell responses to the same peptide pools detected in the donor. These studies describe the first in vivo response to miHA vaccination in a large, outbred animal model without using recipient cells to sensitize the donor. This model provides a platform for ongoing experiments designed to define optimal miHA targets and develop protocols to directly vaccinate the recipient.

  10. [Neurons that encode sound direction].

    Science.gov (United States)

    Peña, J L

    In the auditory system, the inner ear breaks down complex signals into their spectral components, and encodes the amplitude and phase of each. In order to infer sound direction in space, a computation on each frequency component of the sound must be performed. Space specific neurons in the owl s inferior colliculus respond only to sounds coming from a particular direction and represent the results of this computation. The interaural time difference (ITD) and interaural level difference (ILD define the auditory space for the owl and are processed in separate neural pathways. The parallel pathways that process these cues merge in the external nucleus of the inferior colliculus where the space specific neurons are selective to combinations of ITD and ILD. How do inputs from the two sources interact to produce combination selectivity to ITD ILD pairs? A multiplication of postsynaptic potentials tuned to ITD and ILD can account for the subthreshold responses of these neurons to ITD ILD pairs. Examples of multiplication by neurons or neural circuits are scarce, but many computational models assume the existence of this basic operation. The owl s auditory system uses such operation to create a 2 dimensional map of auditory space. The map of space in the owl s auditory system shows important similarities with representations of space in the cerebral cortex and other sensory systems. In encoding space or other stimulus features, individual neurons appear to possess analogous functional properties related to the synthesis of high order receptive fields.

  11. Amplified and Persistent Immune Responses Generated by Single-Cycle Replicating Adenovirus Vaccines

    Science.gov (United States)

    Crosby, Catherine M.; Nehete, Pramod; Sastry, K. Jagannadha

    2014-01-01

    ABSTRACT Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered “single-cycle” adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-γ) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines. IMPORTANCE This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase

  12. Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.

    Directory of Open Access Journals (Sweden)

    Jason S Richardson

    Full Text Available BACKGROUND: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP. The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. METHODOLOGY/PRINCIPAL FINDINGS: Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP. Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. CONCLUSIONS/SIGNIFICANCE: We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the

  13. Molecular mechanisms for protein-encoded inheritance.

    Science.gov (United States)

    Wiltzius, Jed J W; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R; Apostol, Marcin I; Goldschmidt, Lukasz; Soriaga, Angela B; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-09-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  14. Molecular mechanisms for protein-encoded inheritance

    Energy Technology Data Exchange (ETDEWEB)

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David; (Cornell); (HHMI)

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  15. Chromosomal damage induced by human adenovirus type 12 requires expression of the E1B 55-kilodalton viral protein.

    Science.gov (United States)

    Schramayr, S; Caporossi, D; Mak, I; Jelinek, T; Bacchetti, S

    1990-01-01

    Infection of human embryonic kidney cells with adenovirus type 12 results in the induction of damage at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosomes. A previous study by Durnam et al. (D. M. Durnam, P. P. Smith, J. C. Menninger, and J. K. McDougall, Cancer Cells 4:349-354, 1986) indicated that the expression of viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. In the present report we used an adenovirus type 12-adenovirus type 5 recombinant with E1A hybrid sequences as well as viruses with mutations in the adenovirus type 12 E1B genes to map adenovirus type 12 E1 functions involved in the induction of genetic damage. Our results show that the expression of the E1A proteins is not sufficient for this effect. On the other hand, mutations within the E1B 55-kilodalton protein but not the E1B 19-kilodalton protein affect the ability of the virus to induce both specific and random chromosomal damage. Images PMID:2325204

  16. Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

    Science.gov (United States)

    Morelli, A E; Larregina, A T; Smith-Arica, J; Dewey, R A; Southgate, T D; Ambar, B; Fontana, A; Castro, M G; Lowenstein, P R

    1999-03-01

    Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

  17. NAC1, a POZ/BTB protein present in the adult mammalian brain, triggers apoptosis after adenovirus-mediated overexpression in PC-12 cells.

    Science.gov (United States)

    Korutla, Laxminarayana; Neustadter, Jason H; Fournier, Keith M; Mackler, Scott A

    2003-05-01

    POZ/BTB proteins influence cellular development and in some examples act as oncoproteins. However, several POZ/BTB transcription factors have been found in terminally differentiated neurons, where their functions remain unknown. One example is NAC1, a constitutively-expressed protein that can regulate behaviors associated with cocaine use. The present study represents an initial attempt to understand the actions of NAC1 within neurons by using adenoviral-mediated gene transfer into differentiated PC-12 cells. Cell survival in PC-12 cells overexpressing NAC1 was greatly reduced compared with cells infected by a control Ad-GFP. The morphological appearance of the dying cells was consistent with programmed cell death. Fragmentation of genomic DNA occurred in PC-12 cells infected with adenoviruses encoding NAC1 but not control viruses. NAC1 over expression was followed by the down regulation of the anti-apoptotic proteins Bcl-2 and Bcl-2-xl. Concurrently, levels of the pro-apoptotic proteins Bax and p53 increased following NAC1 overexpression. These observations suggest that NAC1expression in PC-12 cells induces apoptosis by altering the expression of these upstream mediators of the execution phase of programmed cell death. These findings raise the possibility that aberrantly regulated NAC1 expression in the mammalian brain may contribute to programmed cell death.

  18. Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice.

    Science.gov (United States)

    Yu, Longzheng; Yamagishi, Junya; Zhang, Shoufa; Jin, Chunmei; Aboge, Gabriel Oluga; Zhang, Houshuang; Zhang, Guohong; Tanaka, Tetsuya; Fujisaki, Kozo; Nishikawa, Yoshifumi; Xuan, Xuenan

    2012-09-01

    A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.

  19. Dynamical encoding of cursive handwriting.

    Science.gov (United States)

    Singer, Y; Tishby, N

    1994-01-01

    A model-based approach to on-line cursive handwriting analysis and recognition is presented and evaluated. In this model, on-line handwriting is considered as a modulation of a simple cycloidal pen motion, described by two coupled oscillations with a constant linear drift along the line of the writing. By slow modulations of the amplitudes and phase lags of the two oscillators, a general pen trajectory can be efficiently encoded. These parameters are then quantized into a small number of values without altering the writing intelligibility. A general procedure for the estimation and quantization of these cycloidal motion parameters for arbitrary handwriting is presented. The result is a discrete motor control representation of the continuous pen motion, via the quantized levels of the model parameters. This motor control representation enables successful word spotting and matching of cursive scripts. Our experiments clearly indicate the potential of this dynamic representation for complete cursive handwriting recognition.

  20. Genetically Encoded Sensors for Metabolites

    Science.gov (United States)

    Deuschle, Karen; Fehr, Marcus; Hilpert, Melanie; Lager, Ida; Lalonde, Sylvie; Looger, Loren L.; Okumoto, Sakiko; Persson, Jörgen; Schmidt, Anja; Frommer, Wolf B.

    2009-01-01

    Background Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels. Methods We therefore developed genetically encoded nanosensors for several metabolites to provide a basic set of tools for the determination of cytosolic and subcellular metabolite levels in real time by using fluorescence microscopy. Results Prototypes of these sensors were successfully used in vitro and also in vivo, i.e., to measure sugar levels in fungal and animal cells. Conclusions One of the future goals will be to expand the set of sensors to a wider spectrum of substrates by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets in conjunction with mutagenesis. This toolbox can then be applied for four-dimensional imaging of cells and tissues to elucidate the spatial and temporal distribution of metabolites as a discovery tool in functional genomics, as a tool for high-throughput, high-content screening for drugs, to test metabolic models, and to analyze the interplay of cells in a tissue or organ. PMID:15688353

  1. Evaluation of a rapid immunochromatographic assay for the detection of rotavirus, norovirus and adenovirus from children hospitalized with acute watery diarrhea.

    Science.gov (United States)

    Kas, Monalisa P; Maure, Tobias; Soli, Kevin W; Umezaki, Masahiro; Morita, Ayako; Bebes, Sauli; Jonduo, Marinjho H; Larkins, Jo-Ann; Luang-Suarkia, Dagwin; Siba, Peter M; Greenhill, Andrew R; Horwood, Paul F

    2013-01-01

    We evaluated the IP-Triple I immunochromatographic rapid test for the detection of rotavirus, norovirus and adenovirus using stool samples from children with diarrhoea. The detection of norovirus and adenovirus was poor compared to polymerase chain reaction assays. However, high sensitivity (92%) and specificity (99%) were obtained for the detection of rotavirus.

  2. NMDA receptors and memory encoding.

    Science.gov (United States)

    Morris, Richard G M

    2013-11-01

    It is humbling to think that 30 years have passed since the paper by Collingridge, Kehl and McLennan showing that one of Jeff Watkins most interesting compounds, R-2-amino-5-phosphonopentanoate (d-AP5), blocked the induction of long-term potentiation in vitro at synapses from area CA3 of the hippocampus to CA1 without apparent effect on baseline synaptic transmission (Collingridge et al., 1983). This dissociation was one of the key triggers for an explosion of interest in glutamate receptors, and much has been discovered since that collectively contributes to our contemporary understanding of glutamatergic synapses - their biophysics and subunit composition, of the agonists and antagonists acting on them, and their diverse functions in different networks of the brain and spinal cord. It can be fairly said that Collingridge et al.'s (1983) observation was the stimulus that has led, on the one hand, to structural biological work at the atomic scale describing the key features of NMDA receptors that enables their coincidence function to happen; and, on the other, to work with whole animals investigating the contributions that calcium signalling via this receptor can have on rhythmical activities controlled by spinal circuits, memory encoding in the hippocampus (the topic of this article), visual cortical plasticity, sensitization in pain, and other functions. In this article, I lay out how my then interest in long-term potentiation (LTP) as a model of memory enabled me to recognise the importance of Collingridge et al.'s discovery - and how I and my colleagues endeavoured to take things forward in the area of learning and memory. This is in some respects a personal story, and I tell it as such. The idea that NMDA receptor activation is essential for memory encoding, though not for storage, took time to develop and to be accepted. Along the way, there have been confusions, challenges, and surprises surrounding the idea that activation of NMDA receptors can

  3. Recombinant mammaglobin A adenovirus-infected dendritic cells induce mammaglobin A-specific CD8+ cytotoxic T lymphocytes against breast cancer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Huixia Cui

    Full Text Available Mammaglobin A (MGBA is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC-induced tumor antigen specific CD8(+ cytotoxic T lymphocytes (CTLs may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs of two HLA-A33(+ healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA. After that, the Ad-MGBA-infected DCs were used to stimulate CD8(+ CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12, but down-regulated interleukin-10 (IL-10 secretion from DCs. Ad-MGBA-infected DC-stimulated CD8(+CTLs displayed the highest cytotoxicity towards HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells compared with other CD8(+CTL populations, and compared with the cytotoxicity towards HLA-A33(-/MGBA(+ breast cancer HBL-100 cells and HLA-A33(-/MGBA(- breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8(+ CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33(-/MGBA(+ HBL-100 and HLA-A33(-/MGBA(-MDA-MB-231 cells. In addition, killing of CD8(+CTLs against breast cancer was in a major histocompability complex (MHC-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity

  4. Adenovirus 5-vectored P. falciparum vaccine expressing CSP and AMA1. Part A: safety and immunogenicity in seronegative adults.

    Directory of Open Access Journals (Sweden)

    Martha Sedegah

    Full Text Available BACKGROUND: Models of immunity to malaria indicate the importance of CD8+ T cell responses for targeting intrahepatic stages and antibodies for targeting sporozoite and blood stages. We designed a multistage adenovirus 5 (Ad5-vectored Plasmodium falciparum malaria vaccine, aiming to induce both types of responses in humans, that was tested for safety and immunogenicity in a Phase 1 dose escalation trial in Ad5-seronegative volunteers. METHODOLOGY/PRINCIPAL FINDINGS: The NMRC-M3V-Ad-PfCA vaccine combines two adenovectors encoding circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1. Group 1 (n = 6 healthy volunteers received one intramuscular injection of 2×10∧10 particle units (1×10∧10 each construct and Group 2 (n = 6 a five-fold higher dose. Transient, mild to moderate adverse events were more pronounced with the higher dose. ELISpot responses to CSP and AMA1 peaked at 1 month, were higher in the low dose (geomean CSP = 422, AMA1 = 862 spot forming cells/million than in the high dose (CSP = 154, p = 0.049, AMA1 = 423, p = 0.045 group and were still positive at 12 months in a number of volunteers. ELISpot depletion assays identified dependence on CD4+ or on both CD4+ and CD8+ T cells, with few responses dependent only on CD8+ T cells. Intracellular cytokine staining detected stronger CD8+ than CD4+ T cell IFN-γ responses (CSP p = 0.0001, AMA1 p = 0.003, but similar frequencies of multifunctional CD4+ and CD8+ T cells secreting two or more of IFN-γ, TNF-α or IL-2. Median fluorescence intensities were 7-10 fold higher in triple than single secreting cells. Antibody responses were low but trended higher in the high dose group and did not inhibit growth of cultured P. falciparum blood stage parasites. SIGNIFICANCE: As found in other trials, adenovectored vaccines appeared safe and well-tolerated at doses up to 1×10∧11 particle units. This is the first demonstration in humans of a

  5. Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses.

    Science.gov (United States)

    Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L; Merritt, Robert; Hackett, Neil R; Rovelink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imi; Crystal, Ronald G

    2004-03-01

    Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing beta-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the beta-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing beta-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to beta-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif

  6. Epidemiology of adenovirus respiratory infections among hospitalized children in Seremban, Malaysia.

    Science.gov (United States)

    Foong Ng, Khuen; Kee Tan, Kah; Hong Ng, Boon; Nair, Pritiss; Ying Gan, Wan

    2015-07-01

    There is scarcity of data regarding epidemiology and clinical aspects of human adenovirus acute respiratory infection (ARI) among children in developing countries. Retrospective data on demographics, clinical presentation, outcomes and laboratory findings of 116 children admitted into Tuanku Jaafar Hospital in Seremban, Malaysia from 2012 to 2013 with documented diagnosis of community-acquired adenovirus ARI were collected and analyzed. Male to female ratio was 1.70. Median age was 14 (1-107) months. The commonest symptoms were fever (94.8%; 110/116), cough (82.8%, 96), rhinorrhea (63.8%; 74), interrupted feeding (66.4%; 77), diarrhea (33.6%; 39) and conjunctivitis (21.6%; 25). Mean temperature on admission was 38.4°C±0.9°C. Among all 116 subjects, 20.7% (24) needed oxygen supplementation, 57.8% (67) required intravenous hydration, 11.2% (13) were admitted into the pediatric intensive care unit and 6.9% (8) required mechanical ventilation. Only 1% (1/87) had positive blood culture (Streptococcus pneumoniae) among 87 who received antibiotic treatment. Case fatality rate was 2.6% (3/116) and 1.7% (2/116) developed bronchiolitis obliterans. Median length of hospital stay was 4 (1-50) days. Adenovirus ARI caused significant morbidity and substantial resource utilization among hospitalized Malaysian children. It should be considered in the differential diagnosis of infants below two years presenting with ARI associated with high fever. Antibiotics should not be prescribed as secondary bacterial infections are uncommon. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Avian Adenoviruses Infections with Special Attention to Inclusion Body Hepatitis/ Hydropericardium Syndrome and Egg Drop Syndrome

    Directory of Open Access Journals (Sweden)

    Hafez Mohamed Hafez*

    2011-04-01

    Full Text Available The first avian adenovirus (AAV associated with clinical disease was isolated from an outbreak of respiratory disease in quail in 1950 (Olson, 1950. Since that time, AAVs have been found in all types and breeds of chickens and from a variety of other avian species. The infections may be asymptomatic or associated with several clinical and pathological conditions. Vertical transmission via the egg is the most common way of transmission. Also horizontal transmission through faeces, contaminated egg trays, crates and trucks play a role in the infection route. Studies have demonstrated the presence of antibodies in healthy poultry, and viruses have been isolated from normal birds. Avian adenoviruses in chickens are the etiological agents of 2 diseases known as inclusion body hepatitis (IBH and hydropericardium syndrome (HP. In some cases each condition is observed separately, however, recently the 2 conditions have frequently been observed as a single entity; therefore, the name hepatitis hydropericardium has been widely used to describe the pathologic condition. The syndrome is an acute disease of young chickens associated with anemia, haemorrhagic disorders, hydropericardium and high mortality. Egg-Drop-Syndrome (EDS is caused also by an adenovirus. The disease is characterised by a severe drop in egg production as well as the production of shell-less, thin-shelled, discoloured or misshapen eggs in apparently healthy birds. Ducks and geese are the natural host of the EDS virus. It was first described in chickens in the 1970s and spread to several countries world wide. The birds usually do not show any other signs of disease, and mortality is not expected. There is no specific treatment of the AAV infections. Active immunization by vaccination using an inactivated is wide spread.

  8. Respiratory syncytial virus, adenoviruses, and mixed acute lower respiratory infections in children in a developing country.

    Science.gov (United States)

    Rodríguez-Martínez, Carlos E; Rodríguez, Diego Andrés; Nino, Gustavo

    2015-05-01

    There is growing evidence suggesting greater severity and worse outcomes in children with mixed as compared to single respiratory virus infections. However, studies that assess the risk factors that may predispose a child to a mixture of respiratory syncytial virus (RSV) and adenoviral infections, are scarce. In a retrospective cohort study, the study investigated the epidemiology of RSV and adenovirus infections and predictors of mixed RSV-adenoviral infections in young children hospitalized with acute lower respiratory infection in Bogota, Colombia, South America, over a 2-year period 2009-2011. Of a total of 5,539 children admitted with a diagnosis of acute lower respiratory infection, 2,267 (40.9%) who were positive for RSV and/or adenovirus were selected. Out the total number of cases, 1,416 (62.5%) infections occurred during the 3-month period from March to May, the first rainy season of Bogota, Colombia. After controlling for gender, month when the nasopharyngeal sample was taken, and other pre-existing conditions, it was found that an age greater than 6 months (OR:1.74; CI 95%:1.05-2.89; P = 0.030) and malnutrition as a comorbidity (OR:9.92; CI 95%:1.01-100.9; P = 0.049) were independent predictors of mixed RSV-adenoviral infections in the sample of patients. In conclusion, RSV and adenovirus are significant causes of acute lower respiratory infection in infants and young children in Bogota, Colombia, especially during the first rainy season. The identified predictors of mixed RSV-adenoviral infections should be taken into account when planning intervention, in order to reduce the burden of acute lower respiratory infection in young children living in the country.

  9. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    Science.gov (United States)

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

  10. Repair of segmental bone defects with bone marrow and BMP-2 adenovirus in the rabbit radius

    Science.gov (United States)

    Cheng, Lijia; Lu, Xiaofeng; Shi, Yujun; Li, Li; Xue, Jing; Zhang, Li; Xia, Jie; Wang, Yujia; Zhang, Xingdong; Bu, Hong

    2012-12-01

    Bone tissue engineering (BTE) is approached via implantation of autogenous mesenchymal stem cells (MSCs), marrow cells, or platelet-rich plasma, etc. To the contrary, gene therapy combining with the bone marrow (BM) has not been often reported. This study was performed to investigate whether a modified BTE method, that is, the BM and a recombinant human bone morphogenetic protein-2 adenovirus (Ad.hBMP-2) gene administering in hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramics could accelerate the healing of segmental defects in the rabbit radius. In our study, ceramics were immersed in the adenovirus overnight, and half an hour before surgery, autologous BM aspirates were thoroughly mixed with the ceramics; at the same time, a 15-mm radius defect was introduced in the bilateral forelimbs of all animals, after that, this defect was filled with the following: (1) Ad.hBMP-2 + HA/β-TCP + autologous BM (group 1); (2) HA/β-TCP + Ad.hBMP-2 (group 2); (3) HA/β-TCP alone (group 3); (4) an empty defect as a control (group 4). Histological observation and μ-CT analyses were performed on the specimens at weeks 2, 4, 8, and 12, respectively. In group 1, new bone was observed at week 4 and BM appeared at week 12, in groups 2 and 3, new bone was observed at week 8 and it was more mature at week 12, in contrast, the defect was not bridged in group 4 at week 12. The new bone area percentage in group 1 was significantly higher than that in groups 2 and 3. Our study indicated that BM combined with hBMP-2 adenovirus and porous ceramics could significantly increase the amount of newly formed bone. And this modified BTE method thus might have potentials in future clinical application.

  11. Intracellular replication-deficient Leishmania donovani induces long lasting protective immunity against visceral leishmaniasis.

    Science.gov (United States)

    Selvapandiyan, Angamuthu; Dey, Ranadhir; Nylen, Susanne; Duncan, Robert; Sacks, David; Nakhasi, Hira L

    2009-08-01

    No vaccine is currently available for visceral leishmaniasis (VL) caused by Leishmania donovani. This study addresses whether a live attenuated centrin gene-deleted L. donovani (LdCen1(-/-)) parasite can persist and be both safe and protective in animals. LdCen1(-/-) has a defect in amastigote replication both in vitro and ex vivo in human macrophages. Safety was shown by the lack of parasites in spleen and liver in susceptible BALB/c mice, immune compromised SCID mice, and human VL model hamsters 10 wk after infection. Mice immunized with LdCen1(-/-) showed early clearance of virulent parasite challenge not seen in mice immunized with heat killed parasites. Upon virulent challenge, the immunized mice displayed in the CD4(+) T cell population a significant increase of single and multiple cytokine (IFN-gamma, IL-2, and TNF) producing cells and IFN-gamma/IL10 ratio. Immunized mice also showed increased IgG2a immunoglobulins and NO production in macrophages. These features indicated a protective Th1-type immune response. The Th1 response correlated with a significantly reduced parasite burden in the spleen and no parasites in the liver compared with naive mice 10 wk post challenge. Protection was observed, when challenged even after 16 wk post immunization, signifying a sustained immunity. Protection by immunization with attenuated parasites was also seen in hamsters. Immunization with LdCen1(-/-) also cross-protected mice against infection with L. braziliensis that causes mucocutaneous leishmaniasis. Results indicate that LdCen1(-/-) can be a safe and effective vaccine candidate against VL as well as mucocutaneous leishmaniasis causing parasites.

  12. A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

    Directory of Open Access Journals (Sweden)

    Montgomery Roy D

    2006-04-01

    Full Text Available Abstract Background Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. Results We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus from cell culture and lymphocystis disease virus (iridovirus and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1. The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. Conclusion The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified

  13. San Martín, C., Correction: Latest Insights on Adenovirus Structure and Assembly. Viruses 2012, 4, 847-877.

    Directory of Open Access Journals (Sweden)

    Carmen San Martín

    2012-12-01

    Full Text Available It has come to my attention that my article "Latest Insights on Adenovirus Structure and Assembly" (Viruses 2012, 4, 847-877 [1] contains an inaccurate statement. On page 864, the caption for Figure 7 reads: "There are four potential cleavage sites in pTP but they have not been experimentally verified". However, three of these sites have been experimentally confirmed in vitro using recombinant AVP and pTP, as described in Webster A, Leith I.R., Hay R.T.: Activation of adenovirus-coded protease and processing of preterminal protein. J. Virol. 1994, 68, 7292-7300 [2].

  14. Novelty's effect on memory encoding.

    Science.gov (United States)

    Rangel-Gomez, Mauricio; Janenaite, Sigita; Meeter, Martijn

    2015-07-01

    It is often thought that novelty benefits memory formation. However, support for this idea mostly comes from paradigms that are open to alternative explanations. In the present study we manipulated novelty in a word-learning task through task-irrelevant background images. These background images were either standard (presented repeatedly), or novel (presented only once). Two types of background images were used: Landscape pictures and fractals. EEG was also recorded during encoding. Contrary to the idea that novelty aids memory formation, memory performance was not affected by the novelty of the background. In the evoked response potentials, we found evidence of distracting effects of novelty: both the N1 and P3b components were smaller to words studied with novel backgrounds, and the amplitude of the N2b component correlated negatively with subsequent retrieval. We conclude that although evidence from other studies does suggest benefits on a longer time scale, novelty has no instantaneous benefits for learning. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. ZEB1 limits adenoviral infectability by transcriptionally repressing the Coxsackie virus and Adenovirus Receptor

    Directory of Open Access Journals (Sweden)

    Lacher Markus D

    2011-07-01

    Full Text Available Abstract Background We have previously reported that RAS-MEK (Cancer Res. 2003 May 1;63(9:2088-95 and TGF-β (Cancer Res. 2006 Feb 1;66(3:1648-57 signaling negatively regulate coxsackie virus and adenovirus receptor (CAR cell-surface expression and adenovirus uptake. In the case of TGF-β, down-regulation of CAR occurred in context of epithelial-to-mesenchymal transition (EMT, a process associated with transcriptional repression of E-cadherin by, for instance, the E2 box-binding factors Snail, Slug, SIP1 or ZEB1. While EMT is crucial in embryonic development, it has been proposed to contribute to the formation of invasive and metastatic carcinomas by reducing cell-cell contacts and increasing cell migration. Results Here, we show that ZEB1 represses CAR expression in both PANC-1 (pancreatic and MDA-MB-231 (breast human cancer cells. We demonstrate that ZEB1 physically associates with at least one of two closely spaced and conserved E2 boxes within the minimal CAR promoter here defined as genomic region -291 to -1 relative to the translational start ATG. In agreement with ZEB1's established role as a negative regulator of the epithelial phenotype, silencing its expression in MDA-MB-231 cells induced a partial Mesenchymal-to-Epithelial Transition (MET characterized by increased levels of E-cadherin and CAR, and decreased expression of fibronectin. Conversely, knockdown of ZEB1 in PANC-1 cells antagonized both the TGF-β-induced down-regulation of E-cadherin and CAR and the reduction of adenovirus uptake. Interestingly, even though ZEB1 clearly contributes to the TGF-β-induced mesenchymal phenotype of PANC-1 cells, TGF-β did not seem to affect ZEB1's protein levels or subcellular localization. These findings suggest that TGF-β may inhibit CAR expression by regulating factor(s that cooperate with ZEB1 to repress the CAR promoter, rather than by regulating ZEB1 expression levels. In addition to the negative E2 box-mediated regulation the minimal

  16. Lethal hemorrhagic alveolitis after adenovirus pneumonia in a lung transplant recipient.

    Science.gov (United States)

    Mayeur, Nicolas; Srairi, Mohamed; Tetu, Laurent; Guilbeau Frugier, Céline; Fourcade, Olivier; Dahan, Marcel

    2012-01-01

    Viral infections are frequent and severe in lung transplant recipients. They frequently occur during the first year after transplantation. We report on a rare case of bilateral adenovirus necrotizing pneumonia with a diffuse alveolar hemorrhage, 4 years after bilateral lung transplantation. The medical evolution was lethal in 72 hours because of respiratory, renal, and cardiac failure. Considering this case and the growing evidence on the severity of adenoviral infections, we call for controlled studies and therapeutic recommendations. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.

    Science.gov (United States)

    Smith, Jason G; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R

    2008-07-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

  18. Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport▿

    Science.gov (United States)

    Smith, Jason G.; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R.

    2008-01-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry. PMID:18448546

  19. Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transp...

  20. Isolation, identification, and complete genome sequence of a bovine adenovirus type 3 from cattle in China

    Directory of Open Access Journals (Sweden)

    Zhu Yuan-Mao

    2011-12-01

    Full Text Available Abstract Background Bovine adenovirus type 3 (BAV-3 belongs to the Mastadenovirus genus of the family Adenoviridae and is involved in respiratory and enteric infections of calves. The isolation of BAV-3 has not been reported prior to this study in China. In 2009, there were many cases in cattle showing similar clinical signs to BAV-3 infection and a virus strain, showing cytopathic effect in Madin-Darby bovine kidney cells, was isolated from a bovine nasal swab collected from feedlot cattle in Heilongjiang Province, China. The isolate was confirmed as a bovine adenovirus type 3 by PCR and immunofluorescence assay, and named as HLJ0955. So far only the complete genome sequence of prototype of BAV-3 WBR-1 strain has been reported. In order to further characterize the Chinese isolate HLJ0955, the complete genome sequence of HLJ0955 was determined. Results The size of the genome of the Chinese isolate HLJ0955 is 34,132 nucleotides in length with a G+C content of 53.6%. The coding sequences for gene regions of HLJ0955 isolate were similar to the prototype of BAV-3 WBR-1 strain, with 80.0-98.6% nucleotide and 87.5-98.8% amino acid identities. The genome of HLJ0955 strain contains 16 regions and four deletions in inverted terminal repeats, E1B region and E4 region, respectively. The complete genome and DNA binding protein gene based phylogenetic analysis with other adenoviruses were performed and the results showed that HLJ0955 isolate belonged to BAV-3 and clustered within the Mastadenovirus genus of the family Adenoviridae. Conclusions This is the first study to report the isolation and molecular characterization of BAV-3 from cattle in China. The phylogenetic analysis performed in this study supported the use of the DNA binding protein gene of adenovirus as an appropriate subgenomic target for the classification of different genuses of the family Adenoviridae on the molecular basis. Meanwhile, a large-scale pathogen and serological epidemiological

  1. Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

    Science.gov (United States)

    Bachmann, Mandy; Breitwieser, Theresa; Lipps, Christoph; Wirth, Dagmar; Jordan, Ingo; Reichl, Udo; Frensing, Timo

    2016-02-01

    Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN. We demonstrate that the expression of the early region protein 1A in these cell lines impairs their ability to activate antiviral genes by the IFN pathway. This property may help in the isolation of newly emerging viruses and the propagation of interferon-sensitive virus strains.

  2. Characterization of fowl adenoviruses associated with hydropericardium syndrome and inclusion body hepatitis in broiler chickens

    OpenAIRE

    2013-01-01

    The present study was carried out for detection and molecular characterization of fowl adenoviruses (FAdVs) associated with hydropericardium syndrome or inclusion body hepatitis in commercial broiler chickens. The FAdVs were detected in liver samples from 33 commercial broiler chicken flocks by polymerase chain reaction (PCR) using hexon gene specific primers. The restriction enzyme analysis using StyI, BsiWI, MluI, AspI, BglI and ScaI enzymes of all the 33 FAdV-positive samples revealed FAdV...

  3. Computational Intelligence and Its Encoding Mechanism

    Institute of Scientific and Technical Information of China (English)

    LIU Man-dan

    2004-01-01

    The origin and characteristics of computational intelligence, and several typical computational intelligence algorithms such as genetic algorithm and DNA computing are described, and the influence of evolution strategies and convergence properties on the encoding mechanism is discussed. A novel genetic algorithm based on degressive carry number encoding is then proposed. This algorithm uses degressive carry number encoding in the evolutionary process instead of commonly used fixed carry number. Finally a novel encoding mechanism and a new algorithm are proposed, which combine modern computational intelligence with the traditional Chinese methodology.

  4. Computational Intelligence and Its Encoding Mechanism

    Institute of Scientific and Technical Information of China (English)

    LIUMan-dan

    2004-01-01

    The origin and characteristics of computational intelligence, and several typical computational intelligence algorithms such as genetic algorithm and DNA computing are described, and the influence of evolution strategies and convergence properties on the encoding mechanism is discussed. A novel genetic algorithm based on degressive carry number encoding is then proposed. This algorithm uses degressive carry number encoding in the evolutionary process instead of commonly used fixed carry number. Finally a novel encoding mechanism and a new algorithm are proposed, which combine modem computational intelligence with the traditional Chinese methodology.

  5. Encoder designed to work in harsh environments

    Energy Technology Data Exchange (ETDEWEB)

    Toop, L.

    2007-05-15

    Dynapar has developed the Acuro AX71 absolute encoder for use on offshore or land-based oil rig operations. It provides feedback on the operation of automated systems such as draw works, racking systems, rotary tables and top drives. By ensuring that automated systems function properly, this encoder responds to a need by the oil and gas industry to keep workers safe and improve efficiency, particularly for operations in rugged situations. The encoder provides feedback from motor systems to controllers, giving information about position and speed of downhole drill bits. This newly developed encoder is better than commonly used incremental encoders which are not precise in strong electrical noise environments. Rather, the absolute encoder uses a different method of reporting to the controller. A digital signal is transmitted constantly as the device operates. It is less susceptible to noise issues. It is highly accurate, tolerant of noise and is not affected by power outages. However, the absolute encoder is generally more delicate in drilling applications with high ambient temperatures and shock levels. Dynapar addressed this issue by developing compact stainless steel housing that is useful for corrosion resistance in marine applications. The AX71 absolute encoder can withstand up to 100 G of mechanical shock and ambient temperatures of up to 60 degrees C. The encoder is ATEX certified without barriers, and offers the high resolution feedback of 4,000 counts of multiturn rotation and 16,000 counts of position. 1 fig.

  6. Cellular encoding for interactive evolutionary robotics

    NARCIS (Netherlands)

    Gruau, F.C.; Quatramaran, K.

    1996-01-01

    This work reports experiments in interactive evolutionary robotics. The goal is to evolve an Artificial Neural Network (ANN) to control the locomotion of an 8-legged robot. The ANNs are encoded using a cellular developmental process called cellular encoding. In a previous work similar experiments ha

  7. A METHOD OF SHAPE ENCODING AND RETRIEVAL

    Institute of Scientific and Technical Information of China (English)

    Huang Xianglin; Song Lei; Shen Lansun

    2002-01-01

    A method of shape encoding and retrieval is proposed in this letter, which uses centripetal code to encode shape and extracts shape's convex for retrieval. For the rotation invariance and translation invariance of the centripetal code and the normalization of convex,the proposed retrieval method is similarity transform resistant, Experimental results confirm this capability.

  8. Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

    Science.gov (United States)

    El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M; Rommelaere, Jean; Marchini, Antonio

    2012-10-01

    In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.

  9. Suppression of gastric cancer growth by adenovirus-mediated transfer of the PTEN gene

    Institute of Scientific and Technical Information of China (English)

    Ying Hang; Yong-Chen Zheng; Yan Cao; Qing-Shan Li; Yu-Jie Sui

    2005-01-01

    AIM: To investigate the tumor-suppressive effect of the phosphatase and tensin homologue deleted from chromosome (PTEN) in human gastric cancer cells th atwere wild type for PTEN.METHODS: Adenoviruses expressing PTEN or luciferase as a control were introduced into gastric cancer cells.The effect of exogenous PTEN gene on the growth and apoptosis of gastric cancer cells that are wtPTEN were examined in vitro and in vivo.RESULTS: Adenovirus-mediated transfer of PTEN (AdPTEN) suppressed cell growth and induced apoptosis significantly in gastric cancer cells (MGC-803, SGC-7901)carrying wtPTEN in comparison with that in normal gastric epithelial cells (GES-1) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase and mitogen-activated protein kinase and cell-cycle arrest at the G2/M phase but not at the G1 phase. Furthermore,treatment of human gastric tumor xenografts (MGC-803,SGC-7901) with Ad-PTEN resulted in a significant (P<0.01)suppression of tumor growth.CONCLUSION: These results indicate a significant tumorsuppressive effect of Ad-PTEN against human gastric cancer cells. Thus, Ad-PTEN may be used as a potential therapeutic strategy for treatment of gastric cancers.

  10. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    Directory of Open Access Journals (Sweden)

    Natascha Krömmelbein

    2016-02-01

    Full Text Available The human cytomegalovirus (HCMV replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.

  11. CAR-associated vesicular transport of an adenovirus in motor neuron axons.

    Science.gov (United States)

    Salinas, Sara; Bilsland, Lynsey G; Henaff, Daniel; Weston, Anne E; Keriel, Anne; Schiavo, Giampietro; Kremer, Eric J

    2009-05-01

    Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

  12. Adenovirus Mediated BIMS Transfer Induces Growth Supression and Apoptosis in Raji Lymphoma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHAO Ya Ning; LI Qiang

    2014-01-01

    Objective To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. Methods BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. Results After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. Conclusion These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells.

  13. Derivation of a myeloid cell-binding adenovirus for gene therapy of inflammation.

    Directory of Open Access Journals (Sweden)

    Michael O Alberti

    Full Text Available The gene therapy field is currently limited by the lack of vehicles that permit efficient gene delivery to specific cell or tissue subsets. Native viral vector tropisms offer a powerful platform for transgene delivery but remain nonspecific, requiring elevated viral doses to achieve efficacy. In order to improve upon these strategies, our group has focused on genetically engineering targeting domains into viral capsid proteins, particularly those based on adenovirus serotype 5 (Ad5. Our primary strategy is based on deletion of the fiber knob domain, to eliminate broad tissue specificity through the human coxsackie-and-adenovirus receptor (hCAR, with seamless incorporation of ligands to re-direct Ad tropism to cell types that express the cognate receptors. Previously, our group and others have demonstrated successful implementation of this strategy in order to specifically target Ad to a number of surface molecules expressed on immortalized cell lines. Here, we utilized phage biopanning to identify a myeloid cell-binding peptide (MBP, with the sequence WTLDRGY, and demonstrated that MBP can be successfully incorporated into a knob-deleted Ad5. The resulting virus, Ad.MBP, results in specific binding to primary myeloid cell types, as well as significantly higher transduction of these target populations ex vivo, compared to unmodified Ad5. These data are the first step in demonstrating Ad targeting to cell types associated with inflammatory disease.

  14. Investigating the role of Acanthamoeba polyphaga in protecting Human Adenovirus from water disinfection treatment.

    Science.gov (United States)

    Verani, Marco; Di Giuseppe, Graziano; Tammaro, Carmine; Carducci, Annalaura

    2016-06-01

    Human adenoviruses are responsible for a wide range of clinical infections and are present in aquatic environments, including river, seawater, drinking-water and sewage. Free-living amoebae (Acanthamoeba) in the same environments may internalize them and other microorganisms can act as a reservoir for the internalized viruses. In this study, we studied the interaction between Acanthamoeba polyphaga and Human Adenovirus type 5 (HAdV 5) to determine whether the amoeba played a role in protecting the internalized viruses from chemical disinfection. The efficacy of sodium hypochlorite disinfection against A. polyphaga and HAdV 5 either singly or in combination was assessed at three different concentrations. Individually, the amoeba were more resistant to chemical disinfection than HAdV 5 and remained alive after exposure to 5mg/l of sodium hypochlorite. In contrast, HAdV 5 lost infectivity following exposure to 2.5mg/l of sodium hypochlorite. When the amoeba and HAdV 5 were co-cultured, infectious virus was found in the cytoplasm of the amoeba at 5mg/l disinfectant concentration. These findings suggest that the A. polyphaga is providing protection for the HAdV 5.

  15. The Role of NK Cell in T Cell Recruitment in Murine Liver Infected with Adenovirus

    Institute of Scientific and Technical Information of China (English)

    游上游; 艾洪武; 黄巍; 张楚瑜

    2003-01-01

    To study the role of natural killer (NK) cells in T cell recruitment in murine liver infected with virus, mice wereintravenously injected daily with anti-NK1.1+ antibody to deplete NK cells. Lymphocytes in the liver tissue of mice infectedwith type 5 adenovirus depleted in the E1 and E3 regions were assessed by fluorometric activated cell sorting (FACS). Ex-pression of chemokine IP-10 and its receptor CXCR3 mRNA in the liver, hepatic lymphocytes and spleen tissue were examined by reverse transcription polymerase chain reaction (RT-PCR). Serum almfine aminotransferase (ALT) was measured asan indicator of liver injury. It was found that infection of adenovims and anfi-Fas monoclonal antibody (mAb) into mice caused liver injury and high expression of interfemn-γ inducible protein-10 (IP-10) mRNA in the liver. Anfi-NK1.1+ mAb, which was intraperitoneally injected into the mice infected with adenovirus, suppresses T cell recruitment and expression of IP-10 mRNA in the hver. Slighter hver injury was also observed. After vires infection, expression of CXCR3 mRNAin spleen and hver tissue was observed at different time. The results suggested that T cell recruitment was initiated by NKcell dependent chemokine IP-10, which induced activated T cells priming in the spleen to the hver of the mouse. NK cells played a key role in T cell recruitment in the liver of mouse infected with adenovims.

  16. Occurrence of adenovirus infection and clinical impact in paediatric stem cell transplant recipients

    Directory of Open Access Journals (Sweden)

    Gabriele Bianco

    2016-10-01

    Full Text Available In this study, the occurrence and clinical impact of adenovirus (AdV infection was investigated in paediatric hematopoietic stem cell transplantation (HSCT recipients. A number of 603 specimens (including whole blood, respiratory and other samples from 181 patients were tested by real-time polymerase chain reaction; clinical outcome was investigated. Overall, 118/603 (19.6% specimens from 21/181 (11.6% patients resulted positive to AdV (including 17.3, 29.9, 17.6, and 15.8% of total number of whole blood, respiratory, urine and other specimens, respectively. On whole blood specimens, viral loads ranged from <600 (limit of detection to >5×106 copies/mL, with a median value 2×104. Multiple specimens were positive in patients in which viral load on whole blood was high. Adenoviral positivity on whole blood was associated to poor prognosis, as death occurred in three of ten (30% patients with persistent positivity on whole blood specimens, also despite the administration of an antiviral agent (cidofovir. Adenovirus infection can account for systemic and/or organ-specific signs/symptoms in approximately 10% of paediatric HSCT recipients. At moment, there is no indication for routine monitor of AdV in these patients, although AdV aetiology of infectious transplant complications should be taken in account.

  17. Adenovirus mediated BIMS transfer induces growth supression and apoptosis in Raji lymphoma cells.

    Science.gov (United States)

    Zhao, Ya Ning; Li, Qiang

    2014-09-01

    To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  18. Immunological effects of a tumor necrosis factor alpha-armed oncolytic adenovirus.

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    Hirvinen, Mari; Rajecki, Maria; Kapanen, Mika; Parviainen, Suvi; Rouvinen-Lagerström, Noora; Diaconu, Iulia; Nokisalmi, Petri; Tenhunen, Mikko; Hemminki, Akseli; Cerullo, Vincenzo

    2015-03-01

    For long it has been recognized that tumor necrosis factor alpha (TNFa) has anticancer characteristics, and its use as a cancer therapeutic was proposed already in the 1980s. However, its systemic toxicity has limited its usability. Oncolytic viruses, selectively cancer-killing viruses, have shown great potency, and one of their most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local versus systemic concentrations. Therefore, the overall magnitude of tumor cell killing results from the combination of oncolysis, transgene-mediated direct effect such as TNFa-mediated apoptosis, and, perhaps most significantly, from activation of the host immune system against the tumor. We generated a novel chimeric oncolytic adenovirus expressing human TNFa, Ad5/3-D24-hTNFa, whose efficacy and immunogenicity were tested in vitro and in vivo. The hTNFa-expressing adenovirus showed increased cancer-eradicating potency, which was shown to be because of elevated apoptosis and necrosis rates and induction of various immune responses. Interestingly, we saw increase in immunogenic cell death markers in Ad5/3-d24-hTNFa-treated cells. Moreover, tumors treated with Ad5/3-D24-hTNFa displayed enhanced presence of OVA-specific cytotoxic T cells. We thus can conclude that tumor eradication and antitumor immune responses mediated by Ad5/3-d24-hTNFa offer a new potential drug candidate for cancer therapy.

  19. Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

    Science.gov (United States)

    Adhya, S; Shneidman, P S; Hurwitz, J

    1986-03-05

    Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

  20. Cryo-EM structures of two bovine adenovirus type 3 intermediates

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    Cheng, Lingpeng; Huang, Xiaoxing; Li, Xiaomin [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xiong, Wei [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Luo-jia-shan, Wuhan, Hubei 430072 (China); Sun, Wei; Yang, Chongwen; Zhang, Kai; Wang, Ying [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Liu, Hongrong [College of Physics and Information Science, Hunan Normal University, Changsha, Hunan 410081 (China); Huang, Xiaojun; Ji, Gang; Sun, Fei [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Zheng, Congyi, E-mail: cctcc202@whu.edu.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Luo-jia-shan, Wuhan, Hubei 430072 (China); Zhu, Ping, E-mail: zhup@ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-02-15

    Adenoviruses (Ads) infect hosts from all vertebrate species and have been investigated as vaccine vectors. We report here near-atomic structures of two bovine Ad type 3 (BAd3) intermediates obtained by cryo-electron microscopy. A comparison between the two intermediate structures reveals that the differences are localized in the fivefold vertex region, while their facet structures are identical. The overall facet structure of BAd3 exhibits a similar structure to human Ads; however, BAd3 protein IX has a unique conformation. Mass spectrometry and cryo-electron tomography analyses indicate that one intermediate structure represents the stage during DNA encapsidation, whilst the other intermediate structure represents a later stage. These results also suggest that cleavage of precursor protein VI occurs during, rather than after, the DNA encapsidation process. Overall, our results provide insights into the mechanism of Ad assembly, and allow the first structural comparison between human and nonhuman Ads at backbone level. - Highlights: • First structure of bovine adenovirus type 3. • Some channels are located at the vertex of intermediate during DNA encapsidation. • Protein IX exhibits a unique conformation of trimeric coiled–coiled structure. • Cleavage of precursor protein VI occurs during the DNA encapsidation process.

  1. The role of Cajal bodies in the expression of late phase adenovirus proteins.

    Science.gov (United States)

    James, Nicola J; Howell, Gareth J; Walker, John H; Blair, G Eric

    2010-04-10

    Cajal bodies (CBs) are subnuclear structures involved in RNA metabolism. Here we show that, following infection of HeLa cells by adenovirus type 5 (Ad5), CBs fragment and form ordered structures, which we have termed "rosettes". Formation of CB rosettes was prevented by inhibition of viral DNA synthesis and preceded expression of the L4-33K protein. CB rosettes localised to the periphery of E2A-72K-containing replication centers and to the edges of ASF/SF2 and hnRNP A1 ring structures that demarcate sites of viral transcription and splicing. At later times of infection, CB rosettes were undetectable. Furthermore, knock-down of p80-coilin (the major structural protein of CBs) by RNA interference reduced the yield of infectious Ad5 and expression of the late proteins IIIa (from L1), hexon (from L3) and fiber (from L5), whereas the E2A-72K protein was unaffected. We conclude that CBs have an important role in the expression of adenovirus major late gene products.

  2. Virology and epidemiology analyses of global adenovirus-associated conjunctivitis outbreaks, 1953-2013.

    Science.gov (United States)

    Zhang, L; Zhao, N; Sha, J; Wang, C; Jin, X; Amer, S; Liu, S

    2016-06-01

    This study aimed to compare the virology and epidemiology of epidemic keratoconjunctivitis (EKC), pharyngoconjunctival fever (PCF) and acute haemorrhagic conjunctivitis (AHC) outbreaks worldwide caused by the human adenovirus (HAdV) from 1953 to 2013. Eighty-three hexon sequences from 76 conjunctivitis outbreaks were analysed and subtyped using Mega 5.05, Clustal X and SimPlot software. Epidemiology was performed for the area, age and seasonal distribution. A phylogenetic analysis indicated that all the isolates could be divided into three subgenetic lineages, without a common ancestor. The major causes of the outbreaks were Ad8, Ad7 and Ad2 co-infection with enterovirus 70 (EV70) in EKC, PCF and AHC, respectively. The epidemiological findings suggested that EKC and AHC were circulating predominantly in Asia during the early winter and spring, whereas PCF was circulating mainly in China, Australia and the United States during the summer. This study suggests that EKC, AHC and PCF outbreaks have different circulating patterns throughout the world and are caused by different adenovirus serotypes. A global surveillance system should be established to monitor conjunctivitis outbreaks in the future.

  3. Promoting lumbar spinal fusion by adenovirus-mediated bone morphogenetic protein-4 gene therapy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jian; ZHAO Dun-yan; SHEN Ai-guo; LIU Fan; ZHANG Feng; SUN Yu; WU Hong-fu; LU Chun-feng; SHI Hong-guang

    2007-01-01

    Objective: To determine whether an adenoviral construct containing bone morphogenetic protein-4 (BMP-4) gene can be used for lumbar spinal fusion. Methods: Twelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter and β-galactosidase gene (Ad-β-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9×108 pfu/ml ) was directly implanted on the surface of L5-L6 lamina in the experimental group, while Ad-β-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection.Results: In 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified.Conclusions: In vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.

  4. Adenovirus with p16 gene exerts antitumor effect on laryngeal carcinoma Hep2 cells.

    Science.gov (United States)

    Yang, Zhengang; Hu, Jingxia; Li, Dajun; Pan, Xinliang

    2016-08-01

    Laryngeal cancer is an uncommon form of cancer. The tumor suppressor P16, known to be mutated or deleted in various types of human tumor, including laryngeal carcinoma, is involved in the formation and development of laryngeal carcinoma. It has been previously reported that the inactivation or loss of P16 is associated with the acquisition of malignant characteristics. The current study hypothesized that restoring wild‑type P16 activity into P16‑null malignant Hep2 cells may exert an antitumor effect. A recombinant adenovirus carrying the P16 gene (Ad‑P16) was used to infect and express high levels of P16 protein in P16‑null Hep2 cells. Cell proliferation and invasion assays and polymerase chain reaction were performed to evaluate the effects of the P16 gene on cell proliferation and the antitumor effect on Hep2 cells. The results demonstrated that the Hep2 cells infected with Ad‑P16 exhibited significantly reduced cell proliferation, invasion and tumor volume compared with untreated or control adenovirus cells. Furthermore, the expression of laryngeal carcinoma‑associated genes, EGFR, survivin and cyclin D1, were measured in Ad‑P16‑infected cells and were significantly reduced compared with control groups. The results of the current study demonstrate that restoring wild‑type P16 activity into P16-null Hep2 cells exerts an antitumor effect.

  5. Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine.

    Science.gov (United States)

    Toro, Haroldo; Suarez, David L; Tang, De-chu C; van Ginkel, Frederik W; Breedlovea, Cassandra

    2011-03-01

    We evaluated protection conferred by mucosal vaccination with replication-competent adenovirus-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdTW68.H5ck). Commercial, layer-type chicken groups were either singly vaccinated ocularly at 5 days of age, singly vaccinated via spray at 5 days of age, or ocularly primed at 5 days and ocularly boosted at 15 days of age. Only chickens primed and boosted via the ocular route developed AI systemic antibodies with maximum hemagglutination inhibition mean titers of 3.9 log2 at 32 days of age. In contrast, single vaccination via the ocular or spray routes maintained an antibody status similar to unvaccinated controls. All chickens (16/16) subjected to ocular priming and boosting with AdTW68.H5ck survived challenge with highly pathogenic AI virus A/chicken/Queretaro/14588-19/95 (H5N2). Single ocular vaccination resulted in 63% (10/16) of birds surviving the challenge followed by a 44% (7/16) survival of single-sprayed vaccinated birds. Birds vaccinated twice via the ocular route also showed significantly lower (P < 0.05) AI virus RNA concentrations in oropharyngeal swabs compared to unvaccinated-challenged controls.

  6. Comparison of Liver Detargeting Strategies for Systemic Therapy with Oncolytic Adenovirus Serotype 5

    Directory of Open Access Journals (Sweden)

    Tien V. Nguyen

    2017-08-01

    Full Text Available Oncolytic viruses would ideally be of use for systemic therapy to treat disseminated cancer. To do this safely, this may require multiple layers of cancer specificity. The pharmacology and specificity of oncolytic adenoviruses can be modified by (1 physical retargeting, (2 physical detargeting, (3 chemical shielding, or (4 by modifying the ability of viral early gene products to selectively activate in cancer versus normal cells. We explored the utility of these approaches with oncolytic adenovirus serotype 5 (Ad5 in immunocompetent Syrian hamsters bearing subcutaneous HaK tumors. After a single intravenous injection to reach the distant tumors, the physically hepatocyte-detargeted virus Ad5-hexon-BAP was more effective than conditionally replicating Ad5-dl1101/07 with mutations in its E1A protein. When these control or Ad5 treated animals were treated a second time by intratumoral injection, prior exposure to Ad5 did not affect tumor growth, suggesting that anti-Ad immunity neither prevented treatment nor amplified anti-tumor immune responses. Ad5-dl1101/07 was next chemically shielded with polyethylene glycol (PEG. While 5 kDa of PEG blunted pro-inflammatory IL-6 production induced by Ad5-dl1101/07, this shielding reduced Ad oncolytic activity.

  7. Alpha interferon-induced antiviral response noncytolytically reduces replication defective adenovirus DNA in MDBK cells.

    Science.gov (United States)

    Guo, Ju-Tao; Zhou, Tianlun; Guo, Haitao; Block, Timothy M

    2007-12-01

    Although alpha interferon (IFN-alpha) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-alpha inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-alpha. We found that IFN-alpha efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin-Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-alpha did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-alpha treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-alpha-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections.

  8. Coinfection of a bearded dragon, Pogona vitticeps, with adenovirus- and dependovirus-like viruses.

    Science.gov (United States)

    Jacobson, E R; Kopit, W; Kennedy, F A; Funk, R S

    1996-05-01

    Four neonate bearded dragons, Pogona vitticeps, from two collections became ill and died. Multiple tissues were collected and processed for light microscopy. In hematoxylin and eosin-stained sections of liver of one lizard, numerous basophilic intranuclear inclusions were observed. In three lizards, intranuclear inclusions were primarily seen within enterocytes in the small intestine. A portion of paraffin-embedded liver of one lizard and small intestine of a second lizard were removed, deparaffinized, and examined by electron microscopy. For the most part, inclusions in the liver consisted of nonenveloped viral particles 60-66 nm in diameter. Smaller nonenveloped virions 15-17 nm in diameter were occasionally seen in association with these particles. In the intestine, inclusions consisted only of 60-70 nm particles. Based on morphology and location, the larger particles were consistent with an adenovirus. Based on size and presence within nuclei of host cells coinfected with the adenovirus-like virus, the smaller viral agent was consistent with members of the genus Dependovirus.

  9. Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months

    Science.gov (United States)

    Pelliccia, Maria; Andreozzi, Patrizia; Paulose, Jayson; D'Alicarnasso, Marco; Cagno, Valeria; Donalisio, Manuela; Civra, Andrea; Broeckel, Rebecca M.; Haese, Nicole; Jacob Silva, Paulo; Carney, Randy P.; Marjomäki, Varpu; Streblow, Daniel N.; Lembo, David; Stellacci, Francesco; Vitelli, Vincenzo; Krol, Silke

    2016-11-01

    Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10-8-10-6 M) or polyethylene glycol (PEG, molecular weight ~8,000 Da, 10-7-10-4 M) increase the half-life of a green fluorescent protein expressing adenovirus from ~48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.

  10. Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

    Institute of Scientific and Technical Information of China (English)

    朱瑾; 吴军; 陈希炜; 易绍萱; 罗高兴; 贺伟峰

    2003-01-01

    Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

  11. Effects of nanoparticle coatings on the activity of oncolytic adenovirus-magnetic nanoparticle complexes.

    Science.gov (United States)

    Tresilwised, Nittaya; Pithayanukul, Pimolpan; Holm, Per Sonne; Schillinger, Ulrike; Plank, Christian; Mykhaylyk, Olga

    2012-01-01

    Limitations to adenovirus infectivity can be overcome by association with magnetic nanoparticles and enforced infection by magnetic field influence. Here we examined three core-shell-type iron oxide magnetic nanoparticles differing in their surface coatings, particle sizes and magnetic properties for their ability to enhance the oncolytic potency of adenovirus Ad520 and to stabilize it against the inhibitory effects of serum or a neutralizing antibody. It was found that the physicochemical properties of magnetic nanoparticles are critical determinants of the properties which govern the oncolytic productivities of their complexes with Ad520. Although high serum concentration during infection or a neutralizing antibody had strong inhibitory influence on the uptake or oncolytic productivity of the naked virus, one particle type was identified which conferred high protection against both inhibitory factors while enhancing the oncolytic productivity of the internalized virus. This particle type equipped with a silica coating and adsorbed polyethylenimine, displaying a high magnetic moment and high saturation magnetization, mediated a 50% reduction of tumor growth rate versus control upon intratumoral injection of its complex with Ad520 and magnetic field influence, whereas Ad520 alone was inefficient. The correlations between physical properties of the magnetic particles or virus complexes and oncolytic potency are described herein.

  12. CAR-associated vesicular transport of an adenovirus in motor neuron axons.

    Directory of Open Access Journals (Sweden)

    Sara Salinas

    2009-05-01

    Full Text Available Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2 that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.

  13. The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro.

    Science.gov (United States)

    Grill, Jacques; Lamfers, Martine L M; van Beusechem, Victor W; Dirven, Clemens M; Pherai, D Shareen; Kater, Mathijs; Van der Valk, Paul; Vogels, Ronald; Vandertop, W Peter; Pinedo, Herbert M; Curiel, David T; Gerritsen, Winald R

    2002-11-01

    The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. We studied the interactions between tumor and adenoviruses using multicellular spheroids grown from primary brain tumor material. Using beta-galactosidase and luciferase reporter genes expressed by replication-defective adenoviruses, we showed that infection was restricted to the first layer of cells. Using a replication-competent adenovirus expressing the luciferase gene, we showed that transgene expression in the spheroid was considerably enhanced and that viral spreading deep into the 3D structure took place. In addition, a tetrazolium salt-based metabolic assay could be used to compare the oncolytic activity of different concentrations of replication-competent adenoviruses. We can conclude that organotypic spheroids offer a versatile in vitro system for studying distribution, spread, and oncolysis by adenoviruses in a clinically relevant model.

  14. The Arabic Diatessaron Project: Digitalizing, Encoding, Lemmatization

    Directory of Open Access Journals (Sweden)

    Giuliano Lancioni

    2016-04-01

    Full Text Available The Arabic Diatessaron Project (henceforth ADP is an international research project in Digital Humanities that aims to collect, digitalise and encode all known manuscripts of the Arabic Diatessaron (henceforth AD, a text that has been relatively neglected in scholarly research. ADP’s final goal is to provide a number of tools that can enable scholars to effectively query, compare and investigate all known variants of the text that will be encoded as far as possible in compliance with the Text Encoding Initiative (TEI guidelines. The paper addresses a number of issues involved in the process of digitalising manuscripts included in the two existing editions (Ciasca 1888 and Marmardji 1935, adding variants in unedited manuscripts, encoding and lemmatising the text. Issues involved in the design of the ADP include presentation of variants, choice of the standard text, applicability of TEI guidelines, automatic translation between different encodings, cross-edition concordances and principles of lemmatisation.

  15. A model for visual memory encoding.

    Directory of Open Access Journals (Sweden)

    Rodolphe Nenert

    Full Text Available Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA. All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN. Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.

  16. A model for visual memory encoding.

    Science.gov (United States)

    Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P

    2014-01-01

    Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.

  17. Adenovirus surveillance, 1982-1993, Japan. A report of the National Epidemiological Surveillance of Infectious Agents in Japan.

    Science.gov (United States)

    Yamadera, S; Yamashita, K; Akatsuka, M; Kato, N; Hashido, M; Inouye, S; Yamazaki, S

    1995-08-01

    The Infectious Agents Surveillance Center, the National Institute of Health, Japan, received 17,265 reports from 1982 to 1993 on cases from whom adenovirus was isolated or detected; 85% from 57 public health institutes and the other 15% from two national hospitals and two commercial diagnostic laboratories. The followings were found. Three major diseases caused by adenovirus were upper respiratory tract infection, gastroenteritis, and conjunctivitis. Patients of upper respiratory tract infection numbered 6,837 (40% of all patients due to adenovirus), the identified serotypes being in order of frequency types 3, 2, 1, and 5. Those of gastroenteritis numbered 1,636 (9.5%). From 40% of the gastroenteritis patients, adenovirus was detected by electron microscopy or immunochemical methods without cultivation. From the remaining 60%, virus was isolated in tissue culture; the serotypes of the isolates resembled those causing upper respiratory tract infection. Patients of conjunctivitis numbered 3,437 (20%), the frequency being in order of types 3, 4, 8, 37, and 19. Conjunctivitis due to types 3 and 4 prevailed every summer; type 3 was isolated often from children with pharyngo-conjunctival fever and the other four types were mostly from adults with epidemic keratoconjunctivitis. Type 3 had a unique feature not seen in other types: it was most frequently isolated, causing upper respiratory tract infection, gastroenteritis, conjunctivitis, and pharyngo-conjunctival fever. Reports on isolation of type 7, which has been reported to cause severe pneumonia in many other countries, were as few as 28 (0.2%).

  18. Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

    Directory of Open Access Journals (Sweden)

    Poornima L N Kotha

    2015-03-01

    Full Text Available Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR, a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

  19. Effective gene-viral therapy for telomerase-positive cancers by selective replicative-competent adenovirus combining with endostatin gene

    Institute of Scientific and Technical Information of China (English)

    Zhang Q; Liu C; Jiang M; Fang G; Liu X; Wu M; Qian Q; Nie M; Sham J; Su C; Xue H; Chua D; Wang W; Cui Z; Liu Y

    2005-01-01

    Gene-viral therapy, which uses replication-selective transgene-expressing viruses to manage tumors, can exploit the virtues of gene therapy and virotherapy and overcome the limitations of conventional gene therapy. Using a human telomerase reverse transcriptase-targeted replicative adenovirus as an antiangiogenic gene transfer vector to target new angiogenesis and making use of its unrestrained proliferation are completely new concepts in tumor management. CNHK300-mE is a selective replication transgene-expressing adenovirus constructed to carry mouse endostatin gene therapeutically. Infection with CNHK300-mE was associated with selective replication of the adenovirus and production of mouse endostatin in telomerase-positive cancer cells. Endostatin secreted from a human gastric cell line, SGC-7901, infected with CNHK300-mE was significantly higher than that infected with nonreplicative adenovirus Ad-mE in vitro (800±94.7 ng/ml versus 132.9±9.9 ng/ml) and in vivo (610±42 ng/ml versus 126 +/- 13 ng/ml). Embryonic chorioallantoic membrane assay showed that the mouse endostatin secreted by CNHK300-mE inhibited angiogenesis efficiently and also induced distortion of pre-existing vasculature. CNHK300-mE exhibited a superior suppression of xenografts in nude mice compared with CNHK300 and Ad-mE. In summary, we provided a more efficient gene-viral therapy strategy by combining oncolysis with antiangiogenesis.

  20. Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters.

    Science.gov (United States)

    Shoji, Masaki; Yoshizaki, Shinji; Mizuguchi, Hiroyuki; Okuda, Kenji; Shimada, Masaru

    2012-01-01

    Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.